International Pharmacopoeia 3rd Ed Vol 1 & 2

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The International
Pharmacopoeia
FOURTH EDITION
Pharmacop’oea Internationalis
Editio Quarta
Volume 1
World Health Organization

Geneva
2006
WHO Library Cataloguing-in-Publication Data
The International pharmacopoeia = Pharmacopoea internationalis. - 4'"’’
ed.
2 V.
1. Pharmacopoeias 2. Pharmaceutical preparations - standards 3. Pharmaceutical preparations -

analysis 4. Dosage forms - standards LWorld Health Organization.
ISBN 92 4 155301 X (LC/NLM classification: QV 738.1)
© World Health Organization 2006
World Health Organization can be obtained from WHO

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The designations employed and the presentation of the material in this publication do not imply
the expression of any opinion whatsoever on the part of the World Health Organization concern-
ing the legal status of any country, territory, city or area or of its authorities, or concerning the
delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines
for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products does not imply that they
are endorsed or recommended by World Health Organization in preference to others of a
the
similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary
products are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization to verify the infor-
mation contained in this publication. However, the published material is being distributed without
warranty of any kind, either express or implied. The responsibility for the interpretation and use
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damages arising from its use.
Printed in Singapore
Contents
Contents of Volume 1
Preface iv
History ix
Acknowledgements xiii
General Notices 1
Monographs 21
Pharmaceutical substances (A to O)
Monographs 713
Pharmaceutical substances (P to Z)
Dosage forms 945
General monographs 945
Capsules 947
Ophthalmic preparations 951
Parenteral preparations 956
Suppositories 960
Tablets 963
Topical semi-solid dosage forms 969
Specific monographs 975
Radiopharmaceuticals 1113
Methods of Analysis 1137
Reagents, test solutions and volumetric solutions 1281
Supplementary information 1439
Index 1467
Preface
The International Pharmacop’oeial' (Ph. Int.) comprises a collection of recom-

mended procedures for analysis and specifications for the determination of
pharmaceutical substances, excipients and dosage forms that is intended to
serve as source material for reference or adaptation by any WHO Member State
wishing to establish pharmaceutical requirements. The pharmacopoeia, or any
part of it, shall have legal status, whenever a national or regional authority
expressly introduces it into appropriate legislation. Further explanation of the
role of The International Pharmacopoeia is provided in the paragraphs entitled
“Scope and function” at the end of this Preface and a summary of its historic
development is provided under History.
This is the fourth edition of The International Pharmacopoeia. It comprises two
volumes; the General Notices and monographs for pharmaceutical substances
(A to O) are to be found in Volume I and the remaining monographs for phar-
maceutical substances together with those for dosage forms and radiopharma-
ceutical preparations, the methods of analysis and the reagent sections are to
be found in Volume 2. The main reason for publishing a new edition at this
time is to consolidate the texts of the five separate volumes of the third edition
and to include those new monographs for antiretroviral substances that were
adopted by the WHO Expert Committee on Specifications for Pharmaceutical
Preparations in October 2004.
In preparing this consolidated edition, the opportunity has been taken to
improve certain aspects of the layout and format of the publication. In this
edition all the monograph texts have been brought together in one main section
and the method texts in another. Each of these major sections has been divided
into appropriate sub-sections and the method texts have been numbered for
ease of cross-reference.
General Notices During preparation of this edition a review was carried
out of the General Notices. Certain additions and amendments have been made
in order to clarify the interpretation of the Pharmacopoeia and to facilitate
application of the requirements by the user. New General Notices have been
added for Definition, Manufacture and Impurities. The new notice on Defini-
tion serves to define dosage forms as being made with active ingredients of
pharmacopoeial quality and to clarify the mandatory status of certain state-
’
Published in accordance with World Health Assembly resolution WHA3.10, WHO Handbook of
Resolutions and Decisions, Vol. 1, 1977, p. 127.
IV
Preface
ments in monographs. The newon Manufacture governs the interpre-

notice
tation of statements included under this heading in monographs such as the
general monographs for dosage forms, the monographs for the different grades
of water and certain other individual monographs, for example Bleomycin
hydrochloride and Desferoxamine mesilate. The need for a notice on Impuri-
ties arose from the inclusion of information at the end of certain of the new
monographs for antiretoviral substances. Such lists of known and potential
impurities that have been shown to be controlled by the tests in a monograph
are likely to be included more widely in future.
Notices that have been significantly revised include those for Labelling,
General Requirements and Tests and Assays. General methods of analysis that
are commonly used in carrying out the tests and assays included in the mono-
graphs of The International Pharmacofoeia are described in the section on
Methods of analysis. In some cases a specific cross-reference to the method
required is provided within the monograph text. Examples include references
to Spectrometry in the infrared region, High performance liquid chromatogra-

phy (HPLC) and Limit test for heavy metals. In other cases, where the relevant
method can be inferred from the title of the test, no explicit cross-reference is
given. Examples include tests for Specific optical rotation, Sulfated ash. Loss on
drying and pH value. A statement has been added to the General notice on
General requirements to assist in the correct interpretation of the monograph
requirements, especially in those cases where there is no cross-reference. It
emphasizes that whether or not a specific cross-reference is included, the
requirements of the monographs of The International Pharmacofoeia are to be
interpreted in relation to the relevant method of analysis.
New monographs New monographs are included for the following
antiretroviral substances: Didanosine, Indinavir sulfate, Nelfinavir mesilate.
Nevirapine, Ritonavir, Saquinavir and Saquinavir mesilate. These monographs
have been developed as part of the WHO
strategy to make quality antiretro-
viral medicines more widely available to HIV-positive patients. Such specifica-
tions support the joint UNICEF-WHO-UN Prequalification project', managed
by WHO. These new monographs provide an element of choice in relation to
test methods used for identification and, where possible, a titration method for
assay, in line with established policy. However, in order to provide adequate
control of impurities, it has been found necessary to place reliance on HPLC.
Revision The monograph for Oral rehydration salts has been revised to
conform to the modified formula as published in the 13th Model List of Essen-
tial Medicines^ and in the Model Formulary 2004^. The revised formula has
a reduced sodium chloride and glucose content providing a solution with a

reduced osmolarity of 245mOsm/l. Due to the improved effectiveness of the
reduced osmolarity ORS solution, especially for children with acute, non-
^
WHO Technical Report Series, No. 933, 2005, p. 13 and, for current project information, consult
the prequalificacion website (http://mednet3.who.int/prequal).
^
WHO Technical Report Series, No. 920, 2003.
^
WHO Model Formulary, Geneva, World Health Organization. 2004.
V
The International Pharmacopoeia
cholera diarrhoea, WHO

and UNICEF now recommend that countries use and
manufacture this formulation in place of the previously recommended ORS,
that is, the one published in the third edition of The International Pharmacopoeia,
which had a total osmolarity of 31 1 mOsm/1.
The general monograph for Parenteral Preparations has been amended to
make reference to the test for bacterial endotoxins as the preferred method for
ensuring the quality of preparations with respect to pyrogens. Individual mono-
graphs no longer include recommended storage temperatures where these are
covered by the normal storage conditions as defined in the General Notices.
Method texts that have been updated include, for example, the text on High
performance liquid chromatography (HPLC). This has been revised to clarify
certain technical terms and to add advice on adjustment of chromatographic
conditions as recommended by the WHO
Expert Committee on Specifications
for Pharmaceutical Preparations.
CD-ROM This fourth edition is published simultaneously both in print and
on CD-ROM. This provides the user of The International Pharmacopoeia with a
choice of form in which to consult the publication depending on the circum-
stances and the type of enquiry. The response from users of the CD-ROM of
the third edition, published in 2004, has demonstrated the usefulness of making
the publication available electronically. The simplified structure of the fourth
edition and the improved functionality of the CD-ROM will facilitate both
reading and especially searching the text.
Future Work is on the preparation of new monographs for anti-
in progress
retroviral substances, for the associated dosage forms and for a number of fixed-
dose combination products for the treatment of HIV/ AIDS and of tuberculosis.
Revision of monographs also continues in order to improve specifications, for
example, by providing better means of impurity control or by the addition of
a dissolution test. It is intended to make additions and revisions to the fourth
edition available, at appropriate intervals, by means of printed Supplements and
updated CD-ROMs. Meanwhile, attention is drawn to the WHO Medicines
website (htpp://www.who.int/medicines), where finalized texts of mono-
graphs adopted by the Expert Committee are provided for information together
with much other detailed information of relevance to all those responsible for
the quality assurance of medicines.
Scope and function

The activities related to The International Pharmacopoeia are an essential element
in the overall quality control and assurance of pharmaceuticals contributing to
the safety and efficacy of medicines.' The selection of monographs for inclu-
sion in The International Pharmacopoeia recognizes the needs of specific disease
programmes and the essential medicines nominated under these programmes;
WHO Technical Report Series, No. 908, 2003, Annex 2.

Preface
it is based primarily on those substances included in the current WHO Model

List of Essential Medicines d The work on The International Pharmacopoeia is carried
out in collaboration with members of the WHO Expert Advisory Panel on the
International Pharmacopoeia and Pharmaceutical Preparations as well as with
specialists from regulatory authorities, from industry and from other institu-
tions (see under Acknowledgements). Clearly defined steps are followed in the
development of new monographs.^
It is emphasized that pharmacopoeial specifications represent only one
element of the quality assurance of medicines. Pharmaceutical substances and

dosage forms for human use, as described in a monograph of The International
Pharmacopoeia, should be manufactured according to the current requirements
of Good Manufacturing Practices (GMP)^ The processes, premises, equipment,
and installations should also comply with the provisions of the product licence
or marketing authorization, relevant regulations and, in the case of products
destined for export, with any binding international norms that would affect
their entry onto the market. In many cases this compliance cannot be verified
by analysing a sample of the final product against a pharmacopoeial mono-
graph. The national, regional or other competent authority will need to ensure
that all relevant provisions have been met by any means at its disposal, includ-
ing use of appropriate certificates, inspection of the manufacturing sites or
testing of samples beyond specifications.
It should be understood that a distinction exists between pharmacopoeial
standards and manufacturers' release specifications. Pharmacopoeial standards

are publicly-available compliance specifications and provide the means for an
independent check of the quality of a product at any time during its shelf-life.
Although release specifications must be compatible with pharmacopoeial spec-
ifications, they may differ in several respects. In order to ensure compliance
with the pharmacopoeia, the manufacturers’ specifications may need to be
more exacting than corresponding pharmacopoeial requirements. The manu-
facturer is entitled to use other analytical methods for routine testing and, more-
over, he may assure himself that the requirements of the pharmacopoeia wiU
be met by means other than routinely performing all of the tests in the mono-
graph. For example, on occasions, in-process controls and manufacturing
process validation studies may have already provided the necessary assurances
with respect to certain aspects of the monograph.
The requirements of the monographs are not framed to detect all possible
impurities. The present tests are designed to determine impurities on which
attention should be focused, to fix the limits of those that are tolerable to a
certain extent, and to indicate methods for ensuring the absence of those that
are undesirable. It is, therefore, not to be presumed that an impurity can be tol-
erated because it has not been precluded by the prescribed tests. In some purity
^
WHO Technical Report Series, No. 933, 2005.
^
WHO Technical Report Series, No. 929, 2005. p. 5.
^ For the current WHO recommendations, consult the WHO Medicines website
(htpp://www.who,int/medicines).
vii
tests, limits are indicated additionally in brackets in percentage terms: as stated

in the General Notices, such limits are given for information only.
Pharmaceutical preparations (dosage forms) that are produced on a large
scale and that will be stored before use should undergo testing to show phys-
icaland chemical stability during storage over the claimed shelf-life.'
The degreeof protection provided by pharmacopoeial standards will
depend not only on their technical content but also to a great extent on how
they are used. The specified tolerances and limits allow for the inherent varia-
tions that occur during production and packaging, as well as for subsequent
degradation within normal handling and storage conditions and for any accept-
able variance of analytical results.
* * *
When pharmacopoeial standards are used to establish the compliance

of products with regulatory requirements, the following principles
apply:
• The interpretation of a monograph must be in accordance with all

general requirements and testing methods, texts, or notices per-
taining to it as found in this edition.
• No further tolerances are to be applied to the limits prescribed.
• A product is not of pharmacopoeial quality unless it complies with
all the requirements stated.
* * *
’ Quality assurance of pharmaceuticals: a compendium of guidelines and related materials. Volume 1.

Geneva, World Health Organization, 1997. For the current WHO recommendations, consult the
WHO Medicines website (htpp://www.who.int/medicines).
viii
History
The history of The International Pharmacop’oeia dates back to 1874 when the need
to standardize terminology and to specify dosages and composition of drugs
led to attempts to produce an international pharmacopoeial compendium. The
first conference, called by the Belgian Government and held in Brussels in 1902,
resulted in the Agreement for the Unification of the Formulae of Potent Drugs,
which was ratified in 1906 by 19 countries. The outcome considerably influ-
enced the subsequent publication of national pharmacopoeias.
A second agreement, the Brussels Agreement, was drawn up in 1925 and
This 41-article agreement stipulated that the League of Nations
ratified in 1929.
would be responsible for the administrative work to produce a unified phar-
macopoeia, and a permanent secretariat of an international organization would
coordinate the work of national pharmacopoeial commissions. General prin-
ciples for the preparation of galenicals, maximal doses, nomenclature, and
biological testing of arsenobenzones were included in the articles of this
agreement, as was a table of dosage strengths and descriptions for 77 drug
substances and preparations.
In response to repeated calls from pharmaceutical experts in various coun-
tries that the Brussels Agreement be revised and extended to cover an interna-
tional pharmacopoeia, the Health Organization of the League of Nations set up

a Technical Commission of Pharmacopoeial Experts in 1937. This first com-
mittee comprised seven experts from Belgium, Denmark, France, Netherlands,
Switzerland, the United Kingdom (Chairman), and the United States of
America.
In 1947 the Interim Commission of WHO took over the work on pharma-
copoeias previously undertaken by the Health Organization of the League of
Nations, and set up an Expert Committee on the Unification of Pharmacopoeias
to continue the work of the League’s Technical Commission. The aim of the
Expert Committee was to produce a draft international agreement for the uni-
fication of pharmacopoeias, modifying and extending the existing Agreement
for the Unification of the formulae of Potent Drugs.
In 1948 the first World Health Assembly approved the establishment of the
Expert Committee by the Interim Commission. In 1951 this became the Expert
Committee on the International Pharmacopoeia; and subsequently, in 1959, the
Expert Committee on Specifications for Pharmaceutical Preparations. The panel
has always been named the WHO Expert Advisory Panel on the International
Pharmacopoeia and Pharmaceutical Preparations.
Article 2 of the WHO Constitution states that one of the functions of the
Organization is “to develop, establish and promote international standards with
respect to food, biological, pharmaceutical and similar products’’. The Interna-
tional Pharmacopoeia falls clearly into this category. In this context also the Third
World Health Assembly in 1950 adopted a resolution to create the International
Nonproprietary Names
(INN) Programme in order to identify pharmaceutical
substances unambiguously on a worldwide basis and to provide a single non-
proprietary name to be used in monographs.
First edition
The Third World Health Assembly, held in May 1950, formally approved the
publication of the Pharmacopoea Internationalis and recommended, in accordance
with Article 23 of the WHO Constitution, “the eventual inclusion of its provi-
sions by the authorities responsible for the pharmacopoeias’’. It was thus rec-
ommended that the Pharmacopoea Internationalis was not intended to be a legal
pharmacopoeia in any country unless adopted by the pharmacopoeial author-
ity of that country. From that moment the World Health Organization consti-
tuted the Permanent International Pharmacopoeia Secretariat.
The first edition, published with the aim of creating a worldwide, unified
pharmacopoeia, relied on collaboration with national pharmacopoeia commis-
sions for its preparation. It was published in two volumes (1951 and 1955) and
a supplement (1959) in English, French and Spanish, and was also translated
into German and Japanese. Altogether, it included 344 monographs on drug
substances, 183 monographs on dosage forms (capsules, injections, tablets and
tinctures) and 84 tests, methods, and general requirements.
A large number of national pharmacopoeias and official lists were examined
and assistance was also obtained from the International Pharmaceutical Feder-
ation (FIP) to determine the selection of substances and products to be described
in the pharmacopoeia. Latin was chosen for the monograph titles because of
its distinction as an international language. Experts collaborated with the WHO
Expert Committee on Biological Standardization with regard to biological
products, and with those working in specific divisions, e.g. malaria, maternal
and child health, mental health, and venereal diseases, to help collate the
required information.
Second edition
The second edition was published in 1967 as Specifications for the Quality Control
of Pharmaceutical Preparations, with a subtitle classifying it as the second edition

of The International Pharmacopoeia.
Owing development of new analytical techniques such as infrared
to the
spectroscopy, chromatography (column, paper and thin-layer), non-aqueous
titration, and radioactivity, the second edition incorporated numerous alter-
ations and constituted a revision of the first edition.
The selection of monographs and appendices was based largely on the avail-
ability, at the time of preparation, of specifications intended for publication in
X
History
national pharmacopoeias and in other volumes of specifications for pharma-

ceutical quality control. Specifications for 162 pharmaceutical preparations not
included in the first edition were introduced in the second edition, while 114
monographs were deleted, based on feedback from the first edition. New ana-
lytical methods were also added. The specifications and methods in the mono-
graphs were tested in a number of national pharmacopoeial and pharmaceutical
quality control laboratories, in pharmaceutical manufacturers’ laboratories, and
at various pharmacopoeial institutes.
Special thanks were expressed to the authorities of the British Pharma-
copoeia and the United States Pharmacopeia.
Third edition
In 1975 the purpose of The International Pharmacofoeia was reconsidered. It was
decided that the publication should focus more on the needs of developing
countries and recommend only simple, classical chemical techniques that had
been shown to be sound. Priority would be given to drugs that were widely
used throughout the world, with emphasis on the therapeutic value of these
drugs. High priority would be accorded to drugs important to WHO
health pro-
grammes, and to those likely to contain impurities arising from degradation or
due to difficulties in their manufacture. Wherever possible, classical procedures
would be used in the analytical methods so that the pharmacopoeia could be
applied without the need for expensive equipment. Where a sophisticated ana-
lytical method was suggested, an alternative, less complex method would also
be proposed.
Since 1979, the drugs appearing in The International Pharmacopoeia have been
selected from the list of essential drugs based on the first report of the WHO
Expert Committee on the Selection of Essential Drugs. Specifications are pro-
vided in the monographs for the identification, purity, and content of the essen-
tial drugs appearing in the WHO Model List of Essential Drugs, and their
updates.
The Third edition eventually consisted of five volumes: Volume 1 contained
general methods of analysis; Volumes 2 and 3, quality specifications for the
majority of essential drug substances in the WHO Model List of Essential
Drugs and Volume 4, information on tests, methods, and general requirements
and quality specifications for pharmaceutical substances, excipients, and dosage
forms. Volume 5, the final volume, contained tests and general requirements
for dosage forms and quality specifications for pharmaceutical substances and
tablets, which practically completed the list of monographs for active pharma-
ceutical substances, and a section on antimalarial drug substances and their
most widely used dosage forms.
Fourth edition
The fourth edition of The International Pharmacopoeia comprises two volumes
published together; Volume I contains the General Notices and many of the
monographs for pharmaceutical substances and Volume 2 contains the remain-
XI
ing monographs for pharmaceutical substances together with those for dosage
forms and radiopharmaceutical preparations, the methods of analysis and the
reagents section and index. This edition consolidates and updates the texts of
the five separate volumes of the third edition and includes new monographs
for antiretroviral substances. The fourth edition is published simultaneously
both in print and on CD-ROM.
Acknowledgements
The specifications included in the fourth edition were developed in collabora-

tion with members of the WHO Expert Advisory Panel on the International
Pharmacopoeia and Pharmaceutical Preparations, other specialists, and the
WHO Collaborating Centres on quality control and quality assurance.
Thanks are also due to the Controller of Her Majesty’s Stationery Office,
the European Pharmacopoeia Commission and the United States Pharma-
copeial Convention, Inc. for providing valuable background material.
The following specialists participated both in person and by correspondence
in the preparation of this edition: Professor H.Y. Aboul-Enein, Bioanalytical
and Drug Development Laboratory, Department of Biological and Medical
Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi
Arabia; Professor I. Addae-Mensah, Department of Chemistry, University of
Ghana, Legon, Accra, Ghana; Professor J.-M. Aiache, Laboratory of Biophar-

macy, Faculty of Pharmacy, University of Clermont-Ferrand, Clermont-Ferrand,
France; Dr K. Akalin, Ministry of Health, Ankara, Turkey; Professor P.I. Akubue,
Department of Pharmacology and Toxicology, University of Nigeria, Nsukka,
Lagos, Nigeria; Dr H. Ali, Novartis Pharma Ltd, Basel, Switzerland; Dr S.L. Ali,
Association of German Pharmacists’ Central Laboratory, Eschborn, Germany;
Professor H.R. Altorfer, Department of Pharmacy, Swiss Federal Institute of
Technology, Zurich, Switzerland; Dr N. Anand, Lucknow, Uttar Pradesh, India;
Dr P. Arends, Skagen Apotek, Skagen, Denmark; Dr A. Artiges, European Direc-
torate for the Quality of Medicines, Gouncil of Europe, Strasbourg, France;
Dr K. Bailey, Bureau of Drug Research, Health and Welfare, Health Protection
Branch, Ottawa, Ontario, Ganada; Professor I. Bayer, Budapest, Hungary;
Dr C. Becerril Martinez, Subdireccion de Normalizacion y Farmacovigilancia,
Mexico, DF, Mexico; Mr J.Y. Binka, Accra, Ghana; Professor C.E Bittencourt,
Brazilian Pharmacopoeia Permanent Revision Committee, Santa Maria RS,
H. Blume, Bad Homburg, Germany; Dr Y. Boer, Laboratory of
Brazil; Professor
theDutch Pharmaceutical Society, The Hague, Netherlands; Dr L. Borka, Oslo
Norway; Dr R. Boudet-Dalbin, Faculty of Pharmaceutical Sciences, Rene
Descartes University, Paris, France; Professor D.D. Breimer, Center for Bio-
Pharmaceutical Sciences, Sylvius Laboratories, Leiden, Netherlands; Dr Briskot,
Bayer AG, Wuppertal, Germany; Dr T. Burat, Drug and Gosmetics Research
Department, Central Institute of Hygiene, Ankara, Turkey; Dr M.N. Caetano
Pisciottano, Department of Pharmacy, Federal University of Pernambuco,
Recife, Brazil; Dr D.H. Calam, Pewsey, Wiltshire, England; Dr R. Carter, Knoll
AG, Basel, Switzerland; Dr E. Charton, European Directorate for the Quality
of Medicines, Council of Europe, Strasbourg, France; Ms Cheah Nuan Ping,

Quality Assurance, Centre for Analytical Science, Health Sciences Authority,
Singapore; Dr G.P. Chiu, The Upjohn Company, Kalamazoo, MI, USA; Dr J.A.
Clements, Scientific and Technical Services Division, Royal Pharmaceutical
Society of Great Britain, London, England; Mrs E.M. Cortes Montejano, Min-
istry of Health and Consumer Affairs, Madrid, Spain; Professor P.E Coville,
School of Pharmacy, University of Otago, Dunedin, New Zealand; Dr R. Cox,
Manager Corporate Compendia and Standards, Abbott Laboratories, Abbott
Park, Illinois, USA; Dr P. Christen, Pharmaceutical Analytical Chemistry Labo-
ratory, Faculty of Sciences, Pharmacy Section, University of Geneva, Geneva,
Switzerland; Professor T.G. Dekker, Research Institute for Industrial Pharmacy,
North-West University, Potchefstroom, South Africa; Dr E. Demant, Bayer,
Pharmaceutical Division, West Haven, CT, USA; Mrs Ding Lixia, National Insti-
tute for the Control of Pharmaceutical and Biological Products, Beijing, People’s
Republic of China; Dr A. Dobrotvorsky, Ministry of Health and Medical Indus-
try State Pharmacopoeia Committee, Moscow, Russian Federation; Professor
E. Doelker, Department of Pharmaceutics, University of Geneva, Geneva,
Switzerland; Dr Doll, Aventis Pharma HG, Frankfurt am Main, Germany; Pro-
fessor J.B. Dressman, Institute for Pharmaceutical Technology, Johann Wolfgang
Goethe University, Frankfurt am Main, Germany; Dr P.O. Emafo, Benin City,
Nigeria; Dr R.R. Engle, Fort Myers, EL, USA; Dr E. Ehrin, Central Laboratory,
Kungens Kurva, Sweden; Dr K. Florey, Princeton, NJ, USA; Dr H. Fukuda,
Society of Japanese Pharmacopoeia, Tokyo, Japan; Professor D. Ganderton,
Gheriton Bishop, England; Mr G. Ghilardi, Novartis Pharma Ltd, Basel, Switzer-
land;Ms E. Gomez, Quality Assurance, Phibro Animal Health Corporation,
USA; Dr K. Goode, GlaxoSmithKline, Ware, Hertfordshire,
Ridgefield Park, NJ,
United Kingdom; Dr L.T. Grady, Drug Standards Division, The United States
Pharmacopeial Gonvention, Inc., Rockville, MD, USA; Dr S.A. Hanna, Bristol-
Myers Squibb, New Brunswick, NJ, USA; Dr H. Hauth, Novartis Pharma Ltd,
Basel, Switzerland; Dr P. Hayes, School of Pharmacy, University of Otago,
Dunedin, New Zealand; Dr W. Hecker, Novartis Pharma Ltd, Basel, Switzer-
land; Dr H. Heimbach, Bayer AG, Wuppertal, Germany; Mr J.-P. Helenport,
Rhone-Poulenc Rorer Doma, Antony Cedex, France; Dr M. Hessel, Messer
Griesheim GmbH, Duisburg, Germany; Mr A. Holbrook, Zeneca Pharmaceu-
ticals, Macclesfield, Gheshire, England; Professor J. Hoogmartens, Laboratory
for Pharmaceutical Ghemistry and Drug Analysis, Leuven, Belgium; Mr R.K.

Howard, Therapeutic Goods Administration Laboratories, Department of
Gommunity Services and Health, Woden, AGT, Australia; Dr Hyung Kook Kim,
Division of Drug Standardization, National Institute of Health, Seoul, Repub-
lic of Korea; Dr R. Ilyas, National Agency of Drug and Food Control, Jakarta,
Indonesia; Professor Jin Shaohong, National Institute for the Control of Phar-
maceutical and Biological Products, Ministry of Health, Beijing, People’s Repub-
lic of China; Dr
Kashemsant, Department of Medical Sciences, Ministry of
P.
Public Health, Nonthaburi, Thailand;Dr A. Kaukinen, Helsinki, Finland; Dr E.

Keller, Dr L. Kelly, Therapeutic Goods
Novartis Pharma Ltd, Basel, Switzerland;
Administration Laboratories, Department of Gommunity Services and Health,
XIV
Acknowledgements
Woden, ACT, Australia; Mr Keuser, Novartis Pharma Ltd, Basel, Switzerland;

I.
Dr J. Kincaid, Merck, Manufacturing Division, Westpoint, PA, USA; Mr R.H.

King, Division of Standards Development, The United States Pharmacopeial
Convention, Inc., Rockville, MD, USA; Mr M. Krummen, Mepha AG, Aesch,
Basel, Switzerland; Dr P. Kucera, Wyeth-Ayerst Pharmaceuticals, New York,
NY, USA; Dr C.S. Kumkumian, Center for Drug Evaluation and Research, Food
and Drug Administration, Rockville, MD, USA; Dr S. Kuttatharmmakul, Head
of Inspection Control Section, The Government Pharmaceutical Organization,
Bangkok, Thailand; Dr TP. Layloff, The United States Pharmacopeial Conven-
tion Inc., Rockville, MD, USA; Mr M.A. Lee, Analytical Sciences Development,
SmithKline Beecham Pharmaceuticals, Worthing, England; Dr P. Lim, Palo Alto,
CA, USA; Professor J. Liptak, Budapest, Hungary; Dr H. Lomska, Head, Phar-
macopoeia Department, State Institute for Drug Control, Czech Republic;
Ms Low Min Yong, Pharmaceutical Laboratory, Centre for Analytical Science,
WHO Collaborating Centre for Drug Quality Assurance, Health Science
Authority, Singapore; Dr H. Ludwig, Novartis Pharma Ltd, Basel, Switzerland;
Dr C.B. Lugt, Artecef BV, Maarssen, Netherlands; Dr G. Maillard, The French
Pharmaceutical Manufacturers Association, Paris, France; Dr M.K. Majumdar,
Jadavpur, Calcutta, India; Dr G. Maldener, Bayer AG, Wuppertal, Germany;
Associate Professor L. Martinec, State Institute for Control of Drugs, Bratislava,
Slovakia; Dr I.N. Matondo, Scientific Liaison Office, Harare, Zimbabwe; Mr
E Maxi, Novartis Pharma Ltd, Basel, Switzerland; Dr I.J. McGilveray, Biophar-
maceutics Section, Health Protection Branch, Ottawa, Ontario, Canada; Dr J.H.
McB Miller, European Directorate for the Quality of Medicines, Council of
Europe, Strasbourg, France; Dr M. Mehmandoust, Evaluateur Pharmaceutique
Analyste, Agence Francaise de Securite Sanitaire des Produits de Same, Saint
Denis, France; Dr S. Messner, Corporate Compendial Liaison Abbott Labora-
tories, Abbott Park, Illinois, USA; Dr N. Miyata, Faculty of Pharmaceutical Sci-
ences and Chemistry, Graduate School, Nagoya City University, Nagoya, Japan;
Mr M.G. Moester, Inspectorate of Health Care, Ministry of Health, Welfare
and Sport, Rijswijk, Netherlands; Dr H. Moller, Aventis Pharma Ltd, Frankfurt
am Main, Germany; Mr G. Mondain-Monval, Air Liquide Same France, Paris,
France; Mrs Z.J. Montbrun de Reinfeld, National Institute of Hygiene Rafael
Rangel, Garacas, Venezuela; Mrs A.B. Moraes da Silva, Escola National de
Saude Publica-Fiocruz, Manguinhos, Rio de Janeiro, Brazil; Dr O. Morin, Direc-
tor, Regulatory and Scientific Affairs, International Federation of Pharmaceuti-
cal Manufacturers Associations, Geneva, Switzerland; Dr H. Muller, Messer

Griesheim GmbH, Duisburg, Germany; Mr R.D. Munro, Therapeutic Goods
Administration, Woden, ACT, Australia; Dr M. Negwer, Berlin, Germany;
Dr Ng Tju Lik, Singapore; Dr J.D. Nicholson, Medicines Testing Laboratory,
Department of Pharmaceutical Sciences, Royal Pharmaceutical Society of Great
Britain, Edinburgh, Scotland; Dr E. Njau, Arusha, United Republic of Tanzania;
Professor L. Ogunlana, Lanpharm Laboratories, Lagos, Nigeria; Professor TL.
Paal, National Institute of Pharmacy, Budapest, Hungary; Dr PR. Pabrai, New
Delhi, India; Dr H. Partenheimer, Novartis Pharma Ltd, Basel, Switzerland; Pro-
fessor X. Perlia, Pharmaceutical Institute, Swiss Federal Institute of Technology,
XV
Zurich, Switzerland; Dr Pham Hoang Ngoc, National Center for Scientific

Research, Institute of Chemistry, Hanoi, VietNam; Dr J. Pogany, Budapest,
Hungary; Ms A. Poompanich, Division of Drug Analysis, Department of
Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand; Miss M.L.
Rabouhans, London, England; Professor M. Rafiee-Tehrani, Industrial Pharmacy
Research Laboratory, Tehran University of Medical Sciences, Tehran, Islamic
Republic of Iran; Dr J.-L. Robert, National Health Laboratory, Luxembourg; Dr
S. Roenninger, E Hoffmann-La Roche, Basel, Switzerland; Dr K. Satiadarma,
Bandung, Indonesia; Dr M. Scheiwe, Mepha AG, Aesch, Basel, Switzerland;

Dr B. Schmauser, Federal Institute for Drugs and Medical Devices, Department
of Pharmaceutical Quality, Bonn, Germany; Dr PJ. Schorn, Offenburg, Germany;
Dr G. Schwartzman, Sarasota, EL, USA; Dr P. Sidhu, Therapeutic Goods
Administration Laboratories, Department of Community Services and Health,
Woden, ACT, Australia; Dr G.N. Singh, Indian Pharmacopoeia Commission,
Central Indian Pharmacopoeia Laboratory, Ghaziabad, India; Dr S. Siiskonen,
International Pharmaceutical Federation (FIP), The Hague, Netherlands; Dr K.
Sinivuo, National Agency for Medicines, Helsinki, Finland; Dr CJ.P. Siregar,
National Quality Control Laboratory of Drug and Food, Ministry of Health,
Jakarta, Indonesia; Dr L. Slamet, Deputy for Therapeutic Product, Narcotic, Psy-
chotropic and Addictive Substance Control, National Agency of Drug and Food
Control, Jakarta, Indonesia; Dr M. Smid, State Institute for Drug Control,
Prague, Czech Republic; Dr R. Soliman, Alexandria, Egypt; Dr J.-M. Spieser,
European Directorate for the Quality of Medicines, Council of Europe, Stras-
bourg, France; Dr H.D. Spitz, International Product Support, The RW. Johnson
Pharmaceutical Research Institute, Raritan, NJ, USA; Dr L. Stefanini-Qresic,
Croatian Institute for Medicines Control, Zagreb, Croatia; Dr A. Sulistiowati,
National Quality Control Laboratory of Drug and Food, Ministry of Health,
Jakarta, Indonesia; Dr S. Sur, Inspectorate for Quality Control of Medicines,
Kiev, Ukraine; Dr Y. Takeda, Society of Japanese Pharmacopoeia, Tokyo, Japan;
Professor Tang Lin-hua, Institute of Parasitic Diseases, Chinese Academy of
Preventive Medicine, Shanghai, People’s Republic of China; Professor K. Thoma,
University of Munich, Munich, Germany; Dr K.M. Thomas, APL Research
Gentre, Hyderabad, India; Dr H.G. ToUe, F. Hoffman-La Roche Ltd, Basel,
Switzerland; Dr H. Tomankova, International Pharmacopoeia Department,
State Institute for Drug Control, Prague, Czech Republic; Dr Torok, Quality
I.
Control Division, National Institute of Pharmacy, Budapest, Hungary; Mrs A.M.

Trapletti, E Hoffman-La Roche Ltd, Basel, Switzerland; Dr EG. Treagust,
SmithKline Beecham Pharmaceuticals, Worthing, West Sussex, England; Ms
Metta Treebamroong, Bureau of Drug and Narcotics, Department of Medical
Sciences, Ministry of Public Health, Nonthaburi, Thailand; Mr R.B. Trigg, Rick-
mansworth, Hertfordshire, England; Professor Trin Van Quy, National Institute
of Drug Quality Control, Hanoi, Viet Nam; Professor Tu Guoshi, Division of
Pharmaceutical Ghemistry, National Institute for the Gontrol of Pharmaceuti-
cal and Biological Products, Ministry of Health, Beijing, People’s Republic of
China; Professor L. Turakka, National Agency for Medicines, Helsinki, Finland;
Dr M. Uchiyama, Japan Pharmacists Education Center, Tokyo, Japan; Dr V.
xvi
Acknowledgements
Uziely, Institute for the Standardization and Control of Pharmaceuticals, Min-

istry of Health, Jerusalem, Israel; Dr J. van Rompay, Quality Assurance, Janssen
Research Foundation, Beerse, Belgium; Mr L, Virgili, Bristol-Myers Squibb
Company, New Brunswick, NJ, USA; Dr J.P, Vora, Bangalore, India; Professor
B. Vrhovac, Department of Medicine, University Hospital Medical School,
Zagreb, Croatia; Dr I. Vukusic, Podravka-Food, Pharmaceuticals and Cosmet-
ics Industries, Zagreb, Croatia; Dr E. Wachberger, E Hoffman-La Roche Ltd,
Basel, Switzerland; Mr Wang Cunzhi, Kunming Pharmaceutical Corporation,

Kunming, People’s Republic of China; Dr Wang Ping, Chinese Pharmacopoeia
Commission, Beijing, People’s Republic of China; Dr B. Warren, Canberra Ana-
lytical Laboratories Pty Ltd, Erindale Centre, ACT, Australia; Mrs M. Wester-
mark, Astra Zeneca, Sodertalje, Sweden; Professor W. Wieniawski, Polish
Pharmaceutical Society, Warsaw, Poland; Dr M, Wierer, European Directorate
for the Quality of Medicines, Strasbourg, France; Dr M Wiggins, Merck & Co.,
Inc., West USA; Dr A. Wilk, Complex-Actives Division,
Point Pennsylvania,
Information and Standards Development, United States Pharmacopeia,
Rockville, MD, USA; Mr G.T. Williams, Dragon Pharmaceuticals Ltd, Merthyr
Tydfil, Wales;Dr R.L. Williams, United States Pharmacopeia, Rockville MD,
USA; Dr Withell, Therapeutic Goods Administration Laboratories, Depart-
J.
ment of Community Services and Health, Woden, ACT, Australia; Dr C. Wong-

pinairat. Bureau of Laboratory Quality Standard, Department of Medical
Sciences, Ministry of Public Health, Nonthaburi, Thailand; Dr Woo Soo Qn,
Singapore; Professor Xiao-Yu Li, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai, People’s Republic of China; Professor Yang

Zhong-Yuan, Guangzhou Municipal Institute for Drug Gontrol, Guangzhou,
People’s Republic of Ghina.
Furthermore, comments were obtained from the International Federation of
Pharmaceutical Manufacturers Associations, Geneva, Switzerland; the World
Self-Medication Industry, Ferney-Voltaire, France; Pharmacopoeial Gom-
missions, national institutes for quality control of drugs, and drug research
laboratories, and from the following WHQ
Collaborating Centres: WHQ
Collaborating Centre for Drug Quality Assurance, Therapeutic Goods Admin-
istration Laboratories, Department of Community Services and Health, Woden,
ACT, Australia; WHQ Collaborating Centre for Drug Quality Assurance,
National Institute for the Control of Pharmaceutical and Biological Products,
Beijing, People’s Republic of China; WHQ Collaborating Centre for Biophar-
maceutical Aspects of Drug Quality Control, Laboratory of Biopharmacy,
Faculty of Pharmacy, University of Clermont-Ferrand, Clermont-Ferrand,
France; the former WHQ Collaborating Centre for Stability Studies of Drugs,
Regional University Hospital, Nantes, France; WHO Collaborating Centre for
Drug Information and Quality Assurance, Budapest, Hungary; WHO Collabo-
rating Centre for Quality Assurance of Essential Drugs, Central Drugs Labora-
tory, Calcutta, India; former
the WHO
Collaborating Centre for Quality
Assurance of Essential Drugs, Directorate General of Drug and Food Control,
Ministry of Health, Jakarta, Indonesia; the former WHO
Collaborating Centre
for Drug Quality Control, State Research Institute for the Standardization and
Control of Drugs, Ministry of Health, Moscow, Russian Federation; WHO Col-

laborating Centre for Drug Quality Assurance, Centre for Analytical Science,
Health Sciences Authority, Singapore; WHO Collaborating Centre for Chemi-
cal Reference Substances, National Corporation of Swedish Pharmacies,
Central Laboratory, Kungens Kurva, Sweden; WHO Collaborating Centre for
International Infrared Reference Spectra, Swiss Federal Institute of Technology,
Zurich, Switzerland; WHO Collaborating Centre for Quality Assurance of
Essential Drugs, Bureau of Drug and Narcotics, Department of Medical
Sciences, Ministry of Public Health, Nonthaburi, Thailand.
The World Health Organization takes this opportunity to express its grati-
tude to all those people and institutions involved in the preparation of this 4*
edition and its 1. Addae-Mensah, Professor J. Hoog-
special thanks to Professor
martens, and Miss M.L. Rabouhans who also served as Chairpersons, and to
Mrs K. Bremer, Molzon, Dr R. Pandjaitan, Professor Jin Shaohong, Ms
Dr J.A.
Metta Treebamroong and Professor Yang Zhong-Yuan who also served as Vice-
Chairpersons of the WHO
Expert Committee on Specifications for Pharma-
ceutical Preparations from 1995 to 2005. Members of the WHO
Secretariat
involved in the elaboration of the publication were Dr S. Kopp (Secretary to
the above Expert Committee), Mrs W. Bonny, Mrs A.N. Lo Conte, Miss M.L.
Rabouhans, Miss M. Schmid and Dr P. Vanbel.
xviii
General Notices
1
1
General Notices: Contents
Monograph nomenclature 3
Chemical formula and relative molecular mass 3
Chemical names 3
Other names 4
Definition 4
Manufacture 4
Description 4
Solubility 4
Category 5
Storage 5
Stability information 6
Labelling 6
Additional information 6
General requirements 6
Identity tests 7
Examination in ultraviolet light 7
Clarity of solution 8
Colourless solution 8
Loss on drying 8
Tests and assays 8
Indicators for visual determination of pH values 9
Accuracy and Precision 9
Calculation of results 10
Impurities 10
Patents and trademarks 10
Reagents, reference substances, test solutions, and volumetric
solutions 1
Reference substances 11
Reference spectra 12
General Notices
Apffaendices to the 13
Abbreviations and symbols 13
Units of measurement 16
Names, symbols, and relative atomic masses of certain elements 19
2
General notices
Monograph nomenclature
The main monograph title is given in Latin and English. A singular Latin form
of the recommended or proposed International Nonproprietary Name (INN) is
applied, unless otherwise indicated.
All names of substances, with certain traditional exceptions, are treated as
second declension neuter substantives (e.g. Ethosuximidum).
The method of nomenclature adopted for salts is the traditional one of
placing the name of the acid component in the nominative case (either second
declension neuter or third declension masculine) and the other component in
(e.g. Codeini phosphas). With compounds that are not derived
the genitive case
from true both components of the title are placed in the nominative case,
acids,
main component as a neuter substantive and using an adjectival
treating the
form of the complementary component in agreement with this substantive
(e.g. Cloxacillinum with "natricus” as the adjectival form of Natrium, thus
Cloxacillinum natricum).
Dosage forms are chosen by placing the Latin name of the dosage form in the
nominative case and the active ingredient in the genitive case, (e.g. AmpiciUini
capsulae, Ephedrini sulfatis injectio). Whenever the dosage form is intended
for reconstitution, this is indicated by using the word “ad” and the appropriate
application in the fourth declension (e.g. AmpiciUini natrici pulvis ad
injectionem).
Chemical formula and relative molecular mass

When the chemical composition of a pharmacopoeial substance is known or
generally accepted, the empirical chemical formula and the relative molecular
mass are given. For organic substances, the graphic formula, when known or
generally accepted, is also given. These formulae and relative molecular masses
are given at the beginning of the monographs and are those of the chemically
pure substances; they are not to be regarded as an indication of the purity of
the substance under test. Elsewhere, in statements of specifications of purity
and strength, and in descriptions of processes for assay, it is evident from the
context that the formulae denote the pure chemical substances.
Chemical names
The chemical names are given in accordance with the rules laid down by the
International Union of Pure and Applied Chemistry (lUPAC). In many cases,
when equally acceptable alternative names may be construed under these rules,
more than one systematic name is given. Such alternative names are given espe-
cially when changes in the interpretation of lUPAC rules occurring in recent
names used for the
years have led to substantial modifications of the chemical
substance.The recognition of substances is further facilitated by a registry
number established by the Chemical Abstracts Service of the American
Chemical Society (CAS No.).
3
Other names
Commonly used synonyms are given to aid identification of the article in
question.
Definition
Statements given under the heading Definition or immediately after the
heading Requirements constitute the official definition of the substance or
dosage form that is the subject of the monograph. They constitute instructions
or requirements with which the article must comply.
A dosage form that is the subject of a specific monograph must be prepared
using an active ingredient that complies with the corresponding substance
monograph. For example, the active ingredient used in preparing Artesunate
tablets must comply with the monograph for Artesunate. Similarly, where an
excipient for which there is a pharmacopoeial monograph is used in preparing
such a dosage form, the excipient must comply with that monograph. For
example, if lactose is used in preparing Artesunate tablets, it must comply with
the monograph for Lactose.
Manufacture
The manufacturing facilities and the manufacturing process for a substance or
dosage form that is the subject of a monograph in the International Pharma-
copoeia must meet the current WHO requirements of Good Manufacturing
Practice’. Where must be manufactured in accordance
applicable, substances
with the WHO "Recommendations on Risk of Transmitting Animal Spongi-
form Encephalopathy Agents via Medicinal Products” reproduced in the section
Supplementary Information.
Statements under the heading manufacture draw attention to particular
aspects of the manufacturing process but are not necessarily comprehensive.
They may be in the form of mandatory instructions to manufacturers or, where
clear from the form of wording used, they may provide guidance. In the general
monographs for dosage forms, information is given that is intended to provide
broad guidelines concerning the main steps to be followed during production,
indicating those that are most important.
Description
Statements given under this heading are not to be interpreted in a strict sense
and are not to be regarded as analytical requirements.
Solubility
Unless otherwise indicated, the approximate solubility of a substance is eval-
uated at 20 °C. The expression “part” describes the number of millilitres (ml)
’
For the current WHO recommendations, consult the WHO Medicines website
(htpp://www.who,int/medicines).
4
General notices
of solvent represented by the stated number of parts in which 1 gram (g) of

solid is soluble.
Very soluble Less than 1 part

Freely soluble From 1 to 10 parts
Soluble From 10 to 30 parts
Sparingly soluble From 30 to 100 parts
Slightly soluble From 100 to 1 000 parts
Very slightly soluble From 1000 to 10000 parts
Practically insoluble More than 10000 parts
Category
The statements given for information are intended to indicate the principal
pharmacological action and therapeutic use or, for excipents, the main phar-
maceutical use. should not be assumed that the substance has no other action
It
or use. The statements are in no way intended to be binding.
Storage
Substances, dosage forms, and other materials must be stored under specified
conditions in order to avoid contamination and deterioration.
(a) Containers
The container and its closure must not interact physically or chemically with
the substance within in any way that would alter its quality. The following
terms include general requirements for the permeability of containers:
Well-closed containers must protect the contents from extraneous matter or
from loss of the substance under normal conditions of handling, shipment, or
storage.
Tightly closed containers must protect the contents from extraneous matter,
from loss of the substance, and from efflorescence, deliquescence, or evapora-
tion under normal conditions of handling, shipment, or storage. If the container
is intended to be opened on several occasions, it must be designed to be
airtight after reclosure.

Hermetically closed containers must protect the contents from extraneous
matter and from loss of the substance, and be impervious to air or any other
gas under normal conditions of handling, shipment, or storage.
In addition, a tamper- evident container is one that is fitted with a device that
reveals clearly whether it has ever been opened.
(b) Protection from light

Substances and dosage forms requiring protection from light should be main-
tained in a light-resistant container that - either by reason of the inherent prop-
which the container is composed, or because a special
erties of the material of
coating has been applied to the container - shields the contents from the effects
5
of light. Alternatively, the container may be placed inside a suitable light-

resistant (opaque) covering and/ or stored in a dark place.
(c) Temperature
Where storage at temperatures other than room temperature (15 to 25 °C or,
depending on the climatic conditions, up to 30 °C) is recommended, this is
stated in the monograph. Such substances and dosage forms should be labelled
accordingly.
Stability information
For substances and dosage forms that deteriorate easily under adverse storage
conditions (such as occur in tropical climates), a warning should be given indi-
cating that degradation is likely to occur in a humid atmosphere and that
decomposition is faster at elevated temperatures. In such cases this warning is
given in the section "Additional information”.
Labelling
Official national and regional labelling requirements should be met.
When the
term “label” is used in the International pharmacopoeia, the label-
ing statements may appear on the container, the package or a leaflet accompa-
nying the package, as decided by the relevant authority.
For dosage forms, the label should state whether an antimicrobial preserv-
ative has been added, its name and concentration, as well as those of other
substances such as buffers and colouring agents.
Further indications should be given, such as the special route of adminis-
tration of a dosage form and, whenever relevant, the shelf-life.
Additional information
Information may be given concerning certain characteristics of a substance such
as polymorphism, hygroscopicity or stability. Information may also be pro-
vided on precautions, special routes of administration, and the usual strengths
for dosage forms'.
General requirements
A substance or a dosage form stated to be of pharmacopoeial quality must
comply with all the requirements set out in The international pharmacopoeia. A
monograph is to be interpreted in accordance with any general notice or mono-
graph or with any general method text that is contained in this edition and that
is applicable to that monograph.
^
WHO
The selection and use of essential medicines. Report of the Expert Committee. Geneva, World
Health Organization, 2005 (WHO Technical Report Series, No. 933). See also the current edition
of theWHO WHO
Model Formulary and other publications available on the Medicines website
(http://www.who.int/medicines).
6
General notices
General methods of analysis that are commonly used in carrying out the
tests and assays included in the monographs of the International Pharma-
copoeia are described in the section on “Methods of Analysis’’. In some cases
a specific cross-reference to the method required is provided within the mono-
graph Examples include references to Spectrometry in the infrared region,
text.
1.14.4 High performance liquid chromatography and 2.2.3 Limit test for heavy
metals. In other cases, where the relevant method can be inferred from the title
of the test, no explicit cross-reference is given. Examples include tests for
Specific optical rotation, Sulfated ash. Loss on drying and pH value. Whether
or not a specific cross-reference is included, the requirements of the mono-
graphs of the International Pharmacopoeia are to be interpreted in relation to
the relevant method of analysis.
Limits are evaluated on the basis of normal analytical practice. Variations in
manufacture and/or compounding, any possible deterioration, and normal ana-
lytical errors are taken into consideration. However, further tolerances beyond
the limits stated are not permitted.
Limits are generally expressed in terms of the equivalent content in per cent
(%) of the chemical formula for a given substance, as determined in the assay.
The requirements for biological substances and preparations such as certain
antibiotics, if determined biologically, are given in International Units (lU)
per milligram (mg). The material is not of pharmacopoeial quality if the upper
fiducial limit of error is less than the stated potency, with fiducial limits of error
based on a probability of 95% (P = 0.95).
Where it is specified that the results are to be calculated with reference to
the dried or anhydrous substance, the determination of loss on drying or water
is to be carried out using the test conditions indicated in the monograph.
Identity tests
Identity tests are provided to verify the identity of the substance described in
the monograph, and analysts will need to decide on the extent of testing that
they are able to carry out based on the instruments available to them.
It is generally recognized that the infrared spectrum provides the best
method of identification because of the uniqueness of a well developed finger-
print region of the spectrum for a given active ingredient. Wherever possible,
infrared spectrum characteristics are used as the principal test of identification.
Further identification tests provided in an individual monograph, considered
together, are intended to provide verification of identity, should the use of an
infrared spectrophotometer be precluded.
It should further be noted that, whenever a melting temperature is provided
under “Identity tests”, only an approximate value is given, since no exact repro-
duction of the quoted temperature is necessary.
Examination in ultraviolet light

When examination in ultraviolet light is required, an ultraviolet lamp having a
maximum output of approximately 254 nm or 365 nm is to be used.
7
Clarity of solution
The determination of the clarity of solution is carried out as described under
1.11 Colour of liquids using a black background. The source of light must be
such that opalescence standard TS2 can be readily distinguished from water. A
solution is considered clear if its opalescence is not more pronounced than that
of opalescence standard TS2.
Colourless solution
A solution is considered colourless not more intensely coloured than any
if it is
of the standard colour solutions BnO, YwO, GnO, or RdO. The matching is made
with the solution of most appropriate hue as described under 1.11 Colour of
liquids.
Loss on drying
In order to determine the loss on drying, l.Og of the substance - unless other-
wise specified - is dried under the conditions indicated.
The desiccants mentioned may be replaced by other substances of equiva-
lent desiccant capacity.
The expression “dry to constant mass” means that the drying process should
be continued until the results of two consecutive weighings do not differ by
more than mg, the second weighing being made after an additional hour
0.5
of drying under the prescribed conditions. The expression “ignite to constant
mass” has a similar meaning, the second weighing following further ignition.
The expression “under reduced pressure” means that the drying process is
carried out over phosphorus pentoxide, at a pressure not exceeding O.dkPa at

room temperature, unless otherwise stated.
Tests and assays

Alternative methods are given to be used in cases where the required instru-
ments are not available.
Tests and assays are normally carried out at room temperature (between 15
and 25 °C, or up to 30 °C in some climatic zones), unless otherwise indicated.
Any glassware used in pharmacopoeial tests and assays should be of
suitable quality.’
When a water-bath is referred to in the text, boiling water of about 100°C
is to be used, unless a specific water temperature is given.
^
The following requirements concerning the quality of glass were established by the International
Organization for Standardization:
— Glass - Hydrolytic resistance of glass grains at 98 °C - Method of test and classification, Ref.
No. ISO 719-1985 (E).
— Glass - Hydrolytic resistance of glass grains at 121 °C - Method of test and classification, Ref.
No, ISO 720-1985 (E).
— Glass-ware - Hydrolytic resistance of the interior surfaces of glass containers - Methods of
test. Ref. No. ISO 4802-1988 (E).
Gemral notices
Unless otherwise specified, all solutions indicated in the tests and assays are
prepared with distilled or demineralized water complying with the monograph
for Purified water.
Indicators for visual determination of pH values

Alternative indicators and indicator papers can be used in the tests and assays
for the determination of the pH
change by visual inspection pro-
value or of its
vided that they have the same sensitivity in the chosen pH range.
Accuracy and Precision

(a) Expression of concentration
The symbol “%” is normally used to express the concentration of solids in
liquids, the number of grams of solute in 100 ml of product (mass per volume).
If indicated, the following expressions are used:
% nt/m for the number of grams of solute in lOOg of product

% v/v for the number of millilitres of solute in 100 ml of product
% v/m for the number of millilitres of solute in lOOg of product.
The concentration expressed as “mol/1” refers to the number of moles of the

stated solute in sufficient water, or other vehicle, to produce lOOOml.
(b) Quantities
In assays and tests with numerical limits, the quantity stated to be taken for
examination is approximate. The amount actually used, which may deviate by
not more than 10 per cent from that stated, is accurately weighed or measured
and the result is calculated from this exact quantity. Regents are measured and
the procedures are carried out with an accuracy commensurate with the degree
of precision implied by the requirement stated for the assay or the limit spec-
ified for the test. In tests where the limit is not numerical (for example, in com-
parative tests such as that for Clarity and colour of solution), the stated quantity
is taken for examination. Quantities are weighed or measured with an accu-
racy commensurate with the indicated degree of precision. For weighings, the
precision corresponds to plus or minus 5 units after the last figure stated. For
example.
a value of 20 is not less than 19.5 and not greater than 20.5,
20.0 is not less than 19.95 and not greater than 20.05,
2.0 is not less than 1.95 and not greater than 2.05,
0.20 is not less than 0.195 and not greater than 0.205.
(c) pH value
pH values are indicated in a manner similar to that given for quantities and
volumes.
(d) Temperature measurement

Temperature measurement is indicated in a manner similar to that given for
quantities.
Storage conditions given in general terms refer to the following equivalent
temperatures;
in a refrigerator 2 to 8 °C
cold or cool 8 to 15 °C
room temperature 15 to 25°C, or up to 30 °C in some climatic zones.
Calculation of results
The results of tests and assays should be calculated to one decimal place more
than indicated in the requirement and then rounded up or down as follows:
— if the last figure calculated is 5 to 9, the preceding figure is increased by 1;

— if the last figure is 4 or less, the preceding figure is left unchanged.
Other calculations - for example, in the standardization of volumetric solutions

- are carried out in a similar manner.
Impurities
In certain tests, the concentration of impurity is given in parentheses either as
a percentage or in parts per millionby weight (ppm). In chromatographic tests
such concentrations are stated as a percentage irrespective of the limit.
In chromatographic tests in which a comparison is made between spots or
peaks in chromatograms obtained with solutions of different concentrations of
the test substance, the percentage given in parentheses indicates an impurity
limit expressed in terms of a nominal concentration of the medicinal substance
itself.
In all cases where an impurity

limit is given in parentheses, the figures given
are approximations for information only; conformity with the requirements is
determined on the basis of compliance or otherwise with the stated test.
A list of all known and potential impurities that have been shown to be con-
trolled by the tests in a monograph may be given for information at the end of
the monograph.
Patents and trademarks

The inclusion in The International Pharmacopoeia of any product subject to actual
any name that is a trade-
or potential patent or similar rights, or the inclusion of
mark in any part of the world does not and shall not be deemed to imply or
convey permission, authority, or licence to exercise any right or privilege pro-
tected by such patent or trademark, including licence to manufacture, without
due permission, authority, or licence from the person or persons in whom such
rights and privileges are vested.
10
General notices
Reference to a particular trade name in the description of certain materials

used in assays and tests does not imply that other equivalent materials are not
also suitable.
Reagents, reference substances, test solutions,

and volumetric solutions
Names of reagents (R and IR), reference substances (RS), test solutions (TS),
volumetric solutions (VS), and culture media (Cm) are described in the section
on Reagents. Reagents used in tests and assays are not intended for therapeu-
tic use.
Reference substances
(a) Chemical
International Chemical Reference Substances are established on the advice of
the WHO Expert Committee on Specifications for Pharmaceutical Preparations.
They are supplied primarily for use in physical and chemical tests and assays
described in the specifications for quality control of drugs published in The
International Pharmacopoeia or proposed in draft monographs.
Directions for use and the analytical data required for the tests specified in
The International Pharmacopoeia are given in the certificates enclosed with the
substances when distributed. More detailed analytical reports on the substances
may be obtained on request from the WHO Collaborating Centre for Chemical
Reference Substances at the address given below.
International Chemical Reference Substances may also be used in tests and
assays not described in The International Pharmacopoeia. However, responsibility
for assessing the suitability of the substances then rests with the user or with
the pharmacopoeia commission or other authority that has prescribed their use.
It is generally recommended that the substances be stored protected from
light and preferably at a temperature of about -)-5 °C . When special storage con-
ditions are required, this on the label or in the accompanying leaflet.
is stated
The Chemical Reference Substances kept at the
stability of the International
Collaborating Centre is monitored by regular re-examination, and materials
that have deteriorated are replaced by new batches when necessary. Lists giving
control numbers for the current batches are issued in the annual reports from
the Centre. The list published in the 2004 annual report is provided in the
section on Supplementary information. The list current at any particular time
is available on the WHO Medicines website or may be obtained on request
from the centre.

Orders for International Chemical Reference Substances should be sent to:
• WHO Collaborating Chemical Reference Substances,

Centre for
Apoteket AB, Centrallaboratoriet, ACL, Prismavagen 2, SE-141 75
Kungens Kurva, Sweden; Fax: -1-46 8 740 60 40; E-mail:
who.apl@apoteket.se
11
International Chemical Reference Substances are supplied only in the standard

packages indicated in the list.
(b) Biological
The primary purpose underlying the establishment of International Biological
Standards is to provide a means of ensuring uniformity throughout the world
in the designation of the activity or specificity of preparations that are used in
the prophylaxis, therapy, or diagnosis of human diseases, which cannot be
expressed directly in terms of chemical and physical quantities'.
Requests for international biological reference materials should be addressed
directly to the following custodian laboratory, together with a brief summary
on the intended use. The catalogue of International Biological Reference Prepa-
rations current at any particular time is available on the WHO Biologicals
website.
• International Laboratory for Biological Standard, National Institute for

Biological Standards and Control (NIBSC) South Mimms, Potters Bar,
Herts EN6 3QH, England; Tel. +44 1707 646399; Telex 21911 NIBSAC
G; Fax +44 1707 646977.
Reference spectra
International Infrared Reference Spectra are intended to be used to confirm the
identity of specific substances. These spectra can be obtained from:
• WHO Collaborating for Chemical Reference Substances,

Centre
Apoteket AB, Centrallaboratoriet, ACL, Prismavagen 2, SE-141 75
Kungens Kurva, Sweden; Fax -i-46 8 740 60 40. E-mail:
who.apl@apoteket.se
^
More information may be found in: Recommendations for the ^preparation, characterization and estab-
lishment of international and other biological reference standards {Revised 2004) Annex 2 to 55* Report
of the WHO Expert Committee on Biological Standardization, Geneva, World Health Organiza-
tion, 2005 (WHO Technical Report Series, No. 932).
12
Appendices to the General Notices
Abbreviations and symbols
[a] Angle of rotation in polarized light.
““
[a]2’ Specific optical rotation, angle of rotation (a) of a liquid or of a
substance in solution, measured in degrees (°) of rotation at the
wavelength of the sodium D-line (589.3 nanometres) and at a tem-
perature of 20 + 0.5 °C. For liquids it is calculated with reference
to a path length of 100 millimetres and divided by the relative
density at 20 °C; for substances in solution it is calculated with
reference to a path length of 100 millimetres of a solution con-
taining 1 gram of the substance per millilitre.
A Absorbance, extent of radiation absorbed by a solution expressed

in logarithm, decimal, of the reciprocal of the transmittance.
A'lcm Specific absorbance, absorbance of a solution containing 1 gram
of substance in 100 millilitres of solution (1% solution) and mea-

sured at a path length of 1 centimetre.
ATCC American Type Culture Collection, 10801 University Boulevard,

Manassas, VA 20110-2209, USA.
CAS Reg. Registration Number established by the Chemical Abstracts

Service of the American Chemical Society.
C.I. Colour Index Number (British).
CIP Collection de Bacteries de I'lnstitut Pasteur, 25 rue du Docteur

Roux, F-75724 Paris Cedex 15, France.
Cm Culture medium (see “Reagents, test solutions, and volumetric

solutions”).
d Relative density, dimensionless, ratio of the density (r) of a sub-

stance to that of a reference substance under specified conditions.
13
d 20
20 Relative density, ratio of the mass of a given volume of the sub-
stance to that of an equal volume of water, both at 20 °C.
nl"/
-0-1 c This symbol has been replaced by A!”™-
lU International Units.
mol/1 Concentration, amount of substance of solute per 1000 millilitres

of solution.
n Refractive index for electromagnetic radiation in a given medium,

dimensionless, ratio of the sine of the angle of incidence of elec-
tromagnetic radiation on a medium to the sine of its angle of
refraction in the medium.
Hd measured at the wavelength of the sodium

Refractive index, value
D-line (589.3 nanometres) and at a temperature of 20 ± 0.5 °C.
NCIMB National Collections of Industrial and Marine Bacteria Ltd, 23 St

Machar Drive, Aberdeen AB24 3RY, Scotland.
NCTC National Collection of Type Cultures, Central Public Health

Laboratory, Colindale Avenue, London NW9 5HT, England.
NCYC National Collection of Yeast Cultures, AFRC Food Research Insti-
tute, Colney Lane, Norwich NR4 7UA, England.
P Probability of an event in statistical estimations.
pH Dimensionless, physical quantity expressing the acidity or alka-

linity of a solution, measured as the negative logarithm of the
hydrogen-ion concentration or hydrogen-ion activity expressed
in moles per litre. The scale, 0-14, represents the acidity and alka-
linity, numbers below 7 an increas-
7 indicating the neutral state,
ing acidity, and numbers above 7 an increasing alkalinity.
P Mass density, mass divided by volume.
P20 Mass density, the mass of one unit volume of the substance,
expressed in kilograms per cubic metre (in The International Phar-
macopoeia as grams per millilitre) measured at a temperature of
20°C.
R Reagent (see "Reagents, test solutions, and volumetric solutions”).
14
Gemral notices
Rf Ratio of fronts, related to fronts. In paper or thin-layer chro-

matography, ratio of the distance travelled by the substance to that
travelled by the mobile phase.
R, In chromatography, ratio of the distances travelled by the sub-

stance to that of the reference substance.
RS Reference substance (see “Reagents, test solutions, and volumet-

ric solutions”).
SI International System of Units {Syst'enie international d'Unites).
TS Test solution (see “Reagents, test solutions, and volumetric

solutions”).
VS Volumetric solution (see “Reagents, test solutions, and volumetric

solutions”).
15
Units of measurement
The names and symbols for units of measurement used by the International
Pharmacofoeia are those of the Systeme international d'Unites (International
System of Units) (SI), a practical system of units that has been developed
through the efforts of the General Conference of Weights and Measures
(CGPM) and other international organizations. The 11th General Conference
(1960) adopted the international abbreviation SI for this system of units'.
The SI units used in the third edition of the International Pliarmacop'oeia, as
well as their multiples and submultiples, were in many cases identical to the
units used for the respective units of measurement in the second edition. In
other cases, however, the SI had introduced differently defined units; this was
especially true for derived units. In such situations, to promote better under-
standing of the procedures and limits related to quality requirements, the third
edition of the International Pharmacopoeia gave, in addition to the SI units, the
units previously used in the second edition, together with appropriate conver-
sion of numerical values. Where included, this information has been retained
in the fourth edition.
The following multiplicative prefixes, which indicate decimal multiples and
submultiples of the SI units, are used in the International Pharmacopoeia:
giga (G) 10'
mega (M) 10'
kilo (k) 10-
centi (c) 10
milli (m) 10
micro (fi) 10
nano (n) 10
pico (P) 10
The use of these prefixes is illustrated by the following units, multiples and
submultiples, that are employed in the third edition of the International
Pharmacopoeia:
^
The definitions of the units used in the International System are given in the brochure “Le
Systeme international d’unites” published by the Bureau International des Poids et Mesures,
(BIPM) Pavillion de Breteuil, F-92310 Sevres. The most recent edition was published in 1998 and
a Supplement in 2000. For more information, consult the BIPM website (http://www.bipm.fr).
16
Gemral notices
Units of length
metre (m)
centimetre (cm)
millimetre (mm)
micrometre (pm)
nanometre (nm)
Units of volume (capacity)
litre (1)
= 1000 cm=*
millilitre (ml) = 1 cm^
microlitre (lil) = 0,001 cm'
Units of temperature
Kelvin (K)
degree Celsius (°C)
Units of mass
kilogram (kg)
gram (g)
milligram (mg)
microgram (lig)
nanogram (ng)
Units of time
year (a)
day (d)
hour (h)
minute (min)
second (s)
millisecond (ms)
microsecond (lis)
Units of pressure
kilopascal (kPa)
pascal (Pa)
The following non-Sl units of pressure are also used in some special cases:
pound-force per square inch (Ibf/in^ or, incorrectly, psi) = 0.69 kPa
millimetre of mercury (mmHg) = 133 Pa
17
Units of radioactivity'^
gigabecquerel (GBq) = 27.03 mCi

megabecquerel (MBq) = 27.03 pCi
becquerel (Bq) = 27.03 pCi
curie (Ci) = 37 GBq
millicurie (mCi) = 37 MBq
microcurie (|^Ci) = 37kBq
Units of electric current
ampere (A)
milliampere (mA)
nanoampere (nA)
Units of electric potential
volt (V)
millivolt (mV)
Unit of resistance
ohm (Cl)
’
The definition of units of radioactivity is given under “Radiopharmaceuticals”.
18
Names, symbols, and relative
atomic masses of certain elements
The relative atomic mass (the term atomic weight was previously used) given
in the table below is scaled relatively to the isotope taken as 12 exactly and
is given to 4 significant figures. Only the elements most commonly encoun-
tered in pharmaceutical analysis are included.
Most elements have more than one naturally occurring isotope and the vari-
ation in the relative abundance of these isotopes influences the precision with
which the relative atomic mass of an element in nature can be quoted. Among
the elements included in the table, the relative atomic mass of samples in nature
of boron, calcium, lead, strontium and sulfur may differby more than one in
the fourth significant figure.
Name Symbol Relative atomic mass
Aluminium A1 26.98
Antimony Sb 121.75*
Arsenic As 74.92
Barium Ba 137.3
Bismuth Bi 209.0
Boron B 10.81
Bromine Br 79.90
Cadmium Cd 112.4
Calcium Ca 40.08
Carbon C 12.01
Cerium Ce 140.1
Chlorine Cl 35.45
Chromium Cr 52.00
Cobalt Co 58.93
Copper Cu 63.55*
Fluorine F 19.00
Gold Au 197.0
Helium He 4.003
Holmium Ho 164.9
Hydrogen H 1.008
Iodine I 126.9
Iron Fe 55.85*
19
Name Symbol Relative atomic mass
Lanthanum La 138.9
Lead Pb 207.2
Lithium Li 6.941*
Magnesium Mg 24.31
Manganese Mn 54.94
Mercury Hg 200.6*
Molybdenum Mo 95.94
Nickel Ni 58.71*
Nitrogen N 14.01
Oxygen O 16.00*
Phosphorus P 30.97
Platinum Ft 195.1*
Potassium K 39.10*
Ruthenium Ru 101.1*
Selenium Se 78.96*
Silicon Si 28.09*
Silver Ag 107.9
Sodium Na 22.99
Strontium Sr 87.62
Sulfur S 32.06
Thorium Th 232.0
Tin Sn 118.7’
Titanium Ti 47.90*
Tungsten (Wolfram) W 183.85*
Uranium U 238.0
Vanadium V 50.94*
Zinc Zn 65.38
Zirconium Zr 91.22
*
Values are considered reliable to ±1 in the last digit or ±3 when followed by an asterisk.
20
Monographs
Pharmaceutical
substances
Monograp’hs for p/harmaceutical substances
Pharmaceutical substances
ACETAZOLAMIDUM
ACETAZOLAMIDE
Molecular formula. C4H6N4O3S2
Relative molecular mass. 222.2
Graphic formula.
Chemical name. N-(5-Sul£amoyl-l,3,4-thiadiazol-2-yl)acetamide; N-[5-

(aminosulfonyl)-l,3,4-thiadiazol-2-yl]acetamide; 5-acetamido-l,3,4-thiadia-
zole-2-sulfonamide; CAS Reg. No. 59-66-5.
Description. A white, or almost white, crystalline powder; odourless.
Solubility. Very slightly soluble in water; slightly soluble in ethanol (-750 g/1)
TS; practically insoluble in ether R.
Category. Carbonic anhydrase inhibitor.
Storage. Acetazolamide should be kept in a well-closed container.
Requirements
Definition. Acetazolamide contains not less than 99.0% and not more than
101.0% of C4H6N4O3S2, calculated with reference to the dried substance.
Identity tests
A. Carry out the examination as described under 1.7 Spectrophotometry in the
The infrared absorption spectrum is concordant with the
infrared region.
spectrum obtained from acetazolamide RS or with the reference sp>ectrum of
acetazolamide.
B. Dissolve 25mg in 5ml of water, add 0.15ml of sodium hydroxide (1 mol/1)

VS and 0.1ml of copper(II) sulfate (80 g/1) TS; a bluish green colour or pre-
cipitate is formed.
23
C. Triturate 0.5 g with a mixture of 5 ml of water and 1 ml of sodium hydrox-

ide (1 mol/1) VS, add 0.2 g of zinc R powder and 0.5 ml of hydrochloric acid
(-420 g/1) TS; hydrogen sulfide is evolved and may be detected by its odour
(proceed with caution), or by the use of filter paper soaked in lead acetate
(80 g/1) TS which turns black on exposure.
Heavy metals. Use l.Og for the preparation of the test solution as described
under 2.2.3 Limit test for heavy metals, Procedure 3; determine the heavy metal
content according to Method A; not more than 20pg/g.
Sulfates. Dissolve l.Og in 40ml of water, warm to 70 °C for 5 minutes, cool

and filter. Proceed with the filtrate as described under 2.2.2 Limit test for
sulfates; the sulfate content is not more than 0.5mg/g.
Sulfated ash. Not more than l.Omg/g.
Loss on drying. Dry to constant weight at 1 05 °C; it loses not more than 5.0 mg/g.
pH value. Shake 1 g with 50 ml of water for 5 minutes; pH of the suspension,

4.0-6.0.
Related substances. Carry out the test as described under 1.14.1 Thin-layer
chromatography, using silica gel R2 as the coating substance and a mixture of
30 volumes of dioxan R, 30 volumes of 2-propanol R, 20 volumes of ammonia
(-35 g/1) TS, 10 volumes of toluene R, and 10 volumes of xylene R as the mobile
phase. Apply separately to the plate 20|il of each of 2 solutions in ethanol
(-750 g/1) TS containing (A) 5.0 mg of the test substance per ml and (B)
O.OSOmg of the test substance per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in air, and examine the chromatogram in
ultraviolet light (254 nm). Any spot obtained with solution A, other than the
principal spot, is not more intense than that obtained with solution B.
Assay. Dissolve about 0.45 g, accurately weighed, in 90 ml of dimethylfor-

mamide R and titrate with tetrabutylammonium hydroxide (0.1 mol/1) VS
determining the endpoint potentiometrically as described under 2.6 Non-
aqueous titration. Method B. Each ml of tetrabutylammonium hydroxide
(0.1 mol/1) VS is equivalent to 22.22 mg of C H 6N 4 O 3 S 2
4 .
ACIDUM ACETICUM
ACETIC ACID
This monograph covers three concentrations, namely glacial acetic acid, acetic acid and
dilute acetic acid.
24
H3C — CO2H
C H4O 2
2 ,
Glacial acetic acid
Relative molecular mass. Glacial acetic acid, 60.05
Chemical name. Acetic acid; CAS Reg. No. 64-19-7.
Description. Clear, colourless liquids. At a temperature below 15 °C Glacial

acetic acid may occur as a translucent, crystalline mass; odour, characteristic
and pungent.
Miscibility. Miscible with water, ethanol (~750g/l) TS, and glycerol R.
Category. Acidifying agent.
Storage. Acetic acid should be kept in a tightly closed container.
Additional information. Glacial acetic acid is flammable and should be

handled with care. Congealing point, about 15 °C.
Relative densities. <^20 - Glacial acetic acid 1.050

- Acetic acid 1.041
- Dilute acetic acid 1.005
Requirements
Glacial acetic acid contains not less than 99 0 %
. m/m and not more than the
equivalent of 100 5 % m/m of C 2 H 4 O 2
. .
Acetic acid contains not less than 32.5% m/m and not more than the equiva-
lent of 33.5% m/m of C 2H 4O 2 .
Dilute acetic acid contains not less than 5.7% m/m and not more than the
equivalent of 6.3% m/m of C 2H O 2 4 .
Identity tests
A. Strongly acid, when diluted.
B. Transfer to a test-tube either 0.1 ml of Glacial acetic acid with 1 ml of water,

or take 1 ml of Acetic acid or of Dilute acetic acid; to each add 1 ml of ethanol
(-750 g/1) TS and 1ml of sulfuric acid (-1760 g/1) TS, and heat the mixture
to boiling; ethyl acetate, perceptible by its odour (proceed with caution), is
produced.
25
C. Dilute either 0.3 ml of Glacial acetic acid with 3 ml of water or1 ml of Acetic
acid with 2 ml of water, or take 3 ml of Dilute acetic acid; neutralize each

of them with ammonia (~100g/l) TS. Then add 0.5 ml of lanthanum nitrate
(30g/l) TS, 0.1ml of iodine TS, and 0.05 ml of ammonia (-100 g/1) TS. Heat
the mixtures carefully to boiling and allow to stand; a dark blue colour
appears.
Heavy metals. Take 3.5 ml of Glacial acetic acid, 10 ml of Acetic acid, or

20ml of Dilute acetic acid. Evaporate each of the volumes to dryness on a
water-bath. To each residue add 1.5ml of hydrochloric acid (0.1 mol/1) VS,
warm gently to dissolve, and determine the heavy metals content as described
under 2.2.3 Limit test for heavy metals. Method A; Glacial acetic acid contains
not more than 6pg/g, Acetic acid not more than 2pg/g, and Dilute acetic acid
not more than 1 pg/g.
Chlorides. Take 3.5 ml and proceed with the test as described under 2.2.1
is not more than 70pg/g.
Limit test for chlorides; the chloride content
Sulfates. Take 2 ml and proceed as described under 2.2.2 Limit test for
sulfates; the sulfate content is not more than 0.24 mg/g.
Non-volatile residue. Evaporate a volume of known mass (not less than

lOg) to dryness on a water-bath and dry at 105 °G; the residue weighs not more
than 0.1 mg/g.
Readily oxidizable substances. Dilute 5 ml of Glacial acetic acid with 10 ml

of water and dilute again 5 ml of this solution with 20 ml of water, or use 5 ml
of Acetic acid diluted with 20ml of water, or use 25ml of Dilute acetic acid.
To each add 0.2 ml of potassium permanganate (0.02 mol/1) VS, and allow to
stand for 30 seconds; the colour is not entirely discharged.
Assay. To about 2g, accurately weighed, of Glacial acetic acid and 50 ml of

water, or about 5g, accurately weighed, of Acetic acid and 50ml of water, or
about 20 g, accurately weighed, of Dilute acetic acid and 30 ml of water, add 3
drops of phenolphthalein/ethanol TS and titrate with carbonate-free sodium
hydroxide (1 mol/1) VS. Repeat the procedure without the acids being exam-
ined and make any necessary corrections.
Each ml of carbonate-free sodium hydroxide (1 mol/1) VS is equivalent to

60.05mg of C2H4O2.
26
ACIDUM ACETYLSALICYLICUM
ACETYLSALICYLIC ACID
Molecular formula. C9H8O4
Graphic formula.
a COOH
OCOCH3
Chemical name. 2-(Acetyloxy)benzoic acid; 2-acetoxybenzoic acid; CAS Reg.

No. 50-78-2.
Description. Colourless crystals or a white, crystalline powder; odourless or

almost odourless.
Solubility. Soluble in about 300 parts of water; freely soluble in ethanol

(-750 g/1) TS; soluble in ether R.
Category. Analgesic; antipyretic.
Storage. Acetylsalicylic acid should be kept in a tightly closed container, pro-

tected from light.
Additional information. Even in the absence of light, Acetylsalicylic acid is

gradually degraded on exposure to a humid atmosphere, the decomposition
being faster at higher temperatures.
Requirements
Definition. Acetylsalicylic acid contains not less than 99.0% and not more
than 100.5% of C 9 H 8O 4 ,
calculated with reference to the dried substance.
Identity tests
A. Heat 0.05g in 2ml of water for several minutes, cool, and add 1-2 drops of
ferric chloride (25 g/1) TS; a violet-red colour is produced, which does not
change on the addition of ethanol (~750g/l) TS.
B. Boil 0.2 g with 4 ml of sodium hydroxide (-80 g/1) TS for about 3 minutes,
cool, and add 5ml of sulfuric acid (-100 g/1) TS; a white, crystalline precip-
27
itate is formed. Filter (keep the filtrate for test C), wash the precipitate with
water and dry at 105 °C. Melting temperature, about 159 °C (salicylic acid).
C. Heat the filtrate from test B with 2 ml of ethanol (-750 g/1) TS and 2 ml of
sulfuric acid (-1760 g/1) TS; ethyl acetate, perceptible by its odour (proceed
with caution), is produced.
Heavy metals. Use l.Og and 25ml of acetone R for the preparation of the
test solution as described under 2.2.3 Limit test for heavy metals, procedure 2;
determine the heavy metal content according to Method A; not more than
20pg/g.
Solution in ethanol. A solution of l.Og in 10ml of ethanol (-750g/l) TS is
clear and colourless.
Solution in alkali. A solution of 0.5g in 10ml of warm sodium carbonate

(50 g/1) TS is clear.
Loss on drying. Dry to constant weight at ambient temperature under

reduced pressure (not exceeding 0.6kPa or about 5 mm of mercury) over silica
gel, desiccant, R; it loses not more than 5.0mg/g.
Salicylic acid. Dissolve 0.50 g in sufficient ethanol (-750g/l) TS to produce

25ml and transfer 10ml to a comparison tube. Dissolve separately 0.040g of
salicylic acid R
in sufficient water to produce 100 ml. Transfer 1 ml of this solu-
tion to a second comparison tube and add to it 10ml of ethanol (-750 g/1) TS.
Add water both tubes to make 50ml, followed by 1 ml of ferric ammonium
to
sulfateTSl, mix and allow to stand for 1 minute. The violet colour of the test
solution is not more intense than that of the reference solution when compared
as described under 1.11 Colour of liquids; the salicylic acid content is not more
than 2.0mg/g.
Assay. To about 0.20 g, accurately weighed, add 50 ml of carbonate-free

sodium hydroxide (0.1 mol/1) VS, and boil under reflux for 10 minutes. Titrate
the excess of alkali with sulfuric acid (0.05 mol/1) TS, using
phenolphthalein/ethanol TS as indicator. Repeat the operation without the
substance being examined and make any necessary corrections. Each ml of
carbonate-free sodium hydroxide (0.1 mol/1) VS is equivalent to 9.008 mg of
CgHa 04 .
28
ACIDUM ALGINICUM
ALGINIC ACID
Chemical name. Alginic acid; CAS Reg. No. 9005-32-7.
Description. A white to yellowish white, fibrous powder; almost odourless.
Solubility. Slightly soluble in water; practically insoluble in most organic sol-

vents; dissolves in solutions of alkali hydroxides.
Category. Tablet binder and disintegrant; viscosity-increasing agent; release-

rate modifier.
Storage. Alginic acid should be kept in a well-closed container.
Additional information. Alginic acid has an equivalent mass of about 240.

Attention should be paid to the microbiological quality since Alginic acid is of
natural origin.
Requirements
Definition. Alginic acid is a polyuronic acid composed of D-mannuronic and
L-guluronic acids and is obtained chiefly from algae belonging to the Phaeo-
phyceae, mainly species of Laminaria.
Identity tests
A. Dissolve 30mg in 5ml of sodium hydroxide (0.1 mol/1) VS and add 1ml of
calcium chloride (55 g/1) TS; a voluminous, gelatinous precipitate is produced.
B. Dissolve 30mg in 5ml of sodium hydroxide (0.1 mol/1) VS and add 1ml of
sulfuric acid (-570 g/1) TS; a heavy, gelatinous precipitate is formed.
C. To 5 mg in a test-tube add 5 ml of water and 1 ml of a freshly prepared solu-

tion of naphthalene- 1,3-diol R containing 1 g in 100ml of ethanol (~750g/l)
TS. Heat the mixture to boiling, boil gently for 3 minutes, and cool to about
15 °C. Transfer the contents of the test-tube to a 30-ml separator with the
aid of 5 ml of water and extract with 15ml of diisopropyl ether R. For
the blank repeat the procedure without the Alginic acid being examined.
The red-violet colour produced in the extract is not darker than that
obtained in the blank.
Heavy metals. For the preparation of the test solution ignite carefully in a cru-
cible l.Og with 2 ml of nitric acid (-1000 g/1) TS and 5 drops of sulfuric acid
(-1760g/l) TS until white vapours evolve, then ignite completely. Cool, add
29
2 ml of hydrochloric acid (-420 g/1) TS, and evaporate slowly on a water-bath

until dry. To the residue adddrop of hydrochloric acid (-420 g/1) TS and
1
10ml of water, then add ammonia (-260 g/1) TS to render the solu-
sufficient
tion slightly alkaline, adjust the pH to 3-4 with acetic acid (-60 g/1) TS, and
dilute to 40ml with water. Determine the heavy metals content as described
under 2.2.3 Limit test for heavy metals. Method A; not more than 40pg/g.
Ash. Carry out the procedure as described under 4.1 Determination of ash and
acid-insoluble ash; not more than 40mg/g.
Loss on drying. Dry to constant mass at 1 05 °C; it loses not more than 0.18 g/g.
pH value. Disperse 3g in 100ml of water, pH 1. 5-3.5.
Acid value. Suspend about 1 g, accurately weighed, in a mixture of 50 ml of

water and 30ml of calcium acetate (0.25 mol/1) VS. Shake thoroughly, allow to
stand for 1 hour, and titrate with sodium hydroxide (0.1 mol/1) VS, using phe-
nolphthalein/ethanol TS as indicator. Repeat the procedure without the Alginic
acid being examined and make any necessary corrections. Proceed with the cal-
culation as given in 4.6 Determination of acid value, using the number of ml
of sodium hydroxide (0.1 mol/1) VS required as a and with reference to the dried
substance; not less than 230.
ACIDUM AMIDOTRIZOICUM
AMIDOTRIZOIC ACID
Amidotrizoic acid, anhydrous
Amidotrizoic acid, dihydrate
n= 2 (di hydrate)
C H 3N 2 O 4
11 9I (anhydrous)
C H 3N 2 O 4
11 9I ,
2 HO
2 (dihydrate)
Relative molecular mass. 613.9 (anhydrous); 649.9 (dihydrate).
30
Chemical name. 3,5-Diacetamido-2,4,6-triiodobenzoic acid; 3,5-bis(acety-

lamino)-2,4,6-triiodobenzoic acid; CAS Reg. No. 117-96-4 (anhydrous).
3,5-Diacetamido-2,4,6-triiodobenzoic acid dihydrate; 3,5-bis(acetylamino)-

2,4,6-triiodobenzoic acid dihydrate; CAS Reg. No. 50978-11-5 (dihydrate).
Other name. Diatrizoic acid.
Description. A white or almost white, crystalline powder; odourless.
Solubility. Very slightly soluble in water and ethanol (-750 g/1) TS; soluble in
dimethylformamide R; sparingly soluble in methanol R; practically insoluble in
ether R; dissolves in solutions of alkali hydroxides.
Category. Used in the preparation of meglumine amidotrizoate as a radio-

contrast medium.
Storage. Amidotrizoic acid should be kept in a well-closed container, pro-

tected from light.
Labelling. The designation on the container of amidotrizoic acid should state

whether the substance is the dihydrate or the anhydrous form.
Requirements
Amidotrizoic acid contains not less than 98 0 . % and not more than the equiv-
alent of 102 0 %
. of C H 9I 3N O 4
11 2 ,
calculated with reference to the dried
substance.
Identity tests
• Either test A alone or tests B, C, and D may be applied.

infrared region. Previously dry the dihydrate of amidotrizoic acid at 105°C
The infrared absorption spectrum is concordant with the spec-
for 4 hours.
trum obtained from amidotrizoic acid RS or with the reference spectrum of
amidotrizoic acid.
B. Carry out the under 1.14.1 Thin-layer chromatography,

test as described
using silica gel R4 and a mixture of 20 volumes
as the coating substance
of chloroform R, 10 volumes of methanol R, and 2 volumes of ammonia
(-260 g/1) TS as the mobile phase. Apply separately to the plate 10 pi of each
of two solutions in a mixture of 0.8g of sodium hydroxide R dissolved in
1000 ml of methanol R containing (A) 1 mg of Amidotrizoic acid per ml and
(B) 1 mg of amidotrizoic acid RS per ml. After removing the plate from the
31
chromatographic chamber, allow it to dry in air, and examine the chro-

matogram in ultraviolet light (254 nm).
The principal spot obtained with solution A corresponds in position, appear-

ance, and intensity with that obtained with solution B.
C. Heat 0.5g in a suitable crucible; violet vapours are evolved.
D. Use 10 mg; it yields the reaction described for the identification of primary
aromatic amines under 2.1 General identification tests, producing a red-
violet precipitate.
Heavy metals. Suspend lOg in 10ml of water and with stirring add slowly
1.5ml of sodium hydroxide (-400 g/1) TS. When dissolved, adjust the pH to
between 7.0 and 7.5 with sodium hydroxide (-80 g/1) TS or hydrochloric acid
(-70g/l) TS, and dilute with water to 20ml. Use 2ml of this solution and deter-
mine the heavy metals content as described under 2.2.3 Limit test for heavy
metals, Method A; not more than 20pg/g. (Keep the remaining solution for
“Iodine and iodides”.)
Halides. Dissolve 2.5 g in a mixture of 20 ml of water and 2.5 ml of ammonia

(-100 g/1) TS. Add 20 ml of nitric acid (-130 g/1) TS, dilute with sufficient water
to produce 100ml, allow to stand for 15 minutes with occasional shaking, and
filter. Eliminate the first 10 ml of the filtrate and proceed with 25ml of the fil-
trate as described under “2.2.1 Limit test for chlorides”; the content of halides,
expressed as chlorides, does not exceed 35pg/g.
Iodine and iodides. Place 4 ml of the solution prepared above for “Heavy
metals” in a 50-ml centrifuge tube and add 20 ml of water, 5 ml of toluene R,
and 5ml of sulfuric acid (~100g/l) TS. Shake and centrifuge; the toluene layer
shows no red colour. Add 2 ml of sodium nitrite (10 g/1) TS, shake well, and
centrifuge. Similarly prepare a reference solution containing 0.5 mg of potas-
sium iodide R in 22 ml of water. Any red colour in the toluene layer is no darker
than that obtained with the reference solution (200pgl/g).
Loss on drying. Dry at 105 °C for 4 hours; anhydrous Amidotrizoic acid loses
not more than lOmg/g and the dihydrate loses not less than 45mg/g and not
more than 70mg/g.
Primary aromatic amines. Dissolve about 0.2 g, accurately weighed, in a

mixture of 5ml of water and 1 ml of sodium hydroxide (~80g/l) TS. Add 4ml
of sodium nitrite (10 g/1) TS and 10 ml of hydrochloric acid (1 mol/1) VS, shake,
and allow to stand for 2 minutes. Add 5 ml of ammonium sulfamate (25 g/1) TS,
32
shake well, allow to stand for 1 minute, add 0.4 ml of 1-naphthol/ethanol TS

and 15ml of sodium hydroxide (~80g/l) TS, and dilute with water to 50ml.
Measure the absorbance at about 485 nm against a solvent cell containing the
reagents prepared in a similar manner; the absorbance is not greater than 0.15.
Assay. Place about 0.3 g, accurately weighed, in a 125-ml conical flask and add
30ml of sodium hydroxide (50g/l) TS and 0.5 g of zinc R powder. Connect the
flask to a reflux condenser and boil for 1 hour. Cool the flask to room temper-
ature, rinse the condenser with 20 ml of water into the flask, and filter the
mixture. Rinse the flask and the filter thoroughly and add the rinse liquids to
the filtrate. Add 5ml of glacial acetic acid R and 1 ml of tetrabromophenolph-
thalein ethyl ester TS and titrate with silver nitrate (0.05 mol/1) VS until the
yellow precipitate just changes to green.
Each ml of silver nitrate (0.05mol/l) VS is equivalent to 10.23mg of

C11H9I3N2O4.
Additional requirement for Amidotrizoic acid for

p>arenteral use
Complies with the monograph for “Parenteral preparations”.
Pyrogens. Carry out the test as described under 3.5 Test for pyrogens, inject-
ing, per kg of the rabbit’s mass, a solution in sterile water R containing 0.6g of
Amidotrizoic acid in not more than 5 ml.
ACIDUM ASCORBICUM
ASCORBIC ACID
Molecular formula. CeHsOe
Graphic formula.
CH,OH
^
I
H-C— OH
OH OH
33
Chemical name. L-Ascorbic acid; CAS Reg. No. 50-81-7.
Description. Colourless crystals or a white or almost white, crystalline

powder; odourless or almost odourless.
Solubility. Freely soluble in water; soluble in ethanol (-750 g/1) TS; practically
insoluble in ether R.
Category. Antiscorbutic.
Storage. Ascorbic acid should be kept in a tightly closed, non-metallic con-

tainer, protected from light.
Additional information. Ascorbic acid in solution deteriorates rapidly in

contact with air; it has an acid taste. Even in the absence of light. Ascorbic acid
is gradually degraded on exposure to a humid atmosphere, the decomposition
Requirements
Definition. Ascorbic acid contains not less than 99.0% and not more than
100.5% of CgHsOg.
Identity tests
A. Dissolve 0.1 g in 2ml of water, add a few drops of nitric acid (~130g/l)
TS and a few drops of silver nitrate (40 g/1) TS; a dark grey precipitate is
produced.
B. Dissolve 0.04 g in 4 ml of water, add 0.1 g of sodium hydrogen carbonate R

and about 20 mg of ferrous sulfate R, shake and allow to stand; a deep violet
colour is produced, which disappears on the addition of 5 ml of sulfuric acid
(-100 g/1) TS.
C. Melting temperature, about 190°C with decomposition.
Specific optical rotation. Use a 50mg/ml solution; [a]c = -1-20.5 to -1-21.5°.
under 2.2.3 Limit test for heavy metals. Procedure 3; determine the heavy
metals content according to Method A; not more than 20pg/g.
Clarity and colour of solution. A solution of 0.50g in 10ml of water is clear

and not more intensely coloured than standard colour solution Rdl when com-
pared as described under 1.11 Colour of liquids.
34
Readily carbonizable substances. Dissolve O.lOg in 10ml of sulfuric acid

(~1760g/l) TS. After 15 minutes the solution is not more intensely coloured
than standard colour solutions Ywl or Gnl when compared as described under
1.11 Colour of liquids.
Assay. Dissolve about 0.20 g, accurately weighed, in a mixture of 25 ml of

carbon-dioxide-free water R and 25 ml of sulfuric acid (~100g/l) TS. Titrate the
solution at once with iodine (0.05 mol/1) VS using starch TS as indicator, added
towards the end of the titration, until a persistent blue colour is obtained. Each
ml of iodine (0.05 mol/1) VS is equivalent to 8.806mg of C^HsOg.
ACIDUM BENZOICUM
BENZOIC ACID
Graphic formula.
COOH
Chemical name. Benzenecarboxylic acid; CAS Reg. No. 65-85-0.
Description. Colourless, light, feathery crystals or a white, microcrystalline

powder; odour, characteristic, faint.
Solubility. Slightly soluble in water; freely soluble in ethanol (~750g/l) TS,

ether R.
Categoiy. Dermatological agent.
Storage. Benzoic acid should be kept in a well-closed container.
Requirements
Definition. Benzoic acid contains not less than 99.0% and not more than
100.5% of C H6O 2
7 ,
calculated with reference to the anhydrous substance.
35
Identity test
Boil 0,1 g with 0.1 g of calcium carbonate R1 and 5ml of water, and filter; add
a few drops of ferric chloride (25g/l) TS to the filtrate; a beige coloured pre-
cipitate is produced.
Melting range. 121-124 °C.
Heavy metals. For the preparation of the test solution use l.Og dissolved in
25ml of acetone R, add 2ml of water and dilute to 40ml with acetone R and
mix; determine the heavy metal content as described under 2.2.3 Limit test for
heavy metals, according to Method A; not more than 20|tg/g.
Chlorinated compounds and chlorides. Dissolve 0.35 g in 5 ml of sodium

carbonate (50g/l) TS, evaporate to dryness, and heat the residue until com-
pletely charred, keeping the temperature below 400 °C. Extract the residue with
a mixture of 10ml of water and 12ml of nitric acid (~130g/l) TS, and filter.
Proceed with the filtrate as described under 2.2.1 Limit test for chlorides; the
chloride content is not more than 0.7 mg/g.
Sulfated ash. Not more than 1.0 mg/g.
Water. Determine as described under 2.8 Determination of water by the Karl

Fischer method. Method A, using about 1 g of the substance and 25 ml of a solu-
tion composed of 1 volume of methanol R and 2 volumes of pyridine R as the
solvent; the water content is not more than 7.0 mg/g.
Readily oxidizable substances. Add 1.5ml of sulfuric acid (-1760 g/1) TS to

100 ml of water, heat to boiling, and add, drop by drop, potassium perman-
ganate (0.02 mol/1) VS until the pink colour persists for 30 seconds. Dissolve
l.Og of the substance being examined in the hot solution and titrate with potas-
sium permanganate (0.02 mol/1) VS to a pink colour that persists for 15 seconds;
not more than 0.5 ml of potassium permanganate (0.02 mol/1) VS is required.
Assay. Dissolve about 0.25g, weighed, in 15ml of ethanol

accurately
(-750 g/1) TS, previously neutralized to phenol red/ethanol TS, add 20 ml of
water and titrate with sodium hydroxide (0.1 mol/1) VS, using phenol red/
ethanol TS as indicator. Repeat the operation without the substance being
examined and make any necessary corrections. Each ml of sodium hydroxide
(0.1 mol/1) VS is equivalent to 12.21 mg of C/HgOj.
36
ACIDUM CITRICUM
CITRIC ACID
Citric acid, anhydrous
Citric acid monohydrate.
CH2 — COjH
CHj — COjH
tt = 0 (anhydrous)
tt = 1 (monohydrate)
CeHaO, (anhydrous)
CaHaO/iHjO (monohydrate)
Relative molecular mass. 192.1 (anhydrous); 210.1 (monohydrate).
Chemical name. Citric acid; 2-hydroxy-l,2,3-propanetricarboxylic acid; CAS

Reg. No. 77-92-9.
Citric acid monohydrate; 2-hydroxy- 1,2, 3-propanetricarboxylic acid monohy-

drate; CAS Reg. No. 5949-29-1.

practically odourless.
Solubility. Very soluble in water; freely soluble in ethanol (-750 g/1) TS; spar-
ingly soluble in ether R.
Category. Acidifying agent; buffer component.
Storage. Citric acid should be kept in a well-closed container.
Labelling. The designation on the container of Citric acid should state

whether it is the monohydrate or the anhydrous form.
Additional information. Citric acid monohydrate effloresces in dry air.
Requirements
not less than 99 5 % and not more than the equivalent of
Citric acid contains .
101 0 % of C^HsO/, calculated with reference to the anhydrous substance.

.
37
Identity test
A 20 mg/ml solution yields the reactions described under 2.1 General identifi-
cation tests as characteristic of citrates.
under 2.2.3 Limit test for heavy metals, Procedure 1; determine the heavy
metals content according to Method A; not more than lOpg/g.
Barium. Dissolve Ig in 7.8 ml of sodium hydroxide (~80g/l) TS and dilute to

10 ml with water. Acidify half of this solution with sulfuric acid (-100 g/1) TS
and allow to stand for at least 1 hour. Compare with the untreated portion of
solution; it remains clear.
Oxalates. Dissolve Ig in 10ml of water, neutralize with ammonia (~100g/l)

TS, add 0.35 ml of hydrochloric acid (2 mol/1) VS, cool, and add 2 ml of calcium
chloride (55 g/1) TS; no turbidity is produced.
Sulfates. Dissolve 0.1 g in 10ml of water, add 1ml of barium chloride (50g/l)
TS to which 1 drop of hydrochloric acid (-420 g/1) TS has been added; no tur-
bidity is produced.

Fischer method. Method A.
— For the anhydrous form use 1 g; the water content is not more than lOmg/g.
— For the monohydrate use 0.15g; the water content is not less than 75mg/g
and not more than 90mg/g.
Assay. Dissolve about 1.5g, accurately weighed, in 50 ml of carbon-dioxide-

free water R and titrate with carbonate-free sodium hydroxide (1 mol/1) VS,
using phenolphthalein/ethanol TS as indicator. Repeat the procedure without
the Citric acid being examined and make any necessary corrections.

64.03mg of CeHaO^.
38
ACIDUM FOLICUM
FOLIC ACID
Molecular formula. CigHigNjOg
Graphic formula.
Chemical name. N-[;?-[[(2-Amino-4-hydroxy-6-pteridinyl)methyl]amino]-

benzoyl]-L-glutamic acid; N- [4- [[(2-amino- l,4-dihydro-4-oxo-6-pteridinyl)-
methyl]amino]benzoyl]-L-glutamic acid; CAS Reg. No. 59-30-3.
Description. A yellow or yellowish orange, crystalline powder; odourless or

almost odourless.
Solubility. Very slightly soluble in water; practically insoluble in ethanol

(-750 g/1) TS, acetone R and ether R.
Category. Haemopoietic.
Storage. Folic acid should be kept in a well-closed container, protected from light.
Requirements
Definition. Folic acid contains not less than 96.0% and not more than 102.0%
of Ci 9 HitJSf 70 s, calculated with reference to the anhydrous substance.
Identity tests
A. The absorption spectrum of a 15 p/ml solution in sodium hydroxide
(0.1 mol/1) when observed between 230nm and 380nm, exhibits 3
VS,
maxima at about 256nm, 283 nm, and 365 nm. The absorbances at these
wavelengths are about 0.82, 0.80 and 0.28, respectively (preferably use 2-
cm cells for the measurements and calculate the absorbances of 1-cm layers).
The ratio of the absorbance of a 1-cm layer at 256 nm to that at 365 nm is
between 2.80 and 3.00.
39
B. Carry out the test as described under 1.14.1 Thin-layer chromatography, using
R1 as the coating substance and a mixture of 2 volumes of 1 -propanol
silica gel
R, volume of ethanol (-750 g/1) TS, and 2 volumes of ammonia (-260 g/1) TS
1
as the mobile phase. Apply separately to the plate 2 pi of each of 2 solutions

in a mixture of 9 volumes of methanol R and 1 volume of ammonia (-260 g/1)
TS containing (A) 0.50 mg of the test substance per ml and (B) 0.50 mg of folic
acid RS per ml. After removing the plate from the chromatographic chamber,
allow it to dry in air, and examine the chromatogram in ultraviolet light
(365 nm). The principal spot obtained with solution A corresponds in posi-
tion, appearance, and intensity with that obtained with solution B.
Sulfated ash. Not more than 2.0mg/g.

Fischer method. Method A, using about 0.15 g of the substance; the water
content is not less than 70mg/g and not more than 90mg/g.
Free amines. The ratio of the absorbance Aj of the test solution T 2 to the
absorbance Ab of the blank solution B,, as measured in the assay, should be
larger than 6.
Assay. Prepare the test solution T by dissolving about 0.050 g, accurately

weighed, in 50 ml of sodium hydroxide (-80 g/1) TS, mixing and diluting with
sodium hydroxide (-80 g/1) TS to 100 ml.
Transfer 30,0ml of the test solution to a 100-ml volumetric flask (test solution
Ti), and a second aliquot of 30.0 ml of the test solution to a second 100-ml
volumetric flask (blank Bi). To both solutions, Ti and blank Bi add 20ml of
hydrochloric acid (-70 g/1) TS and dilute them both with water to volume.
Retain blank solution B]. To 60ml of the test solution T] add 0.5 g of zinc R
powder, and allow to stand, shaking frequently, for 20 minutes. Filter the
mixture through a dry filter paper, discard the first 10ml of the filtrate, and
dilute 10ml of the subsequent filtrate with water to 100ml (test solution T 2).
Into three separate 25-ml volumetric flasks place 5.0ml each of test solution T 2,
of blank solution Biand of water (solution B 2 ), add to each of them 1ml of

water, 1ml of hydrochloric acid (-70 g/1) TS, and 1ml of sodium nitrite (lg/1)
TS, mix well and allow to stand for 2 minutes. Then add to each of them 1 ml
of ammonium sulfamate (5 g/1) TS, mix thoroughly, allow to stand for 2
minutes, add 1ml of Al-(l-naphthyl)ethylenediamine hydrochloride (lg/1) TS,
shake, allow to stand for 10 minutes, and dilute with water to volume.
Measure the absorbance of the test solution T2 and of the blank solution Bj
against a solvent cell containing solution B 2 at the maximum of about 550 nm;
designate these as Ay and Ab, respectively.
40
Carry out a similar procedure using folic acid RS and designate the respective
absorbances as As and Abs-
Calculate the content of C19H19N7O5 in terms of the percentage of anhydrous

Aj - Ab)/(10 As -
substance in the test substance, using the formula: 100(10
Abs),if necessary multiplying the result by the declared content (%) of
C19H19N7OB in the chemical reference substance.
ACIDUM HYDRO CHLORICUM

HYDROCHLORIC ACID
HCl
Chemical name. Hydrochloric acid; CAS Reg. No. 7647-01-0.
Description. A clear, colourless, fuming liquid; odour, pungent.
Miscibility. Miscible with water.
Storage. Hydrochloric acid should be kept in a tightly closed container, prefer-

ably in a cool place.
Additional information. The fumes and odour disappear when the acid is
diluted with 2 volumes of water.
Mass density: p2o = about 1.18g/ml.
Requirements
Hydrochloric acid contains not less than 35 0. % nt/m and not more than the
equivalent of 38 0 % m/nt of HCl.
.
Identity tests
A. It is strongly acid.
B. Use 0.1 ml; it yields the reactions described under 2.1 General identification
tests as characteristic of chlorides.
41
C. Allow a glass stick wetted with ammonia (-100 g/1) TS to come near the
surface of Hydrochloric acid; white fumes are evolved.
Heavy metals. For the preparation of the test solution evaporate 4g to dryness
on a water-bath, add 2ml of acetic acid (-60 g/1) PbTS, dilute to 40ml and mix;
determine the heavy metals content as described under 2.2.3 Limit test for
heavy metals, Method A; not more than 5pg/g.
Arsenic. Dilute 4.3 ml to 10ml with water. Use 1 ml and proceed as described
under 2.2.5 Limit test for arsenic; not more than 2[xg/g.
For the following three tests mix / volume of Hydrochloric acid with Z volumes of water:
Bromides and iodides. To 10ml add 1 ml of chloroform R and add cautiously,
with constant stirring, chlorine TS which has been diluted with

a drop at a time
an equal volume of water; the chloroform remains free from even a transient
yellow, orange, or violet colour.
Free bromine and chlorine. To 10ml add 1ml of potassium iodide (80g/l)
TS and 1 ml of chloroform R, and shake the mixture; the chloroform remains
free from any violet colour for at least 1 minute.
Sulfites. Mix 3 ml with 5 ml of water and add 5 drops of barium chloride

(50 g/1) TS and 2 drops of iodine (0.05 mol/1) VS; no turbidity is produced and
the colour of the iodine is not completely discharged.
Sulfates. To 20ml add 40mg of sodium hydrogen carbonate R and evaporate

to dryness on a water-bath; dissolve the residue in 20ml of water and proceed
as described under 2.2.2 Limit test for sulfates; the sulfate content is not more
than 20[ig/g.
Residue on ignition. Place 10 g in a porcelain dish and evaporate to dryness

on a water-bath. Ignite the residue to constant mass; not more than 0.1 mg/g.
Assay. Add about 1.5ml, accurately weighed, to a tared glass-stoppered flask

containing 20 ml of water; then add 25ml of water and titrate with sodium
hydroxide (1 mol/1) VS, using methyl red/ethanol TS as indicator.
Each ml of sodium hydroxide (1 mol/1) VS is equivalent to 36.46mg of HCl.
42
ACIDUM HYDROCHLORICUM DILUTUM

DILUTE HYDROCHLORIC ACID
Description. A colourless liquid; odourless.
Storage. Dilute hydrochloric acid should be kept in a rightly closed container.
Requirements
Dilute hydrochloric acid contains not less than 9 5 . % m/m and not more than
the equivalent of 10 5 %
. m/m of HCl.
Identity tests
A. It is strongly acid.
B. Use 0.5ml; it yields the reactions described under 2.1 General identification
Mass density. p 2 o = 1-043-1. 049 g/ml.
Heavy metals. For the preparation of the test solution evaporate 4g to dryness
on a water-bath, add 2ml of acetic acid (~60g/l) PbTS, dilute to 40ml and mix;
heavy metals, Method A; not more than 5pg/g.
Arsenic. Dilute 17ml to 20ml with water. Use 2ml and proceed as described
under 2.2.5 Limit test for arsenic; not more than 0.5pg/g.
Bromides and iodides. To 10ml add 1 ml of chloroform R and add cautiously,
with constant stirring, chlorine TS which has been diluted with

a drop at a time
an equal volume of water; the chloroform remains free from even a transient
yellow, orange, or violet colour.
Free bromine and chlorine. To 10 ml add 1ml of potassium iodide (80g/l)

TS and 1 ml of chloroform R, and shake the mixture; the chloroform remains
free from any violet colour for at least 1 minute.
Sulfites. Mix 3 ml with 5 ml of water and add 5 drops of barium chloride

(50g/l) TS and 2 drops of iodine (0.05 mol/1) VS; no turbidity is produced and
the colour of the iodine is not completely discharged.
43
Sulfates. To 90 ml add 40 mg of sodium hydrogen carbonate R and evaporate

to dryness on a water-bath; dissolve the residue in 20ml of water and proceed
than 5pg/g.
Residue on ignition. Place 10 g in a porcelain dish and evaporate to dryness

on a water-bath. Ignite the residue to constant mass; not more than 0.1 mg/g.
Assay. Add about 2ml, accurately weighed, to 20 ml of water and titrate with
sodium hydroxide (1 mol/1) VS, using methyl red/ethanol TS as indicator.
Each ml of sodium hydroxide (1 mol/1) VS is equivalent to 36.46mg of HCl.
ACIDUM lOPANOICUM
lOPANOIC ACID
C11H12I3NO2
Chemical name. 3-Amino-a-ethyl-2,4,6-triiodohydrocinnamic acid; 3-amino-

a-ethyl-2,4,6-triiodobenzenepropanoic acid; CAS Reg. No. 96-83-3.
Description. A light yellowish white powder; odour, faint, characteristic.
Solubility. Practically insoluble in water; soluble in ethanol (~750g/l) TS,

and acetone R; dissolves in solutions of alkali hydroxides.
Category. Radiocontrast medium.
Storage. lopanoic acid should be kept in a tightly closed container, protected

from light. lopanoic acid is gradually affected by light.
44
Requirements
lopanoic acid contains not less than 97 0 % and not more than the equivalent
.
of 101 0 % of C11HJ2I3NO2, calculated with reference to the dried substance.

.
Identity tests
A. Carry out the examination as described under 1.7 Spectrophotometry in
the infrared region. The infrared absorption spectrum is concordant with
the spectrum obtained from iopanoic acid RS or with the reference sfrectrum
of iopanoic acid.
B. Heat strongly 0.05g in a suitable crucible; violet vapours are evolved.
C. Melting temperature, about 155 °C with decomposition.
metals content according to Method A; not more than 20|ig/g.
Iodides. Dissolve 0.8 g in a minimum quantity of sodium hydroxide (lOg/1)

TS, dilute to 10ml with water, add drop by drop nitric acid (~130g/l) TS until
complete precipitation is obtained, then add an excess of 3ml. Filter, and wash
the precipitate with 5 ml of water; to the filtrate add 1 ml of hydrogen perox-
ide (~330g/l) TS and 1ml of chloroform R, and shake. To serve as a reference
solution, treat similarly 2 ml of iodide standard (20 pg I/ml) TS with 3 ml of nitric
acid (-130 g/1) TS and sufficient water to equal the volume of the solution to
be tested. Any red-violet colour in the chloroform layer is no darker than that
obtained from the reference solution.
Loss on drying. Dry at 105 °C for 1 hour; it loses not more than lOmg/g.
Assay. Place about 0.4g, accurately weighed, in a 125-ml conical flask and add
30ml of sodium hydroxide (50 g/1) TS and 0.5 g of zinc R powder. Connect the
flask to a reflux condenser and boil for 30 minutes. Cool the flask to room tem-
perature, rinse the condenser with 20ml of water into the flask, and filter the
mixture. Rinse the flask and the filter thoroughly and add the rinsing to the fil-
trate. Add 5 ml of glacial acetic acid R and 1 ml of tetrabromophenolphthalein
ethyl ester TS and titrate with silver nitrate (0.05 mol/1) VS until the yellow pre-
cipitate just changes to green.
Each ml of silver nitrate (0.05mol/l) VS is equivalent to 9.516mg of

C11H12I3NO2.
45
ACIDUM lOTROXICUM
lOTROXIC ACID
C22H18I6N2O9
Chemical name. 3,3'-[Oxybis(ethyleneoxymethylenecarbonylimino)]bis[2,4,6-

triiodobenzoic acid]; 3,3'-[oxybis[2,l-ethanediyloxy(l-oxo-2,l-ethanediyl)
imino]]-bis[2,4,6,-triiodobenzoic acid]; CAS Reg. No. 51022-74-3.
Description. An almost white powder.
Solubility. Practically insoluble in water, benzene R, and ether R; freely soluble in

methanol R and dimethylformamide R; dissolves in solutions of alkali hydroxides.
Category. Used in the preparation of meglumine iotroxate as a radiocontrast

medium.
Storage. lotroxic acid should be kept in a well-closed container, protected

from light.
Requirements
lotroxic acid contains not less than 98 0 % and not more than
. the equivalent of
102 0 %
. of C 22 H 18 6N 2 O 9
I ,
Identity tests
infrared region.
spectrum obtained from iotroxic acid RS or with the reference spectrum of
iotroxic acid.
B. Heat 0.05 g with 2 ml of sulfuric acid (-1760 g/1) TS in a suitable crucible;

violet vapours are evolved.
46
Heavy metals. For the preparation of the add 3ml of

test solution use l.Og,
meglumine (lOOg/1) TS, and proceed as described under 2,2.3 Limit test for
heavy metals, Procedure 1; determine the heavy metals content according to
Method A; not more than lOpg/g.
Halides. Dissolve lOg in 30ml of meglumine (lOOg/1) TS and titrate
potentiometrically with silver nitrate (0.001 mol/1) VS. Each ml of silver nitrate
(0.001 mol/1) VS is equivalent to 0.1269 mg of I; the content of halides, expressed
as iodides, does not exceed 40pg/g.
Solution in alkali. Dissolve 5g in 5ml of sodium hydroxide (~80g/l) TS and

add 2 ml of water; the solution is not more intensely coloured than standard
colour solution Yw2 when compared as described under 1.11 Colour of liquids.

Fischer method. Method A, using 0.4 g; the water content is not less than
lOmg/g and not more than 30mg/g.
Foreign substances. Carry out the test as described under 1.14.1 Thin-layer
chromatography, using silica gel R6 as the coating substance and a mixture of 62
volumes of chloroform R, 32 volumes of methanol R, 2 volumes of anhydrous
formic acid R, and 6 volumes of water as the mobile phase. Apply separately to
the plate 5 pi of each of two solutions in methanol R containing (A) 0,1 g of
lotroxic acid per ml and (B) 0.5 mg of lotroxic acid per ml. After removing the
plate from the chromatographic chamber, allow it to dry in a current of air at
room temperature, and examine the chromatogram in ultraviolet light (254 nm).
Any spot obtained with solution A, other than the principal spot, is not more
intense than that obtained with solution B.
Primary aromatic amines. Transfer about 1 g, accurately weighed, to a 50-

ml volumetric flask, dissolve in 2.5ml of sodium hydroxide (1 mol/1) VS, and
add 12.5ml of water (= solution A), Dissolve 5mg of 3-amino-2,4,6-triiodoben-
zoic acid RS in 0,2ml of sodium hydroxide (0,1 mol/1) VS and dilute with suf-
ficient water to produce 10 ml. Introduce 2 ml of this solution to a 50-ml
volumetric flask and add 3 ml of water and 10 ml of sodium hydroxide
(0.1 mol/1) VS (= solution B). For the blank solution, transfer 5ml of water to a
50-ml volumetric flask and add 10ml of sodium hydroxide (0.1 mol/1) VS.
Note: Strictly observe the instructions and proceed without delay using the
three solutions concurrently.
Add 25 ml of dimethyl sulfoxide R to each solution, close the flasks, and swirl
to mix. Place them in the dark in an ice-bath and allow to stand for 5 minutes.
47
Continue the procedure in the dark. Add while shaking 2 ml of hydrochloric

acid (-420 g/1) TS and allow to stand again in the ice-bath for 5 minutes. Add
while shaking 2ml of freshly prepared sodium nitrite (20g/l) TS. Using a stop-
watch readable to 1 second, start the timing and allow to stand in the ice-bath
for exactly 5 minutes. Add 1ml of freshly prepared sulfamic acid (80g/l) TS,
start the timing again, shake until no more gas evolves, and allow to stand
in the ice-bath for exactly 5 minutes. Continue to add 2 ml of freshly prepared
N-(l-naphthyl)ethylenediamine hydrochloride/propylene glycol TS, allow to
stand in a water-bath at 22-25 °C for exactly 10 minutes, and dilute to volume
with water.
Proceed immediately with the measurement of the absorbances of solutions

A and B against the blank solution at a wavelength of about 465 nm. The
absorbance of solution A does not exceed that of solution B.
Assay. Carry out the combustion as described under 2.4 Oxygen flask method,
but using about 4 mg of lotroxic acid, accurately weighed, and allowing the
absorbing liquid after rinsing to stand for 20-30 minutes. Titrate the liberated
iodine with sodium thiosulfate (0.02 mol/1) VS.
Each ml of sodium thiosulfate (0.02 mol/1) VS is equivalent to 0.6754mg of

C22Hial6hl209-
Additional requirement for lotroxic acid for parenteral use

ing, per kg of the rabbit’s mass, a solution in sterile water R containing 0.6 g of
lotroxic acid in not more than 5ml.
Additional requirement for lotroxic acid for sterile use

Complies with 3.2.1 Test for sterility of non-injectable preparations.
ACIDUM LACTICUM
LACTIC ACID
H OH
\/
H3C CO2H
CaHÔs
48
Chemical name. Lactic acid; 2-hydroxypropanoic acid; CAS Reg. No.

50-21-5.
Description. A colourless or slightly yellow, clear, syrupy, caustic liquid;

odourless or with a slight characteristic odour.
Miscibility. Miscible with water, ethanol (~750g/l) TS, and ether R.
Category. Used in the preparation of sodium lactate solution as electrolyte.
Storage. Lactic acid should be kept in a tightly closed container.
Additional information. Lactic acid as described is not suitable for parenteral

administration (haemodialysis). Lactic acid is usually a racemate {RS), but the
(+)‘(b')-isomer may predominate; it is hygroscopic.
Requirements
Definition. Lactic acid is a mixture of lactic acid, its condensation products,
and water, the equilibrium between the components being dependent on the
concentration and temperature.
Lactic acid contains not less than 88.0% m/m and not more than the equiva-
lent of 92.0% m/m of CaHÔs.
Identity tests
A. To 5 ml of a solution containing 5 mg of Lactic acid, add 1ml of bromine
TSl and 0.5ml of sulfuric acid (~100g/l) TS and heat on a water-bath until
the colour is discharged, while stirring occasionally with a glass rod (an
odour of acetaldehyde is perceptible). Add 4g of ammonium sulfate R, mix,
and add, drop by drop without mixing, 0.2 ml of a solution containing
lOmg of sodium nitroprusside R per ml of sulfuric acid (~100g/l) TS. Without
prior mixing add 1ml of ammonia (~260g/l) TS and allow to stand for 30
minutes; a dark green ring is produced at the interface of the two liquids.
B. A mixture of 1ml and 9 ml of water shows an acid reaction with pH-

indicator paper R.
C. Relative density, dfo = 1.20 - 1.21.
49
Iron. Use l.Og; the solution complies with the 2.2.4 Limit test for iron; not
more than 40 pg/g.
Calcium. Dissolve 5g in 42 ml of sodium hydroxide (1 mol/1) VS and dilute to

50ml with distilled water. Dilute 5ml to 15ml with distilled water. (Keep the
remaining solution for the “Chlorides” and “Sugars and other reducing sub-
stances” tests.)
To 0,2ml of ethanolic calcium standard (lOOpg/ml Ca) TS add 1ml of ammo-

nium oxalate (50g/l) TS and allow to stand for 1 minute. Add 1ml of acetic
acid (~60g/l) TS and the above-prepared 15ml of test solution. Similarly,
prepare a reference solution but using 10ml of calcium standard (lOpg/ml) TS
and 5ml of water. Allow both solutions to stand for 15 minutes. Any opales-
cence observed in the test solution is not more intense than that of the refer-
ence solution (200pg/g).
Chlorides. Dissolve 0.1 g in 10ml of water, acidify with nitric acid (~130g/l)
TS, and add a few drops of silver nitrate (40g/l) TS; no opalescence is imme-
diately produced.
Sulfates. Take 25ml of the solution prepared for the “Calcium” test and
proceed as described under 2.2.2 Limit test for sulfates; the sulfate content is
not more than 200pg/g.
Sugars and other reducing substances. To 1 ml of the solution prepared for

the “Calcium” test add 1 ml of hydrochloric acid (1 mol/1) VS, heat to boiling,
cool, and add 1.5ml of sodium hydroxide (1 mol/1) VS and 2ml of potassio-
cupric tartrate TS. Heat to boiling; no red or greenish precipitate is produced.
Volatile fatty acids. Heat 5g cautiously in a glass-stoppered flask at 50 °C for

10 minutes; on opening of the flaskno unpleasant odour resembling that of the
lower fatty acids is noticed.
Methanol and methyl esters. Place 2g in a round-bottomed flask and add

10ml of water. Cool in ice, add cautiously a mixture of 7.5ml of water with
22.5ml of potassium hydroxide (-400 g/1) TS, and cool in ice for a further 10-15
minutes. Connect to a suitable condenser and steam distil. Collect the distillate
in a 10-ml graduated flask containing 1ml of ethanol (~750g/l) TS and distil
until a volume of at least 9.5ml is obtained. Dilute to 10ml with water and to
1ml add 5ml of potassium permanganate/phosphoric acid TS and mix. After
15 minutes add 2ml of oxalic acid/sulfuric acid TS, stir with a glass rod until
the solution is colourless, and then add 5 ml of decolorized fuchsin TS. Allow
to stand for 2 hours. The solution is not more intensely coloured than a refer-
ence solution prepared similarly, but using instead of the distillate 1.0ml of a
solution containing lOOpg of methanol R and 0.1ml of ethanol (-750 g/1) TS
per ml (500pg/g of methanol).
50
Citric, oxalic,phosphoric, and tartaric acid. To Ig dissolved in 10ml of

water add 40 ml of calcium hydroxide TS and boil for 2 minutes; no turbidity
is produced.
Ether-insoluble substances. Dissolve 1 g in 25ml of ether R and compare it
with 25 ml of ether R; both solutions are equally clear.
Colour. Lactic acid is not more intensely coloured than standard colour solu-
tion Yw2 when compared as described under 1.11 Colour of liquids.
Assay. Place about 1 g, accurately weighed, in a glass-stoppered flask, and add

10ml of water and 20ml of sodium hydroxide (1 mol/1) VS. Stopper the flask
and allow to stand for 30 minutes. Back-titrate with hydrochloric acid (1 mol/1)
VS, using 0.5ml of phenolphthalein/ethanol TS as indicator.
Each ml of sodium hydroxide (1 mol/1) VS is equivalent to 90.08 mg of CsHÔa.
Additional requirements for Lactic acid for parenteral use

Bacterial endotoxins. Carry out the test as described under 3.4 Test for bac-
terial endotoxins; contains not more than 83.3 lU of endotoxin RS per mg.
ACIDUM NICOTINICUM
NICOTINIC ACID
Molecular formula. C6H5NO2
Graphic formula.
Chemical name. 3-Pyridinecarboxylic acid; CAS Reg. No. 59-67-6.

almost odourless.
51
Solubility. Sparingly soluble in water; freely soluble in boiling water; soluble

in 100 parts of ethanol (~750g/l) TS; practically insoluble in ether R.
Category. Component of vitamin B complex; vasodilator.
Storage. Nicotinic acid should be kept in a well-closed container, protected

from light.
Requirements
Definition. Nicotinic acid contains not less than 99.0% and not more than
101.0% of C6H5NO2, calculated with reference to the dried substance.
Identity tests
• Either test A alone or all 3 tests B, C and D may be applied.

infrared region.
spectrum obtained from nicotinic acid RS or with the reference spectrum of
nicotinic acid.
B. Heat 0.1 g with 0.4g of anhydrous sodium carbonate R; pyridine, percepti-

ble by its odour, is produced.
C. Dissolve lOmg in 10ml of water. To 2ml add 2ml of thiocyanate reagent,

obtained by adding, drop by drop, ammonium thiocyanate (0.1 mol/1) VS to
bromine TSl until the yellow coloration disappears. Then add 3 ml of aniline
(25g/l) TS and shake; a yellow colour is produced.
D. Melting temperature, about 235 °C.
Chlorides. Dissolve 1.25g in a mixture of 2ml of nitric acid (~130g/l) TS and

20ml of water, filter if necessary, and proceed as described under 2.2.1 Limit
test for chlorides; the chloride content is not more than 0.2mg/g.
Loss on drying. Dry to constant weight at 105°C; it loses not more than
lOmg/g.
pH value. pH of a 13mg/ml solution, 3. 0-3. 5.
52
85 volumes of 1 -propanol R, 10 volumes of anhydrous formic acid R, and 5
volumes of water as the mobile phase. For the test solution, dissolve 75 mg in
5.0 ml of water with gentle heating; this constitutes solution A. Prepare a ref-
erence solution containing 0.12mg/ml of nicotinic acid RS; this constitutes solu-
tion B. Apply to the plate 10 pi of solution A using two 5-pl aliquots, allowing
the plate to dry in a current of cold air after the first application; then apply
separately 5 pi of solution B. After removing the plate from the chromato-
graphic chamber, allow it to dry in a current of warm air, and examine the chro-
matogram in ultraviolet light (254nm). Beside the principal spot, not more than
3 spots are obtained with solution A, and they are not more intense than the
spot obtained with solution B.
Assay. Dissolve about 0.25 g, accurately weighed, in 50 ml of carbon-dioxide-

free water R, and titrate with carbonate-free sodium hydroxide (0.1 mol/1) VS,
using phenolphthalein/ethanol TS as indicator. Each ml of carbonate-free
sodium hydroxide (0.1 mol/1) VS is equivalent to 12.31 mg of CsHsNOj.
ACIDUM SALICYLICUM
SALICYLIC ACID
Molecular formula. C/HgOa
Graphic formula.
COOH
Chemical name. 2-Hydroxybenzoic acid; CAS Reg. No. 69-72-7.
Description. Colourless crystals, usually needle-like, or a white, crystalline

powder; odourless.
Solubility. Slightly soluble in water; soluble in 4 parts of ethanol (-750 g/1) TS

and in 3 parts of ether R.
Category. Keratolytic.
53
Storage. Salicylic acid should be kept in a well-closed container.
Requirements
Definition. Salicylic acid contains not less than 99.0% and not more than
101.0% of C/HfiOs, calculated with reference to the dried substance.
Identity test
Dissolve 0.14g in 1 ml of sodium hydroxide (1 mol/1) VS and add 5ml of water;
this solution yields the reaction described under 2.1 General identification tests
as characteristic of salicylates.
Heavy metals. Use 2.0g and 15ml of ethanol (~750g/l) TS for the preparation
of the test solution as described under 2.2.3 Limit test for heavy metals. Pro-
cedure 2; determine the heavy metals content according to Method A; not more
than 20 pg/g.
Chlorides. Dissolve 1.7g in 40 ml of boiling water, cool and filter. Add 2 ml of

TS to the filtrate and proceed as described under 2.2.1 Limit
nitric acid (~130g/l)
Sulfates. Dissolve 2,5 g in 40ml of boiling water, cool, filter, and proceed with
the filtrate as described under 2.2,2 Limit test for sulfates; the sulfate content
is not more than 0.2mg/g.
Solution in ethanol. A solution of l.Og in 10ml of ethanol (~750g/l) TS is
Loss on drying. Dry to constant weight over silica gel, desiccant, R at ambient
temperature; it loses not more than 5.0mg/g.
Assay. Dissolve about 0.3 g, accurately weighed, in 15 ml of neutralized

ethanol TS and add 20 ml of water. Titrate with carbonate-free sodium hydrox-
ide (0.1 mol/1) VS, using phenolphthalein/ethanol TS as indicator. Repeat the
operation without the substance being examined and make any necessary cor-
rections. Each ml of carbonate-free sodium hydroxide (0.1 mol/1) VS is equiva-
lent to 13.81 mg of C/HgOa.
54
ADEPS LANAE
WOOL FAT
Ad^s lanae cum aqua
Hydrous wool fat
Chemical name. Lanolin; CAS Reg. No. 8020-84-6.
Other name. Anhydrous lanolin, lanolin. (In certain countries the name
lanolin is used to describe a formulation containing wool fat, water, and liquid
paraffin.)
Description. Wool fat is a brown-yellow, unctuous mass. Hydrous wool fat

is a yellowish white, unctuous mass. Odour, characteristic.
Solubility. Practically insoluble in water; soluble in ether R; slightly soluble in

boiling ethanol (-750 g/1) TS.
Category. Ointment base.
Storage. Wool fat should be kept in a well-closed container.
Additional information. Melted Wool fat is a yellow, clear or almost clear

liquid. On heating. Hydrous wool fat first separates into two layers, water is
then driven off, and a transparent residue is formed; after cooling, a yellowish,
tenacious, soft mass is produced. Melting point for Wool fat and Hydrous wool
fat after drying (use the residue from the “Wool fat content”), 36-44 °C.
Requirements
Definition. Wool fat is a purified wax-like material obtained from the raw
wool of sheep (Ov/s aries L).
Hydrous wool fat is a mixture of 75% m/m of wool fat and 25% ni/m of water.
Identity tests
A. Dissolve 0.5 g in 5 ml of chloroform R and add 1 ml of acetic anhydride R
and 0.1ml of sulfuric acid (~1760g/l) TS; a green colour is produced.
B. Dissolve 0.5 g in 5 ml of chloroform R and carefully superimpose 5 ml of sul-

furic acid (-1760 g/1) TS; a bright, brown-red ring is gradually formed at the
interface of the two liquids.
Acid value. Wool fat, not more than 1.0; Hydrous wool fat, not more than 0.8.
55
Saponification value. Reflux for 4 hours; Wool fat, 90-105; Hydrous wool
fat, 67-79.
Sulfated ash. Wool fat, not more than 1.5mg/g; Hydrous wool fat, not more
than l.Omg/g.
Loss on drying. Dry at 105°C for 1 hour; Wool fat loses not more than
5,0mg/g; Hydrous wool fat loses not more than 0.32 g/g.
Wool fat content. Heat 30 g of Hydrous wool fat to constant mass on a water-
bath, stirring continuously, and weigh; the residue weighs between 21. 8g and
23.3 g (72.5-77 .5% m/m). (Keep the residue for “Water-absorption capacity",
“Paraffins”, and the melting point in “Additional information”.)
Water-absorption capacity. Place 10 g of Wool fat or Hydrous wool fat after

drying (use the residue from “Wool fat content”) in a mortar. Using a burette,
add water in portions of 0.2-0. 5 ml, stirring vigorously after each addition to
incorporate the water, until visible droplets separate and cannot be absorbed;
not less than 20ml of water is absorbed.
Water-soluble acid and alkaline substances. Melt 5g of Wool fat or 6.7 g

of Hydrous wool fat on a water-bath, add 75 ml of water heated to 90-95 °C,
and shake vigorously for 2 minutes. Cool and filter through a filter-paper pre-
viously moistened with water. To 60ml of the filtrate, which may show some
cloudiness (keep the remaining filtrate for “Water-soluble oxidizable sub-
stances” and “Ammonia”), add 0.25ml of bromothymol blue/ethanol TS; not
more than 0.2 ml of hydrochloric acid (0.02 mol/1) VS or 0.15 ml of sodium
hydroxide (0.02 mol/1) VS is required to change the colour of the indicator (blue-
yellow).
Water-soluble oxidizable substances. To 10 ml of the filtrate retained in the

above test add 1 ml of sulfuric acid (~100g/l) TS and 0.1 ml of potassium per-
manganate (0.02 mol/1) VS, and allow to stand for 10 minutes; the colour is not
completely discharged.
Paraffins. To 40 ml of dehydrated ethanol R add 0.5 g of Wool fat or Hydrous

wool fat after drying (use the residue from “Wool fat content”) and boil; the
solution is clear or not more than opalescent.
Ammonia. To 5 ml of the filtrate from “Water-soluble acid and alkaline sub-
stances” add 0.5 ml of sodium hydroxide (1 mol/1) VS and boil; the vapours do
not turn red litmus paper R to blue.
56
ADEPS SOLIDUS
HARD FAT
Chemical name. Hard fat.
Description. A white, brittle, unctuous mass; almost odourless.
Solubility. Practically insoluble in water; freely soluble in ether R; slightly

soluble in ethanol (-750 g/1) TS.
Category. Suppository base.
Storage. Hard fat should be kept in a well-closed container, protected from

and stored at a temperature not exceeding 5°C below the melting range.
light,
Additional information. On warming, hard fat melts to a colourless to

slightly yellow liquid. On shaking the molten material with an equal quantity
of hot water, forms a white emulsion.
Note-. For certain applications, it may be necessary to comply with more restric-
tive specifications.
Requirements
Definition. Hard fat is a mixture of mono-, di- and triglyceride esters of higher
saturated fatty acids (C 10 H 20 O 2 to C SH 36O 2
1 ).
Melting range. Cool the charged tube at a temperature below 10 °C for 24

hours; 33-40 °C.
Acid value. Not more than 0.5.
Hydroxyl value. Not more than 50.
Iodine value. Not more than 3.
Peroxide value. Not more than 6.
Saponification value. Use 2.0 g; 220-250.
Unsaponifiable matter. Use 5g; not more than 1.0%.
Ash. Not more than 0.5mg/g.
57
Alkaline impurities. Dissolve 2g in a mixture of 1.5 ml of ethanol (-750 g/1)

TS and 3 ml of ether R. Add 0.05 ml of bromophenol blue/ethanol TS; not more
than 0.15ml of hydrochloric acid (0.01 mol/1) VS is required to change the
colour of the solution to yellow.
Decomposition products. Shake 1 g with 1 ml of hydrochloric acid (-420 g/1)

TS for 1 minute, add 1ml of resorcinol/toluene TS, shake for 5 seconds, and
allow to stand for 5 minutes; the aqueous layer is not more intensely coloured
than 1ml of a mixture of 0.4ml of potassium permanganate (0.002mol/l) VS
and 9.6ml of water.
ALBENDAZOLUM
ALBENDAZOLE
C12H15N3O2S
Chemical name. Methyl 5-(propylthio)-2-benzimidazolecarbamate; CAS

Reg. No. 54965-21-8.
Description. A white or almost white powder.
Solubility. Practically insoluble in water; soluble in glacial acetic acid R;

slightly soluble in acetone R, very slightly soluble in ethanol (-750 g/1) TS.
Category. Anthelminthic.
Storage. Albendazole should be kept in a well-closed container, protected

from light.
Additional information. Melting temperature, about 210 °C, with

decomposition.
Requirements
Albendazole contains not less than 98 0 . %
and not more than 101 0 . % of
C] 2 Hi 5 N 302 S, calculated with reference to the dried substance.
58
Identity tests

infrared region.
spectrum obtained from albendazole RS or with the reference spectrum of
albendazole.
B. See the test described below under “Related substances”. The principal spot
obtained with solution B corresponds in position, appearance, and intensity
with that obtained with solution C.
C. Ignite about 0.1 g; fumes are evolved, staining lead acetate paper R black.
D. Add about 0.1 g to 3ml of sulfuric acid (-100 g/1) TS and warm to dissolve.
Add about 1ml of potassium iodobismuthate/acetic acid TS; a reddish
brown precipitate is produced.
Loss on drying. Dry at 105 °C for 4 hours; it loses not more than 5.0mg/g.
6 volumes of dichlorome thane R, 1 volume of ether R, and 1 volume of glacial
acetic acid R as the mobile phase. Apply separately to the plate 10 pi of each
of 5 solutions in a mixture of 9 volumes of dichlorome thane R and 1 volume
of anhydrous formic acid R containing (A) 10.0 mg of Albendazole per ml,
(B) 1.0 mg of Albendazole per ml, (C) 1.0 mg of albendazole RS per ml, (D)
0.05 mg of albendazole RS per ml, and (E) 0.025 mg of albendazole RS per ml.
After removing the plate from the chromatographic chamber, allow it to dry
in a current of warm air, and examine the chromatogram in ultraviolet light
(254 nm).
intense than the principal spot obtained with solution D (0.5%), and only one
spot may be more intense than the principal spot obtained with solution E
(0.25%).
Assay. Dissolve about 0.25 g, accurately weighed, in 3 ml of anhydrous formic

acid R, and add 40ml of glacial acetic acid Rl. Then add 0.2ml of 1-naphthol-
benzein/acetic acidTS and titrate with perchloric acid (0.1 mol/1) VS until a green
colour is obtained as described under 2.6 Non-aqueous titration. Method A.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 26.53 mg of

C12H15N3O2S.
59
ALCOHOL BENZYLICUS
BENZYL ALCOHOL
CH— OH
CyHaO
Chemical name. Benzyl alcohol; benzenemethanol; CAS Reg. No. 100-51-6.
Description. A clear, colourless, oily liquid; odour, slightly aromatic.
Solubility. Soluble in water; miscible with ethanol (-750 g/1) TS, ether R, fatty
and essential oils.
Category. Antimicrobial preservative.
Storage. Benzyl alcohol should be kept in a tightly closed container, protected

from light.
Additional information. Benzyl alcohol is affected by air and light, and

should be protected from exposure to excessive heat.
Requirements
Identity test
Add 2-3 drops to 5 ml of potassium permanganate (25 g/1) TS, and acidify
with 1ml of sulfuric acid (-100 g/1) TS; an odour of benzaldehyde is
perceptible.
Refractive index. «d = 1.538 - 1.541.
Relative density. = 1.043 - 1.050.
Clarity of solution. Shake 2 ml with 60ml of water; the solution is clear.
Sulfated ash. Evaporate 10 ml from a porcelain crucible and ignite to constant

mass; not more than 0.05 mg/g.
60
Acidity. To 10ml add 10ml of ethanol (~750g/l) TS and 1ml of phenolph-

thale in/ ethanol TS; not more than 1 ml of carbonate-free sodium hydroxide
(0.1 mol/1) VS is required to obtain the midpoint of the indicator (pink).
Peroxide value. Not more than 5.
Chlorinated compounds. Mix 2g with 50 ml of amyl alcohol R using a dry

flask, add in small quantities 3g of sodium R {Note-. Proceed with caution),
connect the flask to a reflux condenser, warm gently until the evolution of
hydrogen ceases, and boil gently for 1 hour. Cool the liquid to just below
100 °C and add 50 ml of water, 5 ml of silver nitrate (0.1 mol/1) VS, and 20 ml of
nitric acid (~1000g/l) TS. Titrate the excess of silver nitrate with ammonium
thiocyanate (0.1 mol/1) VS, using ferric ammonium sulfate (45g/l) TS as indica-
tor. Repeat the procedure without the Benzyl alcohol being examined and make
any necessary corrections. The difference between the titrations does not
exceed 0.3ml.
Aldehydes. Transfer about 20 ml, accurately measured, to a 250-ml conical

flask containing 5 ml of a solution containing 3.5 g of hydroxylamine hydrochlo-
ride R in 100ml of ethanol (~600g/l) TS, add 50ml of ethanol (-600 g/1) TS, and
mix. Allow to stand for 10 minutes, add 1 ml of bromophenol blue/ethanol TS,
and titrate with sodium hydroxide (0.1 mol/1) VS to a light green endpoint.
Repeat the procedure without the Benzyl alcohol being examined and make
any necessary corrections.
The net volume of sodium hydroxide (0.1 mol/1) VS consumed does not exceed
4.0ml, corresponding to 2.0mg/g of benzaldehyde. Benzyl alcohol used for par-
enteral administration does not consume more than 1.0 ml, corresponding to
0.5mg/g of benzaldehyde.
ALCOHOL CETYLICUS
CETYL ALCOHOL
H3C — [CH2li4 — CH — OH
2
C16H34O
Chemical name. 1-Hexadecanol; CAS Reg. No. 36653-82-4.
Description. Unctuous, colourless flakes or a white, crystalline mass; odour,

faint and characteristic.
61
Solubility. Practically insoluble in water; soluble in ethanol (-750 g/1) TS and

ether R.
Category. Emulsifying agent; viscosity-increasing agent.
Storage. Cetyl alcohol should be kept in a well-closed container.
Requirements
Definition. Cetyl alcohol is a mixture of solid alcohols consisting mainly of
1-hexadecanol (C16H34O).
Acid value. Not more than 2.
Saponification value. Not more than 2.
Hydroxyl value. Place about 2 g, accurately weighed, in a glass-stoppered 250-

ml flask, and add 2 ml of pyridine R and 10 ml of toluene R. To this mixture
add 10 ml of a solution of acetyl chloride prepared by adding 10 ml of acetyl
chloride R to 90ml of toluene R. Insert the stopper in the flask, and heat in a
water-bath at about 65°C for 20 minutes. Add 25ml of water, stopper the flask,
and shake vigorously for several minutes to decompose the excess acetyl chlo-
while shaking the flask vigorously throughout the titration in order
ride. Titrate
to maintain the contents in an emulsified condition with carbonate-free sodium
hydroxide (1 mol/1) VS, using 0.5ml of phenolphthalein/ethanol TS as indica-
tor, to a permanent pink endpoint. Repeat the procedure without the Cetyl
alcohol being examined and make any necessary corrections.
Multiply the difference in ml between the two titrations of carbonate-free

sodium hydroxide (1 mol/1) VS by 56.1 and divide it by the mass in g of Cetyl
alcohol used; 218-238.
Paraffin. Dissolve 0.5 g in 20ml of neutralized ethanol TS by warming; the

solution is clear and not more intensely coloured than standard colour solution
Bn2 when compared as described under 1.11 Colour of liquids.
62
ALCOHOL CETYLSTEARYLICUS
CETOSTEARYL ALCOHOL
Chemical name. 1-Octadecanol mixture with 1-hexadecanol; CAS Reg. No.
67762-27-0.
Description. A white or yellowish white, unctuous mass, or white flakes or

granules; odour, characteristic and faint.

ether R.
Category. Retarding agent; stiffening agent.
Storage. Cetostearyl alcohol should be kept in a well-closed container,

protected from light.
Requirements
Definition. Cetostearyl alcohol is a mixture of solid aliphatic alcohols con-
sisting mainly of cetyl alcohol and stearyl alcohol.
Saponification value. Not more than 2.
Hydroxyl value. Place 2.0 g in a glass-stoppered 250-ml flask, and add 2 ml of

pyridine R and 10 ml of toluene R. To this mixture add 10 ml of a solution of
acetyl chloride prepared by adding 10 ml of acetyl chloride R to 90 ml of toluene
R. Insert the stopper in the flask, and heat in a water-bath at about 65 °C for
20 minutes. Add 25ml flask, and shake vigorously for
of water, stopper the
several minutes to decompose the excess acetyl chloride. Titrate while shaking
the flask vigorously throughout the titration in order to maintain the contents
in an emulsified condition with carbonate-free sodium hydroxide (1 mol/1) VS,
using 0.5 ml of phenolphthalein/ethanol TS as indicator, to a permanent pink
endpoint. Repeat the procedure without the Cetostearyl alcohol being exam-
ined and make any necessary corrections.
Multiply the difference in ml between the two titrations of carbonate-free

sodium hydroxide (1 mol/1) VS by 56.1 and divide it by the mass in g of
Cetostearyl alcohol used; 208-228.
63
Paraffin. Dissolve 0.5 g in 20ml of neutralized ethanol TS by warming; the

solution is clear and not more intensely coloured than standard colour solution
Bn2 when compared as described under 1.11 Colour of liquids.
ALCOHOLUM
ALCOHOL
Description. A clear, colourless, and mobile liquid; odour, characteristic.
Miscibility. Miscible with water, ether R, and glycerol R.
Category. Solvent; antiseptic.
Storage. Alcohol should be kept in a well-closed container, and stored when-

ever possible at a temperature between 8 and 15 °C.
Labelling. The designation on the container should state the content of

Alcohol in % v/v.
Additional information. Different concentrations of Alcohol are prepared

from alcohol 96% v/v and water at about 20 °C.
Note-. Contraction of volume and rise in temperature occur when mixing

Alcohol with water.
Alcohol Approximate Volume of Alcohol (ml)

% v/v g/1 relative density 96% v/v to be diluted to
djo 1000 ml with water
90 701.4 0.8304 934

80 625.3 0.8610 831
70 561.8 0.8872 727
60 488.0 0.9109 623
50 404.6 0.9320 519
45 341.3 0.9412 468
25 209.0 0.9699 259
20 163.8 0.9754 207
64
Requirements
Alcohol is a mixture of ethanol and water. It contains not less than 98.0% v/v
and not more than the equivalent of 102.0% v/v of the declared content of
ethanol (C 2 H 6 O).
Identity tests
A. To 0.25 a small beaker add 1 ml of potassium permanganate (lOg/1) TS
ml in
and 0.5 ml of
sulfuric acid (0.5 mol/1) VS; cover the beaker immediately with
a filter-papermoistened with a recently prepared solution of 0.1 g of sodium
nitroprusside R and 0.5 g of piperazine hydrate R in 5 ml of water; a dark
blue colour is produced on the filter-paper, which fades after a few minutes.
B. To a few drops add 1 ml of sulfuric acid (-1760 g/1) TS and a few drops of
potassium dichromate (100 g/1) TS; a green colour is developed and an odour
of acetaldehyde is perceptible.
Relative density. According to the labelled concentration, relate to the value

stated under “Additional information”.
Non-volatile residue. Place 100ml in a porcelain dish and heat on a water-

bath until volatilized, dry the residue at 105 °C for 1 hour, and weigh; not more
than 5 mg.
Water-insoluble substances. Dilute a volume of Alcohol with an equal

volume of water; the mixture is clear and, after cooling to 10°C, remains clear
for 30 minutes.
Acidity. Add 20ml of carbon-dioxide-free water R and 3 drops of phenolph-

thalein/ethanol TS to 20 ml of Alcohol; the colour remains unchanged. Titrate
with carbonate-free sodium hydroxide (0.02 mol/1) VS; not more than 0.5ml is
required to obtain the midpoint of the indicator (pink).
Aldehydes and other foreign organic substances. Thoroughly clean a

glass-stoppered cylinder with hydrochloric acid (-250 g/1) TS, rinse with water
and the Alcohol to be examined. Place 20 ml of Alcohol in the cylinder. Cool
the contents to about 15 °C and, by means of a carefully cleaned pipette and
noting the time, add 0.1ml of potassium permanganate (0.02 mol/1) VS. Mix at
once by inverting the stoppered cylinder, and allow to stand at 15 °C for exactly
5 minutes; the pink colour does not entirely disappear.
Fusel oil and allied impurities. Carefully protected from dust, allow 25ml
to evaporate spontaneously from a porcelain dish until it is barely moist; no
foreign odour is perceptible. Add a few drops of sulfuric acid (-1760g/l) TS;
the colour does not change to brown or red.
65
Methanol. To 1 drop add 1 drop of water, 1 drop of phosphoric acid

(~105g/l) TS, and 2 drops of potassium permanganate (25g/l) TS, mix, and
allow to stand for 1 minute. Add, drop by drop, sodium metabisulfite (50g/l)
TS until the permanganate colour is discharged. If a brown colour remains, add
1 drop of phosphoric acid (~105g/l) TS. To the colourless solution, add 5 ml
of freshly prepared disodium chromotropate TS, and heat on a water-bath at

60 °C for 10 minutes; the colour does not change to violet.
Benzene. Record an absorption spectrum of the Alcohol in a 1-cm layer

against water between 220 nm and 350 nm. The absorbance at about 220 nm is
not more than 0.30, at about 230 nm not more than 0.18, at about 240 nm not
more than 0.08, and between 270 and 350 nm not more than 0.02. A curve
drawn through these points is smooth.
ALCURONII CHLORIDUM
ALCURONIUM CHLORIDE
Chemical name. Af,N'-Diallylnortoxiferinium dichloride; CAS Reg. No.

15180-03-7.
Other name. Alcuronium dichloride.
Description. A white to yellow-white, crystalline powder.
Solubility. Soluble in water and ethanol (-750 g/1) TS.
Category. Muscle relaxant.
Storage. Alcuronium chloride should be kept in a tightly closed container.
66
Labelling. The designation Alcuronium chloride for parenteral use indicates

that the substance complies with the additional requirements and may be used
for parenteral administration. Expiry date.
Additional infomiation. CAUTION-. Alcuronium chloride is highly toxic. It

must be handled with care, avoiding contact with the skin and inhalation of
airborne particles. It is hygroscopic.
Requirements
Alcuronium chloride contains not less than 98 0 . %
and not more than 101 0 % .
of C 44 H 50 CI 2 N 4 O 2 calculated with reference to the anhydrous substance.

,
Note-. All tests must be carried out immediately after opening the container, and
as rapidly as possible.
Identity tests
• Either tests A and D or tests B, C, and D may be applied.

infrared region. The infrared absorption spectrum is concordant with the
spectrum obtained from alcuronium chloride RS or with the reference sp’ec-
truni of alcuronium chloride.
B. The absorption spectrum of a 14pg/ml solution in phosphate buffer, pH 7.0

(0.067mol/l) TS, when observed between 230nm and 350nm, exhibits a
maximum at about 293 nm and a minimum at about 237 nm; the absorbance
of a 1-cm layer at the maximum wavelength is about 0.9.
C. See the test described below under “Related substances”. The principal spot
obtained with solution A corresponds in position, appearance, and intensity
with that obtained with solution B.
D. A 20 mg/ml solution yields reaction A described under 2.1 General identifi-

cation tests as characteristic of chlorides.
Specific optical rotation. Use a lOmg/ml solution, measured within 10

minutes of preparation, and calculate with reference to the anhydrous sub-
stance; [a]o = -430 ° to -451 °.
Heavy metals. Use 2.5 g for the preparation of the test solution as described
metals content according to Method B; not more than 20pg/g.
67
Clarity and colour of solution. A solution of O.lOg in 10ml of carbondiox-

ide- free water R is clear and not more intensely coloured than standard colour
solution Yw2 when compared as described under 1.11 Colour of liquids.

Fischer method, Method A, using about 0.5 g of the substance; the water
content is not more than 0.050 g/g.
pH value. pH of a lOmg/ml solution in carbon-dioxide-free water R, 6. 0-8. 5.
Related substances. Carry out the test protected from daylight until the start
of detection as described under 1.14.1 Thin-layer chromatography, using silica
gel R6 as the coating substance (a precoated plate from a commercial source is
suitable) and a mixture of 1 volume of methanol R and 1 volume of ammo-
nium nitrate TS as the mobile phase. Apply separately to the plate 5 pi of each
of 4 solutions in methanol R containing (A) 40 mg of Alcuronium chloride per
ml, (B) 40 mg of alcuronium chloride RS per ml, (C) 0.20 mg of alcuronium chlo-
ride RS per ml, and (D) 0.10 mg of alcuronium chloride RS per ml. Prior to devel-
opment allow the plate to dry in a current of cold air and place in a
chromatographic chamber. After removing the plate from the chromatographic
chamber, allow it again to dry in a current of cold air, and examine the chro-
intense than that obtained with solution C (0.5%), and no more than 3 of these
spots are greater than the spot obtained with solution D (0.25%).
Assay. To about 0.3 g, accurately weighed, add 70 ml of acetic anhydride Rand

place the mixture in an ultrasonic bath for 15 seconds. Titrate the turbid solu-
tion with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous
titration. Method A.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 36.89mg of

C44H50CI2N4O2.
Additional requirements for Alcuronium chloride for

prarenteral use
68
ALLOPURINOLUM
ALLOPURINOL
Molecular formula. C5H4N4O
Graphic formula.
Chemical name. l,5-Dihydro-4/f-pyrazolo[3,4-4/]pyrimidin-4-one; 1-ff-

pyrazolo[3,4-i]pyrimidin-4-ol; CAS Reg. No. 315-30-0.
Description. A white or almost white, microcrystalline powder; odourless or

almost odourless.
Solubility. Very slightly soluble in water and in ethanol (-750 g/1) TS; practi-
cally insoluble in ether R.
Category. Xanthine oxidase inhibitor.
Storage. Allopurinol should be kept in a well-closed container.
Requirements
Definition. Allopurinol contains not less than 98.0% and not more than
101.0% of C5H4N4O, calculated with reference to the dried substance.
Identity test
the spectrum obtained from allopurinol RS or with the reference spectrum of
allopurinol.
B. Dissolve 0.1 g in 10ml of sodium hydroxide (0.1 mol/1) VS and add sufficient
hydrochloric acid (0.1 mol/1) VS to produce 100ml; dilute 10ml to 100ml
with hydrochloric acid (0.1 mol/1) VS and dilute 10 ml of this solution again
to 100 ml with hydrochloric acid (0.1 mol/1) VS. The absorption spectrum of
the resulting solution, when observed between 230 nm and 350 nm, exhibits
a maximum at about 250 nm and a minimum at about 231 nm. The
69
absorbance at the maximum wavelength is about 0.55. The ratio of the

absorbance of a 1-cm layer at 231 nm to that at 250 nm is between 0.52 and
0.62.
C. Dissolve 0.05 g in 5 ml of sodium hydroxide (~80g/l) TS, add 1ml of alka-

line potassio-mercuric iodide TS, heat to boiling, and allow to stand; a
yellow, flocculent precipitate is produced.
5-Omg/g.
chromatography, using cellulose R3 as the coating substance. Prepare the
mobile phase by shaking 200 ml of Tbutanol R with 200 ml of ammonia
(~100g/l) TS. Apply separately to the plate 10 pi of each of 2 freshly prepared
solutions in diethylamine R containing (A) 25 mg of the test substance per ml
and (B) 0.050 mg of 3-aminopyrazole-4-carboxamide hemisulfate RS per ml.
After removing the plate from the chromatographic chamber, allow it to dry in
air, and examine the chromatogram in ultraviolet light (254 nm). Any spot
obtained with solution A, other than the principal spot, is not more intense
than that obtained with solution B.

mamide R, add 2 drops of thymol blue/dimethylformamide TS and titrate with
sodium methoxide (0.1 mol/1) VS to a blue endpoint, as described under 2.6
Non-aqueous titration. Method B. Each ml of sodium methoxide (0.1 mol/1) VS
is equivalent to 13.61 mg of C 5 H 4 N 4 O.
ALUMINII HYDROXIDUM
ALUMINIUM HYDROXIDE
Molecular formula. Al(OH)3
Chemical name. Aluminium hydroxide; CAS Reg. No. 21645-51-2.
70
Description. A white, fine, amorphous powder; odourless.
Solubility. Practically insoluble in water and ethanol (-750 g/1) TS; soluble in
hydrochloric acid (~70g/l) TS and sodium hydroxide (-80 g/1) TS.
Category. Antacid.
Storage. Aluminium hydroxide should be kept in a tightly closed container.
Requirements
Definition. Aluminium hydroxide contains not less than 71.9% and not more
than 94.9% of Al(OH) 3 .
Identity test
Dissolve O.lOg by heating in 5ml of sodium hydroxide (-80 g/1) TS. To the clear
solution add 0.5 g of ammonium chloride R; a white, gelatinous precipitate is
produced.
Heavy metals. For the preparation of the test solution dissolve 0.5g, while
heating, in 5ml of acetic acid (-300 g/1) TS, dilute to 10 ml with water and filter.
Adjust the pH of the filtrate 40 ml with water and mix. Deter-

to 3-4, dilute to
mine the heavy metals content as described under 2.2.3 Limit test for heavy
metals, according to Method A; not more than 60 pg/g.
Arsenic. Use a solution of 3.3 g in 20ml of sulfuric acid (-100 g/1) TS and
35ml of water and proceed as described under 2.2.5 Limit test for arsenic; the
arsenic content is not more than 5 pg/g.
Ammonium salts. Transfer 5.0 g to an ammonia distillation apparatus, add

25ml of sodium hydroxide (-200 g/1) TS and 200ml of water, distil about
100ml, collecting the distillate in 25.0ml of hydrochloric acid (0.1 mol/1) VS.
Titrate the excess acid with sodium hydroxide (0.1 mol/1) VS using methyl
red/ethanol TS as indicator; not less than 22.5ml of sodium hydroxide
(0.1 mol/1) VS is required.
Chlorides. Dissolve O.lOg in 2ml of nitric acid (-130 g/1) TS, boil, cool, dilute
to 10 ml with water and filter. Proceed with 5 ml of the filtrate as described
under 2.2.1 Limit test for chlorides; the chloride content is not more than
lOmg/g.
Sulfates. Dissolve O.lOg in 5ml of hydrochloric acid (-70g/l) TS, boil, cool,
dilute to 1 0 ml with water and filter. Proceed with the filtrate as described under
2.2.2 Limit test for sulfates; the sulfate content is not more than 5mg/g.
71
Neutralizing capacity. Pass a sufficient quantity of the test substance, tritu-

rated if necessary, through a sieve of nominal mesh aperture 150 pm, add
0.50 g to 200 ml of hydrochloric acid (0.05mol/l) VS previously heated to 37 °C,
and stir continuously, maintaining the temperature at 37 °C; the pH of the
solution at 37 °C, after 10, 15, and 20 minutes, is not less than 1.8, 2.3, and
3.0, respectively, and at no time more than 4.0. Add 10ml of hydrochloric acid
(0.5mol/l) VS previously heated to 37 °C, stir continuously for 1 hour, main-
taining the temperature at 37 °C. Titrate the solution with sodium hydroxide
(0.1 mol/1) VS to pH 3.5. The neutralizing capacity is not less than 83.3% of the
theoretical amount when calculated by the formula (1000)(150-y)/A xWx 38.46,
in which y is the number of ml of sodium hydroxide (0.1 mol/1) VS required, A
is the percent of Al(OH )3 obtained in the assay, W
is the quantity, in g, of test
substance taken, and 38.46 is the theoretical value of each g of Al(OH) 3 .
Alkaline impurities. The pH of a 0.04 g/ml suspension in carbon-dioxide-free

water R is not more than 10.0.
Assay. Proceed with about 0.15g, accurately weighed, as described under

2.5 Complexometric titrations for aluminium. Each ml of disodium edetate
(0.05 mol/1) VS is equivalent to 3.900 mg of Al(OH)s.
ALUMINII MAGNESII SILICAS

ALUMINIUM MAGNESIUM SILICATE
Chemical name. Magnesium aluminosilicate; aluminium magnesium silicate;
CAS Reg. No. 1327-43-1.
Other name. Aluminum magnesium silicate.
Description. A creamy white or greyish white powder or flakes; odourless or

almost odourless.
Solubility. Practically insoluble in water and most organic solvents; when

added to water it swells to form a colloidal suspension.
Category. Suspending agent; viscosity-increasing agent; tabletting aid.
Storage. Aluminium magnesium silicate should be kept in a well-closed

container.
Additional information. Several types of aluminium magnesium silicate
occur, of which the powder or flakes vary in shape and size.
72
Requirements
Definition. Aluminium magnesium silicate is a natural, colloidal hydrated
aluminium magnesium silicate, a saponite, freed from gritty particles.
Identity tests
A. In a metal crucible fuse 1 g with 2 g of anhydrous sodium carbonate R. To
the fused mass, add hot water and filter. (Keep the filtrate for test B.) To the
residue remainingon the filter, add 5 ml of hydrochloric acid (~70g/l) TS and

10ml of water, and filter. To the filtrate add 2ml of ammonia (~100g/l) TS;
a white, gelatinous precipitate is produced. Centrifuge (keep the precipitate
for test C), neutralize 2 ml of the supernatant liquid, add 0.2ml of titan
yellow TS and 0.5ml of sodium hydroxide (0.1 mol/1) VS; a bright red tur-
bidity is formed which gradually settles to give a bright red precipitate.
B. Acidify the filtrate from test A with hydrochloric acid (-420 g/1) TS and
evaporate to dryness. Heat the residue with a mixture of lOmg of calcium
fluoride R and a few drops of sulfuric acid (-1760 g/1) TS; a gas is evolved.
Add a few ml of water; it gives a white precipitate.
C. Dissolve the precipitate from test A in 2 ml of hydrochloric acid (-70 g/1) TS

and add 0.5ml of alkaline thioacetamide TS; no precipitate is produced.
Add, drop by drop, sodium hydroxide (-80 g/1) TS; a white, gelatinous pre-
cipitate appears that redissolves on addition of more sodium hydroxide. Add
slowly ammonium chloride (100 g/1) TS; the white, gelatinous precipitate
reappears.
Heavy metals. Shake l.Og with 5ml of hydrochloric acid (-70g/l) TS for 5
minutes and centrifuge. Dilute the supernatant liquid to 10ml with water,
adjust the pH, and determine the heavy metals content as described under 2.2.3
Limit test for heavy metals, Method A; not more than 40pg/g.
Acid -insoluble impurities. To l.Og add 25 ml of hydrochloric acid (-70 g/1)

TS and shake for 5 minutes. Filter through a tared sintered-glass filter, wash the
residue with water, dry to constant mass at 105°C, and weigh; the residue
weighs not more than 20 mg.
Alkalinity. Suspend 1 g in 50ml of water and titrate with hydrochloric acid

(0.1 mol/1) VS to pH 4; not more than 10ml is required.
73
ALUMINII SULFAS
ALUMINIUM SULFATE
M(so4)3,«H20
Relative molecular mass. 342.1 (anhydrous).
Chemical name. Aluminium sulfate (2:3); CAS Reg. No. 10043-01-3 (anhy-
drous). Aluminium sulfate (2:3) hydrate; CAS Reg. No. 17927-65-0 (hydrate).
Description. Colourless, lustrous crystals or crystalline masses, or a white,

crystalline powder; odourless.
Solubility. Soluble in cold water; freely soluble in hot water; practically insol-
uble in ethanol (-750 g/1) TS.
Category. Astringent drug; used in the preparation of aluminium acetate

solution.
Storage. Aluminium sulfate should be kept in a well-closed container.

Aluminium sulfate contains a variable quantity of water of crystallization.
Requirements
Aluminium sulfate contains not less than 51 0 . % and not more than the equiv-
alent of 59 0 % of Al 2 (S 04 ) 3
. .
Identity tests
A. Dissolve 0.2g in 2ml of water and add 0.5ml of hydrochloric acid (~70g/l)
TS and 0.5 ml of alkaline thioacetamide TS; no precipitate is formed. Add
drop by drop sodium hydroxide (-80 g/1) TS; a white, gelatinous precipitate
is formed which dissolves on further addition of sodium hydroxide. Grad-
ually add ammonium chloride (100 g/1) TS; a white, gelatinous precipitate
reappears.
B. A O.lg/ml solution yields reaction A described under 2.1 General identifi-

cation tests as characteristic of sulfates.
Ammonium salts. Heat Ig with 10 ml of sodium hydroxide (1 mol/1) VS on

a water-bath for 1 minute; no odour of ammonia is perceptible.
74
Iron. Use 0.4g; the solution complies with the 2.2.4 Limit test for iron; not
more than lOOpg/g.
Alkali and alkaline-earth metals. Dissolve l.Og in 100ml of water, heat,

and add 0.1 ml of methyl red/ethanol TS and sufficient ammonia (-100 g/1) TS
until the colour of the solution changes to yellow. Dilute to 150ml with water,
heat to boiling, and filter. Evaporate 75 ml of the filtrate to dryness on a water-
bath and ignite to constant mass; the residue weighs not more than 2 mg
(0.4%).
Colour and clarity of solution. A solution of 0.50 g in 10 ml of water is
colourless and not more opalescent than opalescence standard TS2.
pH value. pH of a 20 mg/ml solution in carbon-dioxide-free water R, 2. 5-4.0.
Assay. Dissolve about 0.5 g, accurately weighed, in 20 ml of water, and

proceed as described under 2.5 Complexometric titrations for aluminium.
Each ml of disodium edetate (0.05 mol/1) VS is equivalent to 8.554 mg of

Al2(SO,)s.
AMIKACINI SULFAS
AMIKACIN SULFATE
Amikacin sulfate (non-injectable)
Amikacin sulfate, sterile
Molecular formula. C 22 H 43 N 50 i 3, 2 H 2 S 04
75
Graphic formula.
Chemical name. 0-3-Amino-3-deoxy-a-D-glucopyranosyl-(l—>4)-0-[6-amino-

6-deoxy-a-D-glucopyranosyl-(l—>6)]-AP-(4-amino-L-2-hydroxybutyryl)-2-deoxy-
L-streptamine sulfate (1:2) (salt); (S)-0-3-amino-3-deoxy-a-D-glucopyranosyl-
(1—>6)-0-[6-amino-6-deoxy-a-D-glucopyranosyl-(1^4)]-N'-(4-amino-2-hydroxy-
l-oxobutyl)-2-deoxy-D-streptamine sulfate (1 :2) (salt); CAS Reg. No. 39831-55-5.
Description. A white to yellowish white, crystalline powder; almost

odourless.
Solubility. Very soluble in water; practically insoluble in methanol R, acetone

R or ether R.
Category. Antibacterial drug.
Storage. Amikacin sulfate should be kept in a tightly closed container.
Labelling. The designation sterile Amikacin sulfate indicates that the sub-
stance complies with the additional requirements for sterile Amikacin sulfate
and may be used for parenteral administration or for other sterile applications.
Requirements
Definition. Amikacin sulfate contains not less than 650 International Units per
mg, with reference to the anhydrous substance.
76
Identity tests
A. Dissolve 10 mg in 1 ml of water, add 1 ml of sodium hydroxide (~80g/l) TS
and mix, then add 2ml of cobalt(II) nitrate (lOg/1) TS; a violet colour is
produced.
B. Dissolve 0.05g in 3ml of water and add 4ml of anthrone TS; a bluish violet
colour is produced.
C. A 20 mg/ml solution yields reaction A described under 2.1 General identifi-

Specific optical rotation. Use a lOmg/ml solution and calculate with refer-
ence to the anhydrous substance; [a]o = +69 to -t79°.
Sulfated ash. After ignition moisten the residue with 2 ml of nitric acid
(-1000 g/1) TS and about 0.2 ml of sulfuric acid (-1760 g/1) TS; not more than
lOmg/g.

content is not more than 50mg/g.
Assay. Carry out the assay as described under 3.1 Microbiological assay of
(ATCC 6633) as the test organism,
antibiotics, using either {a) Bacillus subtilis
culture medium Cml with a final pH of 6.5-67, sterile phosphate buffer pH
6.0 TSl, TS2 or TS3, an appropriate concentration of amikacin (usually 5-
20 lU per ml), and an incubation temperature of 32-35 °C, or (b) Staphylococcus
aureus (ATCC 29737) as the test organism, the same culture medium and phos-
phate buffer, an appropriate concentration of amikacin (usually lOIU per ml),
and the same incubation temperature. The precision of the assay is such that
the fiducial limits of error of the estimated potency (P = 0.95) are not less than
95% and not more than 105% of the estimated potency. The upper fiducial
limit of error of the estimated potency (P = 0.95) is not less than 650 lU per mg,
Additional requirements for Amikacin sulfate for sterile use

Storage. Sterile Amikacin sulfate should be kept in a hermetically closed
container.
Sterility. Complies with 3.2.2 Sterility testing of antibiotics, applying the

membrane filtration test procedure and using the sampling plan described under
3.2.1 Test for sterility of non-injectable preparations.
77
Additional requirements for Amikacin sulfate for fârenteral

use
terial endotoxins; contains not more than 0.33 lU of endotoxin RS per mg of
amikacin.
AMIKACINUM
AMIKACIN
Molecular formula. C22H43N5O13
Graphic formula.
Chemical name. 0-3-Amino-3-deoxy-a-D-glucopyranosyl-(l—>4)-0-[6-

amino-6-deoxy-a-D-glucopyranosyl-(1^6)]-Al^-(4-amino-L-2-hydroxybu-
tyryl)-2-deoxy-L-streptamine; (S)-0-3-amino-3-deoxy-a-D-glucopyranosyl-(l
—>6)-0-[6-amino-6-deoxy-a-D-glucopyranosyl-(l—>4)]-N*-(4-amino-2-hydrox-
yoxobutyl)-2-deoxy-D-streptamine;CAS Reg. No. 37517-28-5.
Description. A white, crystalline powder; almost odourless.
Solubility. Sparingly soluble in water.
78
= ,
Storage. Amikacin should be kept in a tightly closed container.
Requirements
Definition. Amikacin contains not less than 900 pg of C 22 H 43N 50,3 per mg,
with reference to the anhydrous substance.
Identity tests
A. Dissolve 10 mg in 1 ml of water, add 1 ml of sodium hydroxide (~80g/l) TS
and mix, then add 2ml of cobalt(II) nitrate (lOg/1) TS; a violet colour is
produced.
B. Dissolve 0.05g in 3ml of water and add 4ml of anthrone TS; a bluish violet
colour is produced.
Specific optical rotation. Use a 20mg/ml solution and calculate with refer-
ence to the anhydrous substance: [a]™ -1-97 to -1-105°.
Sulfated ash. After ignition moisten the residue with 2 ml of nitric acid
(-1000 g/1) TS and about 0.2 ml of sulfuric acid (~1760g/l) TS; not more than
lOmg/g.

pH value. pH of a 10 mg/ml solution in carbon-dioxide-free water R, 9.5-11.5.
antibiotics, using either [a) Bacillus subtilis (ATCC 6633) as the test organism,
culture medium Cml with a final pH phosphate buffer pH
of 6.5-67 sterile
6.0 TSl, TS2 or TS3, an appropriate concentration of amikacin (usually 5-

20pg/ml), and an incubation temperature of 32-35 °C, or (b) Staphylococcus
aureus(ATCC 29737) as the test organism, the same culture medium and phos-
phate buffer, an appropriate concentration of amikacin (usually lOpg/ml), and
the same incubation temperature. The precision of the assay is such that the
fiducial limits of error of the estimated potency (? = 0.95) are not less than 95%
and not more than 105% of the estimated potency. The upper fiducial limit of
error of the estimated potency (P = 0.95) is not less than 900 g per mg, calcu-
lated with reference to the anhydrous substance.
79
AMILORIDI HYDROCHLORIDUM
AMILORIDE HYDROCHLORIDE
Amiloride Iwdrochloride, anhydrous
Axniloride hydrochloride dihydrate
Molecular formula. CsHsClN 70 ,HCl (anhydrous); CsHsClN 70 ,HCl, 2 H 20

(dihydrate).
Graphic formula.
n = O (anhydrous)
n = 2 (dihydrate)
Chemical name. N-Amidino-3,5-diamino-6-chloropyrazinecarboxamide

monohydrochloride; 3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecar-
boxamide monohydrochloride; 3,5-diamino-6-chloro-Ai-(diaminomethylene)
pyrazinecarboxamide monohydrochloride; CAS Reg. No. 2016-88-8 (anhy-
drous). N-Amidino-3,5-diamino-6-chloropyrazinecarboxamide monohydrochlo-
ride dihydrate; 3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecarboxamide
monohydrochloride dihydrate; 3,5-diamino-6-chloro-N-(diaminomethylene)-
pyrazinecarboxamide monohydrochloride dihydrate; CAS Reg. No. 17440-83-4
(dihydrate).
Description. A pale yellow to greenish yellow powder; odourless or almost

odourless.
Solubility. Slightly soluble in water and ethanol (-750 g/1) TS; practically insol-
uble in ether R.
Category. Diuretic.
Storage. Amiloride hydrochloride should be kept in a well-closed container,

80
Requirements
Definition. Amiloride hydrochloride contains not less than 98,0% and not
more than 101.0% of CeHaClNzOjHCl, calculated with reference to the dried
substance.
Identity tests
• Either tests A and C or tests B and C may be applied.

infrared region.
spectrum obtained from amiloride hydrochloride RS or with the reference
spectrum of amiloride hydrochloride.
B. The absorption spectrum of a 5.0|ig/ml solution in hydrochloric acid

(0.1 mol/1) VS, when observed between 230 nm and 380 nm, exhibits
maxima at about 285 nm and 361 nm; the absorbances of a 1-cm layer at the
maximum wavelength of 285nm and 361 nm are about 0.28 and 0.31,
respectively.
C. A 5mg/ml solution yields reaction A described under 2.1 General identifi-

Sulfated ash. Not more than l.Omg/g,

Fischer method. Method A, using about 0.2 g of the substance; for the dihy-
drate the water content is not less than llOmg/g and not more than 130mg/g.
Free acid. Dissolve 1.0 g in a mixture of 50ml of methanol R and 50 ml of

water, and titrate with sodium hydroxide (0.1 mol/1) VS determining the end-
point potentiometrically; not more than 0.3 ml is required.
15 volumes of tetrahydrofuran R and 2 volumes of ammonia (~50g/l) TS as the
mobile phase. Apply separately to the plate 5 pi of each of 2 solutions in a
mixture of 4 volumes of methanol R and 1 volume of chloroform R containing
(A) 0.40 mg of the test substance per ml, (B) 4.0 pg of the test substance per ml.
airand examine the chromatogram in ultraviolet light (365 nm). Any spot
Assay. Dissolve about 0,45 g, accurately weighed, in a mixture of 100ml of

glacial acetic acid Rl, 15ml of dioxan R, and 10ml of mercuric acetate/acetic
81
acid TS, and titrate with perchloric acid (0.1 mol/1) VS, determining the end-
point potentiometrically as described under 2.6 Non-aqueous titration, Method
A. Each ml o£ perchloric acid (0.1 mol/1) VS is equivalent to 26.61mg of
C«HaClN 70 ,HCl.
AMINOPHYLLINUM
AMINOPHYLLINE
Molecular formula. (C 7 HsN 402 ) 2 ,C 2 H 8N 2 (anhydrous) or C 16H 24N 10 O 4
Relative molecular mass. 420.4 (anhydrous)
Graphic formula.
Chemical name. Theophylline compound with ethylenediamine (2 :

1); 3,7-
dihydro-l,3-dimethyl-lH-purine-2,6-dione compound with 1,2-ethanediamine
(2:1); CAS Reg. No. 317-34-0 (anhydrous).
Description. White or slightly yellowish granules or powder; odour, slightly

ammoniacal.
Solubility. Freely soluble in water (the solution may become cloudy in the
presence of carbon dioxide); slightly soluble in ethanol (-750 g/1) TS; practically
Category. Antispasmodic; diuretic; coronary vasodilator.
Storage. Aminophylline should be kept in a tightly closed container, protected

from light.
Additional information. Aminophylline contains a variable quantity of water

of hydration. Upon exposure to air Aminophylline gradually loses ethylenedi-
amine and absorbs carbon dioxide with the liberation of free theophylline. Even
in the absence of light, Aminophylline is gradually degraded on exposure to a
humid atmosphere, the decomposition being faster at higher temperatures.
82
Requirements
Definition. Aminophylline contains not less than 78.0% and not more than
86.0% of theophylline (C7H8N4O2), and not less than 12.8% and not more than
15.0% of ethylenediamine (C2H8N2), both calculated with reference to the
anhydrous substance.
Identity tests
A. Dissolve 1 g in 10 ml of water and add, drop by drop, while shaking 2 ml of
hydrochloric acid (~70g/l) TS. Collect the precipitate on a filter, wash it with
water and dry at 105°C; melting temperature, about 272 °C (theophylline).
Keep the precipitate for test B.
B. To 10 mg of the precipitate obtained from test A, contained in a porcelain

dish, add 1ml of hydrochloric acid (-250 g/1) TS and 0.5ml of hydrogen
peroxide (-60 g/1) TS, and evaporate to dryness on a water-bath. Add 1 drop
of ammonia (-100 g/1) TS; the residue acquires a purple colour which is
destroyed by the addition of a few drops of sodium hydroxide (-80 g/1) TS.
C. Dissolve 0. 05 g in 1 ml of water and add 2 drops of copper(II) sulfate (80 g/1)
TS; a deep violet colour is produced.
D. Warm 0.05 g with 2 ml of sodium hydroxide (-80 g/1) TS and 2 drops of chlo-
roform R; an isocyanide, perceptible by its characteristic odour (proceed
Clarity of solution. A solution of l.Og in 10ml of boiling water is clear or is
not more than slightly opalescent.

Fischer method. Method A, using about 0.15g of the substance and 25ml of
pyridine R as the solvent; the water content is not more than 80mg/g.
Alkalinity. Add 1 drop of thymol blue/ethanol TS to a 10 mg/ml solution pre-

pared in carbon-dioxide-free water R; a green or blue colour is produced.
Assay
For theophylline. Place about 0.25 g, accurately weighed, in a 250-ml conical
flask, add 50ml of water and 8 ml of ammonia (-100 g/1) TS and gently warm
the mixture on a water-bath until complete solution is effected. Add 20.0ml of

silver nitrate (0.1 mol/1) VS, mix, heat to boiling and boil for 15 minutes. Cool
to between 5°C and 10°C for 20 minutes, then filter through a filtering
crucible under reduced pressure and wash the precipitate 3 times with 10-ml
83
portions of water. Acidify the combined filtrate and washings with nitric acid
(~1000g/l) TS, and add an excess of 3 ml of the acid. Cool, add 2 ml of ferric
ammonium sulfate (45g/l) TS, and titrate the excess of silver nitrate with
ammonium thiocyanate (0.1 mol/1) VS. Each ml of silver nitrate (0.1 mol/1) VS
is equivalent to 18.02 mg of C H 8N 4 O 2
7 .
For ethylenediamine. Dissolve about 0.5g, accurately weighed, in 30ml of

water and titrate with hydrochloric acid (0.1 mol/1) VS, using bromocresol
green/ethanol TS as indicator. Repeat the operation without the substance
being examined and make any necessary corrections. Each ml of hydrochloric
acid (0.1 mol/1) VS is equivalent to 3.005mg of C 2 HgN 2 .
Additional requirements for Aminophytline for parenteral use

AMITRIPTYLINI HYDROCHLORIDUM
AMITRIPTYLINE HYDROCHLORIDE
Molecular formula. C2oH23N,HCl
Graphic formula.
HCI
CH(CHj)jN(CH3)j
Chemical name. 10,1 l-Dihydro-N,N-dimethyl-5ff-dibenzo[a,T]cycloheptene-

A^’^-propylamine hydrochloride; 3-(10,ll-dihydro-5H-dibenzo[ia,^cyclohepten-
5-ylidene)-A/',Al-dimethyl-l-propanamine hydrochloride; CAS Reg. No. 549-18-8.
Description. Colourless crystals or a white or almost white powder; odour-

less or almost odourless.
Solubility. Soluble in 1 part of water and in 1.5 parts of ethanol (-750 g/1) TS;
practically insoluble in ether R.
84
Category. Antidepressant.
Storage. Amitriptyline hydrochloride should be kept in a tightly closed con-

Additional information. Amitriptyline hydrochloride has a bitter and

burning taste that is followed by a sensation of numbness. Even in the absence
of light, Amitriptyline hydrochloride is gradually degraded on exposure to a
Requirements
Definition. Amitriptyline hydrochloride contains not less than 99.0% and
not more than 101.5% of C 2 oH 23N,HCl, calculated with reference to the dried
substance.
Identity tests
infrared region.
spectrum obtained from amitriptyline hydrochloride RS or with the refer-
ence spectrum of amitriptyline hydrochloride.
B. A 20mg/ml solution yields reaction B described under 2.1 General identifi-

C. Melting temperature, about 197 °C.
Loss on drying. Dry to constant weight at 60 °C under reduced pressure (not

exceeding 0.6kPa or about 5 mm
of mercury); it loses not more than 5.0mg/g.
pH value. pH of a 10 mg/ml solution, 4. 5-6.0.
85 volumes of cyclohexane R, 15 volumes of ethyl acetate R and 3 volumes of
diethylamine R as the mobile phase. Apply separately to the plate 10 pi of each
of 2 solutions in chloroform R containing (A) 20 mg of the test substance per
ml and (B) 0.20 mg of the test substance per ml. After removing the plate from
the chromatographic chamber, allow it to dry in air, and examine the chro-
matogram in ultraviolet light (254nm). Any spot obtained with solution A,
other than the principal spot, is not more intense than that obtained with
solution B.
85
Assay. Dissolve about 0.3 g, accurately weighed, in 30ml of glacial acetic acid
Rl, add 10ml of dioxan R and 10ml of mercuric acetate/acetic acid TS, and
titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous
titration. Method A. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to
31. 39 mg of C2 oH23N,HC1.
AMODIAQUINI HYDROCHLORIDUM
AMODIAQUINE HYDROCHLORIDE
Molecular formula. C 2 oH 22 ClN 30 ,
2 HCl, 2 H 20
Graphic formula.
Chemical name. 4-[(7-Chloro-4-quinolyl)amino]-a-(diethylamino)-o-cresol

dihydrochloride dihydrate; 4-[(7-chloro-4-quinolinyl)amino]-2-[(diethylamino)-
methyl]phenol dihydrochloride dihydrate; CAS Reg. No. 6398-98-7.
Description. A yellow, crystalline powder; odourless.
Solubility. Soluble in about 22 parts of water; sparingly soluble in ethanol

(-750 g/1) TS; practically insoluble in ether R.
Category. Antimalarial.
Storage. Amodiaquine hydrochloride should be kept in a tightly closed

container.
Requirements
Definition. Amodiaquine hydrochloride contains not less than 98.0% and not
more than 101.5% of C 2 oH 22 ClN 30 2 HCl, calculated with reference to the
,
86
Identity tests
• Either test A alone or all 3 tests B, C, and D may be applied.

reference spectrum of amodiaquine hydrochloride.
B. To 1ml of a 20 mg/ml solution add 0.5 ml of cobaltous thiocyanate TS; a

green precipitate is produced.
C. A 20mg/ml solution yields reaction B described under 2.1 General identifi-

D. Melting temperature, determined without previous drying, about 158 °C

with decomposition.

pH value. pH of a 20 mg/ml solution, 4. 0-4. 8.
chromatography. Prepare a solution of chloroform saturated with ammonia by
shaking chloroform R with ammonia (-260 g/1) TS and separate the chloroform-
layer. Use silica gel R2 as the coating substance and a mixture of 9 volumes of
chloroform saturated with ammonia, and 1 volume of dehydrated ethanol R as
the mobile phase. For the preparation of the test solutions transfer 0.20 g of the
substance being examined to a glass-stoppered test-tube, add 10 ml of chloro-
form saturated with ammonia, shake vigorously for 2 minutes, allow the solids
to settle, and decant the solution to a second tube; this constitutes solution A.
Dilute 1.0ml of solution A with sufficient chloroform saturated with ammonia
to 200 ml; this constitutes solution B. Apply separately to the plate 10 pi of each
of solutions A and B. After removing the plate from the chromatographic
chamber, allow it to dry in air, and examine the chromatogram in ultraviolet
light (254 nm). Any spot obtained with solution A, other than the principal spot,
is not more intense than that obtained with solution B.
Assay. Dissolve about 0.3 g, accurately weighed, in 50 ml of water, make the

solution alkaline with ammonia (-100 g/1) TS, and allow to stand for 30
minutes. Filter, wash the residue with water until the washings are free from
chlorides, and dry to constant weight at 105 °C. Each g of residue is equivalent
to 1.205g of C oH 22 C N 30 2 HC
2 1 ,
1.
87
AMODIAQUINUM
AMODIAQUINE
Molecular formula. C20H22CIN3O
Graphic formula.
Cl
Chemical name. 4-[(7-Chloro-4-quinolyl)amino]-a-(diethylamino)-o-cresol;

4-[(7-chloro-4-quinolinyl)amino]-2-[(diethylamino)methyl]phenol; CAS Reg.
No. 86-42-0.
Solubility. Practically insoluble in water.
Category. Antimalarial drug.
Storage. Amodiaquine should be kept in a tightly closed container.
Requirements
Definition. Amodiaquine contains not less than 97.0% and not more than
103.0% of C 20 H 22 CIN 3 O, calculated with reference to the anhydrous substance.
Identity tests
• Either test A or tests B and C may be applied.

spectrum obtained from the free base of amodiaquine hydrochloride RS or
with the reference spectrum of amodiaquine.
88
B. Dissolve 20 mg in 1.0ml of water and add 0.5ml of ammonium thio-

cyanate/cobalt(II) nitrate TS; a green precipitate is produced.

content is not more than 5.0mg/g.
Related substances. Carry out the test as described under 1.14.1 Thin-layer chro-
matography. Prepare a solution of chloroform saturated with ammonia by shaking
chloroform R with ammonia (-260 g/1) TS and separate the chloroform layer. Use
silica gel R2 as the coating substance and a mixture of 9 volumes of chloroform,
saturated with ammonia, and 1 volume of dehydrated ethanol R as the mobile
phase. For the preparation of the test solution dissolve 0.15g of the substance being
examined in 10ml of chloroform saturated with ammonia (solution A). For the
preparation of the reference solutions transfer 40 mg of amodiaquine hydrochlo-
rideRS to a glass-stoppered test-tube, add 2.0ml of chloroform saturated with
ammonia, and shake vigorously for 2 minutes. Allow the solids to settle, and decant
the solution to a second test-tube (solution B). Dilute 1.0ml of solution B to
200ml with chloroform ammonia (solution C). Apply separately to
saturated with
the plate lOpl of each of solutions A, B and C. After removing the plate from the
chromatographic chamber, allow it to dry in air and examine the chromatogram
in ultraviolet light (254 nm). Any spot obtained with solution A, other than the
principal spot, is not more intense than that obtained with solution C.
Assay. Dissolve about 0.3 g, accurately weighed, in sufficient hydrochloric acid

(0.1 mol/1) VS to produce 200ml; dilute 10.0ml of this solution to 1000ml with
the same medium. Separately prepare a reference solution containing 15 pg of
amodiaquine hydrochloride RS per ml of hydrochloric acid (0.1 mol/1) VS.
Measure the absorbance of a 1-cm layer of both solutions at the maximum at
about 342 nm against a solvent cell containing hydrochloric acid (0.1 mol/1) VS.
Calculate the quantity, in mg, of C 20 H 22 CIN 3 O in the substance being examined
using the formula (355.9/428.8) (20C) (A/Af), in which 355.9 and 428.8 are the
relative molecular masses of amodiaquine and anhydrous amodiaquine
hydrochloride, respectively, C is the concentration, in pg per ml, calculated with
reference to the anhydrous substance of amodiaquine hydrochloride RS in the
reference solution, and A„ and A, are the absorbances of the solution of the sub-
stance being examined and the reference solution, respectively.
AMOXICILLINUM TRIHYDRICUM
AMOXICILLIN TRIHYDRATE
;
cOjH
HO
C,,Hi,N305S,3H20
Chemical name. (-)-6-[2-Amino-2-(;?-hydroxyphenyl)acetamido]-3,3-

dimethyl-7-oxo-4-thia-l-azabicyclo [3.2.0]heptane-2-carboxylic acid trihydrate;
(2S,5i?,6i?)-6-[(i?)-2-amino-2-(4-hydroxyphenyl)acetamido]-3,3-dimethyl-7-
oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate; 6-[[amino
(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo
[3.2.0]heptane-2-carboxylic acid trihydrate; CAS Reg. No. 61336-70-7.
Solubility. Slightly soluble in water and methanol R; very slightly soluble in

ethanol (-750 g/1) TS, ether R, and fatty oils; soluble in dilute acids and dilute
solutions of alkali hydroxides.
Storage. Amoxicillin trihydrate should be kept in a tightly closed container.
Requirements
Amoxicillin trihydrate contains not less than 95 0 . % and not more than the
equivalent of 102 0 % of C16H19N3O5S,
. calculated with reference to the anhy-
drous substance.
Identity tests
• Either test A alone or tests B and C may be applied.

infrared region.
spectrum obtained from amoxicillin trihydrate RS or with the reference spec-
trum of amoxicillin trihydrate.
90

test as described
using silanized silica gel R3 as the coating substance and a mixture of 9
volumes of a solution containing 15.4 g of ammonium acetate R in 100 ml,
the pH of which has been adjusted to 5.0 with glacial acetic acid R, and 1
volume of acetone R as the mobile phase. Apply separately to the plate
1 pi of each of 3 solutions in sodium hydrogen carbonate (40g/l) TS con-
taining (A) 2.5 mg of Amoxicillin trihydrate per ml, (B) 2.5 mg of amoxicillin
trihydrate RS per ml, and (C) a mixture of 2.5 mg of amoxicillin trihydrate
RS and 2.5mg of ampicillin trihydrate RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air until the solvents
have evaporated. Expose the plate to iodine vapours until the spots appear
and examine the chromatogram in daylight.

ance, and intensity with that obtained with solution B. The test is valid only
if the chromatogram obtained with solution C shows two clearly separated
spots.
C. Place about 2mg in a test-tube (150mm x 15mm), moisten with 1 drop of

water, and add about 2 ml of sulfuric acid (-1760 g/1) TS. Mix the contents
of the tube by swirling; the solution remains practically colourless. Place the
tube in a water-bath for 1 minute; a dark yellow colour develops.
Specific optical rotation. Use a 2.0 mg/ml solution in carbon-dioxide-free

water R and calculate with reference to the anhydrous substance; [aj^ =
-t290° to -t315°
Solution in hydrochloric acid and ammonia. Prepare a solution of 1.0 g in

10ml of hydrochloric acid (0.5mol/l) VS. Prepare a second solution of l.Og in
10ml of ammonia (-100 g/1) TS. Examine both solutions immediately.
Neither of these solutions are more opalescent than opalescence standard

TS3.
metals content according to Method A; not more than 20p.g/g.
Sulfated ash. Not more than lOmg/g.

Fischer method, Method A, using about 0.1 g of Amoxicillin trihydrate; the
water content is not less than 0.115g/g and not more than 0.145g/g.
pH value. pH of a 2 mg/ml solution in carbon-dioxide-free water R, 3. 5-5. 5.
91
Related substances. Carry out the test as described under 1.14.4 High per-
formance liquid chromatography, using a stainless steel column (25 cm x
4.6 mm) packed with particles of silica gel, the surface of which has been mod-
ified with chemically bonded octadecylsilyl groups (5 pm). Prepare the follow-
ing pH 5 buffer solution to be used in the mobile phases: to 250 ml of potassium

dihydrogen phosphate (27.2 g/1) TS add sodium hydroxide (~80g/l) TS until a
pH of 5.0 is reached, and dilute the solution with sufficient water to produce
1000 ml. As mobile phase A use a mixture of 99 volumes of buffer solution pH
5.0 and 1 volume of acetonitrile R. As mobile phase B use a mixture of 8
volumes of buffer solution pH 5.0 and 2 volumes of acetonitrile R.
Prepare the following solutions in mobile phase A: solution (A) 1.5 mg of

Amoxicillin trihydrate per ml; solution (B) 0.015mg of amoxicillin trihydrate
RS per ml; and solution (C) 0.15pg of amoxicillin trihydrate RS per ml.
Operate with a flow rate of 1.0ml per minute. As a detector use an ultraviolet
spectrophotometer set at a wavelength of about 254 nm.
Using a 50-pl loop injector, inject solution B. Start the elution isocratically with
the mobile phase mixture used for the equilibration. Immediately after elution
of the amoxicillin peak start a linear gradient elution to reach a ratio of mobile
phase A: B of 0: 100 over a period of 25 minutes. Adjust the sensitivity of the
system so that the height of the principal peak is at least 50% of the full scale
of the recorder. Continue the chromatography with mobile phase B for 15
minutes, then equilibrate the column for 15 minutes with the mobile phase
originally used for the equilibration. The mass distribution ratio for the first
peak (amoxicillin) is 1.3-2. 5. Inject mobile phase A using the 50-pl loop injec-
tor and use the same elution gradient to obtain a blank. Inject solution C using
the 50-pl loop injector. Adjust the system to obtain a peak with a signal-tonoise
ratio of at least 3.
Using the 50-pl loop injector, inject solution A. Measure the areas of the peak
responses obtained in the chromatograms from solutions A and B, and calcu-
late the content of the related substances as a percentage. In the chromatogram
obtained with solution A, the area of any peak, other than the principal peak
and any peak obtained in the blank chromatogram, is not greater than that of
the principal peak obtained with solution B (1%).
Assay. Dissolve about 0.06g, accurately weighed, in sufficient water to

produce 500 ml. Simultaneously, prepare a reference solution containing 0.06g
of amoxicillin trihydrate RS. Transfer 10.0ml of one solution to a 100-ml vol-
umetric flask and 10.0ml of the other solution to a second 100-ml volumetric
flask. To each add 10ml of buffer borate, pH 9.0, TS and 1ml of acetic anhy-
dride/ dioxan TS, mix, allow to stand for 5 minutes at room temperature, and
dilute to volume with water. Transfer two 2.0ml aliquots of each solution to
separate stoppered test-tubes. To one tube containing the test solution, and to
92
the other, containing the reference solution, add 10 ml of imidazole/mercuric

chloride TS, mix, stopper the tubes, and place them in a water-bath at 60 °C
for exactly 25 minutes. Cool the tubes rapidly to 20 °C {solution A), To the
remaining tubes add 10 ml of water and mix {solution B). Without delay, measure
the absorbances of a 1-cm layer at the maximum at about 325nm of both solu-
tions A, using as a blank a mixture of 2.0ml of water and 10ml of imidazole/
mercuric chloride TS placed in the solvent cell. For solutions B use water as a
blank placed in the solvent cell.
From the difference between the absorbances of solutions A and solutions B, cal-
culate the percentage content of C] 6 H] 9 N 306 S by comparison with amoxicillin
trihydrate RS, with reference to the anhydrous substance.
AMPHOTERICINUM B
AMPHOTERICIN B
Amphotericin B for parenteral use
Molecular formula. C 47 H 73 NO 17
Graphic formula.
Chemical name. {\R,BS,5RM,9RAl-RA5SA6R,\7R,\BSA9E,2\E,2BE,25E,27E,

29£,31£,33R,35S,36R,37S)-33-[(3-Amino-3,6-dideoxy-P-D-mannopyranosyl)-
oxy]-l,3,5,6,9,ll,17,37-octahydroxy-15,16,18-trimethyl-13-oxo-14,39-dioxa-
bicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene-36- carboxylic
acid; [1R-(1R*3S*5R‘6R*9R*11R‘15S*,16R*,17R‘18S‘19£,21£,23£,25£,27£,
29£,31£,33R*,35S*,36R*,37S*)]-33-[(3-amino-3,6-dideoxy-P-D-mannopyra-
nosyl)oxy] -1,3, 5, 6, 9, 11, 17,37 -octahydroxy- 15, 16, 18-trimethyl- 13-0X0-14,39-
93
dioxabicyclo[33.3.1]nonatriaconta-l 9,2 1 ,23,25,27,29,31-heptaene-36-

carboxylic acid; (3R,5R,8R,9R,llS43R45S,16R47S,19R,34S,35R,36R,37S)-19-(3-
amino-3,6-dideoxy-|3-D-mannopyranosyloxy)-16-carboxy-3,5,8,9,ll,13,15,35-
octahydroxy-34,36-dimethyl43,17-epoxyoctatriaconta-20,22,24,26,28,30,
CAS Reg. No. 1397-89-3.
32-heptaen-37-olide;
Description. A yellow to orange powder; odourless or almost odourless.
Solubility. Practically insoluble in water, ethanol (-750 g/1) TS, toluene R and
ether R; soluble in 200 parts of dimethylformamide R and in 20 parts of
dimethyl sulfoxide R, slightly soluble in methanol R.
Category. Antifungal drug.
Storage. Amphotericin B should be kept in a tighdy closed container, pro-

tected from light, and stored at a temperature between 2 and 8°C.
Labelling. The designation Amphotericin B for parenteral use indicates that

the substance complies with the altered and additional requirements for
Amphotericin B and may be used for parenteral administration.
Additional information. Even in the absence of light. Amphotericin B is grad-

ually degraded on exposure to a humid atmosphere, the decomposition being
faster at higher temperatures. In diluted solutions it is sensitive to light and is
inactivated at low pH values.
Requirements
Definition. Amphotericin B contains not less than 750 pg per mg, calculated
with reference to the dried substance.
Identity tests
A. Dissolve 25 mg in 5 ml of dimethyl sulfoxide R, add sufficient methanol R
to produce 50 ml, and dilute 2.0 ml to 200ml with methanol R. The absorp-
tion spectrum of the resulting solution, when observed between 300 nm and
450 nm, exhibits 3 maxima at about 362 nm, 381 nm, and 405 nm. The ratio
of the absorbance of a Tcm layer at 362 nm to that at 381 nm is about 0.6;
the ratio of the absorbance at 381 nm to that at 405 nm is about 0.9.
B. Dissolve about 1 mg in 2.0ml of dimethyl sulfoxide R and introduce 5ml of

phosphoric acid (-1440 g/1) TS to form a lower layer; a blue ring is imme-
diately formed at the interface of the two liquids. Mix the two liquids; a
strong blue colour is produced. Add 15 ml of water and mix; the colour of
the solution changes to pale yellow.
Sulfated ash. Not more than 30mg/g.
94

exceeding 0.6kPa or about 5mm of mercury); it loses not more than 50mg/g.
Content of tetraenes. Dissolve 0,05 g, accurately weighed, in 5ml of

dimethyl sulfoxide R, and add sufficient methanol R to produce 50 ml; dilute 4
ml to 50ml with methanol R (solution A). For the reference solutions dissolve
similarly 0.05 g, accurately weighed, of amphotericin B RS instead of the
substance being examined (solution B). Further prepare a solution of 25mg of
nystatin RS, accurately weighed, in 25 ml of dimethyl sulfoxide R, and add
sufficientmethanol R to produce 250 ml; dilute 4 ml to 50 ml with methanol R
(solution C).Measure the absorbances of a 1-cm layer of solutions A, B, and C
at the at about 282 nm and 304 nm, using as a blank a solution of 5 ml
maxima
of dimethyl sulfoxide R diluted to 50ml with methanol R, 4 ml of which are
diluted once again to 50 ml with methanol R.
C at both wavelengths and then apply

Calculate the A( °(m of solutions A, B, and
the following formula: F + 1000(B]A2 - B 2 Ai)/(C 2 Bi - C B 2 ), where Ai and A 2
1
are the Ajlm of the substance being examined at 282 nm and 304 nm, respec-
tively, Bi and B 2 are the of amphotericin B RS at 282 nm and 304 nm,
respectively, C, and C 2 are the of nystatin RS at 282 nm and 304 nm,
respectively, and F is the declared content of tetraenes in amphotericin B RS;
the content of tetraenes in the substance examined is not more than 150mg/g.
Assay. Triturate 0.060 g with dimethylformamide R and add, with shaking,

sufficient dimethylformamide R to produce 100ml. Dilute 10ml to 100ml with
dimethylformamide R and carry out the assay as described under 3.1 Microbi-
ological assay of antibiotics, using Saccharomyces cerevisiae (NCTC 10716, or
ATCC medium Cm3 with a final pH of 6.1,
9763) as the test organism, culture
sterile phosphate buffer pH TSl, an appropriate concentration of ampho-
10.5,
tericinB (usually between
0.5 and lO.Opg/ml), and an incubation temperature
of 29-33 °C. The precision of the assay is such that the fiducial limits of error
of the estimated potency (P = 0.95) are not less than 95% and not more than
105% of the estimated potency. The upper fiducial limit of error of the esti-
mated potency (P = 0.95) is not less than 750 pg per mg, calculated with refer-
ence to the dried substance.
Additional requirements for Amp>hotericin B for fârenteral use

Content of tetraenes. Not more than lOOmg/g.
95
AMPICILLINUM
AMPICILLIN
Ampicillin anhydrous
Ampicillin trihydrate
Molecular formula. C16H19N3O4S (anhydrous); CisHiijN304S,3H20 (trihydrate).
Relative molecular mass. 349.4 (anhydrous); 403.5 (trihydrate).
Graphic formula.
n = 0 (anhydrous)
n = 3 (trihydrate)
Chemical name. (2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetamido]-3,3-

dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid; [2S-[2a,5a,
6(3(S’ *)]] -6- [(aminophenylace tyl)amino]-3,3-dimethyl-7-oxo-4-thia- 1 -azabicyclo
[3.2.0]-heptane-2-carboxylic acid; CAS Reg. No. 69-53-4 (anhydrous).
(2S,5R,6R)-6-[(i^)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-
l-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate; [2S-[2a,5a,6(3(S*)]]-6-
[(aminophenylacetyl)amino]-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
CAS Reg. No. 7177-48-2 (trihydrate).
heptane-2-carboxylic acid trihydrate;
Description. A white or almost white, crystalline powder; odourless or

almost odourless.
Solubility. Slightly soluble in water; practically insoluble in ethanol (-750 g/1)

TS and ether R.
Category. Antibiotic.
Storage. Ampicillin should be kept in a tightly closed container, protected

from light.
Labelling. The designation on the container of Ampicillin should state

whether the substance is in the anhydrous form or is the trihydrate.
96
Additional information. Even in the absence of light, Ampicillin is gradually

degraded on exposure to a humid atmosphere, the decomposition being faster
at higher temperatures.
Requirements
Definition. Ampicillin contains not less than 95.0% and not more than
102.0% of Ck;H] 9 N 304 S, calculated with reference to the anhydrous substance.
Identity tests
• Either test A or test B may be applied.

infrared region. For the anhydrous form the infrared absorption spectrum
is concordant with the spectrum obtained from ampicillin RS or with the
reference spectrum of ampicillin.
For the trihydrate the infrared absorption spectrumis concordant with the
spectrum obtained from ampicillin trihydrate RS or with the reference spec-

trum of ampicillin trihydrate.
B. To 2 mg in a test-tube add 1 drop of water followed by 2 ml of sulfuric acid

(-1760 g/1) TS and mix; the solution is colourless. Immerse the test-tube for
1 minute in a water-bath; the solution remains colourless. Place 2 mg in a
second test-tube, add 1 drop of water and 2 ml of formaldehyde/sulfuric acid
TS and mix; the solutionis colourless to slightly pink. Immerse the test-tube
for 1 minute in a water-bath; an orange-yellow colour is produced.
Specific optical rotation. Use a 2.5mg/ml solution and calculate with refer-
ence to the anhydrous substance: [a]D = -h280 to -h305°.

Fischer method, Method A.
For the anhydrous form use about 0.8 g of the substance; the water content is
not more than 15mg/g.
For the trihydrate use about 0.1 g of the substance; the water content is not less
than 120mg/g and not more than 150mg/g.
pH value. pFl of a 2.5 mg/ml solution, 3. 5-6.0.
Assay. Dissolve about 0.12 g, accurately weighed, in sufficient water to

produce 500 ml. Transfer 10.0 ml of this solution to a 100-ml volumetric flask,
add 10 ml of buffer borate, pH 9.0, TS, 1 ml of acetic anhydride/dioxanTS, allow
to stand for 5 minutes at room temperature and dilute to volume with water.
97
Transfer two 2.0-ml aliquots of this solution into separate stoppered tubes. To
one tube add 10.0ml of imidazole/mercuric chloride TS, mix, stopper the tube,
and place in a water-bath at 60 °C for exacdy 25 minutes. Cool the tube rapidly
to 20 °C (solution A).
To the second tube add 10.0ml of water and mix (solution B).
Without delay, measure the absorbance of a 1-cm layer at the maximum at

about 325 nm, against a solvent cell containing a mixture of 2.0 ml of water and
10.0ml of imidazole/mercuric chloride TS for solution A and water for solu-
tion B.
From the difference between the absorbance of solution A and that of solution
B, calculate the amount of C 16H 19N 3 O 4 S in the substance being tested by com-
parison with ampicillin RS similarly and concurrently examined. In an adequate
calibrated spectrophotometer the absorbance of the reference solution should
be 0.29 + 0.02.
AMPICILLINUM NATRICUM
AMPICILLIN SODIUM
Ampicillin sodium (non-injectable)
Ampicillin sodium, sterile
Molecular formula. C](sHi 8 N Na 04 S

3
Graphic formula.
Chemical name. Sodium (2S,5/?,6R)-6-[(R)-2-amino-2-phenylacetamido]-3,3-

dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate; sodium [2S-
[2a,5a,6P(S*)]]-6-[(aminophenylacetyl)amino]-3,3-dimethyl-7-oxo-4-thia-l-aza
bicyclo[3.2.0]heptane-2-carboxylate; CAS Reg. No. 69-52-3.
Description. A white or almost white powder; odourless.
98
Solubility. Soluble in about 2 parts of water; practically insoluble in ether R.
Category. Antibiotic,
Storage. Ampicillin sodium should be kept in a tightly closed container, pro-

tected from light.
Labelling. The designation sterile Ampicillin sodium indicates that the sub-
stance complies with the requirements for sterile Ampicillin sodium and may
be used for parenteral administration or for other sterile applications.
Additional information. Ampicillin sodium is a crystalline or amorphous

powder; it is very hygroscopic and is deliquescent in air with a relative humid-
ity of 60% or more. Even in the absence of light, Ampicillin sodium is gradu-
ally degraded on exposure to a humid atmosphere, the decomposition being
faster at higher temperatures.
Requirements
Definition. Ampicillin sodium contains not less than 85.0% and not more
than 96.0% of C]5Hi9N304S, calculated with reference to the anhydrous sub-
stance. Furthermore, the sum of the percentage of C15H19N3O4S determined in
the assay and the percentage of iodine-absorbing compounds, both calculated
with reference to the anhydrous substance, is not less than 90.0%,
Identity tests

spectrum obtained from ampicillin sodium RS or with the reference spectrum
of ampicillin sodium.

TS and mix; the solution is colourless. Immerse the test-tube for 1 minute
in a water-bath; a dark yellow colour is produced.
C. When tested for sodium as described under 2.1 General identification tests
yields the characteristic reactions. If reaction B is to be used, ignite a small
quantity and dissolve the residue in acetic acid (-60 g/1) TS.
99
=
Specific optical rotation. Use a 5.0mg/ml solution in acetate standard buffer TS

and calculate with reference to theanhydrous substance; [a]o +260 to +290°.
Clarity of solution. A freshly prepared solution of l.Og in 10ml of water is

clear. A solution of l.Og in 10ml of hydrochloric acid (1 mol/1)VS is also clear.

pH value. pH of a O.lOg/ml solution in carbon-dioxide-free water R, 8.0-10.0.
Iodine-absorbing compounds. Dissolve 0.25 g in sufficient water to produce

100ml. To 10ml of this solution add 0.5ml of hydrochloric acid (1 mol/1) VS
and 10ml of iodine (0.01 mol/1) VS and titrate with sodium thiosulfate (0.02
mol/1) VS, using starch TS as indicator, added towards the end of the titration.
Repeat the procedure without the substance being examined; the difference
between the titrations represents the amount of iodine-absorbing compounds.
Calculate as a percentage the amount of these compounds in the examined sub-
stance, taking into account that each ml of sodium thiosulfate (0.02 mol/1) VS
is equivalent to 0.7392 mg of iodine-absorbing compounds expressed as
C16H19N3O4S.

produce 500 ml. Transfer 10.0 ml of this solution to a 100-ml volumetric flask,
add 10ml of buffer borate, pH 9.0, TS, 1ml of acetic anhydride/dioxan TS,
allow to stand for 5 minutes at room temperature and dilute to volume with
water.
Transfer two 2.0-ml aliquots of this solution into separate stoppered tubes. To
one tube add 10.0 ml of imidazole/mercuric chloride TS, mix, stopper the tube
and place in a water-bath at 60 °C for exactly 25 minutes. Cool the tube rapidly
to 20 °C (solution A).
Without delay measure the absorbance of a 1 -cm layer at the maximum at about
325 nm against a solvent cell containing a mixture of 2.0 ml of water and 10.0 ml
of imidazole/mercuric chloride TS for solution A and water for solution B.
From between the absorbance of solution A and that of solution

the difference
B, calculate as a percentage the amount of C](;H] 9N 304 S in the substance being
tested by comparison with ampicillin RS similarly and concurrently examined.
In an adequately calibrated spectrophotometer the absorbance of the reference
solution should be 0.29 ± 0.02.
100
Additional requirements for Amp>iciltin sodium for sterile use

ampicillin.
Sterility. Complies with 3.2.2 Sterility testing of antibiotics, applying either

the membrane filtration test procedure with added penicillinase TS or the direct
test procedure and using the sampling plan described under 3.2.1 Test for steril-
ity of non-injectable preparations.
AMYLA
STARCHES
Chemical name. Starch; CAS Reg. No. 9005-25-8.
Description. A white to slightly yellowish, fine powder or ovoid granules

whose size and shape are characteristic for each botanical variety; odourless.
Solubility. Practically insoluble in cold water and ethanol (-750 g/1) TS.
Category. Tablet and capsule binder; diluent; disintegrant.
Storage. Starches should be kept in tighdy closed containers.
Labelling. The designation on the container of starches should state the botan-
ical source.
Additional information. Types of starches should not be interchanged since

the properties are characteristic for each one obtained from different botanical
sources and thus their performance may not be identical. Attention should be
paid to the microbiological purity since starches are of natural origin.
Requirements
Definition. Starches consist of polysaccharide granules obtained from the
grains of corn {Zea mays L.), of rice {Oryza sativa L.), of wheat {Triticum aestivum
L.), or from the tubers of the potato {Solatium tuberosum L.).
Identity tests
A. To 1 g add 50 ml of water, heat to boiling for 1 minute, and cool; a thin cloudy
mucilage is obtained from all starches other than potato starch, which gives
a thicker and more translucent mucilage. (Keep the mucilages for test B.)
101
B. To 1 ml of the mucilage obtained in test A add 0.05 ml of iodine (0.005mol/l)
VS and mix; a dark blue colour is obtained which disappears on heating and
reappears on cooling.
Microscopic examination
Corn starch - Angular polyhedral or rounded granules, up to 35 pm in diame-
ter; central hilum consisting of a distinct cavity or several rayed central clefts.
Rice starch - Polyhedral granules, size 2-1 0 pm, either isolated or aggregated
in ovoid masses; central hilum poorly visible.
Wheat starch - Two distinct types of granule, either simple lenticular, 20-
50 pm in diameter, or small spherical, 5-1 0 pm in diameter; hilum and striations
poorly visible.
Potato starch - Simple irregular, ovoid, or spherical granules, up to 100 pm in

size; hilum near the narrower end; striations well marked and concentric.
Iron. Shake 1.5g with 15ml of hydrochloric acid (~70g/l) TS and filter. The
filtrate complies with the 2.2.4 Limit test for iron; not more than lOpg/g.
Sulfated ash
Corn starch - not more than 6mg/g.
Rice starch - not more than lOmg/g.
Wheat starch - not more than 6 mg/g.
Potato starch - not more than 6mg/g.
Loss on drying. Dry to constant mass at 100 °C:
Corn starch - not more than 150 mg/g.
Rice starch - not more than 150mg/g.
Wheat starch - not more than 150 mg/g.
Potato starch - not more than 200 mg/g.
Acidity. To lOg add 100ml of ethanol (~600g/l) TS previously neutralized

using phenolphthalein/ethanol TS, shake for 1 hour, filter, and titrate 50 ml of
the filtrate with sodium hydroxide (0.1 mol/1) VS; not more than 2.0ml is
required to change the colour of the solution.
102
Foreign matter. Using a microscope, not more than traces of cell debris are
present, and there are no granules of any origin other than that stated on the label.
Oxidizing matter. Shake 5g with a mixture of 10 ml of water and 1.2 ml of

acetic acid (-300 g/1) TS until a suspension of homogenous appearance is
obtained. Add 0.5ml of a freshly prepared saturated solution of potassium

iodide R; no blue, brown, or red colour is observed.
Sulfur dioxide. Mix 20 g with 200 ml of water until a suspension of homoge-

nous appearance is obtained. Filter. To 100ml of the clear filtrate add 3 ml of
starch TS and titrate with iodine (0.005mol/l) VS until a permanent blue colour
is obtained; not more than 2.7 ml is required (0.08 mg/g).
AQUA PRO INJECTIONE

WATER FOR INJECTIONS
Description. A clear and colourless liquid; odourless.
Category. Solvent.
Labelling. The designation on the container should indicate that water for
injections is non-sterile.
Storage. Water for injections should be kept in a well-closed container.
Additional information. CAUTION: Water for injections is not sterile. It is

not a final dosage form, but an intermediate product in the manufacture of par-
enteral preparations, either for bulk injectable solutions or for “Sterile water for
injections”.
Requirements
Definition. Water for injections is pyrogen-free. It contains no added
substance.
Manufacture. Water for injections is obtained from potable or Purified water

by distillation in an apparatus of which the parts in contact with the liquid are
of neutral glass, quartz, or suitable metal and fitted with an effective device to
prevent entrainment of droplets. The first portion of the distillate obtained
when the apparatus begins to function is discarded. The distillate is collected
and stored in conditions designed to prevent growth of microorganisms and to

avoid any other contamination.
103
Heavy metals. Use 40ml, adjust the pH with acetic acid (~60g/l) PbTS, and
proceed as described under 2,2,3 Limit test for heavy metals, procedure 1; deter-
mine the heavy metals content according to Method A, allowing to stand for
10 minutes; the colour is not darker than that of 40 ml of the same untreated
Water for injections, the pH of which has been similarly adjusted.
Ammonia. Transfer 50ml to a comparison tube, add 2 ml of alkaline potassio-

mercuric iodide TS, and observe down the vertical axis of the tube in diffused
white background; the colour produced is not more intense than
light against a
that of 50 ml of ammonia-free water R with the addition of 2 ml of dilute
ammonium chloride TS.
Calcium and magnesium. To 100ml add 2 ml of ammonium chloride buffer,

pH 10.0, TS, 50mg of mordant black HR, and 0.5ml of disodium edetate
(0.01 mol/1) VS; a pure blue colour is produced.
Carbon dioxide. To 25ml add 25ml of calcium hydroxide TS; it remains clear.
Chlorides. To 10 ml add 1 ml of silver nitrate (40g/l) TS and allow to stand for

5 minutes; it remains clear and colourless.
Nitrates. Carefully superimpose 5 ml on 5 ml of diphenylamine/sulfuric acid

TS, ensuring that the liquids do not mix; no blue colour appears at the inter-
face of the two liquids.
Sulfates. To 10 ml add 1ml of barium chloride (50g/l) TS and allow to stand

for 5 minutes; it remains clear and colourless.
Oxidizable matter. To 100ml add 10ml of sulfuric acid (-lOOg/1) TS and

0.2 ml of potassium permanganate (0.02 mol/1) VS and boil for 3 minutes; the
colour is not completely destroyed.
Non-volatile residue. Boil 500 ml on a hot plate or over a flame until the
volume is reduced to about 50 ml, then evaporate on a water-bath to dryness.
Dry the residue for 1 hour at 105 °C; not more than 5 mg (0.01 mg/ml).
Acidity or alkalinity. To 10 ml add 2 drops of methyl red/ethanol TS; no red

colour appears. To a further 10 ml portion add 5 drops of bromothymol
blue/ethanol TS; no blue colour develops.
terial endotoxins; contains not more than 0.25 lU of endotoxin RS per ml.
104
AQUA PURIFICATA
PURIFIED WATER
H2O
Chemical name. Water; CAS Reg. No. 7732-18-5.
Description. A clear, colourless liquid; odourless.
Category. Solvent.
Storage. Purified water should be kept in a well-closed container.
Labelling. The designation on the container should indicate the method of

preparation.
Additional information. CAUTION:
— Purified water must not be used for preparations intended for parenteral
administration.
— Purified water intended for ophthalmic preparations must be sterilized imme-

diately before use (see 3.2 Test for sterility and 5.8 Methods of sterilization).
Requirements
Definition. Purified water contains no added substance.
Manufacture. Purified water is prepared by distillation, ion-exchange treat-
ment, reverse osmosis, or other appropriate process from suitable water.
Heavy metals. Use 40 ml, adjust the pH with acetic acid (~60g/l) PbTS and
proceed as described under 2.2.3 Limit test for heavy metals, procedure 1; deter-
mine the heavy metals content according to Method A, allowing to stand for
10 minutes; the colour is not darker than that of 40 ml of the same untreated
Purified water, the pH of which has been similarly adjusted.

light against a white background; the colour produced is not more intense than
105

(0,01 mol/1) VS; a pure blue colour is produced.
Carbon dioxide. To 25 ml add 25ml of calcium hydroxide TS; it remains clear.



Oxidizable matter. To 100ml add 10ml of sulfuric acid (~100g/l) TS and

0.5ml of potassium permanganate (lOg/1) TS and boil for 3 minutes; the colour
is not completely destroyed.
Non-volatile residue. Evaporate 500 ml on a water-bath to dryness and dry

the residue for 1 hour at 105 °C; not more than 5 mg (0.01 mg/ml).
Acidity or alkalinity. To 10 ml add 2 drops of methyl red/ethanol TS; a red

colour does not appear. To a further 10 ml portion add 5 drops of bromothy-
mol blue/ethanol TS; no blue colour develops.
Additional requirement for Purified water for sterile use

AQUA STERILISATA PRO INJECTIONE

STERILE WATER FOR INJECTIONS
Description. A clear and colourless liquid; odourless.
Category. Solvent (for extemporaneous use).
Storage. Sterilewater for injections should be kept in a single dose container

the size of which is not larger than 1 litre.
106
Requirements
Definition. Sterile water for injections is Water for injections that is pyrogen-
free and sterile. It contains no added substance. It is suitably packaged.
Manufacture, (see 5.8 Methods of sterilization).
Heavy metals. Use 40ml, adjust the pH with acetic acid (~60g/l) PbTS, and
proceed as described under 2.2.3 Limit test for heavy metals, procedure 1; deter-
mine the heavy metals content according Method A, allowing to stand for
to
10 minutes; the colour is not darker than that of 40ml of the same untreated
Sterile water for injections, the pH of which has been similarly adjusted.

white background; the colour produced is not more intense than
light against a

(0.01 mol/1) VS; a pure blue colour is produced.
Carbon dioxide. To 25ml add 25ml of calcium hydroxide TS; it remains clear.



Oxidizable matter. To 100ml add 10ml of sulfuric acid (~100g/l) TS and

0.5ml of potassium permanganate (lOg/1) TS and boil for 3 minutes; the colour
is not completely destroyed.
Non-volatile residue. Evaporate 500 ml on a water-bath to dryness and dry

the residue for 1 hour at 105°C; not more than 5mg (0.01 mg/ml).
Acidity or alkalinity. To 10 ml add 2 drops of methyl red/ethanol TS; no red

colour appears. To a further 10 ml portion add 5 drops of bromothymol
blue/ethanol TS; no blue colour develops.
107
terial endotoxins; contains not more than 0.25 lU of endotoxin RS per ml.
Sterility. Complies with 3.2 Test for sterility.
ARGENTI NITRAS
SILVER NITRATE
Molecular formula. AgNOa
Chemical name. Silver(l-i-) nitrate; CAS Reg. No. 7761-88-8.
Description. Colourless or white crystals or white cylindrical rods; odourless.
Solubility. Soluble in 0.5 parts of water; soluble in ethanol (~750g/l) TS.
Category. Antiinfective agent.
Storage. Silver nitrate should be kept in a tightly closed, non-metaUic con-

Additional information. Even in the absence of light, Silver nitrate is gradu-

faster at higher temperatures. On exposure to light and in the presence of
organic matter, it becomes grey or greyish black.
Requirements
Definition. Silver nitrate contains not less than 99.0% and not more than
100.5% of AgNOa.
Identity tests
A. Dissolve 20 mg in 1.0 ml of water, add ammonia (~100g/l) TS, drop by drop,
until the precipitate first formed just dissolves; add about 0.1 ml of formalde-
hyde TS and warm the mixture; glossy metallic silver forms on the wall of
the test-tube.
B. Dissolve 20 mg in 1.0 ml of water and add a few drops of potassium iodide

(~80g/l) TS; a cream-coloured precipitate is produced which is insoluble in
ammonia (-100 g/1) TS and nitric acid (-1000 g/1) TS.
108
C. To 2 ml of a 0.05 g/ml solution add 2 ml of ferrous sulfate (15g/l) TS; it yields

reaction A described under 2.1 General identification tests as characteristic
of nitrates.
Clarity and colour. A solution of 0.4 g in 10 ml of water is clear and colourless.
Acidity or alkalinity. Dissolve 0.4 g in 10 ml of water; to a 2-ml portion add

0.1 ml of bromocresol green/ethanol TS; the colour of the solution is blue. To
another 2-ml portion of the test solution add 0.1 ml of phenol red/ethanol TS;
the colour of the solution is yellow.
Foreign salts. Dissolve 1.2g in 30 ml of water, add 7.5 ml of hydrochloric acid

(~70g/l) TS, shake vigorously, heat on a water-bath for 5 minutes and filter.
Evaporate 20 ml of the filtrate to dryness on a water-bath and dry at105°C;
the residue weighs not more than 2.0 mg.
Bismuth, copper, and lead. Dissolve 1 .0 g in 5 ml of water, add drop by drop

ammonia (-lOOg/1) TS until the precipitate first formed just dissolves; the solu-
tion is clear and colourless.
Assay. Dissolve about 0.3 g, accurately weighed, in 50 ml of water, add 2 ml

of nitric acid (-130 g/1) TS and 4 ml of ferric ammonium sulfate (45g/l) TS.
Titrate ammonium thiocyanate (0.1 mol/1) VS until a reddish yellow colour

with
is produced. Each ml of ammonium thiocyanate (0.1 mol/1) VS is equivalent to
16.99mg of AgNOj.
ARTEMETHERUM
ARTEMETHER
C16H26O5
109
Chemical name. (3i^,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-10-methoxy-

3,6,9-trimethyl-3,12-epoxy-12f/-pyrano[4,3-/]-l,2-benzodioxepin; CAS Reg.
No. 71963-77-4.
Description. White crystals or a white, crystalline powder.
Solubility. Practically insoluble in water; very soluble in dichlorome thane R

and acetone R; freely soluble in ethyl acetate R and dehydrated ethanol R.
Storage. Artemether should be kept in a tightly closed container, protected

from light and stored in a cool place.
Labelling. The designation Artemether for parenteral use indicates that the
substance complies with the additional requirements and may be used for par-
enteral administration.
Additional information. The parenteral form is normally intended for intra-

muscular administration.
Requirements
Artemether contains not less than 97 0 % and not more than the equivalent of
.
102 0 % of C 16H 26 O 5 using Assay method A, and not less than 98 0

. and not . %
more than the equivalent of 102 0 % of C 16H 26 O 5 using Assay method B, both
.
Identity tests
• Either tests A and B or tests B, C, and D may be applied.

infrared region.
spectrum obtained from artemether RS or with the reference spectrum of
artemether.
B. See the test described below under “Related substances test B”. The princi-
pal spot obtained with solution D corresponds in position, appearance, and
intensity with that obtained with solution E.
C. To 30mg add about 1 ml of dehydrated ethanol R and about 0.1 g of potas-

sium iodide R. Heat the mixture on a water-bath; a yellow colour is
produced.
no
D. Dissolve 30 mg in 6.0 ml of dehydrated ethanol R. Place a few drops of the

mixture on a white porcelain dish and add 1 drop of vanillin/sulfuric acid
TSl; a pink colour is produced.
Melting range. 86.0-90.0 °C.
Specific optical rotation. Use a 10 mg/ml solution in dehydrated ethanol R;
[a]g’"^ = +166° to +173°.
Loss on drying. Dry over phosphorus pentoxide R under reduced pressure

(not exceeding 2.67 kPa or 20 mm
of mercury); it loses not more than S.Omg/g.
Related substances
A. Carry out the test as described under 1.14.4 High performance liquid chro-
matography, using the conditions given below under Assay method A.
Inject alternately 20 pi each of solutions A and C.
Measure the areas of the peak responses obtained in the chromatograms

from solutions A and C, and calculate the content of the related substances
as a percentage. In the chromatogram obtained with solution A, the area of
any peak, other than the principal peak, is not greater than that obtained
with solution C (0.5%). Not more than one peak is greater than half the
area of the principal peak obtained with solution C (0.25%). The sum of
the areas of all the peaks, other than the principal peak, is not greater than
twice the area of the principal peak obtained with solution C (1.0%). Dis-
regard any peak with an area less than 0.1 times that of the principal peak
in the chromatogram obtained with solution C.
B. Carry out the test as described under 1.14.1 Thin-layer chromatography,

using silica gel R1 as the coating substance and a mixture of 7 volumes of
lightpetroleum R1 and 3 volumes of ethyl acetate R as the mobile phase.

Apply separately to the plate lOpl of each of the following 5 solutions
in acetone R containing (A) 10 mg of Artemether per ml, (B) 0.05 mg of
Artemether per ml, (C) 0.025mg of Artemether per ml, (D) O.lOmg of
Artemether per ml, and (E) O.lOmg of artemether RS per ml. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air, and
spray with vanillin/sulfuric acid TSl. Examine the chromatogram in
daylight.
Ill
Any spot obtained with solution A, is not more

other than the principal spot,
intense than that obtained with solution B (0.5%). Furthermore, not more
than one such spot is more intense than that obtained with solution C
(0.25%).
Assay
• Either method A or method B may be applied.
A. Determine by 1.14.4 High performance liquid chromatography, using a

stainless steel column (25 cm x 4 mm) packed with particles of silica gel, the
surface of which has been modified with chemically bonded octadecylsilyl
groups (5 pm). As the mobile phase, use a mixture of 62 volumes of
acetonitrile R and 38 volumes of water.
Prepare the following solutions in the mobile phase; solution (A) 10 mg of

Artemether per ml; solution (B) lOmg of artemether RS per ml; and for solu-
tion (C) dilute solution A to obtain a concentration equivalent to 0.05 mg of
Artemether per ml.
Operate with a flow rate of 1.5 ml per minute. As a detector use an

ultraviolet spectrophotometer set at a wavelength of about 216nm.
Inject alternately 20 pi each of solutions A and B.

from solutions A and B, and calculate the percentage content of C 16 H 26O 5
B. Dissolve about 0.050 g of Artemether, accurately weighed, in sufficient dehy-

drated ethanol R to produce 100ml. Dilute 2ml of this solution to 100ml
with hydrochloric acid/ethanol (1 mol/1) VS. Stopper the flask and place it in
a water-bath at 55 °C for 5 hours. Allow to cool to room temperature.
Measure the absorbance of this solution in a 1-cm layer at the maximum at

about 254 nm. Calculate the percentage content of C 16 H 26 O 5 by comparison
with artemether RS, similarly and concurrently examined, and with refer-
Additional requirement for Artemether for p>arenteral use

Complies with the monograph for “Parenteral preparations” and with 5.6 Test
for extractable volume for parenteral preparations, and 5.7 Visual inspection of
particulate matter in injectable preparations.
112
ARTEMISININUM
ARTEMISININ
Chemical name. (3R,5aS,6R,8aS,9i?,12S,12ai?)-Octahydro-3,6,9-trimethyl-

3,12-epoxy-12/f-pyrano[4.3-/]-l,2-benzodioxepin-10(3/^)-one; CAS Reg. No.
63968-64-9.
Description. Colourless needles or a white, crystalline powder.
Solubility. Practically insoluble in water; very soluble in dichloromethane R;

freely soluble in acetone R and ethyl acetate R; soluble in glacial acetic acid R,
methanol R and ethanol (~750g/l) TS.
Storage. Artemisinin should be kept in a well-closed container, protected from

light and stored in a cool place.
Requirements
Artemisinin contains not less than 97 0 % and not more than the equivalent of
.
102 0 % of C 15 H 22 O 5 using Assay method A, and not less than 98 0 % and not
. .
more than the equivalent of 102 0 % of C 15 H 22 O 5 using Assay method B, both

.
Identity tests

spectrum obtained from artemisinin RS or with the reference spectrum of
artemisinin.
113
C. Dissolve 5 mg in about 0.5 ml of dehydrated ethanol R, add about 0.5ml

of hydroxylamine hydrochloride TS2 and 0.25 ml of sodium hydroxide
(-80g/l) TS. Heat the mixture in a water-bath to boiling, cool, add 2 drops
of hydrochloric acid (~70g/l) TS and 2 drops of ferric chloride (50g/l) TS;
a deep violet colour is immediately produced.
D. Dissolve 5mg in about 0.5ml of dehydrated ethanol R, add 1.0ml of potas-

sium iodide (80g/l) TS, 2.5ml of sulfuric acid (-100 g/1) TS and 4 drops of
starch TS; a violet colour is immediately produced.
[a]g'"^ = -t75° to -)-78°
Loss on drying. Dry to constant mass at 80 °C; it loses not more than
5-Omg/g.
Related substances
matography, using a column (10 cm x 4.6mm) packed with
stainless steel
particles of silica gel, the surface of which has been modified with chemi-
cally bonded octadecylsilyl groups (pmm). The mobile phases for gradient
elution consist of a mixture of acetonitrile and water, using the conditions
shown in the following table:
Time Mobile phase A Mobile phase B Comment

(min) (% v/v of acetonitrile) (% v/v of water)
0-17 60 40 Isocratic
17-30 60 => 100 40 ^0 Linear gradient
30-35 100 ^ 60 0 => 40 Return to initial conditions
35-45 60 40 Isocratic - re-equilibration
Prepare the following solutions. For solution (A) use 10 mg of Artemisinin

per ml in a mixture of 8 volumes of acetonitrile R and 2 volumes of water.
114
and for solution (B) use 50 |ig of Artemisinin per ml in a mixture of 6 volumes
of acetonitrile R and 4 volumes of water.
For the system suitability test prepare solution (C) containing 1 mg of

artemisinin RS per ml and 1 mg of artenimol RS per ml in a mixture of 8
volumes of R and 2 volumes of water.
acetonitrile
Operate with a flow rate of 0.6ml per minute. As a detector use an ultravi-
olet spectrophotometer set at a wavelength of about 216nm.
Inject alternately 20 pi each of solutions A, B, and C.

from solutions A and B, and calculate the content of the related substances
with solution B (0.5%). Not more than one peak is greater than half the area
of the principal peak obtained with solution B (0.25%). The sum of the areas
of all peaks, other than the principal peak, is not greater than twice the area
of the principal peak obtained with solution B (1.0%). Disregard any peak
with an area less than peak in the chro-
0.1 times the area of the principal
matogram obtained with solution B. The
not valid unless the relative
test is
retention of a-artenimol compared with artemisinin is about 0.6, and the

resolution between the peaks is not less than 2.0.

using silica gel R1 as the coating substance and a mixture of equal volumes
of light petroleum R1 and ether R as the mobile phase. Apply separately to
the plate 10 pi of each of the following 5 solutions in toluene R containing
(A) 10 mg of Artemisinin per ml, (B) 0.05 mg of Artemisinin per ml, (C)
0.025 mg of Artemisinin per ml, (D) 0.10 mg of Artemisinin per ml, and (E)
0.10 mg of artemisinin RS per ml. After removing the plate from the
chromatographic chamber, allow it to dry in air, spray with anisaldehyde/
sulfuric acid TS, and heat the plate to 105 °C for 7 minutes. Examine the
chromatogram in daylight.
(0.25%).
Assay

stainless steel column (10cm x 4.6mm) packed with particles of silica gel.
115
the surface of which has been modified with chemically bonded octade-
cylsilyl groups (3 pm). As the mobile phase, use a mixture of 6 volumes of
Prepare the following solutions in the mobile phase: solution (A) l.Omg of
Artemisinin per ml; and solution (B) l.Omg of artemisinin RS per ml.
For the system suitability test prepare solution (C) containing 1 mg of

artemisinin RS per ml and 1 mg of artenimol RS per ml in a mixture of 8
volumes of acetonitrile R and 2 volumes of water.
The testnot valid unless the relative retention of a-artenimol compared

is
with artemisinin is about 0.6, and the resolution between the peaks is not
less than 2.0.

from solutions A and B, and calculate the percentage content of C15H22O5
B. Dissolve about 0.05 g of Artemisinin, accurately weighed, in sufficient

ethanol (~750g/l)TS to produce 100ml, and dilute 10ml to 100ml with the
same solvent Accurately transfer 10 ml to a 50-ml volumetric flask, dilute
to volume with sodium hydroxide (0.05 mol/1) VS, mix thoroughly, and
warm to 50 °C in a water-bath for 30 minutes. Cool to room temperature.
Measure the absorbance of a 1-cm layer at the maximum at about 292 nm

against a solvent cell containing a blank prepared with 10 ml of ethanol
(-750 g/1) TS diluted with sufficient sodium hydroxide (0.05mol/l) VS to
produce 50 ml. Calculate the percentage content of C15H22O5 in the sub-
stance being tested by comparison with artemisinin RS, similarly and con-
currently examined, and with reference to the dried substance.
116
ARTEMOTILUM
ARTEMOTIL
C17H28O5
CH 3
Chemical name. (3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-10-ethoxy-3,

6,9-trimethyl-3,12-epoxy-12f7-pyrano[4,3-/]-l>2-benzodioxepin; CAS Reg. No.
75887-54-6.
Other names. Arteether, p-arteether.
Description. A white or almost white, crystalline powder.
Solubility. Practically insoluble in water; sparingly soluble in dichloromethane R,

ethanol (-750 g/1) TS and methanol R; soluble in arachis oil R and sesame oil R.
Storage. Artemotil should be kept in a well-closed container, protected from

light.
Labelling. The designation Artemotil for parenteral use indicates that the
substance complies with the additional requirements and may be used for
parenteral administration.
Additional information. The parenteral form is normally intended for intra-

muscular administration.
Requirements
Artemotil contains not less than 97.0% and not more than the equivalent of
102 0 %
. of C 17 H 28 O 5 calculated with reference to the dried substance.
117
Identity tests
• Either tests A and B or tests B, C, and D may be applied.

infrared region.
spectrum obtained from artemotil RS or with the reference spectrum of
artemotil.
C. To 30mg add about 1ml of dehydrated ethanol R and about 0.1 g of potas-
sium iodide R. Heat the mixture on a water-bath; a yellow colour is
produced.
D. Dissolve 30 mg in 6.0 ml of dehydrated ethanol R. Place a few drops of the

Melting range. 81.0-84.0 °C.
Specific optical rotation. Use a 20 mg/ml solution in dehydrated ethanol R

and calculate with reference to the dried substance; [«]d = -1-155° to -I- 157°

(not exceeding 2.67 kPa or 20 mm of mercury); it loses not more than 5.0mg/g.
Related substances
matography, using the conditions given below under Assay.

118
regard any peak with an area less than 0.1 times the area of the principal
peak in the chromatogram obtained with solution C.

the plate 10 pi of each of the following 5 solutions in toluene R containing
(A) 10 mg of Artemotil per ml, (B) 0.05 mg of Artemotil per ml, (C) 0.025 mg
of Artemotil per ml, (D) O.lOmg of Artemotil per ml, and (E) O.lOmg of
artemotil RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air, and spray with vanillin/ sulfuric acid TSl.
Examine the chromatogram in daylight.

(0.25%).
Assay
Determine by 1.14.4 High performance liquid chromatography, using a stain-
less steel column (25 cm x 4mm) packed with particles of silica gel, the surface
of which has been modified with chemically bonded octadecylsilyl groups

(5 pm). As the mobile phase, use a mixture of 62 volumes of acetonitrile R and
38 volumes of water.
Prepare the following solutions in acetonitrile R: solution (A) 10 mg of Arte-

motil per ml; solution (B) 10 mg of artemotil RS per ml; and for solution (C)
dilute solution A to obtain a concentration equivalent to 0.05 mg of Artemotil
per ml.
spectrophotometer set at a wavelength of about 216nm.
Measure the areas of the peak responses obtained in the chromatograms from
solutions A and B, and calculate the percentage content of C17H28O5 with ref-
erence to the dried substance.
Additional requirement for Artemotil for /parenteral use

for extractable volume for parenteral preparations, and 5.7 Visual inspection of
particulate matter in injectable preparations.
119
ARTENIMOLUM
ARTENIMOL
C15H24O5
Chemical name. (31?,5aS,61?,8aS,91?,10S,121?,12al?)-Decahydro-3,6,9-trimethyl-

3,12-epoxy-12H-pyrano[4,3-/]-l,2-benzodioxepin-10-ol; CAS Reg. No. 81496-81-3.
Other names. Dihydroartemisinin, b-dihydroartemisinin.
Description. Colourless needles or a white or almost white, crystalline

powder.
Solubility. Practically insoluble in water; slightly soluble in acetonitrile R,

ethanol (-750 g/1) TS and dichloromethane R.
Storage. Artenimol should be kept in a well-closed container, protected from

light.
Requirements
Artenimol contains not less than 97 0 % and not more than the equivalent of
.
102 0 % of C 15 H 24 O 5 using Assay method A, and not less than 98 0 % and not
. .
more than the equivalent of 102 0 % of C 15 H 24 O 5 using Assay method B, both

.
Identity tests

infrared region. The infrared absorption spectrum is concordant with
120
the spectrum obtained from artenimol RS or with the reference spectrum of

artenimol.
C. Dissolve 5 mg in about 0.5 ml of dehydrated ethanol R, add about 0.5ml

(~80g/l) TS. Heat the mixture in a water-bath to boiling, cool, add 2 drops
D. Dissolve 5mg in about 0.5ml of dehydrated ethanol R, add 1.0ml of potas-

sium iodide (80g/l) TS, 2.5ml of sulfuric acid (~100g/l) TS, and 4 drops of
starch TS; a violet colour is immediately produced.

(not exceeding 2.67 kPa or 20 mm of mercury); it loses not more than 10.0 mg/g.
Related substances
Prepare fresh solutions and perform the tests without delay.
stainless steel
particles of silica gel, the surface ofwhich has been modified with chemi-
cally bonded (pmm). As the mobile phase for gradi-
octadecylsilyl groups
ent elution, use a mixture of 6 volumes of acetonitrile R and 4 volumes of
water for the first 17 minutes; then run a gradient, which should reach 100%
acetonitrile within 13 minutes.
Prepare the following solutions in methanol R with sonication. For solution

(A) use 10 mg of Artenimol per ml in and for solution (B) use 50 pg of Arten-
imol per ml.
For the system suitability test prepare solution (C) by dissolving 1.0 mg of
artemisinin RS per ml and 1.0 mg of artenimol RS per ml in methanol R with
sonication.
121
Inject alternately 20|il each of solutions A, B, and C.

is
less than 2.0.
Measure the areas of the peak (twin-peak) responses obtained in the chro-
matograms from solutions A and B, and calculate the content of the related
substances as a percentage. In the chromatogram obtained with solution A,
the area of any peak, other than the twin peak, is not greater than that
obtained with solution B (0.5%). Not more than one peak is greater than
half the area of the twin peak obtained with solution B (0.25%). The sum
of the areas of all the peaks, other than the twin peak, is not greater than
twice the area of the twin peak obtained with solution B (1.0%). Disregard
any peak with an area less than 0.1 times the area of the twin peak in the
chromatogram obtained with solution B.

the plate 10 ml of each of the following 5 solutions in toluene R containing
(A) lOmg 0.05mg of Artenimol per ml, (C) 0.025
of Artenimol per ml, (B)
mg O.lOmg of Artenimol per ml, and (E) O.lOmg
of Artenimol per ml, (D)
of artenimol RS per ml. After removing the plate from the chromatographic
(0.25%).
Assay

stainless steel column (10cm x 4.6mm) packed with particles of silica gel,
Artenimol per ml, and solution (B) l.Omg of artenimol RS per ml.
122
For the system suitability test prepare solution (C) containing 1.0 mg of
artemisinin RS per ml and 1.0 mg of artenimol RS per ml in a mixture of 8
acetonitrile
The test is not valid unless the relative retention of a-artenimol compared
less than 2.0.
matograms from solutions A and B, and calculate the percentage content of
C15H22O5 with reference to the dried substance.
B. Dissolve about 0.05 g of Artenimol, accurately weighed, in sufficient ethanol

(-750 g/1) TS to produce 100ml and dilute 10 ml to 100 ml with the same
solvent. Accurately transfer 10 ml to a 50-ml volumetric flask, dilute to
volume with sodium hydroxide (0.05 mol/1) VS, mix thoroughly, and warm
to 50 °C in a water-bath for 30 minutes. Cool to room temperature.

(-750 g/1) TS diluted with sufficient sodium hydroxide (0.05 mol/1) VS to
stance being tested by comparison with artenimol RS, similarly and
concurrently examined, and with reference to the dried substance.
ARTESUNATUM
ARTESUNATE
123
Chemical name. (3R,5aS,6R,8aS,9R,10'S,12R,12ai?)-Decahydro-3,6,9-trimethyl-

,2-benzodioxepin- 1 0-ol, hydrogen succinate;
3, 1 2-epoxy-l 2H-pyrano [4,3-/]- 1
CAS Reg. No. 182824-33-5.
Description. A fine, white crystalline powder.
Solubility. Very slightly soluble in water; very soluble in dichlorome thane R;

freely soluble in ethanol (-750 g/1) TS and acetone R.
Storage. Artesunate should be kept in a well-closed container, protected from

light and stored in a cool place.
Requirements
Artesunate contains not less than 96 0 % and not more than the equivalent of
.
102 0 % of C]9H2 s 08 using Assay method A, and not less than 99 0 % and not
. .
more than the equivalent of 101 0 % of C19H28O8 using Assay method B, both
.
Identity tests

infrared region.
spectrum obtained from artesunate RS or with the reference spectrum of
artesunate.

ethyl acetate R and 95 volumes of toluene R as the mobile phase. Apply
separately to the plate 2 pi of the following 2 solutions in toluene R con-
taining (A) O.lOmg of Artesunate per ml, and (B) O.lOmg of artesunate RS
per ml. After removing the plate from the chromatographic chamber, allow
it to dry in air, spray with anisaldehyde/methanol TS, and heat the plate
to 120 °C for 5 minutes. Examine the chromatogram in ultraviolet light
(254 nm).

124
C. Dissolve 0.1 g of Artesunate in 40ml of dehydrated ethanol R, shake, and

filter. To half of the filtrate (keep the remaining filtrate for test D) add about
0.5ml of hydroxylamine hydrochloride TS2 and 0.25 ml of sodium hydrox-

ide (~80g/l) TS, Heat the mixture in a water-bath to boiling, cool, add 2
drops of hydrochloric acid (~70g/l) TS and 2 drops of ferric chloride (50g/l)
TS; a light red-violet colour is produced.
D. Evaporate the remaining filtrate from test C on a water-bath to a volume of

about 5 ml. Place a few drops of the mixture on a white porcelain dish, add
1 drop of vanillin/sulfuric acid TSl; a reddish-brown colour is produced.
Specific optical rotation. Use a 10 mg/ml solution in dichlorome thane R;
[a]g>"'' = -H4.5° to +6.5°.

Fischer method. Method A, using 2g of Artesunate; the water content is not
more than 5 mg/g.
pH value. pH of an aqueous suspension containing lOmg/g, 3. 5-4. 5.
Related substances

area of the principal peak obtained with solution C (0.5%). The sum of the
areas of all peaks, other than the principal peak, is not greater than twice
125
the area of the principal peak obtained with solution C (2.0%). Disregard
any peak with an area less than 0.1 times the area of the principal peak in
the chromatogram obtained with solution C.

light petroleum Rl, 36 volumes of ethyl acetate R and 1 volume of glacial

of the following 3 solutions in dichloromethane R containing (A) 5.0 mg of
Artesunate per ml, (B) 0.05 mg of Artesunate per ml, and (C) 0.025 mg of
Artesunate per ml. After removing the plate from the chromatographic
than one such spot is more intense than that obtained with solution C (0.5%).
Assay

stainless steel column (12.5 cm x 3.5mm) packed with particles of silica gel,
cylsilylgroups (5pm). As the mobile phase, use a mixture of equal volumes
of acetonitrile R and buffer pH 3.0 (dissolve 1.36g of potassium dihydrogen
phosphate R in 1000ml of water and adjust the pH to 3.0 with phosphoric
acid (-1440 g/1) TS).
Prepare the following solutions in acetonitrile R: solution (A) 4.0 mg of

Artesunate per ml; solution (B) 4.0mg of artesunate RS per ml; and for solu-
tion (C) dilute solution A to obtain a concentration equivalent to 0.04 mg of
Artesunate per ml.
Operate with a flow rate of 0.6ml per minute. Maintain the column
temperature at 30 °C and use an ultraviolet spectrophotometer set at a
wavelength of about 216nm.

from solutions A and B, and calculate the percentage content of C]9H280s
126
B. Dissolve about 0.25 g of Artesunate, accurately weighed, in 25 ml of neu-

tralized ethanol TS and titrate with sodium hydroxide (0.05 mol/1) VS, using
2 drops of phenolphthalein/ethanol TS as indicator.
Each ml of sodium hydroxide (0.05 mol/1) VS is equivalent to 19.22 mg of

Ci9H280g.
ATENOLOLUM
ATENOLOL
NHj
CH3
H,C
A and enantiomer
C14H22N2O3
Chemical name. 2-[p-[2-Hydroxy-3-(isopropylamino)propoxy]phenyl]aceta-

mide (racemate); CAS Reg. No. 29122-68-7.
Solubility. Sparingly soluble in water; soluble in ethanol (-750 g/1) TS; slightly
soluble in dichloromethane R.
Category. Cardiovascular agent; |3-adrenoreceptor blocking agent.
Storage. Atenolol should be kept in a tightly closed container.
Requirements
Atenolol contains not less than 99 0 . % and not more than 101 0 % . of
C 14 H 22 N 2 O 3 ,
Identity tests

127
the spectrum obtained from atenolol RS or with the reference spectrum of

atenolol.
B. The absorption spectrum of a O.lOmg/ml solution in methanol R, when

observed between 230 nm and 350 nm, exhibits 2 maxima at about 275 nm
and 282nm. The ratio of the absorbance at 275nm to that at 282 nm is
C. Carry out the test as described under 1.14.1 Thin-layer chromatography,

methanol R and 1 volume of ammonia (~260g/l) TS as the mobile phase.
Apply separately to the plate lOpl of each of 2 solutions in methanol R con-
taining (A) 10 mg of Atenolol per ml, and (B) 10 mg of atenolol RS per ml.
in air, and examine the chromatogram in ultraviolet light (254 nm).


20ml of water, and proceed as described under 2.2.1 Limit test for chlorides;
the chloride content is not more than l.Omg/g.
Loss on drying. Dry to constant mass at 105 °C; it loses not more than 5.0 mg/g.
Related substances. Carry out the test as described under 1.14.4 High
performance liquid chromatography, using a stainless steel column (15 cm x
4.6 mm) packed with particles of silica gel, the surface of which has been
modified with chemically bonded octadecylsilyl groups (5 pm). Prepare the
following solution to be used as the mobile phase: dissolve l.Og of sodium
octanesulfonate R and 0.4g of tetrabutylammonium hydrogen sulfate R in
1000 ml of a mixture of 80 volumes of a 3.4 mg/ml solution of potassium dihy-
drogen phosphate R, the pH of the solution adjusted to 3.0 with phosphoric acid
(~1440g/l), 18 volumes of methanol R, and 2 volumes of tetrahydrofuran R.
Prepare the following solutions: for solution (A) dissolve 10 mg of Atenolol in

5ml of mobile phase; for solution (B) dissolve 0.05g of Atenolol in 0.10ml of
dimethyl sulfoxide R, if necessary applying gentle heat by placing the flask in
a water-bath for a few seconds, and dilute with sufficient mobile phase to
produce 25 ml; for solution (C) dilute 0.5 ml of solution A with sufficient mobile
phase to produce 100ml; and for solution (D) dissolve 0.05g of atenolol for
column validation RS in 0.10ml of dimethyl sulfoxide R, if necessary applying
128
gentle heat by placing the flask in a water-bath for a few seconds, and dilute
with sufficient mobile phase to produce 25 ml.
Operate with a flow rate of 1,0ml per minute. As a detector use an ultraviolet
Inject 10 pi of solution C. Adjust the sensitivity of the system so that the height
of the principal peak is at least 50% of the full scale of the recorder.
Inject 10 pi of solution D. The tracing obtained is similar to that of the speci-

men chromatogram provided with atenolol for column validation RS, where
the peak due to the bis-ether precedes and is separated from the tertiary amine
which normally appears as a doublet. If necessary, adjust the concentration of
sodium octanesulfonate R in the mobile phase: a higher concentration would
increase the retention time of the tertiary amine.
Inject alternately 10 pi each of solutions A and C. Continue the recording of the

chromatogram for four times the retention time of the principal peak.
solutions A and C, and calculate the content of the related substances as a per-
centage. In the chromatogram obtained with solution A, the area of any peak,
other than the principal peak, is not greater than half the area of the principal
peak obtained with solution C (0.25%). The sum of the areas of all the peaks,
other than the principal peak, is not greater than that of the principal peak
obtained with solution C (0.5%). Disregard any peak with an area less than 0.1
times that of the principal peak obtained with solution C. If the content of
bisether in Atenolol is greater than 0.15%, repeat the chromatography with
10ml of solution B to confirm its compliance.
Rl, and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-
aqueous titration. Method A, determining the end-point potentiometrically.

C14H22N2O3.
ATROPINI SULFAS
ATROPINE SULFATE
Molecular formula. (Ci7H23N03)2,H2S04,H20
129
Graphic formula.
Chemical name. laf/,5a/f-Tropan-3a-ol (+)-tropate (ester) sulfate (2:1) (salt)

monohydrate; (+)-eWo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl a-(hydroxymethyl)
benzeneacetate sulfate (2:1) (salt) monohydrate; CAS Reg. No. 5908-99-6.
Description. Colourless crystal or a white, crystalline powder; odourless.
Solubility. Soluble in less than 1 part of water; freely soluble in ethanol

(-750 g/1) TS; practically insoluble in ether R and benzene R.
Categoiy. Cholinergic blocking agent (parasympatholytic).
Storage. Atropine sulfate should be kept in a tightly closed container, pro-

tected from light.
Additional information. Atropine sulfate is very poisonous; it effloresces in

dry air; it is slowly affected by light.
Requirements
Definition. Atropine sulfate contains not less than 98.5% and not more than
101.0% of (Ci7H23N03)2,H2S04, Calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from atropine sulfate RS or with the reference spectrum of
atropine sulfate.
B. Mix 1 mg with 5 drops of fuming nitric acid R and evaporate to dryness on
a water-bath.To the cooled residue add 2 ml of acetone R and 3-4 drops of

potassium hydroxide/methanol TS; a deep violet colour is produced.
130

D. Dissolve 0.6g in 30 ml of carbon-dioxide-free water R and add 2 ml of

sodium hydroxide (~80g/l) TS. Filter, wash the precipitate with water and
dry at 100°C. Melting temperature, about 116°C (atropine base).
Optical rotation. Use a solution containing the equivalent of O.lOg/ml of the

dried substance, in a 200-mm tube; optical rotation = -0.50 to -1-0.10° (distinc-
tion from hyoscyamine).
Loss on drying. Dry to constant weight at 120 °C; it loses not less than
25mg/g and not more than 40mg/g.
Acidity. Dissolve l.Og in 20 ml of carbon-dioxide-free water R and titrate with

sodium hydroxide (0.02 mol/1) VS, using methyl red/ethanol TS as indicator; not
more than 0.3 ml is required to obtain the midpoint of the indicator (orange).
Readily oxidizable substances. To 10ml of a lOmg/ml solution add 0.1ml

of potassium permanganate (0.02 mol/1) VS; the colour is not completely dis-
charged at the end of 3 minutes.
6 volumes of ethylmethylketone R, 3 volumes of methanol R and 1 volume of
ammonia (~100g/l) TS as the mobile phase. Apply to the plate 10 pi of a solu-
tion in methanol R containing 12.5 mg/ml of the test substance. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air, spray
with potassium iodobismuthate TS2 and examine the chromatogram in day-
light. No spot is obtained, other than the principal spot.
Assay. Dissolve about 0.6g, accurately weighed, in 30ml of glacial acetic acid
aqueous titration. Method A. Each ml of perchloric acid (0.1 mol/1) VS is equiv-
alent to 67.68 mg of (Ci7H23N03)2,H2S04.
Additional requirements for Atropnne sulfate for

parenteral use
Complies with the monograph for “Parenteral preparations’’.
131
Additional requirement for Atrofîne sulfate for sterile use

AZATHIOPRINUM
AZATHIOPRINE
Molecular formula. C9H7N7O2S
Graphic formula.
Chemical name. 6-[(l-Methyl-4-nitroimidazol-5-yl)thio]purine; 6 -[(l-methyl-

4-nitro-liT-imidazol-5-yl)thio]-liT-purine; CAS Reg. No. 446-86-6.
Description. A pale yellow powder; odourless.
Solubility. Practically insoluble in water; very slightly soluble in ethanol

(-750 g/1) TS; sparingly soluble in dilute mineral acids; soluble in dilute solu-
tions of alkali hydroxides.
Category. Immunosuppressive drug.
Storage. Azathioprine should be kept in a well-closed container, protected

from light.
Additional information. Azathioprine decomposes in strong solutions of

alkali hydroxides. CAUTION: Azathioprine must be handled with care, avoid-
ing contact with the skin and inhalation of airborne particles.
Requirements
Definition. Azathioprine contains not less than 98.0% and not more than
101.5% of C 9 H 7N 7 O 2 S, calculated with reference to the dried substance.
132
Identity tests

infrared region.
spectrum obtained from azathioprine RS or with the reference spectrum of
azathioprine.
C. Heat 20mg with 100ml of water and filter. To 5ml of the nitrate add 1 ml of
hydrochloric acid (-420 g/1) TS, 10 mg of zinc R powder, and allow to stand for
5 minutes; the solution becomes yellow. Filter, cool in ice, add 0.1 ml of sodium
nitrite (100 g/1) TS and 0.1 g of sulfamic acid R, and shake until the bubbles
disappear. Add 1ml of 2-naphthol TSl; a pale pink precipitate is produced.
Loss on drying. Dry at 105 °C under reduced pressure (not exceeding 0.6kPa
or about 5mm of mercury) for 5 hours; it loses not more than lOmg/g.
Acidity or alkalinity. Shake 0.5 g with 25ml of water for 15 minutes, and
filter; to 20ml of the filtrate add 0.15ml of methyl red/ethanol TS; not
more than 0.10 ml of hydrochloric acid (0.02 mol/1) VS or 0.10ml of sodium

hydroxide (0.02 mol/1) VS is required to obtain the midpoint of the indicator
(orange).
Related substances. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using cellulose R3 (a precoated plate from a commercial source is suit-
able) and Tbutanol R saturated with ammonia (-100 g/1) TS as the mobile phase.
Apply separately to the plate 10 pi of each of 3 solutions in ammonia (-100 g/1) TS
containing (A) lOmg of the test substance per ml, (B) O.lSmg of the test substance
per ml, and (C) O.lSmg of azathioprine RS per ml. After removing the plate from
the chromatographic chamber, allow it to dry in air and examine the chromatogram

mamide R and titrate with tetrabutylammonium hydroxide (0.1 mol/1) VS,
(0.1 mol/1) VS is equivalent to 27.73mg of C 9H N 7 O
7 2 S.
133
BACITRACINUM
BACITRACIN
Bacitracin (non-injectable)
Bacitracin, sterile
Chemical name. Bacitracin; CAS Reg. No. 1405-87-4.
Description. A white or pale brownish yellow powder; odourless or with a

faint characteristic odour.
Solubility. Freely soluble in water, methanol R and ethanol (-750 g/1) TS; prac-
tically insoluble in acetone R and ether R.
Category. Antiinfective drug.
Storage. Bacitracin should be kept in a tightly closed container, protected from

and stored at a temperature not exceeding 15 °C. If it is intended for par-
light,
enteral administration, the container should be sterile and sealed so as to

exclude microorganisms.
Labelling. The designation sterile Bacitracin indicates that the substance com-
plies with the additional requirements for sterile Bacitracin and may be used
for parenteral administration or for other sterile applications.
Additional information. Bacitracin is hygroscopic. Its solutions deteriorate

rapidly at room temperature. Even in the absence of light, it is gradually
Requirements
Definition. Bacitracin is a polypeptide produced by the growth of an organ-
ism of the licheniformis group of Bacillus subtilis. The main components are
Bacitracin A, B,, and B2 .
Bacitracin contains not less than 55 International Units per mg, calculated with
reference to the dried substance.
Identity test
Carry out the test as described under 1.14.1 Thin-layer chromatography, using
silica R1 as the coating substance and a mixture of 60 volumes of Tbutanol
gel
R, 10 volumes of water, 6 volumes of pyridine R, 15 volumes of glacial acetic
acid R, and 5 volumes of ethanol (~750g/l) TS as the mobile phase. Apply sepa-
134
lately to the plate 1 disodium edetate (lOg/1) TS con-

pi of each of 2 solutions in
taining (A) 6.0 mg of the test substance per ml and (B) 6,3 mg of bacitracin zinc
RS per ml. A third spot (C) is made by applying 1 pi of each of solutions A and
B at the same point of application, allowing to dry between the two loadings.
air, spray itwith triketohydrindene/pyridine/butanolTS, and heatitat 110°C for
10 minutes. Allow to cool, and examine the chromatogram in daylight. The spots
obtained with solution A correspond in position, appearance, and intensity with
that obtained with solution B. A single spot is obtained with solution C.

or about 5mm of mercury) for 3 hours; it loses not more than 50mg/g.
pH value. Shake l.Og with 10ml of carbon-dioxide-free water R; the pH is
Bacitracin F and related substances. Prepare a solution containing 30 mg in

100ml of sulfuric acid (0.05mol/l) VS. The ratio of the absorbance at 290nm
to that at 252 nm is not greater than 0.15.
Assay. Dissolve 0.05 g, accurately weighed, in 5 ml of water and add 0.5 ml of

hydrochloric acid (~70g/l) TS and sufficient water to produce 100ml. Allow to
stand at room temperature for 30 minutes and carry out the assay as described
under 3.1 Microbiological assay of antibiotics, using Micrococcus luteus (NCTC
7743 or ATCC 10240) as the test organism, culture medium Cml with a final
pH of either 7. 0-7.1 or 6.5-6.6, sterile phosphate buffer TS of pH either 7.0 or
6.0, an appropriate concentration of bacitracin (usually 1-4 lU per ml), and an
incubation temperature of either 35-39 °C or 32-35 °C. The precision of the
assay is such that the fiducial limits of error of the estimated potency {P = 0.95)
arenot less than 95% and not more than 105% of the estimated potency. The
upper fiducial limit of error of the estimated potency {P = 0.95) is not less than
55 lU per mg, calculated with reference to the dried substance.
Additional requirements for Bacitracin for sterile use

Storage. Sterile Bacitracin should be kept in a hermetically closed container,
protected from light, and stored at a temperature not exceeding 15°C.
Bacteritilendotoxins. Carry out the test as described under 3.4 Test for bacterial
endotoxins; contains not more than 0.01 lU of endotoxin RS per mg of bacitracin.

135
The International Pharmacofoeia
BACITRACINUM ZINCUM
BACITRACIN ZINC
Bacitracin zinc (non-injectable)
Bacitracin zinc, sterile
Chemical name. Bacitracin zinc; CAS Reg. No. 1405-89-6.
Description. A white or pale brownish yellow powder; odourless or with a

Solubility. Soluble in 900 parts of water and in 500 parts of ethanol (-750 g/1)
TS; very slightly soluble in ether R.
Storage. Bacitracin zinc should be kept in a tightly closed container, protected

from light, and stored at a temperature not exceeding 25 °C. If it is intended for
parenteral administration, the container should be sterile and sealed so as to
exclude microorganisms.
Labelling. The designation sterile Bacitracin zinc indicates that the substance
complies with the additional requirements for sterile Bacitracin zinc and may
be used for other sterile applications.
Additional information. Bacitracin zinc is hygroscopic.
Requirements
is a zinc complex of bacitracin, a polypeptide
Definition. Bacitracin zinc
produced by the growth of an organism of the licheniforniis group of Bacillus
The main components are Bacitracin A, B, and B 2
subtilis. .
Bacitracin zinc contains not less than 55 International Units of bacitracin per
mg, calculated with reference to the dried substance.
Identity tests
A. Carry out the test as described under 1.14.1 Thin-layer chromatography,
1-butanol R, 10 volumes of water, 6 volumes of pyridine R, 15 volumes of
glacial acetic acid R, and 5 volumes of ethanol (-750 g/1) TS as the mobile
phase. Apply separately to the plate 1 pi of each of 2 solutions in disodium
edetate (lOg/1) TS containing (A) 6.0mg of the test substance per ml and (B)
6.0 mg of bacitracin zinc RS per ml. A third spot (C) is made by applying
136
1 1^1 of each of solutions A and

B at the same point of application, allowing
the plate to dry between the two loadings. After removing the plate from
the chromatographic chamber, allow it to dry in air, spray it with triketo-
hydrindene/pyridine/butanol TS, and heat it at 110 °C for 10 minutes. Allow
to cool, and examine the chromatogram in daylight. The spots obtained
with solution A correspond in position, appearance, and intensity with that
obtained with solution B. A single spot is obtained with solution C.
B. Ignite 30 mg; dissolve half of the residue in 1.0 ml of hydrochloric acid

(~70g/l) TS and add 1.0ml of potassium ferrocyanide (45g/l) TS; a white
precipitate is produced. Dissolve the remaining residue in 1.0ml of sulfuric
acid (~100g/l) TS, add 0.05ml of copper(II) sulfate (lg/1) TS and 2.0ml of
ammonium mercurithiocyanate TS; a violet precipitate is produced.

pH value. Shake l.Og with 10ml of carbon-dioxide-free water R and filter; pH

of the filtrate, 6.0-7. 5.
Bacitracin F and related substances. Prepare a solution containing 30 mg in

100ml of sulfuric acid (0.05mol/l) VS. The ratio of the absorbance at 290nm
to that at 252 nm is not greater than 0.15.
Zinc. Dissolve 0.20 g in 5ml of acetic acid (~60g/l) TS and add 50 ml of water,
50 mg of xylenol orange indicator mixture R, and sufficient methenamine R to
produce a red solution. Add 2.0g of methenamine R in excess and titrate with
disodium edetate (0.01 mol/1) VS until the colour changes to yellow. Each ml of
disodium edetate (0.01 mol/1) VS is equivalent to 0.6537 mg of Zn; the zinc
content is not less than 40mg/g and not more than 60mg/g, calculated with
Assay. Suspend 0.05 g, accurately weighed, in 5 ml of water and add 0.5ml of

hydrochloric acid (~70g/l) TS and sufficient water to produce 100 ml. Allow to
stand at room temperature for 30 minutes and carry out the assay as described
under 3.1 Microbiological assay of antibiotics, using Micrococcus luteus (NCTC
pH of either 7. 0-7.1 or 6.5-6.6, sterile phosphate buffer TS of pH either 7.0 or
6.0, an appropriate concentration of bacitracin (usually 1-4 lU per ml), and an
incubation temperature of either 35-39 °C or 32-35 °C. The precision of the
assay is such that the fiducial limits of error of the estimated potency (P = 0.95)
are not less than 95% and not more than 105% of the estimated potency. The
upper fiducial limit of error of the estimated potency (P = 0.95) is not less than
55 lU of bacitracin per mg, calculated with reference to the dried substance.
137
Additional requirements for Bacitracin zinc for sterile use

Storage. Sterile Bacitracin zinc should be kept in a hermetically closed con-
tainer, protected from light, and stored at a temperature not exceeding 25 °C.

membrane filtration test procedure. Dissolve the test substance in peptone
(1 g/1) TSl to which disodium edetate R has been added. Use the sampling plan
described under 3.2.1 Test for sterility of non-injectable preparations.
BARII SULFAS
BARIUM SULFATE
Molecular formula. BaS 04
Chemical name. Barium sulfate (1 :

1); CAS Reg. No. 7727-43-7.
Description. A white, heavy, fine powder; free from grittiness; odourless.
Solubility. Practically insoluble in water and in organic solvents; very slightly

soluble in acids and in solutions of alkali hydroxides.
Storage. Barium sulfate should be kept in a well-closed container.
Additional information. Barium sulfate is inclined to caking.
Requirements
Identity tests
A. Boil 0.2 g in a solution of 5.0 g of sodium carbonate R dissolved in 5ml of
water for 5 minutes, then add 10 ml of water and filter (keep the precipitate
for test B). To 5ml of the filtrate add 5ml of hydrochloric acid (~70g/l) TS;
this solution yields reaction A described under 2.1 General identification
tests as characteristic of sulfates.
B. Wash the precipitate from test A with 3 successive small quantities of water.
To add 5 ml of hydrochloric acid (-70 g/1) TS, filter, and to the
the residue
add 0.3ml of sulfuric acid (-100 g/1) TS; a white precipitate is pro-
filtrate
duced which is insoluble in sodium hydroxide (-80 g/1) TS.
138
g
Sedimentation. Place 5.0 g, previously sifted, in a glass-stoppered 50-ml grad-

uated cylinder, having the 50-ml graduation mark 11-14 cm from the base. Add
sufficient water to produce 50 ml, shake for 5 minutes and allow to stand for 15
minutes; the barium sulfate does not settle below the 15-ml graduation mark.
Heavy metals. For the preparation of the test solution boil 4g with 6ml of
acetic acid (-60g/l) PbTS and 44 ml of water for 10 minutes, filter, allow to cool
and dilute to 50 ml with water. Determine the heavy metals content in 25 ml
of the filtrate as described under 2.2.3 Limit test for heavy metals, according to
Method A; not more than lOpg/g.
Arsenic. Transfer 0.5 to a long-necked combustion flask, add 30 ml of water

and 2 ml of nitric acid (~1000g/l) TS, insert a small funnel into the neck of the
flasks and heat in an inclined position on a water-bath for 2 hours. Allow to
cool, adjust to the original volume with water, and filter. Wash the residue three
times with 5 ml of water, combine the filtrate and washings, add 1 ml of sulfu-
ric acid (-1760 g/1) TS, and evaporate on a water-bath until white fumes are
evolved. Dissolve the residue in 10ml of sulfuric acid (-100 g/1) TS, add 10ml
of water, and proceed as described under 2.2.5 Limit test for arsenic; the arsenic
content is not more than 2|ig/g.
Soluble barium salts. Boil 10 g with 20 ml of water and 30ml of acetic acid
(-60 g/1) TS for 5 minutes, filter, allow to cool and dilute to 50 ml with water.
To a 10-ml portion of this solution add 1ml of sulfuric acid (-100 g/1) TS and
to a second 10-ml portion add 1 ml of water. When compared after 1 hour, the
two solutions remain equally clear.
Phosphates. To l.Og add 3 ml of nitric acid (-130 g/1) TS and 7 ml of water and
heat on a water-bath for 5 minutes. Filter and dilute the filtrate to 10 ml with
water. Add 5 ml of ammonium molybdate/vanadate TS and allow to stand for 5
minutes; any yellow colour produced is not more intense than that of a refer-
ence solution prepared similarly using 10 ml of phosphate standard (5|tg/ml) TS.
Oxidizable sulfur compounds. Shake l.Og with 5 ml of water for 30 seconds

and filter. To the filtrate add 0.1ml of starch TS, 0.1 g of potassium iodide R,
1 ml of freshly prepared potassium iodate (3.6mg/l) TS, and 1 ml of hydrochlo-
ric acid (1 mol/1) VS, and shake well; the colour produced is more intense than
that of a solution prepared in a similar way, but omitting the potassium iodate.
Acid-soluble substances. Boil 5g with 15ml of acetic acid (-300g/l) TS and

10ml of water for 5 minutes. Filter, evaporate the filtrate to dryness on a water-
bath, and dry to constant weight at 105°C; the residue weighs not more than
15 mg.
Loss on ignition. Ignite l.Og at 600 °C; it loses not more than 20mg/g.
139
Acidity or alkalinity. Heat 5.0 g with 20ml of carbon-dioxide-free water R

on a water-bath for 5 minutes and filter. To 10ml of the filtrate add 0.05ml of
bromothymol blue/ethanol TS; not more than 0.5ml of hydrochloric acid
(0.01 mol/1) VS or 0.5ml of carbonate-free sodium hydroxide (0.01 mol/1) VS is
required to obtain the midpoint of the indicator (green).
BECLOMETASONI DIPROPIONAS
BECLOMETASONE DIPROPIONATE
Molecular formula. C28H37CIO7
Graphic formula.
C =0
CH,
Chemical name. 9-Chloro-lip,17|21-trihydroxy-16|3-methylpregna-l,4-diene-

3,20-dione 17,21-dipropionate; 9-chloro-liP-hydroxy-16P-methyl-17,21-bis(l-
oxopropoxy)pregna-l,4-diene-3,20-dione; CAS Reg. No. 5534-09-8.
Description. A white to creamy white powder; odourless.
Solubility. Practically insoluble in water; soluble in 60 parts of ethanol

(-750 g/1) TS.
Category. Antiasthmatic drug.
Storage. Beclometasone dipropionate should be kept in a weU-closed con-

140
Requirements
Definition. Beclometasone dipropionate contains not less than 96,0% and
not more than 104.0% of C28H37CIO7, calculated with reference to the dried
substance.
Identity tests

spectrum obtained from beclometasone dipropionate RS or with the refer-
ence spectrum of beclometasone dipropionate.

using kieselguhr R1 as the coating substance and a mixture of 1 volume of
propylene glycol R and 9 volumes of acetone R to impregnate the plate,

dipping it about 5 mm into the liquid. After the solvent has reached a height
of at least 16cm, remove the plate from the chromatographic chamber and
allow it to stand at room temperature until the solvent has completely evap-
orated. Use the impregnated plate within 2 hours, carrying out the chro-
matography in the same direction as the impregnation. Use a mixture of 4
volumes of cyclohexane R and 1 volume of toluene R as the mobile phase.
Apply separately to the plate 2 pi of each of 2 solutions in a mixture of 9
volumes of chloroform R and 1 volume of methanol R containing (A)
2.5 mg of the test substance per ml and (B) 2.5 mg of beclometasone dipro-
pionate RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air until the solvents have evaporated, heat it at
120 °C for 15 minutes, spray the hot plate with sulfuric acid/ethanol TS, and
then heat at 120 °C for 10 minutes. Allow to cool, and examine the chro-
matogram in daylight and in ultraviolet light (365 nm). The principal spot
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; [a]o = -H 88

to-h94°.
5.0mg/g.
95 volumes of dichloroe thane R, 5 volumes of methanol R, and 0.2 volumes of
water as the mobile phase. Apply separately to the plate 10 pi of each of 2 solu-
141
tions in a mixture of 9 volumes of chloroform R and 1 volume of methanol R

containing (A) 15 mg of the test substance per ml and (B) 0.30 mg of the test
substance per ml. After removing the plate from the chromatographic chamber,
allow it to dry in air until the solvents have evaporated and heat at 105°C for
10 minutes; allow it to cool, spray it with blue tetrazolium/sodium hydroxide
TS, and examine the chromatogram in daylight. Any spot obtained with solu-
tion A, other than the principal spot, is not more intense than that obtained
with solution B.
Assay
• The solutions must be protected from light throughout the assay.
Dissolve about 20 mg, accurately weighed, in sufficient aldehyde-free ethanol

(~750g/l) TS produce 100ml. Dilute 20ml of this solution with sufficient
to
aldehyde-free ethanol (~750g/l) TS to produce 100ml. Transfer 10.0ml of
the diluted solution to a 25-ml volumetric flask, add 2.0ml of blue tetra-
zolium/ethanol TS, and displace the air in the flask with oxygen-free nitrogen
R. Immediately add 2.0 ml of tetramethylammonium hydroxide/ethanol TS and
again displace the air with oxygen-free nitrogen R. Stopper the flask, mix the
contents by gentle swirling, and allow to stand for 1 hour in a water-bath at
30 °C. Cool rapidly, add sufficient aldehyde-free ethanol (-750 g/1) TS to
produce 25ml, and mix. Measure the absorbance of a 1-cm layer at the
maximum at about 525 nm against a solvent cell containing a solution prepared
by treating 10 ml of aldehyde-free ethanol (-750 g/1)
TS in a similar manner.
Calculate the amount of C 2 sH 37 C 107 in
the substance being tested by compari-
son with beclometasone dipropionate RS, similarly and concurrently examined.
BENTONITUM
BENTONITE
Chemical name. Bentonite; CAS Reg. No. 1302-78-9.
Description. A greyish white to cream-coloured, very fine, homogeneous

powder; odourless.
Solubility. Practically insoluble in water and most organic solvents; when

added to water it swells to approximately 12 times its volume.
Category. Suspending agent; viscosity-increasing agent.
Storage. Bentonite should be kept in a tightly closed container.
142
Additional information. Bentonite may also contain calcium, magnesium,

and iron. Attention should be paid to the microbiological quality since Ben-
tonite is of mineral origin.
Requirements
Definition. Bentonite is a natural, colloidal, hydrated aluminium silicate.
Identity tests
A. In a metal crucible fuse 0.5 g with 0.4 g of anhydrous sodium carbonate R.
Add hot water to the residue and filter. (Keep the filtrate for test B.) Add a
few drops of hydrochloric acid (~420g/l) TS to the residue on the filter, dilute
to 5 ml with water, and filter. To 2 ml of the filtrate add 2 ml of ammonium
chloride (100 g/1) TS and 2ml of ammonia (-100 g/1) TS; a white, gelatinous
precipitate is produced which is soluble in hydrochloric acid (-420 g/1) TS,
acetic acid (-300 g/1) TS, and sodium hydroxide (-80 g/1) TS, but insoluble
in ammonia (-260 g/1) TS.
B. Acidify the filtrate from test A with hydrochloric acid (-420 g/1) TS and
evaporate to dryness. Heat the residue with a mixture of lOmg of calcium
fluoride R and a few drops of sulfuric acid (-1760 g/1) TS; a gas is evolved
which, if bubbled into water, gives a white precipitate.
Loss on drying. Dry to constant mass at 105°C; it loses not less than 50mg/g
Alkalinity. Shake 2g with 100ml of carbon-dioxide-free water R for 5

minutes using a stoppered flask. To 5ml of the suspension add 0.1ml of
thymolphthalein/ethanol TS; a bluish colour is produced. Add 0.1ml of
hydrochloric acid (0.1 mol/1) VS and allow to stand for 5 minutes; the solution
becomes colourless.
Sedimentation volume. Mix 6g with 0.3 g of freshly calcined light magne-

sium oxide R and add progressively 200 ml of water. Shake for 1 hour, place
100 ml of the suspension in a graduated cylinder, and allow to stand for 24
hours; the volume of the supernatant liquid is not larger than 2 ml.
Swelling power. Transfer 100ml of sodium laurilsulfate (lOg/1) TS to a glass-

stoppered cylinder with a volume of 100ml and add, in 20 portions, 2g of Ben-
tonite at intervals of at least 2 minutes, permitting each portion to settle before
adding the next. Allow to stand for 2 hours; the apparent volume of the sedi-
ment at the bottom of the cylinder is not less than 22 ml.
Fineness of powder. Triturate 2g in a mortar with 20ml of water. Allow to

swell, disperse evenly with a pestle, and dilute with water to 100 ml. Pour the
143
suspension through a sieve with the nominal aperture size of 75 pm (sieve no.
75), and wash the sieve thoroughly with water; no grit is felt when the fingers
are rubbed over the wire mesh of the sieve.
BENZALKONII CHLORIDUM
BENZALKONIUM CHLORIDE
Chemical name. Alkylbenzyldimethylammonium chloride; alkyldimethyl
(phenylmethyl)ammonium chloride; CAS Reg. No. 8001-54-5.
Description. A white or yellowish white powder, thick gel, or gelatinous

pieces; odourless or a slight aromatic odour.
Solubility. Very soluble in water and ethanol (-750 g/1) TS; freely soluble in
acetone R; practically insoluble in ether R.
Category. Antimicrobial preservative; surfactant.
Storage. Benzalkonium chloride should be kept in a tightly closed container,

Additional information. Benzalkonium chloride is hygroscopic.
Requirements
Definition. Benzalkonium chloride is a mixture of alkylbenzyldimethylam-
monium chlorides, the alkyl groups having chain lengths of Cb to Cib.
Benzalkonium chloride contains not less than 95 0 % and not more than the
.
equivalent of 104 0 % of alkylbenzyldimethylammonium chlorides, calculated

.
as C 22 H 40 CIN (relative molecular mass 354.0) and with reference to the anhy-
drous substance.
Identity tests
A. Shake a solution of 0.1 g in 100ml of water; it foams strongly.
B. To 5 ml of sodium hydroxide (-80 g/1) TS add 0.1 ml of bromophenol blue

TS and 5 ml of chloroform R and shake; the chloroform layer is colourless.
Dissolve lOmg in 1ml of carbon-dioxide-free water R, add 0.1ml to the
solution above, and shake; the chloroform layer becomes blue.
144
C. A solution of 10 mg/ml in a mixture of equal volumes of water and ethanol

(~750g/l) TS yields reaction A described under 2.1 General identification

Fischer method, Method A, using 0. 1 g; the water content is not more than 150 mg/g.
Ammonium compounds. Dissolve 0.1 g in 5ml of water, add 3ml of sodium

hydroxide (1 mol/1) VS, and heat to boiling. Place a moistened strip of red litmus
paper R over the solution; no blue colour appears on the paper.
Assay. Dissolve about 2g, accurately weighed, in sufficient water to produce

100 ml. Transfer 25 ml to a separating funnel, add 25 ml of chloroform R, 10 ml
of sodium hydroxide (0.1 mol/1) VS, and 10ml of a freshly prepared solution of
50 mg of potassium iodide R per ml. Shake well, allow to separate, and discard
the chloroform layer. Shake the aqueous layer with three quantities, each of
10ml, of chloroform R and discard the chloroform layers. To the aqueous layer
add 40ml of hydrochloric acid (-420 g/1) TS, allow to cool, and titrate with
potassium iodate (0.05 mol/1) VS until the deep brown colour is discharged. Add
2 ml of chloroform R and continue the titration, shaking vigorously, until the
chloroform layer no longer changes colour. Carry out a blank titration on a
mixture of 10 ml of the freshly prepared potassium iodide solution as described
above, 20ml of water, and 40 ml of hydrochloric acid (-420 g/1) TS and make
any necessary corrections.
Each ml of potassium iodate (0.05 mol/1) VS is equivalent to 35.40 mg of C22H40CIN.
BENZATHINI BENZYLPENICILLINUM
BENZATHINE BENZYLPENICILLIN
Benzathine benzvlpenicillin (non-injectable)
Benzathine benzylpenicillin, sterile
Molecular formula. (Ci6Hi8N204S)2,Ci6H2oN2 (anhydrous)
145
Graphic formula.
Chemical name. iV,N'-Dibenzylethylenediamine compound with (2S,5R,6R)-

3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-l-azabicyclo[3.2.0]heptane-
2-carboxylic acid (1:2); N,W'-bis(phenylmethyl)-l,2-ethanediamine compound
with [2S-(2a,5a,6P)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylic acid (1:2); N,A/'-dibenzylethylenedi-
amine salt of benzylpenicillin; CAS Reg. No. 1538-09-6 (anhydrous).
Other name. Penicillin G benzathine.
Description. A white powder; odourless or almost odourless.
Solubility. Very slightly soluble in water; sparingly soluble in ethanol

Storage. Benzathine benzylpenicillin should be kept in a tightly closed con-

Labelling. The designation sterile Benzathine benzylpenicillin indicates that

the substance complies with the additional requirements for sterile Benzathine
benzylpenicillin and may be used for parenteral administration or for other
sterile applications.
Additional information. Benzathine benzylpenicillin contains a variable

amount of water of crystallization.
Requirements
Definition. Benzathine benzylpenicillin contains not less than 96.0% and not
more than 100.5% of total penicillins calculated as (Ci 6 HigN 204 S) 2 ,C] 5 H 2 oN 2 ,
and not less than 24.0% and not more than 27.0% of C 16H 20 N 2 ,
both calculated
146
Identity tests
A. To 2 mg add 1 drop of water followed by 2 ml of sulfuric acid
in a test-tube
(-1760 g/1) TS and mix; the solution is almost colourless. Immerse the test-
tube for minute in a water-bath; the solution remains almost colourless.

1
Place 2 mg in a second test-tube, add 1 drop of water and 2 ml of formalde-

hyde/sulfuric acid TS and mix; the solution is almost colourless and after a
few minutes the colour changes to yellow-brown. Immerse the test-tube for
1 minute in a water-bath; a reddish brown colour is produced.
B. Shake 0.1 g with 2 ml of sodium hydroxide (1 mol/1) VS for 2 minutes, extract

the mixture with 2 quantities, each of 3 ml of ether R, evaporate the com-
bined extracts, and dissolve the residue in 1 ml of ethanol (~375g/l) TS. Add
5 ml of trinitrophenol (7 g/1) TS, heat at 90 °C for 5 minutes, and allow to
cool slowly. Collect the precipitate and recrystallize it from hot ethanol
(-150 g/1) TS that contains 10 mg/ml of trinitrophenol R; melting tempera-
ture, about 214 °C (picrate).

pH value. pH of a saturated solution containing about 0.05g in 10ml of

carbon-dioxide-free water R, 5. 0-7.5.
Assay
A. For total penicillins. Dissolve about 0.065 g, accurately weighed, in 10ml of
dimethylformamide R and dilute with sufficient water to produce 1000 ml.
Transfer two 2.0-ml aliquots of this solution into separate stoppered tubes.
To one tube add 10.0 ml of imidazole/mercuric chloride TS, mix, stopper
the tube and place it in a water-bath at 60 °C for exactly 25 minutes. Cool
the tube rapidly to 20 °C (solution A).
Without delay measure the absorbance of a 1-cm layer at the maximum at about
325nm against a solvent cell containing a mixture of 2.0ml of water and 10.0ml
From the difference between the absorbance of solution A and that of solu-
tion B, calculate theamount of (C] 6 H] 8N 204 S) 2 ,CifiH 2 oN 2 in the substance
being tested by comparison with 0.050g of benzylpenicillin sodium RS
similarly and concurrently examined, taking into account that each mg of
benzylpenicillin sodium RS (C] 6 Hi 7 N 204 S) is equivalent to 1.275 mg of
(CifiHigN 204 S) 2 ,CisH 2 oN 2 In an adequately
. calibrated spectrophotometer,
the absorbance of the reference solution should be 0.62 ± 0.03.
147
B. For C 16H 20N 2 To about Ig, accurately weighed, add 30 ml of sodium chlo-
.
ride (400g/l) TS and 10ml of sodium hydroxide (-150 g/1) TS, shake well,
and extract with four quantities, each of 50 ml of ether R. Wash the com-
bined extracts with three quantities, each of 10 ml of water, extract the com-
bined washings with 25 ml of ether R, and add the extract to the main ether
solution. Evaporate the ether solution to a low bulk, add 2 ml of dehydrated
ethanol R and evaporate to dryness. To the residue add 50ml of glacial
acetic acid R and titrate with perchloric acid (0.1 mol/1) VS, using 1 ml of 1-
naphtholbenzein/acetic acid TS as indicator. Repeat the operation without
the substance being examined; the difference between the titrations repre-
sents the amount of perchloric acid required to neutralize the liberated base.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 12.02 mg of C 16H 20N 2 .
Additional requirements for Benzathine benzytpeniciltin for

sterile use
Storage. Sterile Benzathine benzylpenicillin should be kept in a hermetically

closed container, protected from light.
benzylpenicillin.

Additional requirements for Benzathine henzylfaenicillin for

pfarenteral use
BENZNIDAZOLUM
BENZNID AZOLE
NO2
C12H12N4O3
148
Chemical name. N-Benzyl-2-nitroimidazole-l -acetamide; N-benzyl-2-nitro-

1-imidazole-acetamide; CAS Reg. No. 22994-85-0.
Description. A yellowish powder; odourless or almost odourless.
Solubility. Practically insoluble in water; sparingly soluble in acetone R;

slightly soluble in methanol R; very slightly soluble in ethanol (~750g/l) TS.
Category. Antiprotozoal drug.
Storage. Benznidazole should be kept in a well-closed container, protected

from light.
Requirements
Benznidazole contains not less than 98 5 % and not more than the equivalent
.
of 101 5 % of C, 2 Hi 2 N 403 calculated with reference to the dried substance.

.
,
Identity tests

infrared region.
spectrum obtained from benznidazole RS or with the reference spectrum of
benznidazole.
C. Melting temperature, about 190°C.
40 volumes of chloroform R, 40 volumes of ethyl acetate R, 15 volumes of
methanol R, and 5 volumes of glacial acetic acid R as the mobile phase. Apply
separately to the plate 20 pi of each of 3 solutions in acetone R containing (A)
25 mg of Benznidazole per ml, (B) 25 mg of benznidazole RS per ml, and (C)
125 pg of benznidazole RS per ml. After removing the plate from the chro-
149
matographic chamber, allow it to dry in air until the solvents have evaporated,
and heat at 110°C for 10 minutes. Allow it to cool and examine the chro-
intense than that obtained with solution C (0.5%).
Assay. Dissolve about 0.2 g, accurately weighed, in 75 ml of acetic anhydride

R, and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 26.03 mg of C12H12N4O3.
BENZOCAINUM
BENZO CAINE
Graphic formula.
Chemical name. Ethyl p-aminobenzoate; ethyl 4-aminobenzoate; CAS Reg.

No. 94-09.7.
Other name. Ethyl aminobenzoate.
Description. Colourless crystals or a white, crystalline powder; odourless.
Solubility. Very slightly soluble in water; soluble in 6 parts of ethanol

(-750 g/1) TS and 5.5 parts of ether R.
Category. Local anaesthetic.
Storage. Benzocaine should be kept in a well-closed container, protected from

light.
150
Additional information. Benzocaine causes local numbness after being

placed on the tongue.
Requirements
Definition. Benzocaine contains not less than 98.0% and not more than
Identity tests
A. Dissolve O.lOg in 5ml of water, add 3 drops of hydrochloric acid (~70g/l)
TS and 5 drops of iodine TS; a brown precipitate is produced.
B. Heat 0.05 g with 2 drops of acetic acid (-300 g/1) TS and 4 drops of sulfuric
acid (~1760g/l) TS; ethyl acetate, perceptible by its odour (proceed with
caution), is produced.
C. About 0.05 g yields the reaction described for the identification of primary
aromatic amines under 2.1 General identification tests, producing an orange-
red precipitate.
Heavy metals. Use l.Og and ethanol (-750 g/1) TS for the preparation of the
test solution as describedunder 2.2.3 Limit test for heavy metals. Procedure 2;
determine the heavy metals content according to Method A; not more than
lOpg/g.
Solution in ethanol. A solution of l.Og in 10ml of ethanol (-750 g/1) TS is

reduced pressure (not exceeding O.hkPa or about 5 mm
of mercury) over silica
gel, desiccant, R or phosphorus pentoxide R; it loses not more than lOmg/g.
Acidity or alkalinity. To a solution of 0.5g in 10ml of neutralized ethanol TS

add 10ml of carbon-dioxide-free water R and 2 drops of phenolphthalein/
ethanol TS; no pink colour is produced. Add 0.5ml of carbonate-free sodium
hydroxide (0.01 mol/1) VS; a pink colour is produced.
Assay. Carry out the assay as described under 2.7 Nitrite titration, using about
0.3 g, accurately weighed, dissolved in 50ml of hydrochloric acid (-70g/l) TS
and titrate with sodium nitrite (0.1 mol/1) VS. Each ml of sodium nitrite
(0.1 mol/1) VS is equivalent to 16.52 mg of C9H11NO2.
151
BENZOYLIS PEROXIDUM CUM AQUA

HYDROUS BENZOYL PEROXIDE
C14H10O4J XH2O
Chemical name. Dibenzoyl peroxide; CAS Reg. No. 94-36-0.
Description. A white, amorphous or granular powder.
Solubility. Practically insoluble in water; soluble in acetone R; soluble in

dichlorome thane R with separation of water; slightly soluble in ethanol
(-750 g/1) TS.
Category. Keratolytic agent.
Storage. Hydrous Benzoyl peroxide should be kept in a container that has been
treated to reduce static discharge and that has a device for the release of excess
pressure. Store at a temperature between 2 and 8°C, protected from light.
Additional information. CAUTION-. Hydrous Benzoyl peroxide may

explode at temperatures higher than 60 °C or if its water content is too low. It
may burst into flame in the presence of reducing substances. Unused material
must not be returned to the original container but destroyed by treating with
sodium hydroxide (-80 g/1) TS to a point where no iodine is liberated after acid-
ifying with hydrochloric acid (-70g/l) TS and adding a crystal of potassium
iodide R.
Hydrous Benzoyl peroxide loses water rapidly on exposure to air. It must be

handled with care, avoiding contact with the skin and mucous membranes and
inhalation of airborne particles.
Requirements
Hydrous Benzoyl peroxide contains not less than 70 0 % and not more than
.
77 0 % of C14H10O4, and not less than 20 0 % of water.

. .
Note-. Before carrying out any tests, thoroughly mix the entire sample.
152
Identity tests

reference spectrum of benzoyl peroxide.

toluene R, 2 volumes of dichloromethane R, and 1 volume of glacial acetic
acid R mobile phase. Apply separately to the plate 5 ml of each of 2

as the
solutions in methanol R containing (A) lO.Omg of Hydrous Benzoyl perox-
ide per ml, and (B) a solution of hydrous benzoyl peroxide R containing the
equivalent of 10.0 mg of benzoyl peroxide per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air, and examine the
chromatogram in ultraviolet light (254 nm).

C. Dissolve about 25 mg in 2 ml of acetone R, add 1ml of diethylphenylenedi-

amine sulfate TS, and mix; a red colour is produced which turns rapidly to
dark violet within 5 minutes.
D. To 1 g add 5 ml of ethanol (-750 g/1) TS, 5ml of sodium hydroxide (~80g/l) TS,
and 10ml of water. Boil the mixture under a reflux condenser for 20 minutes
and cool. To 1 ml of the resulting solution add 0.5 ml of ferric chloride (65 g/1)
TS; a dull yellow precipitate is produced which is soluble in ether R.
Chlorides. Dissolve a quantity containing the equivalent of 0.5 g of anhydrous

Benzoyl peroxide in 15 ml of acetone R. Add, while stirring, 50 ml of nitric acid
(0.05mol/l) VS, allow to stand for 10 minutes, and residue with

filter. Wash the
two combining the
quantities, each of 10 ml, of nitric acid (0.05 mol/1) VS,
filtrate and the washings. Dilute this solution to 100 ml with nitric acid
(0.05mol/l) VS. Using 2.5ml of this solution, proceed as described under 2.2.1
Limit test for chlorides; the chloride content does not exceed 4mg/g.

Fischer method. Method A, using 5.0 ml of solution A as prepared below under
“Assay”. Add 3ml of a solution containing O.lOg of potassium iodide R in
dimethylformamide R. Stir for 5 minutes before starting the titration. Repeat
the procedure using 5 ml of dimethylformamide R in place of solution A and
make any necessary corrections. Calculate the content of water as a percentage.
Acidity. Dissolve a quantity containing the equivalent of 1.0 g of anhydrous

Benzoyl peroxide in 25 ml of acetone R, add 75 ml of water, and filter. Wash
153
the residue with two quantities of 10 ml of water. Combine the filtrate and
washings, and titrate with sodium hydroxide (0.1 mol/1) VS, using 0.25 ml of
phenolphthalein/ethanol TS as indicator, until the change in colour is observed.
Repeat the procedure without the substance being examined. The difference
between the titrations represents the amount of sodium hydroxide required;
not more than 1.25ml of sodium hydroxide (0.1 mol/1) VS.
40 volumes of light petroleum Rl, 20 volumes of toluene R, 15 volumes of
acetone R, and 1 volume of glacial acetic acid R as the mobile phase. Apply
separately to the plate 5 pi of each of 4 freshly prepared solutions in acetone R
containing (A) a quantity equivalent to 40 mg of anhydrous Benzoyl peroxide
per ml, (B) 0.4 mg of anhydrous Benzoyl peroxide per ml, (C) 0.6mg of benzoic
acid R per ml, and for solution (D) mix 0.4ml of benzyl benzoate R with 5 ml
of acetone R and dilute to 10 ml with the same solvent. To 1.0ml of this solu-
tion add 1.0 ml of solution A and dilute to 10 ml with acetone R. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air for 20
minutes, and examine the chromatogram in ultraviolet light (254 nm).
Any spot corresponding to benzoic acid obtained with solution A is not more
intense than that obtained with solution C (1.5%). Any spot obtained with
solution A, other than the principal spot and the spot corresponding to benzoic
acid, is not more intense than that obtained with solution B (1%). The test is
not valid unless the chromatogram obtained with solution D shows two clearly
separated principal spots.
Assay. Immediately before testing dissolve 2.5 g in sufficient dimethylfor-

mamide R to produce 100ml {solution A). To 5.0 ml of solution A add 20ml of
acetone R and 5 ml of potassium iodide (300 g/1) TS. Mix, allow to stand for 1
minute, and titrate with sodium thiosulfate (0.1 mol/1) VS until the solution is
colourless. Repeat the procedure using 5 ml of dimethylformamide R in place
of solution A and make any necessary corrections.
Each ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to 12.11 mg of

C14H10O4.
BENZYLIS BENZOAS
BENZYL BENZOATE
154
Graphic formula.
Chemical name. Phenylmethyl benzoate; CAS Reg. No. 120-51-4.
Description. A clear, colourless, oily liquid; odour, faintly aromatic.
Miscibility. Practically immiscible with water and glycerol R; miscible with

ethanol (-750 g/1) TS and ether R.
Category. Scabicide (topical use).
Storage. Benzyl benzoate should be kept in a tightly closed and well-filled

container, protected from light.
Additional information. Benzyl benzoate may slowly decompose on contact

with air.
Requirements
Definition. Benzyl benzoate contains not less than 98.0% and not more than
100.5% of C 14 H 12 O 2 .
Identity tests
Boil 2g with 25 ml of potassium hydroxide/ethanol TS2 for 10 minutes, evap-
orate the ethanol on a water-bath, cool, extract with 2 successive quantities,
each of 15 ml of ether R, and proceed as follows:
A. Evaporate the ethereal layer on a water-bath; heat 1 drop of the oily liquid
with 5 ml of sodium carbonate (50 g/1) TS and 1ml of potassium perman-

ganate (0.02 mol/1) VS; an odour of benzaldehyde is discernible.
B. To the aqueous layer add 10 ml of sulfuric acid (-100 g/1) TS; a white, crys-
tallineprecipitate is produced. Wash and dry the precipitate; melting
temperature, about 123 °C (benzoic acid).
Congealing temperature. Not below 17.0 “C.
Refractive index, no = 1.568 - 1.570.
Mass density. P 20 = 1.116 - 1.120g/ml.
155
Chlorinated compounds. For the preparation of the test solution dissolve

0.30g in 15ml of ethanol (~750g/l) TS and add 6ml of sodium hydroxide
(~80g/l) TS, Warm the solution for 5 minutes on a water-bath. Cool, transfer
to a comparison tube, and add 3 ml of nitric acid (-130 g/1) TS. For the refer-
ence solution transfer separately 2.0ml of hydrochloric acid CITS and 4ml of
nitric acid (-130 g/1) TS to a comparison tube, and dilute to 25 ml with water.
To both tubes add 0.5ml of silver nitrate (40 g/1) TS. Stir immediately with a
glass rod and set aside for 5 minutes, protected from direct sunlight. The opales-
cence produced from the test liquid is not stronger than that produced from
the reference solution; not more than 0.33mg/g of chlorine.
Acidity. Add 5 ml of the test liquid to 5 ml of neutralized ethanol TS, and

titrate with carbonate-free sodium hydroxide (0.1 mol/1) VS, phenolph-
thale in/ ethanol TS being used as indicator; not more than 0,3 ml is required to
obtain the midpoint of the indicator (pink).
Assay. Add about 2.0g, accurately weighed, to 40ml of potassium hydrox-

ide/ethanol (0.5 mol/1) VS and boil under reflux for 1 hour. Cool, and titrate
with hydrochloric acid (0.5 mol/1) VS, using phenolphthalein/ethanol TS as indi-
cator. Repeat the operation without the test liquid being examined and make
any necessary corrections. Each ml of potassium hydroxide/ethanol (0.5 mol/1)
VS is equivalent to 106.1 mg of C14H12O2.
BENZYLIS HYDROXYBENZOAS
BENZYL HYDROXYBENZOATE
o
CmFIoOs
Chemical name. Benzyl /2-hydroxybenzoate; phenylmethyl 4-hydroxyben-

zoate; CAS Reg. No. 94-18-8.
Description. A white to creamy white, crystalline powder; odourless or

almost odourless.
156

ether R; dissolves in solutions of alkali hydroxides.
Storage. Benzyl hydroxybenzoate should be kept in a weU-closed container.
Requirements
Benzyl hydroxybenzoate contains not less than 99 0 . % and not more than the
equivalent of 101 0 . %
of C14H12O3.
Identity tests
A. The absorption spectrum of a lOpg/ml solution in ethanol (-750 g/1) TS,
when observed between 230 nm and 350 nm, exhibits a maximum at about
260 nm; the absorbance of a 1-cm layer at this wavelength is about 0.76.
B. Dissolve 0.1 g in 2ml of ethanol (~750g/l) TS, boil, and add 0.5ml of
mercury/ nitric acid TS; a precipitate gradually separates and the supernatant
liquid becomes red.
Acidity. Dissolve 0.2g in 10ml of ethanol (-375g/l) TS previously neutralized

using methyl red/ethanol TS. Titrate with sodium hydroxide (0.1 mol/1) VS; not
more than 0.1ml is required to obtain the midpoint of the indicator (orange).
Assay. To about 0.12g, accurately weighed, add 20ml of sodium hydroxide

(-80 g/1) TS, and boil gently under reflux for 30 minutes. Cool, and extract with
three quantities, each of 20ml, of dichloroethane R. Wash the combined
extracts with 20 ml of sodium hydroxide (0.1 mol/1) VS and add the wash liquids
to the main aqueous phase, discarding the organic phase. To the aqueous solu-
tion add 25ml of potassium bromate (0.0333 mol/1) VS, 6ml of potassium
bromide (100 g/1) TS, and 10ml of hydrochloric acid (-420g/l) TS and immedi-
ately stopper the flask. Shake for 15 minutes and allow to stand for 15 minutes.
Add 25ml of potassium iodide (100 g/1) TS and shake vigorously. Titrate the
liberated iodine with sodium thiosulfate (0.1 mol/1) VS, using starch TS as indi-
cator, added towards the end of the titration. Repeat the procedure without the
Benzyl hydroxybenzoate being examined and make any necessary corrections.
The volume of potassium bromate (0.0333 mol/1) VS used is equivalent to half
of the volume of sodium thiosulfate (0.1 mol/1) VS required for the titration.
Each ml of potassium bromate (0.0333 mol/1) VS is equivalent to 7.608mg of

C14H12O3.
157
BENZYLPENICILLINUM KALICUM
BENZYLPENICILLIN POTASSIUM
Benzylpenicillin potassium (non-injectable)
Benzylpenicillin potassium, sterile
Molecular formula. Ci 6 H, 7 KN 204 S
Graphic formula.
Chemical name. Potassium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylac-

etamido)-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate; potassium [2S-
(2a,5a,6P)]-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-l-azabicyclo[3.
2.0]heptane-2-carboxylate; CAS Reg. No. 113-98-4.
Description. A white or almost white, crystalline powder; odourless or with

a faint characteristic odour.
Solubility. Very soluble in water; practically insoluble in ether R.
Storage. Benzylpenicillin potassium should be kept in a tightly closed con-

Labelling. The designation sterile Benzylpenicillin potassium indicates that

the substance complies with the additional requirements for sterile Ben-
zylpenicillin potassium and may be used for parenteral administration or for
other sterile applications.
Additional information. Benzylpenicillin potassium is moderately hygro-

scopic; it is readily decomposed by acids, alkalis and oxidizing agents. Even
in the absence of light, Benzylpenicillin potassium is gradually degraded on
exposure to a humid atmosphere, the decomposition being faster at higher
temperatures.
158
Requirements
Definition. Benzylpenicillin potassium contains not less than 96.0% and
not more than 102.0% of C16H17KN2O4S, calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from benzylpenicillin potassium RS or with the reference
spectrum of benzylpenicillin potassium.

(-1760 g/1)TS and mix; the solution is colourless. Immerse the test-tube for
TS and mix; the solution is brownish yellow. Immerse the test-tube for 1
minute in a water-bath; a reddish brown colour is produced.
C. Ignite a small quantity, dissolve the residue in water and filter; on addition
of 2 ml of sodium hydroxide (-80 g/1) TS to the filtrate it yields the reaction
described under 2.1 General identification tests, as characteristic of
potassium.
Specific optical rotation. Use a 20mg/ml solution; [a]o = + 270 to + 300°.
Clarity and colour of solution. A solution of 0.20 g in 10 ml of water is clear

and colourless.
lOmg/g.
pH value. pH of a 20mg/ml solution in carbon-dioxide-free water R, 5. 0-7. 5.
Light-absorbing impurities. Using a freshly prepared 1.9mg/ml solution in

water, measure the absorbances of a 1-cm layer at 280 nm and at 325 nm; the
absorbance at each of these wavelengths does not exceed 0.10.
Assay. Dissolve about 50 mg, accurately weighed, in sufficient water to

produce 1000 ml. Transfer two 2.0-ml aliquots of this solution into separate
stoppered tubes. To one tube add 10.0ml of imidazole/mercuric chloride TS,
mix, stopper the tube and place in a water-bath at 60 °C for exactly 25 minutes.
Cool the tube rapidly to 20 °C (solution A).
159
B, calculate the amount of C, 6H] 7 KN 204 S in the substance being tested by com-
parison with benzylpenicillin sodium RS similarly and concurrently examined,
taking into account that each mg of benzylpenicillin sodium RS
(C,(;Hi 7 N Na 04 S)
2 is equivalent to 1.045mg of benzylpenicillin potassium
(C 16 H 17 KN 2 O 4 S). In an adequately calibrated spectrophotometer the absorbance
of the reference solution should be 0.62 + 0.03.
Additional requirements for Benzylpenicillin potassium for

sterile use
benzylpenicillin.

BENZYLPENICILLINUM NATRICUM
BENZYLPENICILLIN SODIUM
Benzylpenicillin sodium (non-injectable)
Benzylpenicillin sodium, sterile
Molecular formula. Ci 6 Hi 7 N 2 Na 04 S
Graphic formula.
160
Chemical name. Sodium (2S,5R,6/^)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-

4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate; sodium [2S-(2a,5a,6P)]-3,
3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-l-azabicyclo[3.2.0]-heptane-
2-carboxylate; CAS Reg. No, 69-57-8.

Solubility. Soluble in about 0.5 part of water; practically insoluble in ether R.
Storage. Benzylpenicillin sodium should be kept in a tightly closed container,

protected from light, and stored at a temperature not exceeding 25 °C.
Labelling. The designation sterile Benzylpenicillin sodium indicates that the

substance complies with the additional requirements for sterile Benzylpenicillin
sodium and may be used for parenteral administration or for other sterile
applications.
Additional information. Benzylpenicillin sodium is hygroscopic; it is readily

decomposed by acid, alkalis and oxidizing agents. Even in the absence of light,
Benzylpenicillin sodium is gradually degraded on exposure to a humid atmos-
phere, the decomposition being faster at higher temperatures.
Requirements
Definition. Benzylpenicillin sodium contains not less than 96.0% and not more
than 102.0% of Ci 6 H, 7N 2 Na 04 S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from benzylpenicillin sodium RS or with the reference
spectrum of benzylpenicillin sodium.

(-1760 g/1)TS and mix; the solution is colourless. Immerse the test-tube for
TS and mix; the solution is brownish yellow. Immerse the test-tube for 1
minute in a water-bath; a reddish brown colour is produced.
161
yields the characteristic reaction. If reaction B is to be used, ignite a small
quantity and dissolve the residue in acetic acid (~60g/l) TS.
Specific optical rotation. Use a 20mg/ml solution; [a]o = + 280 to + 310°.

and colourless.
Loss on drying. Dry to constant weight at 105°C; it loses not more than lOmg/g.
Light-absorbing impurities. Using a freshly prepared 1.8mg/ml solution in

water, measure the absorbances of a 1-cm layer at 280nm and at 325nm; the
absorbance at each of these wavelengths does not exceed 0.10.

Cool the tube rapidly to 20 °C (solution A).

the difference
B, calculate the amount of Ci 6 H] 7 N 2 Na 04 S in the substance being tested by
comparison with benzylpenicillin sodium RS similarly and concurrently exam-
ined. In an adequately calibrated spectrophotometer the absorbance of the ref-
erence solution should be 0.62 ± 0.03.
Additional requirements for Benzylfaenicillin sodium for

sterile use
Bacterial endotoxins. Carry out the test as described under 3.4 Test for bacterial en-
dotoxins; contains not more than 0.01 lU of endotoxin RS per mg of benzylpenicillin.

162
BEPHENII HYDROXYNAPHTHOAS
BEPHENIUM HYDROXYNAPHTHOATE
Graphic formula.
COO“
OH
Chemical name. Benzyldimethyl(2-phenoxyethyl)ammonium 3-hydroxy-2-

naphthoate (1 1); N,Af-dimethyl-N-(2-phenoxyethyl)benzenemethanaminium
:
salt with 3-hydroxy-2-naphthalenecarboxylic acid (1 1); CAS Reg. No.

:
3818-50-6.
Description. A yellow to greenish yellow, crystalline powder; odourless or

almost odourless.
Solubility. Practically insoluble in water, ether R and benzene R; soluble in 50

parts of ethanol (-750 g/1) TS.
Category. Anthelmintic.
Storage. Bephenium hydroxynaphthoate should be kept in a tightly closed

container.
Additional information. Even in the absence of light. Bephenium hydroxy-

naphthoate is gradually degraded on exposure to a humid atmosphere, the
decomposition being faster at higher temperatures.
Requirements
Definition. Bephenium hydroxynaphthoate contains not less than 99.0% and
not more than 101.0%, of C28H29NO4, calculated with reference to the dried
substance.
Identity tests
163

the spectrum obtained from bephenium hydroxynaphthoate RS or with the
reference spectrum of bephenium hydroxynaphthoate.
B. See the test described below under “Related substances”. The principal spots
obtained with solution B at 254 nm correspond in position, appearance, and
intensity with those obtained with solution C.
Chlorides. For the preparation of the test solution, boil 0.7 g with 30 ml of
water, cool in ice and filter. Add 10ml of nitric acid (-130 g/1) TS to the filtrate,
and proceed as described under 2.2.1 Limit test for chlorides; the chloride
content is not more than 0.35 mg/g.
10 mg/g.
Related substances. Carry out the test described under 1.14.1 Thin-layer
chromatography, using silica gel R4 as the coating substance and a mixture
of 5 volumes of 1 -butanol R, 4 volumes of water and 1 volume of acetic acid
(-300 g/1) TS as the mobile phase. Apply separately to the plate 5 pi of each of
3 solutions in methanol R containing (A) 40 mg of the test substance per ml, (B)
0.40 mg of the test substance per ml, and (C) 0.40 mg of bephenium hydroxy-
naphthoate RS per ml. After removing the plate from the chromatographic
light (254nm and 365 nm). At 254nm two principal spots are visible with each
of the solutions A, B, and C, whereas at 365 nm only the spots closer to the
solvent front fluoresce. Any additional spot visible with solution A other than
the two principal spots, is not more intense in appearance in both lights than
the spot obtained closer to the starting line of solution B.
Afterwards, spray the plate first with sodium molybdotungstophosphate TS,
then with sodium carbonate (200 g/1) TS, and examine the chromatogram in
daylight. Two principal spots are obtained with each of the solutions A, B, and
C. Any additional spot obtained with solution A, disregarding those that may
have been visible in ultraviolet light, is not more intense than the principal spot
closer to the solvent front obtained with solution B.
Assay. Dissolve about 0.4 g, accurately weighed, in 30 ml of glacial acetic

acid Rl, and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6
Non-aqueous titration. Method A. Each ml of perchloric acid (0.1 mol/1) VS is
equivalent to 44.35mg of C28H29NO4.
164
BETAMETHASONI VALERAS
BETAMETHASONE VALERATE
Molecular formula. C27H37FO<s
Graphic formula.
CH,OH
'
I
CO
Chemical name. 9-Fluoro-l lp,17,21-trihydroxy-16p-methylpregna-l,4-diene-

3,20-dione 1 7 -valerate; 9-fluoro-l 1 1 -dihydroxy- 1 6P-methyl- 1 7 [(1 -oxopentyl)
P,2 -
oxyl]pregna-l,4-diene-3,20-dione; CAS Reg. No. 2152-44-5.
Description. A white or creamy white powder; odourless.
Solubility. Practically insoluble in water; soluble in ethanol (-750 g/l)TS; freely

soluble in acetone R.
Category. Antiinflammatory drug.
Storage. Betamethasone valerate should be kept in a tightly closed container,

Requirements
Definition. Betamethasone valerate contains not less than 96.0% and not
more than 104.0% of C 27H 37 FO 6 ,
substance.
Identity tests
• Either tests A and B or tests C, D and E may be applied.

infrared region.
spectrum obtained from betamethasone valerate RS or with the reference
spectrum of betamethasone valerate.
165
B. Dissolve 20 mg in 20 ml of ethanol (-750 g/1) TS and dilute 2 ml to 20 ml with

the same solvent. To 2 ml of this solution placed in a stoppered test-tube
add 10ml of phenylhydrazine/sulfuric acid TS, mix, heat in a water-bath at
60 °C for 20 minutes, and cool immediately. The absorbance of a 1-cm layer
at the maximum at about 423 nm is not more than 0.25.
C. See the test described below under “Related steroids”. The principal spots
obtained with solutions A and C correspond in position with that obtained
with solution B. In addition, the principal spot obtained with solution A cor-
responds in appearance and intensity with that obtained with solution B.
D. Carry out the combustion as described under 2.4 Oxygen flask method,
using 7 mg of the test substance and a mixture of 0.5 ml of sodium hydrox-
VS and 20ml of water as the absorbing liquid. When the
ide (0.01 mol/1)
processis complete, add 0.1 ml of a mixture of 0.1 ml of a freshly prepared
sodium aKzarinsulfonate (1 g/1) TS and 0.1 ml of zirconyl nitrate TS; the red
colour of the solution changes to clear yellow.
E. Heat 0.05g with 2.0ml of potassium hydroxide/ethanol TSl in a water-bath

for 5 minutes. Cool, add 2.0 ml of sulfuric acid (-100 g/1) TS, and boil gently
for 1 minute; a pleasant odour of ethyl valerate is perceptible.
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; [aj^ = -l-75
to -h81 °.
Sulfated ash. Weigh 0.1 g and ignite on a platinum dish; not more than
2-Omg/g.
5.0mg/g.
Related steroids. Carry out the test as described under 1.14.1 Thin-layer
95 volumes of dichloroe thane R, 5 volumes of methanol R, and 0.2 volumes of
containing (A) 15 mg of the test substance per ml and (B) 15mg of betametha-
sone valerate RS per ml; also apply to the plate 2 pi of a third solution (C) com-
posed of a mixture of equal volumes of solutions A and B and 1 pi of a fourth
solution (D) containing 0.15 mg of the test substance per ml in the same solvent
mixture used for solutions A and B. After removing the plate from the chro-
matographic chamber, allow it to dry in air until the solvents have evaporated.
Then heat it at 105°C for 10 minutes, allow it to cool, spray it with blue
tetrazolium/sodium hydroxide TS, and examine the chromatogram in daylight.
intense than that obtained with solution D.
166
Assay
Dissolve about 20mg, accurately weighed, in sufficient aldehyde-free ethanol

(-750 g/1) TS to produce 100 ml. Dilute 20 ml of this solution with sufficient
aldehyde-free ethanol (~750g/l) TS to produce 100ml. Transfer 10.0ml of the
diluted solution to a 25-ml volumetric flask, add 2.0ml of blue tetrazolium/
ethanol TS, and displace the air in the flask with oxygen-free nitrogen R. Imme-
diately add 2.0 ml of tetramethylammonium hydroxide/ethanol TS and again
displace the air with oxygen-free nitrogen R. Stopper the flask, mix the con-
tents by gentle swirling, and allow to stand for 1 hour in a water-bath at 30 °C.
Cool rapidly, add sufficient aldehyde-free ethanol (-750 g/1) TS to produce
25ml, and mix. Measure the absorbance of a 1-cm layer at the maximum at
about 525 nm against a solvent cell containing a solution prepared by treating
10ml of aldehyde-free ethanol (-750 g/1) TS in a similar manner. Calculate the
amount of C 27H 37 FO 6 in the substance being tested by comparison with
betamethasone valerate RS, similarly and concurrently examined.
BETAMETHASONUM
BETAMETHASONE
Molecular formula. C22H29FO5
Graphic formula.
Chemical name. 9-Fluoro-l ip,17,21-trihydroxy-16P-methylpregna-l,4-diene-

3,20-dione; CAS Reg. No. 378-44-9.
Solubility. Practically insoluble in water; sparingly soluble in ethanol (-750 g/1)

TS.
167
Category. Adrenoglucocorticoid.
Storage. Betamethasone should be kept in a tightly closed container, protected

from light.
Requirements
Definition. Betamethasone contains not less than 96.0% and not more than
104.0% of C22H29FO5 calculated with reference to the dried substance.
Identity tests
• Either tests A, B and C, or tests B, C and D may be applied.

infrared region.The infrared absorption spectrum is concordant with the
spectrum obtained from betamethasone RS or with the reference spectrum of
betamethasone (recrystallization from chloroform R of the test substance
and the reference substance might be necessary to obtain the same crys-
talline form).

add 10 ml of phenylhydrazine/sulfuric acid TS, mix, heat in a water-bath at
at the maximum at about 450 nm is not more than 0.30 (preferably use 2-
cm cells for the measurement and calculate the absorbance of a 1-cm layer).
with solution B. In addition the appearance and intensity of the principal
spot obtained with solution A corresponds with that obtained with
solution B.
ide (0.01 mol/1)
processis complete, add 0.1 ml to a mixture of 0.1 ml of a freshly prepared
sodium aHzarinsulfonate (1 g/1) TS and 0.1 ml of zirconyl nitrate TS; the red
Specific optical rotation. Use a 5.0 mg/ml solution in dioxan R; [a]c°'^ = -1-114
to-tl22°.
Sulfated ash. Weigh 0.1 g and use a platinum dish; not more than 5.0mg/g.
168
Loss on drying. Dry to constant weight at 1 00 °C under reduced pressure (not

exceeding 0.6kPa or about 5mm of mercury); it loses not more than 5.0mg/g.
of 77 volumes of dichlorome thane R, 15 volumes of ether R, 8 volumes of
methanol R, and 1.2 volumes of water as the mobile phase. Apply separately
to the plate 1 pi of each of 2 solutions in a mixture of 9 volumes of chloroform
R and 1 volume of methanol R containing (A) 15 mg of the test substance per
ml and (B) 15 mg of betamethasone RS per ml; also apply to the plate 2 pi of a
third solution (C) composed of a mixture of equal volumes of solutions A and
B and 1 pi of a fourth solution (D) containing O.lSmg of the test substance per
ml in the same solvent mixture used for solutions A and B. After removing the
plate from the chromatographic chamber, allow it to dry in air until the sol-
vents have evaporated and heat at 105 °C for 10 minutes, allow to cool, spray
with blue tetrazoKum/sodium hydroxide TS, and examine the chromatogram
in daylight. Any spot obtained with solution A, other than the principal spot,
is not more intense than that obtained with solution D.
Assay

aldehyde-free ethanol (~750g/l)TS to produce 100ml. Transfer 10.0ml of the
tents by gentle swirling and allow to stand for 1 hour in a water-bath at 30 °C.
25ml and mix. Measure the absorbance of a 1-cm layer at the maximum at
10 ml of aldehyde-free ethanol (-750 g/1) in a similar manner. Calculate the
amount of C22H29FO5 in the substance being tested by comparison with
betamethasone RS, similarly and concurrently examined.
169
BIPERIDENI HYDROCHLORIDUM
BIPERIDEN HYDROCHLORIDE
Molecular formula. C2iH2S)NO,HCl
Graphic formula.
Chemical name. a-5-Norbornen-2-yl-a-phenyl-l-piperidinepropanol hydro-

chloride; a-bicyclo [2.2. 1 ]hept-5-en-2-yl-a- phenyl- 1 -piperidine propanol hydro-
chloride; CAS Reg. No. 1235-82-1.
Description. A white, crystalline powder; odourless.
Solubility. Slightly soluble in water, ethanol (~750g/l) TS and ether R; spar-

ingly soluble in methanol R.
Category. Antiparkinsonism drug.
Storage. Biperiden hydrochloride should be kept in a well-closed container,

Requirements
Definition. Biperiden hydrochloride contains not less than 98.0% and not
more than 101.0% of C 2 iH 29 NO,HCl, calculated with reference to the dried
substance.
Identity tests
• Either tests A and D or tests B, C and D may be applied.

infrared region.
spectrum obtained from biperiden hydrochloride RS or with the reference
spectrum of biperiden hydrochloride.
170
B. Dissolve 20mg in 5ml of phosphoric acid (-1440 g/1) TS and allow to stand;
a green colour is produced.
C. Dissolve O.lOg in 50ml of water and to 5ml add bromine

of this solution
TSl drop by drop; a yellow precipitate is formed which dissolves on
shaking. Upon the addition of more bromine TSl, a permanent precipitate
is produced.
D. A 20 mg/ml solution yields reaction B described under 2.1 General identifi-
Loss on drying. Dry for 3 hours at 105°C; it loses not more than 5mg/g.
chromatography, using a plate coated with a suspension of silica gel R1 in
sodium hydroxide (0.5 mol/1) VS, and as the mobile phase a mixture of 96.5
volumes of toluene R and 3.5 volumes of methanol R. Apply separately to the
plate 1 0 pi of each of 2 solutions in methanol R containing (A) 20 mg of the test
substance per ml and (B) 0.10 mg of the test substance per ml. After removing
the plate from the chromatographic chamber, allow it to dry in air, spray it with
potassium iodobismuthate TS2, and examine the chromatogram in daylight.
Rl, warming slightly to effect solution, add 10ml of mercuric acetate/acetic
acid TS and 0.15ml of 1-naphtholbenzein/acetic acid TS as indicator. Titrate
with perchloric acid (0.1 mol/1) VS, as described under 2.6 Non-aqueous
34.79 mg of C 2 ,H 29NO,HCl.
BIPERIDENUM
BIPERIDEN
Molecular formula. C21H29NO
171
Graphic formula.
Chemical name. a-5-Norbornen-2-yl-a-phenyl-l-piperidinepropanol; a-

bicyclo[2.2.1]hept-5-en-2-yl-a-phenyl-l-piperidinepropanol; CAS Reg. No.
514-65-8.
Solubility. Practically insoluble in water; soluble in ether R; sparingly soluble

in ethanol (-750 g/1) TS.
Storage. Biperiden should be kept in a well-closed container, protected from

light.
Requirements
Definition. Biperiden contains not less than 98.0% and not more than 101.0%
of C 21 H 29NO, calculated with reference to the dried substance.
Identity tests
• Either test A alone or tests B, C and D may be applied.

the spectrum obtained from biperiden RS or with the reference spectrum of
biperiden.
B. Dissolve 20mg in 5ml of phosphoric acid (-1440 g/1) TS and allow to stand;
C. To 0.20 g add 80ml of water, 0.5 ml of hydrochloric acid (-70 g/1) TS, and
warm until dissolved. Cool and to 5 ml of this solution add bromine TSl drop
by drop; a yellow precipitate is formed which dissolves on shaking. Upon
the addition of more bromine TSl, a permanent precipitate is produced.
D. Melting temperature, about 114°C.
172
Loss on drying. Dry for 3 hours at 105 °C; it loses not more than lOmg/g.
chromatography, using a plate coated with a suspension of silica gel R1 in
sodium hydroxide (0.5 mol/1) VS, and as the mobile phase a mixture of 96.5
volumes of toluene R and 3.5 volumes of methanol R. Apply separately to the
plate 5 pi of each of 2 solutions in chloroform R containing (A) 40 mg of the
test substance per ml and (B) 0.20 mg of the test substance per ml. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air, spray it
with potassium iodobismuthate TS2, and examine the chromatogram in day-
light. Any spot obtained with solution A, other than the principal spot, is not
more intense than that obtained with solution B.
Rl, add 0.15ml of 1-naphtholbenzein/acetic acid TS as indicator and titrate
titration, Method A. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to
31.15mg of C 21 H 29 NO.
Additional requirement for Biperiden for parenteral use

Complies with the monograph for ‘‘Parenteral preparations”.
BLEOMYCINI HYDROCHLORIDUM
BLEOMYCIN HYDROCHLORIDE
Bleomycin hjrdrochloride (non-injectable)
Bleomycin hydrochloride, sterile
Chemical name. Bleomycin hydrochloride; CAS Reg. No. 67763-87-5.
Molecular formula. Bleomycin A 2 hydrochloride: C 55 H 84N] 702 iS 3 ,Cl.

Bleomycin B 2 hydrochloride: C55H84N2o02iS2,HCl.
Relative molecular mass. Bleomycin A 2 hydrochloride: 1452; Bleomycin B 2

hydrochloride: 1461.
173
Graphic formulas for the bleomycin A2/B2 bases.
Chemical names for the bleomycin A2/B2 bases. Bleomycin A 2 hydrochlo-

ride: N'-[3-(Dimethylsul£onio)-propyl]bleomycinamide chloride; [3-[2'-[2-
[(2S,3R)-2-[(2S,3S,4/^)-4-[(2S,31^)-2-[6-amino-2-[(lS)-l-[[(2S)-2-amino-2-carbam
oylethyl]amino]-2-carbamoylethyl]-5-methyl-4-pyrimidinecarboxamido]-3-[[2-
0-(3-0-carbamoyl-a-D-mannopyranosyl)-a-L-gulopyranosyl]oxy]-3-imidazol-
4-ylpropionamido]-3-hydroxy-2-methylvaleramido]-3-hydroxybutyramido]
ethyl][2,4'-bithiazole]-4-carboxamido]propyl]dimethylsulfonium chloride; CAS
Reg. No. 49830-49-1.
Bleomycin B 2 hydrochloride: N’-(Guanidinobutyl)bleomycinamide hydrochlo-

ride; (pS)-4-amino-P-[[(2h)-2-amino-2-carbamoylethyl]amino]-6-[[(lS,2i^)-2-[[2-
0-(3-0-carbamoyl-a-D-mannopyranosyl)-a-L-gulopyranosyl]oxy] - 1 - [[(1 i?,2S,3
S)-3-[[(l S,21?)-l -[[2- [4- [(4-guanidinobutyl) carbamoyl] [2,4'-bithiazol]-2'-
yl]ethyl]carbamoyl]-2-hydroxypropyl]carbamoyl]-2-hydroxy-l-
methylbutyl]carbamoyl]-2-imidazol-4-ylethyl]carbamoyl]-5-methyl-2-pyrimidi
nepropionamide hydrochloride; A/*-[4-[(aminoiminomethyl)amino]butyl]
bleomycinamide hydro-chloride; CAS Reg. No. 55658-44-1.
Description. A white to yellowish white powder.
Solubility. Freely soluble in water and in methanol R; slightly soluble in

ethanol (-750 g/1) TS; practically insoluble in acetone R and ether R.
Category. Cytotoxic drug.
174
Storage. Bleomycin hydrochloride should be kept in a tightly closed container.
Labelling. The designation sterile Bleomycin hydrochloride indicates that the

substance complies with the additional requirements for sterile Bleomycin
hydrochloride and may be used for parenteral administration or for other sterile
applications. CAUTION: Bleomycin hydrochloride must be handled with care,
avoiding contact with the skin and inhalation of airborne particles.
Requirements
Definition. Bleomycin hydrochloride is the hydrochloride salt of a mixture of
substances produced by the growth of Strep>tomyces verticillus. The main com-
ponents of the mixture are bleomycin A 2 and bleomycin B 2 .
Bleomycin hydrochloride contains, when tested according to assay A, not less

than 1500 and not more than 2000 International Units of bleomycin A 2/B 2 per
mg, calculated with reference to the dried substance.
Further,Bleomycin hydrochloride contains, when tested according to assay B,

not than 55.0% and not more than 70.0% of bleomycin A 2 and not less
less
than 25.0% and not more than 32.0% of bleomycin B 2 the total of bleomycin
;
A2 and bleomycin B 2 is not less than 85%. The content of bleomycin A5 is not
more than 7.0%, of bleomycin B^ not more than 1.0%, and of demethyl-
bleomycin A 2 not more than 3.0%.
Identity tests
A. Dissolve about 5 mg in 10 ml of water, add 5 pi of copper(ll) sulfate (160g/l)
TS and dilute with water to 100ml; the absorption spectrum exhibits
maxima at about 242 nm and 290 nm, and a minimum at about 268 nm.
B. A 10 mg/ml solution yields reaction B described under 2.1 General identifi-


pH value. pH of a 5.0 mg/ml solution, 4. 5-6.0.
Copper content. Transfer 75 mg, accurately weighed, to a 60-ml separating

funnel and dissolve in 10ml of hydrochloric acid (0.1 mol/1) VS. Transfer 10ml
of copper standard TS2 to an additional separating funnel. To both funnels add
10 ml of zinc bis(dibenzyldithiocarbamate) TS and shake vigorously for 1
minute. Allow the layers to separate. Filter the lower layer through 1 g of anhy-
drous sodium sulfate R to remove excess water. Measure the absorbances of a
175
1-cm layer at the maximum at about 435 nm, using a solvent cell containing
carbon tetrachloride R.
Calculate the content of copper in mg/g from the formula {Ac x \5)l(As x W)
where Ac is the absorbance of the substance to be examined, A, is the
absorbance of copper standard TS2, and W

is the weight in mg of the substance
to be examined; the copper content is not more than 0.2 mg/g.
Assay
A. Microbiological assay. Carry out the assay as described under 3.1 Micro-
biological assay of antibiotics, using Mycobacterium smegmatis (ATCC 607) as
the test organism. Prepare the inoculum as follows: the test organism is
grown for 40-48 hours at a temperature of 27 °C on the surface of culture

medium Cm8. Using 3 ml of saline TS wash the growth into a flask con-
taining 100 ml of culture medium Cm9 and 50 g of glass beads, and incu-
bate at 25-27 °C for 5 days with constant mechanical agitation using an
orbital shaker. The resulting suspension should be used for no longer than
14 days, and kept at a temperature below 5°C. For the preparation of inoc-
ulated plates use 0.5ml of the suspension or a suitable volume previously
determined using test plates with culture medium Cm8 at a temperature of
27 °C. Prepare the reference solution in phosphate buffer, pH 7.0, TS, dilut-
ing the International Reference Preparation of bleomycin A 2 /B 2 to an appro-
priate concentration (usually between 10 and 200 pg per ml). The precision
of the assay is such that the fiducial limits of error of the estimated potency
(P = 0.95) are not less than 95% and not more than 105% of the estimated
potency. The upper fiducial limit of error of the estimated potency (P = 0.95)
is not less than 1500 lU and the lower fiducial limit is not more than
2000 lU of bleomycin A 2 /B 2 per mg, calculated with reference to the dried

substance.
B. Content of the bleomycin components. Carry out the test as described under
1.14.4 High performance liquid chromatography, using a column 25 cm long
and 4.6mm in internal diameter packed with particles of silica gel, 5-10 pm
in diameter, the surface of which has been modified with chemically bonded
octadecylsilyl groups. As the mobile phase for a linear gradient develop-
ment, start with a mixture of 9 volumes of 1-pentanesulfonic acid TS and
1 volume of methanol R, both previously filtered and deaerated, and end
with a composition of 6 volumes of 1-pentanesulfonic acid TS and 4

volumes of methanol R, using a suitable linear rate of change of mobile
phase so as to reach the final composition in 60 minutes. (If needed, add
the following to the mobile phase to obtain satisfactory chromatography:
1.86g of disodium edetate R per litre.) As detector use an ultraviolet spec-
trophotometer at a wavelength of about 254 nm, fitted with a low- volume
flow cell (8-20 pi is suitable). Inject 5 pi of a solution of the test substance
in water containing the equivalent of 5 lU of bleomycin per ml. Proceed
176
with the gradient elution, pumping the mobile phase mixture at the condi-
tion mentioned above for about 80 minutes or until the demethylbleomycin
A2 is eluted.
The elution order of the bleomycin components is the following: void volume,
bleomycin acid, bleomycin A2, bleomycin B2, bleomycin A5, bleomycin B4, and
demethylbleomycin A2.
Calculate in % the content of each bleomycin component, comparing the ratios

of the individual areas of the peaks with that of the total area of all the bleomycins.
Additional requirements for Bleomycin hydrochloride for

sterile use
Storage. Sterile Bleomycin hydrochloride should be kept in a hermetically

closed container.
Manufacture. The method of manufacture is validated to demonstrate that

the product, if tested, would comply with the following test.
Histamine-like substances. Carry out the test as described under 3.6 Test
for histamine-like substances (vasodepressor substances) using 1ml per kg
of body mass of a solution in saline TS containing a quantity equivalent to
500 lU per ml.
BactericJ endotoxins. Carry out the test as described under 3.4 Test for bacterial
endotoxins; contains not more than 10.0 lU of endotoxin RS per mg of bleomycin.
Sterility. Complies with 3 2.2 Sterility testing of antibiotics, applying the

.
3 2.1 Test for sterility of non-injectable preparations.
.
BLEOMYCINI SULFAS
BLEOMYCIN SULFATE
Bleoitwdn sulfate (non-injectable)
Bleomycin sulfate, sterile
Chemical name. Bleomycin sulfate; CAS Reg. No. 9041 - 93 - 4 .
Molecular formula. Bleomycin A2 sulfate: C55Hs4Ni702iS3,H2S04; Bleomycin

B2 sulfate: C55Ha4N2o02iS2,H2S04
177
Relative molecular mass. Bleomycin Aj sulfate: 1514; Bleomycin sulfate:

1524.
Graphic formulas for the bleomycin A2/B2 bases.
A
Chemical names for the bleomycin 2/B 2 bases. Bleomycin A 2 sulfate:
W*-[3-(Dimethylsulfonio)propyl]bleomycinamide hydrogen sulfate; [3-[2'-[2-
[(2S,3R)-2-[(25,3S,4R)-4-[(2S,3R)-2-[6-amino-2-[(lS)-l-[[(2S)-2-amino-2-carbam
oylethyl]amino]-2-carbamoylethyl]-5-methyl-4-pyrimidinecarboxamido]-3-[[2-
0-(3-0-carbamoyl-a-D-mannopyranosyl)-a-L-gulopyranosyl]oxy]-3-imidazol-
4-ylpropionamido]-3-hydroxy-2-methylvaleramido]-3-hydroxybutyramido]
ethyl][2,4'-bithiazole]-4-carboxamido]propyl]dimethylsulfonium hydrogen
sulfate.
Bleomycin B 2 sulfate: N'-(Guanidinobutyl)bleomycinamide; (PS)-4-amino-P-

[[(2S)-2-amino-2-carbamoylethyl]amino]-6-[[(lh,2i?)-2-[[2-0-(3-0-carbamoyl-
a-D-mannopyranosyl)-a-L-gulopyranosyl]oxy]-l-[[(lR,2S,3S’)-3-[[(lS,2R)-l-[[2-
[4-[(4-guanidinobutyl) carbamoyl] [2, 4'- bithiazol] -2 '-yl] ethyl] carbamoy 11-
2-hydroxy propyl] carbamoyl] -2 -hydroxy- 1 -me thy Ibutyl] carbamoyl]-
2-imidazol-4-ylethyl]carbamoyl]-5-methyl-2-pyrimidinepropionamide sulfate
(salt); N'-[4-[(aminoiminomethyl)amino]butyl]bleomycinamide sulfate (salt).
Description. A white or cream-coloured, amorphous powder.
Solubility. Very soluble in water.
178
Storage. Bleomycin sulfate should be kept in a tightly closed container.
Labelling. The designation sterile Bleomycin sulfate indicates that the sub-
stance complies with the additional requirements for sterile Bleomycin sulfate
andmay be used for parenteral administration or for other sterile applications.
CAUTION: Bleomycin sulfate must be handled with care, avoiding contact
with the skin and inhalation of airborne particles.
Requirements
Definition. Bleomycin sulfate is the sulfate salt of a mixture of substances pro-
duced by the growth of Streptomyces verticillus. The main components of the
mixture are bleomycinA 2 and bleomycin B 2 .
Bleomycin sulfate contains, when tested according to assay A, not less than
1500 and not more than 2000 International Units of bleomycin A 2 /B 2 per mg,
Further,Bleomycin sulfate contains, when tested according to assay B, not less

than 55.0% and not more than 70.0% of bleomycin A 2 and not less than 25.0%
and not more than 32.0% of bleomycin B 2 the total of bleomycin A 2 and
;
bleomycin B 2 is not less than 85%. The content of bleomycin As is not more
than 7.0%, of bleomycin B 4 not more than 1.0%, and of demethylbleomycin
A2 not more than 3.0%.
Identity tests
A. Dissolve about 5 mg in 10 ml of water, add 5 pi of copper(II) sulfate (160g/l)
TS, and dilute with water to 100ml; the absorption spectrum exhibits
maxima at about 242 nm and 290 nm, and a minimum at about 268 nm.
B. A lOmg/ml solution yields reaction A described under 2.1 General identifi-


Copper content. Transfer 75 mg, accurately weighed, to a 60-ml separating

funnel and dissolve in 10ml of hydrochloric acid (0.1 mol/1) VS. Transfer 10ml
of copper standard TS2 to an additional separating funnel. To both funnels add
10 ml of zinc bis(dibenzyldithiocarbamate) TS and shake vigorously for 1
minute. Allow the layers to separate. Filter the lower layer through 1 g of anhy-
drous sodium sulfate R to remove excess water. Measure the absorbances of a
179
1-cm layer at the maximum at about 435 nm, using a solvent cell containing
carbon tetrachloride R.
Calculate the content of copper in mg/g from the formula {Ac x \5)l(As x W)
where Ac is the absorbance of the substance to be examined, A, is the
absorbance of copper standard TS2, and W

is the weight in mg of the substance
to be examined; the copper content is not more than 0.2 mg/g.
Assay
A. Microbiological assay. Carry out the assay as described under 3.1 Micro-
biological assay of antibiotics, using Mycobacterium smegmatis (ATCC 607) as
the test organism. Prepare the inoculum as follows: the test organism is
grown for40-48 hours at a temperature of 27 °C on the surface of culture

medium Cm8. Using 3 ml of saline TS wash the growth into a flask con-
taining 100 ml of culture medium Cm9 and 50 g of glass beads, and incu-
bate at 25-27 °C for 5 days with constant mechanical agitation using an
orbital shaker. The resulting suspension should be used for no longer than
14 days, and kept at a temperature below 5°C. For the preparation of inoc-
ulated plates use 0.5ml of the suspension or a suitable volume previously
determined using test plates with culture medium Cm8 at a temperature of
27 °C. Prepare the reference solution in phosphate buffer, pH 7.0, TS, dilut-
ing the International Reference Preparation of bleomycin A 2 /B 2 to an appro-
priate concentration (usually between 10 and 200 pg per ml). The precision
of the assay is such that the fiducial limits of error of the estimated potency
(P = 0.95) are not less than 95% and not more than 105% of the estimated
potency. The upper fiducial limit of error of the estimated potency (P = 0.95)
is not less than 1500 lU and the lower fiducial limit is not more than
2000 lU of bleomycin A 2 /B 2 per mg, calculated with reference to the dried

substance.
B. Content of the bleomycin components. Carry out the test as described under
1.14.4 High performance liquid chromatography, using a column 25 cm long
and 4.6mm in internal diameter packed with particles of silica gel, 5-10 pm
in diameter, the surface of which has been modified with chemically bonded
octadecylsilyl groups. As the mobile phase for a linear gradient develop-
ment, start with a mixture of 9 volumes of 1-pentanesulfonic acid TS and
1 volume of methanol R, both previously filtered and deaerated, and end
with a composition of 6 volumes of 1-pentanesulfonic acid TS and 4

volumes of methanol R, using a suitable linear rate of change of mobile
phase so as to reach the final composition in 60 minutes. (If needed, add the
following to the mobile phase to obtain satisfactory chromatography:
1.86g of disodium edetate R per litre.) As detector use an ultraviolet spec-
trophotometer at a wavelength of about 254 nm, fitted with a low- volume
flow cell (8-20 pi is suitable). Inject 5 pi of a solution of the test substance
in water containing the equivalent of 5 lU of bleomycin per ml. Proceed
180
with the gradient elution, pumping the mobile phase mixture at the condi-
tion mentioned above for about 80 minutes or until the demethylbleomycin
A2 is eluted.
The elution order of the bleomycin components is the following: void volume,
bleomycin acid, bleomycin A2, bleomycin B2, bleomycin A5, bleomycin B4, and
demethylbleomycin A2.
Calculate in % the content of each bleomycin component, comparing the ratios

of the individual areas of the peaks with that of the total area of all the
bleomycins.
Additional requirements for Bleomycin sulfate for sterile use

Storage. Sterile Bleomycin sulfate should be kept in a hermetically closed
container.

for histamine-like substances (vasodepressor substances) using 1 ml per kg
of body mass of a solution in saline TS containing a quantity equivalent to

500 lU per ml.
bleomycin.

BUPIVACAINI HYDRO CHLORIDUM

BUPIVACAINE HYDROCHLORIDE
Molecular formula. Ci8H2sN20,HCl,H20
181
Graphic formula.
CH3(CH,>3CH, 0
CNH
H3C
Chemical name. 1 -Butyl-2', 6'-pipecoloxylidide monohydrochloride monohy-

drate; l-butyl-Ai-(2,6-dimethylphenyl)-2-piperidinecarboxamide monohy-
drochloride monohydrate; CAS Reg. No. 73360-54-0.
Solubility. Soluble in 25 parts of water and in 8 parts of ethanol (-750 g/1) TS;
slightly soluble in ether R.
Storage. Bupivacaine hydrochloride should be kept in a well-closed container.
Requirements
Definition. Bupivacaine hydrochloride contains not less than 98.5% and not
more than 101.0% of C] 8H 28 N 20 ,HC 1
,
substance.
Identity tests
infrared region.
spectrum obtained from bupivacaine hydrochloride RS or with the reference
spectrum of bupivacaine hydrochloride.
B. Dissolve 0.15g in 10ml of water and add 20ml of trinitrophenol (7 g/1) TS.
Heat the mixture to boiling, allow to cool and, if necessary, scrape the inner
surface of the beaker to induce crystallization; wash the precipitate rapidly
with a small quantity of water, followed by successive quantities of
methanol R and ether R, using 2 ml each time; melting temperature about
194 °C (bupivacaine picrate).
C. A 2 mg/ml solution yields reaction B, described under 2.1 General identifi-

Copper. To 0.25 g in 10ml of water, add 0.25 ml of disodium edetate

(0.05mol/l) VS, and allow to stand for 2 minutes; add 0.2g of copper-free citric
182
acid R, 1ml of ammonia (~100g/l) TS and 1ml of sodium diethyldithiocarba-

mate (0.8 g/1) TS and extract with 10 ml of carbon tetrachloride R for 2 minutes.
The colour of the extract is not deeper than that of the extract obtained when
10 ml of a mixture of 3 volumes of copper (II) sulfate (80 g/1) TS and 397 volumes
of water are similarly treated.
Iron. Ignite l.Og with Ig of anhydrous sodium carbonate FeR; cool and dis-
solve the residue in 5 ml of hydrochloric acid (-250 g/1) FeTS and 30 ml of water.
Treat the solution as described under 2.2.4 Limit test for iron, using 0.5ml of
iron standard FeTS; the iron content is not more than lOpg/g.
Absorption in the ultraviolet region. The absorption spectrum of a

0.4mg/ml solution in hydrochloric acid (0.01 mol/1) VS, when observed between
230 nm and 350 nm, exhibits 2 maxima at about 263 nm and 271 nm. The
absorbance of a Tcm layer at the maximum wavelength of 263 nm is not less
than 0.53 and not more than 0.58, and at the maximum wavelength of 271 nm
isnot less than 0.43 and not more than 0.48 (preferably use 2-cm cells for the
measurements and calculate the absorbances of 1-cm layers).
chromatography, using silica gel R1 as the coating substance and ethanol
2 solutions in methanol R containing (A) 50 mg of the test substance per ml and
(B) 0.50 mg of the test substance per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in air, spray with potassium iodobis-

muthate TS2, and examine the chromatogram in daylight. Any spot obtained
with solution A, other than the principal spot, is not more intense than that
obtained with solution B.
Assay. Dissolve about 0.65g, accurately weighed, in 30 ml of glacial acetic acid

Rl, add 10 ml of mercuric/acetic acid TS, and titrate with perchloric acid
(0.1 mol/1) VS as described under 2.6 Non-aqueous titration. Method A. Each
ml of perchloric acid (0.1 mol/1) VS is equivalent to 32.49mg of CisH28N20,HCl.
Additional requirements for Bupnvacaine hydrochloride for

p>arenteral use
183
BUSULFANUM
BUSULFAN
Molecular formula. C6H14O6S2
Graphic formula.
HO
II
OH
II
H— C — S — 0-ICH,).— 0-S-C-H
II II
HO OH
Chemical name. 1,4-Butanediol dimethanesulfonate; tetramethylene
dimethanesulfonate; CAS Reg. No. 55-98-1.
Other name. Myelosanum.
Description. A white, crystalline powder.
Solubility. Very slightly soluble in water; sparingly soluble in acetone R;

slightly soluble in ethanol (~750g/l) TS.
Storage. Busulfan should be kept in a well-closed container, protected from light.
Additional information. CAUTION: Busulfan must be handled with care,

Requirements
Definition. Busulfan contains not less than 98.5% and not more than 100.5%
of C 6H 14 O 6S 2 ,
Identity tests
A. Heat 0.1 g with 10 ml of water and 5 ml of sodium hydroxide (1 mol/1) VS until
a clear solution is obtained; an intense odour of methanesulfonic acid is per-
ceptible. Cool the solution and divide it into two equal portions for test B.
184
B. To one portion of the solution prepared in test Aadd 0.05 ml of potassium

permanganate (lOg/1) TS; the purple colour changes to violet, then to blue,
and finally to emerald-green. Acidify the second portion of the solution pre-
pared in test A with 2ml of sulfuric acid (-100 g/1) TS, add 0.05ml of potas-
sium permanganate (10 g/1) TS and shake; the colour of the permanganate
is slowly discharged.
C. To a test-tube transfer O.lOg of the test substance, suspend it in 1.0ml of

copper edetate TS and 0.5ml of ammonia (-260 g/1) TS, then add 0.5ml of
hydrogen peroxide (-60 g/1) TS; this constitutes solution 1 Similarly, prepare
.
a blank without the test substance; this constitutes solution 2. Place both
tubes in a water-bath for 5 minutes, cool and add 1 .0 ml of hydrochloric acid
(-70g/l)TS and 4.0 ml of barium chloride (50g/l)TS; solution 2 remains clear
and an opalescence is produced in solution 1, which changes to a white pre-
cipitate after a few minutes.
Melting range. 115-118°C.

Assay. To about 0.25 g, accurately weighed, add 25 ml of water and boil gently
under reflux for 30 minutes. Wash the condenser with a small quantity of water,
cool, and titrate with carbonate-free sodium hydroxide (0.1 mol/1) VS, using
phenolphthalein/ethanol TS as indicator. Repeat the operation without the
substance being examined and make any necessary corrections. Each ml of
carbonate-free sodium hydroxide (O.lmol/1) VS is equivalent to 12.32 mg of
C6EI14O6S2.
BUTYLHYDROXYANISOLUM
BUTYLATED HYDROXYANIS OLE
OH
C11H16O2
185
Chemical name. rert-Butyl-4-methoxyphenol; (l,l-dimethylethyl)-4-

methoxyphenol; CAS Reg. No, 25013-16-5.
Other name. BHA.
Description. A white or almost white, crystalline powder or a yellowish

white solid; odour, faint and characteristic.
Solubility. Practically insoluble in water; freely soluble in ethanol (-750 g/1)

TS, ether R, propylene glycol R, and arachis oil R; dissolves in solutions of alkali
hydroxides.
Category. Antioxidant.
Storage. Butylated hydroxyanisole should be kept in a well-closed container,

Requirements
Definition. Butylated hydroxyanisole contains a variable amount of 3-tert-
butyl-4-me thoxy phenol.
Identity tests
A. Dissolve 0.1 g in 10ml of ethanol (-750 g/1) TS and add 4ml of sodium
tetraborate (lOg/1) TS and 1ml of 2,6-dichloroquinone chlorimide/ethanol
TS; a blue colour is produced (distinction from butylated hydroxytoluene).
B. Dissolve a few crystals in 10ml of ethanol (~750g/l) TS and add 0.1ml of

potassium ferricyanide (10 g/1) TS and 0.5 ml of ferric ammonium sulfate
TS2; a green to blue colour is produced.
Solution in methanol. A solution of Ig in 10 ml of methanol R is clear and

not more intensely coloured than standard colour solution Yw3 when com-
Hydroquinone. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using silica gel R1 as the coating substance and a mixture of 4
volumes of chloroform R and 1 volume of ethyl acetate R as the mobile phase.

Apply separately to the plate 3 ml of each of two solutions in ether R contain-
ing (A) 50mg of Butylated hydroxyanisole per ml, and (B) O.lOmg of hydro-
quinone R per ml. After removing the plate from the chromatographic chamber.
186
allow it to dry in air for a few minutes, spray with phosphomolybdic

acid/ethanol TS, and while still damp expose it to the vapours of ammonia
(-260 g/1) TS. Examine the chromatogram in daylight as soon as the yellow
background has disappeared.
The spot obtained with solution B is more intense than any corresponding spot
obtained with solution A.
3-ferf-Butyl-4-methoxyphenol. Carry out the test as described under 1.14.1

Thin-layer chromatography, using silica gel R1 as the coating substance and
chloroform R as the mobile phase. Apply separately to the plate 2 pi of each of

three solutions in ether R containing (A) 25 mg of Butylated hydroxyanisole per
ml, (B) 2.5mg of Butylated hydroxyanisole per ml, and (C) 0.125mg of Buty-
lated hydroxyanisole per ml. After removing the plate from the chromato-
graphic chamber, allow itspray it with ferric chloride/potassium
to dry in air,
ferricyanide TS, and examine the chromatogram in daylight.
The blue-violet spot at R^-35 obtained with solution A is not more intense than
the principal spot obtained with solution B. Any other spot obtained with solu-
tion A is not more intense than the spot obtained with solution C.
BUTYLHYDROXYTOLUENUM
BUTYLATED HYDROXYTOLUENE
OH
(H3C)3C •c(ch3:
C15H24O
Chemical name. 2,6-Di-tert-butyl-y-cresol; 2,6-bis(l,l-dimethylethyl)-4-

methyl-phenol; CAS Reg. No. 128-37-0.
Other name. BHT.

powder; odour, faint and characteristic.
187

TS, acetone R, ether R, and arachis oil R.
Category. Antioxidant.
Storage. Butylated hydroxytoluene should be kept in a well-closed container,

Requirements
Identity tests
A. Dissolve 0.1 g in 10ml of ethanol (-750 g/1) TS and add 4ml of sodium
tetraborate (lOg/1) TS and a few crystals of 2,6-dichloroquinone chlorimide
R; no more than a faint blue colour is produced (distinction from butylated
hydroxyanisole).
B. Dissolve lOmg in 2ml of ethanol (-750 g/1) TS. Add 1ml of testosterone
propionate/ethanol TS and 2 ml of sodium hydroxide (-80 g/1) TS. Warm
in a water-bath at80 °C for 10 minutes, and allow to cool; a blue colour is
produced.
Congealing temperature. Not lower than 69.2 °C.
CALAMINUM
CALAMINE
Chemical name. Calamine; CAS Reg. No. 8011-96-9.
Description. A fine, amorphous pink or reddish brown powder; odourless.
Solubility. Practically insoluble in water; soluble with effervescence in mineral

acids.
Category. Antipruritic drug.
Storage. Calamine should be kept in a well-closed container.
Additional information. Attention should be paid to the microbiological

quality since Calamine is of natural origin.
188
Requirements
Definition. Calamine is zinc oxide with a small proportion of ferric oxide.
Calamine contains not less than 98 0 . %

and not more than the equivalent of
100 5 % of ZnO,
. calculated with reference to the ignited substance.
Identity tests
A. Shake 1 g with 10 ml of hydrochloric acid (~70g/l) TS and filter. To 5 ml of
the filtrate add 0.3ml of sodium hydroxide (~80g/l) TS; a white precipitate
is formed. Add a further 2 ml of sodium hydroxide (~80g/l) TS; the precip-
itate dissolves. Add 10ml of ammonium chloride (lOOg/1) TS; the solution
remains clear. Add 0.1 ml of sodium sulfide TS; a white, flocculent precipi-
tate is formed.
B. To 1 g add 10ml of hydrochloric acid (~70g/l) TS, heat to boiling, and filter.
To the filtrate add a few drops of ammonium thiocyanate (75g/l) TS; a
reddish colour is produced.
Calcium or magnesium. Digest 1 g in 25ml of hydrochloric acid (~70g/l) TS

for 30 minutes and filter. To the filtrate, add slowly ammonia (-100 g/1) TS until
the precipitate first formed redissolves, then add an excess of 5 ml of ammonia
(-100 g/1) TS. To 10ml of this solution add 2 ml of ammonium oxalate (25 g/1)
TS; not more than a slight turbidity is produced. To a further 10ml portion add
2 ml of disodium hydrogen phosphate (100 g/1) TS; not more than a slight
turbidity is produced.
Lead. Dissolve 2g in a mixture of 20 ml of water and 5 ml of glacial acetic acid

R, filter, and add 0.1ml of potassium chromate (100 g/1) TS to the filtrate; the
solution remains clear for 5 minutes.
Acid-insoluble substances. Dissolve 2.0g in 50ml of hydrochloric acid

(-70 g/1) TS, and filter. Wash the residue with water and dry to constant mass
at 105 °C; the residue weighs not more than 40 mg (2.0%).
Alkaline substances. Digest 1 g with 20 ml of water and warm on a water-

bath for 15 minutes. Filter and add 2 drops of phenolphthalein/ethanol TS to
the filtrate; if a red colour is produced, titrate with sulfuric acid (0.05 mol/1) VS;
not more than 0.2 ml of acid is required to discharge the colour.
Ethanol-soluble dyes. Shake 1 g with 10 ml of ethanol (-710 g/1) TS and filter;
the filtrate is colourless.
Water-soluble dyes. Shake Ig with 10 ml of water and filter; the filtrate is
colourless.
189
Loss on ignition. Weigh 2.0 g and ignite at 500 °C to constant mass; it loses
Assay. Add to about 1.5g, accurately weighed, 50 ml of sulfuric acid (0.5 mol/1)
VS, heat gently until no further precipitation occurs, and filter. Wash the residue
with hot water until the last washing is neutral to litmus paper R. Combine
the wash liquid and the filtrate, add 2.5 g of ammonium chloride R, cool, and
back-titrate with sodium hydroxide (1 mol/1) VS using methyl orange/ethanol
TS as indicator.
Each ml of sulfuric acid (0.5mol/l) VS is equivalent to 40.69mg of ZnO.
CALCII CARBONAS
CALCIUM CARBONATE
Molecular formula. CaCOs
Chemical name. Calcium carbonate (1 :

1); CAS Reg. No. 471-34-1.
Description. A white, fine, microcrystalline powder; odourless.
Solubility. Practically insoluble in water and ethanol (-750 g/1) TS. It dissolves
with effervescence in acetic acid (-60 g/1) TS, hydrochloric acid (-70g/l) TS, and
nitric acid (-130 g/1) TS.
Category. Antacid.
Storage. Calcium carbonate should be kept in a well-closed container.
Requirements
Definition. Calcium carbonate contains not less than 98.0% and not more
than 100.5% of CaCOg, calculated with reference to the dried substance.
Identity tests
A. Dissolve 20mg in 0.3ml of hydrochloric acid (~70g/l) TS and 2ml of water,
and filter. The filtrate yields the reactions described under 2.1 General iden-
tification tests as characteristic of calcium.
190
B. To O.lOg add 1.0ml of acetic acid (~300g/l) TS; a gas evolves that is colour-
less and odourless. Pass the evolved gas into calcium hydroxide TS; a white
precipitate is produced immediately.
Heavy metals. Dissolve 5g in 80ml of acetic acid (~60g/l) TS; when effer-
vescence ceases, boil the solution for 2 minutes, allow to cool, dilute to 100ml
with acetic acid (-60 g/1) TS and, if through a sintered glass filter
necessary, filter
(retain the filter for the test of substances insoluble in acetic acid). Determine
the heavy metals content in 20 ml of the filtrate (keep the remaining filtrate for
the limit test for barium), as described under 2.2.3 Limit test for heavy metals,
according to Method A; not more than 30pg/g.
Arsenic. Use a solution of 3.3g in 35ml of hydrochloric acid (~70g/l) TS and

proceed as described under 2.2.5 Limit test for arsenic; the arsenic content is
Barium. To 10ml of the filtrate retained from the limit test for heavy metals
add 10 ml of calcium sulfate TS (solution A). Mix a further 10 ml of the filtrate
with 10 ml of water (solution B). After not less than 15 minutes, solution A is
not more opalescent than solution B.
Iron. Dissolve 0.20 gin 10 ml of hydrochloric acid (~70g/l) TS and dilute to 40 ml

with water. Proceed with the 2.2.4 Limit test for iron; not more than 200 pg/g.
Magnesium and alkali metals. Dissolve l.Og in 10ml of hydrochloric acid

(-70 g/1) TS, boil for 2 minutes and add 20 ml of water, 1 g of ammonium chlo-
ride R, and 0.1 ml of methyl red/ethanol TS. Add ammonia (-100 g/1) TS drop
by drop until the solution changes colour, and then add a further 2 ml. Heat to
boiling and add 40 ml of hot ammonium oxalate (50 g/1) TS. Allow to stand for
4 hours, dilute to 100 ml with water and filter. To 50 ml of the filtrate add
0.25ml of sulfuric acid (~100g/l) TS and evaporate to dryness on a water-bath.
Ignite the residue to constant weight at 600 °C; not more than 5 mg.
Substances insoluble in acetic acid. Wash the filter retained from the test
for heavy metals with 4 successive quantities, each of 5 ml of hot water, and
dry at 105 °C for 1 hour; the residue weighs not more than 10 mg.
Loss on drying. Dry to constant weight at 200 °C; it loses not more than 20 mg/g.
Assay. Dissolve about 0.15g, accurately weighed, in a mixture of 3ml of

hydrochloric acid (-70g/l) TS and 20ml of water, boil for 2 minutes, allow to
cool, and dilute to 50 ml with water. Proceed with the titration as described
under 2.5 Complexometric titrations for calcium. Each ml of disodium edetate
(0.05mol/l) VS is equivalent to 5.004 mg of CaCOg.
191
CALCII FOLINAS
CALCIUM FOLINATE
Molecular formula. C 2 oH 2 iCaN 707 5 H 20 ,
Graphic formula.
Chemical name. Calcium N-[/?-[[(2-amino-5-£ormyl-5,6,7,8-tetrahydro-4-

hydroxy-6-pteridinyl)methyl]amino]benzoyl]-L-glutamate (1 :
1) pentahydrate;
calcium N-[4-[[(2-amino-5-formyl-l,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)-
methyl]amino]benzoyl]-L-glutamate (1:1) pentahydrate; CAS Reg. No. 6035-
45-6 (pentahydrate).
Other name. Leucovorin calcium.
Solubility. Very soluble in water; practically insoluble in ethanol (-750 g/1) TS.
Storage. Calcium folinate should be kept in a well-closed container, protected

from light.
Additional information. CAUTION: Calcium folinate must be handled with

care, avoiding contact with the skin and inhalation of airborne particles.
Requirements
Definition. Calcium folinate contains not less than 95.0% and not more
than 105.0% of C 2 oH iCaN 707
2 ,
calculated with reference to the anhydrous
substance.
192
Identity tests
• Either tests A and C or tests B, C and D may be applied.

infrared region.
spectrum obtained from calcium folinate RS or with the reference spectrum of
calcium folinate.
B. Dissolve20mg in 3.0ml of water, add 0.5ml of hydrochloric acid (~70g/l)

TS and 0.5ml of sodium nitrite (lOOg/1) TS. Shake for 2 minutes and
add 1.5ml of 2-naphthol TSl; a yellow-brown precipitate appears and the
solution turns green.
C. Dissolve 20 mg in 2.0ml of water and add 1.0 ml of ammonium oxalate

(25g/l) TS; a white precipitate is produced, which is insoluble in acetic
acid (-300 g/1) TS and ammonia (-260 g/1) TS, but is soluble in hydrochloric
acid (-70 g/1) TS.
D. Dissolve 20 mg in 5 ml of water and add 1.0 ml of silver nitrate (40 g/1) TS;
a white, curdy precipitate is produced. Add a few drops of nitric acid
(-130 g/1) TS; the precipitate dissolves.

content is not less than 0.080g/g and not more than 0.150g/g.
Assay
• Use freshly deionized water throughout the procedure, and perform the
assay in low-actinic glassware or protect the solutions containing calcium
folinate from light. Complete the assay without prolonged interruption.
Carry out the test as described under 1.14.4 High performance liquid
chromatography, using a column 30 cm long and 4 mm
in internal diameter,
packed with particles of porous silica gel or ceramic, 5-10 pm in diameter, the
groups.
As the mobile phase, use a mixture of 15ml of tetrabutylammonium hydrox-

ide/methanol TS with 835 ml of water, add 125 ml of acetonitrile R, adjust the
pH to 7.5 + 0.1 with sodium dihydrogen phosphate (275 g/1) TS, dilute with
water to 1000 ml, and filter. Adjust the concentration of acetonitrile, if
necessary.
Dilute the following solution for use in the preparation of the test solutions:
To 15ml of tetrabutylammonium hydroxide/methanol TS add 900ml of water.
193
adjust the pH to 7.5 ± 0.1 with sodium dihydrogen phosphate (275g/l) TS,
dilutewith water to 1000ml, and mix. Weigh accurately a quantity of calcium
folinate RS, dissolveit in the above solution and dilute with the same solution
to contain about 175 pg per ml (solution A). Dissolve 20 mg of the substance to

be examined in a sufficient volume of the above solution to produce 100 ml,
and mix (solution B). For the system suitability test, dissolve a quantity of folic
acid RS in the above solution and dilute with the same solution to contain
about 175 pg per ml. Mix 1 part of this solution with 4 parts of solution A
(solution C).
Operate at a flow rate of 1-2 ml per minute. As detector use an ultraviolet spec-
trophotometer at a wavelength of about 254 nm, fitted with a suitable recorder.
Make 6 replicate injections, each of 15 pi of solution C. The resolution factor

between calcium folinate and folic acid should be not less than 3.6, with a rel-
ative standard deviation for the calcium folinate peak of not more than 2.0%.
The relative retention times for calcium folinate and folic acid are 1.0 and about
1.6, respectively.
Then inject 15 pi of each of solutions A and B. Measure the peak responses

at the corresponding retention times and calculate the quantity, in %, of
C oH2 iCaN 707
2 ,
using the following formula: 100(0. lC)(r[/rs) in which C is the
concentration in pg per ml of calcium folinate RS in solution A, and Vy and %
are thepeak responses obtained from solutions B and A, respectively.
Additional requirements for Calcium folinate for

parenteral use
CALCII GLUCONAS
CALCIUM GLUCONATE
Molecular formula. (C<sHi] 07) 2 Ca,H 20
194
Graphic formula.
H H OH H
I I I I
HOCH, C C- C c- COO Ca HjO

I I I I
OH OH H OH 2
Chemical name. Calcium D-gluconate (1:2) monohydrate; CAS Reg. No.

299-28-5.
Description. White, crystalline granules or a white, crystalline powder; odourless.
Solubility. Slowly soluble in water; freely soluble in boiling water; practically

insoluble in dehydrated ethanol R and ether R.
Category. Calcium source.
Storage. Calcium gluconate should be kept in a tighdy closed container, pro-

tected from light.
Additional information. Even in the absence of light. Calcium gluconate is

Requirements
Definition. Calcium gluconate contains not less than 98.0% and not more
than 102.0% of (C 6 Hn 07) 2 Ca,H 20 calculated as the monohydrate.
,
Identity tests
A. A 20mg/ml solution yields the reactions described under 2.1 General iden-
B. To 1ml of a 30 mg/ml solution add 1 drop of ferric chloride (25g/l) TS; a

yellow colour is produced.
C. To 5 ml of a warm 0.1 g/ml solution add 0.7 ml of glacial acetic acid Rand 1 ml
of freshly distilled phenylhydrazine R, heat on a water-bath for 30 minutes,
allow to cool, and scrape the inner surface of the tube to induce crystalliza-
tion. Collect the crystals, dissolve in 1 0 ml of hot water, add a small amount of
charcoal R, and filter. Allow the filtrate to cool, and scrape the inner surface of
the tube; a white, crystalline precipitate is produced; melting temperature,
about 200 °C with decomposition (phenylhydrazide of gluconic acid).
195
Chlorides and other halides. Dissolve 0.50 g in a mixture of 2 ml of nitric

g/1) TS and 20 ml of water, and proceed as described under 2.2.1 Limit
acid (-130
Magnesium and alkaline metals. Dissolve l.Og in 100ml of boiling water,

add 10 ml of ammonium chloride (100 g/1) TS, 1ml of ammonia (-260 g/1) TS
and, drop by drop, 50 ml of hot ammonium oxalate (25 g/1) TS. Allow to stand
for 4 hours, dilute to 200ml with water and filter. Evaporate 100ml of the fil-
trate to dryness and ignite; the residue weighs not more than 2.0 mg.
Sulfates. Dissolve 5.0 g in 40 ml of boiling water, cool and filter. Proceed with
the filtrate as described under 2.2.2 Limit test for sulfates; the sulfate content
is not more than 0.1 mg/g.
Glucose and sucrose. Dissolve 0.5 g in 10 ml of hot water, add 2 ml of

hydrochloric acid (~70g/l) TS, and boil for about 2 minutes. Cool, add 15ml of
sodium carbonate (50 g/1) TS, allow to stand for 5 minutes, and filter. Add 5 ml
of the clear filtrate to about 2 ml of potassio-cupric tartrate TS, and boil for
1 minute; neither a red turbidity nor any precipitate is produced.

and not more intensely coloured than standard colour solution Ywl when com-
Assay. Dissolve about 0.5 g, accurately weighed, in 20 ml of hot water

containing 2 ml of hydrochloric acid (-70 g/1) TS, allow to cool and dilute to
100ml with water. Proceed with the titration as described under 2.5 Com-
plexometric titrations for calcium. Each ml of disodium edetate (0.05 mol/1)
VS is equivalent to 22.42mg of (C 6 Hn 07 ) 2 Ca,H 20 .
Additional requirements for Calcium gluconate for

f'arenteral use
terial endotoxins; contains not more than 1 67 lU of endotoxin RS per g.
196
CALCII HYDROGENOPHOSPHAS
CALCIUM HYDROGEN PHOSPHATE
Calcium hydrogen phosphate, anhydrous
Calcium hydrogen phosphate dihydrate
CaHP 04 (anhydrous)
CaHP 04 2 H 20 (dihydrate)
,
ChemiccJ name. Calcium phosphate (1 :

1); CAS Reg. No. 7757-93-9 (anhydrous).
Calcium phosphate (1:1) dihydrate; CAS Reg. No, 7789-77-7 (dihydrate).
Other name. Dibasic calcium phosphate.
Solubility. Practically insoluble in cold water and ethanol (-750 g/1) TS; soluble
in dilute acids.
Category. Tablet and capsule diluent.
Storage. Calcium hydrogen phosphate should be kept in a well-closed container.
Labelling. The designation on the container of Calcium hydrogen phosphate

should state whether it is the dihydrate or the anhydrous form.
Requirements
Calcium hydrogen phosphate contains not less than 30 9 % and not more than.
the equivalent of 31 7 % of calcium, Ca, calculated with reference to the ignited

.
substance.
Identity tests
A. To 0.2g add a mixture of 10ml of hydrochloric acid (~70g/l) TS and 10ml of
water, and heat to dissolve. To 10ml of this solution add 2.5ml of ammonia
(-100 g/1) TS (keep the remaining solution for test B); it yields reaction A
described under 2.1 General identification tests as characteristic of calcium.
B. Acidify the remaining solution from test A with nitric acid (-130g/l) TS; it
yields reaction A described under 2.1 General identification tests as charac-

teristic of orthophosphates.
197
10ml filter if necessary, and add ammonia
of hydrochloric acid (~70g/l) TS,
(~100g/l) TS until a precipitate is formed. Add just sufficient hydrochloric acid
(~70g/l) TS to dissolve the precipitate and determine the heavy metals content
as described under 2.2.3 Limit test for heavy metals, Method A; not more than
40pg/g.
Arsenic. Use a solution of l.Og in 35ml of hydrochloric acid (~70g/l) TS and

Barium. Dissolve 1.25g in 10 ml of hydrochloric acid (~70g/l) TS, filter if nec-

essary, and add ammonia (~100g/l)TS until a precipitate is formed. Add just suf-
ficient hydrochloric acid (~70g/l) TS to dissolve the precipitate and dilute with
water to 25ml. Place a 10-ml portion in each of two separate matched tubes. To
one portion add 0.5ml of sulfuric acid (-100 g/1) TS, and to the other 0.5ml of
water; the solutions remain equally clear when viewed after 15 minutes.
Carbonates. To 1 g add 5ml of carbon-dioxide-free water R and 2ml of

hydrochloric acid (-420 g/1) TS and shake; no effervescence is produced.
Chlorides. Dissolve 0.1 g in a mixture of 2 ml of nitric acid (-130 g/1) TS and

the chloride content is not more than 2.5 mg/g.
Fluorides. Prepare and store all solutions in plastic containers.
Weigh 2.0 g of the test sample into a beaker and add 20ml of water and 2.0ml
of hydrochloric acid (-250 g/1) TS. Using a magnetic stirrer and a plastic-coated
stirring bar, stir until the sample has dissolved. Then add 50 ml of sodium citrate
(250 g/1) TS and dilute to 100 ml with water. Use a fluoride-ion-sensitive elec-
trode and a silver/silver chloride reference electrode system, connected to a
potentiometer capable of indicating reproducibly a minimum of ±0.2 mV. Insert
the previously rinsed and dried electrodes into the solution, stir for 5 minutes,
and read the potential in mV.
Prepare a standard solution of fluoride ion containing 1.1 052 mg sodium fluo-
ride R 50ml of sodium citrate (250 g/1) TS
per ml. To 20 ml of this solution add
and dilute with water to produce 100 ml (100 pg F/ml). For the estab-
sufficient
lishment of a standard curve, place 50 ml of sodium citrate (250 g/1) TS in a
beaker, add 2 ml of hydrochloric acid (-250 g/1) TS, and dilute to 100ml with
water. Stir as described above for 15 minutes, insert the electrodes, and read
the potential in mV. Continue to stir, and at 5-minute intervals add 100 pi,
100 pi, 300 pi, and 500 pi of fluoride ion standard solution (lOOpg F/ml), equiv-
alent to the cumulative fluoride ion concentration of 0.1, 0.2, 0.5, and 1.0 pg/ml.
198
reading the potential 5 minutes after each addition. Plot the logarithms of the
cumulative fluoride ion concentration versus potential.
Determine the concentration of fluoride ion in the solution being examined,

reading off from the standard curve the value of mV correlating with the pg of
F/ml, and divide by the sample mass taken to obtain the content in the sample;
Sulfates. Dissolve O.lOg in 5ml of hydrochloric acid (~70g/l) TS, and proceed
than 5mg/g.
Acid-insoluble substances. To 5g add a mixture of 40ml of water and 10ml

of hydrochloric acid (-420 g/1) TS, heat until no more dissolves, and dilute to
100 ml with water. Filter any residue, wash with hot water until the washing
is free of chlorides, dry the residue at 105 °C for 1 hour, and weigh; not more
than 2mg/g.
Loss on ignition. Ignite 1.0 g to constant mass between 800 and 825 °C. The
anhydrous form loses not less than 66mg/g and not more than 85mg/g. The
dihydrate loses not less than 0.245 g/g and not more than 0.265 g/g.
Assay. To about 0.2 g, accurately weighed, add a mixture of 1 ml of hydrochlo-

ricacid (-420 g/1) TS and 5 ml of water, use gende heat to dissolve, and add
125ml of water. Proceed with the titration as described under 2.5 Complexo-
metric titrations for calcium.
Each ml of disodium edetate (0.05 mol/1) VS is equivalent to 2.004 mg of Ca.
CALCII PHOSPHAS
CALCIUM PHOSPHATE
Chemical name. Calcium phosphate (3 2) mixture with calcium phosphate
:
(1 1); CAS Reg. No. 7758-87-4 [Ca 3 (P 04) 2 ]; CAS Reg. No. 7757-93-9 (CaHP04 ).
:
Other name. Tribasic calcium phosphate.
Description. A white, amorphous powder; odourless or almost odourless.
hydrochloric acid (-70g/l) TS and nitric acid (-130 g/1) TS.
Category. Tablet diluent.
199
Storage. Calcium phosphate should be kept in a well-closed container.
Additional information. At relative humidities between 15% and 65%, the

equilibrium moisture content at 25°C is about 2%, but at relative humidities
above 75% small additional amounts of moisture are absorbed.
Requirements
Definition. Calcium phosphate is a mixture consisting mainly of Ca 3 (P 04)2
together with CaHP 04 .
Calcium phosphate contains not less than 34 0 %

and not more than the equiv-
.
alent of 40 0 % of calcium, Ca, calculated with reference to the ignited

.
substance.
Identity tests
A. Dissolve 0.05 g in 1 ml of hydrochloric acid (~70g/l) TS by gentle warming
and add 4 ml of water and 0.5 g of sodium acetate R. It yields reaction A

described under 2.1 General identification tests as characteristic of calcium.
B. To 0.5 g add 2 ml of nitric acid (~130g/l) TS and heat gently. This solution
10ml of hydrochloric acid (~70g/l) TS. Heat to boiling, cool, dilute to 40ml
with water, and mix. Determine the heavy metals content as described under
2.2.3 Limit test for heavy metals. Method A; not more than 30pg/g.
Arsenic. Use a solution of 3.3g in 35ml of hydrochloric acid (~70g/l) TS, heat
to dissolve and proceed as described under 2.2.5 Limit test for arsenic; the
Barium. Mix with 10ml of water, heat, and add, drop by drop,
0.5 g
hydrochloric acid (-420 g/1) TS until solution is effected. Add an excess of 2
drops of acid, to the filtrate add 1 ml of potassium sulfate (0.1 g/1) TS;
filter, and
no turbidity appears within 15 minutes.
Carbonates. Suspend 5g in 30ml of carbon-dioxide-free water R and add

slowly 10 ml of hydrochloric acid (-70 g/1) TS; not more than a slight efferves-
cence is observed. (Keep the solution for “Acid-insoluble substances’’.)

the chloride content is not more than 1.4mg/g.
200
Fluorides. Prepare and store all solutions in plastic containers.
Weigh 2.0 g of the test sample into a beaker, add 20ml of water and 3ml of
hydrochloric acid (-250 g/1) TS. Using a magnetic stirrer and a plastic-coated
stirring bar, stir until the sample has dissolved. Then add 50 ml of sodium citrate
(250 g/1) TS and dilute to 100 ml with water. Use a fluoride-ion-sensitive elec-
trode and a silver/silver chloride reference electrode system, connected to a
potentiometer capable of indicating reproducibly a minimum of +0.2 mV. Insert
the previously rinsed and dried electrodes into the solution, stir for 5 minutes,
and read the potential in mV.
Prepare a standard solution of fluoride ion containing 1.1052 mg sodium fluo-

ride R per ml. To 20 ml of this solution add 50ml of sodium citrate (250 g/1) TS
and dilute with sufficient produce 100 ml (lOOpg F/ml). For the estab-
water to
lishment of a standard curve, place 50 ml of sodium citrate (250 g/1) TS in a
beaker, add 3 ml of hydrochloric acid (-250 g/1) TS, and dilute to 100ml with
water. Stir as described above for 15 minutes, insert the electrodes, and read
the potential in mV. Continue to stir, and at 5-minute intervals add 100 pi,
100 pi, 300 pi, 500 pi, and 500 pi of fluoride ion standard solution (lOOpg F/ml),
equivalent to the cumulative fluoride ion concentration of 0.1, 0.2, 0.5, 1.0, and
1.5pg/ml, reading the potential 5 minutes after each addition. Plot the loga-
rithms of the cumulative fluoride ion concentration versus potential.
Determine the concentration of fluoride ion in the solution being examined,

reading off from the standard curve the value of mV correlating with the pg of
F/ml, and divide by the sample mass taken to obtain the content in the sample;
Sulfates. Dissolve 0.1 g in 5ml of hydrochloric acid (-70g/l) TS, and proceed
as describedunder 2.2.2 Limit test for sulfates; the sulfate content is not more
than 8mg/g.
Acid-insoluble substances. Filter the solution prepared for “Carbonates”,

wash the residue with water, and dry to constant mass at 105 °C; the residue
weighs not more than 15mg (0.3%).
Loss on ignition. Ignite 1.0 g at 800 °C for 30 minutes; it loses not more than
80mg/g.
Assay. To about 0.15g, accurately weighed, add a mixture of 5 ml of

hydrochloric acid (-420 g/1) TS and 3 ml of water, use gentle heat to dissolve,
and add 125 ml of water. Proceed with the titration as described under 2.5
Complexometric titrations for calcium.
Each ml of disodium edetate (0.05mol/l) VS is equivalent to 2.004 mg of Ca.
201
CALCII STEARAS
CALCIUM STEARATE
{^sC [CH2 ]i 6 C02)2Ca
C3eH7oCa04
Chemical name. Calcium stearate; calcium octadecanoate; CAS Reg. No.

1592-23-0.
Description. A white to yellowish white, fine, bulky powder; odour, slight,
characteristic.
Solubility. Practically insoluble in water, ethanol (-750 g/1) TS, acetone R, and
ether R.
Category. Tablet and capsule lubricant.
Storage. Calcium stearate should be kept in a well-closed container.
Additional information. The degree of lubrication depends on the particular

form and size of the material.
Requirements
Definition. Calcium stearate consists of calcium salts mainly of stearic acid
and palmitic acid in variable proportions.
Calcium stearate contains not less than 9 0 % and not more than the equiva-
.
10 5 % of CaO, calculated with reference to the dried substance.

lent of .
Identity tests
A. Heat Ig with a mixture of 25 ml of water and 5 ml of hydrochloric acid
(-420 g/1) TS; fatty acids are liberated and float as an oil on the surface
of the liquid. The aqueous layer yields the reactions described under 2.1
General identification tests as characteristic of calcium.
B. Mix 25g with 200ml of hot water, add 60ml of sulfuric acid (~100g/l) TS,
and heat the mixture until the separated fatty acids layer is clear. Wash it
with boiling water until free from sulfates, transfer it to a beaker, and warm
on a water-bath until the water separates and the fatty acids are clear. Allow
to cool, pour off the water layer, melt the fatty acids, and filter into a dry
beaker. Dry at 105°C for 20 minutes; congealing temperature, not lower
than 54 °C.
202
Loss on drying. Heat at 105 “C for 2 hours and weigh; repeat the heating using
2-hour increments until a constant mass is obtained; not more than 40mg/g,
Assay. To about 1.2 g, accurately weighed, add 50ml of hydrochloric acid

(0.1 mol/1) V, and heat to boiling for 10 minutes or until the separated fatty acids
layer is clear, adding water if necessary to maintain the original volume. Cool,
filter, and wash the filter and the flask thoroughly with water until the washing
is free from acid when tested with litmus paper R. Neutralize the filtrate with
sodium hydroxide (1 mol/1) VS against litmus paper R and proceed with the
titration as described under 2.5 Complexometric titrations for calcium.
Each ml of disodium edetate (0.05mol/l) VS is equivalent to 2.804 mg of CaO.
CALCII SULFAS
CALCIUM SULFATE
CaS04,2H20
Chemical name. Calcium sulfate (1:1) dihydrate; CAS Reg. No. 10101-41-4.
Description. A white to almost white, fine powder; odourless or almost

odourless.
Solubility. Slightly soluble in water; more soluble in dilute mineral acids; prac-
tically insoluble in most organic solvents.
Storage. Calcium sulfate should be kept in a well-closed container.
Requirements
Calcium sulfate contains not less than 98 0
. % and not more than the equiva-
lent of 101.0% of CaS 04 ,
Identity tests
Dissolve 1 g in 20 ml of a solution prepared by mixing equal volumes of water
and hydrochloric acid (~420g/l) TS. Heat to boiling for 2 minutes, cool, and
filter if necessary. Use this solution for the following tests:
203
A. The solution yields the reactions described under 2.1 General identification
tests as characteristic of calcium.
B. The solution yields the reactions described under 2.1 General identification
Heavy metals. To l.Og add 10ml of water and 20ml of hydrochloric acid
(~70g/l) TS, heat to boiling until dissolved, cool, and adjust the pH as described
metals content according to Method A; not more than 20|tg/g.
Clarity of solution. Dissolve Ig in a mixture of 45ml of water and 5ml of

hydrochloric acid (-420 g/1) TS, heating to 50 °C for 5 minutes; the solution is
clear.
Loss on drying. Dry to constant mass at a temperature not lower than

250°C; it loses not less than 190mg/g and not more than 230mg/g.
pH value. Slurry 20 g with 80 ml of carbon-dioxide-free water R, allow to

setde, and filter, 6. 0-7. 6.
Assay. To about 0.3 g, accurately weighed, add a mixture of 100ml of water

and 6ml of hydrochloric acid (~70g/l) TS, heat to boiling until dissolved, and
allow to cool. Proceed with the titration as described under 2.5 Gomplexo-
metric titrations for calcium.
Each ml of disodium edetate (0.05 mol/1) VS is equivalent to 6.807 mg of CaS 04 .
CAPTOPRILUM
CAPTOPRIL
H CH,
.N^ ,CO,H
C,H, 5 N 03 S
Chemical name. l-[(2S)-3-Mercapto-2-methylpropionyl]-L-proline; l-[(2S)-3-

mercapto-2-methyl-l-oxopropyl]-L-proline; CAS Reg. No. 62571-86-2.
204
=
Solubility. Freely soluble in water, dichloromethane R, and methanol R.
Categoiy. Cardiovascular agent; angiotensin-converting enzyme inhibitor.
Storage. Captopril should be kept in a tightly closed container, protected from

light.
Additional information. Captopril may exist in different polymorphic forms.
Requirements
Captopril contains not less than 98 0 % and not more than 102 0 %
. . of
C9H15NO3S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from captopril RS or with the reference spectrum of
captopril.

test as described
using silica gel R4 and a mixture of 75 volumes of
toluene R, 25 volumes of glacial acetic acid R, and 1 volume of methanol R
as the mobile phase. Apply separately to the plate 2 pi of each of 2 solutions

in dichloromethane R containing (A) 5.0 mg of Captopril per ml, and (B)
5.0 mg of captopril RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in air, and spray with 5,5t- dithiobis-
2-nitrobenzoic acid/methanol TS. Examine the chromatogram in ultraviolet
light (254 nm).

C. Dissolve 25 mg in 2 ml of ethanol (-750 g/1) TS, add a few crystals of sodium

nitrite R and 10 ml of sulfuric acid (-100 g/1) TS, and shake; a red colour is
produced.
Specific optical rotation. Use a lOmg/ml solution in dehydrated ethanol R

and calculate with reference to the dried substance; [a]o -125 ° to -134 °C.
205

formance liquid chromatography, using a stainless steel column (12.5 cm x
4 mm) packed with particles of silica gel, the surface of which has been modi-
fied with chemically bonded octadecylsilyl groups (5 pm). Prepare the follow-
ing solution to be used as the mobile phase: mix 0.05 volumes of phosphoric
acid (-1440 g/1) TS with 50 volumes of methanol R and 50 volumes of water.
Prepare the following solutions in the mobile phase: solution (A) 0.5 mg of Cap-
topril per ml; solution (B) 10 pg of Captopril per ml; and for solution (C) dis-
solve lOpg of Captopril in the mobile phase, add 1 ml of iodine (0.05 mol/1) VS,
and dilute to100 ml with the mobile phase; further dilute 10 ml of this solution
to 100 ml with the mobile phase.
Inject 20 B and adjust the sensitivity of the system so that the

pi of solution
is not less than 40% of the full scale of the recorder.
height of the principal peak
Inject 20 pi of solution C. The
test is not valid unless three peaks are obtained
and the resolution between the last two eluting principal peaks is at least 2.0.
Inject alternately 20 pi each of solutions A and B. Continue the chromatogra-

phy for three times the retention time of the principal peak obtained with
solution A.
solutions A and B, and calculate the content of the related substances as a per-
other than the principal peak, is not greater than half the area of the principal
peak obtained with solution B (1.0%). The sum of the areas of all the peaks,
other than the principal peak, is not greater than the area of the peak obtained
with solution A (2.0%). Disregard any peak with a retention time of less than
1.4 minutes or with an area less than 0.1 times that of the peak obtained with
solution B.
Assay. Dissolve about 0.3g, accurately weighed, in 100ml of water, add 10ml
of sulfuric acid (~190g/l) TS and 1 g of potassium iodide R. Mix and titrate with
206
potassium iodate (0.01 mol/1) VS, using starch TS as indicator. Repeat the oper-
ation without the substance being examined. The difference between the titra-
tions represents the amount of potassium iodate required.
Each ml of potassium iodate (0.01 mol/1) VS is equivalent to 13.04 mg of

C,H, 5 N 03 S.
CARBAMAZEPINUM
CARBAMAZEPINE
Graphic formula.
CONHj
Chemical name. 5H-Dibenz[/’,/]azepine-5-carboxamide; CAS Reg, No.

298-46-4.
Description. A white to yellowish white, crystalline powder; odourless or

almost odourless.
Solubility. Practically insoluble in water and ether R; soluble in ethanol

(-750 g/1) TS.
Category. Antiepileptic drug.
Storage. Carbamazepine should be kept in a tightly closed container.
Requirements
Definition. Carbamazepine contains not less than 98.0% and not more than
102 0
. % of C 15 H 12N 2 O, calculated with reference to the dried substance.
Identity tests
• Either test A or tests B, C and D may be applied.
207

The infrared absorption spectrum obtained from the test
infrared region.
substance without pretreatment is concordant with the spectrum obtained
from carbamazepine RS or with the reference spectrum of carbamazepine.
obtained with solution C corresponds in position, appearance, and intensity
with that obtained with solution D.
C. Expose a small amount of the test substance to ultraviolet light (365 nm); an
intense blue fluorescence is observed.
D. Heat with 2ml of nitric acid (~1000g/l) TS

0.1 g in a water-bath for 3
minutes; an orange-red colour is produced.
5-Omg/g.
Acidity or alkalinity. Stir l.Og with 20ml of carbon-dioxide-free water R for

15 minutes and filter. To 10ml of the filtrate add 0.1ml of phenolphthalein/
ethanol TS and titrate with carbonate-free sodium hydroxide (0.01 mol/1) VS;
not more than 0.5ml is required to obtain a pink colour. Add 0.15ml of methyl
red/ethanol TS and titrate with hydrochloric acid (0.01 mol/1) VS; not more than
1.0 ml is required to obtain a red colour.
86 volumes of toluene R and 14 volumes of methanol R as the mobile phase.
Apply separately to the plate 2 pi of each of 5 solutions in a mixture of equal
volumes of ethanol (-750 g/1) TS and chloroform R containing (A) 0.050 g of the
test substance per ml, (B) 0.050 mg of iminodibenzyl R per ml, (C) 5.0 mg of
the test substance per ml, (D) 5.0 mg of carbamazepine RS per ml, and (E)
5.0 pg of carbamazepine RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in air, spray it with potassium dichro-
mate TS3, and examine the chromatogram in daylight. Any spot obtained with
solution A, other than the principal spot, is not more intense than that obtained
with solution B. Then heat the plate at 140 °C for 15 minutes and examine the
208
chromatogram in ultraviolet light (254 nm). Any additional spot obtained with
solution A is not more intense than that obtained with solution E.
Assay. Dissolve about 0.1 g, accurately weighed, in sufficient ethanol (~750g/l)

TS to produce 100ml. Dilute 10ml of this solution to 100ml with the same
solvent, and again dilute 10ml of this dilution to 100ml with ethanol (~750g/l)
TS. Measure the absorbance of a 1-cm layer of the resulting solution at the
maximum at about 285 nm. Calculate the amount of C 15 H 12N 2 O in the sub-
stance being tested by comparison with carbamazepine RS, similarly and
concurrently examined. In an adequately calibrated spectrophotometer the
absorbance of the reference solution should be 0.49 ± 0.02.
CARBIDOPUM
CARBIDOPA
Molecular formula. CioHi 4N 204 ,H 20
Graphic formula.
Chemical name. (-)-L-a-Hydrazino-3,4-dihydroxy-a-methylhydrocinnamic

acid monohydrate; (S)-a-hydrazino-3,4-dihydroxy-a-methylbenzenepropanoic
acid monohydrate; CAS Reg. No. 38821-49-7 (monohydrate).
Description. A white to creamy white powder; odourless or almost

odourless.
Solubility. Slightly soluble in water; very slightly soluble in ethanol (-750 g/1)
Storage. Carbidopa should be kept in a well-closed container, protected from

light.
209
Requirements
Definition. Carbidopa contains not less than 99.0% and not more than
101.0% of C10H14N2O4, calculated with reference to the anhydrous substance.
Identity tests
infrared region.
spectrum obtained from carbidopa RS or with the reference spectrum of
carbidopa.
B. To 5 mg add 1ml of water, 1ml of pyridine R, and 5 mg of 4-nitrobenzoyl

chloride R, mix and allow to stand for 3 minutes; the solution remains
colourless, but after boiling changes to a pale yellow colour. While shaking,
add 0.1ml of sodium carbonate (200 g/1) TS; an orange colour is produced.
Specific optical rotation. Use a 10 mg/ml solution in aluminium chloride TS

and calculate with reference to the anhydrous substance; [a]o = -22.5 to -26.5 °.

Methyldopa and 3-O-Methylcarbidopa. Carry out the test as described

under 1.14.4 High performance liquid chromatography, using a stainless steel
column 20 cm long and 4 mm
in internal diameter packed with particles of silica
gel, 10 pm in diameter, the surface of which has been modified with chemically
bonded octylsilyl groups. As the mobile phase, use a mixture of 98 volumes of

potassium dihydrogen phosphate (13.6g/l) TS and 2 volumes of methanol R at
a flow rate of 1.5ml per minute. As detector use an ultraviolet spectropho-
tometer at a wavelength of about 282 nm, fitted with a low-volume flow cell
(10 pi is suitable).
Prepare the following solutions in hydrochloric acid (0.1 mol/1) VS containing

(A) 0.050 mg of methyldopa RS, 0.050 mg of (-)-3-(4-hydroxy-3-
methoxyphenyl)-2-hydrazino-2-methylalanine RS and 0.10 mg of (-)-3-(4-
hydroxy-3-methoxyphenyl)-2-methylalanine RS per ml, the last serving as an

internal standard, (B) 10 mg of the test substance per ml, and (C) 10 mg of the
test substance and 0.10 mg of the internal standard per ml.
210
In the chromatogram obtained with solution A the peaks, excluding the solvent
peak, are due to {a) methyldopa, ib) (-)-3-(4-hydroxy-3-methoxyphenyl)-2-
methylalanine and (c) (-)-3-(4-hydroxy-3-methoxyphenyl)-2-hydrazino-2-
methylalanine in order of their emergence. The ratios of the areas of the peaks
{a) and (c) to the area of the peak due to the internal standard are greater than
the corresponding ratios in the chromatogram obtained with solution C.
Assay. Dissolve about 0.3 g, accurately weighed, in 25.0 ml of perchloric acid

(0.1 mol/1) VS with the aid of a minimum of heat Titrate the excess perchloric
acid with sodium acetate/glacial acetic acid (0.1 mol/1) VS, determining the end-
point potentiometrically as described under 2.6 Non-aqueous titration. Method
A. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 22.62 mg of
C10H14N2O4.
CARBO ACTIVATUS
CHARCOAL, ACTIVATED
Description. Fine, black powder, free from grittiness; odourless.
Solubility. Practically insoluble in water and in all usual solvents.
Category. General-purpose antidote; pharmaceutic aid.
Storage. Activated Charcoal should be kept in a well-closed container.
Additional information. Activated Charcoal is a tasteless powder.
Requirements
Identity test. Heat a small quantity of the test substance to redness; it burns
slowly without a flame.
Heavy metals. Boil Ig with a mixture of 20ml of hydrochloric acid (~70g/l)

TS and 5 ml of bromine TSl for 5 minutes, filter, and wash with 50 ml of boiling
water. Evaporate the combined filtrates to dryness on a water-bath and add to
the residue 1ml of hydrochloric acid (1 mol/1) VS, 20ml of water, and 5ml of
sulfurous acid TS. Boil the solution until all the sulfur dioxide has been expelled,
filter if necessary, and dilute with water to 50 ml. Use 10 ml as the test solution
and determine the content of heavy metals as described under 2.2.3 Limit test
for heavy metals, according to Method A; not more than lOOpg/g.
Cyanides. In a distillation apparatus, heat 5g carefuUy with 50 ml of water

and 2g of tartaric acid R. Collect about 25ml of distillate in a mixture of 10ml
211
of water and 2 ml of sodium hydroxide (1 mol/1) VS and dilute to 50 ml with

water. To 25ml add 0.05 g of ferrous sulfate R and heat until boiling starts. Cool
in a water-bath at 70°C and acidify with 10ml of hydrochloric acid (~250g/l)
TS; no green or blue colour develops.
Sulfides. To 1 g in a small conical flask, add 20ml of water and 5ml of

hydrochloric acid (-250 g/1) TS; the escaping vapours do not darken a strip of
filter paper moistened with lead acetate (80 g/1) TS.
Zinc. To 1 g add 25ml of nitric acid (~130g/l) TS and heat to boiling for 5
minutes; filter through sintered glass and wash with 10ml of hot water. Deter-
mine the content of zinc either by method (A) or by atomic absorp-
a dithizone
tion spectrophotometry (B):
A. To 10 ml of the clear solution obtained as described above add successively

3.0 ml of water, 3.0ml of sodium acetate (60 g/1) TS, 5.0ml of
cyanide/oxalate/thiosulfate TS, and 5.0ml of a freshly prepared 30mg/ml
solution of dithizone R in carbon tetrachloride R, Mix thoroughly for 2-3
minutes. Separate the dithizone-layer and place in a suitable comparison
tube. To 0.5ml of zinc standard (20pg/ml Zn) TS add 9.5ml of water and
treat it in the same manner as above. The solution of the test substance
shows by reflection a more intense violet colour and, by transmitted light,
a not more intense violet colour than the reference solution.
B. Dilute appropriately the solution obtained as described above and proceed

as described under 1.8 Atomic spectrometry: emission and absorption.
Fluorescent substances. In an apparatus for intermittent extraction, treat

lOg with 100ml of cyclohexane R1 for 2 hours. Collect the cyclohexane extract,
adjust the volume to 100 ml, and examine in ultraviolet light (365 nm). The flu-
orescence of the solution is not more intense than that of a solution contain-
ing 0.083 mg of quinine R in 1000ml of sulfuric acid (0.005mol/l) VS.
Ethanol-soluble substances. In a flask fitted with a reflux condenser, heat

2g with 50 ml of ethanol (-750 g/1) TS. Boil for 10 minutes, filter immediately,
cool, and readjust the volume to 50ml with ethanol (-750 g/1) TS; the filtrate is
not more intensely coloured than reference solution Ywl. Evaporate 40ml of
the filtrate, dry the residue at 105°C, and weigh; not more than 8mg (5mg/g).
Acid-soluble substances. Boil 1 g with a mixture of 20 ml of water and 5 ml

of hydrochloric acid (-420 g/1) TS for 5 minutes, filter into a tared porcelain cru-
cible, and wash the residue with 10 ml of hot water, adding the washings to
the filtrate. To the combined filtrate and washings add 1 ml of sulfuric acid
(-1760 g/1) TS, evaporate to dryness, and ignite to constant weight; not more
than 35mg/g.
212
Alkali-soluble coloured matter. Heat 0.25 g with 10 ml of sodium hydrox-

ide (~80g/l) TS for 1 minute, cool and filter. Dilute the filtrate to 10ml with
water; the colour is not more intense than reference solution Gn2.
Acidity or alkalinity. To 2g add 40 ml of water and heat to boiling for 5

minutes. Cool, restore to the original volume with freshly boiled and cooled
water and filter. Reject the first 20ml of filtrate. The filtrate does not induce any
colour change in red or blue litmus paper R. To 10 ml of the filtrate add 0.25ml
of bromothymol blue/ethanol TS and 0.25ml of sodium hydroxide (0.02 mol/1)
VS; the solution is blue. Add 0.75ml of hydrochloric acid (0.02mol/l) VS; the
solution turns yeUow.
Adsorbing power
A. Place 1 g, previously dried at 120°C for 4 hours, in a solution of lOOmg of
strychnine sulfate R in 50 ml of water and shake for 5 minutes; filter, reject-
ing the first 10 ml of filtrate. To a 10-ml portion of the filtrate add 1 drop of
hydrochloric acid (-420 g/1) TS and 5 drops of potassio-mercuric iodide TS;
no turbidity is produced.
B. To each two glass-stoppered 100-ml flasks transfer 50 ml of methylth-

of
ioninium chloride (lg/1) TS. To one of the flasks add 0.25g, accurately
weighed, of the test substance, insert the stopper in the flask and shake for
5 minutes. Filter the contents of each flask, rejecting the first 20ml of each
filtrate. Transfer 25-ml portions of the filtrates to two 250-ml volumetric

flasks. Add to each flask 50 ml of sodium acetate
g/1) TS, mix, and add (60
from a burette 35.0 ml of iodine (0.05 mol/1) VS, swirling the mixture during
the addition. Stopper the flasks and allow them to stand for 50 minutes,
shaking them vigorously at 10-minute intervals. Dilute each mixture with
water to volume, mix, allow to stand for 10 minutes, and filter, rejecting the
first 30ml of each filtrate. Titrate the excess iodine in a 100-ml aliquot of
each filtrate with sodium thiosulfate (0.1 mol/1) VS, adding 3ml of starch
TS towards the end of the titration. Calculate the number of ml of iodine
(0.05 mol/1) VS consumed in each titration; the difference between the two
volumes is not less than 0.7ml.
213
CARBOMERUM
CARBOMER
Chemical name. Acrylic acid polymer with sucrose polyalkyl ether; car-
bomer; CAS Reg. No. 9007-20-9.
Description. A white, fluffy powder; odour, slight, characteristic.
Solubility. After neutralization with alkali hydroxides or amines, soluble in

water, ethanol (~750g/l) TS, and glycerol R.
Category. Suspending agent.
Storage. Carbomer should be kept in a tightly closed container.
Additional information. Carbomer is very hygroscopic.
Requirements
Definition. Carbomer is a synthetic high molecular mass polymer of allyl acid
copolymerized with polysucrose.
Carbomer contains not less than 56 0 % and not more than the equivalent of
.
—
68 0 % of carboxylic acid groups ( COOH), calculated with reference to the
.
dried substance.
Identity tests
A. Disperse 0.5g in 50ml of water. To 10ml add a few drops of thymol
blue/ethanol TS; the colour of the dispersion is orange. To a further 10ml
add a few drops of cresol red/ethanol TS; the colour is yellow. (Keep the
dispersion for test B.)
B. Adjust the pH of the dispersion from test A to about 7.5 with sodium
hydroxide (1 mol/1) VS; a very viscous gel is produced.
Yield value. Prepare a gel as follows: Carefully add 2.5 g to 500ml of water
containing 0.25g of sodium chloride R in a 1000-ml beaker, stirring continu-
ously at 990-1010 revolutions per minute, the stirrer shaft set to one side of
the beaker and near to the bottom at an angle of 60 ° from the vertical. Add
Carbomer being examined slowly at a uniform rate over 45-90 seconds, ensur-
ing that any loose aggregates of powder are broken up. Continue to stir for 15
minutes, remove the stirrer, and allow the beaker containing the dispersion to
stand in a water-bath at a temperature of 24.8-25.2 °C for 30 minutes. Insert
the stirrer to a depth such that air is not drawn into the dispersion and, while
214
stirring at 290-310 revolutions per minute, add 0.2ml of phenolphthalein/

ethanol TS and 1.5 ml of bromothymol blue/ethanol TS. Add rapidly below the
surface 5 ml of sodium hydroxide (-200 g/1) TS and stir for 2-3 minutes until
neutralization is reached, indicated by a uniform blue colour. Adjust the pH to
7. 3-7. 8 potentiometrically, using glass and calomel electrodes, either adding
more sodium hydroxide (-200 g/1) TS or preparing a new mucilage using less
sodium hydroxide for the neutralization. Return the neutralized mucilage to
the water-bath maintained at 25 °C for 1 hour.
The apparatus consists of two clear soda-glass plates, 100 mm x 100 mm x

3 mm. Rub together by hand fine carborundum paste using two opposing faces
of the plates to obtain an even and matt surface. With a diamond marker
engrave the plates to show centre and corner alignments and four sample loca-
tion points equidistant from the plate centre and the four corners.
Place the plates in a water-bath at 24.8-25.2 °C to settle, and dry them rapidly
before use. Apply 0.1 g of the mucilage to the matt surface of one of the plates
at each location for the sample. Align the second plate and lower it carefully,
matt side downwards, onto the lower plate. Top the apparatus with a weight
so that the combined mass applied equals lOOg. Allow the assembly to stand
for 10 minutes and measure the diameters of the 4 zones of each sample using
a strip of paper calibrated in mm; the mean diameter of the zones does not
exceed 2. 0-2. 2 cm.
Loss on drying. Dry at 80 °C for 1 hour; it loses not more than 20mg/g.
Assay. Slowly add 0.4g, accurately weighed and previously dried at 80 °C for
1 hour, to 400 ml of water while mixing with a magnetic stirrer until completely
dissolved. At reduced stirring speed, titrate potentiometrically, using glass and

calomel electrodes, with sodium hydroxide (0.2 mol/1) VS. After each addition
of sodium hydroxide and before recording the pH of the solution, allow to stir
for 1 minute.

carboxylic acid groups ( — COOH).
CARMELLOSUM NATRICUM
CARMELLOSE SODIUM
Chemical name. Cellulose carboxymethyl ether, sodium salt; CAS Reg. No.
9004-32-4.
215
Other name. Carboxymethylcellulose sodium.
Description. A white to faintly yellowish powder or granules; odourless.
Solubility. Easily dispersed in water giving a colloidal solution; practically

insoluble in acetone R, ethanol (-750 g/1) TS, ether R, and toluene R.
Category. Suspending agent; tablet binder and disintegrant; viscosity-

increasing agent.
Storage. Carmellose sodium should be kept in a tightly closed container.
Labelling. The designation on the container of Carmellose sodium should

state its viscosity.
Additional information. Carmellose sodium is hygroscopic after drying. This

substance is not necessarily suitable for the manufacture of parenteral preparations.
Requirements
Definition. Carmellose sodium is the sodium salt of a partially substituted
polycarboxymethyl ether of cellulose.
Carmellose sodium contains not less than 6 5 . % and not more than the equiv-
alent of 10 8 % of Na, calculated with reference
. to the dried substance.
Identity tests
A. Sprinkle 1.0 g of powdered Carmellose sodium onto 90 ml of carbon-
dioxide-free water R at 40-50 °C, stir vigorously until a colloidal solution is
produced, cool, and dilute to 100 ml with carbon-dioxide-free water R.
Transfer 0.5 ml to a test-tube (keep the remaining solution for “Chlorides”,
“Clarity and colour of solution”, and “pH value”), add 1 ml of water and 5
drops of 1-naphthol TSl, mix, and carefully introduce down the side of the
tube 2 ml of sulfuric acid (~1760g/l) TS to form a lower layer; a red-violet
colour develops at the interface.
B. To the sulfated ash, add 1ml of hydrochloric acid (-420 g/1) TS, evaporate
to dryness on a water-bath, and dissolve the residue in 20ml of water. Use
5 ml, keeping the remaining solution for “Heavy metals”; it yields reaction
B described under 2.1 General identification tests as characteristic of sodium.
Heavy metals. Use 12 ml of the solution remaining from identity test B and
heavy metals. Method A; not more than 40|t.g/g.
216
Chlorides. Use 10 ml of the solution prepared for identity test A and proceed
as described under 2.2.1 Limit test for chlorides; the chloride content is not
more than 2.5mg/g.
Clarity and colour of solution. The solution prepared in identity test A is

not more opalescent than opalescence standard TS3 and not more intensely
coloured than standard colour solution Yw2 when compared as described
under 1.11 Colour of liquids.
Sulfated ash. Use 1.0 g and a mixture of equal volumes of sulfuric acid
(-1760 g/1) TS and water. Calculate the result with reference to the dried sub-
stance; 0.200 g/g - 0.333 g/g corresponding to a content of Na equivalent to 6.5-
10.8%. (Keep the residue for identity test B.)
Loss on drying. Dry to constant mass at 105 °C; itloses notmore than lOOmg/g.
pH value. pH of the solution prepared for identity test A, 6. 0-8. 5.
CELLACEFATUM
CELLACEFATE
Chemical name. Cellulose acetate phthalate; cellulose acetate 1,2-
benzenedicarboxylate; CAS Reg. No. 9004-38-0.
Other names. Cellulose acetate phthalate; cellacephate.
Description. A white, free-flowing powder or colourless flakes; odourless or

with a faint odour of acetic acid.
Solubility. Practically insoluble in water and ethanol (~750g/l) TS; freely

soluble in acetone R; soluble in dioxan R; dissolves in dilute solutions of alkali.
Category. Enteric coating agent for solid oral dosage forms.
Storage. Cellacefate should be kept in a well-closed container, and stored in

a cool and dry place.
Additional information. Cellacefate is hygroscopic.
Requirements
Definition. Cellacefate is a cellulose, some of the hydroxyl groups of which
are esterified by phthaloyl groups and others by acetyl groups.
217
30 0 % and not more than the equivalent of

Cellacefate contains not less than .
40 0 % of phthaloyl groups (CaHsOj, relative molecular mass = 149.1) and not

.
lessthan 17 0 % and not more than the equivalent of 26 0 % of acetyl groups

. .
(C2H3O, relative molecular mass = 43.05), both calculated with reference to the
Identity tests
A. To lOmg add 1.0ml of ethanol (~750g/l) TS and 1ml of sulfuric acid
(~1760g/l) TS, and warm; ethyl acetate, perceptible by its odour {proceed
B. Transfer lOmg to a small test-tube, add lOmg of resorcinol R and 0.5ml of

sulfuric acid (~1760g/l) TS, and mix. Heat in a liquid bath at 160 °C for 3
minutes. Cool and pour the solution into a mixture of 25 ml of sodium
hydroxide (1 mol/1) VS and 200ml of water; a vivid green fluorescence is
observed in the solution.
C. Dissolve 0.1 g in 1 ml of acetone R and pour onto a clear glass plate; as the
solvent evaporates, a glossy, clear film remains.
Free acid. Shake l.Og of finely powdered material with 100ml of carbon-
dioxide-free water R for 5 minutes and filter. Wash the flask and the filter with
two quantities, each of 10 ml, of carbon-dioxide-free water R. Combine the fil-
trate and washings, add 0.1 ml of phenolphthalein/ethanol TS, and titrate with
carbonate-free sodium hydroxide (0.1 mol/1) VS until a faint pink colour is
obtained. Repeat the procedure without the Cellacefate being examined and
make any necessary corrections.
Each ml of carbonate-free sodium hydroxide (0.1 mol/1) VS is equivalent to

8.306mg of phthalic acid. Not more than 60mg/g (6.0%) is found, calculated
as phthalic acid and with reference to the anhydrous substance.

Fischer method. Method A, using 0.5 g and 20 ml of a mixture of equal volumes
of dehydrated methanol R and chloroform R; the water content is not more
than 50mg/g (5.0%).
Assays
A. Phthaloyl groups. Dissolve about 0.4 g, accurately weighed, in 20 ml of
ethylene glycol monomethyl ether R previously neutralized using 0.1ml of
phenolphthalein/ethanol TS. Titrate with carbonate-free sodium hydroxide
(0.1 mol/1) VS until a faint pink colour is obtained.
218
Calculate the content of phthaloyl groups in %: —

(100 - a)m
— -1.795h
where ti is the number of ml of carbonate-free sodium hydroxide (0.1 mol/1) VS
used, a is the content of water in %, m is the mass of Cellacefate in g, and S
is the content of free acid in %.
B. Acetyl groups. To about 0.1 g, accurately weighed, add 25ml of carbonate-

free sodium hydroxide (0.1 mol/1) VS and heat on a water-bath under a reflux
condenser for 30 minutes. Cool, add 0.1 ml of phenolphthalein/ethanol TS,
and titrate with hydrochloric acid (0.1 mol/1) VS until the colour is dis-
charged. Repeat the procedure without the Cellacefate being examined and
make any necessary corrections.
Calculate the content of acetyl groups in %:

43(n
,
(100 -
-n)
a)nt
— (0.578P-H 0.5185)
where number of ml of hydrochloric acid (0.1 mol/1) VS used for the

«2 is the
blank, «i is the number of ml of hydrochloric acid (0.1 mol/1) VS used for Cel-
lacefate, a is the content of water in %, ni is the mass of Cellacefate in g, P is
the content of phthaloyl groups in %, and S is the content of free acid in %.
CELLULOSUM MICRO CRYSTALLINUM

MICRO CRYSTALLINE CELLULOSE
Chemical name. Cellulose; CAS Reg. No. 9004-34-6.
Description. A white or almost white, fine crystalline or granular powder;

odourless.
Solubility. Practically insoluble in water and most organic solvents; slightly

soluble in dilute solutions of sodium hydroxide.
Category. Tablet and capsule diluent; suspending agent; disintegrant.
Storage. Microcrystalline cellulose should be kept in a well-closed container.
Additional information. Microcrystalline cellulose is usually defined by its
particle size which ranges between 20 and 150 pm.
219
Requirements
Definition. Microciystalline cellulose is partially depolymerized cellulose pre-
pared from alpha cellulose.
Identity tests
A. Place 20 g on an air-jet sieve with a screen having a nominal aperture of
38 pm and shake for 5 minutes. If more than Ig is retained on the screen,
mix 30 g with 270 ml of water; otherwise, mix 45 g with 255 ml of water.
Perform the mixing in a high-speed blender (18000rev/min) for 5 minutes.
Transfer 100 ml of the mixture to a 100-ml graduated cylinder and allow to
stand for 3 hours; a white, opaque, bubble-free dispersion is obtained
without any supernatant liquid.
B. Dissolve 0.05g in 10ml of copper tetramine hydroxide TS; it dissolves com-

pletely without any residue. Add 5 ml of ethanol (-750 g/1) TS; a precipitate
is produced.
Heavy metals. To l.Og add 4ml of magnesium sulfate/sulfuric acid TS, mix,
and heat cautiously to dryness on a water-bath. Progressively heat to ignition,
not exceeding a temperature of 800 °C, and continue to heat until a white to
greyish residue is obtained. Moisten the residue with 1 drop of hydrochloric
acid (-250 g/1) TS and continue as described under 2.2.3 Limit test for heavy
metals. Procedure 3; determine the heavy metals content according to Method
A; not more than lOpg/g.
Water-soluble substances. Shake 5g with 80ml of water for 10 minutes.

Filter into a tared dish, evaporate to dryness on a water-bath, dry at 105°C for
1 hour, and weigh; the residue weighs not more than 2.0mg/g.
pH value. Shake 2 g with 100 ml of carbon-dioxide-free water R for 5 minutes;

pH of the supernatant liquid, 5. 0-7. 5.
Organic impurities. Place 10 mg on a watch-glass and add 0.05 ml of a freshly

prepared solution of 0.1 g of phloroglucinol R in 5 ml of hydrochloric acid
(-420 g/1) TS; no red colour appears.
Starch and dextrins. Shake 0.1 g with 5 ml of water and add 0.2 ml of iodine
(0.05 mol/1) VS; no blue or red-brown colour develops.
220
CERA CARNAUBA
CARNAUBA WAX
Chemical name. Carnauba wax; CAS Reg. No. 8015-86-9.
Description. Pale yellow to light brown, moderately coarse powder, flakes,

or irregular lumps of hard, brittle wax; odour, characteristic and free from
rancidity.
Solubility. Practically insoluble in water; soluble in toluene R; slightly soluble

in boiling ethanol (-750 g/1) TS.
Category. Polishing agent for coated tablets; viscosity-increasing agent for

ointments; release-rate modifier for oral formulations.
Storage. Carnauba wax should be kept in a well-closed container.
Requirements
Definition. Carnauba wax is obtained from the leaves of Copernicia cerifera
Mart (Pam. Palmae).
Ash. Weigh 2.0 g and use an open porcelain or platinum dish. Heat over a
flame; it volatilizes without emitting an acrid odour. Ignite; the residue weighs
not more than 2.5mg/g.
Acid value. Use 3g; not more than 8.
Saponification value. Use about 3 g, accurately weighed, add 25 ml of xylene

R, and dissolve by warming. To this solution add 50 ml of ethanol (-750 g/1) TS
and proceed with the determination of saponification. Attach a reflux con-
denser and heat for 2 hours; 75-95.
Iodine value. 5-14.
CERA CETYLA
CETYL ESTERS WAX
Chemical name. Ci 4_i 8 Fatty acids Ci 4 _ia alkyl esters; CAS Reg. No. 85566-
24-1.
221
Other name. Synthetic spermaceti.
Description. White to almost white, somewhat translucent flakes (5 pm to

several millimetres in the largest dimension), with a crystalline structure and a
pearly lustre when caked; odour, faint, mild, aromatic.
ether R; slightly soluble in hexane R.
Category. Stiffening agent.
Storage. Cetyl esters wax should be kept in a well-closed container, protected

from heat.
Additional information. Cetyl esters wax has a mass density of about

0.83 g/ml at 50 °C.
Requirements
Definition. Cetyl esters wax is a mixture consisting primarily of esters of sat-
urated fatty alcohols (C ]4 to C]s) and saturated fatty acids (C 14 to C,s).
Saponification value. 109-120.
Paraffin. To Ig add 50 ml of boiling ethanol (-750 g/1) TS; the wax is com-
pletely dissolved.
CETOMACROGOLUM 1000
CETOMACROGOL 1000
H3C [CHjlis-j-O CH2 CH2
(C2H40)«C,,H340
Chemical name. Polyethylene glycol monohexadecyl ether; a-hexadecyl-co-

hydroxypoly(oxy-l,2-ethanediyl); CAS Reg. No. 9004-95-9.
222
Description. A cream-coloured, waxy, unctuous mass, pellets, or flakes;

when heated, it melts to a brownish yellow, clear liquid; odourless or almost
odourless.
Solubility. Soluble in water, ethanol (~750g/l) TS, and acetone R; practically

insoluble in light petroleum R.
Category. Nonionic surfactant.
Storage. Cetomacrogol 1000 should be kept in a well-closed container, pro-

tected from heat
Requirements
Definition. Cetomacrogol 1000 is a condensation product of linear fatty alco-
hols with ethylene oxide, prepared under controlled conditions in order to
obtain the required ether with the polyethylene glycol of the desired molecu-
lar mass.
Identity tests
A. Dissolve 0.1 g in 5ml of water and add 10ml of hydrochloric acid (~70g/l)
TS, 10ml of barium chloride (50g/l) TS, and 10ml of phosphomolybdic acid
(80g/l) TS; a greenish yellow precipitate is produced.
B. Dissolve 0.1 g in 5ml and add gradually tannic acid (50g/l) TS; a
of water
precipitate is formed which dissolves on further addition of tannic acid
solution.
Melting point. Not lower than 38 °C.
Refractive index. At 60°C, no = 1.448 - 1.452.
Alkalinity. Dissolve 2g in 20ml of carbon-dioxide-free water R, add 1 drop of

phenolphthalein/ethanol TS, and titrate with hydrochloric acid (0.1 mol/1) VS;
not more than 0.5 ml is required to obtain a pink colour.
Hydroxyl value. Use 10 g. Method A; 40.0-52.5.
Saponification value. Use lOg; not more than 1.0.

Fischer method, Method A, using 2.5 g; the water content is not more than
lOmg/g.
223
CETRIMIDUM
CETRIMIDE
Chemical name. Trimethyltetradecylammonium bromide mixture with
dodecyltrimethylammonium bromide and hexadecyltrimethylammonium
bromide; cetrimide; CAS Reg. No. 8044-71-1.
Description. A white or almost white, voluminous, free-flowing powder;

odour, slight, characteristic.
Solubility. Freely soluble in water and ethanol (-750 g/1) TS; practically insol-
uble in ether R.
Storage. Cetrimide should be stored in a well-closed container.
Requirements
Definition. Cetrimide is a mixture consisting mainly of tetradecyltrimethy-
lammonium bromide together with smaller amounts of dodecyltrimethylam-
monium bromide and hexadecyltrimethylammonium bromide.
Cetrimide contains notless than 96 0 % and not more than the equivalent of
.
101 0 % of alkyltrimethylammonium bromides, calculated as CuHasBrN (rela-

.
tivemolecular mass = 336.4) and with reference to the dried substance.
Identity tests
A. Dissolve 5 mg in 5 ml of phosphate buffer, pH 8.0, TS. Dip a strip of methyl
green/iodomercurate paper R into the solution. Similarly prepare a blank
solution without the Cetrimide being examined. After 5 minutes withdraw
the strip of paper from the tube; the solution to be tested shows a darker
greenish blue colour than the blank solution.
B. Dissolve 0.2 g in 10ml of carbon-dioxide-free water R and shake; a volumi-

nous froth is produced. (Keep the mixture for test C.)
C. The solution prepared above yields reaction A described under 2.1 General
identification tests as characteristic of bromides.
Amines and amine salts. Dissolve 5 g in 30 ml of a mixture of 1 volume of

hydrochloric acid (1 mol/1) VS and 99 volumes of methanol R and add 100ml
of 2-propanol R. Slowly pass a stream of nitrogen R through the solution. Grad-
ually add 15ml of tetrabutylammonium hydroxide (0.1 mol/1) VS and titrate
224
potentiometrically, recording a titration curve; the volume of titrant added

between the two points of inflexion is not larger than 2.0 ml.
Loss on drying. Dry at 105 °C for 2 hours; it loses not more than 20mg/g.
Acidity or alkalinity. Dissolve 1 g in 50 ml of carbon-dioxide-free water R and

add 0.1ml of bromocresol purple/ethanol TS; not more than 0.1ml of
hydrochloric acid (0.1 mol/1) VS or 0.1 ml of sodium hydroxide (0.1 mol/1) VS is
required to obtain the midpoint of the indicator (grey).
Assay. Dissolve about 2g, accurately weighed, in 100 ml of water. Transfer

25ml to a separating funnel, add 25ml of chloroform R, 10ml of sodium
hydroxide (0.1 mol/1) VS, and 10ml of a freshly prepared solution containing
5 g of potassium iodide R in 100 ml of water. Shake well, allow to separate, and
discard the chloroform layer. Shake the aqueous layer with three quantities,
each of 10ml, of chloroform R, and discard the chloroform layers. Add 40ml
of hydrochloric acid (-420 g/1) TS, allow to cool, and titrate with potassium
iodate (0.05 mol/1) VS until the deep brown colour is almost discharged. Add
2 ml of chloroform R and continue the titration, shaking vigorously, until the
colour of the chloroform layer no longer changes. Repeat the procedure with
a mixture of 10 ml of the above freshly prepared solution of potassium iodide,
20 ml of water, and 40 ml of hydrochloric acid (-420 g/1) TS and make any nec-
essary corrections.
Each ml of potassium iodate (0.05 mol/1) VS is equivalent to 33.64 mg of

Ci/HsaBrH.
CHLORALI HYDRAS
CHLORAL HYDRATE
OH
CI3C OH
C2H3CI3O2
Chemical name. 2,2,2-Trichloroethane-l,l-diol; CAS Reg. No. 302-17-0.
225
Description. Colourless, transparent or white crystals; odour, aromatic,

pungent and characteristic.
Solubility. Very soluble in water; freely soluble in ethanol (-750 g/1) TS and
ether R.
Category. Premedication.
Storage. Chloral hydrate should be kept in a tightly closed container.
Additional information. Melting temperature, about 55 °C; when exposed to

air it slowly volatilizes.
Requirements
Chloral hydrate contains not less than 98 5 . % and not more than 101 0 % of .
C2H3CI3O2.
Note: Prepare the following test solution for use in “Identity tests A and B”, and
for “Clarity and colour”. Dissolve 2.5 g in sufficient carbon-dioxide-free water
R to produce 25 ml.
Identity tests
A. To 1.0ml of the test solution add 2.0ml of sodium sulfide TS; a yellow
colour develops which quickly becomes reddish brown. On standing, a red
precipitate may be produced.
B. Transfer 10ml of the test solution to a conical flask and add 10ml of 1-
ethylquinaldinium iodide (15 g/1) TS that has previously been filtered through
a 0.45-pm filter. Then add 60 ml of 2-propanol R, 5 ml of monoethanolamine
(0.1 mol/1) VS, and 15ml of water. Mix, and heat in a water-bath at 60°C
for 15 minutes; a blue colour develops.

Chloral alcoholate. Warm l.Og with 10ml of sodium hydroxide (-80 g/1) TS.
Filter the upper layer and add iodine (0.05 mol/1) VS a drop at a time until a
yellow colour is obtained; no precipitate is produced within 1 hour.
Clarity and colour of solution. The test solution is clear and colourless.
226
Monograp’hs for p/harmaceutkal substances
pH value. pH of a O.lOg/ml solution in carbon-dioxide-free water R, 3. 5-5.5.
Assay. Dissolve about 4 g, accurately weighed, in 10 ml of carbon-dioxide-free

water R and add 30.0 ml of carbonate-free sodium hydroxide (1 mol/1) VS.
Allow the mixture to stand for 2 minutes and titrate with sulfuric acid
(0.5mol/l) VS, using phenolphthalein/ethanol TS as indicator. Repeat the pro-
cedure without the Chloral hydrate being examined and make any necessary
corrections.

0.1654gof C 2 H 3 CI 3 O 2 .
CHLORAMBUCILUM
CHLORAMBUCIL
Molecular formula. CHH19CI2NO2
Graphic formula.
ICICH2CHj)2N CHjCHjCHjCOOH
Chemical name. 4-[p-[Bis(2-chloroethyl)amino]phenyl]butyric acid; 4-[bis(2-

chloroethyl)amino]benzenebutanoic acid; CAS Reg. No. 305-03-3.
Description. A white or almost white, crystalline or slightly granular powder.
Solubility. Practically insoluble in water; freely soluble in ethanol (-750 g/1) TS

and acetone R.
Storage. Chlorambucil should be kept in a well-closed container, protected

from light.
Additional information. CAUTION: Chlorambucil must be handled with

227
Requirements
Definition. Chlorambucil contains not less than 98.0% and not more than
101.0% of C14H19CI2NO2, calculated with reference to the anhydrous substance.
Identity tests
A. Place 20 mg in a test-tube, add 0.20ml of potassium dichromate TS2, cover
the tube with a piece of filter-paper moistened with sodium nitroprusside
(8.5g/l) TS and 0.05ml of piperidine R. Heat the tube over a small flame; a
blue spot appears on the filter-paper.
B. Dissolve 0.05g in 5ml of acetone R, and dilute with water to 10ml. Add
0.05ml of sulfuric acid (~100g/l) TS, then add 0.20ml of silver nitrate
(0.1 mol/1) VS; no opalescence is observed immediately (absence of chloride
ion). Warm the solution on a water-bath; an opalescence develops (presence
of ionizable chlorine).
C. Mix 0.4 g with 10ml of hydrochloric acid (~70g/l) TS and allow to stand for
30 minutes, shaking occasionally. Filter, wash the residue with 2 quantities,
each of 10 ml of water, and dry at ambient temperature under reduced pres-

sure (not exceeding 0.6kPa or about 5 mm of mercury) over phosphorus pen-
toxide R for 3 hours; melting temperature, about 146°C.

Related substance. Carry out the test as described under 1.14.1 Thin-layer
chromatography, using silica gel R2 and allowing the
coated plate to dry at room temperature Use as the mobile phase
for 24 hours.
a mixture of 8 volumes of toluene R, 5 volumes of methanol R, 4 volumes of
heptane R, and 4 volumes of ethylmethylketone R. Apply separately to the
plate 10 pi of each of 2 solutions in acetone R containing (A) 20 mg of the test
the plate from the chromatographic chamber, allow it to dry in air and examine
the chromatogram in ultraviolet light (254 nm). Any spot obtained with solu-
with solution B.
Assay. Dissolve about 0.2 g, accurately weighed, in 10ml of acetone R, add

10ml of water, and titrate with carbonate-free sodium hydroxide (0.1 mol/1) VS
using phenolphthalein/ethanol TS as indicator. Repeat the operation without
228
the substance beingexamined and make any necessary corrections. Each ml of

CmHi,C12N02.
CHLORAMPHENICOLI NATRII SUCCINAS

CHLORAMPHENICOL SODIUM SUCCINATE
Ci5Hi5Cl2N2NaOs
Chemical name. A mixture in variable proportions of {2R,3R)-2-{2,2-

dichloroacetamido)-3-hydroxy-3-(4-nitrophenyl)propyl succinate (3 isomer)
and of sodium (17i,2R)-2-(2,2-dichloroacetamido)-3-hydroxy-l-(4-nitrophenyl)
propyl succinate (1 isomer); [/?-(/?*, i?*)]-mono[2-[(2,2-dichloroacetyl)amino]-3-
hydroxy-3-(4-nitrophenyl)propyl] ester, butanedioic acid, monosodium salt; D-

ffergo-(-)-2,2-dichloro-Al-[P-hydroxy-a-(hydroxymethyl) -/!’-nitrophenethyl]
acetamide a-(sodium succinate); CAS Reg. No. 982-57-0.
Description. A white or yellowish white powder.
Solubility. Very soluble in water; freely soluble in ethanol (-750 g/1) TS.
Categoiy. Antibacterial drug.
Storage. Chloramphenicol sodium succinate should be kept in a tightly closed

Labelling. The designation Chloramphenicol sodium succinate for parenteral

use indicates that the substance complies with the additional requirements and
may be used for parenteral administration. Expiry date.
229
Additional information. Chloramphenicol sodium succinate is hygroscopic.

Even in the absence of light, Chloramphenicol sodium succinate gradually
degrades when exposed to a humid atmosphere; decomposition is more rapid
Requirements
Chloramphenicol sodium succinate contains not less than 98 0 % and not more .
than the equivalent of 102 0 % of C] 5 Hi 5 Cl 2 N 2 NaOs, calculated with reference

.
to the anhydrous substance.
Identity tests
spectrum obtained from chloramphenicol sodium succinate RS or with
the reference spectrum of chloramphenicol sodium succinate.
B. See the test described below under “Chloramphenicol and chloramphenicol

disodium disuccinate, test B”. The two principal spots obtained with solu-
tion A correspond in position and appearance with those obtained with
solution B. The and B
positions of the spots obtained with solutions A are
different from that of the principal spot obtained with solution C.
C. Dissolve 10 mg in 2.0 ml of ethanol (-750 g/1) TS, add 0.2 g of zinc R powder,
1.0ml of sulfuric acid (~100g/l) TS, and allow to stand for 10 minutes. Filter.
To the filtrate add 0.5ml of sodium nitrite (10 g/1) TS, and allow to stand for
2 minutes. Then add l.Og of urea R and a solution containing 10 mg of
2-naphthol R in 2 ml of sodium hydroxide (-80 g/1) TS; a red colour is
produced. Repeat the test omitting the zinc R powder; no red colour is
produced.
D. Dissolve 5 mg in 5 ml of water and add a few drops of silver nitrate (40 g/1)
TS; no precipitate is produced. Heat 0.05 g with 2.0 ml of potassium hydrox-
ide/ethanol TSl on a water-bath for 15 minutes, add 15mg of charcoal R,

shake, and filter. The filtrate yields reaction A described under 2.1 General
identification tests as characteristic of chlorides.
E. When tested for sodium as described under 2.1 General identification tests,
it yields the characteristic reactions. If reaction B is to be used, prepare a

20mg/ml solution.
ence to the anhydrous substance; = -1-5.0° to -h8.0°G.
230
Clarity of solution. A solution of 1.0 g in 3.0 ml of carbon-dioxide-free water

R is clear.

Fischer method, Method A, using about 0.5 g of Chloramphenicol sodium suc-
cinate; the water content is not more than 0.20 g/g.
pH value. pH of a 0.25 g/ml solution in carbon-dioxide-free water R, 6. 4-7.0.
Chloramphenicol and chloramphenicol disodium disuccinate

matography, using a column (25cm x 4.6mm) packed with
stainless steel
which has been modified with chemi-
particles of silica gel, the surface of
cally bonded As the mobile phase, use a
octadecylsilyl groups (5 pm).
mixture of 55 volumes of water, 40 volumes of methanol R, and 5 volumes
of phosphoric acid (~20g/l) TS.
Prepare the following solutions in the mobile phase: solution (A) 0.25 mg of
Chloramphenicol sodium succinate per ml; solution (B) 5.0 pg of chloram-
phenicol RS per ml; solution (C) 5.0 pg of chloramphenicol disodium disuc-
cinate RS per ml; and for solution (D) dissolve 25 mg of Chloramphenicol
sodium succinate in the mobile phase, add 0.5 mg of chloramphenicol RS
and 0.5 mg of chloramphenicol disodium disuccinate RS and dilute to
100 ml with the mobile phase.
olet spectrophotometer set at a wavelength of about 275 nm.
Using a 20-pl loop injector inject solution D. Inject alternately solutions A,

B, C, and D. The test is not valid unless the two peaks in the chromatogram
obtained with solution D, corresponding to those in the chromatograms
obtained with solutions B and C, are clearly separated from the peaks cor-
responding to the two principal peaks in the chromatogram obtained with
solution A. If necessary, adjust the methanol content of the mobile phase.

from solutions A, B, and C, and calculate the content of the related sub-
stances as a percentage. In the chromatogram obtained with solution A, the
area of any peak corresponding to chloramphenicol is not greater than that
of the principal peak obtained with solution B (2.0%). The area of any peak
corresponding to chloramphenicol disodium disuccinate is not greater than
that of the principal peak obtained with solution C (2.0%).
231

dichlorome thane R, 14 volumes of methanol R, and 1 volume of acetic acid

(~60g/l) TS as the mobile phase. Apply separately to the plate 2 pi of each
of 3 solutions in acetone R containing (A) 10 mg of Chloramphenicol sodium
succinate per ml, (B) 10 mg of chloramphenicol sodium succinate RS per ml,
and (C) 10 mg of chloramphenicol RS per ml. Then apply separately 10 pi
of solution (A) as prepared above and 1 pi of solution (D) containing
0.20 mg of chloramphenicol RS per ml of acetone R. After removing the
vents have evaporated, and examine the chromatogram in ultraviolet light
(254 nm).
Any spot obtained with the second application of solution A, other than the
principal spot, is not more intense than that obtained with solution D (2.0%).
Assay. Dissolve about 0.2 g, accurately weighed, in sufficient water to produce

500 ml; dilute 5.0 ml of this solution to 100 ml with water. Measure the
absorbance of the diluted solution in a 1-cm layer at the maximum at about
276nm and calculate the percentage content of Ci5H]5Cl2N2Na08 using the
absorptivity value of 22.0 (A!°(^ = 220), and with reference to the anhydrous
substance.
Additional requirements for Chloramphenicol sodium

succinate for parenteral use
Storage. SterileChloramphenicol sodium succinate should be kept in a sterile,

tightly closed, and tamper-evident container, protected from light.
Sterility. Complies with 3.2.2 Sterility testing of antibiotics, Membrane filtra-
tion test procedure.
CHLORAMPHENICOLI PALMITAS
CHLORAMPHENICOL PALMITATE
Molecular formula. C27H42CI2N2O6
232
Graphic formula.
c CH20C0 (CHj),.,CH3
I
NHCOCHClj
Chemical name. D-f/z«o-(-)-2,2-Dichloro-N-[P-hydroxy-a-(hydroxymethyl)-

/?-nitrophenethyl]acetamide a-palmitate; R’)]-2-[(dichloroacetyl)amino]-
3-hydroxy-3-(4-nitrophenyl)propyl hexadecanoate; CAS Reg. No. 530-43-8.
Description. A fine, white, unctuous, crystalline powder; odour, faint.

TS; soluble in ether R.
Storage. Chloramphenicol palmitate should be kept in a tightly closed con-

Additional information. If Chloramphenicol palmitate is to be used to

prepare a dosage form in the solid state, it should contain at least 90% of poly-
morph B. If a liquid dosage form is to be prepared, e.g., a suspension, the
method of preparation should be such as to ensure that at least 90% of the
chloramphenicol palmitate is present as polymorph B in the final product.
Requirements
Definition. Chloramphenicol palmitate contains not less than 98.0% and not
more than 102.0% of C27H42CI2N2O6, calculated with reference to the dried
substance.
Identity tests
chloroform R, 1 volume of methanol R, and 0.1 volume of water as the
mobile phase. Apply separately to the plate 10 pi of each of 2 solutions in
acetone R containing (A) 10 mg of the test substance per ml and (B) 10 mg
of chloramphenicol palmitate RS per ml. After removing the plate from the
chromatographic chamber, allow it to dry in air and examine the chro-
matogram in ultraviolet light (254 nm). The principal spot obtained with
solution A corresponds in position, appearance, and intensity with that
233
B. Dissolve lOmg in 4ml of ethanol (~750g/l) TS, add 1.0ml of sulfuric acid
(-100 g/1) TS and 0.05g of zinc R powder, and allow to stand for 10 minutes.
Decant the supernatant liquid or filter if necessary. Cool the resulting solu-
tion in ice and add 0.5ml of sodium nitrite (100 g/1) TS and, after 2 minutes,
l.Og of urea R, followed by 1.0ml of 2-naphthol TSl and 2.0ml of sodium
hydroxide (-400 g/1) TS; a red colour develops. Repeat the test omitting the
zinc R powder; no red colour is produced.

[a]g>"c = -t22.5 to-t25.5°.

reduced pressure (not exceeding 0.6kPa or about 5 mm of mercury) over phos-
phorus pentoxide R; it loses not more than 5.0mg/g.
Acidity. Dissolve l.Og by warming to 35 °C with 5 ml of a mixture of equal

volumes of ethanol (-750 g/1) TS and ether R, previously neutralized to phe-
nolphthalein/ethanol TS. Titrate with sodium hydroxide (0.1 mol/1) VS, using
phenolphthalein/ethanol TS as indicator, until, on gentle shaking, a pink colour
persists for 30 seconds; not more than 0.4ml is required.
Free chloramphenicol. Dissolve, with the aid of gentle heat, l.Og in 80ml of
xylene R, cool, and extract with 3 quantities, each of 15 ml, of water; discard the
xylene and dilute the combined aqueous extracts to 50 ml with water. Extract
the solution with 10 ml of carbon tetrachloride R, allow to separate, discard the
carbon tetrachloride, and centrifuge a portion of the aqueous solution. Measure
the absorbance of the clear supernatant liquid in a 1 -cm layer at the maximum
atabout 278 nm, using as the blank a solution obtained by repeating the proce-
dure without the substance being examined; the absorbance of this blank solu-
tion should not exceed 0.05. Calculate the content of free chloramphenicol,
using the absorptivity value of 29.8 (A!°(ln = 298); not more than 0.45 mg/g.
Assay. Dissolve about 0.03 g, accurately weighed, in sufficient dehydrated

ethanol R to produce 100ml; dilute 10ml of this solution to 100ml with the
same solvent. Measure the absorbance of the diluted solution in a 1-cm layer
at the maximum at about 271 nm and calculate the content of C 27 H 42 CI 2 N 2 O 5
using the absorptivity value of 17.8 (AS°cm = 178).
234
CHLORAMPHENICOLUM
CHLORAMPHENICOL
Molecular formula. C11H12CI2N2O5
Graphic formula.
OH H
OjN C C CHÔH
^ ^
H NHCOCHClj
Chloramphenicol
Chemical name. D-f//«o-(-)-2,2-Dichloro-N-[P-hydroxy-a-(hydroxymethyl)-

/?-nitrophenethyl]acetamide; [jR-(l?*l?*)]-2,2-dichloro-iV-[2-hydroxy-l-(hydrox-
ymethyl)-2-(4-nitrophenyl)ethyl]acetamide; CAS Reg. No. 56-75-7.
Description. Colourless to greyish white or yellowish white, needle-like crys-

tals or elongated plates or a crystalline powder; odourless.
Solubility. Slightly soluble in water; freely soluble in ethanol (-750 g/1) TS and
propylene glycol R; slightly soluble in ether R.
Storage. Chloramphenicol should be kept in a well-closed container, protected

from light.
Additional information. Chloramphenicol has a bitter taste. A solution in

dehydrated ethanol R is dextrorotatory and a solution in ethyl acetate R is
levorotatory.
Requirements
Definition. Chloramphenicol contains not less than 97.0% and not more than
102.0% of C11H12CI2N2O5 calculated with reference to the dried substance.
Identity tests
235

infrared region.
spectrum obtained from chloramphenicol RS or with the reference spectrum
of chloramphenicol.

[a]“"= = +18.5 to +21.5°.
Free chlorides. For the preparation of the test solution shake 0.50 g with
20ml of water and 10 ml of nitric acid (-130 g/1) TS for 1 minute, filter, and
wash the filter with 5 ml of water. Proceed with the filtrate as described under
2.2.1 Limit test for chlorides; the chloride content is not more than 0.5mg/g.
Solution in ethanol. A solution of 0.50g in 10ml of ethanol (-750 g/1) TS is
clear.
lOmg/g.
pH value. Shake 0.05g with lOml of carbon-dioxide-free water R; pH of the

suspension, 5. 0-7. 5.
9 volumes of chloroform R and 1 volume of methanol R as the mobile phase.
Apply separately to the plate 5 pi of each of 3 freshly prepared solutions in
ethanol (-750 g/1) TS containing (A) 20 mg of the test substance per ml, (B)
0.20 mg of the test substance per ml, and (C) 0.20 mg of chloramphenicol RS
per ml. After removing the plate from the chromatographic chamber, allow it
to dry in air until the solvents have evaporated, heat at 105°C for 5 minutes,
and examine the chromatogram in ultraviolet light (254 nm). Any spot obtained

produce 100ml; dilute 10.0ml of this solution to 100ml with the same solvent.
Measure the absorbance of a 1-cm layer of the diluted solution at the maximum
at about 278 nm. Calculate the amount of C11H12CI2N2O5 in the substance being
236
tested by comparison with chloramphenicol RS, and concurrently

similarly
examined. In an adequately calibrated spectrophotometer, the absorbance of
the reference solution should be 0,60 + 0.03.
CHLORHEXIDINI DIACETAS
CHLORHEXIDINE DIACETATE
Molecular formula. C 22 H 3 oCl 2 Nio, 2 C 2 H 402
Graphic formula.
NH NH
Chemical name. l,l'-Hexamethylenebis[5-(;2-chlorophenyl)biguanide] diac-

etate; N,N"-bis(4-chlorophenyl)-3,12-diimino-2,4,ll,13-tetraazatetradecanedi-
imidamide diacetate; CAS Reg. No. 56-95-1.
Description. A white or yellowish white, microcrystalline powder; odourless

or almost odourless.
Solubility. Soluble in 55 parts of water and in 15 parts of ethanol (~750g/l)

TS; very slightly soluble in glycerol R.
Category. Disinfectant.
Storage. Chlorhexidine diacetate should be kept in a well-closed container,

Requirements
Definition. Chlorhexidine diacetate contains not less than 97.5% and not
more than 101.0% of C 22 H 3oCl2Nio, 2 C 2 H 402 ,
calculated with reference to the
dried substance.
237
g
Identity tests
A. Dissolve 0.1 g in 10ml of methanol R by warming, and add a mixture of
2ml of sodium hydroxide (-150 g/1) TS and 2ml of bromine TSl; a deep red
colour is produced.
B. Dissolve 0.1 g in10ml of water and add, with shaking, 0.15ml of copper(II)
chloride/ammonia TS; a purple precipitate is produced immediately. Con-
tinue to add 0.5ml of copper(II) chloride/ammonia TS; the colour of the pre-
cipitate changes to blue.
C. Heat gently 0.2 with 1 ml of ethanol (~750g/l) TS and 1 ml of sulfuric acid

(~1760g/l) TS; ethyl acetate, perceptible by its odour (proceed with caution),
is produced.
35mg/g.
Chloraniline. Dissolve 0.20 g in 30ml of water. Add with mixing 5ml of

(1 mol/1) VS, 1 ml of sodium nitrite (35 g/1) TS, 2 ml of
hydrochloric acid ammo-
nium sulfamate (50g/l) TS, and shake. Then add 5ml of freshly prepared W-(l-
naphthyl)ethylenediamine hydrochloride(l g/1) TS, 1ml of ethanol (~750g/l)
TS, and sufficient water to produce 50 ml. Allow to stand for 30 minutes. Treat
similarly 30ml of a solution containing 0.10 mg R that has been
of chloraniline
slightly acidified with hydrochloric acid (-70 g/1) TS. The colour produced in
the test solution is not more intense than that of the reference solution when
compared as described in 1.11 Colour of liquids (0.5mg/g).
chromatography, using silica gel R4 as the coating substance and preparing a
slurry as follows: To 8g of silica gel R4 add 16ml of water containing Ig of

sodium formate R and coat the plates with a layer 0.5mm thick. Use a mixture
of 50 volumes of chloroform R, 50 volumes of ethanol (-750 g/1) TS and 7
volumes of formic acid (-1080 g/1) TS as the mobile phase. Apply to the plate
in the form of a band, 4 cm wide, 20 pi of a solution in acetic acid (-90 g/1) TS
containing 72 mg of the test substance per ml (solution A). After removing the
plate from the chromatographic chamber, allow it to dry in air and examine
the chromatogram in ultraviolet light (254nm). Score a rectangular area around
each group of bands above and below the principal band, quantitatively trans-
fer the enclosed areas of silica gel to a glass-stoppered test-tube, add 5 ml of
methanol R, shake for 15 minutes, centrifuge, and measure the absorbance of
the clear supernatant liquid in a 1-cm layer at the maximum at about 256 nm.
For the blank solution treat in a similar manner equivalent sized areas of silica
gel removed from the coating adjacent to the areas previously removed. Prepare
solution B in the following manner; Dissolve 0.14g of the test substance in suf-
238
ficient acetic acid (~90g/l) TSproduce 100 ml and dilute 200 pi of this solu-
to
tion to 50 ml with methanol R. The absorbance obtained from the eluted solu-
tion A is not greater than the absorbance obtained from solution B.

Rl, add 0.15ml of 1-naphtholbenzein/acetic acid TS as indicator, and titrate
with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous
15.64mg of C 22 H 3 oCl 2 Nio, 2 C 2 H 402 .
CHLORHEXIDINI DIHYDROCHLORIDUM
CHLORHEXIDINE DIHYDROCHLORIDE
Molecular formula. C22H3oCl2Nio,2HCl
Graphic formula.
Chemical name. l,l'-Hexamethylenebis[5-(;7-chlorophenyl)biguanide] dihy-

drochloride; Ai,N"-bis(4-chlorophenyl)-3,12-diimino-2,4,ll,13-tetraazatetrade-
canediimidamide dihydrochloride; CAS Reg. No. 3697-42-5.
Solubility. Sparingly soluble in water; soluble in 450 parts of ethanol (-750 g/1)
TS.
Category. Disinfectant.
Storage. Chlorhexidine dihydrochloride should be kept in a well-closed con-

239
Requirements
Definition. Chlorhexidine dihydrochloride contains not less than 98.0% and
not more than 101.0% of C 22 H 3oCl2Nio, 2 HCl, calculated with reference to the
dried substance.
Identity tests
A. Dissolve 20mg in 10ml of methanol R by warming, and add a mixture of
2ml of sodium hydroxide (-150 g/1) TS and 2ml of bromine TSl; a deep red
colour is produced.
B. Dissolve 0.1 g in10ml of water and add, with shaking, 0.15ml of copper(II)
chloride/ammonia TS; a purple precipitate is produced immediately. Con-
tinue to add 0.5ml of copper(II) chloride/ammonia TS; the colour of the pre-
cipitate changes to blue.
C. Dissolve 0.1 g in 50 ml of nitric acid (-130 g/1) TS; the solution yields reac-
tion A
described under 2.1 General identification tests as characteristic of
chlorides.
Loss on drying. Dry to constant weight at 130 °C; it loses not more than
20mg/g.
Chloraniline. Dissolve 0.20 g in 30ml of water. Add with mixing 5ml of

hydrochloric acid(1 mol/1) VS, 1 ml of sodium nitrite (35 g/1) TS, 2 ml of ammo-
nium sulfamate (50g/l) TS and shake. Then add 5ml of freshly prepared iV-(l-
naphthyl)ethylenediamine hydrochloride(l g/l)TS, 1 ml of ethanol (-750 g/1) TS,
and sufficient produce 50 ml. Allow to stand for 30 minutes. Treat sim-
water to
ilarly 30ml O.lOmg of chloraniline R that has been
of a solution containing
slightly acidified with hydrochloric acid (-70g/l) TS. The colour produced in
the test solution is not more intense than that of the reference solution when
compared as described in 1.11 Colour of liquids (0.5mg/g).
slurry as follows: To 8g of silica gel R4 add 16ml of water containing Ig of
sodium formate R and coat the plates with a layer, 0.5mm thick. Use a mixture
of 50 volumes of chloroform R, 50 volumes of ethanol (-750 g/1) TS, and 7
volumes of formic acid (-1080g/l) TS as the mobile phase. Prepare solution A
by dissolving 1.1 g of the test substance in 35ml of hydrochloric acid (-330 g/1)
TS, add 100 ml of 2-propanol R, cool in ice, make alkaline with sodium hydrox-
ide (-200 g/1) TS, cool in ice, add 200ml of ice-cooled water, and extract with
100 ml of chloroform R. Dry the chloroform extract over anhydrous potassium
carbonate R, filter, evaporate the chloroform almost to dryness under a stream
240
of nitrogen R, add 50 ml of methanol R, evaporate to dryness under a stream

of nitrogen R, and dry the residue at 65 °C for 30 minutes; dissolve 0.56g of
the dried residue in sufficient acetic acid (~90g/l) TS to produce 100ml. Apply
to the plate in the form of a band, 4 cm wide, 20 pi of solution A. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air and
examine the chromatogram in ultraviolet light (254nm). Score a rectangular
area around each group of bands above and below the principal band, quanti-
tatively transfer the enclosed areas of silica gel to a glass-stoppered test-tube,
add 5 ml of methanol R, shake for 15 minutes, centrifuge, and measure the
absorbance of the clear supernatant liquid in a 1-cm layer at the maximum at
about 256 nm. For the blank solution treat in a similar manner equivalent-sized
areas of silica gel removed from the coating adjacent to the areas previously
removed. Prepare solution B in the following manner: Dissolve 0.1 Ig of the
dried residue in sufficient acetic acid (~90g/l) TS to produce 100ml and dilute
200 pi of this solution to 50 ml with methanol R. The absorbance obtained from
the eluted solution A is not greater than the absorbance obtained from solu-
tion B.
Rl, add 10 ml of mercuric acetate/acetic acid TS and titrate with perchloric acid
(0.1 mol/1) VS, determining the endpoint potentiometrically as described under
2.6 Non-aqueous titration. Method A. Each ml of perchloric acid (0.1 mol/1) VS
is equivalent to 14.46mg of C22H3oCl2Nio,2HCl.
CHLORMETHINI HYDROCHLORIDUM
CHLORMETHINE HYDROCHLORIDE
Molecular formula. C5HnCl2N,HCl
Graphic formula.
CKCHjljNICHjljCI • HCI
CHj
Chemical name. 2,2'-Dichloro-A/'-methyldiethylamine hydrochloride; 2-
chloro-W-(2-chloroethyl)-iV-methylethanamine hydrochloride; CAS Reg. No.

55-86-7.
241
Solubility. Very soluble in water; soluble in ethanol (~750g/l) TS.
Category. Antineoplastic.
Storage. Chlormethine hydrochloride should be kept in a tightly closed con-

tainer and stored at a cool temperature.
Additional information. CAUTION: Chlormethine hydrochloride is vesi-

cant, it must be handled with care avoiding contact with the skin and inhaling
airborne particles. It is hygroscopic.
Requirements
Definition. Chlormethine hydrochloride contains not less than 98.0% and not
more than 101.0% of CsHnChNjHCl, calculated with reference to the anhy-
drous substance.
Identity tests
A. Dissolve 0.05 g in 5 ml of water and add 0.02 ml of potassio-mercuric iodide
TS; a cream-coloured precipitate is produced.
B. Add 0.1 g to 1ml of sodium thiosulfate (0.1 mol/1) VS contained in a test-

tube. Shake, allow to stand for 2 hours, and add 1 drop of iodine TS; the
colour of free iodine remains.
Clarity of solution. A solution of O.lOg in 10ml of carbon-dioxide-free water

R is clear.

Fischer method. Method A, using about 1 g of the substance; the water content
pH value. pH of a 2.0mg/ml solution, 3. 0-5.0.
Assay. To about 0.20 g, accurately weighed, add 15ml of potassium hydrox-

ide/ethanol (1 mol/1) VS and 15ml of water. Boil under a reflux condenser for 2
hours, and evaporate the solution to half its volume on a water-bath. Dilute to
150 ml with water, add 3 ml of nitric acid (~1000g/l) TS and 50 ml of silver
nitrate (0.1 mol/1) VS. Shake vigorously and filter. Wash the precipitate with
water and titrate the excess of silver nitrate in the combined filtrate and wash-
ings with ammonium thiocyanate (0.1 mol/1) VS, using 2.5ml of ferric ammo-
242
nium sulfate (45g/l) TS as indicator. Each ml of silver nitrate (0.1 mol/1) VS is
equivalent to 6.417mg of C 5 HuCl 2 N,HCl.
CHLOROBUTANOLUM
CHLOROBUTANOL
Chlorobutanol, anhydrous
Chlorobutanol hemihydrate
OH
H3C —— C
I
CH3 • nHjO
CCI3
tt = 0 (anhydrous)
n = \ (hemihydrate)
C4H7CI3O (anhydrous)
C 4 H 7 Cl 30 ,|-H 20 (hemihydrate)
Relative molecular mass. 177.5 (anhydrous); 186.5 (hemihydrate).
Chemical name. l,l,l-Trichloro-2-methyl-2-propanol; CAS Reg. No. 57-15-8

(anhydrous).
l,l,l-Trichloro-2-methyl-2-propanol hemihydrate; CAS Reg. No. 6001-64-5

(hemihydrate).
Description. Colourless crystals or a white, crystalline powder; odour, char-

acteristic, camphoraceous.
Solubility. Slightly soluble in water; very soluble in ethanol (~750g/l) TS and

ether R; soluble in glycerol R.
Storage. Chlorobutanol should be kept in a tightly closed container, and

stored in a cool place.
Labelling. The designation on the container of Chlorobutanol should state

whether it is the hemihydrate or the anhydrous form.
243
Additional information. Anhydrous Chlorobutanol melts at about 95 °C and

Chlorobutanol hemihydrate melts at about 77 °C, both determined without
previous drying.
Requirements
Chlorobutanol contains not less than 98 0 . %
and not more than the equivalent
of 101 0. %
of C4H7CI3O, calculated with reference to the anhydrous substance.
Identity tests
A. Shake 20 mg with 3 ml of sodium hydroxide (1 mol/1) VS, add 5 ml of water,
then slowly add 2 ml of iodine TS; iodoform, perceptible by its odour, is
produced and a yellowish precipitate is formed.
B. To 20mg add 1ml of pyridine R and 2ml of sodium hydroxide (~400g/l)

TS. Heat in a water-bath and shake. Allow to stand; the pyridine layer
becomes red.
Solution in ethanol. A solution of 5g in 10ml of ethanol (~750g/l) TS is not

more opalescent than opalescence standard TS2 and not more intensely

Fischer method. Method A. For the anhydrous form, use 2g; the water content
is not more than lOmg/g. For the hemihydrate, use 0.3 g; the water content is
not less than 45mg/g and not more than 60mg/g.
Acidity. Dissolve 2g in 20 ml of ethanol (-750 g/1) TS and titrate with sodium

hydroxide (0.01 mol/1) VS, using 0.1ml of bromothymol blue/ethanol TS as
indicator; not more than 1.0 ml is required to produce a blue colour.
Assay. Dissolve about 0.1 g, accurately weighed, in 20ml of ethanol (~750g/l)

TS, add 10 ml of sodium hydroxide (-80 g/1) TS, heat in a water-bath for 5
minutes, and cool. Add 20 ml of nitric acid (-130 g/1) TS, 25.0 ml of silver nitrate
(0.1 mol/1) VS, and 2 ml of dibutyl phthalate R, and shake vigorously. Add 2 ml
of ferric ammonium sulfate (45 g/1) TS and titrate with ammonium thiocyanate
(0.1 mol/1) VS until an orange colour is obtained. Repeat the procedure without
the Chlorobutanol being examined and make any necessary corrections.
Each ml of silver nitrate (0.1 mol/1) VS is equivalent to 5.916mg of C4H7CI3O.
244
CHLOROCRESOLUM
CHLOROCRESOL
OH
Cl
C7H7CIO
Chemical name. 4-Chloro-m-cresol; 4-chloro-3-methylphenol; CAS Reg. No.

59-50-7.
Description. Colourless or almost colourless crystals or a white, crystalline

powder; odour, characteristic.
Solubility. Slightly soluble in water; very soluble in ethanol (-750 g/1) TS;
freely soluble in ether R, fatty oils, and sodium hydroxide (-80 g/1) TS.
Storage. Chlorocresol should be kept in a well-closed container, protected

from light.
Requirements
Chlorocresol contains not less than 98 0 . % and not more than the equivalent
of 101 0 % of
. C H;C 10
7 .
Identity tests
A. To 0.5g of a fine powder, add 10ml of carbon-dioxide-free water R, shake
for 2 minutes, and filter. Add 0.1ml of ferric chloride (25 g/1) TS; a bluish
colour is produced.
B. Mix 0.05 g with 0.5 g of anhydrous sodium carbonate R and ignite with a
strong flame. Cool, add a mixture of 5 ml of water and 5 ml of nitric acid
(-130 g/1) TS to the residue, and filter. Add 1 ml of silver nitrate (0.1 mol/1)
VS to the filtrate; a white precipitate is produced.
245
Non-volatile residue. Place 2g in a porcelain dish and heat on a water-bath

until volatilized. Dry the residue at 105 °C and weigh; not more than l.Omg/g.
Assay. Place about 0.07 g, accurately weighed, in a ground-glass-stoppered

flask and dissolve in 30 ml of glacial acetic acid R. Add 25 ml of potassium
bromate (0.0167 mol/1) VS, a solution composed of 3 g of potassium bromide R
dissolved in 20 ml of water, and 10 ml of hydrochloric acid (-420 g/1) TS. Allow
to stand protected from light for 15 minutes. Add 1 g of potassium iodide R
and 100 ml of water. Titrate with sodium thiosulfate (0.1 mol/1) VS, shaking vig-
orously, and add starch TS as indicator towards the end of the titration. Repeat
the procedure without the Chlorocresol being examined and make any neces-
sary corrections.
Each ml of potassium bromate (0.0167mol/l) VS is equivalent to 3.565 mg of

C 7 H 7 CIO.
CHLOROQUINI PHOSPHAS
CHLOROQUINE PHOSPHATE
Molecular formula. Ci8H26ClN3,2H3P04
Graphic formula.
• 2H3PO,
NHCHICHjljNICj
CH3
Chemical name. 7-Chloro-4-[[4-(diethylamino)-l-methylbutyl]amino]quino-

line phosphate (1:2); NT(7-chloro-4-quinolinyl)-iV',N’-diethyl-l,4-pentanedi-
amine phosphate (1 :2); CAS Reg. No. 50-63-5.
Description. A white or almost white, crystalline powder: odourless or

almost odourless.
Solubility. Soluble in 4 parts of water; very slightly soluble in ethanol

(-750 g/1) TS and ether R.
Category. Antimalarial; antiamoebic.
246
Storage. Chloroquine phosphate should be kept in a well-closed container,

Additional information. Chloroquine phosphate has a bitter taste; it is

slowly discoloured by light. Chloroquine phosphate may exist in 2 polymor-
phic forms differing in melting temperature, one of which melts at about
194 °C, the other at about 215 °C; mixtures of the 2 forms melt between
194 °C and 215 °C.
Requirements
Definition. Chloroquine phosphate contains not less than 98.0% and not
more than 101.0% of Ci 8 H 26 ClN 2 H
3, 3P 04 ,
substance.
Identity tests
A. The absorption spectrum of a lOpg/ml solution in hydrochloric acid
(0.01 mol/1) VS, when observed between 240 nm and 360 nm, exhibits 3
maxima about 257 nm, 329 nm, and 343 nm. The absorbances at those
at
wavelengths are about 0.29, 0.32, and 0.37, respectively (preferably use
2-cm cells for the measurements and calculate the absorbances of Tcm
layers). The ratio of the absorbance of a 1-cm layer at 257 nm to that at
343 nm is between 0.77 and 0.85, and the ratio of the absorbance at 329 nm
to that at 343 nm is between 0.86 and 0.95.
B. To 1ml of a 20mg/ml solution add 3ml of nitric acid (-130 g/1) TS; yields
of orthophosphates.
C. To a solution of 0.05g in 20 ml of water add 5 ml of trinitrophenol (7 g/1) TS.

Filter, wash the precipitate with water until the filtrate iscolourless, and dry
the precipitate over silica gel, desiccant, R. Melting temperature, about
207 °C (picrate).
20mg/g.
pH value. pH of a O.lOg/ml solution, 3. 5-4. 5.
5 volumes of chloroform R, 4 volumes of cyclohexane R, and 1 volume of
of 2 solutions containing (A) 40 mg of the test substance per ml and (B) 0.80 mg
of the test substance per ml. After removing the plate from the chromatographic
247
Assay. Dissolve about 0.23 g, accurately weighed, in 20 ml of glacial acetic acid

R1 with the aid of heat (preferably heat under reflux condenser), cool and add
20ml of dioxan R. Titrate with perchloric acid (0.1 mol/1) VS as described under
is equivalent to 25.79mg of C]8H26C1N3,2H3P04.
Additional requirement for Chtoroquine phosp'hate for

parenteral use
Complies with the monograph for "Parenteral preparations”.
CHLOROQUINI SULFAS
CHLOROQUINE SULFATE
Molecular formula. CisH 2 sClN 3 ,H 2 S 04 ,H 20
Graphic formula.
HjSO^ • HjO
Chemical name. 7-Chloro-4-[[4-(diethylamino)-l-methylbutyl]amino]quino-

linesulfate (1:1) monohydrate; V*-(7-chloro-4-quinolinyl)-N',Ai‘-diethyl-l,4-
pentanediamine sulfate (1:1) monohydrate; CAS Reg. No. 6823-83-2.
Solubility. Soluble in 3 parts of water; practically insoluble in ethanol

(-750 g/1) TS; sparingly soluble in ether R.
248
Storage. Chloroquine sulfate should be kept in a well-closed container pro-

tected from light.
Additional information. Chloroquine sulfate has a bitter taste.
Requirements
Definition. Chloroquine sulfate contains not less than 98.0% and not more
than 101.0% of C] 8 H 26 C 1 N 3 ,H 2 S 04 ,
substance.
Identity tests
(0.01 mol/1) VS, when observed between 240 nm and 360 nm, exhibits 3
maxima about 257 nm, 329 nm, and 343 nm. The absorbances at those
at
wavelengths are about 0.39, 0.44, and 0.46, respectively (preferably use 2-
cm cells for the measurements and calculate the absorbances of 1-cm layers).
The ratio of the absorbance of a 1-cm layer at 257 nm to that at 343 nm is
between 0.83 and 0.98 and the ratio of the absorbance at 329 nm to that at
343 nm is between 0.94 and 1.03.
B. A 0.05g/ml solution yields reaction A described under 2.1 General identifi-

C. To a solution of 0.05g in 20 ml of water add 5ml of trinitrophenol (7 g/1) TS. Filter,
wash the precipitate with water until the filtrate is colourless and dry the pre-
cipitate over silica gel, desiccant, R. Melting temperature, about 207 °C (picrate).
Loss on drying. Dry to constant weight at 105°C under reduced pressure (not
exceeding 0.6kPa or about 5mm of mercury); itloses not less than 30mg/g and
pH value. pH of a O.lOg/ml solution, 4. 0-5.0.
of 2 solutions containing (A) 40 mg of the test substance per ml and (B) 0.80 mg
249
R1 with the aid of heat (preferably under a reflux condenser), cool, and add
20ml of dioxan R. Titrate with perchloric acid (0.1 mol/1) VS as described under
is equivalent to 41.8 mg of CibH 26C1N3,H2S04.
Additional requirement for Chloroquine sulfate for

prarenteral use
CHLORPHENAMINI HYDROGENOMALEAS
CHLORPHENAMINE HYDROGEN MALEATE
Molecular formula. Ci6Hi9ClN2,C4H404 or C20H23CIN2O4
Graphic formula.
Chemical name. 2-[/7-Chloro-a-[2-(dimethylamino)ethyl]benzyl]pyridine male-

ate (1 :
1); Y-(4-chlorophenyl)-N,N-dimethyl-2-pyridinepropanamine (Z)-2-
butenedioate (1 ;
1); CAS Reg. No. 113-92-8.
Other name. Chlorpheniramine hydrogen maleate.
Solubility. Soluble in 4 parts of water; soluble in ethanol (-750 g/1) TS; slightly
soluble in ether R.
Category. Antihistaminic.
Storage. Chlorphenamine hydrogen maleate should be kept in a tightly closed

250
Additional information. Even in the absence of light, Chlorphenamine

hydrogen maleate is gradually degraded on exposure to a humid atmosphere,
the decomposition being faster at higher temperatures.
Requirements
Definition. Chlorphenamine hydrogen maleate contains not less than 98.0%
and not more than 101.0% of C] 6 H, 9 C 1 N 2 ,C 4 H 404 calculated with reference
,
to
the dried substance.
Identity tests

spectrum obtained from chlorphenamine hydrogen maleate RS or with
the reference spectrum of chlorphenamine hydrogen maleate.
B. Dissolve 1 mg in 5 ml of water. To 1 ml of this solution add 1 ml of buffer

phthalate, pH 3.5, TS and 1 ml of cyanogen bromide TS. Allow to stand for
10 minutes with intermittent, gentle shaking. Add 2 ml of a solution com-
posed of 1 ml of aniline R diluted to 25 ml with dichloroe thane R, and shake;
an orange-yellow colour is produced in the lower layer.
C. Dissolve 0.5g in 5ml of water, add 0.2ml of sulfuric acid (~100g/l) TS and
extract 4 times with ether R, using 25 ml each time. Combine the ethereal
extracts, dry them over anhydrous sodium sulfate R, filter, and evaporate
the filtrate ina current of warm air; melting temperature of the residue,
about 132 °C (maleic acid).
5-Omg/g.
5 volumes of ethyl acetate R, 3 volumes of methanol R, and 2 volumes of acetic
acid (~60g/l) TS as the mobile phase. Apply separately to the plate 2 pi of each
ml and (B) O.lOmg of the test substance per ml. After removing the plate from
251
other than the two principal spots due to chlorphenamine and maleic acid, is
not more intense than that obtained with solution B.
Assay. Dissolve about 0,4 g, accurately weighed, in 30ml of glacial acetic acid
alent to 19.54 mg of CisHi 9C1N2,C4H404.
Additional requirements for Chlorphenamine hydrogen

maleate for parenteral use
Bacterial endotoxins. Carry out the test as described under 3,4 Test for bac-
CHLORPROMAZINI HYDROCHLORIDUM
CHLORPROMAZINE HYDROCHLORIDE
Molecular formula. C17H19CIN2S, HCl
Graphic formula.
Cl
• HCl
Chemical name. 2-Chloro-10-[3-(dimethylamino)propyl]phenothiazine mono-

hydrochloride; 2-chloro-N,N-dimethyl-10f/-phenothiazine-10-propanamine
monohydrochloride; CAS Reg. No. 69-09-0.
Description. A white or slightly creamy white, crystalline powder; odour, slight.
Solubility. Soluble in 0.4 part of water; freely soluble in ethanol (~750g/l) TS;
Category. Neuroleptic.
252
Storage. Chlorpromazine hydrochloride should be kept in a tightly closed con-

Additional information. Chlorpromazine hydrochloride has a very bitter

taste. It darkens on prolonged exposure to light. Even in the absence of light,
Chlorpromazine hydrochloride is gradually degraded on exposure to a humid

atmosphere, the decomposition being faster at higher temperature.
Requirements
Definition. Chlorpromazine hydrochloride contains not less than 98.0% and
not more than 101.0% of C] 7 Hi 9 ClN 2 S,HCl, calculated with reference to the
dried substance.
Identity tests

spectrum obtained from chlorpromazine hydrochloride RS or with the ref-
erence spectrum of chlorpromazine hydrochloride.

using kieselguhr R1 as the coating substance and a mixture of 10 volumes
of 2-phenoxyethanol R, 5 volumes of macrogol 400R, and 85 volumes of
acetone R to impregnate the plate. After the solvent has reached the top of
the plate, remove the plate from the chromatographic chamber, and use it
immediately. As the mobile phase use a mixture of 2 volumes of diethy-

lamine R and 100 volumes of light petroleum R1 saturated with 2-
phenoxyethanol R. Apply separately to the plate 2 pi of each of 2 solutions
inchloroform R containing (A) 2.0 mg of the test substance per ml and (B)
2.0mg of chlorpromazine hydrochloride RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air, and examine the
chromatogram in ultraviolet light (365 nm), observing the fluorescence pro-
duced after about 2 minutes. Spray the plate with sulfuric acid/ethanol TS
and examine the chromatogram in daylight. The principal spot obtained
with solution A corresponds in position, appearance, and intensity with that
C. A O.lg/ml solution yields reaction B described under 2.1 General identifi-

253
5-Omg/g,
pH value. pH of a freshly prepared O.lOg/ml solution, 4. 0-5.0.
80 volumes of cyclohexane R, 10 volumes of acetone R, and 10 volumes of
of 2 freshly prepared solutions in a mixture of 95 volumes of methanol R and
5 volumes of diethylamine R containing (A) 20 mg of the test substance per ml
and (B) 0.50 mg of the test substance per ml. After removing the plate from the
chromatographic chamber allow it to dry in air, and examine the chromatogram
in ultraviolet light (254 nm). Ignore any spot on the base line. Any spot obtained
Assay. Dissolve about 0.7 g, accurately weighed, in 200 ml of acetone R, add

10ml of mercuric acetate/acetic acid TS and 3ml of methyl orange/acetone TS,
and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-
alent to 35.53 mg of Ci 7 H] 9 ClN 2 S,HCl.
Additional requirements for Chlorpromazine hydrochloride

for p>arenteral use
CHLORTALIDONUM
CHLORTALIDONE
Molecular formula. C14H11CIN2O4S
254
Graphic formula.
Chemical name. 2-Chloro-5-(l-hydroxy-3-oxo-l-isoindolinyl)benzenesulfon-

amide; 2-chloro-5-(2,3-dihydro-l-hydroxy-3-oxo-lff-isoindol-l-yl)benzenesul-
fonamide; CAS Reg. No, 77-36-1.
Description. A white to yellowish white, crystalline powder; odourless or

almost odourless.
Solubility. Practically insoluble in water and ether R; soluble in methanol R;

Category. Diuretic.
Storage. Chlortalidone should be kept in a well-closed container.
Requirements
Definition. Chlortalidone contains not less than 98.0% and not more than
102.0% of C] 4 Hi]C 1 N 204 S, calculated with reference to the dried substance.
Identity tests
• Either tests A and B or tests B and C may be applied.

infrared region.
spectrum obtained from chlortalidone RS or with the reference spectrum of
chlortalidone.
C. Dissolve 20mg in 1ml of sulfuric acid (~1760g/l) TS; an intense yellow

colour is produced. Warm the mixture in a water-bath and add 10 mg of
1-naphthol R; a red-violet colour is produced.
255
Solution in alkali. A solution of 1.0 g in 10ml of sodium hydroxide (-200 g/1)

TS is clear and not more intensely coloured than standard colour solution Yw2
when compared as described under 1.11 Colour of liquids.
5.0mg/g.
15 volumes of 1-butanol R and 3 volumes of ammonia (-17 g/1) TS as the mobile
phase. Apply separately to the plate 10|il of each of 3 solutions in methanol R
containing (A) 10 mg of the test substance per ml, (B) 10 mg of chlortalidone RS
per ml, and (C) 0.10 mg of 2-(4-chloro-3-sulfamoylbenzoyl)benzoic acid RS per
ml. After removing the plate from the chromatographic chamber, allow it to
dry in air, and examine the chromatogram in ultraviolet light (254 nm). Any
spot obtained with solution A, other than the principal spot, is not more intense
than that obtained with solution C.
Assay. Dissolve about 0.3 g, accurately weighed, in 50 ml of pyridine R and

titrate with tetrabutylammonium hydroxide (0.1 mol/1) VS, determining the
endpoint potentiometrically as described under 2.6 Non-aqueous titration.

Method B. Each ml of tetrabutylammonium hydroxide (0.1 mol/1) VS is equiv-
alent to 33.88 mg of C 14 H CIN 2 O
11 4 S.
CHLORTETRACYCLINI HYDROCHLORIDUM
CHLORTETRACYCLINE HYDROCHLORIDE
Chlortetracycline hydrochloride (non-injectable)
Chlortetracycline hydrochloride, sterile
Molecular formula. C22H23ClN20a,HCl
256
Graphic formula.
• HCI
Chemical name. (4S,4aS,5aS,6S,12aS)-7-Chloro-4-(dimethylamino)-l,4,4a,5,

5a,6,ll,12a-octahydro-3,6,10,12,12a-pentahydroxy-6-methyl-l,l l-dioxo-2-
naphthacenecarboxamide monohydrochloride; [4S-(4a,4aa,5aa,6P,12aa)]-7-
chloro-4-(dimethylamino)-l,4,4a,5,5a,6,ll,12a-octahydro-3,6,10,12,12a-penta
hydroxy-6-methyl-l,ll-dioxo-2-naphthacenecarboxamide monohydrochloride;
CAS Reg. No. 64-72-2.
Description. Yellow crystals or a yellow, crystalline powder; odourless.
Solubility. Soluble in about 100 parts of water and in about 250 parts of
ethanol (-750 g/1) TS; practically insoluble in acetone R and ether R.
Storage. Chlortetracycline hydrochloride should be kept in a tightly closed

Labelling. The designation sterile Chlortetracycline hydrochloride indicates

that the substance complies with the additional requirements for sterile
Chlortetracycline hydrochloride and may be used for parenteral administration

or for other sterile applications.
Additional information. Chlortetracycline hydrochloride has a bitter taste.

Even in the absence of light, Chlortetracycline hydrochloride is gradually
Requirements
Definition. Chlortetracycline hydrochloride contains not less than 900 Inter-
national Units of Chlortetracycline per mg, calculated with reference to the
dried substance.
257
=
Identity tests
using a kieselguhr coating prepared as follows: To 25g of kieselguhr R1 add
50ml of a mixture of 2.5ml of glycerol R and 47.5ml of disodium edetate
(0.1 mol/1) VS previously adjusted to pH 7 with ammonia (~100g/l) TS. Coat
the plates with this mixture, and allow them to dry at room temperature
for about 70-90 minutes, or until sufficiently dry to give a satisfactory sep-
aration.As the mobile phase, take 200 ml of a mixture of 2 volumes of ethyl
acetate R, 2 volumes of chloroform R, and 1 volume of acetone R, Shake
with 25 ml of disodium edetate (0.1 mol/1) VS previously adjusted to pH 7
with ammonia (~100g/l) TS, allow to settle, and use the lower layer. Apply
separately to the plate 1 pi of each of 3 solutions in methanol R containing
(A) 0.50 mg of the test substance per ml, (B) 0.50 mg of chlortetracycline
hydrochloride RS per ml, and (C) a mixture of 0.50 mg of chlortetracycline
hydrochloride RS per ml, 0.50 mg of oxytetracycline hydrochloride RS per
ml, and 0.50 mg of tetracycline hydrochloride RS per ml. After removing the
plate from the chromatographic chamber, allow it to dry in air, expose it to
the vapour of ammonia (-260 g/1) TS, and examine the chromatogram in
ultraviolet light (365 nm). The principal spot obtained with solution A cor-
responds in position, appearance, and intensity with that obtained with
solution B. The test is not valid unless the chromatogram obtained with
solution C shows 3 clearly separated spots.
B. Dissolve lOmg 10ml of phosphate buffer, pH 7.6, TS, heat at 100°C for
in
1 minute and examine the solution in ultraviolet light (365 nm); a strong blue
fluorescence is observed.
C. To about 1 mg add 2 ml of sulfuric acid (-1760 g/1) TS; a blue to bluish green
colour is produced, which changes to brown on the addition of about 1 ml
of water.
D. A 0.05 g/ml solution yields reaction B described under 2.1 General identifi-
Specific optical rotation. Dissolve 0.125g in sufficient water to produce

25.0ml, and allow to stand in the dark for 30 minutes. Measure the rotation
at 25 °C and calculate with reference to the dried substance; [aj^ -235 to
-250°.
258

or about 5mm of mercury) over phosphorus pentoxide R for 3 hours; it loses
Absorption in the ultraviolet region. Dissolve 10 mg in sufficient sulfuric

acid (0.5 mol/1) VS to produce 100 ml. Dilute 10 ml of this solution to 100 ml with
sulfuric acid (0.5 mol/1) VS. Place 10ml of the resulting solution in a test-tube
25 mm in diameter and 200 mm long and immerse in a water-bath for 8 minutes.
Cool, replace the water lost by evaporation, and measure the absorbance of a
1-cm layer at 274 nm; not less than 0.70 and not more than 0.76.
antibiotics, using either {a) Bacillus piumilus (NCTC 8241 or ATCC 14884) as the
test organism, culture medium Cml with a final pH of 6. 5-6. 6, sterile phos-
phate buffer, pH 4.5 TS, an appropriate concentration of chlortetracycline
(usually between 2 and 20 lU per ml), and an incubation temperature of 35-
39°C, or (b) Bacillus cereus (ATCC 11778) as the test organism, culture medium
Cml with a final pH of 5. 9-6.0, sterile phosphate buffer, pH 4.5 TS, an appro-
priate concentration of chlortetracycline (usually between 0.05 and 0.2 lU), and
an incubation temperature of 29-33 °C. The precision of the assay is such that
the fiducial limits of error of the estimated potency (P = 0.95) are not less than
95% and not more than 105% of the estimated potency. The upper fiducial
limit of error of the estimated potency (P = 0.95) is not less than 900 lU of
chlortetracycline per mg, calculated with reference to the dried substance.
Additional requirements for Chlortetracycline Hydrochloride

for sterile use
Storage. Sterile Chlortetracycline hydrochloride should be kept in a hermeti-
cally closed container, protected from light.
Sterility. Complies with 3.2.2 applying the

Sterility testing of antibiotics,
259
CICLOSPORINUM
CICLOSPORIN
C62HiiiNi]Oi2
Relative molecular mass. 1203
Chemical name. Cyclo[[(£)-(2S,3R,47?)-3-hydroxy-4-methyl-2-(methylamino)-

6-octenoyl]-L-2-aminobutyryl-W-methylglycyl-iV-methyl-L-leucyl-L-valyl-
Nmethyl-L-leucyl-L-alanyl-D-alanyl-A/-methyl-L-leucyl-N-methyl-L-leucyl-Nm
eth.yl-L-valyl]; cyclosporin A; CAS Reg. No. 59865-13-3.
Other name. Cyclosporin.

and dichloromethane R.
Category. Immunosuppressant drug.
Storage. Ciclosporin should be kept in a well-closed container, protected from

light.
Requirements
Ciclosporin contains not less than 98 5 % and not more than 101 5 %
. . of
C 62 H 111 N O 12
11 ,
Identity tests

spectrum obtained from ciclosporin RS or with the reference spectrum of
ciclosporin.
260
=
B. See the test described below under “Related substances” and under “Assay”.
The principal peak obtained with solution A corresponds in retention time
to that obtained with solution B.
C. Dissolve 5 mg in 5 ml of methanol R, and 1 drop of potassium permanganate

(lOg/1) TS, and allow to stand; the blue-red colour is gradually discharged.
Specific optical rotation. Use a 5.0 mg/ml solution in methanol R and calcu-
late with reference to the dried substance; [a]™ -185 to -1931-
Clarity and colour of solution in ethanol. A solution of l.Og in 10ml of

ethanol (-750 g/1) TS is clear and not more intensely coloured than standard
colour solution Yw3 or Rdl when compared as described under 1.11 Colour of
liquids.

Related substances. Carry out the test as described below under “Assay”.
Inject alternately 20 pi each of solutions A and C. Continue the recording of the

chromatogram for 1.7 times the retention time of the principal peak.
is not greater than 0.7 times the area of the prin-
other than the principal peak,
cipalpeak obtained with solution C (0.7%), and the sum of these areas is not
greater than 1.5 times the area of the principal peak of the chromatogram
obtained with solution C (1.5%).
Assay. Determine as described under 1.14.4 High performance liquid chro-

matography, using a stainless steel column (25 cm x 4mm) packed with parti-
cles of silica gel, the surface of which has been modified with chemically
bonded octadecylsilyl groups (3-5 pm). The column is connected to the injec-
tion port by a steel capillary tube about 1 m long with an internal diameter of
0.25 mm. Maintain the temperature of the column and of the steel capillary at
80 °C. As the mobile phase, use a mixture of 52 volumes of water, 43 volumes
of acetonitrile R, 5 volumes of rert-butyl methyl ether R, and 0.1 volume of
phosphoric acid (-1440 g/1) TS.
261
Prepare the following solutions in a solvent mixture of equal volumes of ace-

tonitrile R and water; solution (A) 1.2 mg of Ciclosporin per ml; solution (B)
1.2 mg of ciclosporin RS per ml; for solution (C) dilute 2.0 ml of solution B to
200 ml with the solvent mixture; and for solution (D) dissolve 3 mg of
ciclosporin U RS in 2.5ml of the solvent mixture and add 2.5ml of solution B.
Operate with a flow rate of about 1.5ml per minute. As a detector use an ultra-
violetspectrophotometer set at a wavelength of about 210 nm.
Inject 20 pi of solution D. The assay is valid only if the relative standard devi-
ation of the area of the principal peak not more than 1.0%, unless the reso-
is
lution between the two is 1.0 and 1.8. The assay is not valid
principal peaks
unless the retention time of the principal peak is between 25 and 30 minutes.
solutions A and B, and calculate the percentage content of C62HinNi,0,2.
CIMETIDINUM
CIMETIDINE
Molecular formula. CioHieNgS
Graphic formula.
NCN
II
CHjSCHjCHjNHCNHCHj
Chemical name 2-Cyano- 1 -methyl-3- [2- [[(5-methylimidazol-4-yl)me thyl] -thio]

.
ethyl] guanidine; A/"-cyano-iV-methyl-N'-[2-[[(5-methyl-lH-imidazol-4-yl)methyl]

thiojethyljguanidine; l-cyano-2-methyl-3-[2-[[(5-methylimidazol-4-yl)methyl]
thiojethyljguanidine; A/-cyano-A/'-methyl-N"-[2-[[(5-methyl-lH-imidazol-4-yl)
methyljthiojethyljguanidine; CAS Reg. No. 51481-61-9.
Description. A white to off-white powder; odourless or with a faint odour.
Solubility. Sparingly soluble in water; very soluble in methanol R.
262
Category. Antiulcer drug.
Storage. Cimetidine should be kept in a well-closed container.
Additional information. Cimetidine exists in three polymorphic forms. The

polymorph specified in the monograph corresponds to the crystal form of cime-
tidine RS.
Requirements
Definition. Cimetidine contains not less than 98.5% and not more than
101.0% of CioHifiN^S, calculated with reference to the dried substance.
Identity tests
infrared region.The infrared absorption spectrum obtained from the solid
statewithout prior solvent treatment is concordant with the spectrum sim-
ilarlyobtained from cimetidine RS or with the reference sfectrum of cimeti-
dine; no shoulder or peak is discernible at 1180cm“* (confirmation of
polymorphic form).
B. Melting temperature, about 142 °C.
Heavy metals. Use 1.0 g for the preparation if the test solution as described
lO.Omg/g.
pH value. pH of a 5.0mg/ml solution in carbon-dioxide-free water R, 8. 0-9. 5.
formance liquid chromatography, using a column 25 cm long and 4.6mm inter-
nal diameter, packed with particles of porous silica gel or ceramic, 5-10 pm in
diameter, the surface of which has been modified with chemically bonded
octadecylsilyl groups. Prepare the following solvent mixture: Dilute 1 ml of
glacial acetic acid R with sufficient water to produce 200ml. To 190 ml of this
solution add 10ml of ammonium acetate (2g/l) TS. As the mobile phase use a
degassed and filtered mixture of 84 volumes of the above solvent mixture and
16 volumes of acetonitrile R. For the system suitability test prepare a solution
containing 18pg of cimetidine RS and 24 pg of caffeine RS per ml of the above
solvent mixture (solution A). Further prepare a solution of the substance to be
263
examined containing 18 |tg per ml of solvent mixture (solution B). Operate with
a flow rate of about 1 ml per minute. As detector use an ultraviolet spec-
trophotometer at a wavelength of about 228 nm, fitted with a suitable recorder.
Make 6 replicate injections, each of 10 pi of solution A. Measure the peak
responses; the relative standard deviation of the ratio of the responses from
cimetidine to the sum of all the responses in the chromatogram, excluding any
from the solvent mixture, is not more than 2.0%, and the resolution between
caffeine and cimetidine is not less than 3.0. The relative retention times are
about 1.0 for caffeine and 1.4 for cimetidine. Then inject 10 pi of solution B and
measure the peak responses: the ratio of the response from cimetidine to the
sum of all the responses in the chromatogram, excluding any from the solvent
mixture, is not less than 0.99.

Rl, and titrate with perchloric acid (0.1 mol/1) VS, determining the endpoint
potentiometricaUy as described under 2.6 Non-aqueous titration. Method A.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 25.23 mg of CioHieN^S.
Additional requirement for Cimetidine for parenteral use

CIPROFLOXACINI HYDRO CHLORIDUM

CIPROFLOXACIN HYDROCHLORIDE
C,7H,sFN303,HC1,H20
Chemical name. l-Cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-7-(l-piperazinyl)-

3-quinolinecarboxylic acid monohydrochloride monohydrate; CAS Reg. No.
86393-32-0.
Description. A pale yellow, crystalline powder.
264
Solubility. Soluble in water; slightly soluble in methanol R; very slightly

soluble in ethanol (-750 g/1) TS; practically insoluble in acetone R and
dichlorome thane R.
Categoiy. Antibacterial.
Storage. Ciprofloxacin hydrochloride should be kept in a tightly closed con-

Requirements
Ciprofloxacin hydrochloride contains not less than 98 0 % and not more than
.
102 0 %
. of CijHiaFNsOjjHCl, calculated with reference to the anhydrous
substance.
Identity tests
infrared region.
spectrum obtained from ciprofloxacin hydrochloride RS or with the refer-
ence spectrum of ciprofloxacin hydrochloride.

using silica gel R4 as the coating substance and a mixture of 4 volumes
of methanol R, 4 volumes of dichlorome thane R, 2 volumes of ammonia

(-260 g/1) TS, and 1 volume of acetonitrile Ras the mobile phase. Apply sep-
arately to the plate as 1-cm bands, 5 pi of each of 2 solutions containing (A)
10 mg of Ciprofloxacin hydrochloride per ml, and (B) 10 mg of ciprofloxacin
hydrochloride RS per ml. Place an evaporating-dish containing 50 ml of
ammonia (-260 g/1) TS in the chromatographic chamber. Expose the plate
to the ammonia vapour in the closed chamber for 15 minutes. Withdraw
the plate and transfer to another chromatographic chamber containing the
mobile phase to develop. After removing the plate from the chromato-
graphic chamber, allow it to dry in air for about 15 minutes, and examine
the chromatogram in ultraviolet light (254nm and 365 nm).


Heavy metals. For the preparation of the test solution dissolve 0.25 g in water
and dilute to 30 ml with the same solvent. Carry out the prefiltration. Deter-
mine the heavy metals content in the filtrate as described under 2.2.3 Limit test
for heavy metals. Method B; not more than 20pg/g.
265
Clarity and colour of solution. A solution of 0.25 g in 10ml of carbondiox-

ide-free water R is clear and not more intensely coloured than standard colour
solution Gn4 when compared as described under 1.11 Colour of liquids.

Fischer method, Method B, using about 0.2 g of the substance; the water
content is between 0.047 g/g and 0.067 g/g.
Fluoroquinolonic acid. Carry out the test as described under 1.14.1 Thin-
layer chromatography, using silica gel R4 as the coating substance and a mixture
of 4 volumes of methanol R, 4 volumes of dichloromethane R, 2 volumes of
ammonia (-260 g/1) TS, and 1 volume of acetonitrile R as the mobile phase.
Apply separately to the plate 5 pi of each of 2 solutions containing (A) 10 mg
of Ciprofloxacin hydrochloride per ml, and for solution (B) dissolve 10 mg of
fluoroquinolonic acid RS in a mixture of 0.10ml of ammonia (~100g/l) TS and
90ml of water, and dilute to 100ml with water. Dilute 2.0ml of this solution
to 10 ml with water. Place an evaporating-dish containing 50 ml of ammonia
(-260 g/1) TS in the chromatographic chamber. Expose the plate to the ammonia
vapour in the closed chamber for 15 minutes. Withdraw the plate and transfer
to another chromatographic chamber containing the mobile phase to develop.
air for about 15 minutes, and examine the chromatogram in ultraviolet light
(254 nm).
The spot corresponding to fluoroquinolonic acid in the chromatogram obtained

with solution A is not more intense than that obtained with solution B (0.2%).
Inject 50 pieach of solutions A and F. Record the chromatogram for twice the
retention time of ciprofloxacin.
solutions A and F, and calculate the content of the related substances as a per-
centage. In the chromatogram obtained with solution A, the areas of the peaks
corresponding to the ethylenediamine compound and the by-compound A are
not greater than the corresponding peaks in the chromatogram obtained with
solution F (0.2%); the area of any other peak is not greater than the area of the
peak corresponding to the ethylenediamine compound in the chromatogram
obtained with solution F (0.2%); the sum of the areas of all the peaks, other
than the principal peak, is not greater than 2.5 times the area of the peak cor-
responding to the ethylenediamine compound in the chromatogram obtained
266
with solution F (0.5%). Disregard any peak with an area less than 0.25 times
the area of the peak corresponding to the ethylenediamine compound in the
chromatogram obtained with solution F (0.05%).

matography, using a stainless steel column (25cm x 4.6mm) packed with par-
ticles of silica gel, the surface of which has been modified with chemically
bonded octadecylsilyl groups (5 pm). As the mobile phase, use a mixture of 87

volumes of phosphoric acid (~2.45g/l) TS, adjusted to a pH of 3.0 with tri-
ethylamine R and 13 volumes of acetonitrile R.
Prepare the following solutions in the mobile phase to produce 50ml: solution
(A) contains 0.50 mg of Ciprofloxacin hydrochloride per ml; solution (B) con-
tains 0.50 mg of ciprofloxacin hydrochloride RS per ml; solution (C) contains
O.OSOmg of 1 -cyclopropyl-1 ,4-dihydro-4-oxo-7 -(1 -piperazin- 1 -yl)quinoline-
3-carboxylic acid RS per ml (desfluoro compound); solution (D) contains
O.OSOmg of 7-[(2-aminoethyl)amino]-l-cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-
quinoline-3-carboxylic acid RS (ethylenediamine compound) per ml; solution
(E) contains O.OSOmg of 7-chloro-l-cyclopropyl-l,4-dihydro-4-oxo-6-(piper-
azin-l-yl)quinoline-3-carboxylic acid RS (by-compound A) per ml. For solution
(F) mix 0.1ml of solution A with 1.0ml of solution C, 1.0ml of solution D,
1.0 ml of solution E, and dilute to 50 ml with the mobile phase.
spectrophotometer set at a wavelength of about 278 nm. Maintain the temper-
column at 40 °C.
ature of the
Inject 50|il of solution F. The following order of elution is obtained: desfluoro

compound, ethylenediamine compound, ciprofloxacin and by-compound A.
The retention time of ciprofloxacin is about 9 minutes. Adjust the sensitivity
of the system so that the height of the peak due to the ethylenediamine com-
pound is at least 40% of the full scale of the recorder. The assay is not valid
unless the resolution between the peaks corresponding to the desfluoro com-
pound and the ethylenediamine compound is at least 1.3, and the resolution
between the peaks corresponding to ciprofloxacin and the by-compound A is
at least 3,0. Inject lOpl of solution B. The assay is not valid unless the relative
standard deviation of the peak area of ciprofloxacin is at most 1.0%.
solutions A and B, and calculate the percentage content of C, 7 HisFN 303 ,HCl.
267
CIPROFLOXACINUM
CIPROFLOXACIN
C17H18FN303
Chemical name. l-Cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-7-(l-piperazinyl)-

3-quinolinecarboxylic acid; CAS Reg. No. 85721-33-1.
Description. A white to pale yellow, crystalline powder.

(-750 g/1) TS and dichloromethane R.
Category. Antibacterial.
Storage. Ciprofloxacin should be kept in a well-closed container, protected

from light.
Additional information. Ciprofloxacin exists in different polymorphic forms.
Requirements
Ciprofloxacin contains not less than 98 0 . % and not more than 102 0 % of
.
C 17 H 18FN 3 O 3 ,
Identity test
Carry out the examination as described under 1.7 Spectrophotometry in
the infrared region. The infrared absorption spectrum is concordant with the
spectrum obtained from ciprofloxacin RS or with the reference spectrum of
ciprofloxacin.
Heavy metals. For the preparation of the test solution dissolve 0.5g in acetic
acid (~60g/l) TS and dilute to 30ml with the same solvent. Carry out the pre-
filtration. To the filtrate add 2.0ml of water and determine the heavy metals
268
content as described under 2.2.3 Limit test for heavy metals, Method B; not
more than 20 Hg/g.
Clarity and colour of solution. A solution of 0.25g in 20ml of hydrochlo-

ric VS is clear and not more intensely coloured than standard
acid (0.1 mol/1)
colour solution Gn4 when compared as described under 1.11 Colour of liquids.
Loss on drying. Dry to constant mass at 120 °C under reduced pressure (not
exceeding 0.6kPa or 5mm of mercury); it more than lOmg/g.
loses not
Fluoroquinolonic acid. Carry out the test as described under 1.14.1 Thin-
layer chromatography, using silica gel R4 as the coating substance and a mixture
of 4 volumes of methanol R, 4 volumes of dichloromethane R, 2 volumes of
ammonia (-260 g/1) TS, and 1 volume of acetonitrile R as the mobile phase.
Apply separately to the plate 5|il of each of 2 solutions containing (A) 10 mg
of Ciprofloxacin per ml of acetic acid (-60 g/1) TS, and for solution (B) dissolve
10 mg of fluoroquinolonic acid RS in a mixture of 0.10 ml of ammonia
(-100 g/1) TS and 90 ml of water, and dilute to 100 ml with water. Dilute 2.0ml
of this solution to 10 ml with water. Place an evaporating-dish containing 50 ml
of ammonia (-260 g/1) TS in the chromatographic chamber. Expose the plate to
the ammonia vapour in the closed chamber for 15 minutes. Withdraw the plate
and transfer to another chromatographic chamber containing the mobile phase
to develop. After removing the plate from the chromatographic chamber, allow
it to dry in air for about 15 minutes, and examine the chromatogram in ultra-
violet light (254 nm).
The spot corresponding to fluoroquinolonic acid in the chromatogram obtained

with solution A is not more intense than that obtained with solution B (0.2%).
Related substances. Carry out the test as described under 1.14.4 High
performance liquid chromatography, using a stainless steel column (25 cm x
4.6mm) packed with base-deactivated octadecylsilyl silica gel for chromatog-
raphy (5pm). As the mobile phase, use a mixture of 87 volumes of phosphoric
acid (-2.8 g/1) TS adjusted to a pH of 3.0 with triethylamine R and 13 volumes
of acetonitrile R.
Prepare the following solutions in the mobile phase. For solution (A) add
0.2 ml of phosphoric acid (-105 g/1) TS to 25 mg of Ciprofloxacin, dilute to
50ml, and treat in an ultrasonic bath until a clear solution is obtained. For
solution (B) dilute 0.10ml of solution A to 50ml. For solution (C) use 2.5mg of
Tcyclopropyl-l,4-dihydro-4-oxo-7-(piperazin-Tyl)quinoline-3-carboxylic acid
RS (desfluoro compound) and dilute to 50 ml (this solution is also used to
prepare solutionF), further dilute 1.0ml of this solution to 50ml with the
mobile phase. For solution (D) use 2.5mg of 7-[(2-aminoethyl)amino]-l-
269
cyclopropyl-6-fluoro-l,4-dihydro-4-oxo-quinoline-3-carboxylic acid RS (ethyl-

enediamine compound) and dilute to 50 ml (this solution is also used to prepare
solution F), further dilute 1.0ml of this solution to 50ml with the mobile phase.
For solution (E) use 2.5mg of 7-chloro-l-cyclopropyl-l,4-dihydro-4-oxo-6-
(piperazin-l-yl)quinoline-3-carboxylic acid RS (by-compound A) and dilute to
50ml (this solution is also used to prepare solution F), further dilute 1.0ml of
this solution to 50ml with the mobile phase. For solution (F) mix 0.1 ml of solu-
tion A with 1.0 ml of each of solutions C, D, and E, prior to dilution as described
above, and dilute to 50 ml.
spectrophotometer set at a wavelength of about 278 nm. Maintain the temper-
column at 40 °C.
ature of the
Inject alternately 50 pi each of solutions B, C, D, E, and F. The retention time

of ciprofloxacin is about 9 minutes. Adjust the sensitivity of the system so that
the height of the peak due to the ethylene diamine compound obtained with
solution F is at least 40% of the full scale of the recorder. The test is not valid
unless the resolution between the peaks corresponding to the desfluoro com-
pound and the ethylenediamine compound in the chromatogram obtained with
solution F is at least 1.3, and the resolution between the peaks corresponding
to ciprofloxacin and the by-compound A is at least 3.0.
Inject alternately 50 pi each of solutions A, D, and E. Record the chromatogram

for twice the retention time of ciprofloxacin.
solutions A, D, and E, and calculate the content of the related substances as a
percentage. In the chromatogram obtained with solution A, the areas of the
peaks corresponding to the ethylenediamine compound and by-compound A
are not greater than the corresponding peaks obtained with solutions D and E
(0.2%). The area of any other peak is not greater than the area of the peak
obtained with solution D (0.2%). The sum of the areas of all the peaks, other
than the principal peak, is not greater than 2.5 times the area of the peak in the
chromatogram obtained with solution D (0.5%). Disregard any peak with an
area less than 0.25 times the area of the peak obtained with solution D (0.05%).

C17H18FN3O3.
270
CISPLATINUM
CISPLATIN
Cl NH,
Cl NH3
Cl2H6N2Pt
Chemical name. ds-Diamminedichloroplatinum; (SP-4-2)-diamminedichloro-

platinum; CAS Reg. No. 15663-27-1.
Description. White to yellowish crystals or a yellow powder.
Solubility. Slightly soluble in water; sparingly soluble in dimethylformamide

R; practically insoluble in methanol R.
Storage. Cisplatin should be kept in a tightly closed container, protected from

and stored at a temperature between 2 and 8°C.
light,
Additional information. CAUTION: Cisplatin must be handled with care,

When heated, it blackens at about 270 °C with decomposition.
Requirements
Cisplatin contains not less than 96 0 % and not more than
. the equivalent of
102 0 %
. of Cl 2 H 6N 2 Pt, calculated with reference to the anhydrous substance.
Identity tests

infrared region.
spectrum obtained from cisplatin RS or with the reference spectrum of
cisplatin.
271
C. Place 0.05 g in a glass dish, add 2 ml of sodium hydroxide (~80g/l) TS, and
evaporate to dryness on a water-bath. Dissolve the residue in a mixture of
0.5ml of nitric acid (-lOOOg/1) TS and 1.5ml of hydrochloric acid (-420g/l)
TS, and again evaporate to dryness; the residue is orange. Again dissolve
the residue in 0.5 ml of water and add 0.5 ml of ammonium chloride
(lOOg/1) TS; a yellow, crystalline precipitate is produced.
Clarity and colour of solution. Dissolve 25 mg in 25ml of a solution of

0.22 g of sodium chloride R dissolved in 25ml of carbon-dioxide-free water R;
the solution is clear and not more intensely coloured than standard colour Gn3
when compared as described under 1.11 Colour of liquids. (Keep the solution
for the “pH value”.)

Fischer method, Method A, using 0.5 g of Cisplatin; the water content is not
more than lOmg/g.
pH value. Measure without delay the pH of the solution prepared for the
“Clarity and colour of solution”; 4. 5-6.0.
chromatography, using cellulose R2 previously activated by heating at 150 °C
as the coating substance and a mixture of 1 volume of water and 9 volumes of
acetone R as the mobile phase. Apply separately to the plate 2.5 pi of each of
two solutions in a mixture of equal volumes of dimethylformamide R and
water containing (A) 2 mg of Cisplatin per ml and (B) 2 mg of cisplatin RS per
ml. Also apply 5 pi of each of two solutions in dimethylformamide R contain-
ing (C) 20 mg of Cisplatin per ml and (D) 0.4 mg of Cisplatin per ml. After
removing the plate from the chromatographic chamber, allow it to dry in a
current of warm air, spray it with stannous chloride/ hydrochloric acid TS, and
allow to dry again. Examine the chromatogram in daylight.
With solution C no spot occurs in front of the principal spot. Any other spot
obtained with solution C, other than the principal spot, is not more intense
than that obtained with solution D.
Ultraviolet absorbance ratio. Prior to use, clean all glassware with a mixture
of 3 volumes of hydrochloric acid (-420 g/1) TS and 1 volume of nitric acid
(~1000g/l) TS, rinse thoroughly with water, and dry. Do not use any dichro-
mate solution for cleaning, or acetone or pressurized air for drying. Protect the
test solutions from light, and use them within 1 hour of preparation.
272
Transfer about 98.5mg, accurately weighed, to a 100-ml volumetric flask and

dissolve in sufficient hydrochloric acid (0.1 mol/1) VS to produce 100ml. Stir
with a magnetic bar at a high speed for 5 minutes or place in an ultrasonic bath
for 10 seconds or until completely dissolved.
The ratio of the absorbance measured in a 1-cm layer against hydrochloric acid
(0.1 mol/1) VS at the maximum wavelength of about 301 nm to that at the
minimum wavelength of about 246 nm is not less than 4.5.
Silver. Determine by atomic absorption spectrophotometry “1.8 Atomic spec-

trometry: emission and absorption” at a wavelength of 328 nm using a silver
hollow cathode lamp, an air-acetylene flame, and a slit width of 0.5 nm. Dis-
solve 0.1 g in 15ml of nitric acid (-1000 g/1) TS while heating at 80°C and dilute
with water to 25ml. As a reference solution use silver standard (5pg Ag/ml)
TS; not more than 250 pg of Ag per g.
Assay. Dissolve about 25 mg, accurately weighed, in sufficient hydrochloric

acid (-70 g/1) TS to produce 25 ml. Dilute 1.0 ml of this solution with the same
acid to 25 ml. Transfer 5 ml to a glass-stoppered 25-ml volumetric flask and add
10ml of hydrochloric acid (-70g/l) TS. Place 15ml of hydrochloric acid (-70g/l)
TS in a second flask to serve as a blank. Add 2.5ml of stannous
chloride/hydrochloric acid TSl and dilute to volume with hydrochloric acid
(-70 g/1) TS. Mix and allow to stand for 30 minutes.

against a solvent cell containing the blank. Calculate the amount of Cl 2 HgN 2 Pt
in Cisplatin being examined by comparison with cisplatin RS, similarly and
concurrently examined.
CLINDAMYCINI PHOSPHAS
CLINDAMYCIN PHOSPHATE
C,8H34ClN20aPS
273
Chemical name. (2S-trans)-Methyl 7-chloro-6,7,8-trideoxy-6-(l-methyl-fWtt.‘;-

4-propyl-L-2-pyrrolidinecarboxamido)-l-thio-L-f/zreo-a-D-gia/i3cro-octopyra-
noside 2-(dihydrogen phosphate); CAS Reg. No. 24729-96-2.
Solubility. Freely soluble in water; very slightly soluble in ethanol (-750 g/1)
TS and acetone R.
Storage. Clindamycin phosphate should be kept in a tightly closed container.
Labelling. The designation Clindamycin phosphate for parenteral use indi-

cates that the substance complies with the additional requirements and may
be used for parenteral administration. Expiry date.
Additional information. Clindamycin phosphate is slightly hygroscopic.
Requirements
Clindamycin phosphate contains not less than 95 0 % and not more than
.
100 5 % of C18H34CIN2O8PS, calculated with reference to the anhydrous

.
substance.
Identity tests

infrared region.
spectrum obtained from clindamycin phosphate RS or with the reference
spectrum of clindamycin phosphate.

1-butanol R, 2 volumes of water, and 2 volumes of glacial acetic acid R as
the mobile phase. Apply separately to the plate 5 pi of each of 3 solutions
in methanol R containing (A) 2.0 mg of Clindamycin phosphate per ml, (B)
2.0mg of clindamycin phosphate RS, and for solution (C) dissolve lOmg of
lincomycin hydrochloride RS in 5 ml of solutionB. After removing the plate
from the chromatographic chamber, allow it to dry at 105 °C for 30 minutes,
and spray with potassium permanganate (1 g/1) TS. Examine the chro-
matogram in daylight.
274
=
The with solution A corresponds in position, appear-

principal spot obtained
ance, and intensity to that obtained with solution B. The test is not valid
unless the chromatogram obtained with solution C shows two clearly sep-
arated spots.
C. Dissolve lOmg in 2ml of hydrochloric acid (~70g/l) TS and heat direcdy in

a flame for 1 minute; a disagreeable sulfurous odour is perceptible. Cool,
add 4 ml of sodium carbonate (75g/l) TS and 0.5ml of sodium nitroprusside
(45g/l) TS; a violet-red ring is formed that fades quickly.
D. Boil g under a reflux condenser with a mixture of 5 ml of sodium hydrox-

0.1
ide (-400 g/1) TS and 5 ml of water for 90 minutes. Cool and add 5 ml
of nitric acid (-1000 g/1) TS. Extract with three 15-ml quantities of
dichlorome thane R, and discard the extracts. Filter the aqueous layer
through a paper filter; the filtrate yields reaction B described under 2.1
General identification tests as characteristic of orthophosphates.
ence to the anhydrous substance; [a]™ -1-115 ° to -1-130 °.
Clarity and colour of solution. A solution of 0.040 g in 10 ml of carbon-

dioxide-free water R is clear and colourless.

Fischer method. Method A, using 0.5 g of the substance; the water content is
not more than 0.060 g/g.
Inject alternately 20 pi each of solutions A and D. Continue the recording of

the chromatogram until clindamycin is eluted.
solutions A and D, and calculate the content of the related substances as a per-
other than the principal peak or any peak corresponding to the solvent, is not
greater than 2.5 times the area of the principal peak obtained with solution D
(2.5%). The sum of the areas of all the peaks, other than the principal peak or
any peak corresponding to the solvent, is not greater than 4 times the peak cor-
responding to clindamycin phosphate obtained with solution D (4.0%).

matography, using a stainless steel column (25cm x 4.6mm) packed with par-
275
bonded octadecylsilyl groups (5-1 0pm). As the mobile phase, use a mixture of
8 volumes of potassium dihydrogen phosphate (13.6g/l) TS adjusted to pH 2.5
with phosphoric acid (~105g/l) TS and 2 volumes of acetonitrile R.
Prepare the following solutions in the mobile phase: solution (A) 3.0 mg of Clin-
damycin phosphate per ml; solution (B) 3.0 mg of clindamycin phosphate RS
per ml; for solution (C) dissolve 5 mg of lincomycin hydrochloride RS and
15.0 mg of clindamycin hydrochloride RS in 5.0 ml of solution B and dilute with
sufficient mobile phase to produce 100 ml; and for solution (D) dilute 1.0 ml of
solution B with sufficient mobile phase to produce 100 ml.
Inject 20 pi of solution C.
The assay is not valid unless the first peak (lincomycin) is clearly separated from
the solvent peak, and the resolution between the second peak (clindamycin
phosphate) and the third peak (clindamycin) is at least 6.0. The assay is valid only
if the symmetry factor of the clindamycin phosphate peak is not greater than 1.5.
Inject 20 pi of solution B. If necessary adjust the integrator parameters.
Measure the areas of the peak responses obtained with solutions A and B, and
calculate the percentage content of C 18 H 34CIN 2 OSPS.
Additional requirements for Clindamycin fahosfahate for

prarenteral use
Bacterial endotoxins. Carry out the test as described under 3.4 Test for bacterial
endotoxins; contains not more than 0.6IU of endotoxin RS per mg of clindamycin.
Sterility. Complies with 3.2.2 applying the

Sterility testing of antibiotics,
membrane filtration test procedure and using a solution in water containing
150 mg of Clindamycin phosphate per ml.
276
CLOFAZIMINUM
CLOFAZIMINE
Molecular formula. C27H22CI2N4
Graphic formula.
Cl
Chemical name. 3-(;7-Chloroanilino)-10-(/:^-chlorophenyl)-2,10-dihydro-2-(iso-

propylimino)phenazine; N,5-bis(4-chlorophenyl)-3,5-dihydro-3-[(l-methylethyl)
imino]-2-phenazinamine; CAS Reg. No. 2030-63-9.
Description. A reddish brown, fine powder; odourless or almost odourless.
Solubility. Practically insoluble in water; slightly soluble in ethanol (-750 g/1)

TS and in ether R.
Category. Antileprosy drug.
Storage. Clofazimine should be kept in a well-closed container.
Additional information. Clofazimine melts at about 217 °C.
Requirements
Definition. Clofazimine contains not less than 98.0% and not more than
101.0% of C27H22CI2N4, calculated with reference to the dried substance.
Identity tests

277
spectrum obtained from clofazimine RS or with the reference spectrum of

clofazimine.
B. The absorption spectrum of a 5.0pg/ml solution in hydrochloric

acid/methanol (0.01 mol/1) VS, when observed between 230nm and 600 nm,
exhibits 2 maxima at about 283nm and 487 nm. The absorbances of a 1-cm
layer at these wavelengths are about 0.65 and 0.32, respectively.
C. Dissolve about 2 mg in 3 ml of acetone R and add 0.1 ml of hydrochloric acid

(-420 g/1) TS; an intense violet colour is produced. Add 0.5 ml of sodium
hydroxide (-200 g/1) TS; the colour changes to orange-red.
5-Omg/g.
chromatography, using silica gel R6 (a precoated plate from a commercial
source is suitable), exposed immediately before use to ammonia vapour by sus-
pending the plate for 30 minutes in a chromatographic chamber containing a
shallow layer of ammonia (~17g/l) TS. As the mobile phase, to be used in a
separate chamber, prepare a mixture of 85 volumes of dichloromethane R and
4 volumes of 1 -propanol R. Apply separately to the plate 5 |il of each of 3 solu-
tions in chloroform R containing (A) 20 mg of the test substance per ml, (B)
0.16mg of the test substance per ml, and (C) O.lOmg of the test substance per
ml. Allow the mobile phase to ascend 12 cm above the line of application. After
removing the plate from the chromatographic chamber, allow it to dry in air
for 5 minutes, and replace it in the chamber. Allow the mobile phase to ascend
again 12 cm, remove the plate from the chamber, dry it in air for 5 minutes,
with solution A, other than the principal spot, is not more intense than the spot
obtained with solution C, except that 2 such spots are not more intense than
that obtained with solution B.
Assay. Dissolve about 0.4 g, accurately weighed, in 20ml of chloroform R, add

50 ml of acetone R, and titrate with perchloric acid (0.1 mol/1) VS as described
under 2.6 Non-aqueous titration. Method A. Each ml of perchloric acid
(0.1 mol/1) VS is equivalent to 47.34mg of C27H22CI2N4.
278
CLOMIFENI CITRAS
CLOMIFENE CITRATE
Molecular formula. C26H28ClN0,C6Ha07
Graphic formula.
CHjCOOH
HO — C — COOH
I
CHjCOOH
Chemical name. 2-[p’-(2-Chloro-l,2-diphenylvinyl)phenoxy]triethylamine citrate
(1 : 2-[4-(2-chloro-l,2-diphenylethenyl)phenoxy]-N,N-diethylethanamine 2-
1);
hydroxy-l,2,3-propanetricarboxylate (1:1); CAS Reg. No. 50-41-9.
Description. A white to pale yellow powder; odourless.
Solubility. Slightly soluble in water; freely soluble in methanol R; sparingly

soluble in ethanol (-750 g/1) TS; practically insoluble in ether R.
Category. Ovulation inducer.
Storage. Clomifene citrate should be kept in a well-closed container, protected

from light.
Additional information. Clomifene citrate is a mixture of the £ and Z geo-

metric isomers.
Requirements
less than 97.0% and not more than
Definition. Clomifene citrate contains not
101.0% of C26H28ClN0,CgHs07, and not less than 30.0% and not more than
50.0% of the Z-isomer, both calculated with reference to the anhydrous
substance.
Identity tests
279

spectrum obtained from clomifene citrate RS or with the reference spectrum
of clomifene citrate.
B. The spectrum of a 25pg/ml solution in hydrochloric

ultraviolet absorption
when observed between 220nm and 350nm, exhibits
acid (0.1 mol/1) VS,
maxima about 235 nm and 292 nm; the absorbances of a 1-cm layer at
at
these wavelengths are about 0.79 and 0.44, respectively.
C. A lOmg/ml solution yields reaction B described under 2.1 General identifi-

Solution in methanol. A solution of l.Og in 30ml of methanol R is clear and

colourless.

is not more than lOmg/g.
Z-isomer. Carry out the test as described under 1.14.1 Thin-layer chro-
volumes of chloroform R, 10 volumes of methanol R, and 1 volume of water
as the mobile phase. Dissolve lOOmg of the test substance in 50ml of a mixture
of 3 volumes of chloroform R and 1 volume of ethanol (-750 g/1) TS (solution
A), and dissolve 50 mg of clomifene citrate Z-isomer RS in 50 ml of the same
solvent mixture (solution B). With a syringe apply 100 pi of solution A to one
side and 100 pi of solution B to the other side, keeping the centre of the plate
to serve as a blank. After removing the plate from the chromatographic
light (254 nm). Mark the bands of the Z-isomer with a pencil, scratch off sep-
arately the bands of silica gel produced by solutions A and B, as well as a band
of a similar size from the centre of the plate for the blank, and transfer them
to separate test-tubes. Add 10ml of ethanol (-750 g/1) TS to each tube, shake
vigorously and then centrifuge or filter. Measure the absorbances of a 1-cm
layer of the filtered solutions at 240 nm against a solvent cell containing ethanol
(-750 g/1) TS. Calculate the content of Z-isomer in the following manner:
Deduct the absorbance of the blank solution from the absorbances of the test
and reference solutions and apply the formula: (Aj) (Wf) (1000)/(A2) (Wf), where
Ai is the absorbance of the test substance, A 2 the absorbance of clomifene citrate
Z-isomer RS, the weight in mg of the test substance, and W
2 the weight in
280
mg of clomifene citrate Z-isomer RS; the content of the Z-isomer is not less
than 300mg/g and not more than 500mg/g.
Assay. Dissolve about l.Og, accurately weighed, in 30ml of glacial acetic acid
aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/1) VS is equiv-
alent to 59.81 mg of C26H2 sC1N0,C6H807.
CLOXACILLINUM NATRICUM
CLOXACILLIN SODIUM
Cloxadllin sodium (non-injectable)
Cloxadllin sodium, sterile
Molecular formula. Ci 9 Hi 7 ClN 3 Na 05 S,H 20
Graphic formula.
COONa
Chemical name. Sodium (2S,5R,6R)-6-[3-(o-chlorophenyl)-5-methyl-4-isoxa-

zolecarboxamido]-3, 3-dime thyl-7-oxo-4-thia- l-azabicyclo[3. 2. 0]heptane-2-
carboxylate monohydrate; sodium [2S-(2a,5a,6P)]-6-[[[3-(2-chlorophenyl)-5-
methyl-4-isoxazolyl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo-
[3.2.0]heptane-2-carboxylate monohydrate; monosodium [3-(o-chlorophenyl)-
5-methyl-4-isoxazolyl]penicillin monohydrate; CAS Reg. No. 7081-44-9.
Solubility. Soluble in 2.5 parts of water and in 30 parts of ethanol (-750 g/1)
TS.
281
Storage. Cloxacillin sodium should be kept in a tightly closed container, pro-

tected from light.
Labelling. The designation sterile Cloxacillin sodium indicates that the sub-
stance complies with the additional requirements for sterile Cloxacillin sodium
Additional information. Cloxacillin sodium is hygroscopic. Even in the

absence of light, Cloxacillin sodium is gradually degraded on exposure to a
Requirements
Definition. Cloxacillin sodium contains not less than 90.0% of total penicillins
calculated as C 19H 18 CIN 3 O 5 S, and with reference to the anhydrous substance.
Identity tests

spectrum obtained from cloxacillin sodium RS or with the reference spectrum
of cloxacillin sodium.
B. Place 2 mg into a test-tube, add 2 mg of disodium chromotropate R and 2 ml

of sulfuric acid (~1760g/l) TS. Immerse the tube in a suitable bath at 150 °C
for 3-4 minutes; a purple colour is produced.
C. Ignite 20 mg and dissolve the residue in acetic acid (~60g/l) TS. The solu-
tion yields reaction B described under 2.1 General identification tests as
characteristic of sodium.
Specific optical rotation. Use a 10 mg/ml solution, and calculate with refer-
ence to the anhydrous substance; [a]§^“‘' = +163 to -tl72°.

Chlorine. Carry out the combustion as described under the 2.4 Oxygen flask
method, but using 0.040 g of the test substance and 20ml of sodium hydrox-
ide (1 mol/1) VS as the absorbing liquid. When the process is complete, add
2.5ml of nitric acid (-130 g/1) TS, 2.5ml of water, and 20ml of silver nitrate
282
(0.01 mol/1) VS and titrate with ammonium thiocyanate (0.01 mol/1) VS, using
ferric ammonium sulfate (45g/l) TS as indicator. Repeat the operation without
the substance being tested. Each ml of silver nitrate (0.01 ml/1) VS is equivalent
to 0.3546mg of Cl; the chlorine content is 70-75mg/g.

stoppered tubes. To one tube add 10ml of imidazole/mercuric chloride TS, mix,
stopper the tube and place in a water-bath at 60 °C for exactly 25 minutes. Cool
the tube rapidly to 20 °C (solution A).
Without delay measure the absorbance of a 1-cm layer at the maximum at

about 343 nm against a solvent cell containing a mixture of 2.0 ml of water and
tion B.
B, calculate theamount of C] 9 H] 7 ClN 3Na 05 S in the substance being tested by
comparison with cloxacillin sodium RS, similarly and concurrendy examined.
In an adequately calibrated spectrophotometer the absorbance of the reference
Additional requirements for Cloxacillin Sodium for sterile use

terial endotoxins; contains not more than 40 lU of endotoxin RS per mg of
cloxacillin.

the membrane procedure with added penicillinase TS or the direct
filtration test
test procedure. Use the sampling plan described under 3.2.1 Test for sterility of
non-injectable preparations.
CODEINI PHOSPHAS
CODEINE PHOSPHATE
Codeine phosphate hemihydrate
Codeine phosphate sesquihydrate
Molecular formula. C18H21NO3, H3PO4, IH2O (hemihydrate); C18H21NO3,

H3PO4, (sesquihydrate).
283
:
Relative molecular mass. 406.4 (hemihydrate); 424.4 (sesquihydrate).
Graphic formula.
n = I (hemihydrate)
n = 1|- (sesquihydrate)
Chemical name. 7,8-Didehydro-4,5a-epoxy-3-methoxy-17-methylmorphinan-

6a-ol phosphate (1 1) (salt) hemihydrate; CAS Reg. No. 41444-62-6 (hemihydrate).
:
7,8-Didehydro-4,5a-epoxy-3-methoxy-17-methylmorphinan-6a-ol phosphate (1
1) (salt) sesquihydrate; CAS Reg. No. 5913-76-8 (sesquihydrate).
Description. Small, colourless crystals or a white, crystalline powder;

odourless.
Solubility. Soluble in 4 parts of water; slightly soluble in ethanol (-750 g/1) TS;
Category. Antitussive; analgesic.
Storage. Codeine phosphate should be kept in a tightly closed container, pro-

tected from light.
Labelling. The designation on the container should state if the Codeine phos-
phate is the hemihydrate or the sesquihydrate.
Additional information. Codeine phosphate effloresces in dry air.
Requirements
Definition. Codeine phosphate contains not less than 98.0% and not more
than 101.0% of C]sH 2 iN 03 ,H 3 P 04 ,
substance.
284
Identity tests
A. Dissolve 5 mg in 1ml of sulfuric acid (-1760 g/1) TS, add 1 drop of ferric chlo-
ride (25 g/1) TS, and warm on a water-bath; a blue colour produced, which
is
changes to red on the addition of 1 drop of nitric acid (~130g/l) TS,
B. Dissolve Img 0.5ml of selenious acid/sulfuric acid TS; a green colour is

in
produced, which rapidly changes to blue, then slowly to dark yellow-green.
C. Neutralize a 20 mg/ml solution with ammonia (-100 g/1) TS; it yields reac-
tion B described under 2.1 General identification tests as characteristic of
orthophosphates.
D. To 5 ml of a 0.2g/ml solution add 1 ml of ammonia (-100 g/1) TS, cool and

scratch the inside of the test-tube to induce crystallization. Wash the pre-
cipitate with ethanol (-750g/l) TS and dry at 105 °C. Melting temperature,
about 156°C (codeine base).
ence to the dried substance; [a]c = -98 to -102°.
Chlorides. Dissolve 0.70g in a mixture of 2ml of nitric acid (-130g/l) TS and

Sulfates. Dissolve 0,50 g in 20ml of water and proceed as described under

2.2.2 Limit test for sulfates; the sulfate content is not more than 1 mg/g.

and not more intensely coloured than standard colour solution Yw2 when com-
Loss on drying. Dry to constant weight at 105°C: Codeine phosphate (hemi-

hydrate) loses not more than 30 mg/g. Codeine phosphate sesquihydrate loses
not less than 50mg/g and not more than 70mg/g.
pH value. pH of a 0.04 g/ml solution, 4. 2-5,0.
Related alkaloids. Carry out the test as described under 1.14.1 Thin-layer
72 volumes of ethanol (-750 g/1) TS, 30 volumes of cyclohexane R and 6
volumes of ammonia (-260 g/1) TS as the mobile phase. Apply separately to the
plate 10 pi of each of 2 solutions in a mixture of 4 volumes of hydrochloric acid
(0.01 mol/1) VS and 1 volume of ethanol (-750 g/1) TS containing (A) 50mg of
the test substance per ml and (B) 0.66mg of the test substance per ml. After
removing the plate from the chromatographic chamber, allow it to dry in air,
spray with potassium iodobismuthate TS2, and examine the chromatogram in
285
daylight. Any spot obtained with solution A, other than the principal spot, is

alent to 39.74mg of Ci 8 H 2 iN 03 ,H 3 P 04 .
CODEINUM MONOHYDRICUM
CODEINE MONOHYDRATE
Molecular formula. Ci 8 H 2 iN 03 ,H 20
Graphic formula.
Chemical name. 7,8-Didehydro-4,5a-epoxy-3-methoxy-17-methylmorphinan-

6a-ol monohydrate; CAS Reg. No. 6059-47-8.
ether R.
Category. Antitussive; analgesic.
Storage. Codeine monohydrate should be kept in a tightly closed container,

Additional information. Codeine monohydrate effloresces slowly in dry air.
286
Requirements
Definition. Codeine monohydrate contains not less than 99.0% and not more
than 101.0% of C18H21NO3, calculated with reference to the dried substance.
Identity tests
A. Dissolve 5mg in 1ml of sulfuric acid (~1760g/l) TS, add 1 drop of ferric chlo-
and warm on a water-bath; a blue colour is produced, which
ride (25 g/1) TS,
changes to red on the addition of 1 drop of nitric acid (~130g/l) TS.
B. Dissolve Img 0.5ml of selenious acid/sulfuric acid TS; a green colour is

in
produced, which rapidly changes to blue, then slowly to dark yellow-green.
Specific optical rotation. Use a 20 mg/ml solution in ethanol (-750 g/1) TS

and calculate with reference to the dried substance; [a]o = -142 to -146°.
Clarity and colour of solution. A solution of 0.050g in 10ml of water is
pH value. pH of a 5.0 mg/ml solution in carbon-dioxide-free water R, not less

than 9.0.
72 volumes of ethanol (-750 g/1) TS, 30 volumes of cyclohexane R, and 6
volumes of ammonia (-260 g/1) TS as the mobile phase. Apply separately to the
plate 1 0 pi of each of 2 solutions in a mixture of 4 volumes of hydrochloric acid
(0.01 mol/1) VS and 1 volume of ethanol (-750 g/1) TS containing (A) 50mg of
the test substance per ml and (B) 0.66 mg of the test substance per ml. After
spray with potassium iodobismuthate TS2, and examine the chromatogram in
daylight. Any spot obtained with solution A, other than the principal spot, is

Rl, add 10 ml of acetic anhydride R, and titrate with perchloric acid (0.1 mol/1)
VS as described under 2.6 Non-aqueous titration. Method A. Each ml of per-
chloric acid (0.1 mol/1) VS is equivalent to 29.94 mg of C18H21NO3.
287
COFFEINUM
CAFFEINE
Caffeine anhydrous
Caffeine monohydrate
Molecular formula. C8H10N4O2 (anhydrous); C8HioN402,H20 (monohydrate).
Graphic formula.
• nHÔ
n = 0 (anhydrous)
n = 1 (monohydrate)
Chemical name. 3,7-Dihydro-l,3,7-trimethyl-lf/-purine-2,6-dione; CAS Reg.

No. 58-08-2 (anhydrous). 3,7-Dihydro-l,3,7-trimethyl-lf/-purine-2,6-dione
monohydrate; CAS Reg. No. 5743-12-4 (monohydrate).
Description. Silky, colourless crystals or a white, crystalline powder;

odourless.
TS; slightly soluble in ether R.
Category. Central nervous stimulant.
Storage. Caffeine should be kept in a well-closed container.
Labelling. The designation on the container of Caffeine should state whether

the substance is the monohydrate or is in the anhydrous form.
Additional information. Caffeine monohydrate is efflorescent in air.
Requirements
Definition. Caffeine contains not less than 98.5% and not more than 101.0%
of C8H10N4O2, calculated with reference to the dried substance.
288
Identity tests

infrared region. For Caffeine monohydrate, the substance must be previ-
ously dried to constant weight at 80 °C. The infrared absorption spectrum
is concordant with the spectrum obtained from caffeine RS or with the ref-
erence spectrum of caffeine.
B. To 10 mg, contained in a porcelain dish, add 1ml of hydrochloric acid

(-250 g/1) TS and 0.5ml of hydrogen peroxide (~60g/l) TS, and evaporate to
dryness on a water-bath. Add 1 drop of ammonia (-100 g/1) TS; the residue
acquires a purple colour, which disappears upon the addition of 2-3 drops
of sodium hydroxide (-80 g/1) TS.
C. To a saturated solution add a few drops of iodine TS; the solution remains
clear. Add a few drops of hydrochloric acid (-70 g/1) TS; a brown precipi-
tate is produced. On neutralization with sodium hydroxide (-80 g/1) TS, the
precipitate dissolves.
D. Melting temperature, after drying at 80 °C, about 236 °C.
Clarity and colour of solution. A solution of 5.0g in 10ml of boiling water

is clear and colourless.
Loss on drying. Dry to constant weight at 80 °C: Caffeine (anhydrous) loses

not more than 5.0mg/g. Caffeine monohydrate loses not less than 50mg/g and
of 4 volumes of 1 -butanol R, 3 volumes of chloroform R, and 1 volume of
ammonia (-260 g/1) TS as the mobile phase. Prepare 2 solutions in a mixture of
6 volumes of chloroform R and 4 volumes of methanol R containing (A) 20 mg
of the test substance per ml and (B) 0.20 mg of the test substance per ml. Apply
separately to the plate 10 pi of solution A and 5 pi of solution B. After remov-
examine the chromatogram in ultraviolet light (254 nm). Any spot obtained
289
Assay. Dissolve about 0.18g, accurately weighed, in 10ml of acetic anhydride

R and 20ml of toluene R, and titrate with perchloric acid (0.1 mol/1) VS as
described under 2.6 Non-aqueous titration, Method A. Each ml of perchloric
acid (0.1 mol/1) VS is equivalent to 19.42 mg of CSH 10N 4 O 2 .
COLCHICINUM
COLCHICINE
Graphic formula.
Chemical name. (S)-N-(5,6,7,9-Tetrahydro-l,2,3,10-tetramethoxy-9-oxobenzo

[fl]heptalen-7-yl)acetamide; CAS Reg. No. 64-86-8.
Description. Pale yellow to pale greenish yellow crystals, amorphous scales

or a powder; odourless or almost odourless.
Solubility. Soluble in water; freely soluble in ethanol (-750 g/1) TS; slightly
soluble in ether R.
Category. Antigout drug.
Storage. Colchicine should be kept in a tightly closed container, protected

from light.
Additional information. Colchicine is an alkaloid obtained from Cokhicunt

autumnale L. (Earn. Liliaceae). It darkens on exposure to light. CAUTION:
Colchicine is extremely poisonous and must be handled with care.
290
=
Requirements
Definition. Colchicine contains not less than 97.0% and not more than
103.0% of C 22 H 25 NO 6 ,
calculated with reference to the anhydrous and solvent-
free substance.
Identity tests

infrared region.
spectrum obtained from colchicine RS or with the reference spectrum of
colchicine.
B. The absorption spectrum of a lOpg/ml solution in ethanol (-750 g/1) TS,

when observed between 230 nm and 400 nm, exhibits 2 maxima at about
243 nm and 350 nm.
C. Dissolve 30 mg in 1 ml of ethanol (-750 g/1) TS and add 1 drop of ferric chlo-

ride (25 g/1) TS; a red colour is immediately produced.
D. Mix 1 mg with about 0.2 ml of sulfuric acid (~1760g/l) TS; a lemon-yellow
colouris produced. Add about 0.1 ml of nitric acid (-1000 g/1) TS; the colour
changes to greenish blue, rapidly becoming reddish and finally yellow or

almost colourless. Then add a few drops of sodium hydroxide (-80 g/1) TS;
the colour changes to red.
ence to the dried and solvent-free substance; [a]n -425 to -460°.
Content of solvent and water. Dry at 130 °C for 4 hours, using about 0.5g
of the substance, and determine the loss of weight. Weigh 0.3 g of the dried
material and determine the water content as described under 2.8 Determina-
tion of water by the Karl Fischer method. Method A, using pyridine R as the
solvent; the sum of the loss of weight and of the water content, both expressed
in mg/g, is not less than 115mg/g and not more than 145mg/g.
Colchiceine. Dissolve 0.050 g in 4 ml of water, add 0.2 ml of ferric chloride

(25 g/1) TS, dilute to 6 ml with water, and mix; the colour produced in the test
solution is not more intense than that of the standard colour solution produced
by mixing 2 ml of iron colour TS, 1 ml of cobalt colour TS, 2 ml of copper colour
TS and 0.70ml of hydrochloric acid (-70 g/1) TS, when compared as described
291
chromatography, using a suitable aluminium oxide containing a substance that
fluoresces at about 254 nm as the coating substance and a mixture of 25
volumes of chloroform R, 20 volumes of acetone R, and 0.4 volumes of
ammonia (-260 g/1) TS as the mobile phase. Apply separately to the plate 2 pi
of each of 2 solutions in ethanol (-750 g/1) TS containing (A) 50 mg of the test
substance per ml and (B) 2.5mg of the test substance per ml. After removing
from the chromatographic chamber, allow it to dry in air, and examine

the plate
with solution B.
Assay. Dissolve about 0.05g, accurately weighed, in a mixture of 10 ml of

acetic anhydride R and 20 ml of toluene R, and titrate with perchloric acid
2.6 Non-aqueous titration. Method A. Each ml of perchloric acid (0.02 mol/1)
VS is equivalent to 7.988 mg of C 22H 25 NO 5 .
COLECALCIFEROLUM
COLECALCIFEROL
Molecular formula. C27H44O
Graphic formula.
Chemical name. (5Z,7£)-9,10-Secocholesta-5,7,10(19)-trien-3|3-ol; CAS Reg.

No. 67-97-0.
Other name. Cholecalciferol.
292
Solubility. Practically insoluble in water; soluble in ethanol (~750 g/1) TS, ether
R.
Category. Vitamin, antirachitic.
Storage. Colecalciferol should be kept in a hermetically closed container, in

an inert atmosphere, protected from light and stored in a cool place.
Additional information. Even in the absence of light, Colecalciferol is grad-

Requirements
Definition. Colecalciferol contains not less than 95.0% and not more than
105.0% of C27H44O.
Identity tests

infrared region.
spectrum obtained from colecalciferol RS or with the reference spectrum of
colecalciferol.
B. Dissolve 1 mg in 1 ml of dichloroethane R and add 4 ml of antimony trichlo-

ride TS; a yellowish orange colour is produced.
C. Dissolve 5 mg in 5 ml of chloroform R, add 0.3 ml of acetic anhydride R and

0.1ml of sulfuric acid (-1760 g/1) TS, and shake vigorously; a bright red
colour is produced which changes rapidly through violet to blue and finally
to green.
Specific optical rotation. Use a freshly prepared lOmg/ml solution in alde-

hyde-free ethanol (-750 g/1) TS; [a]n = -tl05 to -1-112°.
7-Dehydrocholesterol. Dissolve 0.04g in 2ml of ethanol (-750 g/1) TS, using

a glass-stoppered test-tube, and add 1 ml of digitonin TS; the solution produced
remains clear for 12 hours.
Assay. With the aid of heat, dissolve about 20 mg, accurately weighed, in suf-
ficient aldehyde-free ethanol (-750 g/1) TS to produce 100ml; dilute 5.0ml of
this solution to 100 ml with the same solvent. Measure without delay the
absorbance of a 1-cm layer of the diluted solution at the maximum at about
265 nm. Calculate the amount of C27H44O in the substance being examined by
293
comparison with cole calciferol RS, similarly and concurrently examined. In an

adequately calibrated spectrophotometer the absorbance of the reference solu-
tion should be 0.48 + 0.03.
CYANOCOBALAMINUM
CYANOCOBALAMIN
Molecular formula. CssHasCoNwOMP
Graphic formula.
Chemical name. a-(5,6-Dimethylbenzimidazol-2-yl)cobamide cyanide; CAS

Reg. No. 68-19-9,
Other name. Vitamin B 12 .
Description. Dark red crystals or a red, crystalline powder; odourless.
Solubility. Soluble in 80 parts of water; soluble in ethanol (~750g/l) TS; prac-

tically insoluble in acetone R, and ether R.
Category. Haemopoietic.
Storage. Cyanocobalamin should be kept in a tightly closed container, pro-

tected from light.
294
Additional information. The anhydrous form of Cyanocobalamin is highly

hygroscopic.
Requirements
Definition. Cyanocobalamin contains not less than 96.0% and not more than
102.0% of C63H88C0N14O14P, calculated with reference to the dried substance.
Identity tests
A. The absorption spectrum of a 20pg/ml solution, when observed between
230 nm and 600 nm, exhibits 3 maxima at about 278 nm, 361 nm and
550 nm; the ratio of the absorbance of a 1-cm layer at 361 nm to that at
278nm is between and 1.90, and the ratio of the absorbance
1.70 at 361 nm
to that at 550 nm is between 3.15 and 3.45.
B. Mix 1 mg with about 10 mg of potassium sulfate R and 2 drops of sulfuric

acid (~100g/l) TS, heat the mixture carefully to redness until fused. Cool,
break up the mass with a glass rod, and dissolve in 3 ml of water by boiling.
Add 1 drop of phenolphthalein/ethanol TS and, drop by drop, sodium
hydroxide (~80g/l) TS until the solution is just pink. Add 0.5 g of sodium
acetate R, 0.5ml of acetic acid (~60g/l) TS, and 0.5ml of l-nitroso-2-naph-
thol-3, 6-disodium disulfonate (2g/l) TS; a red or orange-red colour appears
immediately. Add 0.5ml of hydrochloric acid (-250 g/1) TS and boil for 1
minute; the red colour persists.
Clarity of solution. A solution of 20mg in 10ml of water is clear.
of mercury); it loses not more than 120mg/g.
Pseudocyanocobalamin. Dissolve Img in 20 ml of water. Transfer the solu-

tion to a small separator, add 5 ml of a mixture of equal volumes of carbon
tetrachloride R and freshly distilled e-cresol R, and shake well for about 1
minute. Allow to separate, draw off the lower layer into a second small sepa-
rator,add a mixture of 2.5ml of sulfuric acid (~570g/l) TS and 2.5ml of water,
shake well and allow to separate completely (the separation of the layers may
be facilitated by centrifuging). Prepare a reference solution containing 1.5ml of
potassium permanganate (0.002 mol/1) VS in 250 ml of water. The separated
upper layer of the test solution is colourless or not more intensely coloured than
the reference solution when compared as described under 1.11 Colour of
liquids.
chromatography, using equal parts of silica gel R1 and kieselguhr R1 as the
coating substance and a mixture of 15 volumes of chloroform R, 10 volumes
295
ofmethanol R, and 3 volumes of ammonia (-100 g/1) TS as the mobile phase.

Carry out all operations protected from light. Apply separately to the plate
lOpl of each of 3 solutions containing (A) S.Omg of the test substance per ml, (B)
0.20mg of the test substance per ml, and (C) O.lOmg of the test substance per
dry in air, and examine the chromatogram in daylight. Any spot obtained with
solution A, other than the principal spot not more intense than that obtained
is
with solution B. Not more than one spot obtained with solution A, other than
the principal spot, is more intense than that obtained with solution C.

produce 1000ml. Determine the absorbance of this solution in a 1-cm layer at
the maximum at about 361 nm and calculate the content of CgaHaaCoNuOi^P,
using the absorptivity value of 20.7 (EJcm = 207).
CYCLOPHOSPHAMIDUM
CYCLOPHOSPHAMIDE
Molecular formula. CjHisChNaOahHaO
Graphic formula.
*
'i’^N(CH^ CH,, Cl)^
k^NH
I
Chemical name. 2-[Bis(2-chloroethyl)amino]tetrahydro-2/f-l,3,2-oxazaphos-

phorine 2-oxide monohydrate; A(N-bis(2-chloroethyl)tetrahydro-2/i-l,3,2-
oxazaphosphorin-2-amine 2-oxide monohydrate; CAS Reg. No. 6055-19-2
(monohydrate).
Other name. Cyclophosphanum.
Solubility. Soluble in water; freely soluble in ethanol (-750 g/1) TS; slightly
soluble in ether R.
296
Storage. Cyclophosphamide should be kept in a tightly closed container and

stored at a temperature between 2 and 30°C.
Additional information. CAUTION; Cyclophosphamide must be handled

with care, avoiding contact with the skin and inhalation of airborne particles.
Requirements
Definition. Cyclophosphamide contains not less than 98.0% and not more
than 101.0% of C7H15CI2N2O2P, calculated with reference to the anhydrous
substance.
Identity tests
A. Dissolve 0.1 g in 10ml of water and add 5 ml of silver nitrate (40g/l) TS; no
precipitate is produced. Boil; a white precipitate is produced, which is insol-
uble in nitric acid (-130 g/1) TS but soluble in ammonia (~100g/l) TS from
which it is reprecipitated by the addition of nitric acid (-130 g/1) TS.
B. Dissolve 20mg in 1ml of sulfuric acid (~100g/l) TS and heat until white
fumes are evolved. After cooHng, add 5 ml of water and shake. Neutralize
with ammonia (-100 g/1) TS, then acidify with nitric acid (-130 g/1) TS; this
test yields reaction A described under 2.1 General identification tests as char-
acteristic of orthophosphates.
Melting range. 49-53 °C, determined without previous drying.

and colourless.

pH value. pH of a 20 mg/ml solution in carbon-dioxide-free water R, deter-

mined 30 minutes after its preparation, 4. 0-7.0.
50 volumes of benzene R, 25 volumes of chloroform R, and 25 volumes of
methanol R as the mobile phase. Apply separately to the plate 10 pi of each
of 2 solutions: (A) 25 mg of the test substance per ml of chloroform R and (B)
0.125g of the test substance dissolved in 5.0ml of water, boiled under a reflux
condenser for 30 minutes, and then cooled to 20 °C. After removing the plate
from the chromatographic chamber, allow it to dry in air and spray it with
triketohydrindene/methanol TS. Examine the chromatogram in daylight. With
297
solution B, a pale violet spot is obtained at an Revalue between 0.10 and 0.25;
other spots could also appear. With solution A, a brown-violet spot is obtained
with an R, value between 3.50 and 5.50, and no other spot is obtained above
this spot.
Assay. To about weighed, add 20 ml of potassium hydroxide/

0.2 g, accurately
ethanol (0.5 mol/1) VS. Boil under a reflux condenser for 1 hour, cool, then add
30ml of water, 3ml TS and 20.0ml of silver nitrate

of nitric acid (~1000g/l)
(0.1 mol/1) VS. Shake the flask, add 5ml of diethyl phthalate R and titrate the
excess of silver nitrate with ammonium thiocyanate (0.1 mol/1) VS, using 5 ml
of ferric ammonium sulfate (45g/l) TS as indicator. Repeat the operation
without the substance being examined and make any necessary corrections.
Each ml of silver nitrate (O.lmol/1) VS is equivalent to 13.05mg of C 7 H 15 CI 2 N 2 O 2 P.
CYTARABINUM
CYTARABINE
Graphic formula.
OH
Chemical name. l-|3-D-Arabinofuranosylcytosine; 4-amino-l-|3-D-arabinofu-

ranosyl-2(lH)-pyrimidone; CAS Reg. No. 147-94-4.
Solubility. Freely soluble in water; slightly soluble in ethanol (-750 g/1) TS.
298
Storage. Cytarabine should be kept in a well-closed container, protected from

light, and stored at a temperature not exceeding 15 °C.
Additional information. CAUTION; Cytarabine must be handled with care,

Requirements
Definition. Cytarabine contains not less than 99.0% and not more than
100.5% of CgHisNsOs, calculated with reference to the dried substance.
Identity tests
infrared region.
spectrum obtained from cytarabine RS or with the reference spectrum of
cytarabine.
B. The absorption spectrum of a lOpg/ml solution in hydrochloric acid

(0.1 mol/1) VS, when observed between 230nm and 350nm, exhibits a
maximum at about 280 nm; the absorbance of a 1-cm layer at this wave-
length is about 0.55.
Specific optical rotation. Use a lOmg/ml solution; = -tl54 to -1-160°.
Sulfated ash. Not more 5.0mg/g.
Loss on drying. Dry to constant weight at 60 °C under reduced pressure

(not exceeding 0.6 kPa or about 5 mm
of mercury); it loses not more than
lOmg/g.
13 volumes of ethylmethylketone R, 4 volumes of acetone R, and 3 volumes
of water as the mobile phase. Apply separately to the plate 5|il of each of 3
solutions containing (A) 40 mg of the test substance per ml, (B) 0.20 mg of
undine per ml, and (C) 0.20 mg of the test substance per ml. After removing
R
the platefrom the chromatographic chamber, allow it to dry in air, and examine
tion A with an 1?^ value of about 1.1, compared with the spot obtained with
solution B, is not more intense than that obtained with solution B. Any other
than that obtained with solution C.
299
with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous

24.32 mg of C9H13N3O5.
DACARBAZINUM
DACARBAZINE
H
C.H,„N,0
Chemical name. 5-(3,3-Dimethyl-l-triazeno)imidazole-4-carboxamide; 5-(3,3-

dimethyl-l-triazenyl)-l/i-imidazole-4-carboxamide; CAS Reg. No. 4342-03-4.
Description. A colourless or pale yellow, crystalline powder.
Solubility. Slightly soluble in water and ethanol (-750 g/1) TS.
Storage. Dacarbazine should be kept in a tightly closed container, protected

from light, and stored at a temperature not exceeding 8°C.
Additional information. CAUTION: Dacarbazine must be handled with

Requirements
Dacarbazine contains not less than 97 0 %
and not more than 102 0
. . % of
CgHioNÔ, calculated with reference to the dried substance.
Identity tests

300
spectrum obtained from dacarbazine RS or with the reference spectrum of

dacarbazine.
B. The absorption spectrum of a 6pg/ml solution in hydrochloric acid

(0.1 mol/1) when observed between 230nm and 350nm, exhibits a
VS,
maximum about 323 nm and a pronounced shoulder at 275 nm. The
at
absorbance of a 1-cm layer at the maximum wavelength of 323 nm is about
0.64.
C. Dissolve 25 mg in 5 ml of water, add 1 drop of cobalt(II) chloride (30g/l) TS

and 1 drop of ammonia (-100 g/1) TS; a violet-red solution is produced.
D. Dissolve 25 mg in 5ml of hydrochloric acid (-70 g/1) TS, add about 0.2 g of
zinc R powder and allow to stand for 5 minutes. Filter, and to the filtrate
add 3 drops of sodium nitrite (10 g/1) TS and 0.5 ml of ammonium sulfamate
(5 g/1) TS. After the reaction has subsided add 5 drops of N-(l-naphthyl)eth-
ylenediamine hydrochloride/ethanol TS; a deep red solution is produced.
Clarity and colour of solution. A solution of 0.20 g in 10ml of citric acid

(20 g/1) TS is clear and not more intensely coloured than standard colour
Loss on drying. Dry at 60 °C to constant mass under reduced pressure (not

chromatography, using silica gel R2 as the coating substance and 5 volumes of
1-butanol R, 2 volumes of water and 1 volume of acetic acid (~300g/l) TS as
the mobile phase. Apply separately to the plate 5 pi of each of the 3 following
solutions in methanol R containing (A) 0.04 g of Dacarbazine per ml, (B) 0.4 mg
of dacarbazine related compound A RS per ml, and (C) 0.4 mg of dacarbazine
related compound B RS per ml. After removing the plate from the chromato-
graphic chamber, allow it to dry in air, and examine the chromatogram in ultra-
intense or greater in size than that obtained with solution B (1%) and solution
C (1%).
Assay
301
Dissolve about 30 mg, accurately weighed, in sufficient hydrochloric acid

(0.1 mol/1) VS to produce 50ml of stock solution. For solution Si dilute 1.0ml
of the stock solution to 100 ml with hydrochloric acid (0.1 mol/1) VS. For solu-
tion S 2 dilute a further 1.0 ml aliquot of the stock solution to 100 ml with phos-
phate buffer, pH 7.0, TS. Measure the absorbance of a 1-cm layer of solution
S] at the maximum at about 323 nm against a solvent cell containing hydrochlo-
ric acid (0.1 mol/1) VS. Measure the absorbance of a 1-cm layer of solution S 2
at the maximum at about 329 nm against a solvent containing phosphate
cell
buffer, pH 7.0, TS. Calculate the percentage content of CgHioNeO.
DACTINOMYCINUM
DACTINOMYCIN
C62Hs6hI 12 O 16
Chemical name. Actinomycin D; CAS Reg. No. 50-76-0.
Description. An orange-red to red, crystalline powder.
Solubility. Soluble in water at 10 °C and slightly soluble in water at 37 °C;

freely soluble in ethanol (-750 g/1) TS and methanol R; very slightly soluble in
ether R.
Categoiy. Cytotoxic drug.
Storage. Dactinomycin should be kept in a tightly closed container, protected

from light, and stored at a temperature not exceeding 40 °C.
Additional information. Dactinomycin is hygroscopic and is affected by

light and heat.
302
CAUTION. Dactinomycin must be handled with care, avoiding contact with

the skin and inhalation of airborne particles.
Requirements
Dactinomycin contains not less than 95 0 % and not more than the equivalent
.
of 103 0 % of C62H86N,20 5, calculated with reference to the dried substance.

.
i
Identity tests
A. The absorption spectrum of a 25pg/ml solution in methanol R, when
and 445 nm. The absorbance of a 1-cm layer at the maximum wavelength
of 445 nm is about 0.83; the ratio of the absorbance at 240 nm to that at

test as described
using silica gel R4 and a mixture of 4 volumes of
1-butanol R, 2 volumes of water, and 1 volume of methanol R as the mobile
phase. Apply separately to the plate 10 pi of each of two solutions in acetone

R containing (A) 10 mg of Dactinomycin per ml and (B) 10 mg of dactino-
mycin RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air, and examine the chromatogram in ultravio-
let light (254 nm).

C. Add 1 mg to a solution of 10 mg of paraformaldehyde R in 1 ml of sulfuric

acid (-1760 g/1) TS; a red-violet colour is produced.
late with reference to the dried substance; [a]§^°‘" = -292 to -317°.

or 5mm of mercury) for 3 hours; it loses not more than 50mg/g.
pH value. pH of a saturated solution, 5. 5-7.0.
Assay. Carry out the test as described under 1.14.4 High performance liquid
chromatography, using a column, length 30 cm, internal diameter 3.9 mm,
packed with porous silica gel or ceramic microparticles having a diameter of
303
5-10|im, the surface of which has been modified with chemically bonded
octadecylsilyl groups.
As the mobile phase, use a mixture of 46 volumes of acetonitrile R, 25 volumes

of sodium acetate (0.04 mol/1) VS and 25 volumes of acetic acid (0.07 mol/1) VS,
filter through a membrane filter (porosity of 1 pm or finer) and degas the result-
ing solvent mixture. {Note: The concentration of acetonitrile may have to be

adjusted to provide a suitable chromatogram and elution time.)
Prepare the following solutions immediately before use in the above-mentioned

mobile phase, and store them protected from light. Weigh accurately for solu-
tion (A) about 1.2 mg of Dactinomycin per ml, and for solution (B) about
1.2 mg of dactinomycin RS per ml.
spectrophotometer at a wavelength of about 254 nm. Make three replicate
pi, to determine the peak responses. The rel-
injections of solution B, each of 20
ative standard deviation of the peaks is not more than 1.0%. Inject 20 pi of each
of solutions A and B.
Measure the areas of the peak responses. (The retention time for dactinomycin
isabout 25 minutes.) Calculate the content in % of C52Hg6Ni20]5 using the fol-
lowing formula: {M 2/M ) (A/A 2 ) 100, in which Mi and
1 M
2 are the concentra-
tions in mg per ml, of Dactinomycin being examined and the reference

,
solution, and Ai and A 2 are the areas of the peak responses of Dactinomycin
and the reference substance, respectively.
Additional requirements for Dactinomycin for p>arenteral use

terial endotoxins; contains not more than lOO.OlU of endotoxin RS per mg.

membrane filtration test procedure and using a solution in sterile water R con-
taining 20 mg of Dactinomycin per ml.
DAPSONUM
DAPSONE
304
Graphic formula.
Chemical name. 4,4'-Sulfonyldianiline; 4,4'-sulfonylbis[benzenamine]; 4,4'-

diaminodiphenylsulfone; CAS Reg. No, 80-08-0,
Description. A white or creamy white, crystalline powder; odourless.
TS; soluble in acetone R.
Category. Antileprotic.
Storage. Dapsone should be kept in a tightly closed container, protected from

light.
Additional information. Even in the absence of light, Dapsone is gradually

Requirements
Definition. Dapsone contains not less than 99.0% and not more than 101.0%
of C 12 H 12N 2 O 2 S, calculated with reference to the dried substance.
Identity tests
A. The absorption spectrum of a 5.0pg/ml solution in methanol R, when
observed between 230nm and 350nm, exhibits maxima at about 260nm
and 295 nm; the absorbances of a 1-cm layer at the maximum wavelength
of 260 nm and 295 nm are about 0.72 and 1.20, respectively.
aromatic amines under 2.1 General identification tests, producing a vivid red
precipitate.
305
15mg/g.
chromatography, but using an unlined chamber, silica gel R3 as the coating sub-
stance, and a mixture of 8 volumes of toluene R and 4 volumes of acetone R
saturated with water as the mobile phase. Apply separately to the plate 10 pi
of each of 5 solutions in methanol R containing (A) lOmg of the test substance
per ml, (B) lOmg of dapsone RS per ml, (C) O.lSmg of the test substance per
ml, (D) 20pg of the test substance per ml and (E) O.lOmg of 4,4'-thiodianiline
RS per ml. The solution of 4,4'-thiodianiline RS should be freshly prepared.
Pour the mobile phase into the chamber and insert the plate immediately, to
avoid prior saturation of the chamber. After removing the plate from the chro-
matographic chamber, spray it with 4-dimethylaminocinnamaldehyde TS2.
Heat the plate at 100 °C and examine the chromatogram in daylight. The spot
obtained with solution C is more intense than any spot obtained with solution
A, other than the principal spot, and in addition, not more than 2 among those
secondary spots are more intense than the spot obtained with solution D.
Moreover, there is no visible spot corresponding in position and appearance
with that obtained with solution E.
0.25g, accurately weighed, dissolved in a mixture of 15ml of water and 15ml
of hydrochloric acid (~70g/l) TS and titrate with sodium nitrite (0.1 mol/1) VS.
Each ml of sodium nitrite (0.1 mol/1) VS is equivalent to 12.42 mg of
C12H12N2O2S.
DEFEROXAMINI MESILAS
DEFEROXAMINE MESILATE
Molecular formula. C25H4sN608,CH403S
Graphic formula.
0 0 0 0 0
II II II II II
H2N(CHj)5NC(CHj)jCNH(CHj)5NC(CHj)jCNH(CHj)5NCCH3 . CH3SO3H
OH OH OH
306
Chemical name. N-[5-[3-[(5-Aminopentyl)hydroxycarbamoyl]propionamido]

pentyl]-3-[[5-(N-hydroxyacetamido)pentyl]carbamoyl]propionohydroxamic
acid monomethanesulfonate (salt); N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]
amino]-l,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydrox-
ybutane diamide monomethanesulfonate (salt); CAS Reg. No. 138-14-7.
Other name. Desferrioxamine mesylate.
Description. A white to yellowish white powder; odourless or almost

odourless.
Solubility. Soluble in 5 parts of water; soluble in ethanol (-750 g/1) TS; slightly
soluble in methanol R; practically insoluble in ether R.
Category. Antidote to iron poisoning.
Storage. Deferoxamine mesilate should be kept in a well-closed container,

protected from light, and stored at a temperature not exceeding 4°C.
Requirements
Definition. Deferoxamine mesilate contains not less than 98.0% and not more
than 102.0% of C 25 H 4 gN 60 g,CH 403 S, calculated with reference to the anhy-
drous substance.
Manufacture. The production method must be evaluated to determine the

potential for formation of alkyl mesilates, which is particularly likely to occur
if the reaction medium contains lower alcohols. Where necessary, the produc-
tion method is validated to demonstrate that alkyl mesilates are not detectable
in the final product.
Identity tests
A. Dissolve 5 mg in 5 ml of water, add 2 ml of trisodium orthophosphate (2 g/1)
TS, mix, then add 1 ml of sodium l,2-naphthoquinone-4-sulfonate (5 g/1) TS;
a blackish brown colour is produced.
B. The titrated solution obtained in the assay is reddish brown in colour. To
5 ml of the titrated solution add 2 ml of benzyl alcohol R and shake; the

colour is extracted. To a further 5 ml of the titrated solution add 2 ml of ether
R and shake; the colour is not extracted.
307
Chlorides. Dissolve 0.7 g in a mixture of 2 ml of nitric acid (-130 g/1) TS, and
proceed as described under 2.2.1 Limit test for chlorides; the chloride content
Sulfates. Dissolve 0.85g in 40ml of water, and proceed as described under

2.2.2 Limit test for sulfates; the sulfate content is not more than 0.6mg/g.
Clarity and colour of solution. A solution of 1.0 g in 10 ml of water is clear;

measure the absorbance of the solution in a 1-cm layer at 420nm; not more
than 0.10.

Fischer method, Method A, using about 1 g of the substance; the water content
is not more than 20 mg/g.
Assay. Dissolve about 0.3 g, accurately weighed, in 15 ml of water and add

2 ml of sulfuric acid (0.05 mol/1) VS. Titrate slowly with ferric ammonium
sulfate (0.1 mol/1) VS, determining the endpoint potentiometrically using a plat-
inum electrode and a calomel reference electrode. Each ml of ferric ammonium
sulfate (0.1 mol/1) VS is equivalent to 65.68 mg of C25H48N60g,CH403S. (Keep
the titrated solution for identity test B.)
DEHYDROEMETINI DIHYDROCHLORIDUM
DEHYDROEMETINE DIHYDROCHLORIDE
Molecular formula. C29H3 bN 204,2HC1
308
Graphic formula.
Chemical name. (+)-2,3-Didehydroemetine dihydrochloride; (+)-2,3-didehy-

dro-6',7',10,ll-tetramethoxyemetan dihydrochloride; (+)-(llbR*)-3-ethyl-
l,6,7,llb-tetrahydro-9,10-dimethoxy-l-[[(lbS*)-l,2,3,4-tetrahydro-6,7-dimeth
oxy-l-isoquinolyl]methyl]-4/f-benzo[ia]quinolizine dihydrochloride; CAS Reg.
No. 3317-75-7.
Description. A white to yellowish, crystalline powder; odourless.
Solubility. Sparingly soluble in water; soluble in methanol R.
Category. Antiamoebic drug.
Storage. Dehydroemetine dihydrochloride should be kept in a tightly closed

container.
Requirements
Definition. Dehydroemetine dihydrochloride contains not less than 98.0%
and not more than 101.0% of C 29H 3aN 204 2 HCl,
,
calculated with reference to
Identity tests
A. The absorption spectrum of a 0.040mg/ml solution in hydrochloric acid
maximum at about 282 nm. The absorbance of a 1-cm layer at this wave-
B. Sprinkle a small quantity of the powdered substance on the surface of 1 ml

of sulfuric acid (~1760g/l) TS containing 5 mg of molybdenum tri oxide R;
a bright green colour is produced.
309


70mg/g.
pH value. pH of a 30 mg/ml solution, 3.5-5. 0.
9 volumes of ethyl acetate R and 1 volume of diethylamine R as the mobile
phase. Apply separately to the plate 5 pi of each of 3 solutions in methanol R
containing (A) 20mg of the test substance per ml, (B) O.lOmg of the test sub-
stance per ml, and (C) O.lOmg of emetine hydrochloride RS per ml. After
spray it with mercuric acetate/acetic acid TS, heat it at 120 °C for 10 minutes,
obtained with solution B or solution C, as appropriate.
Rl, add 10 ml of mercuric acetate/acetic acidTS, and titrate with perchloric acid
ml of perchloric acid (0.1 mol/1) VS is equivalent to 27.58mg of
C2,H3sN204,2HC1.
DEXAMETHASONI ACETAS
DEXAMETHASONE ACETATE
Dexamethasone acetate, anhydrous
Dexamethasone acetate monohydrate
Molecular formula. C 24 H 31 FO 6 (anhydrous); C 24 H 3 iF 06 ,H 20 (monohydrate).
310
1
Graphic formula.
0
II
Chemical name. 9-Fluoro- 1 13, 17,2 1 -trihydroxy-1 6a-methylpregna- 1 ,4-diene-

3,20-dione 21-acetate; 21-(acetyloxy)-9-fluoro-lip,17-dihydroxy-16a-methyl-
pregna-l,4-diene-3,20-dione; CAS Reg. No. 1177-87-3 (anhydrous).
9-Fluoro- 1 1 P, 1 7,2 1 -trihydroxy- 1 6a-methylpregna- 1 ,4-diene-3,20-dione 21-

acetate monohydrate; 21-(acetyloxy)-9-fluoro-l lP,17-dihydroxy-16a-methyl-
pregna-l,4-diene-3,20-dione monohydrate; CAS Reg. No. 55812-90-3
(monohydrate).
Description. A white or almost white powder, odourless.

(-750 g/1) TS; slightly soluble in ether R.
Storage. Dexamethasone acetate should be kept in a tightly closed container,

Labelling. The designation on the container ofDexamethasone acetate should

state whether the substance is the monohydrate or is in the anhydrous form.
Requirements
Definition. Dexamethasone acetate contains not less than 96.0% and not
more than 104.0% of C 24H 31 FO 5 ,
substance.
Identity tests
• Either tests A, B, C and E, or tests B, C, D and E may be applied.

infrared region. For the anhydrous form the infrared absorption spectrum is
311
=
concordant with the spectrum obtained from dexamethasone acetate RS or

with the reference spectrum of dexamethasone acetate. For the monohydrate
the infrared absorption spectrum is concordant with the spectrum obtained
from dexamethasone acetate monohydrate RS or with the reference spectrum

of dexamethasone acetate monohydrate.

60 °C for 20 minutes and cool immediately. The absorbance of a 1-cm layer
at the maximum at about 423 nm is not less than 0.42 (preferably use 2-cm
cells for the measurement and calculate the absorbance of a 1-cm layer).
C. See the test described below under "Related steroids”. The principal spots
spot obtained with solution A corresponds with that obtained with solu-
tion B.
ide (0.01 mol/1) VS and 20ml of water as the absorbing liquid. When the
process is complete, add 0.1ml to a mixture of 0.1ml of freshly prepared
sodium alizarinsulfonate (1 g/1) TS and 0.1 ml of zirconyl nitrate TS; the red
E. Heat 0.05 g with 2 ml of potassium hydroxide/ethanol (0.5 mol/1) VS in a

water-bath for 5 minutes. Cool, add 2 ml of sulfuric acid (-700 g/1) TS, and
boil gently for 1 minute; ethyl acetate, perceptible by its odour (proceed
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; [a]o -1-82
to-H88°.
of mercury). For the anhydrous form use about
0.5 g of the substance; it loses not more than 5.0mg/g. For the monohydrate
use about 0.15g of the substance; it loses not less than 35mg/g and not more
than 45mg/g.
77 volumes of dichloromethane R, 15 volumes of ether R, 8 volumes of
312
methanol R, and volumes of water as the mobile phase. Apply separately

1.2
ml and (B) 15mg of dexamethasone acetate RS per ml; also apply to the plate
2 pi of a third solution (C) composed of a mixture of equal volumes of solu-
tions A and B and 1 pi of a fourth solution (D) containing O.lSmg of the test
substance per ml in the same solvent mixture as used for solutions A and B.
air until the solvents have evaporated and heat at 105°C for 10 minutes; allow
to cool, spray with blue tetrazolium/sodium hydroxide TS, and examine the
chromatogram in daylight. Any spot obtained with solution A, other than the
principal spot, is not more intense than that obtained with solution D.
Assay

aldehyde-free ethanol (-750 g/1) TS to produce 100ml. Transfer 10.0ml of
the diluted solution to a 25-ml volumetric flask, add 2.0 ml of blue tetra-
zolium/ethanol TS and displace the air with oxygen-free nitrogen R. Immedi-
ately add 2.0ml of tetramethylammonium hydroxide/ethanol TS and again
tents by gentle swirling and allow to stand for 1 hour in a water-bath at 30 °C.
amount of C 24H 31 FO 6 in the substance being tested by comparison with dex-
amethasone acetate RS, similarly and concurrently examined.
DEXAMETHASONI NATRII PHOSPHAS

DEXAMETHASONE SODIUM PHOSPHATE
Molecular formula. C22H2sFNa20gP
313
Graphic formula.
CHjOPO(ONa)j
Chemical name. 9-Fluoro- 1 1 (3, 17,2 1 -trihydroxy-1 6a-methylpregna- 1 ,4-diene-

3,20-dione 21-(dihydrogen phosphate) disodium salt; 9-fluoro-lip,17-
dihydroxy-16a-methyl-21-(phosphonooxy)pregna-l,4-diene-3,20-dione disodium
salt; CAS Reg. No. 2392-39-4.

a slight odour of ethanol.
Solubility. Freely soluble in water; slightly soluble in ethanol (-750 g/1) TS;
Category. Adrenal hormone.
Storage. Dexamethasone sodium phosphate should be kept in a tightly closed

Additional information. Dexamethasone sodium phosphate is very hygro-

scopic. Even in the absence of light, it is gradually degraded on exposure to a
Requirements
Definition. Dexamethasone sodium phosphate contains not less than 96.0%
and not more than 103.0% of C22H2sFNa208P, calculated with reference to the
anhydrous and ethanol-free substance.
Identity tests
using silica gel R1 as the coating substance and a freshly prepared mixture
of 3 volumes of 1 -butanol R, 1 volume of acetic anhydride R, and 1 volume
of water as the mobile phase. Apply separately to the plate 2 pi of each of
4 solutions in methanol R containing (A) 2.5 mg of the test substance per
ml, (B) 2.5 mg of dexamethasone sodium phosphate RS per ml, (C) a mixture
of equal volumes of solutions A and B, and (D) equal volumes of solution
314
A and a solution of 2.5mg of prednisolone sodium phosphate RS per ml of

methanol R. After removing the plate from the chromatographic chamber,
allow it to dry in air until the solvents have evaporated, spray it with
a mixture of 10ml of sulfuric acid (~1760g/l) TS and 90ml of ethanol
(-750 g/1) TS, heat it at 120 °C for 10 minutes, allow it to cool, and examine
the chromatogram in ultraviolet light (365 nm). The principal spot obtained
obtained with solution B. The principal spot obtained with solution C
appears as a single compact spot, whereas the chromatogram of solution D
shows 2 closely running spots.
B. Place 0.5ml of chromic acid TS in a small test-tube and heat in a water-bath

for 5 minutes; the solution wets the sides of the tube but there is no greasi-
ness. Add about 3 mg of the test substance and again heat in a water-bath
for 5 minutes; the solution no longer wets the sides of the tube.
C. Heat carefully 0.04g with 2ml of sulfuric acid (~1760g/l) TS until white
fumes are evolved, add drop by drop nitric acid (-1000 g/1) TS until oxida-
tion is complete, and cool. Add 2 ml of water, heat until white fumes
are again evolved, cool, add 10 ml of water, and neutralize with ammonia
(-100 g/1) TS, using pH-indicator paper R. Keep half of the solution for test
D. The remaining solution yields reaction A described under 2.1 General
identification tests as characteristic of orthophosphates.
D. The solution prepared in test C yields reaction B described under 2.1 General
identification tests as characteristic of sodium.
ence to the anhydrous and ethanol-free substance; [a]™ °'^ = -1-74 to -1-82°.
Clarity of solution. A solution of O.lOg in 10ml of carbon-dioxide-free water

R is clear.

Fischer method, method A, using about 0.3 g of the substance. The sum of the
contents of water and ethanol (described below), both calculated in mg/g, is
not more than 160 mg/g.
Ethanol. Carry out the test as described under 1.14.5 Gas chromatography,
using 3 solutions in water containing (1) a mixture of 10 pi of 1-propanol R per
ml serving as an internal standard and 10 pi of dehydrated ethanol R per ml,
(2) O.lOg of the test substance per ml, and (3) a mixture of O.lOg of the test
substance and 10 pi of the internal standard per ml. It may be necessary to
adjust the content of dehydrated ethanol R in solution (1) to produce a peak of
similar height to the corresponding peak in the chromatogram obtained with
solution (2).
315
For the procedure use a column 1.5 m long and 4 mm

in internal diameter
packed with porous polymer beads (particle size 80-100 pm from a commer-
cial source, is suitable). Maintain the column at 135 °C, use nitrogen R as the
carrier gas and a flame ionization detector.
Calculate the content of ethanol in mg/g, assuming the weight per ml at 20 °C

to be 0.790g; not more than 80mg/g.
pH value. pH of a lOmg/ml solution in carbon-dioxide-free water R, 7.5-10.5.
Free dexamethasone and other related substances. Carry out the test as
described under 1.14.1 Thin-layer chromatography, using silica gel R1 as the
coating substance and methanol R as the mobile phase. Apply separately to the
plate 2 pi of each of 2 solutions in methanol R containing (A) 10 mg of the test
substance per ml, and (B) 0.20 mg of dexamethasone RS per ml. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air for 5
minutes, spray it with a solution of 3 g of zinc chloride R in 10 ml of methanol
R, heat it at about 125 °C for 1 hour, and examine the chromatogram in ultra-
violet light (365 nm). Any spot obtained with solution A, other than the prin-
cipal spot, is not more intense than that obtained with solution B.

200 ml. Dilute 5 ml to 250 ml with water and measure the absorbance of this
solution in a 1-cm layer at the maximum at about 241 nm. Calculate the content
of C22H28FNa20sP, using the absorptivity value of 29.7 (Aj°m = 397).
Additional requirements for Dexamethasone sodium

fhosfahate for jaarenteral use
DEXAMETHASONUM
DEXAMETHASONE
316
Graphic formula.
Chemical name. 9-Fluoro- 1 1 P, 17,2 1 -trihydroxy-1 6a-methylpregna- 1 ,4-diene-

3,20-dione; CAS Reg. No. 50-02-2.

powder; odourless.

TS.
Storage. Dexamethasone should be kept in a tightly closed container, pro-

tected from light.
Requirements
Definition. Dexamethasone contains not less than 96.0% and not more than
104.0% of C22H29FO5, calculated with reference to the dried substance.
Identity tests
• Either tests A, B and C, or tests B, C and D may be applied.

infrared region.
spectrum obtained from dexamethasone RS or with the reference spectrum of
dexamethasone.

at the maximum at about 423 nm is not less than 0.42 (preferably use 2-cm
317

spot obtained with solution A corresponds with that obtained with solu-
tion B.
ide (0.01 mol/1)
processis complete add 0.1ml to a mixture of 0.1ml of freshly prepared
sodium aKzarinsulfonate (1 g/1) TS and 0.1 ml of zirconyl nitrate TS; the red
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; [aj^ = +72

to+WT
Loss on drying. Dry to constant weight at 100°C under reduced pressure

(not exceeding 0.6 kPa or about 5 mm of mercury); it loses not more than
5.0mg/g.
ml and (B) 15 mg of dexamethasone RS per ml; also apply to the plate 2 pi of a
third solution (C) composed of a mixture of equal volumes of solutions A and
B and 1 pi of a fourth solution (D) containing 0.15 mg of the test substance per
ml in the same solvent mixture as used for solutions A and B. After removing
the plate from the chromatographic chamber, allow it to dry in air until the sol-
vents have evaporated and heat at 105 °C for 10 minutes; allow to cool, spray
with blue tetrazolium/sodium hydroxide TS, and examine the chromatogram
is not more intense than that obtained with solution D.
Assay

aldehyde-free ethanol (~750g/l) TS to produce 100ml. Transfer 10.0ml of the
318
tents by gentle swirling, and allow to stand for 1 hour in a water-bath at 30 °C,
amount of C 22 H 29FO 5 in the substance being tested by comparison with dex-
amethasone RS, similarly and concurrently examined.
DEXTROMETHORPHANI HYDROBROMIDUM
DEXTROMETHORPHAN HYDROBROMIDE
Molecular formula. C]8H25N0,HBr,H20
Graphic formula.
Chemical name. (-i-)-3-Methoxy-17-methyl-9a,13a-14a-morphinan hydro-

bromide monohydrate; (-)-)-as-l,3,4,9,10,10a-hexahydro-6-methoxy-ll-
methyl-2/f-10,4a-iminoethanophenanthrene hydro bromide monohydrate;
CAS Reg. No. 6700-34-1 (monohydrate).

almost odourless.
Solubility. Sparingly soluble in water; freely soluble in ethanol (-750 g/1) TS;
Category. Antitussive drug.
Storage. Dextromethorphan hydrobromide should be kept in a well-closed

container.
319
Requirements
Definition. Dextromethorphan hydrobromide contains not less than 98.0%
and not more than 101.0% of CisH 25 NO,HBr, calculated with reference to the
Identity tests
• Either tests A and E or tests B, C, D and E may be applied.
A. Dry under reduced pres-

a small quantity of the test substance for 4 hours
sure (not exceeding O.bkPa or about 5 mm
of mercury) over phosphorus pen-
toxide and carry out the examination as described under 1.7
R,
Spectrophotometry in the infrared region. The infrared absorption spectrum
is concordant with the spectrum obtained from dextromethorphan hydro-
bromide RS similarly prepared or with the reference spectrum of dex-

tromethorphan hydrobromide.
B. The absorption spectrum of a O.lOmg/ml solution in sodium hydroxide

maximum at 280 nm; the absorbance of a Tcm layer at this wavelength is
about 0.59.
C. Dissolve 0.05 g in 2 ml of sulfuric acid (~100g/l) TS. Add 1ml of

mercury/nitric acid TS drop by drop while shaking; a white, crystalline pre-
cipitate in the form of platelets is produced, and the solution does not imme-
diately turn red. Heat on a water-bath for about 10 minutes; a yellow to red
colour develops.
D. Melting temperature, about 125 °C with decomposition.
E. To a 5mg/ml solution add 0.25ml of nitric acid (-130 g/1) TS; this testyields
reaction B described under 2.1 General identification tests as characteristic
of bromides.
Specific optical rotation. Use a 20mg/ml solution in hydrochloric acid

(0.1 mol/1) VS and calculated with reference to the anhydrous substance;
[a]g>"^ = -t28.0 to-t30°.

320
pH value. Dissolve 0.4g in carbon-dioxide -free water R using gentle heat,

dilute to 20 ml with the same solvent and measure the pH at 20 °C; the value
lies between 5.2 and 6.5.
Dimethylaniline. Dissolve 0.5 g in 15 ml of water using gentle heat, cool, and

add 4 ml of acetic acid (~60g/l) TS, 1 ml of sodium nitrite (lOg/1) TS, and suffi-
cient water to produce 25 ml. Prepare similarly a reference solution containing
5 pg of N,N-dimethylaniline R in 25 ml. The colour produced in the test solu-
tion is not more intense than that produced in the reference solution when com-
pared as described under 1.11 Colour of liquids; the dimethylaniline content is
not more than lOpg/g.
Phenolic substances. To 5mg add 1 drop of hydrochloric acid (~70g/l) TS,

1 ml of water, and 0.2 ml of ferric chloride (50g/l) TS. Mix, add 0.2 ml of potas-
sium ferricyanide (50g/l) TS, dilute to 5 ml with water, shake well, and allow
to stand for 15 minutes; the solution is yellowish brown and shows no green-
ish or blue colour.
Rl, and add 10ml of mercuric acetate/acetic acid TS, warming slightly if nec-
essary to effect solution. Titrate with perchloric acid (0.1 mol/1) VS as described
under 2.6 Non-aqueous titration, Method A. Each ml of perchloric acid
(0.1 mol/1) VS is equivalent to 35.23mg of C 18 H 25NO, HBr.
DIAZEPAMUM
DIAZEPAM
Molecular formula. C16H13CIN2O
Graphic formula.
321
Chemical name. 7-Chloro-l,3-dihydro-l-methyl-5-phenyl-2H-l,4-benzodi-

azepin-2-one; CAS Reg. No. 439-14-5.

almost odourless.
Solubility. Very slightly soluble in water; soluble in ethanol (-750 g/1) TS.
Category. Tranquillizer.
Storage. Diazepam should be kept in a well-closed container, protected from

light.
Requirements
Definition. Diazepam contains not less than 99.0% and not more than
101.0% of C16H13CIN2O, calculated with reference to the dried substance.
Identity tests
• For tests B and C use low-actinic glassware and measure within 30 minutes.

reference spectrum of diazepam.
B. The absorption spectrum of an 8.0pg/ml solution in hydrochloric acid

(0.1 mol/1)VS, when observed between 230 nm and 350 nm, exhibits maxima
at about 241 nm and 286nm; the absorbances of a 1-cm layer at the
maximum wavelengths of 241 nm and 286nm are about 0.80 and 0.38,
respectively (preferably use 2-cm cells for the measurements and calculate
the absorbances for 1-cm layers).
C. The absorption spectrum of a 0.030mg/ml solution in hydrochloric acid

VS,
maximum about 362 nm; the absorbance of a 1-cm layer at this wave-
at
length is about 0.44 (preferably use 2-cm cells for the measurement and cal-
culate the absorbance of a 1-cm layer).
using 20 mg of the test substance and 5 ml of sodium hydroxide (-80 g/1) TS
as the absorbing liquid. When the process is complete, acidify with sulfuric
acid (-100 g/1) TS and boil gently for 2 minutes; the solution yields reaction
322
A, described under 2.1 General identification tests as characteristic of

chlorides.

exceeding O.bkPa or about 5mm of mercury); it loses not more than 5.0mg/g.
Related substances. Carry out the test in subdued light as described under
1.14.1 Thin-layer chromatography, using silical gel R2 as the coating substance
and a mixture of 1 volume of dehydrated ethanol R and 24 volumes of ethyl
acetate R as the mobile phase. Apply separately to the plate 10 pi of each of 2
freshly prepared solutions in chloroform R containing (A) 0.20 g of the test sub-
stance per ml and (B) 0.10 mg of 5-chloro-2-methylaminobenzophenone RS per

potentiometrically as described under 2.6 Non-aqueous titration. Method A.
CisH.sClNÔ.
Additional requirements for Diazep>am for p>arenterat use

terial endotoxins; contains not more than 11.6IU of endotoxin RS per mg.
323
DIAZOXIDUM
DIAZOXIDE
Graphic formula.
Chemical name. 7-Chloro-3-methyl-2H-l,2,4-benzothiadiazine 1,1 -dioxide;
CAS Reg. No. 364-98-7.
Solubility. Practically insoluble in water and ether R; freely soluble in

dimethylformamide R; slightly soluble in ethanol (-750 g/1) TS.
Category. Antihypertensive.
Storage. Diazoxide should be kept in a well-closed container.
Requirements
Definition. Diazoxide contains not less than 98.0% and not more than
101.0% of C8H7CIN2O2S, calculated with reference to the dried substance.
Identity tests
infrared region.
trum obtained from diazoxide RS or with the reference spectrum of diazoxide.
324
5-Omg/g,
Related substances. Carry out the test as described under 1,14.1 Thin-layer
17 volumes of ethyl acetate R, 4 volumes of methanol R, and 3 volumes of
of each of 3 solutions in sodium hydroxide (0.1 mol/1) VS containing (A) 15mg
of the test substance per ml, (B) 0.15 mg of the test substance per ml and (C)
0.1 5 mg of diazoxide RS per ml. After removing the plate from the chromato-
graphic chamber, allow it to dry in air until the odour of ammonia is no longer
detectable, and examine the chromatogram in ultraviolet light (254 nm). Any
Assay. Dissolve 0.45 g, accurately weighed, in 100 ml of a mixture of 2

volumes of dimethylformamide R and 1 volume of water, and titrate with
sodium hydroxide (0.1 mol/1) VS, determining the endpoint potentiometrically.
CsH.ClNÔ^S.
DICLOXACILLINUM NATRICUM
DICLOXACILLIN SODIUM
Molecular formula. C] 9 Hi 6 Cl 2 N 3 Na 05 S,H 20
Graphic formula.
COONa
Chemical name. Monosodium (2S,5jR,6/?)-6-[3-(2,6-dichlorophenyl)-5-

methyT4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-l -azabicyclo
[3.2.0]heptane-2-carboxylate monohydrate; monosodium [2S-(2a,5a,6P)]-
6-[[[3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl]carbonyl]amino]-3,3-
dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate monohydrate;
325
monosodium [3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl]penicillin mono-

hydrate; CAS Reg. No. 13412-64-1 (monohydrate).
Solubility. Freely soluble in water and methanol R; soluble in ethanol

(-750 g/1) TS.
Storage. Dicloxacillin sodium should be kept in a tightly closed container, pro-

tected from light.
Additional information. Even in the absence of light, Dicloxacillin sodium is

Requirements
Definition. Dicloxacillin sodium contains not less than 88.0% of total peni-
cillins calculated as dicloxacillin free acid (C] 9 H, 7 Cl 2 N 305 S) and with reference
Identity tests
spectrum obtained from dicloxacillin sodium RS or with the reference spec-
trum of dicloxacillin sodium.
B. To lOmg of paraformaldehyde R dissolved in 1ml of sulfuric acid

(-1760 g/1) TS add about Img of the test substance; a colourless solution
is produced. Heat the solution in a water-bath for 2 minutes and cool; the
solution remains colourless (distinction from cloxacillin sodium).
C. Ignite 20 mg and dissolve the residue in acetic acid (-60 g/1) TS. The solu-
Specific optical rotation. Use a 10 mg/ml solution, and calculate with refer-
ence to the anhydrous substance; [ajS’ = -h 128 to -tl43°.

326
Chlorine. Carry out the combustion as described under 2.4 Oxygen flask
method, but using 25 mg of the test substance and 10 ml of sodium hydroxide
(0.1 mol/1) VS as the absorbing liquid. When the process is complete, transfer
the resulting solution to a titration vessel, heat on a water-bath for 30 minutes,
cool to room temperature, add 20 ml of nitric acid (-130 g/1) TS, and titrate with
silver nitrate (0.01 mol/1) VS, determining the endpoint potentiometrically using
a silver/silver chloride electrode system. Repeat the operation without the sub-
stance being tested. Each ml of silver nitrate (0.01 mol/1) VS is equivalent to
0.3546 mg of Cl. Calculate the total content of chlorine in mg/g and subtract
from it the content of free chlorides as determined below; the content of chlo-
rine is between 130 mg/g and 142 mg/g.
Free chlorides. Dissolve about 0.12g, accurately weighed, in 10ml of sodium

hydroxide (0.1 mol/1) VS, add 20 ml of water, and heat on a water-bath for 30
minutes. Cool to room temperature, add 20ml of nitric acid (~130g/l) TS, and
titrate with silver nitrate (0.01 mol/1) VS, determining the endpoint potentio-
metrically using a silver/silver chloride electrode system. Repeat the operation

without the substance being tested. Each ml of silver nitrate (0.01 mol/1) VS is
equivalent to 0.3546mg of Cl; the content of free chlorides is not more than
5 mg/g.

Cool the tube rapidly to 20 °C (solution A). To the second tube add 10.0ml of
water and mix (solution B).

tion B.
B, calculate the amount of CigHieChNsNaOsS in the substance being tested by
comparison with sodium RS, similarly and concurrently examined.
dicloxacillin
DICOUMAROLUM
DICOUMAROL
327
Graphic formula.
OH OH
Chemical name. 3,3'-Methylenebis[4-hydroxycoumarin]; 3,3'-methylenebis

[4-hydroxy-2H-l-benzopyran-2-one]; CAS Reg, No. 66-76-2,
Description. A white or creamy white, crystalline powder; odour, character-

istic, faint.
Solubility. Practically insoluble in water, ethanol (-750 g/1) TS, and ether R.
Category. Anticoagulant.
Storage. Dicoumarol should be kept in a well-closed container, protected from

light.
Requirements
Definition. Dicoumarol contains not less than 98.5% and not more than
101.0% of C19H12O6, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from dicoumarol RS or with the reference spectrum of
dicoumarol.
B. Fuse 0.2 g with 0.2 g of potassium hydroxide R, cool, stir with 5 ml of water,
filter and acidify the filtrate with hydrochloric acid (-250 g/1) TS; a white,
crystalline precipitate is obtained (salicylic acid). Retain the filtrate for test
C.
C. To 1ml of the filtrate from test B add 5 ml of water and a mixture of 1 drop
of ferric chloride (25 g/1) TS and 2 drops of hydrochloric acid (-70 g/1) TS; a
violet colour is produced.
328
5-Omg/g,
Acidity. Shake 0.5g with 10ml of carbon-dioxide-free water R for 1 minute

and filter; titrate the filtrate with sodium hydroxide (0.1 mol/1) VS, methyl
red/ethanol TS being used as indicator; not more than 0.1ml is required to
obtain the midpoint of the indicator (orange).
Assay. Dissolve about 0.35 g, accurately weighed, in 40 ml of 1-butylamine R,

add 5 drops of azo violet TS and titrate with lithium methoxide (0.1 mol/1) VS
to a deep-blue endpoint, as described under 2.6 Non-aqueous titration. Method
B. Each ml of lithium methoxide (0.1 mol/1) VS is equivalent to 16.82 mg of
C19H12O5.
DIDANOSINUM
DIDANOSINE
o
C 10 H N 4 O 3
12
Chemical name. 9- [(2/?,5S)-5-(hy droxymethyl)tetrahydrofuran-2-yl] - 1 ,9-dihydro-
6H-purin- 6 -one; 9-(2,3-dideoxy-p-D-g/yc«ro-pentofuranosyl)-l,9-dihydro-6Tf-purin-

6 -one; 2',3'-dideoxyinosine (DDl); CAS Reg. No. 69655-05-6.
Description. A white to almost white powder.
Solubility. Sparingly soluble in water; slightly soluble in methanol R and

ethanol (95 per cent) R.
Category. Antiretroviral (Nucleoside Reverse Transcriptase Inhibitor).
Storage. Didanosine should be kept in a tightly closed container.
329
Requirements
Didanosine contains not less than 98 5 . %
CioHi 2 N 403 calculated with reference to the dried substance.
^
Identity test
• Either tests A and B, or test C may be applied.
A. Carry out test A.l. or, where UV detection is not available, test A. 2.
A.l. Carry out the test as described under 1.14.1 Thin-layer chromatogra-
phy, using silica gel R6 as the coating substance and a mixture of
67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10
volumes of methanol R and 3 volumes of ammonia (-260 g/1) TS as
the mobile phase. Apply separately to the plate 5 pi of each of 2 solu-
tions in methanol containing (A) 5 mg of the test substance per ml and
(B) 5 mg of didanosine RS per ml. After removing the plate from the
chromatographic chamber, allow it to dry exhaustively in air or in
a current of cool air. Examine the chromatogram in ultraviolet light
(254 nm).
The principal spot obtained with solution A corresponds in position,

appearance, and intensity with that obtained with solution B.
A.2. Carry out the test as described under 1.14.1 Thin-layer chromatogra-
volumes of methanol R and 3 volumes of ammonia (-260 g/1) TS as the
mobile phase. Apply separately to the plate 5 pi of each of 2 solutions
in methanol containing (A) 5 mg of the test substance per ml and (B)
5 mg of didanosine RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry exhaustively in air or in a current
of cool air. Spray with vanillin/sulfuric acid TSl. Heat the plate for a
few minutes at 120 °C. Examine the chromatogram in daylight.

B. The absorption spectrum of a lOpg/ml solution in methanol R, when

observed between 210nm and 300 nm, exhibits one maximum at about
250 nm; the specific absorbance (Alcm) is between 435 to 485.
C. Carry out the examination as described under 1.7 Spectrophotometry in the

infrared region.
spectrum obtained from didanosine RS or with the reference spectrum of
didanosine.
330
If the spectra are not concordant, use didanosine RS. Dissolve the sample in a
small amount of methanol R, evaporate to dryness and carry out the IR spec-
trum with the residue as mentioned above. Treat didanosine RS in the same
way. The infrared absorption spectrum is concordant with the spectrum
obtained from didanosine RS.
ence to the dried substance; [a]c = -24 to -28°.
Loss on drying. Dry for 4 hours at 105 °C; it loses not more than 5.0mg/g.
Related substances
Prepare fresh solutions and perform the tests without delay
Carry out the test as described under 1.14.4 High performance liquid chro-
matography, using a stainless steel column (25 cm x 4.6mm), packed with
octadecylsilyl base-deactivated silica gel for chromatography R (5 pm).
Maintain the column temperature at 20-25 °C.
The mobile phases for gradient elution consist of a mixture of aqueous

phase (Mobile phase A) and methanol (Mobile phase B), using the following
conditions:
Mobile phase A: A 0.05 M solution of ammonium acetate R adjusted to pH 8.0

using ammonia (-260 g/1) TS.
Mobile phase B: Methanol R.
Time (min) Mobile phase A (% v/v) Mobile phase B (% v/v)
0 92 8
18 92 8
25 70 30
45 70 30
50 92 8
60 92 8
Prepare the following solutions in a mixture of 92 volumes of mobile phase A

and 8 volumes of mobile phase B (dissolution solvent).
331
For solution (1) dissolve 5.0 mg of hypoxanthine R in the dissolution solvent

and dilute to 100.0ml with the same solvent. Dilute 1.0ml to 20.0ml with the
same solvent. For solution (2) dissolve 5 mg of didanosine for system suitabil-
ity RS (containing impurities A to F) in the dissolution solvent and dilute to
10ml with the same solvent. For solution (3) dissolve 25mg of the test sub-
stance in the dissolution solvent and dilute to 50.0ml with the same solvent.
For solution (4) dilute 5.0 ml of solution (3) to 50.0 ml with the dissolution
solvent. Then dilute 5.0 ml of this solution to 50.0 ml with the same solvent.
Use the chromatogram supplied with didanosine for system suitability RS and
the chromatogram obtained with solution (2) to identify the peaks due to impu-
rities A to F.
Inject20 pi of solution (2). The test is not valid unless the resolution factor
between the peaks due to impurity (C) (2'-deoxyinosine) and impurity D (3'-
deoxyinosine) is greater than 2.5, if necessary reduce the amount of methanol
in the mobile phase and adjust the proportion of aqueous phase pH 8.0
accordingly.
Inject separately 20 pi of solution (4) in replicate injections in the chromato-

graphic system. The relative standard deviation for peak areas of didanosine in
replicate injections of solution (4) is not more than 5.0%.
Inject separately 20 pi each of solutions (1) and (3) and 20 ml of dissolution

solvent in the chromatographic system. Examine the mobile phase chro-
matogram for any extraneous peaks and disregard the corresponding peaks
observed in the chromatogram obtained with solution (3).
In the chromatogram obtained with solution (2), the following peaks are eluted
with reference to didanosine (retention time
at the following relative retention
about 13-15min): impurity A about 0.3; impurity B about 0.4; impurity C about
0.44; impurity D about 0.48; impurity E about 0.5; impurity F about 0.8; impu-
rity I about 1.4; impurity G about 1.6; impurity H about 2.0.
In the chromatogram obtained with solution (3) the area of any peak corre-
sponding to impurity A (hypoxanthine) is not greater than the area of the prin-
cipal peak obtained with solution (1) (0.5%). The area of any individual peak
corresponding to impurities B, C, D, E, F or G is not greater than 0.2 times the
area of the principal peak obtained with solution (4) (0.2%). The area of any
other impurity peak is not greater than 0.1 times the area of the principal peak
obtained with solution (4) (0.1 %). The sum of the areas of all peaks, other than
the principal peak, is not greater than the area of the principal peak obtained
332
with solution (4) (1.0%). Disregard any peak with an area less than 0.05 times
the area of the principal peak obtained with solution (4) (0.05%).
Assay
Dissolve about 0.200 g, accurately weighed, in 50 ml of glacial acetic acid R1
aqueous titration; Method A determining the end point potentiometrically.

C10H12N4O3.
Impurities
The following list of known and potential impurities that have been shown to
be controlled by the tests in this monograph is given for information.
A. l,7-dihydro-6f/-purin-6-one (hypoxanthine)
B. R1 = R2 = OH, R3 = H
9-P-D-ribofuranosyl-l,9-dihydro-6H-purin-6-one (inosine)
C. R1 = R3 = H, R2 = OH
9-(2-deoxy- P-D-ef)'t/2ra-pentofuranosyl)-l,9-dihydro-6JT-purin-6-one
(2'-deoxyinosine)
D. R1 = OH, R2 = R3 = H
9-(3-deoxy-P-D-€/'pf/zra-pentofuranosyl)-l,9-dihydro-6H-purin-6-one
(3'-deoxyinosine)
E. R1 + R2 = O, R3 = H
9-(2,3-anhydro-P-D -ribofuranosyl)-l,9-dihydro-6H-purin-6-one
(2',3'-anhydroinosine)
333
F. R=H
9-(2,3-dideoxy-(3-D-g/ycero-pent-2-enofuranosyl]-l,9-dihydro-6F?-purin-6-one;
(2',3'-didehydro-2',3'-dideoxyinosine)
NH„
R. kX> 'N
G. R= OH
9-(2,3-dideoxy-P-D-g/ycero-pentofuranosyl)-9H-purin-6-amine
(2',3'-dideoxyadenosine)
H. R=H
9-(2,3,5-trideoxy-P-D-g/yi;ero-pentofuranosyl)-9H-purin-6-amine
(2',3',5'-trideoxyadenosine)
I. 9-(2,3-dideoxy-P-D-g/yc«/-o-pent-2-eno£uranosyl)-9H-purin-6-amine
(2',3'-dideoxy-2',3'-didehydroadenosine)
J. structure as shown for impurities B to E where R1 = R2 = H, R3 = CO-CH3

9-(5-0-acetyl-2,3-dideoxy-P-D-g/ycero-pento£uranosyl)-l,9-dihydro-6ff-purin-
6-one (didanosine acetate)
K. structure as shown for impurity F where R = CO-CH3

9-(5-0-acetyl-2,3-dideoxy-P-D-g/y«to-pent-2-enofuranosyl)-l,9-dihydro-6H'-
purin-6-one (2',3'-didehydrodidanosine acetate)
334
and epimer at C*
L. 9-[2,3-0-[(lRS)-l-methoxyethylene]-P-D-ribofuranosyl]-l,9-dihydro-6f/-
purin-6-one (2',3'-0-(l-methoxyethyKdene)inosine; (“dioxalane”)
M. mixture of 9-(3,5-di-0-acetyl-2-bromo-2-deoxy-P-D-arabinofuranosyl)-l,9-
dihydro-6/i-purin-6-one and 9-(2,5-di-0-acetyl-3-bromo-3-deoxy-P-D-
xylofuranosyl)-l,9-dihydro-6i4'-purin-6-one (“bromoesters”)
DIETHYLCARBAMAZINI
DIHYDROGENOCITRAS
DIETHYLCARBAMAZINE
DIHYDROGEN CITRATE
Molecular formula. CioH 2 iN 30 ,C 6 Hs 07 or Ci(jH29N308
335
Graphic formula.
CH-COOH
^
I
HO-C-COOH
I
CH COOH
Chemical name. N,N-Diethyl-4-methyl-l-piperazinecarboxamide citrate
(1 :
1); iV,N-diethyl-4-methyl-l-piperazinecarboxamide 2 -hydroxy- 1,2,3-
propanetricarboxylate (1;1); CAS Reg. No, 1642-54-2,
Description. A white, crystalline powder; odourless or almost odourless.
Solubility. Very soluble in water; soluble in 35 parts of ethanol (-750 g/1) TS;
Category. Filaricide.
Storage. Diethylcarbamazine dihydrogen citrate should be kept in a tightly

closed container, protected from light.
Additional information. Diethylcarbamazine dihydrogen citrate is hygro-

scopic; it has an acid and bitter taste. Even in the absence of light, Diethylcar-
bamazine dihydrogen citrate is gradually degraded on exposure to a humid
atmosphere, the decomposition being faster at higher temperatures.
Requirements
Definition. Diethylcarbamazine dihydrogen citrate contains not less than
98.0% and not more than 101.0% of CioH 2 iN 30 ,CsHa 07 calculated with
,
ref-
erence to the anhydrous substance.
Identity tests
• Either tests A and D or tests B and C may be applied.
A. Dissolve 0.05 g in 25 ml of water. Add 1 ml of sodium hydroxide (-80 g/1) TS

and 4 ml of carbon disulfide R, and shake for 2 minutes. Separate the
aqueous layer. Centrifuge the lower layer if necessary, and filter through a
dry filter, collecting the filtrate in a small flask provided with a glass stopper.
Carry out the examination of the filtered solution using carbon disulfide R
as the blank as described under Spectrophotometry in the infrared
1.7
region. The is concordant with the spectrum
infrared absorption spectrum
obtained from diethylcarbamazine dihydrogen citrate RS treated similarly
or with the reference spectrum of diethylcarbamazine base.
336
B. Dissolve 0.5g in 10ml of water, add 10ml

sodium hydroxide (1 mol/l)VS,
of
and extract with 4 successive quantities, each of 5 ml of chloroform R. Retain
the aqueous layer for test C. Wash the combined chloroform extracts with
water, filter through a plug of cotton wool, and evaporate the chloroform.
Add 1 ml of ethyl iodide R to the residue, and heat gently under a reflux
condenser for 5 minutes. Cool, separate the viscous yellow oil, and dissolve
it in ethanol (-750 g/1) TS. Add, with continuous stirring, sufficient ether R
to precipitate the quaternary ammonium salt, and filter. Dissolve the pre-
cipitate in ethanol (-750 g/1) TS, reprecipitate with ether R, and dry at
105 °C; melting temperature, about 152 °C (l-diethylcarbamoyl-4-
methylpiperazine ethiodide).
C. The aqueous layer from test B yields reaction B described under 2.1 General
identification tests as characteristic of citrates.
D. Melting temperature, after drying at 80 °C, about 137 °C.

is not more than lOmg/g.
iV-Methylpiperazine. Carry out the test as described under 1.14.1 Thin-layer

6 volumes of ethanol (~750g/l) TS, 3 volumes of glacial acetic acid R and 1
volume of water as the mobile phase. Apply separately to the plate 5pl of each
of 2 solutions in methanol R containing (A) 50 mg of the test substance per ml
and (B) 0.050 mg of N-methylpiperazine R per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air, spray with a mixture
of 3 volumes of platinic chloride (60g/l) TS, 97 volumes of water and 100
volumes of potassium iodide (60 g/1) TS, and examine the chromatogram in
daylight. The spot obtained with solution B is more intense than any spot,
corresponding in position and appearance, obtained with solution A.

alent to 39.14mg of CioH 2 iN 30 ,C 6 Hs 07 .
337
DIETHYLTOLUAMIDUM
DIETHYLTOLUAMIDE
N
IN CH3
'CH,
CH3
C,2Hi7NO
Chemical name. N,N-Diethyl-w-toluamide; N,W-diethyl-3-methylbenza-

mide; CAS Reg. No. 134-62-3.
Description. Colourless or faintly yellow liquid.
Solubility. Practically immiscible in water and glycerol R; miscible with

Category. Insect repellent.
Storage. Diethyltoluamide should be kept in a tightly closed container.
Additional information. CAUTION: Diethyltoluamide is an irritant to eyes

and mucous membranes.
Requirements
Diethyltoluamide contains not less than 97.0% and not more than 103.0% of
C 12 H 17NO, calculated with reference to the anhydrous substance.
Identity tests

infrared region.
spectrum obtained from diethyltoluamide RS or with the reference spectrum
of diethyltoluamide.
B. Refractive index, n™ = 1.520 - 1.524.
338
C. To about 2 ml, add 25ml TS and heat under

of hydrochloric acid (~250g/l)
a reflux condenser for hour. Neutralize the solution with sodium hydrox-
1
ide (-200 g/1) TS, cool, and extract with three quantities, each of 30ml, of
ether R. (Keep the aqueous layer for test D.) Carefully evaporate the ether
layer to dryness on a water-bath, and dissolve the residue in 5 ml of sodium
nitrite (lOOg/1) TS. Allow to stand at 5°C for 10 minutes, add 10ml of water,
and extract with 20 ml of ether R. Evaporate the ether layer and add to
the residue l.Og of phenol R. Cool and add about 1ml of sulfuric acid
(-1760 g/1) TS; an intense green solution is produced. Pour the mixture into
water; the colour turns to red. Add sodium hydroxide (-80 g/1) TS; the
colour changes to green.
D. Acidify the aqueous layer obtained in test C with hydrochloric acid (-70 g/1)
TS, extract with two quantities, each of 20 ml of ether R, and carefully evap-
orate the ether layer. Dry the residue at 60 °C; the melting temperature of
the residue is about 108 °C.
Mass density. p 2 o = 0.996-1.002.

Acidity. Dissolve 1 0.0 g in 50 ml of neutralized ethanol TS, titrate with sodium
hydroxide (0.01 mol/1) VS using phenolphthalein/ethanol TS as indicator; not

more than 4.0ml of sodium hydroxide (0.01 mol/1) VS is required to obtain the
midpoint of the indicator (pink).
Assay. Carry out Method A as described under 2.10 Determination of nitro-

gen, using about 0.3 g, accurately weighed, and 7 ml of nitrogen-free sulfuric
acid (-1760g/l) TS, and proceed with the distillation. Titrate with sulfuric acid
(0.05 mol/1) VS using methyl red/ethanol TS as indicator. Repeat the procedure
without the Diethyltoluamide being examined and make any necessary
corrections.
Each ml of sulfuric acid (0.05mol/l) VS is equivalent to 19.13mg of C^HuNO.
339
DIGITOXINUM
DIGITOXIN
Graphic formula.
OH OH OH
Chemical name. 3P-[(0-2,6-Dideoxy-P-D-rii’o-hexopyranosyl-(l—>4)-0-2,6-

dideoxy-P-D-nl?o-hexopyranosyl-(ip4)-2,6-dideoxy-p-D-ni’o-hexopyranosyl)-
oxy]-14-hydroxy-5P-card-20(22)-enolide; CAS Reg. No. 71-63-6.
Description. A white or almost white, microcrystalline powder; odourless.

TS.
Category. Cardiotonic.
Storage. Digitoxin should be kept in a well-closed container, protected from

light.
Additional information. CAUTION: Digitoxin is extremely poisonous and

should be handled with care.
Requirements
Definition. Digitoxin contains not less than 95.0% and not more than 105.0%
of C 41 H 64 O 13 ,
Identity tests
• Either tests A, B and D or tests B, C and D may be applied.
340

infrared region.
spectrum obtained from digitoxin RS or with the reference spectrum of
digi toxin.

of formamide R and 90 volumes of acetone R to impregnate the plate,
dipping it about 5 mm beneath the surface of the liquid. After the solvent
has reached a height of at least 15 cm, remove the plate from the chro-
matographic chamber and allow to stand for at least 5 minutes. Use the
impregnated plate within 2 hours, carrying out the chromatography in the
same direction as the impregnation. As the mobile phase, use a mixture of
50 volumes of xylene R, 50 volumes of ethylme thy Ike tone R and 4 volumes
of formamide R. Apply separately to the plate 3 pi of each of 2 solutions (A)
of the test substance, and (B) of digitoxin RS, each prepared by dissolving
50 mg in a mixture of equal volumes of chloroform R and methanol R to
produce 10 ml and then diluting 1 ml to 5 ml with methanol R. Develop the
plate for a distance of 12 cm. After removing the plate from the chromato-
graphic chamber, allow it to dry at 115°C for 20 minutes, cool, spray with
a mixture of 15 volumes of a solution of 25 g of trichloroacetic acid R in
100 ml of ethanol (-750 g/1) TS and 1 volume of a freshly prepared 30 mg/ml
solution of tosylchloramide sodium R, and then heat the plate at 115°C for
5 minutes. Allow to cool, and examine the chromatogram in daylight and
in ultraviolet light (365 nm). The principal spot obtained with solution A cor-
solution B.
C. Dissolve 1 mg in 1ml of ethanol (-750g/l) TS by heating gently. Cool the

solution and add 1 ml of dinitrobenzene/ethanol TS and 1 ml of potassium
hydroxide (1 mol/1) VS; a violet colour develops and then fades.
D. Dissolve Img in 2 ml of a solution prepared by mixing 0.5ml of ferric chlo-

ride (25 g/1) TS and 100 ml of glacial acetic acid R; cautiouslyadd 1 ml of sul-
furic acid (-1760 g/1) TS to form a lower layer; a brown ring, but no red
colour, is produced at the junction of the two liquids, and after some time the
acetic acid layer acquires a blue colour (distinction from allied glycosides).
Specific optical rotation. Use a lOmg/ml solution in chloroform R and cal-
culate with reference to the dried substance; [aj^ = -1-16.5 to -1-18.5°.
20mg/g.
341
Gitoxin. Dissolve about 5 mg, accurately weighed, in 1 ml of methanol R and

dilute to 25 ml with a mixture of equal volumes of hydrochloric acid (-250 g/1)
TS and glycerol R.Allow to stand for 1 hour. The absorbance of a 1-cm layer
of this solution at 352 nm, when measured against a solvent cell containing a
mixture of equal volumes of hydrochloric acid (-250 g/1) TS and glycerol R, is
not more than 0.28 (preferably use 2-cm cells for the measurement and calcu-
late the absorbance of a 1-cm layer); the gitoxin content is about 50mg/g.
Assay. Dissolve about 0.05 g, accurately weighed, in sufficient methanol R to

produce 25ml; dilute 5.0 ml of this solution to 100ml with methanol R. Place
5.0ml of the dilute solution to be tested in a 25-ml volumetric flask, add 15ml
of alkaline trinitrophenol TS, and dilute to 25 ml with methanol R. Set aside for
30 minutes, protected from light, and measure the absorbance in a 1-cm layer
at the maximum at about 490 nm against a solvent cell containing a solution
prepared by diluting 15 ml of alkaline trinitrophenol TS to 25 ml with methanol
R. Calculate the amount of C41H64O13 in the substance being tested by com-
parison with digitoxin RS, similarly and concurrently examined.
Additional requirements for Digitoxin for prarenteral use

terial endotoxins; contains not more than lll.OlU of endotoxin RS per mg.
DIGOXINUM
DIGOXIN
Graphic formula.
342
Chemical name. 3(3-[(0-2,6-Dideoxy-|3-D-nfe-hexopyranosyl)-(l—>4)-0-2,6-

dideoxy-P-D-^•/^'o-hexopyranosyl-(l|34)-2,6-dideoxy-P-D-^ô-hexopyranosyl)-
oxy]-12P,14-dihydroxy-5P-card-20(22)-enolide; CAS Reg. No, 20830-75-5.

powder; odourless.
Solubility. Practically insoluble in water and ether R; freely soluble in pyridine

R; slighdy soluble in ethanol (-750 g/1) TS.
Category. Cardiotonic.
Storage. Digoxin should be kept in a well-closed container, protected from

light.
Additional information. CAUTION: Digoxin is extremely poisonous and

should be handled with care.
Requirements
Definition. Digoxin contains not less than 95.0% and not more than 103.0%
of CîH^Ôh, calculated with reference to the dried substance.
Identity tests
• Either tests A, B and D or tests B, C and D may be applied.

infrared region.
spectrum obtained from digoxin RS or with the reference sfectrum of digoxin.
B. See the test described below under “Related substances”. In daylight the
principal spot obtained with solution A corresponds in position, appearance,
and intensity with that obtained with solution B.
C. Dissolve 1mg in 1ml of ethanol (~750g/l) TS by heating gendy. Cool the

solution, add 1ml of dinitrobenzene/ethanol TS and 1ml of potassium
hydroxide (1 mol/1) VS; a violet colour develops and then fades.
D. Dissolve Img in 2 ml of a solution prepared by mixing 0.5ml of ferric chlo-

ride (25 g/1) TS and 100 ml of glacial acetic acid R; cautiously add 1ml of
sulfuric acid (~1760g/l) TS to form a lower layer; a brown ring, but no
red colour, is produced at the junction of the two liquids, and after some
time the acetic acid layer acquires a blue colour (distinction from allied
glycosides).
343
Specific optical rotation. Use a O.lOg/ml solution in pyridine R; =

+13.6 to +14.2°.

reduced pressure (not exceeding 0.6kPa or about 5 mm of mercury); it loses not
more than lOmg/g.
Gitoxin. Dissolve about 5mg, accurately weighed, in 1 ml of ethanol (~675g/l)

TS by heating gently, and dilute to 25ml with a mixture of equal volumes of
hydrochloric acid (-250 g/1) TS and glycerol R. Allow to stand for 1 hour. The
absorbance of a 1-cm layer of this solution at 352 nm, when measured against
a solvent cell containing a mixture of equal volumes of hydrochloric acid
(-250 g/1) TS and glycerol R, is not more than 0.22 (preferably use 2-cm cells for
the measurement and calculate the absorbance of a 1-cm layer); about 40mg/g.
chromatography, using kieselguhr R1 as the coating substance and a mixture
of 10 volumes of formamide R and 90 volumes of acetone R to impregnate the
plate, dipping it about 5mm beneath the surface of the liquid. After the solvent
has reached a height of at least 15 cm, remove the plate from the chromato-
graphic chamber and allow to stand for at least 5 minutes. Use the impregnated
plate within 2 hours, carrying out the chromatography in the same direction
as the impregnation. As the mobile phase, use a mixture of 50 volumes of
xylene R, 50 volumes of ethylmethylketone R, and 4 volumes of formamide R.
Apply separately to the plate 1 pi of each of 3 solutions in a mixture of equal
volumes of methanol R and chloroform R containing (A) 5.0mg of the test sub-
stance per ml, (B) 5.0 mg of digoxin RS per ml and (C) 0.25 mg of digitoxin RS
per ml. Develop the plate for a distance of 12 cm. After removal of the plate
from the chromatographic chamber, allow it to dry at 115°C for 20 minutes,
cool, spray with a mixture of 15 volumes of a solution of 25 g of trichloroacetic
acid R in 100ml of ethanol (-750g/l) TS and 1 volume of a freshly prepared
30mg/ml solution of tosylchloramide sodium R, and then heat the plate at
115°C for 5 minutes. Allow to cool, and examine the chromatogram in day-
light and in ultraviolet light (365 nm). Any spot obtained with solution A in
ultraviolet light, other than the principal spot, is not more intense than that
obtained with solution C.

produce 25ml; dilute 5.0 ml of this solution to 100ml with methanol R. Place
5.0ml of the dilute solution to be tested in a 25-ml volumetric flask, add 15ml
of alkaline trinitrophenol TS, and dilute to 25 ml with methanol R. Set aside for
30 minutes protected from light, and measure the absorbance in a 1-cm layer
at the maximum of about 490 nm against a solvent cell containing a solution
prepared by diluting 15 ml of alkaline trinitrophenol TS to 25 ml with methanol
344
R. Calculate the amount of C41H64O14 in the substance being tested by com-

parison with digoxin RS, similarly and concurrently examined.
Additional requirements for Digoxin for p>arenteral use

DILOXANIDI FUROAS
DILOXANIDE FUROATE
Molecular formula. C14H11CI2NO4
Graphic formula.
Chemical name. 2,2-Dichloro-4'-hydroxy-N-methylacetanilide 2-furoate

(ester); 4-[(dichloroacetyl)methylamino]phenyl 2-furancarboxylate; 2,2-
dichloro-N-(4-hydroxyphenyl)-Al-methylacetamide 2-furoate; CAS Reg. No.
3736-81-0.

(-750 g/1) TS and in 130 parts of ether R.
Storage. Diloxanide furoate should be kept in a well-closed container, pro-

tected from light.
Requirements
Definition. Diloxanide furoate contains not less than 98.0% and not more
than 102.0% of C14H11CI2NO4, calculated with reference to the dried substance.
345
Identity tests

infrared region.
spectrum obtained from diloxanide furcate RS or with the reference spectrum
of diloxanide furoate.
B. The absorption spectrum of a 7.0pg/ml solution in ethanol (-750 g/1) TS,

258 nm; the absorbance of a Tcm layer at this wavelength is about 0.49.
C. Carry out the combustion as described under 2.4 Oxygen flask method,
using 20mg of the test substance and 10ml of sodium hydroxide (1 mol/1)
VS as the absorbing liquid. When the process is complete, acidify with nitric
acid (-130 g/1) TS; the solution yields reaction A, described under 2.1
General identification tests as characteristic of chlorides.
5-Omg/g.
Free acidity. Shake 3.0 g with 50 ml of carbon-dioxide-free water R, filter and

wash the residue with 3 quantities, each of 20ml of carbon-dioxide-free water
R. Titrate the combined filtrate and washings with sodium hydroxide (0.1 mol/1)
VS, phenolphthalein/ethanol TS being used as indicator; not more than 1.3ml
is required.
24 volumes of dichlorome thane R and 1 volume of methanol R as the mobile
phase. Apply separately to the plate 5 pi of each of 2 solutions in chloroform
R containing (A) O.lOg of the test substance per ml and (B) 2.5mg of the test
allow it to dry in air and examine the chromatogram in ultraviolet light
(254 nm). Any spot obtained with solution A, other than the principal spot, is
Assay. Dissolve about 0.3 g, accurately weighed, in 50ml of anhydrous pyri-

dine R and titrate with tetrabutylammonium hydroxide (0.1 mol/1) VS deter-
mining the endpoint potentiometrically as described under 2.6 Non-aqueous
titration. Method B. Each ml of tetrabutylammonium hydroxide (0.1 mol/1) VS
is equivalent to 32.82 mg of C14H11CI2NO4.
346
DIMERCAPROLUM
DIMERCAPROL
Molecular formula. C3H5OS2
Graphic formula.
CH,CHCH,OH
1 I
SH SH
Chemical name. 2,3-Dimercapto-l-propanol; CAS Reg. No. 59-52-9.
Description. A clear, colourless or slightly yellow liquid, with an unpleasant,

mercaptan-like odour.
Miscibility. Miscible with 20 parts of water; miscible with ethanol (-750 g/1)
TS and methanol R.
Category. Antidote for arsenic, gold, and mercury poisoning.
Storage. Dimercaprol should be kept in a small, well-filled and tightly closed

container, protected from light, and stored at a temperature not exceeding 5 °C.
Requirements
Definition. Dimercaprol contains not less than 98.5% w/w and not more than
101.5% w/w ofC,3H80S2.
Identity tests
A. Mix 0.05 ml of cobalt(Il) chloride (30 g/1) TS with 5 ml of water and add
0.05 ml of the test liquid; a yellow-brown colour is produced.
B. Dissolve 0.1 ml in 4 ml of water and add a few drops of lead acetate (80 g/1)
TS; a yellow precipitate is formed.
Refractive index, = 1.568 - 1.574.
Relative density. 1^20 = 1-239 - 1.259.
Halides. Dissolve 2.0 g in 25ml of potassium hydroxide/ethanol TSl and heat

under a reflux condenser for 2 hours. Evaporate the ethanol in a current of warm
air, add 20 ml of water, and cool. Add a mixture of 10 ml of hydrogen peroxide
347
N
(-330 g/1) TS and 40 ml of water, boil gently for 10 minutes, cool, and filter
rapidly. Add 10 ml of nitric acid (-130 g/1) TS and 5 ml of silver nitrate (0.1 mol/1)
VS and titrate with ammonium thiocyanate (0.1 mol/1) VS, using ferric am-
monium TS as indicator. Repeat the operation without the test
sulfate (45 g/1)
liquid being examined. The difference between the titrations does not exceed
1.0ml.
pH value. pH of a 0.5g/ml solution in carbon-dioxide-free water R, 4. 6-6.8.
Assay. Dissolve about0.12g, accurately weighed, in20ml of hydrochloric acid

(0.1 mol/1) VS and titrate rapidly with iodine (0.05 mol/1) VS, using starch TS as
indicator. Repeat the operation without the test liquid being examined and
make any necessary corrections. Each ml of iodine (0.05 mol/1) VS is equivalent
to 6.21 1 mg of C3H8OS2.
Additional requirement for Dimercaprol for parenteral use

DINATRII EDETAS
DISODIUM EDETATE
NaOsC — CH2.^ yCH2 — COjNa
N — CHj — CHa — • 2HaO
HO2C — CHg-^ ^CHa — COaH
CioHi4N2Na20g,2H20
Chemical name. Disodium dihydrogen (ethylenedinitrilo)tetraacetate dihy-

drate; N,Ai'-l,2-ethanediylbis[A/-(carboxymethyl)glycine] disodium salt, dihy-
drate; CAS Reg. No. 6381-92-6.
Other name. Edetate disodium.
Solubility. Soluble in water; slightly soluble in ethanol (-750 g/1) TS; practi-
Category. Stabilizer; chelating agent.
348
Storage. Disodium edetate should be kept in a well-closed container.
Additional information. Solutions of disodium edetate should not come into

contact with metal.
Requirements
Disodium edetate contains not less than 98 5 % and not more than the equiv-
.
alent of 101 0 % of Ci„H„N 2 Na 203 2 H 20

.
,
.
Identity tests

spectrum obtained from disodium edetate R or with the reference spectrum
of disodium edetate.
B. To 3 drops of ferric chloride (25g/l) TS add 3 drops of ammonium

thiocyanate (75g/l) TS; to the deep red solution produced add 0.05 g of
Disodium edetate; the colour is discharged, leaving a yellowish solution.
(Keep this solution for test D.)
C. Dissolve 2g in 25ml of water, add 2ml of lead nitrate (100 g/1) TS, shake,
and add 6ml of potassium iodide (80 g/1) TS; no yellow precipitate is
observed.
D. To the solution from test B, add ammonia (~100g/l) TS, drop by drop, until
an alkaline reaction is obtained with pH-indicator paper R. Add 5 ml of
ammonium oxalate (25 g/1) TS; no precipitate is produced (distinction from
sodium calcium edetate).
pH value. pH of a 0.05 g/ml solution, 4. 0-5. 5.
Assay. Dissolve 0.5 g, accurately weighed, in sufficient water to produce

300 ml. Add 2g of methenamine R and 2 ml of hydrochloric acid (-70 g/1) TS.
Titrate with lead nitrate (0.1 mol/1) VS to which 50 mg of xylenol orange
indicator mixture R has been added.
Each ml of lead nitrate (0.1 mol/1) VS is equivalent to 37.22 mg of

CioHi4N2Na20g,2H20.
349
DINITRO GENII OXIDUM

DINITROGEN OXIDE
N2O
Chemical name. Nitrous oxide; CAS Reg. No. 10024-97-2.
Other name. Nitrous oxide.
Description. A colourless gas; odourless.
Solubility. One volume dissolves in about 1.5 volumes of water at a pressure

of lOl.SkPa and a temperature of 20 °C.
Category. Inhalational anaesthetic gas.
Storage. Dinitrogen oxide should be kept as compressed gas or liquid at very

low temperatures, in appropriate containers complying with the safety regula-
tions of the national authority. Valves or taps should not be lubricated with oil
or grease.
Labelling. An ISO standard' requires that cylinders containing Dinitrogen

oxide intended for medical use should bear the name of the contents in legible
and permanent characters and, preferably, also the molecular formula N 2 O.
Additional information. In the analysis of medicinal gases certain tests are

not intended for hospital pharmacists. They are applicable solely by laborato-
ries equipped with specialized apparatus.
Requirements
Dinitrogen oxide contains not less than 98.0% v/v of NO2 in the gaseous
phase, when sampled at 15 °C.
Note-. If the analysis is performed on a cylinder, keep the cylinder of the gas
to be examined at room temperature for at least 6 hours before carrying out
the tests. Keep the cylinder in the vertical position with the outlet valve
uppermost.
’
International Standard 3>2. Gas cylinders for medical use - marking for identification content. International
Organization for Standardization, Switzerland, 1977.
350
The test for carbon monoxide should be carried out on the first portion of gas
drawn from the container and the tests for nitrogen monoxide and nitrogen
dioxide immediately thereafter.
Identity tests

reference spectrum of dinitrogen oxide.
B. Place a glowing splinter of wood into the gas; the splinter bursts into
flame.
C. Shake the gas with alkaline pyrogallol TS; it is not absorbed and the solu-
tion does not become brown (distinction from oxygen).
D. Mix the gas with an equal volume of nitrogen monoxide R; no red fumes
are produced (distinction from oxygen).
Carbon monoxide
• Either test A, test B, or test C may be applied.
• The tests should be carried out on the first portion of gas released from the
container.
A. The apparatus (Fig. 6) consists of the following parts connected in series:
— a U-tube (Ul) containing desiccant silica gel R impregnated with

chromium trioxide R;
— a wash bottle (FI) containing 100 ml of potassium hydroxide (-400 g/1)

TS;
— a U-tube (U2) containing pellets of potassium hydroxide R;
—a U-tube (U3) containing phosphorus pentoxide R dispersed on previ-

ously granulated, fused pumice;
— a U-tube (U4) containing 30 g of recrystallized iodine pentoxide R in

granules, previously dried at 200 °C and kept at a temperature of 120 °C
(T) during the test. The iodine pentoxide is packed in the tube in 1-cm
columns separated by 1-cm columns of glass wool to give an effective
length of 5 cm;
351
Figure 6Apparatus for the determination of carbon monoxide in medicinal gases

.
Measurements in mm.
Reproduced with the permission of the European Pharmacopoeia Commission,
European Directorate for the Quality of Medicines, Council of Europe.
— a reaction tube (F2) containing 2.0 ml of potassium iodide (160g/l) TS

and 0.15ml of starch TS.
Flush the apparatus with 5.0 litres of argon R. If necessary, discharge the
blue colour in tube F2 containing potassium iodide (160g/l) TS by adding a
volume of freshly prepared sodium thiosulfate (0.002 mol/1) VS.
sufficient
Continue flushing with argon R until not more than 0.045 ml of sodium thio-
sulfate (0.002 mol/1) VS is required after the passage of 5.0 litres of argon R.
from the container through the apparatus. Flush
Pass 5.0 litres of the test gas
by passing 1.0 litre
the last traces of liberated iodine into the reaction tube
of argon R through the apparatus. Titrate the liberated iodine with sodium
thiosulfate (0.002 mol/1) VS. Repeat the procedure using 5.0 litres of argon
R.
The difference between the volumes of sodium thiosulfate (0.002 moI/1) VS

used in the is not more than 0.25ml (5pl/l).
titrations
B. Carry out the test as described under 1.14.5 Gas chromatography, using a
stainless steel column (2 m x 4 mm) packed with a 0.5-nm molecular sieve
(e.g. xl3, obtainable from a commercial source). Maintain the column at
80 °C, and the injection port and the detector at room temperature. Use
helium R as the carrier gas at a flow rate of 60 ml per minute, and a helium
ionization detector.
352
Use the following gases: (1) the test gas; and (2) a mixture containing 5|tl
of carbon monoxide R in 1 litre of dinitrogen oxide R as the reference gas.
Inject a suitable volume of both gases and (2). Adjust the volume, as well
(1)
as the conditions specified above, to produce a peak response for carbon
monoxide obtained with the reference gas (2) that gives a height of not less
than 5% on the recorder.

from injections 1 and 2 and calculate the content of carbon monoxide in the
test gas (1) by comparing with the peak response for carbon monoxide
obtained from the reference gas (2); not more than 5pl/l.
C. Determine the content using a carbon monoxide detector tube. Pass the
required volume of the test gas through the tube, the calibration of which
is verified according to the manufacturer’s instructions.
The gas supply is connected to a pressure regulator and needle valve.

Connect the flexible tubing fitted with a Y-piece to the valve and adjust
the flow of the test gas to purge the tubing to an appropriate flow. Fit the
carbon monoxide detector tube to the metering pump according to the man-
ufacturer’s instructions. Connect the open end of the tube to the short leg
of the tubing and pump a suitable volume of the test gas through the tube.
Read the value corresponding to the length of the coloured layer or the
intensity of the colour on the graduated scale; not more than 5 pl/1.
Note: For the following tests - Nitrogen

monoxide and nitrogen dioxide, Carbon
dioxide Test A, Halogens and hydrogen sulfide, and Acidity and alkalinity -
pass the gas to be tested through the appropriate reagent contained in a her-
metically closed flat-bottomed glass cylinder, with dimensions such that 50ml
of liquid reaches a height of 12-14 cm, that is fitted with (a) a delivery tube ter-
minated by a capillary 1 mm
in internal diameter and placed within 2 mm of
the bottom of the cylinder; and (b) an outlet tube.
Prepare the reference solutions in identical cylinders.
Nitrogen monoxide and nitrogen dioxide

• This test should be performed after release of the 5.0 litres of gas as
described above under “Carbon monoxide, test A”.
A. Pass the test gas through two of the cylinders connected in series as
described above under “Carbon monoxide, test A”. To obtain the liquid
phase invert the gas cylinder; the liquid vaporizes on leaving the valve.
353
To 50 ml of water add 1.2 ml of sulfuric acid (~1760g/l) TS and dilute with

sufficient water toproduce 100ml. To 15ml of this solution add 375mg
of potassium permanganate R, mix, and transfer to the first cylinder
{solution A).
Dissolve Ig of sulfanilic acid R in a mixture of 180 ml of water and 10 ml

of glacial acetic acid R (solution 1). Separately dissolve 0.2g of N-(l-naph-
thyl)ethylenediamine hydrochloride R in a mixture of 4 ml of glacial acetic
acid R and 5 ml of water, heat gently, and dilute to 200ml with water (solu-
tion 2). Mix 1 volume of solution 2 with 9 volumes of solution 1 and trans-
fer 20 ml of this mixture to the second cylinder {solution B).
Connect the outlet tube of the first cylinder to the delivery tube of the
second cylinder containing solution B. Pass 2.5 litres of the test gas through
the reagents at a rate of 15.0 litres per hour.
Prepare a reference solution by adding 0.25ml of a solution containing

61.6pg/ml of sodium nitrite R in water to 20 ml of solution B as prepared
above. Allow the test solution and reference solution to stand for 10 minutes.
Examine the gaseous and the liquid phases separately.
For both gaseous and liquid phases, any red colour produced from the solu-
tion of the test gas is not more intense than that from the reference solu-
tion (2pl/l of NO- -NO
1
2 ).
B. Determine the content using a nitrogen monoxide and nitrogen dioxide detec-
tor tube. Pass the required volume of the test gas through the tube, the cali-
bration of which is verified according to the manufacturer’s instructions.

Connect the with a Y-piece to the valve and adjust the
flexible tubing fitted
flow of the purge the tubing to an appropriate flow. Fit the nitro-
test gas to
gen monoxide and nitrogen dioxide detector tube to the metering pump fol-
lowing the manufacturer’s instructions. Connect the open end of the tube
to the short leg of the tubing and pump a suitable volume of the test gas
through the tube. Read the value corresponding to the length of the coloured
layer or the intensity of the colour on the graduated scale; not more than
2pl/l.
Carbon dioxide
• Either test A, test B, or test C may be applied.
A. Pass 1.0 litre 50ml of a clear solution of barium

of the test gas through
hydroxide (0.15mol/l) VS. Similarly prepare a reference solution by adding
354
1.0ml of a 1.1mg/ml solution of sodium hydrogen carbonate R in carbon-

dioxide-free water R to 50ml of barium hydroxide (0.15mol/l) VS.
Any turbidity in the solution after the passage of the test gas is not more
intense than that of the reference solution (300pl/l).
B. Carry out the test as described under 1.14.5 Gas chromatography, using a
stainless steel column (3.5 m x 2mm) packed with ethylvinyl-benzenedi-
vinylbenzene copolymer. Maintain the column at 40 °C and the detector at
90 °C. Use helium R as the carrier gas at a flow rate of 15ml per minute,
and a thermal conductivity detector.
Use the following gases: (1) the test gas; and (2) a mixture containing 300 pg
of carbon dioxide R in 1 litre of dinitrogen oxide R as the reference gas.
Inject a suitable volume of both gases (1) and (2). Adjust the volume, as well
as the conditions specified above, to obtain a peak response for carbon
dioxide obtained with the reference gas (2) of a height of not less than 35%
on the recorder.

from the injections of gases 1 and 2 and calculate the content of carbon
dioxide in the test gas (1) by comparing with the peak response for carbon
dioxide obtained from the reference gas (2); not more than 300 pi of COj
per litre.
C. Determine the content using a carbon dioxide detector tube. Pass the
The gas supply is connected to a suitable pressure regulator and needle

valve. Connect the flexible tubing fitted with a Y-piece to the valve and
adjust the flow of the test gas to purge the tubing to an appropriate flow.
Fit the carbon dioxide detector tube to the metering pump according to the
manufacturer’s instructions. Connect the open end of the tube to the short
leg of the tubing and pump a suitable volume of the test gas through the
tube. Read the value corresponding to the length of the coloured layer or
the intensity of the colour on the graduated scale; not more than 300pl/l.
Halogens and hydrogen sulfide. Pass 20.0 litres of the test gas through a
mixture of 1 ml of silver nitrate (40g/l) TS and 49 ml of water at a flow rate not
exceeding 15 litres per hour.
Prepare the reference solution as follows: to 1.0 ml of silver nitrate (40g/l) TS

add 40ml of chloride standard (5pg/ml) TS and 0.15ml of nitric acid (~130g/l)
TS, dilute to 50 ml with water, and allow to stand protected from light for 5
355
minutes. For the blank solution, repeat the procedure passing the test gas
through 50 ml of water.
Compare a 100-mm layer of the solution as described under 1.11 Colour of

liquids.
The solution of the test gas does not darken when compared with the blank.
Any opalescence is not more intense than that of the reference solution (10|ig
Cl per litre of dinitrogen oxide).
Water
A. The apparatus consists of either an electrolytic hygrometer as described

below, an appropriate humidity detector tube, or a capacity hygrometer.
The measuring cell consists of a thin film of phosphoric anhydride placed

between two coiled platinum wires which act as electrodes. The water
vapour in Dinitrogen oxide is absorbed by the phosphoric anhydride to form
phosphoric acid which acts as an electrical conductor.
Before introducing the test gas into the device, allow the gas to stabilize at
room temperature and make sure that the temperature is constant through-
out the apparatus. Apply a continuous voltage across the electrodes to
produce electrolysis of the water and regeneration of phosphoric anhydride.
Measure the resulting electric current, which is proportional to the water
content in the test gas. (This is a self-calibrating system that obeys Faraday’s
law.)
Calculate the content of water; not more than 60 pg/1.
B. Determine the content using a water vapour detector tube. Pass the required
volume of the test gas through the tube, the calibration of which is verified
according to the manufacturer's instructions.

Fit the water vapour detector tube to the metering pump according to the
leg of the tubing and pump a suitable volume of the test gas through the
tube. Read the value corresponding to the length of the coloured layer or
the intensity of the colour on the graduated scale; not more than 60pl/l.
356
Acidity and alkalinity. Pass 2.0 litres of the test gas through a mixture of
0.10ml of hydrochloric acid (0.01 mol/1) VS and 50ml of carbon-dioxide-free
water R.
For reference solution i, use 50 ml of carbon-dioxide-free water R. For reference

solution Z, use a mixture of 0.20ml of hydrochloric acid (0.01 mol/1) VS and
50 ml of carbon-dioxide-free water R.
To each solution add 0.1 ml of methyl red/ethanol TS; the intensity of the colour
in the test gas solution isbetween that of reference solutions 1 and 2.
Assay. Determine as described under 1.14.5 Gas chromatography, using a

stainless steel column (2 m x 2 mm) packed with silica gel for chromatography
R (250 — 355 pm). Maintain the column at 60°C and the detector at 130 °C. Use
helium R as the carrier gas at a flow rate of 50 ml per minute, and a thermal
conductivity detector.
Use the following gases: (1) the test gas; and (2) dinitrogen oxide R as the
reference gas.
Inject a suitable volume of both gases (1) and (2). Adjust the volume, as well
as the conditions specified above, to produce a peak response for dinitrogen
oxide obtained with reference gas (2) that gives a height of not less than 35%
on the recorder.
the injections of gases (1) and (2), and calculate the percentage content of dini-
trogen oxide.
DIPHENOXYLATI HYDRO CHLORIDUM

DIPHENOXYLATE HYDROCHLORIDE
Molecular formula. C3oH.s2N202,HCl
357
Graphic formula.
CN
Chemical name. Ethyl l-(3-cyano-3,3-diphenylpropyl)-4-phenylisonipecotate

monohydrochloride; ethyl l-(3-cyano-3,3-diphenylpropyl)-4-phenyl-4-
piperidinecarboxylate monohydrochloride; CAS Reg. No. 3810-80-8.
Solubility. Sparingly soluble in water, acetone R and ethanol (-750 g/1) TS;
Category. Antidiarrhoeal drug.
Storage. Diphenoxylate hydrochloride should be kept in a well-closed container.
Requirements
Definition. Diphenoxylate hydrochloride contains not less than 98.0% and
not more than 101.0% of C 3oH 32 N 202 ,HCl, calculated with reference to the
dried substance.
Identity tests
• Either tests A and E or tests B, C, D and E may be applied.

spectrum obtained from diphenoxylate hydrochloride RS or with the refer-
ence spectrum of diphenoxylate hydrochloride.
B. The absorption spectrum of a 0.50 mg/ml solution in a mixture of 1 volume

of hydrochloric acid (1 mol/1) VS and 99 volumes of methanol R, when
observed between 230 nm and 350 nm, exhibits maxima at about 252 nm,
258 nm, and 265 nm; the absorbances of a 1-cm layer at these wavelengths
are about 0.55, 0.65 and 0.50, respectively.
C. Dissolve 25 mg in 5 ml of water and add 0.1 ml of potassio-mercuric iodide

TS; a cream-coloured precipitate is produced.
358
E. A 20mg/ml solution yields reaction B described under 2.1 General identifi-

cation tests as characteristic of chlorides,
5-Omg/g.
92 volumes of chloroform R, 3 volumes of methanol R, and 5 volumes of glacial
the chromatographic chamber, allow it to dry in air, expose it to the vapour of
iodine, and examine the chromatogram in daylight. Any spot obtained with
with solution B.
ml of perchloric acid (0.1 mol/1) VS is equivalent to 48.91 mg of C3oH32N202,HCl.
DITHRANOLUM
DITHRANOL
C14H10O3
Chemical name. 1,8,9-Anthratriol; CAS Reg. No. 1143-38-0.
Other name. Anthralin.
359
Description. A yellow or brownish yellow, crystalline powder.
Solubility. Practically insoluble in water; soluble in dichlorome thane R; spar-

ingly soluble in acetone R; slightly soluble in ethanol (~750g/l) TS and ether R.
Storage. Dithranol should be kept in a tightly closed container, protected from

light.
Requirements
Dithranol contains not less than 98 5
. % and not more than 101 0 % of .
C 14 H 10 O 3 ,
Identity tests

spectrum obtained from dithranol RS or with the reference spectrum of
dithranol.
B. The absorption spectrum of a lOpg/ml solution in dichloromethane R, when

observed between 250 nm and 450 nm, exhibits 3 maxima at about 256nm,
288 nm, and 356 nm. The absorbance of a 1 -cm layer at the maximum wave-
length at 356nm is about 0.46 and at 288nm about 0.49.
C. Carry out the test as described under 1.14.1 Thin-layer chromatography,

of hexane R and dichloromethane R as the mobile phase. Apply separately
to the plate 10 pi of each of 3 solutions in dichloromethane R containing (A)
1.0 mg of Dithranol per ml, (B) 1.0 mg of dithranol RS, and for solution (C)
dissolve 5 mg of dantron R in 5 ml of solution B. After removing the plate
from the chromatographic chamber, allow it to dry in air. Place the plate in
a chamber saturated with ammonia vapour until the spots appear. Examine
the chromatogram in daylight.

ance, and intensity with that obtained with solution B. The test is not valid
unless the chromatogram obtained with solution C shows two clearly sep-
arated spots.
360
Chlorides. Dissolve 2.5g in a mixture of 2.0ml of nitric acid (-130 g/1) TS and
30ml of water, and proceed as described under 2.2,1 Limit test for chlorides;
Loss on drying. Dry to constant mass at 105°C; it loses not more than 5 mg/g.
pH value. Shake 1.5g with 30ml of carbon-dioxide-free water R for 1 minute

and filter; pH of the filtrate, 6.0-7. 6.
Related substances
matography, using a stainless steel column (25cm x 4.6mm) packed with
particles of porous silica (5pm). As the mobile phase, use a mixture of 82
volumes of hexane R, 5 volumes of dichloromethane R, and 1 volume of
glacial acetic acid R.
Prepare the following solutions. For solution (A) dissolve 0.20 g of Dithra-
nol in 20 ml of dichloromethane R, add 1.0 ml of glacial acetic acid R, and
dilute to 100ml with hexane R. For solution (B) dissolve lO.Omg of each of
anthrone R, dantron R, 9,9'-bisanthracene-10,10'(9H,9'f/)‘ dione RS, and
dithranol RS in dichloromethane R, and dilute to 10.0 ml with the same
solvent. To 1.0 ml of this solution add 19 ml of dichloromethane R and
1.0 ml of glacial acetic acid R, and dilute to 50 ml with hexane R.
olet spectrophotometer set at a wavelength of 260 nm.
Inject 20 pi each of solutions A and B. Continue the chromatography for

1.5 times the retention time of the peak due to 9,9'-bisanthracene-
10,10'(9H,9'H)-dione obtained with solution B. Adjust the sensitivity of the
system so that the height of the peak due to dithranol in the chromatogram
obtained with solution B is about 70% of the fuU scale of the recorder. The
peaks are eluted in the following order: dithranol, dantron, anthrone and
9,9'-bisanthracene-10,10'(9H,9'H)-dione. The test is not valid unless, in the
chromatogram obtained with solution B, the resolution between the peaks
due to dithranol and dantron is greater than 2.0.

as a percentage. In the chromatogram obtained with solution A, the area
of any peak corresponding to anthrone, dantron or 9,9'-bisanthracene-
10,10'(9ff,9'f/)-dione is not greater than that of the corresponding peak in
the chromatogram obtained with solution B (1.0%). The area of any peak.
361
other than the principal peak and any peaks due to anthrone, dantron or
is not greater than that of the peak
9,9'-bisanthracene-10,10'(9f/,9'fl)-dione,
due to dithranol in the chromatogram obtained with solution B (1.0%).
B. Carry out the test as described under 1.14.4 High performance liquid chro-
matography, using a stainless steel column (20 cm x 4.6mm) packed with
cally bonded octadecylsilyl groups (5 pm). As the mobile phase, use a
mixture of 60 volumes of water, 40 volumes of tetrahydrofuran R, and 2.5
volumes of glacial acetic acid R.
Dithranol per ml; and for solution (B) dissolve 0.5 mg of l-hydroxy-9-
anthrone RS and 0.5 mg of dithranol RS per ml, and dilute 1.0 ml of this solu-
tion to 20ml with the mobile phase.
Operate with a flow rate of about 0.9ml per minute. As a detector use an
ultraviolet spectrophotometer set at a wavelength of 254 nm.
Inject 20 pi each of solutions A and B. Continue the chromatography for 3

times the retention time of the peak due to dithranol. The test is not valid
unless, chromatogram obtained with solution B, the resolution
in the
between the peaks due to l-hydroxy-9-anthrone and dithranol is greater
than 2.5.

any peak corresponding to l-hydroxy-9-anthrone is not greater than that of
the corresponding peak in the chromatogram obtained with solution B
(2.5%).
The total content of related substances as determined in tests A and B is not

more than 3.0%.
Assay. Dissolve about 0.2 g, accurately weighed, in 50ml of anhydrous pyri-

dine R and titrate under an atmosphere of nitrogen with tetrabutylammonium
hydroxide (0.1 mol/1) VS as described under 2.6 Non-aqueous titration. Method
B, determining the end-point potentiometrically.
Each ml of tetrabutylammonium hydroxide (0.1 mol/1) VS is equivalent to

22.62mg of Ci^HjoOs.
362
DOPAMINI HYDROCHLORIDUM
DOPAMINE HYDROCHLORIDE
Molecular formula. CsHuNOjjHCl
Graphic formula.
Chemical name. 4-(2-Aminoethyl)pyrocatechol hydrochloride; 4-(2-amino-

ethyl)-l,2-benzenediol hydrochloride; CAS Reg. No. 62-31-7.

powder; odourless.
Solubility. Freely soluble in water; soluble in methanol R; practically insolu-

ble in ether R and toluene R.
Category. Cardiovascular drug; sympathomimetic.
Storage. Dopamine hydrochloride should be kept in a well-closed container,

Requirements
Definition.Dopamine hydrochloride contains not less than 98.0% and not
more than 101.0% of C 8 HnN 02 ,HCl, calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from dopamine hydrochloride RS or with the reference
spectrum of dopamine hydrochloride.
B. The absorption spectrum of a 0.020mg/ml solution in hydrochloric acid

363
of a 1-cm layer at 280nm is about 0.54.
C. Dissolve 0.05 g in 5 ml of water and add while stirring 10 ml of 4-amino-

antipyrine TS2; a red colour is produced.

Clarity and colour of solution. A solution of l.Og in 10ml of water is clear

and colourless.
Loss on drying. Dry to constant weight at 105 °C; it loses not more than 5.0 mg/g.
chromatography using silica gel R1 as the coating substance and a mixture of
13 volumes of chloroform R, 9 volumes of methanol R, and 4 volumes of acetic
acid (-300 g/1) TS as the mobile phase. Apply separately to the plate 10 pi of
each of 2 solutions in methanol R containing (A) 30 mg of the test substance
per ml and (B) 0.3 mg of dopamine hydrochloride RS per ml. After removing
the plate from the chromatographic chamber, allow it to dry at room temper-
ature for several minutes, then spray it evenly with a freshly prepared mixture
of 2 volumes of ferric chloride (50 g/1) TS and 1 volume of potassium ferri-
cyanide (50 g/1) TS. Examine the chromatogram in daylight. Apart from the
principal spot, not more than 3 spots are obtained with solution A, and the esti-
mated sum of impurities is not larger than that estimated from the chro-
matogram obtained with solution B.
Rl, add 10 ml of mercuric acetate/acetic acid TS, and titrate with perchloric acid
(0.1 mol/1) VS as described under 2.6 Non-aqueous titration, Method A. Each
ml of perchloric acid (0.1 mol/1) VS is equivalent to 18.96mg of CaHnNOjjHCl.
Additional requirements for Dopamine hydrochloride for

parenteral use
terial endotoxins; contains not more than 1 6.67 lU of endotoxin RS per mg.
364
DOXORUBICINI HYDROCHLORIDUM
DOXORUBICIN HYDROCHLORIDE
Molecular formula. C27H29NO„,HCl
Graphic formula.
OH 0
Chemical name. (8S,10S)-10-[(3-Amino-2,3,6-trideoxy-a-L-/}w-hexopyra-

nosyl)oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,ll-trihydroxy-l-methoxy-5,12-
naphthacenedione hydrochloride; (8S-d.s‘)-10-[(3-amino-2,3,6-trideoxy-a-L-
/yjco-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,ll-trihydroxy-8-(hydroxy-
acetyl)-l-methoxy-5,12-naphthacenedione hydrochloride; CAS Reg. No.
25316-40-9.
Description. A red-orange, crystalline powder.
Solubility. Soluble in water and methanol R; practically insoluble in ether R.
Storage. Doxorubicin hydrochloride should be kept in a tightly closed

container.
Additional information. Doxorubicin hydrochloride is hygroscopic; it is poi-

sonous. CAUTION; Doxorubicin hydrochloride must be handled with care,
Requirements
Definition. Doxorubicin hydrochloride contains not less than 97.0% and not
more than 102.0% of C 27 H 29NOn,HCl, calculated with reference to the anhy-
drous substance.
365
Identity tests
A. The absorption spectrum of a 20pg/ml solution in methanol R, when
observed between 220 nm and 600 nm, is qualitatively similar to that of a
20 pg/ml solution of doxorubicin hydrochloride RS in methanol R (maxima
occur at about 233 nm, 253 nm, 290 nm, 477 nm, 495 nm and 530 nm;
minima occur at about 245 nm, 280 nm and 350 nm). The absorbances of the
solutions at the respective maxima do not differ from each other by more
than 3%. The absorbance of a 1-cm layer at the wavelengths of the main
maxima are about 1.32, 0.88, 0.30, 0.46, 0.44 and 0.24, respectively.
obtained with solution A corresponds in position, appearance and intensity
C. Dissolve 2 mg in 2 ml of methanol R, add 2 ml of water and 0.05ml of

sodium hydroxide (~80g/l) TS; the orange-red colour of the solution turns
to blue-violet.

80 volumes of chloroform R, 20 volumes of methanol R and 5 volumes of glacial
of 4 solutions in methanol R containing (A) 2.0mg of the test substance per ml,
(B) 2.0 mg of doxorubicin hydrochloride RS per ml, (C) 20 mg of the test sub-
stance per ml, and (D) 0.40 mg of the test substance per ml. After removing the
the chromatogram in daylight. Any spot obtained with solution C, other than
the principal spot, is not more intense than that obtained with solution D.
Assay. Dissolve about 20 mg, accurately weighed, in sufficient methanol R to

produce 100 ml; dilute 10 ml of this solution to 100 ml with the same solvent.
at about 495nm. Calculate the amount of C 27 H 29 NO,],HCl in the substance
being tested by comparison with doxorubicin hydrochloride RS, similarly and
366
DOXYCYCLINI HYCLAS
DOXYCYCLINE HYCLATE
Molecular formula. (C22H24N20 b,HC1)2,C2H(s0,H20
Graphic formula.
HCI
Chemical name. (4S,4aR,5S,5aR,6R,12aS)-4-(Dimethylamino)-l,4,4a,5,5a,6,

ll,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-l,l l-dioxo-2-naph-
thacenecarboxamide monohydrochloride, compound with ethyl alcohol (2 1), :
mono-hydrate; [4S-(4a,4aa,5a,5aa,6a, 12aa)]-4-(dimethylamino)-l,4,4a,5,

5a,6,ll,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-l,ll-dioxo-2-
naphthacenecarboxamide monohydrochloride, compound with ethanol (2 1), :
monohydrate; CAS Reg. No. 24390-14-5.
Description. A yellow, crystalline powder.
Solubility. Soluble in 3 parts of water and in 4 parts of methanol R; practically

Storage. Doxycycline hyclate should be kept in a tightly closed container, pro-

tected from light.
Additional information. Even in the absence of light, Doxycycline hyclate is

Requirements
Definition. Doxycycline hyclate contains not less than 880 International Units
of doxycycline per mg, calculated with reference to the anhydrous and ethanol-
free substance.
367
Identity tests
A. Dissolve 5 mg in 2 ml of sulfuric acid (-1760 g/1) TS; an intense yellow colour
is produced.
B. Dissolve 5 mg in 2.0 ml of water and add 0.05 ml of ferric chloride (-25 g/1)
TS; a dark red-brown colour is produced.
C. Dissolve 5 mg in 2.0 ml of water and add 0.25ml of alkaline potassio-

mercuric iodide TS; a fine crystalline, lightyellow precipitate is formed.
D. A 20mg/ml solution yields reaction B described under 2.1 General identifi-


Ethanol. Carry out the test as described under 1.14.5 Gas chromatography,
using 3 solutions in water containing (1) a mixture of 0.50 pi of dehydrated
ethanol R per ml, (2) 10 mg of the test substance per ml, and (3) a mixture of
lOmg of the test substance per ml with 0.50 pi of the internal standard.
For the procedure use a column 1.5 m long and 4mm in internal diameter
packed with porous polymer beads (particle size 80-1 00 pm from a commer-
cial source is suitable). Maintain the column at 135 °C, use nitrogen R as the
carrier gas and a flame ionization detector.
Calculate the content of ethanol in mg/g, assuming the weight per ml at 20 °C

to be 0.790g; not less than 43 mg/g and not more than 60mg/g.
pH value. pH of a lOmg/ml solution, 2. 0-3.0.
Absorption in the region. The absorption spectrum of a

ultraviolet
lOpg/ml solution in a mixture of 1 volume of hydrochloric acid (1 mol/1) VS
and 99 volumes of methanol R exhibits a maximum at about 349 nm. The
absorbance of a 1-cm layer at this maximum is not less than 0.28 and not more
than 0.31 for the anhydrous and ethanol-free substance.
Light -absorbing impurities. Prepare a lOmg/ml solution in a mixture of 1

volume of hydrochloric acid (1 mol/1) VS and 99 volumes of methanol R, and
measure the absorbance of a 1-cm layer at 490 nm; the absorbance does not
exceed 0.12 for the anhydrous and ethanol-free substance.
368
Assay. Carry out the assay under 3.1 Microbiological assay of

as described
antibiotics, using Bacillus cereus (NCTC 10320 or ATCC 11778) as the test organ-
ism, culture medium CmlO with a final pH of 6,6, potassium dihydrogen phos-
phate (13.6g/l) TS as a buffer,an appropriate concentration of doxycycline
(usually between 0.2 and 2.0 lU per ml), and an incubation temperature of 35-
39 °C. The precision of the assay is such that the fiducial limits of error of the
estimated potency (P = 0.95) are not less than 95% and not more than 105%
of the estimated potency. The upper fiducial limit of error of the estimated
potency (P = 0.95) is not less than 880 lU of doxycycline per mg, calculated with
reference to the anhydrous and ethanol-free substance.
EDROPHONII CHLORIDUM
EDROPHONIUM CHLORIDE
Molecular formula. CioHigClNO
Graphic formula.
Chemical name. Ethyl(w-hydroxyphenyl)dimethylammonium chloride; N-

ethyl-3-hydroxy-N,N-dimethylbenzenaminium chloride; CAS Reg. No. 116-
38-1,
Solubility. Soluble in 0.5 parts of water and in 5 parts of ethanol (~750g/l) TS;
Category. Diagnostic agent.
Storage. Edrophonium chloride should be kept in a well-closed container, pro-

tected from light.
Requirements
Definition. Edrophonium chloride contains not less than 98.5% and not more
than 101.0% of C 10H 16 CINO, calculated with reference to the dried substance.
369
Identity tests
B. The absorption spectrum of a lOpg/ml solution in sodium hydroxide

VS, when observed between 230 nm and 350 nm, exhibits maxima
(0.1 mol/1)
at about 240 nm and 294 nm; the absorbances of a 1-cm layer at these
wavelengths are about 0.55 and 0.17, respectively.
C. Dissolve 0.05g in 2 ml of water and add 0.05 ml of ferric chloride (25g/l) TS;
a reddish violet colour is produced.
D. Melting temperature, about 168°C with decomposition.
E. A 20 mg/ml solution yields reaction A described under 2.1 General identifi-

Loss on drying. Dry at ambient temperature under reduced pressure (not

exceeding O.fikPa or about 5mm of mercury) over phosphorus pentoxide R for
24 hours; it loses not more than 5.0mg/g.
pH value. pH of a O.lOg/ml solution, 4. 0-5.0.
Dimethylaminophenol. Dissolve 0.1 g in 10ml of water, add 5ml of phos-

phate buffer, pH 8.0, TS, and extract with 2 quantities, each of 20 ml of chlo-
roform R.
Wash with 2 quantities, each of 10ml of water, and

the extracts successively
extract with 10ml of sodium hydroxide (0.1 mol/1) VS. Measure the absorbance
of the sodium hydroxide extract using a Tcm layer at 293nm; not greater than
0.25.

Rl, add 10ml of mercuric acetate/acetic acid TS and 0.25ml of quinaldine
red/ethanol TS, and titrate with perchloric acid (0.1 mol/1) VS as described under
2.6 Non-aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/1) VS
is equivalent to 20.17 mg of CjoHkjCINO.
370
EMETINI HYDROCHLORIDUM
EMETINE HYDROCHLORIDE
Emetine hydrochloride pentahydrate
Emetine hydrochloride heptahydrate
Molecular formula. C29H4oN204,2HCl,5H20 (pentahydrate); C29H40N2O4,

2HC1,7H20 (heptahydrate).
Relative molecular mass. 643.6 (pentahydrate); 679.7 (heptahydrate).
Graphic formula.
2HCI • nHjO
0CH3
0CH3
n =r 5 (pentahydrate)
n = 7 (heptahydrate)
Chemical name. Emetine dihydrochloride pentahydrate; 6',7', 10,11 -tetram-

ethoxyemetan dihydrochloride pentahydrate; CAS Reg. No. 79300-07-5
(pentahydrate).
Emetine dihydrochloride heptahydrate; 6',7',10,11-tetramethoxyemetan dihy-

drochloride heptahydrate; CAS Reg. No. 79300-08-6 (heptahydrate).
Description. A white or very slightly yellow, crystalline powder; odourless.
Solubility. Freely soluble in water and ethanol (-750 g/1) TS.
Storage. Emetine hydrochloride should be kept in a tightly closed container,

Labelling. The designation on the container of Emetine hydrochloride should

state whether the substance is the pentahydrate or the heptahydrate.
371
Additional information. Emetine hydrochloride is the hydrochloride of an

alkaloid obtained from ipecacuanha or prepared by methylation of cephaeline
or by synthesis. Even in the absence of light, Emetine hydrochloride is gradu-
Requirements
Definition. Emetine hydrochloride contains not less than 98.0% and not more
than 101.5% of C29H4oN204,2HCl, calculated with reference to the dried
substance.
Identity tests

spectrum obtained from emetine hydrochloride RS or with the reference spec-
trum of emetine hydrochloride.
B. See the test described below under “Related alkaloids”. The principal spot
C. Sprinkle 5mg of the test substance on the surface of 1ml of ammonium

molybdate/sulfuric acid TS; a bright green colour is produced.
ence to the dried substance; [a]D’
‘"
= +16 to +19°.
Loss on drying. Dry to constantweight at 105°C; Emetine hydrochloride

pentahydrate loses not less than llOmg/g and not more than 150mg/g. Emetine
hydrochloride heptahydrate loses not less than 150mg/g and not more than
190mg/g.
Acidity. Dissolve 0.10 g in 10 ml of carbon-dioxide-free water R and titrate

with sodium hydroxide (0.02 mol/1) VS, using methyl red/ethanol TS as indi-
cator; not more than 0.5ml is required to obtain the midpoint of the indicator
(orange).
372
100 volumes of chloroform R, 20 volumes of ethylene glycol monomethyl ether
R, 5 volumes of methanol R, 2 volumes of water and 0.5 volumes of diethy-
lamine R as the mobile phase. Apply separately to the plate 10 pi of each of 4
solutions in a mixture of 1 volume of ammonia (-17 g/1) TS and 99 volumes of
methanol R containing (A) 0.50mg of the test substance per ml, (B) lOpg of
cephaeline hydrochloride R per ml, (C) 5.0 pg of the test substance per ml and
(D) 0.50 mg of emetine hydrochloride RS per ml. After removing the plate from
the chromatographic chamber, allow it to dry in air until the solvents have
evaporated, spray it with iodine/chloroform TS, heat it at 60 °C for 15 minutes,
with solution A is not more intense than the corresponding spot obtained with
solution B. Any other spot obtained with solution A, is not more intense than
that obtained with solution C,
Rl, add 10ml of mercuric acetate/acetic acid TS and titrate with perchloric
acid (0.1 mol/1) VS as described under 2.6 Non-aqueous titration. Method A.
C 29 H 4oN20 „ 2 HC 1 .
EPHEDRINI HYDRO CHLORIDUM

EPHEDRINE HYDROCHLORIDE
Molecular formula. CioHi6NO,HCl
Graphic formula.
Chemical name. (-)-Ephedrine hydrochloride; [R-(i?*,S*)]‘®‘[l‘(ni6thy-

lamino)ethyl]benzenemethanol hydrochloride; CAS Reg. No. 50-98-6.
Solubility. Soluble in 4 parts of water; soluble in ethanol (~750g/l) TS; practi-

373
Storage. Ephedrine hydrochloride should be kept in a well-closed container,

Additional information. Ephedrine hydrochloride darkens on exposure to

light.
Requirements
Definition. Ephedrine hydrochloride contains not less than 99.0% and not
more than 101.0% of CioHisNOjHCl, calculated with reference to the dried
substance.
Identity tests
A. The absorption spectrum of a 0.50 mg/ml solution, when observed between
230 nm and 350 nm, exhibits maxima at about 251 nm, 257 nm, and 263 nm;
the absorbances of a 1-cm layer at these wavelengths are about 0.37, 0.48,
and 0.36, respectively.
B. Dissolve lOmg in 1ml of water and add 0.1 ml of copper(II) sulfate (80g/l)
TS, followed by 2 ml of sodium hydroxide (~80g/l) TS; a violet colour is
produced. Add 1 ml of ether R and shake; a purple colour is produced in the

ethereal layer and a blue colour in the aqueous layer.
C. Dissolve 0.05g in 5 ml of water. Add a few drops of sodium hydroxide

(~80g/l) TS and 4 ml of potassium ferricyanide (50g/l) TS, and heat; an odour
of benzaldehyde is perceptible.
D. A 0.05g/ml solution yields reaction A described under 2.1 General identifi-

Specific optical rotation. Use a 50mg/ml solution; [a]c = -33.0 to -35.5°.
Sulfates. Dissolve 0.050 g in 40 ml of water and add 1.5 ml of hydrochloric acid

(~70g/l) TS and 1 ml of barium chloride (50g/l) TS; no turbidity develops within
10 minutes.
Clarity and colour of solution. A solution of 1.0 g in 10 ml of water is clear,
or not more opalescent than opalescence standard TS2, and colourless.
374
5-Omg/g,
Acidity and alkalinity. Dissolve l.Og in 10ml of water and add 0.1ml of
methyl red/ethanol TS; not more than 0.1ml of sodium hydroxide (0.1 mol/1)
VS or 0.1ml of hydrochloric acid (0.1 mol/1) VS is required to obtain the mid-
point of the indicator (orange).
Assay. Dissolve about 0.2 g, accurately weighed, in 10ml of warm mercuric

acetate/acetic acid TS, add 50 ml of acetone R and 1ml of methyl
orange/acetone TS and
as indicator, titrate with perchloric acid (0.1 mol/1) VS
as described under 2.6 Non-aqueous titration. Method A. Each ml of perchlo-
ric acid (0.1 mol/1) VS is equivalent to 20.17mg of CioHi 5 NO,HCl.
EPHEDRINI SULFAS
EPHEDRINE SULFATE
Molecular formula. (CioHi 5 NO) 2 ,H 2 S 04
Graphic formula.
Chemical name. (-)-Ephedrine sulfate (2:1) (salt); [R-(R* S*)]-a-[l-(methy-

lamino)ethyl]benzenemethanol sulfate (2:1) (salt); CAS Reg. No. 134-72-5.
Solubility. Freely soluble in water; sparingly soluble in ethanol (-750 g/1) TS.
Storage. Ephedrine sulfate should be kept in a well-closed container, protected

from light.
Additional information. Ephedrine sulfate darkens on exposure to light.

Even in the absence of light, it is gradually degraded on exposure to a humid
375
Requirements
Definition. Ephedrine sulfate contains not less than 98.0% and not more than
101.0% of (CioHi 5 NO) 2 ,H 2 S 04 calculated with reference
,
to the dried substance.
Identity tests
230 nm and 350 nm, exhibits maxima at about 251 nm, 257 nm, and 262 nm;
the absorbances of a Tcm layer at these wavelengths are about 0.61, 0.76,
and 0.61, respectively.
B. Dissolve lOmg in 1ml of water and add 0.1 ml of copper(ll) sulfate (80g/l)

C. Dissolve 0.05 g in 5 ml of water. Add a few drops of sodium hydroxide

(~80g/l) TS and 4 ml of potassium ferri cyanide (50g/l) TS, and heat; an odour
D. A 20mg/ml solution yields reaction A described under 2.1 General identifi-

E. Melting temperature, about 245 °C with decomposition.
ence to the dried substance; [a]c = -30.5 to -32.5°.
Chlorides. A quantity of 0.20 g of the test substance shows no more turbid-

ity than 0.40 ml of hydrochloric acid (0.02 mol/1) VS when subjected to the pro-
cedure described under 2.2.1 Limit test for chlorides; the chloride content is not
more than 1.4mg/g.
Clarity and colour of solution. A solution of 1.0 g in 10 ml of water is clear,
or not more opalescent than opalescence standard TS2, and colourless.
20mg/g.
Acidity and alkalinity. Dissolve l.Og in 10ml of water and add 0.1ml of
methyl red/ethanol TS; not more than 0.1ml of sodium hydroxide (0.1 mol/1)
VS or 0.1ml of hydrochloric acid (0.1 mol/1) VS is required to obtain the mid-
376
Assay. Dissolve about 0.3 g, accurately weighed, in 10 ml of water, add about

3g of sodium chloride R to saturate the solution, then add 5 ml of sodium
hydroxide (1 mol/1) VS and extract with 4 volumes, each of 25 ml, of chloro-
form R. Wash the combined chloroform extracts with 10 ml of a saturated solu-
tion of sodium chloride R, and filter through purified cotton saturated with
chloroform R. Shake the aqueous wash solution with 10 ml of chloroform R
and add it to the main chloroform extract. Add 0.25ml of methyl red/ethanol
TS and titrate with perchloric acid/dioxan (0.1 mol/1) VS, as described under 2.6
Non-aqueous titration. Method A. Each ml of perchloric acid/dioxan (0.1 mol/1)
VS is equivalent to 21.43mg of (C,oH] 5 NO) 2 ,H 2 S 04 .
EPHEDRINUM
EPHEDRINE
Ephedrine, anhydrous
Ephedrine hemihydrate
Molecular formula. CioHisNO (anhydrous); CioHisNO, yH20 (hemihydrate).
Graphic formula.
n = 0 (anhvdrous)
n = 1/2 (hemihydrate)
Chemical name. (-)-Ephedrine; [R-(R*,S*)]-a-[l-(methylamino)ethyl]benzen-

emethanol; CAS Reg. No. 299-42-3 (anhydrous).
(-)-Ephedrine hemihydrate; [7?-(/?*,S*)]-a-[l-(methylamino)ethyl]benzen-

emethanol hemihydrate; CAS Reg. No. 50906-05-3 (hemihydrate).

with a slight, aromatic odour.
Solubility. Soluble in water; very soluble in ethanol (~750g/l) TS; freely

soluble in ether R.
377
Storage. Ephedrine should be kept in a well-closed container, protected from

light.
Labelling. The designation on the container should state whether the sub-
stance is the hemihydrate or is in the anhydrous form.
Additional information. Solutions of Ephedrine in chloroform R may

become turbid, especially when the substance contains more than 10 mg of
water per g. Even in the absence of light, Ephedrine is gradually degraded on
exposure to a humid atmosphere, the decomposition being faster at higher tem-
peratures. Anhydrous Ephedrine melts at about 38 °C, whereas Ephedrine
hemihydrate melts at about 42 °C.
Requirements
Definition. Ephedrine contains not less than 98.5% and not more than
101.0% of CjoHisNO, calculated with reference to the anhydrous substance.
Identity tests
A. The absorption spectrum of a 0.50 mg/ml solution in hydrochloric acid
(0.1 mol/1) VS, when observed between 230nm and 350nm, exhibits 3
maxima at about 251 nm, 257 nm, and 263 nm.
B. Dissolve lOmg in 1ml of water and add 0.1 ml of copper(II) sulfate (80g/l)

C. Dissolve 0.05 g in 5 ml of water. Add a few drops of sodium hydroxide

(~80g/l) TS and 4 ml of potassium ferri cyanide (50g/l) TS, and heat; an odour
Specific optical rotation. Dissolve about 2.25g, accurately weighed, in 15ml

of hydrochloric acid (~70g/l) TS and dilute to 50 ml with water. Calculate the
result with reference to the anhydrous substance; [a]o = -41 to -43°.

Sulfates. Dissolve 1.2g in 20 ml of water and proceed as described under 2.2.2

Limit test for sulfates; the sulfate content is not more than 0.4 mg/g.
378
H

Fischer method, Method A, For the anhydrous form use about 2g of the sub-
stance; the water content is not more than lOmg/g. For the hemihydrate use
about 1 g of the substance; the water content is not less than 45 mg/g and not
more than 55 mg/g.
Assay. Dissolve about 0.5 g, accurately weighed, in 5 ml of ethanol (-750 g/1)

TS, add 50.0ml of hydrochloric acid (0.1 mol/1) VS and titrate with sodium
hydroxide (0.1 mol/1) VS, using methyl red/ethanol TS as indicator. Each ml of
hydrochloric acid (0.1 mol/1) VS is equivalent to 16.52 mg of CioHuNO.
EPINEPHRINI HYDRO GENOTARTRAS

EPINEPHRINE HYDROGEN TARTRATE
Molecular formula. CgHiaNOsjC^HeOg or C 13 H 19NO 9
Graphic formula.
COOH
H — C — OH
I
HO-C —
COOH
Chemical name. (-)-(R)-3,4-Dihydroxy-a-[(methylamino)methyl]benzyl

alcohol L-(-t)-tartrate (1:1) (salt); (-)-(/?)-4-[l-hydroxy-2-(methylamino)ethyl]-
l,2-benzenediol[/?-(/?*,/i!*)]-2,3-dihydroxybutanedioate (1:1) (salt); (-)-a-3,4-
dihydroxyphenyl-|3-(methylamino)ethanol L-(-t)-tartrate; CAS Reg. No.
51-42-3.
Other name. Adrenaline tartrate. (In certain countries the name Adrenaline is
a trademark. In those countries this name may be used only when applied to
the product issued by the owners of the trademark.)
Description. A white to greyish white, crystalline powder; odourless.
379
=
Category. Sympathomimetic.
Storage. Epinephrine hydrogen tartrate should be kept in a tightly closed con-

Additional information. Epinephrine hydrogen tartrate gradually darkens in

colour on exposure to air and light. Even in the absence of light, Epinephrine
hydrogen tartrate is gradually degraded on exposure to a humid atmosphere,
Requirements
Definition. Epinephrine hydrogen tartrate contains not less than 98.0% and
not more than 101.0% of CgHiaNOaiC^HgOg, calculated with reference to the
dried substance.
Identity tests
A. The absorption spectrum of a O.lOmg/ml solution in hydrochloric acid
(0.01 mol/1) VS, when observed between 230nm and 350nm exhibits a
at
B. Dissolve lOmg in 10ml of water and transfer 1 ml to a flask containing

10ml of buffer phthalate, pH 3.4, TS; another buffer having the same pH
may also be used. Add 0.5ml of iodine (0.1 mol/1) VS, and allow to stand
for 5 minutes. Add 2 ml of sodium thiosulfate (0.1 mol/1) VS, and allow to
stand for 1 minute; a strong red colour is produced (distinction from
levarterenol, which gives a clear solution with a pink tinge).
C. The filtrate obtained when carrying out the determination of specific optical
rotation (see below) yields reaction B described under 2.1 General identifi-
cation tests, as characteristic of tartrates.
Specific optical rotation. Dissolve about 0.5 g in 20 ml of water containing

about 0.1 g of sodium metabisulfite R, add a slight excess of ammonia
(~100g/l) TS, and allow to stand in the cold for 1 hour. Filter (keep the filtrate
wash the precipitate with 3 quantities, each of
for identity test C, see above),
2 ml of water, followed by 5 ml of ethanol (-750 g/1) TS and 5 ml of ether R.
Dry the ambient temperature under reduced pressure (not exceed-
precipitate at
ing O.bkPa or about 5mm of mercury) for 3 hours. Use a 20 mg/ml solution of
epinephrine base in hydrochloric acid (0.5mol/l) VS; [a]™ -50 to -53°.

and colourless.
380

exceeding 0.6kPa or about 5mm of mercury) over silica gel, desiccant, R for 3
hours; it loses not more than 5.0mg/g.
Adrenalone. The absorbance of a 1-cm layer of a 4.0mg/ml solution in

hydrochloric acid (0.1 mol/1) VS at 310nm is not more than 0.2 (preferably use
2-cm cells for the measurement and calculate the absorbance of a 1-cm layer).
Levarterenol. Dissolve lOmg in 1 ml of water, add 4ml of buffer borate, pH

9.6, TS, or another buffer having the same pH, mix, add 1 ml of a freshly pre-
pared solution of sodium l,2-naphthoquinone-4-sulfonate (5g/l) TS, mix, and
allow to stand for 30 minutes. Add 0.2ml of benzalkonium chloride TSl, mix,
add 15ml of toluene R, previously washed with buffer borate, pH 9.6, TS and
filtered through a dry filter-paper, shake for 30 minutes and allow to separate,
centrifuging if necessary. Any red or purple colour in the toluene-layer is
not more intense than that produced by treating a solution of 0.40 mg of

levarterenol hydrogen tartrate R and 9.0 mg of epinephrine hydrogen tartrate R
in 1ml of water in a similar manner, when compared as described under 1.11
Colour of liquids.
Rl, warming slightly if necessary, and titrate with perchloric acid (0.1 mol/1) VS,
as described under 2.6 Non-aqueous titration. Method A. Each ml of perchlo-
ric acid (0.1 mol/1) VS is equivalent to 33.33mg of C9Hi3N03,C4H605.
Additional requirements for Efnnefhrine hydrogen tartrate for

parenteral use
EPINEPHRINUM
EPINEPHRINE
381
Graphic formula.
OH
I
C — CHjNHCHj
I
Chemical name. (-)-(i^)-3,4-Dihydroxy-a-[(methylamino)methyl]benzyl

alcohol; (-)-(i?)-4-[l-hydroxy-2-(methylamino)ethyl]-l,2-benzenediol; (-)-cx-
3,4-dihydroxyphenyl-P-methylaminoethanol; CAS Reg. No. 51-43-4.
Other name. Adrenaline. (In certain countries the name Adrenaline is a trade-
mark. In those countries this name may be used only when applied to the
product issued by the owners of the trademark.)
Description. A white or almost white, microcrystalline powder; odourless.

(-750 g/1) TS, ether R and acetone R.
Category. Sympathomimetic.
Storage. Epinephrine should be kept in a hermetically closed container, pro-

tected from light.
Additional information. Epinephrine gradually darkens in colour on expo-

sure to air and light. Even in the absence of light, Epinephrine is gradually
Requirements
Definition. Epinephrine contains not less than 98.5% and not more than
Identity tests
(0.01 mol/l)VS, when observed between 230 run and 350 nm exhibits a
maximum about 280 run; the absorbance of a 1-cm layer at this wave-
at
length is about 0.45 (preferably use 2-cm cells for the measurement and cal-
B. Dissolve lOmg in 10ml of hydrochloric acid (0.01 mol/1) VS and transfer

1 ml to a flask containing 10ml of buffer phthalate, pH 3.4, TS; another
buffer having the same pH may also be used. Add 0.5ml of iodine (0.1 mol/1)
382
VS and allow to stand for 5 minutes. Add 2 ml of sodium thiosulfate

VS, and allow to stand for 1 minute; a strong red colour is pro-
(0.1 mol/1)
duced (distinction from levarterenol, which gives a clear solution with a pink
tinge).

(1 mol/1) VS; [a]g>’^ = -50 to -53 °.

exceeding O.dkPa or about 5mm of mercury) over silica gel, desiccant, R for 18
hours; it loses not more than lOmg/g.
pH value. pH of a 5.0mg/ml solution in carbon-dioxide-free water R, above

7.5.
Adrenalone. The absorbance of a 1-cm layer of a 2.0mg/ml solution in

hydrochloric acid (0.1 mol/1) VS at 310nm is not more than 0.2 (preferably use
Levarterenol. Dissolve 5.0 mg in 1ml of tartaric acid (5g/l) TS, add 4 ml of

buffer borate, pH 9.6, TS, or another buffer having the same pH, mix, add 1 ml
of a freshly prepared solution of sodium l,2-naphthoquinone-4-sulfonate (5g/l)
TS, mix, and allow to stand for 30 minutes. Add 0.2 ml of benzalkonium chlo-
ride TSl, mix, add 15ml of toluene R, previously washed with buffer borate,
pH 9.6,TS and filtered through a dry filter-paper, shake for 30 minutes and
allow to separate, centrifuging if necessary. Treat a solution of 0.40mg of
levarterenol hydrogen tartrate R and 9.0 mg of epinephrine hydrogen tartrate R
in ml of water in a similar manner. Any red or purple colour in the toluene
1
layer is not more intense than that of the reference solution when compared
as described under 1.11 Colour of liquids.

alent to 18.32 mg of C9H13NO3.
Additional requirements for Efnnefhrine for parenteral use

383
ERGOCALCIFEROLUM
ERGOCALCIFEROL
Graphic formula.
Chemical name. (5Z,7£,22£)-9,10-Secoergosta-5,7,10(19),22-tetraene-3[3-ol; 24-

methyl-9,10-secocholesta-5,7,10(19),22-tetraene-3(3-ol; CAS Reg. No. 50-14-6.
Other name. Vitamin Dj.
Description. Colourless or slightly yellowish crystals or a white or slightly

yellowish, crystalline powder; odourless or almost odourless.

TS, acetone R and ether R.
Category. Vitamin, antirachitic.
Storage. Ergocalciferol should be kept in a hermetically closed container,

preferably in an inert atmosphere, such as nitrogen, protected from light and
stored at a temperature between 2 and 8°C.
Additional information. Ergocalciferol is affected by air and by light. Even

in the absence of light, it is gradually degraded on exposure to a humid atmos-
Requirements
Definition. Ergocalciferol contains not less than 95.0% and not more than
105.0% of C28H44O.
384
Identity tests
A. Carry out the examination as described under 1.7 Spectrophotometry in the in-
frared region. The infrared absorption spectrum is concordant with the spectrum
obtained from ergocalciferol RS or with the reference spectrum of ergocalciferol.
B. Dissolve about 2 mg in 10 ml of ethanol (-750 g/1) TS. To 1.0 ml add care-

fully 5ml of sulfuric acid (-1760 g/1) TS and mix; a red colour is produced
(distinction from colecalciferol, which gives a yellow colour).
C. Dissolve 5 mg in 5 ml of chloroform R, add about 0.5ml of acetic anhydride

R and about 0.1ml of sulfuric acid (~1760g/l) TS, shake well; the colour of
the solution changes immediately from red to violet, then to blue, and finally
to dark green.
D. Dissolve about Img in 40 ml of dichloroethane R. To 1ml of this solution

add 4 ml of antimony trichloride TS; an orange colour is produced, which
gradually becomes pink.
Melting range. 1 12-1 17 °C, determined without previous grinding or drying.
Specific optical rotation. Dissolve 0.2 g rapidly and without heating in suf-
ficient aldehyde-free ethanol (~750g/l) TS to produce 25ml. Determine the
rotation within 30 minutes of preparation; [ajD” "”
= -1-103 to -1-107°.
Ergosterol. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using silica gel R1 as the coating substance, and as the mobile
phase use a mixture of equal volumes of cyclohexane R and peroxide-free ether
R, the mixture containing 0.10 mg of butylated hydroxytoluene R per ml. Apply
separately to the plate 10 pi of the following solutions prepared immediately
before use in a mixture of dichloroethane R containing lOmg of squalane R and
0.10 mg of butylated hydroxytoluene R per ml: (A) 0.050 g of the test substance
per ml, (B) 0.050 g of ergocalciferol RS per ml, (C) 0.10 mg of ergosterol R per
ml; also apply to the plate 20 pi of solution (D) consisting of a mixture of equal
volumes of solutions B and C. Develop the plate at once in the dark. After
and spray it three times with antimony trichloride TS. Wait after spraying 3-4
minutes, then examine the chromatogram in daylight. The principal spot
obtained with solution A is initially orange-yeUow and then becomes brown;
it corresponds in position, appearance and intensity with that obtained with
solution B. Any violet spot obtained with solution A with an Rf value slightly
less than that of the principal spot is not more intense than the spot obtained
with solution C. The chromatogram obtained with solution A shows no addi-
tional spots compared with the chromatograms obtained with solutions B and
C. The test is not valid unless the chromatogram obtained with solution D
shows two clearly separated spots.
385
Reducing substances. Dissolve O.lOg in 10ml of aldehyde-free ethanol

(-750 g/1) TS, add 0.5 ml of blue tetrazolium/ethanol TS and 0.5 ml of tetram-
ethylammonium hydroxide/ethanol TS. Allow to stand for exactly 5 minutes
and then add 1ml of glacial acetic acid R. Measure the absorbance of a 1-cm
layer at the maximum at about 525 nm against a solvent cell containing a solu-
tion prepared by treating 10 ml of aldehyde-free ethanol (-750 g/1) TS in a
similar manner. The absorbance is not greater than that obtained by repeating
the operation with 10 ml of a solution containing 0.2pg/ml of hydroquinone R
in aldehyde-free ethanol (~750g/l) TS.
Assay. Dissolve rapidly and without heating 0.05 g, accurately weighed, in suf-
ficient aldehyde-free ethanol (-750 g/1) TS to produce 100ml; dilute 5.0ml of
this solution to 250ml with the same solvent. Measure within 30 minutes
of preparation the absorbance of a 1-cm layer of the diluted solution at the

maximum at about 265 nm. Calculate the amount of C2SH44O in the substance
being examined by comparison with ergocalciferol RS, similarly and con-
currently examined. In an adequately calibrated spectrophotometer the
absorbance of the reference solution should be 0.48 + 0.03.
ERGOMETRINI HYDROGENOMALEAS
ERGOMETRINE HYDROGEN MALEATE
Molecular formula. Ci9H23N302,C4H404 or C23H27N3O6
Graphic formula.
II ;
HC COOH
HC — COOH
Chemical name. 9, 1 0-Didehydro-Al- [(S')-2-hydroxy- 1 -me thylethyl] -6-methy-

lergoline-8P-carboxamide maleate (1:1) (salt); 9,10-didehydro-W-[(S)-2-
hydroxy-l-methylethyl]-6-methylergoline-8P-carboxamide (Z)-2-butanedioate
(1:1) salt; CAS Reg. No. 129-51-1.
386
Description. A white or faintly yellow, crystalline powder; odourless.
Solubility. Sparingly soluble in water and ethanol (-750 g/1) TS; practically
Category. Oxytocic.
Storage. Ergometrine hydrogen maleate should be kept in a hermetically

closed container, preferably in an inert atmosphere, such as nitrogen, protected
from light and stored in a cool place.
Additional information. Ergometrine hydrogen maleate darkens in colour on

exposure to light. Even in the absence of light, Ergometrine hydrogen maleate
Requirements
Definition. Ergometrine hydrogen maleate contains not less than 98.0% and
not more than 101.0% of C] 9 H 23N 302 ,C 4 H 404 calculated with reference to the
dried substance.
Identity tests
A. Dissolve 15mg in 5ml of water; a blue fluorescence is produced.
B. See the test described below under "Related alkaloids”. The principal spot
C. Dissolve 2mg in 20ml of water; to 1ml of this solution add 2ml of 4-

dimethylaminobenzaldehyde TSl and allow to stand for about 5 minutes;
a deep blue colour is produced.
D. Dissolve 2 mg in 1 ml of water, and add 1 drop of bromine TSl; the colour

of the reagent is discharged.
ence to the dried substance; [a]o = -i-50 to -i-56°.
Clarity and colour of solution. A solution of O.lOg in 10ml of carbon-

dioxide-free water R is clear and not more intensely coloured than standard
colour Yw3 when compared under 1.11 Colour of liquids.
as described

exceeding O.bkPa or about 5mm of mercury); it loses not more than 20mg/g.
387
pH value. pH of a lOmg/ml solution in carbon-dioxide-free water R, 3. 0-5.0.
Place a sufficient volume of mobile phase to develop the chromatograms and
a beaker containing 25 ml of ammonia (-260 g/1) TS into the chromatographic
chamber, and equilibrate for 30 minutes. Apply separately to the plate 5 pi of
each of 3 solutions in methanol R containing (A) 4.0 mg of the test substance
per ml, and (B) 0.12 mg of the test substance per ml, and (C) 0.1 2 mg of
ergometrine hydrogen maleate RS per ml. After removing the plate from the
chromatographic chamber, allow it to dry in air until the solvents have evapo-
rated, spray it with 4-dimethylaminobenzaldehyde TS2, and examine the chro-
matogram in daylight. Any spot obtained with solution A, other than the

R1 and 10 ml of acetic anhydride R, and titrate with perchloric acid (0.05 mol/1)
VS, as described under 2.6 Non-aqueous titration, Method A. Each ml of per-
chloric acid (0.05mol/l) VS is equivalent to 22.07 mg of Ci9H23N302,C4H404.
Additional requirements for Ergometrine hydrogen maleate

for parenteral use
ERGOTAMINI TARTRAS
ERGOTAMINE TARTRATE
Molecular formula. (C33H35N505)2,C4H60e; or C/oHjeNioOie
388
Graphic formula.
COOH
j
H C OH
I
HO C H
COOH
Chemical name. Ergotamine L-(+)-tartrate (2 :

1) (salt); 12'-hydroxy-2'-methyl-
5'a-(phenylmethyl)ergotaman-3',6',18-trione[R-(R*,R*)]-2,3-dihydroxybutane-
dioate (2:1) (salt); CAS Reg. No. 379-79-3.
Description. Colourless crystals or a greyish white to yellowish white, crys-

talline powder; odourless.
uble in ether R, benzene R, and light petroleum R.
Category. Sympatholytic.
Storage. Ergotamine tartrate should be kept in a hermetically closed container,

preferably in a inert atmosphere, such as nitrogen, protected from light and
stored in a cool place.
Additional information. Even in the absence of light, Ergotamine tartrate is

Requirements
Definition. Ergotamine tartrate contains not less than 98.0% and not more
than 101.0% of (C 33 H 35 N 50 s) 2 ,C 4 H(; 06 calculated with reference to the dried
,
substance.
Identity tests
A. See the test described below under "Related alkaloids”. The principal spot
389
=
B . Dissolve 1 mg in a mixture of 5 ml of glacial acetic acid R and 5 ml of ethyl acetate

R. To 1 ml of this solution add 1 ml of sulfuric acid (-1760 g/1) TS, with contin-
uous shaking and cooling; a blue colour with a red tinge develops. Add 0.1 ml
of ferric chloride (25g/l) TS, previously diluted with an equal volume of water;
the red tinge becomes less apparent and the blue colour more pronounced.
Specific optical rotation of ergotamine base

• Prepare and use the solution rapidly, in subdued light.
Place about 0.35g, accurately weighed, in a separator, dissolve it in 25 ml of tar-
taric acid (lOg/1) TS, add 0.5g of sodium hydrogen carbonate R, and mix gently.
Add 10ml of ethanol-free chloroform R, shake vigorously, allow to separate,
and filter the chloroform-layer through a small filter-paper previously moist-
ened with ethanol-free chloroform R into a 50-ml volumetric flask. Repeat the
extraction of the aqueous layer with three 10-ml portions of ethanol-free chlo-
roform R, passing the extracts through the same filter. Place the flask in a water-
bath at 20 °C for 10 minutes and adjust to 50ml with the same solvent. Mix
the solution and determine the optical rotation at 20 °C, preferably using tubes
that can be controlled thermostatically. Separately, measure 25.0ml of the solu-
tion, evaporate on a water-bath, and dry to constant weight at 95 °C under
reduced pressure (not exceeding 0.6kPa or about 5 mm
of mercury). Calculate
the specific optical rotation of the ergotamine base from the weight of the
residue and the observed rotation; [a]™ -150 to -160°.
Clarity and colour of solution. Add 25 mg of tartaric acid R to 50 mg of the

test substance and dissolve at 20 °C in 10ml of water; the solution is clear and
not more intensely coloured than standard colour solution Yw2 when com-
Loss on drying. Weigh the substance as rapidly as possible and dry to con-
stant weight at 95°C under reduced pressure (not exceeding 0.6kPa or about
5 mm of mercury); it loses not more than 50mg/g.
Place a sufficient volume of mobile phase to develop the chromatograms and
a beaker containing 25 ml of ammonia (-260 g/1) TS into the chromatographic
chamber, and equilibrate for 30 minutes. Apply separately to the plate 5|il of
each of 3 solutions in the mobile phase containing (A) 5.0 mg of the test sub-
stance per ml, (B) 0.25 mg of the test substance per ml, and (C) 0.25 mg of ergo-
tamine tartrate RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air until the solvents have evaporated, spray it with
4-dimethylaminobenzaldehyde TS2, and examine the chromatogram in day-
390
Assay. Dissolve about 0.3 g, accurately weighed, in 15 ml of a mixture of 6

volumes of acetic anhydride R and 100 volumes of glacial acetic acid Rl. Titrate
with perchloric acid (0.05mol/l) VS as described under 2.6 Non-aqueous
32. 83 mg of (C 33 H 35N 505 )2
,C 4 H« 0 ,.
ERYTHROMYCINI ETHYLSUCCINAS
ERYTHROMYCIN ETHYLSUCCINATE
Erythromycin ethylsuccinate for parenteral use
Molecular formula. C4,3H7,3NO]6
Graphic formula.
Chemical name. Erythromycin 2'-(ethylsuccinate); erythromycin 2'(ethyl

butanedioate); [37?-(3/?‘,4S*,5S*,6R*,71?*,9R*,llR*,12R*,13S‘,14R’)]-4-[(2,6-
dideoxy-3-C-methyl-3-0-methyl-a-L-nTo-hexopyranosyl)oxy]-14-ethyl-
7,12,13-trihydroxy-3,5,7,9,ll,13-hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylam
ino)-P-D-xy/o-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione 2'-(ethyl
butanedioate); CAS Reg. No. 1264-62-6.
Description. A white or slightly yellow, crystalline powder; odourless or

almost odourless.
Solubility. Very slightly soluble in water; soluble in ethanol (~750g/l) TS and

macrogol 400 R.
391
Labelling. The designation Erythromycin ethylsuccinate for parenteral use

indicates that the substance complies with the additional requirement for
Erythromycin ethylsuccinate and may be used for parenteral administration.
Storage. Erythromycin ethylsuccinate should be kept in a tighdy closed con-

Requirements
Definition. Erythromycin ethylsuccinate contains not less than 740 Interna-
tional Units of erythromycin per mg, calculated with reference to the anhy-
drous substance.
Identity tests

spectrum obtained from erythromycin ethylsuccinate RS or with the refer-
ence spectrum of erythromycin ethylsuccinate.
B. To 5mg add 2ml of sulfuric acid (~1760g/l) TS and shake gently; a reddish
brown colour is produced.
C. Dissolve 3 mg in 2 ml of acetone R and add 2 ml of hydrochloric acid

(-420 g/1) TS; an orange colour is produced, which changes to orange-red
and finally to purplish red. Add 2 ml of chloroform R and shake; the chlo-
roform layer becomes blue.
D. To 5 mg add 5 ml of xanthydrol TS and heat on a water-bath; a red colour

is produced.

pH value. Shake 0.5 g with 50ml of carbon-dioxide-free water R; pH of the

suspension, 6.0-8. 5.
Assay. Dissolve 50 mg in sufficient methanol R to produce 100ml and carry

out the assay as described under 3.1 Microbiological assay of antibiotics, using
392
3
Bacillus fruniilus (NCTC 8241 or ATCC 14884) as the test organism, culture
medium Cml with a final pH of 8. 0-8.1, sterile phosphate buffer, pH 8,0 TSl
or TS2, an appropriate concentration of erythromycin (usually between 5 and
25 lU per ml), and an incubation temperature of 35-39 °C. The precision of the
assay such that the fiducial limits of error of the estimated potency (P = 0.95)
is
740 lU of erythromycin per mg, calculated with reference to the anhydrous
substance.
Additional requirement for Erythromycin Ethylsuccinate for

parenteral me
ERYTHROMYCINI LACTOBIONAS
ERYTHROMYCIN LACTOBIONATE
Cs/Hs/N 0 ,C 1
1
2 H 22 O 12
Chemical name. Erythromycin lactobionate (1 :

1) (salt); erythromycin
mono(4-0-|3-D-galactopyranosyl-D-gluconate) (salt); CAS Reg. No. 3847-29-8.
393
Description. White or slightly yellow crystals or a white, crystalline powder;

odour, faint.
Solubility. Freely soluble in water, ethanol (-750 g/1) TS and methanol R;

slightly soluble in acetone R; practically insoluble in ether R.
Storage. Erythromycin lactobionate should be kept in a tighdy closed con-

Additional information. Each mg of erythromycin lactobionate is equivalent

to 0,6722mg of erythromycin.
Requirements
Definition. Erythromycin lactobionate contains not less than 600 lU of
erythromycin per mg, calculated with reference to the anhydrous substance.
Identity tests

infrared region.
spectrum obtained from erythromycin lactobionate RS or with the reference
spectrum of erythromycin lactobionate.
B. See the test described below under “Related substances”. One of the two
principal spots obtainedwith solution A corresponds in position with the
principal spot obtained with solution B. The other principal spot corre-
sponds in position with the principal spot obtained with solution D.
C. Dissolve 20 mg in 2.0 ml of water and cautiously add about 1 ml of sulfuric
acid (-1760 g/1) TS to form a lower layer; a red-brown ring appears at the
interface of the two liquids. Shake; a dark red-brown solution is produced.
D. Dissolve about 10 mg in 5 ml of hydrochloric acid (-250 g/1) TS; a yellowish

green colour develops.

or not more opalescent than opalescence standard TSl, and colourless or not
more intensely coloured than standard colour solution Ywl when compared as
described under 1.11 Colour of liquids.
394

Fischer method, Method A, using about 0.5 g of Erythromycin lactobionate; the
water content is not more than 0.050 g/g.
pH value. pH of a 0.05 g/ml solution in carbon-dioxide-free water R, 6.0-7. 5.
chromatography, using silanized silica gel R3 as the coating substance and a
mixture of 5 volumes of methanol R and 3 volumes of ammonium acetate
(50g/l) TS as the mobile phase. Apply separately to the plate 10 pi of each of
4 solutions in methanol R containing (A) 3 mg of Erythromycin lactobionate
per ml, (B) 2mg of erythromycin RS per ml, (C) O.lOmg of erythromycin RS
per ml, and (D) 0.66 mg of lactobionic acid R per ml. After removing the plate
have evaporated, spray with anisaldehyde TS, heat at 110°C for 5 minutes,
and allow to cool. Examine the chromatogram in daylight.
Disregard the spot corresponding to lactobionic acid. Any spot obtained with
solution A, other than the principal spot and any spot with a lower /Upvalue,
is not more intense than that obtained with solution B. Any spot obtained with
solution A, other than the principal spot and any spot with a higher /Upvalue,
is not more intense than that obtained with solution C.
Assay. Carry out the determination as described under 3.1 Microbiological

assay of antibiotics, using either (a) Bacillus fumilus (NCTC 8241 or ATCC
14884) as the test organism, culture medium Cml with a final pH of 8. 0-8.1,
sterilephosphate buffer, pH 8.0, TS, an appropriate concentration of ery-
thromycin (usually between 5 and 25 lU per ml), and an incubation tempera-
ture of 35-39 °C, or (b) Micrococcus luteus (ATCC 9341) as the test organism,
culturemedium Cml with a final pH of 8.0-8. 1, sterile phosphate buffer, pH
8.0,TSl, or TS2, an appropriate concentration of erythromycin (usually
between 0.5 and 1.51U per ml), and an incubation temperature of 32-35 °C.
The precision of the assay is such that the fiducial limits of error of the esti-
mated potency (P = 0.95) are not less than 95% and not more than 105% of
the estimated potency.
ERYTHROMYCINI STEARAS
ERYTHROMYCIN STEARATE
Molecular formula. C37H67N0i3,CiaH3s02
395
Graphic formula.
Chemical name. Erythromycin stearate (salt); erythromycin octadecanoate

(salt); [3R-(3R*4S*,5S*,6R*,7/^*,9R*,iy^*12R*,13S*,14R*)]-4-[(2,6-dideoxy-3-C-
methyl-3-0-methyl-a-L-n^'o-hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-
3,5,7,9,ll,13-hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-|3-D-x)'/o-hexo
pyranosyl]oxy]oxacyclotetradecane-2,10-dione octadecanoate (salt); CAS Reg.
No. 643-22-1.
Description. Colourless or slightly yellow crystals or a white or slightly

yellow powder; odourless or almost odourless.
Solubility. Practically insoluble in water; soluble in ethanol (-750 g/1) TS;

soluble in methanol R, and ether R; these solutions may be opalescent.
Storage. Erythromycin stearate should be kept in a tightly closed container,

Requirements
Definition. Erythromycin stearate contains not less than 550 International
Units of erythromycin per mg or not less than 77.0% of C37Hg7N0i3,Ci8H3602,
both calculated with reference to the anhydrous substance.
Identity tests

396
spectrum obtained from erythromycin stearate RS or with the reference spec-

trum of erythromycin stearate.
B. Dissolve 3 mg in 2 ml of acetone R and add 2 ml of hydrochloric acid

(-420 g/1) TS; an orange colour is produced, which changes to red and then
to deep purplish red. Add 2 ml of chloroform R and shake; the chloroform
layer becomes purple.
C. To 5 mg add 5 ml of xanthydrol TS and heat on a water-bath; a red colour

is produced.
D. Heat gently with 5ml of hydrochloric acid (~70g/l) TS and 10ml of

0.1 g
water remove
until the solution boils; oily globules rise to the surface. Cool,
the fatty layer and heat it with 3ml of sodium hydroxide (0.1 mol/1) VS;
allow to cool; the solution sets to a gel. Add 10 ml of hot water and shake;
the solution froths. To 1 ml add 1 ml of calcium chloride (-55 g/1) TS; a gran-
ular precipitate, insoluble in hydrochloric acid (-250 g/1) TS, is produced.
Erythromycin stearate. Shake 0.5 g with 30 ml of chloroform R. If the solu-

tion is clear, add 50 ml of glacial acetic acid R1 previously neutralized with per-
,
chloric acid (0.1 mol/1) VS, and continue with the titration as described below.
If the solution is opalescent, shake with an additional 2 quantities, each of

25 ml of chloroform R, filter each extract, wash the filter with chloroform R,
and evaporate the combined filtrate and washings on a water-bath to about
30ml. Add 50 ml of glacial acetic acid Rl, previously neutralized with perchlo-
ric acid (0.1 mol/1) VS, and titrate with perchloric acid (0.1 mol/1) VS, determin-
ing the endpoint potentiometrically. Each ml of perchloric acid (0.1 mol/1) VS is
equivalent to 101. 8mg of C 37 Hs 7 N0 3 ,C, 8 H 3602 the erythromycin stearate
i ;
content is not less than 0.770g/g.

Fischer method, Method A, using about 0.6g of the substance; the water
Free stearic acid. Dissolve 0.4 g in 50 ml of neutralized ethanol TS and titrate

with sodium hydroxide (0.1 mol/1) VS, determining the endpoint potentiometri-
cally. Calculate the volume of sodium hydroxide (0.1 mol/1) VS required for each
g of the substance and subtract the volume of perchloric acid (0.1 mol/1) VS
required for each g of the substance in the test for erythromycin stearate. Each ml
of the difference is equivalent to 28.45 mg of C,8H3(;02; not more than 185mg/g.
Sodium stearate. Moisten 2.0g in a platinum dish with a small quantity of

sulfuric acid (-1760 g/1) TS, ignite gently, again moisten with sulfuric acid
(-1760 g/1) TS, ignite at about 800 °C, cool, and weigh. Each g of residue is
equivalent to 4.317g of Ci8H35Na02; not more than 60mg/g.
397
Total stearic acid, stearates and water. Using the results of the above four
determinations, add together the percentages of free stearic acid, erythromycin
stearate, sodium stearate (all calculated with reference to the undried substance)
and the water content; the total is not less than 98.0% and not more than
103.0%.
Assay. Dissolve 50 mg in sufficient methanol R to produce 100ml and carry

out the assay as described under 3.1 Microbiological assay of antibiotics, using
Bacillus puntilus (NCTC 8241 or ATCC 14884) as the test organism, culture
medium Cml with a final pH of 8. 0-8.1, sterile phosphate buffer, pH 8.0 TSl
or TS2, an appropriate concentration of erythromycin (usually between 5 and
550 lU of erythromycin per mg, calculated with reference to the anhydrous
substance.
ERYTHROMYCINUM
ERYTHROMYCIN
Molecular formula. Cs/HejNOja
Graphic formula.
Chemical name. [3P-(3P*,4S‘,5S*,6P*,7R*,9P*,llP*,12P*,13S*,14P’)]-4-[(2,6-

Dideoxy-3-C-methyl-3-0-methyl-a-L-nPo-hexopyranosyl)oxyl]-14-ethyl-
7,12,13-trihydroxy-3,5,7,9,ll,13-hexamethyl-6-[[3,4,6-trideoxy-3-(dimethy-
398
lamino)-|3-D-:>ry/o-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione; CAS
Reg. No. 114-07-8.
Description. White or slightly yellow crystals or powder; odourless or almost

odourless.
Solubility. Soluble in 1000 parts of water but less soluble in hot water; freely
soluble in ethanol (-750 g/1) TS and ether R.
Storage. Erythromycin should be kept in a tighdy closed container, protected

from light.
Additional information. Erythromycin is slightly hygroscopic.
Requirements
Definition. Erythromycin is produced by the growth
a mixture of substances
of certain strains of Strep’toniyces erythreus. The main component of the mixture
is erythromycin A with lesser amounts of erythromycins B and C.
• The molecular formula, the relative molecular mass, and the chemical name
given above relate to erythromycin A only.
Erythromycin contains not less than 870 International Units per mg, calculated
Identity tests

The infrared absorption is concordant with the spec-
the infrared region.
trum obtained from erythromycin RS or with the reference spectrum of
erythromycin.
B. To 5mg add 2ml of sulfuric acid (-1760 g/1) TS and shake gently; a reddish
C. Dissolve 3 mg in 2 ml of acetone R and add 2 ml of hydrochloric acid

(-420 g/1) TS; an orange colour is produced, which changes to red and then
to deep purplish red. Add 2 ml of chloroform R and shake; the chloroform
layer becomes purple.
D. To 5 mg add 5 ml of xanthydrol TS and heat on a water-bath; a red colour

is produced.
399
Specific optical rotation. Use a 20 mg/ml solution in dehydrated ethanol R,

allow to stand for 30 minutes, measure the angle of rotation, and calculate with
reference to the anhydrous substance; [a]n = -71 to -78°.

Fischer method, method A, using about 1 g of the substance; the water content
is not more than lOOmg/g.
pH value. Dissolve 0.1 g in 50ml of a mixture composed of 1 volume of

methanol R and 19 volumes of carbon-dioxide-free water R; the pH isbetween
8.0 and 10.5.
antibiotics, using Bacillus pumilus (NCTC 8241 or ATCC 14884) as the test
organism, culture medium Cml with a final pH of 8. 0-8.1, sterile phosphate
buffer, pH 8.0 TS or TS2, an appropriate concentration of erythromycin (usually
between 5 and 25 lU per ml), and an incubation temperature of 35-39 °C. The
precision of the assay is such that the fiducial limits of error of the estimated
potency (P = 0.95) are not less than 95% and not more than 105% of the esti-
mated potency. The upper fiducial limit of error of the estimated potency (P =
0.95) is not less than 870 lU per mg, calculated with reference to the anhydrous
substance.
ETHAMBUTOLI HYDROCHLORIDUM
ETHAMBUTOL HYDROCHLORIDE
Molecular formula. CioH 24 N 202 2 HCl ,
Graphic formula.
CH,OH
^
H
t r
I I
CHjCHj c NHCHjCHjNH C CHjCHj - 2HCI

i
i
H CHjOH
Chemical name. (-H)-(S,S)-2,2'-(Ethylenediimino)di-l-butanol dihydrochlo-

ride; [S-(P*P’^]-2,2'-(l,2-ethanediyldiimino)bis[l -butanol] dihydrochloride;
CAS Reg. No. 1070-11-7.
400
Solubility. Soluble in 1 part of water; soluble in ethanol (-750 g/1) TS; practi-
Category. Antibacterial (tuberculostatic).
Storage. Ethambutol hydrochloride should be kept in a well-closed container.
Requirements
Definition. Ethambutol hydrochloride contains not less than 98.0% and not
more than 100.5% of CioH24N202,2HCl, calculated with reference to the dried
substance.
Identity tests

spectrum obtained from ethambutol hydrochloride RS or with the reference
spectrum of ethambutol hydrochloride.
B. See the test described below under '‘2(R-Aminobutanol)”. The principal spot

Specific optical rotation. Use a O.lOg/ml solution; [«]d = +5-0 to 7.0°.
Heavy metals. Use 1.0 g for the preparation Of the test solution as described
5-Omg/g.
401
2(i?-Aminobutanol). Carry out the test as described under 1.14.1 Thin-layer

11 volumes of ethyl acetate R, 7 volumes of glacial acetic acid R, 1 volume of
hydrochloric acid (-420 g/1) TS, and 1 volume of water as the mobile phase.
Apply separately to the plate 2 pi of each of 3 solutions in methanol R con-
taining (A) 50 mg of the test substance per ml, (B) 0.50 mg of the test substance
per ml, and (C) 0.50 mg of 2-aminobutanol R per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air, heat at 105 °C for 5
minutes, cool, spray with triketohydrindene/cadmium TS, heat at 90 °C for 5
minutes, and examine the chromatogram in daylight. The spot obtained with
solution C is more intense than any spot, corresponding in position and appear-
ance, obtained with solution A.
(0.1 mol/1) VS as described under 2.6 Non-aqueous titration. Method A. Each ml
of perchloric acid (0.1 mol/1) VS is equivalent to 13.86mg of CioH24N202,2HCl.
ETHANOLUM
ETHANOL
HjC — CHj — OH
C2H,0
Chemical name. Ethyl alcohol; ethanol; CAS Reg. No. 64-17-5.
Other name. Absolute alcohol, dehydrated alcohol.
Description. A colourless, clear and mobile liquid; odour, characteristic.
Miscibility. Miscible with water and ether R.
Storage. Ethanol should be kept in a well-closed container, and stored when-

ever possible at a temperature between 8 and 15 °C.
Additional information. Ethanol is flammable, burning with a blue smoke-

less flame. Hygroscopic. Boiling point, about 79 °C.
402
Requirements
Ethanol contains not less than 98.8% v/v and not more than the equivalent of
100.0% v/v of C 2 H 6O, corresponding to not less than 98.1% m/m and not
more than the equivalent of 100 0 %
. m/m of CjHeO.
Identity tests
A. Place 0.25ml in a small beaker, add 1 ml of potassium permanganate (lOg/1)
TS and 0.5ml of sulfuric acid (0.5mol/l) VS, and cover the beaker immedi-
ately with a filter-paper moistened with a recently prepared solution of
0.1 g of sodium nitroprusside R and 0.5 g of piperazine hydrate R in 5 ml of
water; a dark blue colour is produced on the filter-paper, that fades after a
few minutes.
B. To a few drops add 1 ml of sulfuric acid (-1760 g/1) TS and a few drops of
potassium dichromate (100 g/1) TS; a green colour is produced and an odour
of acetaldehyde is perceptible.
Relative density, dll = 0.7904 - 0.7935.
Non-volatile residue. Place 100 ml in a porcelain dish and heat on a water-

bath until volatilized, dry the residue at 105 °C for 1 hour, and weigh; not more
than 5 mg.
Water-insoluble substances. Dilute a volume of Ethanol with an equal

volume of water; the mixture is clear and, after cooling to 10 °C, it remains clear
for 30 minutes.
Acidity. Add 20ml of carbon-dioxide-free water R and 3 drops of phenolph-

thalein/ethanol TS to 20 ml of Ethanol; the colour remains unchanged. Titrate
required to obtain the midpoint of the indicator (pink).
Aldehydes and other foreign organic substances. Thoroughly clean

a glass-stoppered cylinder with hydrochloric acid (-250 g/1) TS, rinse with
water and the Ethanol to be examined. Place 20 ml of Ethanol in the cylinder.
Cool the contents to about 15 °C and, by means of a carefully cleaned pipette,
add 0.1ml of potassium permanganate (0.02mol/l) VS, noting the time of
the addition. Mix at once by inverting the stoppered cylinder, and allow
to stand at 15 °C for exactly 5 minutes; the pink colour does not entirely
disappear.
Fusel oil and allied impurities. Allow 25ml to evaporate spontaneously

from a porcelain dish, carefully protected from dust, until the surface of the
dish is barely moist; no foreign odour is perceptible, and on the addition of a
few drops of sulfuric acid (-1760 g/1) TS, no red or brown colour develops.
403
Methanol. To 1 drop add 1 drop of water, 1 drop of phosphoric acid

(~105g/l) TS, and 2 drops of potassium permanganate (25g/l) TS. Mix, allow
to stand for 1 minute, and add, drop by drop, sodium metabisulfite (50g/l) TS
until the permanganate colour is discharged. If a brown colour remains, add
1 drop of phosphoric acid (~105g/l) TS. To the colourless solution add 5 ml
of freshly prepared disodium chromotropate TS, and heat on a water-bath at
60 °C for 10 minutes; no violet colour appears.
Benzene. Record an absorption spectrum of the Ethanol in a 1-cm layer

against water between 220 nm and 350 nm. The absorbance at about 220 nm is
not more than 0.30, at about 230 nm not more than 0.18, at about 240 nm not
more than 0.08, and at about 270 to 350 nm not more than 0.02. A curve drawn
through these points is smooth.
ETHER ANAESTHESICUS
ANAESTHETIC ETHER
Graphic formula.
Chemical name. Ethyl ether; l,l'-oxybis[ethane]; diethyl ether; CAS Reg. No.
60-29-7.
Description. A clear, colourless, volatile, very mobile liquid; odour, characteristic.
TS.
Category. General anaesthetic.
Storage. Anaesthetic Ether should be kept in a securely closed, dry container,

protected from light, at a temperature not exceeding 15°C, and in quantities of
not more than 1 kg.
Labelling. The designation on the container of anaesthetic Ether must state;
“Highly flammable. Do not use near an open flame or other sources of heat
that may cause ignition.” The name and quantity of any antioxidant added
must be stated.
404
Additional information. Anaesthetic Ether may contain a suitable antioxi-

dant. It must not be used for anaesthesia if it has been removed from its
original container for longer than 24 hours. Ether remaining in partially used
containers may deteriorate rapidly. CAUTION; Vapours of ether mixed with
air, oxygen, or nitrous oxide in certain concentrations are explosive. Do not use
an open flame at any time during the testing procedure.
Requirements
Distillation range
• It is dangerous to determine the distillation range of the test liquid if it does
not comply with the test for Peroxides.
Use a suitable heating device, and take precautions to avoid superheating the
distillation flask above the level of the test liquid; it distils completely between
34.0 and 35.0 °C.
Relative density. d\o = 0.713 - 0.716.
Non-volatile residue
• It is dangerous to determine the non-volatile residue of the test liquid if it
does not comply with the test for Peroxides.
Allow 50ml to evaporate, dry the residue at 105°C for 1 hour and weigh; it
leaves not more than 20pg/ml.
Acidity. Place 10ml of ethanol (~750g/l) TS in a 50-ml glass-stoppered flask,

add 0.5ml of phenolphthalein/ethanol TS and just sufficient carbonate-free
sodium hydroxide (0.02 mol/1) VS to produce a pink colour that persists after
shaking the mixture for 30 seconds; add 25ml of the test liquid, mix gently, and
add carbonate-free sodium hydroxide (0.02 mol/1) VS until the pink colour per-
sists after shaking the mixture for 30 seconds; not more than 0.4ml of addi-
tional carbonate-free sodium hydroxide is required.
Peroxides. To 10ml of the test liquid add 2.0ml of vanadium/sulfuric acid TS

and shake. Separately prepare a reference solution by diluting 1.0ml of hydro-
gen peroxide (~60g/l) TS to 100 ml with water. To 0.10 ml of this reference solu-
tion add 2.0 ml of vanadium/sulfuric acid TS. The colour produced in the
aqueous layer in the tube containing the test liquid, when viewed transversely
against a white background, is not more intense than that of the reference solu-
tion when compared as described under 1.11 Colour of liquids.
Acetone and aldehydes. Transfer 2 ml of alkaline potassio-mercuric iodide

TS to a glass-stoppered test-tube of about 12ml capacity and about 1.5cm
diameter, and add 10 ml of the test liquid. Shake the tube vigorously for 10
405
seconds and allow to stand in the dark for 5 minutes; no turbidity is produced.
If the test liquid does not comply with this requirement, distil 40 ml, previously
ensuring that it complies with the test for Peroxides, until only 5 ml remains;
repeat the test on the distillate.
ETHINYLESTRADIOLUM
ETHINYLESTRADIOL
Graphic formula.
OH
Chemical name. 19-Nor-17a-pregna-l,3,5(10)-trien-20-yne-3,17-diol; 17-

ethynyl-estra-l,3,5,(10)-triene-3,17P-diol; CAS Reg. No. 57-63-6.
Description. A white to slighdy yellowish white, crystalline powder;

odourless.

TS; soluble in acetone R, and dioxan R.
Category. Estrogen.
Storage. Ethinylestradiol should be kept in a well-closed container, protected

from light.
Additional information. Ethinylestradiol may exist in 2 polymorphic forms

one of which melts at about 183 °C, the other, metastable, at about 143 °C.
Requirements
Definition. Ethinylestradiol contains not less than 97.0% and not more than
406
Identity tests

spectrum obtained from ethinylestradiol RS or with the reference spectrum of
ethinylestradiol. If the spectrum obtained from the solid state of the test
substance is not concordant with the spectrum obtained from the reference
substance, compare the spectra of solutions in chloroform R containing
30mg/ml, using a path length of 0.2mm.

test as described
propylene glycol R and 9 volumes of acetone R to impregnate the plate,
dipping it about 5 mm
beneath the surface of the liquid. After the solvent
has reached a height of at least 16cm, remove the plate from the chro-
matographic chamber and allow it to stand at room temperature until the
solvent has completely evaporated. Use the impregnated plate within 2
hours, carrying out the chromatography in the same direction as the impreg-
nation. Use toluene R as the mobile phase. Apply separately to the plate
2|xl of each of 2 solutions in a mixture of 9 volumes of chloroform R and
1 volume of methanol R containing (A) 1.0
mg of the test substance per ml,
and (B) 1.0 mg of ethinylestradiol RS per ml. Develop the plate for a dis-
tance of 15 cm. After removing the plate from the chromatographic
chamber, allow it to dry in air until the solvents have evaporated, heat at
120 °C for 15 minutes, spray with 4-toluenesulfonic acid/ethanol TS, and
then heat at 120 °C for 5-10 minutes. Allow to cool, and examine the chro-
matogram in daylight and in ultraviolet light (3 65 nm). The principal spot
Specific optical rotation. Use a 4.0 mg/ml solution in pyridine R and calcu-
latewith reference to the dried substance; = -27.0 to -30.0°.
lOmg/g.
Estrone. Carry out the test as described under 1.14.1 Thin-layer chromatog-
raphy, using silica gel R1 as the coating substance and a mixture of 92 volumes
of dichloroethane R, 8 volumes of methanol R, and 0.5 volumes of water as
the mobile phase. Apply separately to the plate 5pl of each of 2 freshly pre-
pared solutions in a mixture of 9 volumes of chloroform R and 1 volume of
methanol R containing (A) 20 mg of the test substance per ml, and (B) 0.20 mg
of estrone RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air until the odour of the solvent is no longer
detectable; then heat at 110 °C for 10 minutes. Spray the hot plate with sulfu-
407
ric acid/ethanol TS, heat again at 110°C for 10 minutes, and examine the
chromatogram in ultraviolet light (365 nm). The spot obtained with solution B
is more intense than any spot, corresponding in position and appearance,
obtained with solution A,
Assay. Dissolve about 0.05 g, accurately weighed, in sufficient dehydrated

ethanol R to produce 100ml, and dilute 10.0ml of this solution to 50.0ml with
the same solvent. Measure the absorbance of a 1-cm layer of the diluted solu-
tion at the maximum at about 281 nm. Calculate the amount of C 20 H 24 O 2 in the
substance being tested by comparison with ethinylestradiol RS, similarly and
ETHIONAMIDUM
ETHIONAMIDE
Molecular formula. C8H10N2S
Graphic formula.
H5
S:=^c—-NHj
Chemical name. 2-Ethylthioisonicotinamide; 2-ethyl-4-pyridinecarboth-

ioamide; CAS Reg. No. 536-33-4.
Description. Small yellow crystals or a yellow, crystalline powder; odour,

slight.
Solubility. Practically insoluble in water; soluble in methanol R; sparingly

soluble in ethanol (-750 g/1) TS; slighdy soluble in ether R.
Storage. Ethionamide should be kept in a tightly closed container, protected

from light, and stored in a cool place.
Additional information. Ethionamide darkens on exposure to light.
408
Requirements
Definition. Ethionamide contains not less than 98.0% and not more than
101.0% of C 8 H 10N 2 S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from ethionamide RS or with the reference sfectrum of
ethionamide.
B. Mix 0.05g with O.lOg of 2,4-dinitrochlorobenzene R, then transfer lOmg of

thismixture to a test-tube and heat until melted. Cool and add 3ml of potas-
sium hydroxide/ethanol TSl; a red to orange-red colour is produced.
C. Heat 0.1 g with 5ml of hydrochloric acid (1 mol/1) VS; the vapours evolved
blacken lead acetate paper R.
Apply separately to the plate lOpl of each of 3 solutions in acetone R contain-
ing (A) 20 mg of the test substance per ml, (B) O.lOmg of the test substance per
ml, and (C) 0.04 mg of the test substance per ml. After removing the plate from
the chromatographic chamber, allow it and examine the chro-
to dry in air
other than the principal spot, is not more intense than that obtained with solu-
tion B. Not more than one of any such spots is more intense than that obtained
with solution C.

alent to 16.62 mg of C 8H 10N 2 S.
409
ETHOSUXIMIDUM
ETHOSUXIMIDE
Molecular formula. CjHuNOj
Graphic formula.
Chemical name. 2-Ethyl-2-methylsuccinimide; 3-ethyl-3-methyl-2,5-pyrro-

lidinedione; CAS Reg. No. 77-67-8.
Description. A white or almost white powder or waxy solid; odourless or

with a faint characteristic odour.
Solubility. Freely soluble in water; very soluble in ethanol (-750 g/1) TS and
ether R.
Category. Anticonvulsant.
Storage. Ethosuximide should be kept in a tightly closed container, protected

from light.
Additional information. Even in the absence of light, Ethosuximide is grad-

Requirements
Definition. Ethosuximide contains not less than 99.0% and not more than
100.5% of CH
7 11 NO 2, calculated with reference to the anhydrous substance.
Identity tests

spectrum obtained from ethosuximide RS or with the reference spectrum of
ethosuximide. If the spectrum obtained from the solid state of the test sub-
410
stance is not concordant with the spectrum obtained from the reference sub-
stance, dissolve a small amount of the substance in ethanol (-750 g/1) TS,
evaporate to dryness on a water-bath, and prepare a potassium bromide disc
as described in Method 3, Then compare the spectrum obtained from the
disc with that of the reference substance.
B. Heat 0.1 g with 0.2g of resorcinol R and 2 drops of sulfuric acid (~1760g/l)
TS at 140 °C for 5 minutes, allow to cool, add 5 ml of water, make alkaline
with sodium hydroxide (-80 g/1) TS, and pour a few drops into a large
volume of water; a bright green fluorescence is obtained.
Cyanides. Dissolve 1 g in 10ml of ethanol (-750g/l) TS, add 3 drops of ferrous
sulfate (15g/l)TS, 1ml of sodium hydroxide (-80 g/1) TS and a few drops of
TS. Warm gently, and acidify with sulfuric acid (-lOOg/1)
ferric chloride (25 g/1)
TS; no blue precipitate or blue colour is produced within 15 minutes.

Acidity. Dissolve 5.0 g in 50 ml of water by warming on a water-bath for

5 minutes. Cool and titrate with sodium hydroxide (0.1 mol/1) VS, using
bromocresol green/ethanol TS as indicator; not more than 0.7 ml is required to
obtain the midpoint of the indicator (green).
9 volumes of chloroform R and 1 volume of acetone R as the mobile phase.
Apply separately to the plate 10 pi of each of 2 solutions in ethanol (-750g/l)
TS containing (A) 50 mg of the test substance per ml and (B) 0.050 mg of the
test substance per ml. After removing the plate from the chromatographic
chamber allow it to dry in air, and examine the chromatogram in ultraviolet

mamide R, add 3 drops of azo violet TS and titrate with tetrabutylammonium
hydroxide (0.1 mol/1) VS to a blue endpoint, as described under 2.6 Non-
(O.lmol/1) VS is equivalent to 14.12mg of C7Hi]N02.
411
ETHYLCELLULOSUM
ETHYLCELLULOSE
Chemical name. Cellulose ethyl ether; CAS Reg. No. 9004-57-3.
Description. A white to light tan, free-flowing powder.
Solubility. Practically insoluble in water, glycerol R, and propylene glycol R.

When containing less than 46.5% of ethoxy groups, it is freely soluble in
tetrahydrofuran R. When 46.5% of ethoxy groups, it
containing not less than
is freely soluble in ethanol (-750 g/1) TS, methanol R, toluene R, and ethyl
acetate R.
Category. Film-coating agent; tablet binder; release-rate modifier for oral

formulations.
Storage. EthylceUulose should be kept in a well-closed container.
Labelling. The designation on the container of EthylceUulose should state its
viscosity.
Requirements
Definition. EthylceUulose is an ethyl ether of cellulose.
EthylceUulose contains not less than 44 0 % and not more than

. the equivalent
of 51 0 . % of ethoxy ( — OC H
2 5) groups.
Identity tests
A. Dissolve 5 g in 95 g of a mixture by mass of 80 parts of toluene R and 20
parts of ethanol (-750 g/1) TS; a slightly yellow, clear, stable solution is pro-
duced. (Keep the solution for test B.)
B. Pour a few ml of the above solution onto a glass plate and allow the solvent
to evaporate; a thin, though continuous, clear film is formed. Remove the
film from the plate; it is flammable.
412
Assay. Carry out the assay as described under 2.9 Determination of methoxyl,
using about 0.05 g, previously dried and accurately weighed.

(-OC2H5).
ETHYLIS HYDROXYBENZOAS
ETHYL HYDROXYBENZOATE
C9H10O3
Chemical name. Ethyl /^-hydroxybenzoate; ethyl 4-hydroxybenzoate; CAS

Reg. No. 120-47-8.
Other name. Ethylparaben.
Description. Small colourless crystals or a white, crystalline powder; odour-

Solubility. Very slightly soluble in water; sparingly soluble in boiling water;

freely soluble in ethanol (-750 g/1) TS and ether R.
Storage. Ethyl hydroxybenzoate should be kept in a well-closed container.
Additional information. Ethyl hydroxybenzoate is normally used in combi-

nation with other hydroxybenzoates.
Requirements
Ethyl hydroxybenzoate contains not less than 99.0% and not more than the
equivalent of 101 0 . % of C 9H 10 O 3 calculated with reference to the dried
substance.
413
Identity tests
A. Complies with the test under “Melting range”.
B. To add 5 ml of sodium hydroxide (~80g/l) TS, and heat in a water-bath

0,5 g
for 5 minutes. After cooling, add 6ml of sulfuric acid (-190 g/1) TS, collect
the precipitateon a filter, wash thoroughly with a small amount of water,
and dry over silica gel, desiccant, R. Melting temperature, about 214 °C.
Loss on drying. Dry at 80 °C under reduced pressure (not exceeding O.hkPa

or about 5 mm
of mercury) for 2 hours; it loses not more than 5.0mg/g.
Acidity. Dissolve 0.2g in 5ml of ethanol (-750 g/1) TS, add 5ml of carbon-
dioxide-free water R, and titrate with sodium hydroxide (0.1 mol/1) VS, using
0.1ml of bromocresol green/ethanol TS as indicator; not more than 0.1ml is
Assay. Place about 0.08 g, accurately weighed, in a ground-glass-stoppered

flask, add 25 ml of sodium hydroxide (-80 g/1) TS, and boil gently under a reflux
condenser for 30 minutes. Allow to cool, add 25ml of potassium bromate

(0.0333mol/l) VS, 5ml of potassium bromide (125 g/1) TS, and 40ml of glacial
acetic acid R. Cool in ice-water and add 10ml of hydrochloric acid (-420g/l)
TS. Stopper the flask immediately and allow to stand for 15 minutes. Add
30ml of potassium iodide (80 g/1) TS, close the flask, and mix. Titrate with
sodium thiosulfate (0.1 mol/1) VS, using 2ml of starch TS as indicator, added
towards the end of the titration. Repeat the procedure without the Ethyl
hydroxybenzoate being examined and make any necessary corrections.
One volume ofsodium thiosulfate (0.1 mol/1) VS corresponds to two volumes

of potassium bromate (0.0333 mol/1) VS. Each ml of potassium bromate
(0.0333mol/l) VS is equivalent to 5.540 mg of C9H10O3.
414
ETOPOSIDUM
ETOPOSIDE
C29H32O13
Chemical name. 4'-Demethylepipodophyllotoxin 9-(4,6-0-ethylidene-|3-

Dglucopyranoside); [57?-[5a,5a|3,8aa,9|3(R*)]]-9-[(4,6-0-ethylidene-b-P-glu-
copyranosyl)oxy]-5,8,8a,9-tetrahydro-5-(4-hydroxy-3,5-dimethyoxyphenyl)
furo[3',4':6,7]naphtho[2,3-i]-l,3-dioxol-6(5aH)-one; CAS Reg. No. 33419-42-0.
Solubility. Practically insoluble in water; sparingly soluble in methanol R;

slightly soluble in ethanol (-750 g/1) TS and dichloromethane R.
Storage. Etoposide should be kept in a tightly closed container.
Labelling. The designation Etoposide for parenteral use indicates that the
enteral administration. Expiry date.
Additional information. CAUTION: Etoposide must be handled with care,

Requirements
Etoposide contains not less than 98 0.% and not more than the equivalent of
102 0 %
. of C 29 H 320 , 3, calculated with reference to the dried substance.
415
=
Identity tests

the spectrum obtained from etoposide RS or with the reference spectrum of
etoposide.
B. See the test described below under “Related substances”. The principal band
C. Transfer about 5 mg to a test-tube and dissolve in 5 ml of glacial acetic acid

R, add about 0.1ml of ferric chloride (50g/l) TS, and mix. Cautiously add
about 2ml of sulfuric acid (~1760g/l) TS. Withoutmmng allow to stand for
about 30 minutes; a pink to reddish brown ring develops at the interface
and the upper layer is yellow.
D. Dissolve 5mg in 5ml of methanol R, add 5ml of hydrochloric acid (~70g/l)

TS, and evaporate to dryness on a water-bath. To the residue add 20 ml of
water and 10 ml of dichlorome thane R, and shake vigorously. Allow to sep-
arate and to 1.0 ml of the aqueous layer add 2.0ml of anthrone TS2 and mix;
a blue-green colour is produced.
Specific optical rotation. Dissolve O.OSOg in 10ml of a mixture of 1 volume

of methanol R and 9 volumes of dichloromethane R; [a]c -106° to -114°,
Solution in methanol/dichloromethane. Dissolve 0.6g in20mlof a mixture

of 1 volume of methanol R and 9 volumes of dichloromethane R; the solution
isclear and not more intensely coloured than standard colour solution 2 of the
most appropriate hue, when compared as described under 1.11 Colour of
liquids.
or about 5 mm
of mercury) for 4 hours; it loses not more than 30mg/g.
100 volumes of dichloromethane R, 20 volumes of acetone R, 8 volumes of
416
glacial acetic acid R, volumes of water as the mobile phase. Apply sep-
and 1.5
arately to the plate 5 pi, spread to form 10-mm bands, of each of 5 solutions in
a mixture of 1 volume of methanol R and 9 volumes of dichloromethane R con-
taining (A) 0.050 g of Etoposide per ml, (B) 5 mg of Etoposide per ml, (C) 5 mg
of etoposide RS per ml, (D) 0.25mg of etoposide RS per ml, and (E) O.lOmg of
etoposide RS per ml. After removing the plate from the chromatographic
chamber, dry it in a current of warm air for 5 minutes. Spray with a mixture
of 1 volume of sulfuric acid (-1760 g/1) TS and 9 volumes of ethanol (~750g/l)
TS, and heat at 140 °C for 15 minutes. Cover the plate immediately with a glass
plate of the same size. Examine the chromatogram in daylight.
Any band obtained with solution A, other than the principal band, is not more
intense than that obtained with solution D. Furthermore, not more than two
such bands are more intense than the band obtained with solution E (0.2%).

matography, using a stainless steel column (25 cm x 4.6mm) packed with par-
bonded octadecylsilyl groups (10 pm). Prepare a diluted solution of acetic acid
as follows to be used in the mobile phase and for the preparation of solution
C: to 96ml of water add 4 ml of glacial acetic acid R.As the mobile phase, use
a mixture of 76 volumes of the diluted acetic acid solution and 24 volumes of
acetonitrile R.
Prepare the following solutions in methanol R: solution (A) 1.0 mg of Etoposide

per ml; solution (B) 1.0 mg
of etoposide RS per ml; and for solution (C) add
0.1 ml of the above and 0.1 ml of phenolph-
diluted acetic acid solution described
thale in/ ethanol TS
ml of solution A, then add sodium hydroxide (1 mol/1)
to 10
VS until the solution becomes faintly pink (about 0.15ml), allow to stand for 15
minutes, and add 0.1 ml of the diluted acetic acid solution described above.
Inject 10 pi of solution C. Allow the chromatography to continue until the peak

corresponding to phenolphthalein is eluted. Phenolphthalein has a retention
time relative to etoposide of about 2.7. Disregard any peak due to phenolph-
thalein.The assay is not valid unless the chromatogram shows two principal
peaks and the resolution between these peaks is at least 1.5. If necessary, reduce
the concentration of acetonitrile R in the mobile phase or reduce the flow rate
to achieve the required resolution.
solutions A and B, and calculate the percentage content of C 29 H 32 O 13 .
417
Additional requirements for Etofaoside for parenteral use

terial endotoxins; contains not more than 2.01U of endotoxin RS per mg.
FERROSI FUMARAS
FERROUS FUMARATE
Molecular formula. C 4 H 2 pe 04
Graphic formula.
HC C=0
0=C — CH
II I
Chemical name. lron(2-t-) fumarate (1 :

1); iron(2-t-) (£)-2-butenedioate (1 :
1);
CAS Reg. No. 141-01-5.
Description. A fine, reddish orange or reddish brown powder.
Solubility. Slightly soluble in water; very slightly soluble in ethanol (-750 g/1)
TS.
Category. Iron supplement.
Storage. Ferrous fumarate should be kept in a well-closed container.
Requirements
Definition. Ferrous fumarate contains not less than 93.0% and not more than
101.0% of C 4 H 2 Fe 04 calculated with reference
,
Identity tests
A. Dissolve with heating 0.4g in 10ml of hydrochloric acid (1 mol/1) VS and
cool. (Keep 1.0 ml of this solution for test B.) To the remaining solution add
15ml of sodium hydroxide (1 mol/1) VS. Separate the dark precipitate by
To the filtrate add 0.2ml of phenolphthalein/ethanol TS and
filtration.
418
sufficient hydrochloric acid (1 mol/1) VS until the pink colour disappears. To

2.0 ml of the resulting solution add 2.0 ml of copper(ll) acetate (45g/l) TS;
a white, crystalline precipitate is produced.
B. The solution prepared in test A yields reaction A described under 2.1 General
identification tests as characteristic of ferrous salts.
C. Mix 0.5 g with Ig of resorcinol R. Transfer 0.5 g of the mixture to a crucible,

add about 0.15ml of sulfuric acid (-1760 g/1) TS and heat gently; a deep red,
semi-solid mass is produced. Add the mass to a large volume of water; an
orange-yellow solution is obtained which exhibits no fluorescence.
Heavy metals. Ignite l.Og gently until free from carbon, dissolve in 5 ml of
hydrochloric acid (-420 g/1) TS by heating on a water-bath, and evaporate
to dryness. Dissolve the residue in a mixture of 15ml of hydrochloric acid
(-420 g/1) TS, 4ml of nitric acid (-1000 g/1) TS, and 6ml
of water, boil gently
for 1 minute, cool, and extract the iron with 3 portions of ether R, each of
20 ml. Make a fourth extraction with a further 20 ml of ether R if the acid layer
ismore than slightly yellow and reject the extracts. Heat the acid solution gently
to remove the ether, add 1 g of citric acid PbR, make alkaline with ammonia
(-lOOg/1) PbTS, add 1ml of potassium cyanide PbTS and dilute to 40ml with
water; proceed to determine the heavy metals content as described under 2.2.3
Limit test for heavy metals, according to Method A; not more than lOOpg/g.
Arsenic. Mix 0.2 g with 1.5g of anhydrous sodium carbonate R, add 10ml of
bromine AsTS and mix thoroughly. Evaporate to dryness on a water-bath,
gently ignite, and dissolve the cooled residue in 20 ml of brominated hydrochlo-
ric acid AsTS and 10 ml of water. Transfer to a small flask, add sufficient stan-
nous chloride AsTS to remove the yellow colour, connect to a condenser and
distil 22ml; proceed with the distillate as described under 2.2.5 Limit test for
arsenic; the arsenic content is not more than 5pg/g.
Ferric iron. Dissolve about 3g, accurately weighed, in a mixture of 100 ml of

water and 10 ml of hydrochloric acid (-420 g/1) TS by heating to boiling until
dissolved. Cool rapidly and add 3g of potassium iodide R, stopper the flask,
swirl to mix, and allow to stand in the dark for 15 minutes. Titrate the liber-
ated iodine with sodium thiosulfate (0.1 mol/1) VS, using starch TS as indica-
tor, added towards the end of the titration. Repeat the operation without the
substance being examined and determine the difference in the volume of

sodium thiosulfate (0.1 mol/1) VS required for the titration. Each ml of sodium
thiosulfate (0.1 mol/1) VS is equivalent to 5.585 mg of ferric iron; the ferric iron
Sulfates. Boil 0.15g with 8ml of hydrochloric acid (-70g/l) TS and 20ml of
water, cool in ice and filter; proceed with the filtrate as described under 2.2.2
Limit test for sulfates; the sulfate content is not more than 2mg/g.
419
lOmg/g.
Assay. Dissolve about 0.3g, accurately weighed, in 7,5ml of sulfuric acid

(~100g/l) TS, heating gently. Cool, add 25ml of water, and immediately titrate
with ceric ammonium sulfate (0.1 mol/1) VS, using 0.1ml of o-phenanthroUne
TS as indicator. Each ml of eerie ammonium sulfate (0.1 mol/1) VS is equivalent
to 16.99mg of C4H2Fe04.
FERRO SI SULFAS
FERROUS SULFATE
Ferrous sulfate, exsiccated
Ferrous sulfate heptahydrate
Molecular formula. FeS04,nH20 (exsiccated); FeS04, 7H2O (heptahydrate).
Relative molecular mass. 151.9 (anhydrous); 278.0 (heptahydrate).
Chemical name. Iron (2+) sulfate (1 :

1); CAS Reg. No, 7720-78-7 (anhydrous).
Iron (2-I-) sulfate (1 :

1) heptahydrate; CAS Reg. No. 7782-63-0 (heptahydrate).
Description. Exsiccated Ferrous sulfate is a greyish white powder; Ferrous

sulfate heptahydrate has pale blue-green prisms or is a pale green, crystalline
powder; both forms are odourless.
Solubility. Exsiccated Ferrous sulfate is slowly but almost completely soluble

in carbon-dioxide-free water R; practically insoluble in ethanol (-750 g/1) TS.
Ferrous sulfate heptahydrate is freely soluble in water; very soluble in boiling
water; practically insoluble in ethanol (-750 g/1) TS.
Category. Haemopoietic (iron-deficiency anaemia).
Storage. Ferrous sulfate should be kept in a well-closed container.
Labelling. The designation on the container of Ferrous sulfate should state

whether the substance is in the exsiccated form or is the heptahydrate.
Additional information. Exsiccated Ferrous sulfate is Ferrous sulfate

deprived of part of its water of crystallization by drying at a temperature of
40 °C, Ferrous sulfate heptahydrate is efflorescent in dry air. Even in the
absence of light. Ferrous sulfate is gradually degraded on exposure to a humid
atmosphere, the decomposition being faster at higher temperatures. The
420
crystals rapidly oxidize becoming brown. Both forms have a metallic and astrin-
gent taste.
Requirements
Definition. Exsiccated Ferrous sulfate contains not less than 80.0% and not
more than 90.0% of FeS 04 Ferrous sulfate heptahydrate contains not less than
.
98.0% and not more than 105.0% of FeS 04 7 H 20 ,

.
Identity tests
A. A 20 mg/ml solution yields the reactions described under 2.1 General iden-
tification tests as characteristic of ferrous salts.
B. A 20mg/ml solution yields reaction A described under 2.1 General identifi-

Heavy metals. Dissolve l.Og in 10ml of hydrochloric acid (-250 g/1) TS, add
2 ml of hydrogen peroxide (-330 g/1) TS, and evaporate to 5 ml. Allow to cool,
dilute to 20 ml with hydrochloric acid (-250 g/1) TS, transfer the solution to a
separating funnel and shake for 3 minutes with 3 successive quantities, each of
20 ml, of a solution composed of 100 ml of freshly distilled methylisobutylke-
tone R shaken with 1 ml of hydrochloric acid (-250 g/1) TS. Allow the layers to
separate, evaporate the aqueous layer volume, allow to cool and
to half its
dilute to 50ml with water. Neutralize 25ml

TS and determine the
to litmus
heavy metals content as described under 2.2.3 Limit test for heavy metals,
according to Method A, multiplying the result by a factor of 2; not more than
50pg/g.
Alkaline salts. Dissolve 1 g in 10 ml of water and oxidize by warming with a

few drops of nitric acid (-lOOOg/1) TS, make alkaline with ammonia (-lOOg/1)
TS, filter, evaporate the filtrate, ignite the residue and weigh; not more than
l.Omg/g.
Arsenic. Use a solution of 3.3 g in 35 ml of water and proceed as described

under 2.2.5 Limit test for arsenic; the arsenic content is not more than 3pg/g.
Insoluble matter. Dissolve l.Og in lOml of water, filter, wash the filter with
water and dry it at 105 °C; the content of insoluble matter is not more than
5.0mg/g.
pH value. pH of a 0.05 g/ml solution, 3. 0-4.0.
Assay. For exsiccated Ferrous sulfate dissolve about 0.3 g, accurately weighed,
in a mixture of 30 ml of water and 20 ml of sulfuric acid (-100 g/1) TS and titrate
with ceric ammonium sulfate (0.1 mol/1) VS, using 2 drops of o-phenanthroline
421
TS as indicator. Each ml of ceric ammonium sulfate (0.1 mol/1) VS is equivalent

to 15.19mg of FeSO^.
For Ferrous sulfate heptahydrate dissolve 2.5 g of sodium hydrogen carbonate

R in a mixture of 150ml of water, 10.0ml of sulfuric acid (-1760 g/1) TS, and
5.0ml of phosphoric acid (~1440g/l) TS. When the effervescence ceases, add
about 0.5 g of the test substance, accurately weighed, and when solution is
complete titrate with ceric ammonium sulfate (0.1 mol/1) VS, using 2 drops of
o-phenanthroline TS as indicator. Each ml of ceric ammonium sulfate (0.1 mol/1)
VS is equivalent to 27.80mg of FeS 04 7 H 20
,
.
FLUCYTOSINUM
FLUCYTOSINE
Molecular formula. C4H4FN3O
Graphic formula.
NHj
Chemical name. 5-Fluorocytosine; 4-amino-5-fluoro-2(lH)-pyrimidinone;

CAS Reg. No. 2022-85-7.

almost odourless.
Solubility. Sparingly soluble in water; slightly soluble in ethanol (-750 g/1) TS;
Storage. Flucytosine should be kept in a tightly closed container, protected

from light.
Additional information. Flucytosine melts at about 295 °C.
422
Requirements
Definition. Flucytosine contains not less than 98.5% and not more than
101.0% of C4H4FN3O, calculated with reference to the dried substance.
Identity tests

the spectrum obtained from flucytosine RS or with the reference spiectrum of
flucytosine.
B. The absorption spectrum of a 5.0pg/ml solution in hydrochloric acid

(0.1 mol/1) VS, when observed between 230 nm and 350 nm, exhibits a
C. See the test described below under “Fluorouracil”. The principal spot
D. Dissolve 0.05g in 5ml of water and add 0.15ml of bromine TSl; the colour
is discharged or almost discharged.
Sulfated ash. Not more than 1 mg/g.
15 mg/g.
Fluorouracil. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using silica gel R6 as the coating substance (a precoated plate from
a commercial source is suitable) and a mixture of 70 volumes of nitromethane
R, 20 volumes of ethanol (-750 g/1) TS, and 10 volumes of lithium chloride
(lOg/1) TS as the mobile phase. Apply separately to the plate 1 [il of each of 2
solutions in a solvent mixture composed of 15 volumes of methanol R and 10
volumes of water, containing (A) lOmg of the test substance per ml and (B)
lOmg of flucytosine RS per ml; then apply also 10 pi of each of the 2 follow-
ing solutions in the above solvent mixture containing (C) 20 mg of the test
substance per ml and (D) 20 pg of fluorouracil RS per ml. Develop the plate in
an unsaturated chromatographic chamber. After removing the plate from the
chromatographic chamber, allow it to dry in a current of air and examine the
423
chromatogram in ultraviolet light (254 nm). The spot obtained with solution D
is more intense than any corresponding spot obtained with solution C.
Assay. Dissolve about 0.3 g, accurately weighed, in a mixture of 50 ml of acetic

anhydride R and 100ml of glacial acetic acid R1 with warming, if necessary,
and titrate with perchloric acid (0.1 mol/1) VS, determining the endpoint poten-
tiometrically as described under 2.6 Non-aqueous titration, Method A. Each ml
of perchloric acid (0.1 mol/1) VS is equivalent to 12.91 mg of C4H4FN3O.
Additional requirement for Flucytosine for p>arenteral use

FLUDROCORTISONI ACETAS
FLUDROCORTISONE ACETATE
Graphic formula.
CH,OCOCH 3
I
CO
Chemical name. 9-Fluoro-ll(3,17,21-trihydroxypregn-4-ene-3,20-dione 21-

acetate; 21-(acetyloxy)-9-fluoro-lip,17-dihydroxypregn-4-ene-3,20-dione; CAS
Reg. No. 514-36-3.

almost odourless.

424
Storage. Fludrocortisone acetate should be kept in a well-closed container,

Additional information. Fludrocortisone acetate is hygroscopic.
Requirements
Definition. Fludrocortisone acetate contains not less than 96.0% and not more
than 104.0% of C2.^H3]F06, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from fludrocortisone acetate RS or with the reference spec-
trum of fludrocortisone acetate.

allow it to stand at room temperature until the solvent has completely evap-
orated. Use the impregnated plate within 2 hours and carry out the chro-
matography in the same direction as the impregnation. As the mobile phase,
use a mixture of 75 volumes of toluene R and 25 volumes of chloroform R.
Apply separately to the plate 2 pi of each of 2 solutions in a mixture of 9
volumes of chloroform R and 1 volume of methanol R containing (A)
2.5 mg of the test substance per ml and (B) 2.5 mg of fludrocortisone acetate
RS per ml. Develop the plate for a distance of 15 cm. After removing the
plate from the chromatographic chamber, allow it to dry in air until the
solvents have evaporated, heat it at 120 °C for 15 minutes, spray it with
sulfuric acid/ethanol TS, and then heat it at 120 °C for 10 minutes. Allow it
to cool, and examine the chromatogram in daylight and in ultraviolet light
C. Heat 0.5 ml of chromic acid TS in a small test-tube in a water-bath for 5

minutes; the solution wets the sides of the tube but there is no greasiness.
Add about 3 mg of the test substance and again heat in a water-bath for 5
minutes; the solution no longer wets the sides of the tube.
Specific optical rotation. Use a lOmg/ml solution in dioxan R; [a]o

"‘^
= -1-148
to -h156°.
425
Loss on drying. Dry to constant weight at 105 °C; it loses not more than lOmg/g.
Ultraviolet absorption. Absorbance of a 1-cm layer of a lOpg/ml solution in

dehydrated ethanol R at about 240 nm; 0.39-0.42.
95 volumes of dichloroethane R, 5 volumes of methanol R, and 0.2 volumes of
substance per ml. After removing the plate from the chromatographic chamber
allow it to dry in air until the solvents have evaporated; then heat it at 105°C
for 10 minutes, allow it to cool, and examine the chromatogram in ultraviolet
Assay

aldehyde-free ethanol (-750 g/1)TS to produce 50 ml. Transfer 10.0ml of the
25ml and mix. Measure the absorbance of a 1-cm layer at the maximum at
amount of C23H31FO5 in the substance being tested by comparison with flu-
drocortisone acetate RS, similarly and concurrently examined.
FLUORESCEINUM NATRICUM
FLUORESCEIN SODIUM
Molecular formula. C2oHioNa20s
426
Graphic formula.
Chemical name. Fluorescein disodium salt; 2-(6-hydroxy-3-oxo-3/i-xanthen-

9-yl)benzoic acid disodium salt; 3',6'-dihydroxyspiro[isobenzofuran-l(3/f),9'-
[9H]xanthene]-3-one disodium salt; CAS Reg. No. 518-47-8.
Description. An orange-red powder; odourless.
Solubility. Soluble in 1.5 parts of water; soluble in ethanol (-750 g/1) TS.
Category. Diagnostic agent in ophthalmology.
Storage. Fluorescein sodium should be kept in a well-closed container, pro-

tected from light.
Additional information. Fluorescein sodium is hygroscopic.
Requirements
Definition. Fluorescein sodium contains not less than 98.0% and not more
than 100.5% of C 2 oHioNa 205 ,
Identity tests
A. A solution in water is strongly fluorescent, even in extreme dilution; the flu-
orescence disappears when the solution is made acid and reappears when
it is made alkaline.
B. Ignite 20 mg and dissolve the residue in acetic acid (-60 g/1) TS. The solu-
C. Dissolve Img
in 2ml of water, place 0.05ml of this solution on a piece of
yellow spot is produced. Expose the moist paper to the vapour
filter-paper; a
of bromine R
for 1 minute and then to the vapour of ammonia (-260 g/1)
TS; the yellow colour of the spot changes to deep pink.
427
Chlorides. Dissolve 0.07 g in a mixture of 2ml of nitric acid (~130g/l) TS and

20ml of water, and proceed as described under 2,2,1 Limit test for chlorides;
Sulfates. Dissolve 0.05g in 20ml of water and proceed as described under

2.2.2 Limit test for sulfates; the sulfate content is not more than lOmg/g.
Zinc. Dissolve O.lOg in 10ml of water, add 2ml of hydrochloric acid (~420g/l)
TS, filter, and add 0.1ml of potassium ferrocyanide (45g/l) TS; no turbidity or
precipitate is produced immediately.
Chloroform-soluble matter. Dissolve 0.20 g in 10 ml of sodium hydroxide

(0.1 mol/1) VS and extract with 10ml of chloroform R. Allow to separate, dry
the chloroform layer over anhydrous sodium sulfate R, and filter. Measure the
absorbance of the filtrate in a 1-cm cell at a maximum of 480 nm against a
solvent cell containing chloroform R; the absorbance does not exceed 0.10.
Ethanol-insoluble matter. Boil 0.2g with 20ml of ethanol (-750g/l) TS

for 1 minute, filter through a sintered glass filter, wash the filter with ethanol
(-750 g/1) TS until the filtrate is almost colourless, dry the filter at 105 °C for 1
hour and weigh; the residue is not more than 2.0 mg.
100 mg/g.
pH value. pH of a 20 mg/ml solution in carbon-dioxide-free water R, 7. 0-9,0.
Dimethylformamide. Carry out the test as described under 1.14. 5 Gas chro-
matography. Prepare an internal standard consisting of a mixture of 20 pi of
dimethylacetamide R in 100 ml of water. Inject the following 3 solutions: (1) a
mixture of 2 pi of dimethylformamide R in 10 ml of water and containing
10ml of the solution of the internal standard; (2) for the determination of the
retention time of the substance being examined, dissolve l.Og of the test sub-
stance in 10 ml of water, add with stirring 10 ml of hydrochloric acid (0.5 mol/1)
VS, allow to stand for 15 minutes, centrifuge, and then dissolve O.lOg of
trisodium orthophosphate R in 5 ml of the supernatant liquid; (3) dissolve
l.Og of the test substance in 10ml of the solution of the internal standard, add
with stirring 10ml of hydrochloric acid (0.5mol/l) VS, allow to stand for 15
minutes, centrifuge, and then dissolve O.lOg of trisodium orthophosphate R in
5 ml of the supernatant liquid.
For the procedure, use a glass column 1.5m long and4mm in internal diame-
ter packed with an adequate quantity of an adsorbent composed of 1 g of
macrogol lOOOR supported on 9g of acid-washed, silanized diatomaceous
support R and maintained at 120 °C. Use nitrogen R as the carrier gas and a
flame ionization detector.
428
In the chromatogram obtained with solution 3, the ratio of the area of any peak
due to dimethylformamide to the area of the peak due to the internal standard
is not greater than the corresponding ratio for the chromatogram obtained with
solution 1,
Resorcinol. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using silica gel R1 as the coating substance (a precoated plate from
a commercial source is suitable) and a mixture of 6 volumes of hexane R and
4 volumes of ethyl acetate R as the mobile phase. Apply separately to the plate
5 pi of each of the 2 following solutions: (A) dissolve 1.0 g of the test substance
in 10 ml of water, add slowly with constant stirring 10 ml of hydrochloric acid
(0.5mol/l) VS, allow to stand for 15 minutes, centrifuge, and use the super-
natant liquid; mg of resorcinol R in 10 ml of water. After remov-
(B) dissolve 2.5
expose it to the vapour of iodine R for 30 minutes. Examine the chromatogram
in daylight. The spot obtained with solution B is more intense than any corre-
sponding spot obtained with solution A.
chromatography, using silica gel R1 as the coating substance (a precoated plate
from a commercial source is suitable) and a mixture of 8 volumes of chloro-
form R and 2 volumes of methanol R as the mobile phase. Apply separately to
the plate 5 pi of each of 2 solutions in hydrochloric acid/methanol (0.1 mol/1)
VS containing (A) 10 mg of the test substance per ml and (B) 20 pg of the test
allow it to dry in air, and expose the plate to the vapour of iodine R for 30
minutes. Examine the chromatogram in daylight. Any spot obtained with solu-
with solution B.
Assay. Dissolve about weighed, in 20ml of water, add 5 ml

0.5 g, accurately
of hydrochloric acid (~70g/l) TS,and extract with 4 volumes, each of 20ml, of
a solvent mixture of 2-butanol R and chloroform
composed of equal volumes
R. Separate and combine the extracts, wash with 10 ml of water, extract the
washings with 5 ml of the above solvent mixture, and add to the combined
extracts. Evaporate the mixed extracts to dryness on a water-bath in a current
of air, dissolve the residue in 10 ml of ethanol (-750 g/1) TS, evaporate to dryness
on a water-bath and dry to constant weight at 105 °C. Each g of residue is equiv-
alent to 1.132g of C 2 oHioNa 205 .
Additional requirement for Fluorescein sodium for sterile use

429
FLUOROURACILUM
FLUOROURACIL
Molecular formula. C4H3FN2O2
Graphic formula.
Chemical name. 5-Fluorouracil; 5-fluoro-2,4(lFi,3/i)-pyrimidinedione; CAS

Reg. No. 51-21-8.
Storage. Fluorouracil should be kept in a tightly closed container, protected

from light.
Additional information. Fluorouracil melts at about 282 °C with decompo-

sition. CAUTION: Fluorouracil must be handled with care, avoiding contact
Requirements
Definition. Fluorouracil contains not less than 98.5% and not more than
101.0% of C4H3FN2O2, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from fluorouracil RS or with the reference spectrum of
fluorouracil.
430
B. The absorption spectrum of a lOpg/ml solution in acetate buffer, pH 4.7,

TS, when observed between 220 nm and 350 nm, is qualitatively similar to
that of a lO.Opg/ml solution of fluorouracil RS in acetate buffer, pH 4.7, TS
(a maximum occurs at about 266 nm and a minimum occurs at about
232 nm). The absorbances of the solutions at the maximum do not differ
from each other by more than 3%. The absorbance of a 1-cm layer at
266 nm is about 0.54.
C. Heat 0.5 ml of chromic acid TS in a small test-tube in a water-bath for 5

Add about 3 mg of the test substance arid again heat in a water-bath for 5
D. Dissolve 0.05g in 5ml of water, add 1 ml of bromine TSl; the colour of the
bromine is discharged.

from a commercial source is suitable) and a mixture of 70 volumes of ethyl
acetate R, 15 volumes of methanol R, and 15 volumes of water as the mobile
phase. Apply separately to the plate 5 pi of each of 2 solutions in a mixture of
equal volumes of water and methanol R containing (A) 20 mg of the test sub-
stance per ml and (B) 0.050 mg of fluorouracil RS per ml. After removing the
with solution B.
Fluorine content. Carry out the combustion as described under 2.4 Oxygen
flask method, using 7 mg of the test substance, and adding about 15 mg of
sodium peroxide R and 15ml of sodium hydroxide (0.1 mol/1) VS as the absorb-
ing liquid. When the process is complete, allow the flask to stand for not less
than 10 minutes with intermittent shaking, then dilute the contents to 100 ml
with water. Proceed with 5.0ml as described under 2.4 Oxygen flask method
for the determination of fluorine; not more than 55 pg of F.
431

mamide R, add 0,25ml of thymol blue/dimethylformamide TS and titrate with
tetrabutylammonium hydroxide (0.1 mol/1) VS to a blue endpoint as described
under 2.6 Non-aqueous titration, Method B. Each ml of tetrabutylammonium
hydroxide (0.1 mol/1) VS is equivalent to 13.01 mg of C 4 H 3 FN 2 O 2 .
Additional requirements for Fluorouracil for faarenteral use

FLUPHENAZINI DECANOAS
FLUPHENAZINE DECANOATE
Molecular formula. C32H44F3N3O2S
Graphic formula.
Chemical name. 4- [3-[2-(Trifluoromethyl)phenothiazin- 1 0-yl]propyl]- 1 -piper-

azineethanol decanoate (ester); 4-[3-[2-(tri£luoromethyl)-10i4'-phenothiazin-10-
yl]propyl]-l-piperazineethanol decanoate (ester); CAS Reg. No. 5002-47-1.
Description. A pale yellow viscous liquid or a yellow, crystalline, oily solid;

odour, faint, ester-like.
Miscibility. Immiscible with water; miscible with dehydrated ethanol R and

ether R.
432
Storage. Fluphenazine decanoate should be kept in a well-closed container,

Additional information. Even in the absence of light, Fluphenazine

decanoate is gradually degraded on exposure to a humid atmosphere, the
Requirements
Definition. Fluphenazine decanoate contains not less than 98.5% and not
more than 101.5% of C 32 H 44 F 3N 3 O 2 S, calculated with reference to the dried
substance.
Identity tests
infrared region.
spectrum obtained from fluphenazine decanoate RS or with the reference
sjaectrum of fluphenazine decanoate.

tt-tetradecane R and 95 volumes of hexane R to impregnate the plate,
has reached the top of the plate, remove the plate from the chromatographic
chamber and allow to stand at room temperature until the solvents have
completely evaporated. Use the impregnated plate immediately, carrying
out the chromatography in the same direction as the impregnation. As the
mobile phase, use a mixture of 90 volumes of methanol R and 10 volumes
of water. Apply separately to the plate 1 pi of each of 2 solutions in ethanol
(-750 g/1) TS containing (A) 20 mg of the test substance per ml and (B)
20 mg of fluphenazine decanoate RS per ml. After removing the plate from
C. Dissolve 5mg ml of sulfuric acid (-1760 g/1) TS and allow

in 2 to stand for
5 minutes; a reddish brown colour is produced.
Sulfated ash. Not more than 2,0mg/g.

exceeding 0.6kPa or about 5mm of mercury); it loses not more than lOmg/g.
433
80 volumes of acetone R, 30 volumes of cyclohexane R and 5 volumes of
of each of 2 solutions in methanol R containing (A) 25 mg of the test substance
per ml and (B) 0.25 mg of fluphenazine hydrochloride RS per ml. After remov-
examine the chromatogram in ultraviolet light (254 nm). Then spray the plate
with sulfuric acid (-635 g/1) TS and examine the chromatogram in daylight.
Using either method of visualization, any spot obtained with solution A, other
than the principal spot, is not more intense than that obtained with solution B.
Rl, and titrate with perchloric acid (0.1 mol/1) VS, as described under 2.6 Non-
alent to 25.59 mg of C 32 H 44F 3N 3 O 2 S.
Additional requirement for Fluphenazine decanoate for

parenteral use
FLUPHENAZINI ENANTAS
FLUPHENAZINE ENANTATE
Molecular formula. C29H38F3N3O2S
Graphic formula.

azineethanol heptanoate (ester); 4-[3-[2-(tri£luoromethyl)-10Ff-phenothiazin-10-
yl]propyl]-l-piperazineethanol heptanoate (ester); CAS Reg. No. 2746-81-8.
434
Description. A pale yellow, viscous liquid or a yellow, crystalline, oily solid;

odour, faint, ester-like.
Miscibility. Immiscible with water; miscible with dehydrated ethanol R, and

ether R.
Storage. Fluphenazine enantate should be kept in a well-closed container, pro-

tected from light.
Requirements
Definition. Fluphenazine enantate contains not less than 98.5% and not more
than 101.5% of C 29 H 38F N 3 O 2 S,
3 calculated with reference to the dried
substance.
Identity tests
spectrum obtained from fluphenazine enantate RS or with the reference sfrec-
truni of fluphenazine enantate.

«-tetradecane R and 95 volumes of hexane R to impregnate the plate,
has reached the top of the plate, remove the plate from the chromatographic
chamber and allow it to stand at room temperature until the solvents have
completely evaporated. Use the impregnated plate immediately, carrying
out the chromatography in the same direction as the impregnation. As the
mobile phase, use a mixture of 90 volumes of methanol R and 10 volumes
of water. Apply separately to the plate 1 pi of each of 2 solutions in ethanol
(-750 g/1) TS containing (A) 20 mg of the test substance per ml and (B)
20 mg of fluphenazine enantate RS per ml. After removing the plate from
C. Dissolve 5 mg in 2 ml of sulfuric acid (-1760 g/1) TS and allow to stand for

5 minutes; a reddish brown colour is produced.
435

80 volumes of acetone R, 30 volumes of cyclohexane R, and 5 volumes of
of each of 2 solutions in methanol R containing (A) 25 mg of the test substance
per ml and (B) 0.25 mg of fluphenazine hydrochloride RS per ml. After remov-
examine the chromatogram in ultraviolet light (254 nm). Then spray the plate
with sulfuric acid (-635 g/1) TS and examine the chromatogram in daylight.
Using either method of visualization, any spot obtained with solution A, other
than the principal spot, is not more intense than that obtained with solution B.

alent to 27.49 mg of C 29 H 38F 3N 3 O 2 S.
Additional requirement for Ftufhenazine enantate for

p>arenteral use
FLUPHENAZINI HYDROCHLORIDUM
FLUPHENAZINE HYDROCHLORIDE
Molecular formula. C22H26p3N30S,2HCl
Graphic formula.
N (CHJ.OH
/
2HCI
436

azineethanol dihydrochloride; 4-[3-[2-(trifluoromethyl)-10f/-phenothiazin-10-
yl]propyl]-l-piperazineethanol dihydrochloride; CAS Reg. No. 146-56-5.
Solubility. Soluble in 10 parts of water; sparingly soluble in ethanol (-750 g/1)

Storage. Fluphenazine hydrochloride should be kept in a well-closed con-

Additional information. Even in the absence of light, Fluphenazine

hydrochloride is gradually degraded on exposure to a humid atmosphere, the
Requirements
Definition. Fluphenazine hydrochloride contains not less than 98.5% and not
more than 101.5% of C22H26F3hl30S,2HCl, calculated with reference to the
dried substance.
Identity tests
infrared region.
spectrum obtained from fluphenazine hydrochloride RS or with the refer-
ence spectrum of fluphenazine hydrochloride.
B. Carry out the test in subdued light as described under 1.14.1 Thin-layer
of 15 volumes of formamide R, 5 volumes of 2-phenoxyethanol R, and 180
volumes of acetone R to impregnate the plate, dipping it about 5mm
beneath the surface of the liquid. After the solvent has reached the top of
the plate, remove the plate from the chromatographic chamber and allow
to stand at room temperature until the solvents have completely evaporated.
Use the impregnated plate immediately, carrying out the chromatography
in the same direction as the impregnation. As the mobile phase, use a
mixture of 2 volumes of diethylamine R and 100 volumes of light petroleum
R1 saturated with 2-phenoxyethanol R. Apply separately to the plate 2 pi of
each of 2 solutions in chloroform R containing (A) 2.0 mg of the test sub-
stance per ml and (B) 2.0 mg of fluphenazine hydrochloride RS per ml. After
removing the plate from the chromatographic chamber, allow it to dry in
air, and examine the chromatogram in ultraviolet light (365 nm), observing
437
the fluorescence produced after about 2 minutes. Spray the plate with sul-
furic acid/ethanol TS and examine the chromatogram in daylight. The prin-
cipal spot obtained with solution A corresponds in position, appearance, and
intensity with that obtained with solution B.
C. Dissolve 5mg in 5ml of sulfuric acid (-1760 g/1) TS; an orange colour is pro-
duced which becomes brownish red on warming.
D. Heat 0.5 ml of chromic acid TS in a small test-tube in a water-bath for 5

Add about 3 mg of the test substance and again heat in a water-bath for 5
E. A 0.05 g/ml solution yields reaction B described under 2.1 General identifi-
80 volumes of acetone R, 30 volumes of cyclohexane R and 5 volumes of
of each of 2 solutions in sodium hydroxide/methanol TS containing (A) lOmg
of the test substance per ml and (B) 0.10 mg of the test substance per ml. After

acid Rl, add 10 ml of mercuric acetate/acetic acid TS and titrate with perchloric
acid (0.1 mol/1) VS, as described under 2.6 Non-aqueous titration. Method A.
C22H26F3N30S,2HC1.
FUROSEMIDUM
FUROSEMIDE
438
Graphic formula.
Chemical name. 4-Chloro-N-furfuryl-5-sul£amoylanthranilic acid; 5-

(aminosulfonyl)-4-chloro-2-[(2-furanylmethyl)amino]benzoic acid; CAS Reg.
No. 54-31-9.

Category. Diuretic.
Storage. Furosemide should be kept in a well-closed container, protected from

light.
Requirements
Definition. Furosemide contains not less than 98.0% and not more than
101.0% of C12H11CIN2O5S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from furosemide RS or with the reference sp'ectrum of
furosemide.
B. Dissolve 5 mg in 10 ml of methanol R. Transfer 1 ml of this solution to a flask,
add 10 ml of hydrochloric acid (-70 g/1) TS and heat under a reflux condenser
for 15 minutes. Cool and add 15ml of sodium hydroxide (1 mol/1) VS and
5 ml of sodium nitrite (1 g/1) TS. Allow to stand for 3 minutes, then add 2 ml
of ammonium sulfamate (25 g/1) TS and mix. Add 1ml of Ai-(1 -naphthyl)
ethylenediamine hydrochloride (5 g/1) TS; a red-violet colour is produced.
C. Dissolve 25mg 2.5ml of ethanol (-750 g/1) TS and add, drop by drop,
in
about 2 ml of 4-dimethylaminobenzaldehyde TSl; a transient green colour
is produced, which becomes deep red.
439
4-Chloro-5-sulfamoylanthranilic acid. Dissolve 0.1 g in 25 ml of methanol

R. To 1ml add 3 ml of dimethylformamide R, 12 ml of water, and 1ml of
hydrochloric acid (1 mol/1) VS. Cool, add 0.5ml of sodium nitrite (lOg/1) TS,
shake, and allow to stand for 5 minutes. Add 1 ml of ammonium sulfamate
(25g/l) TS, shake, and allow to stand for 3 minutes. Add 1 ml of Ai-(1 -naphthyl)
ethylenediamine hydrochloride (5g/l) TS and sufficient water to produce 25 ml.
Measure the absorbance of a 1-cm layer of the resulting solution at a maximum
of about 530 nm, using as a blank a solution prepared by treating a mixture of
1 ml of methanol R and 3 ml of dimethylformamide R in a similar manner; the
absorbance is not greater than 0.12 (0.3% of free primary aromatic amines
expressed as 4-chloro-5-sulfamoylanthranilic acid) (preferably use 2-cm cells for
the measurement and calculate the absorbance of a 1 -cm layer).
1 volume of toluene R, 1 volume of xylene R, 3 volumes of dioxan R, 3 volumes
of 2-propanol R and 2 volumes of ammonia (-260 g/1) TS as the mobile phase.

Apply separately to the plate 5 pi of each of 2 solutions in acetone R contain-
ing (A) 20 mg of the test substance per ml and (B) 0.30 mg of the test substance
to dry in air, and examine the chromatogram in ultraviolet light (365 nm). Any

mamide R, add 3 drops of bromothymol blue/dimethylformamide TS, and
titrate with sodium hydroxide (0.1 mol/1) VS to a blue endpoint. Repeat the
operation without the substance being examined and make any necessary cor-
rections. Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to 33.08mg
of C 12 H CIN O
11 2 5 S.
Additional requirements for Furosemide for faarenteral use

terial endotoxins; contains not more than 3.6IU of endotoxin RS per mg.
440
GALLAMINI TRIETHIODIDUM
GALLAMINE TRIETHIODIDE
Molecular formula. C30H60I3N3O3
Graphic formula.
Chemical name. [v-Phenenyltris(oxyethylene)]tris[triethylammonium] triio-
dide; 2,2',2"-[l,2,3-benzenetriyltris(oxy)]tris[Ai,N,A/-triethylethanaminium tri-
iodide; l,2,3-tris(2-diethylaminoethoxy)benzene triethiodide; CAS Reg. No.

65-29-2.
Solubility. Very soluble in water; sparingly soluble in ethanol (~750g/l) TS;

Storage. Gallamine triethiodide should be kept in a tightly closed container,

Additional information. Gallamine triethiodide is hygroscopic.
Requirements
Definition. Gallamine triethiodide contains not less than 98.0% and not more
than 101.0% of C30H60I3N3O3, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from gallamine triethiodide RS or with the reference spec-
trum of gallamine triethiodide.
441
B. The absorption spectrum of a lOpg/ml solution in hydrochloric acid

(0.01 mol/1) VS, when observed between 220 nm and 350 nm exhibits a
maximum atabout 225 nm; the absorbance of a 1-cm layer at this wave-
length is between 0,50 and 0.55.
C. Dissolve 0.05 g in 5 ml of water and add 1ml of potassio-mercuric iodide

TS; a yellow precipitate is produced.
D. A 0.01 g/ml solution yields reaction A described under 2.1 General identifi-
cation tests as characteristic of iodides.
Clarity and colour of solution. A freshly prepared solution of 0.20 g in

10 ml of carbon-dioxide free water R is clear and not more intensely coloured
than standard colour solution Ywl when compared as described under 1.11
Colour of liquids.
Loss on drying. Dry to constant weight at 105°C; it loses not more than 15mg/g.
Acidity or alkalinity. To 50 ml of water add 0.2 ml of methyl red/ethanol TS

and adjust to pH 6 by adding either sulfuric acid (0.01 mol/1) VS or sodium
hydroxide (0.02 mol/1) VS until the colour is orange-yellow. Add l.Og of the
substance being examined and shake to dissolve; not more than 0.2 ml of either
sulfuric acid (0.01 mol/1) VS or sodium hydroxide (0,02 mol/1) VS is required to
restore the original orange-yellow colour.
chromatography, using cellulose R1 as the coating substance and a mixture of 17
volumes of glacial acetic acid R, 17 volumes of water and 66 volumes of 1-
butanol R as the mobile phase. Apply separately to the plate 10 pi of each of 2
solutions in ethanol (-750 g/1) TS containing (A) 5.0 mg of the test substance per
ml and (B) 0.05 mg of the test substance per ml. Allow the mobile phase to
ascend 10 cm. After removing the plate from the chromatographic chamber, dry
it in a current of warm air and spray it with potassium iodoplatinate TS. An elon-
gated blue spot, which may appear to be double, is obtained on the chro-
matogram from test solution A. Any spot above the principal spot obtained with
solution A is not more intense than the principal spot obtained with solution B.
Assay. Dissolve about 0.5 g, accurately weighed, in 40ml of acetone R,

and add 15ml of mercuric acetate/acetic acid TS. Titrate with perchloric acid
is equivalent to 29.72 mg of C30H60I3N3O3.
442
GELATINA
GELATIN
Chemical name. Gelatin; CAS Reg. No. 9000-70-8.
Description. Faintly yellow to amber-coloured sheets, flakes, granules, or

powder; practically odourless; in solution it has a slight, characteristic, bouil-
lon-like odour.
Solubility. Practically insoluble in most organic solvents. In cold water it

swells and softens, absorbing 5-10 times its own mass of water. After swelling,
soluble in hot water, in acetic acid (~300g/l) TS, and in a hot mixture of glyc-
erol R and water.
Category. Encapsulating agent; tablet binder; coating agent; suspending agent;

viscosity-increasing agent.
Storage. Gelatin should be kept in a well-closed container.
Additional information. These specifications do not necessarily apply to

gelatin for parenteral use or other particular application. Attention should be
paid to the microbiological quality since gelatin is of natural origin.
The type of gelatin may be distinguished by the following test:
Dissolve g in 100 ml of hot water. Place aliquots of 5ml into six separate test-
1
tubes and add 5 ml of a buffer to each tube, using buffers of pH 4.0, 5.0, 6.0,
7.0, 8.0, and 9.0 (citrate buffer, pH 4.0, TS, phosphate buffer, pH 4.0, TS, or
phthalate buffer, pH 4.0, TS; acetate buffer, pH 5.0, TS; phosphate/citrate
buffer, pH 6.0, TS or acetate buffer, pH 6.0, TS; phosphate buffer, pH 7.0, TS;
phosphate buffer, pH 8.0, TS or buffer borate, pH 8.0, TS; buffer borate, pH
9.0, TS). Cool the test-tubes and allow them to stand at 4°C for 24 hours; the
type of gelatin is recognized by the resulting opalescence - a maximum opales-
cence appearing at pH 5.0 indicates gelatin type B, while a maximum opales-
cence between pH 7.0 and pH 9.0 indicates gelatin type A.
Requirements
Definition. Gelatin is a purified protein obtained either by the partial acid
hydrolysis (type A) or by the partial alkali hydrolysis (type B) of animal colla-
gen. It can exist as a mixture of both types.
Identity tests
A. Dissolve 1 g in carbon-dioxide-free water R, heat to about 55 °C, and dilute
to 100ml with the same solvent. Keep the solution at this temperature
443
throughout the following test (retain the solution for test C): to 2 ml add
0.05ml of copper(II) sulfate (160g/l) TS, mix, and add 0.5ml of sodium
hydroxide (~80g/l) TS; a violet colour is produced.
B. Transfer 0.5g to a test-tube, add 10ml of water, and allow to stand for 10
minutes. Heat at 60 °C for 15 minutes and keep the tube in a vertical posi-
tion at 0°C for 6 hours. Invert the tube; the content does not immediately
flow out.
C. Acidify 2 ml of the solution prepared for test A and add 0.5ml of potassium
dichromate (lOOg/1) TS; a yellow precipitate is formed.
Arsenic. Use a solution of l.Og in a mixture of 2.5ml of sulfuric acid

(-1760 g/1) TS, 2.5 ml of nitric acid (~1000g/l) TS, and a slight excess of bromine
TSl, allow to stand for 30 minutes, and boil under a reflux condenser for 1
hour. Proceed with the test as described under 2.2.5 Limit test for arsenic; the
Odour and water-insoluble substances. Dissolve 1 g in 40ml of hot water;

no disagreeable odour is perceptible. Observe the solution through a layer of
2 cm; only a slight opalescence appears.
Sulfated ash. Use 2.0g; not more than 30mg/g.
Loss on drying. Weigh lOg and dry to constant mass at 105°C; it loses not
more than 150mg/g.
Sulfur dioxide. Dissolve 20 g in 150 ml of hot water using a round-bottom

flask with a long neck. Add 5ml of phosphoric acid (-1440 g/1) TS and 1 g of
sodium hydrogen carbonate R, and without delay connect the flask to a con-
denser. {Note. Excessive foaming can be reduced by adding a few drops of an
antifoaming agent.) Distil 50ml, allowing the distillate to be collected under a
50ml surface of iodine (0.05 mol/1) VS. Acidify the distillate with a few drops
of hydrochloric acid (-70 TS, add 2 ml of barium chloride (50 g/1) TS, and
g/1)
heat on a water-bath until the liquid is nearly colourless. If any, filter the pre-
cipitated barium sulfate, wash, ignite, and weigh. Repeat the procedure without
the Gelatin being examined and make any necessary corrections. The content
of barium sulfate isnot more than 109. 3mg, which corresponds to not more
than 1.5mg/g of sulfur dioxide.
444
GENTAMICINI SULFAS
GENTAMICIN SULFATE
Gentamicin sulfate (non-injectable)
Gentamicin sulfate, sterile
Chemical name. Gentamicin sulfate; CAS Reg. No. 1405-41-0.
Graphic formula.
Description. A white to cream-coloured powder; odourless.
Solubility. Soluble in water; practically insoluble in ethanol (-750 g/1) TS, and
ether R.
Storage. Gentamicin sulfate should be kept in a tightly closed container, pro-

tected from light.
Labelling. The designation sterile Gentamicin sulfate indicates that the sub-
stance complies with the additional requirements for sterile Gentamicin sulfate
Additional information. Gentamicin sulfate is hygroscopic. Even in the

absence of light, it is gradually degraded on exposure to a humid atmosphere,
Requirements
Definition. Gentamicin sulfate is the sulfate salt of gentamicin fractions Gi,
C and
2, Gia produced by the growth of Micromonosp’ora purpurea.
445
Gentamicin sulfate contains not less than 590 International Units of gentam-
icin per mg, calculated with reference to the anhydrous substance.
Identity tests
using silica gel R5 as the coating substance (a precoated plate from a com-
mercial source is suitable); shake together 1 volume of chloroform R, 1
volume of methanol R, and 1 volume of ammonia (-260 g/1) TS, allow to

separate and use the lower layer as the mobile phase. Apply separately to
the plate 1 pi of each of 2 solutions containing (A) 20 mg of the test sub-
stance per ml and (B) 20 mg of gentamicin sulfate RS per ml. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air, spray
it with triketohydrindene/pyridine/acetone TS, and heat it at 105°C for 2
minutes. Examine the chromatogram in daylight. The
3 principal spots
obtained with solution A correspond with the 3 principal spots obtained
with solution B.
B. A lOmg/ml solution yields reaction A described under 2.1 General identifi-

Specific optical rotation. Use a O.lOg/ml solution, and calculate with refer-
ence to the anhydrous substance: = -tl07 to -tl21 °.

pH value. pH of a 40 mg/ml solution, 3.5-5.5.
antibiotics, using either Bacillus pumilus (NCTC 8241; ATCC 14884), Bacillus sub-
(ATCC 6633), or Staphylococcus aureus (ATCC 6538P) as the test organism,
tilis
culture medium Cml with a final pH of 7.8, sterile phosphate buffer pH 8.0 TSl
or TS2, an appropriate concentration of gentamicin (usually between 2 and
590 lU per mg, calculated with reference to the anhydrous substance.
446
Additional requirements for Gentamicin sulfate for sterile use

Sterility. Complies with 3,2.2 Sterility testing of antibiotics, applying the
Additional requirements for Gentamicin sulfate for

p'arenteral use
gentamicin.
GLIBENCLAMIDUM
GLIBENCLAMIDE
Molecular formula. C 2 ,?H 2 sClN 305 S
Graphic formula.
Chemical name. l-[[p-[2-(5-Chloro-o-anisamido)ethyl]phenyl]sulfonyl]-3-cyclo-

hexylurea; 5-chloro-A/-[2-[4-[[[(cyclohexylamino)carbonyl]amino]sulfonyl]phenyl]
-ethyl]-2-methoxybenzamide; l-[4-[2-(5-chloro-2-methoxybenzamido)ethyl]-
phenylsulfonyl]-3-cyclohexylurea; CAS Reg. No. 10238-21-8.
Other name. Glyburide.

almost odourless.
Solubility. Practically insoluble in water and ether R; slightly soluble in ethanol

(-750 g/1) TS and methanol R.
447
Category. Antidiabetic agent.
Storage. Glibenclamide should be kept in a well-closed container.
Requirements
Definition. Glibenclamide contains not less than 98.5% and not more than
101.0% of C 2 .?H 28 C 1 N 305 S, calculated with reference to the dried substance.
Identity tests
A. Garry out the examination as described under 1.7 Spectrophotometry in the

infrared region.
spectrum obtained from glibenclamide RS or with the reference spectrum of
gUbenclamide.
B. The absorption spectrum of a O.lOmg/ml solution in hydrochloric

acid/methanol (0.01 mol/1) VS, when observed between 230 nm and 350 nm,
exhibits a maximum at about 300 nm and a less intense maximum at about
275nm; the absorbance of a Tcm layer at 300nm is about 0.63.
G. Melting temperature, about 172 °G.
under 2.2.3 Limit test for heavy metals. Procedure 3; not more than 20pg/g.
Loss on drying. Dry to constant weight at 105°G; it loses not more than
lOmg/g.
Related substances. Garry out the test as described under 1.14.1 Thin-layer
45 volumes of chloroform R, 45 volumes of cyclohexane R, 5 volumes of
ethanol (~750g/l) TS, and 5 volumes of glacial acetic acid R as the mobile phase.
Apply separately to the plate 10 pi of each of 2 solutions in chloroform R
Assay. Dissolve about 0.5g, accurately weighed, in 100ml of hot neutralized

ethanol TS, and titrate with carbonate -free sodium hydroxide (0.1 mol/1) VS, using
448
phenolphthalein/ethanol TS as indicator. Repeat the operation without the sub-

stance being examined and make any necessary correction. Each ml of carbonate-
free sodium hydroxide (0.1 mol/1) VS is equivalent to 49.40 mg of C 23 H 28 CIN 3 O 5 S.
GLUCOSUM
GLUCOSE
Glucose, anhydrous
Glucose monohydrate
Molecular formula. C 6 H 12 O 6 (anhydrous); C6Hi20(;,H20 (monohydrate).
Graphic formula.
n = 0 (anhydrous)
n = 1 (monohydrate)
Chemical name. a-D-Glucopyranose; CAS Reg. No. 492-62-6 (anhydrous), a-

D-Glucopyranose monohydrate; CAS Reg. No. 14431-43-7 (monohydrate).
Other name. Dextrose.
Description. Colourless crystals or a white, crystalline or granular powder;

odourless.
Solubility. Soluble in about 1 part of water; slightly soluble in ethanol

(-750 g/1) TS; more soluble in boiling water and boiling ethanol (-750 g/1) TS.
Category. Nutrient; fluid replenisher.
Storage. Glucose should be kept in a well-closed container.
Labelling. The designation on the container of Glucose should state whether

the substance is the monohydrate or is in the anhydrous form. If the material is
not intended for parenteral use a designation “for oral use only” should be added.
449
Additional information. Glucose has a sweet taste.
Requirements
Definition. Glucose contains not less than 99.0% and not more than 101.5%
of C6H12O5, calculated with reference to the anhydrous substance.
Identity tests
A. When heated it melts, swells up and burns, evolving an odour of burnt sugar.
B. Add a few drops of a 0.05g/ml solution to 5ml of hot potassio-cupric tar-

trate TS; a copious red precipitate is produced.
Specific optical rotation. Dissolve lO.Og in 50 ml of water, add 0.2 ml of

ammonia (~100g/l) TS, and sufficient water to produce 100 ml, and allow to
stand for 30 minutes. Calculate the result with reference to the anhydrous sub-
stance; [a]o = -1-52.5 to -1-53.0°.
metals content according to Method A; not more than 5 pg/g.
Arsenic. Use a solution of 10 g in 35ml of water and proceed as described

under 2.2.5 Limit test for arsenic; not more than 1 pg/g.


Limit test for sulfates; the sulfate content is not more than 0.2mg/g.
Less-soluble sugars and dextrins. Boil Ig with 30ml of ethanol (~710g/l)

TS and cool; a clear solution is produced.
Soluble starch. Dissolve 2.5 g in 25 ml of water, boil the solution for 1 minute,
cool and add 0.1 ml of iodine (0.1 mol/1) VS; no blue colour is produced.
Sulfites. Dissolve 2.5g in 25ml of water, add 0.1ml of iodine (0.1 mol/1) VS
and a few drops of starch TS; a blue colour is produced.

and not more intensely coloured than standard colour solution Gn3 when com-
450

Fischer method. Method A. For the anhydrous form use about Ig of the sub-
stance; the water content is not more than lOmg/g. For the monohydrate use
about 0.15g of the substance; the water content is not less than 70mg/g and
Acidity. Dissolve 5.0 g in 50 ml of carbon-dioxide-free water R and titrate with

carbonate-free sodium hydroxide (0.02 mol/1) VS, using phenolphthalein/
ethanol TS not more than 0.5 ml is required to obtain the midpoint
as indicator;
of the indicator (pink).
Assay. Dissolve about 0.10 g, accurately weighed, in 50 ml of water, add

25.0ml of iodine (0.05mol/l) VS and 10ml of sodium carbonate (50g/l) TS.
Allow to stand for 20 minutes in the dark and add 15ml of sulfuric acid
(~100g/l) TS. Titrate the excess of iodine with sodium thiosulfate (0.1 mol/1)
VS, using starch TS as indicator. Repeat the operation without the substance
being examined and make any necessary corrections. Each ml of iodine
(0.05 mol/1) VS is equivalent to 9.008 mg of C(jHi20e.
Additional requirements for Glucose for p>arenteral use

GLYCEROLI MONOSTEARAS
GLYCERYL MONOSTEARATE
C21H42O4
Chemical name. Monostearin; ocatadecanoic acid monoester with 1,2,3-

propanetriol; CAS Reg. No. 31566-31-1.
Description. A white or yellowish white, hard waxy mass or unctuous

powder or flakes; odourless or slight, agreeable, fatty odour.
Solubility. Practically insoluble in water; soluble in ether R, benzene R, and

ethanol (-750 g/1) TS at 60 ‘’C.
Category. Emulsifying agent; cream and ointment base.
451
Storage. Glyceryl monostearate should be kept in a tightly closed container,

Additional information. Glyceryl monostearate may contain a suitable

antioxidant. Self-emulsifying glyceryl monostearate contains additional emul-
sifying agents.
Requirements
Definition. Glyceryl monostearate is a mixture of mono-, di- and triglycerides
of stearic and palmitic acids.
Glyceryl monostearate contains not less than the equivalent of 35 0 . % of mono-

glycerides, calculated as C 20 H 40 O and
4, not more than the equivalent of 6.0%
of free glycerol.
Identity tests
A. Melting temperature, not lower than 55 °C.
B. Dip a strip of filter-paper in a freshly prepared solution containing 9.5ml of

sodium nitroprusside (8.5 g/1) TS and 0.5ml of piperidine R. Place the moist-
ened filter-paper into the evolving vapours over the mouth of the test-tube
containing 1 g of Glyceryl monostearate and heat with 2 ml of phosphoric
acid (-1440 g/1) TS; a deep blue colour develops on the paper.
C. Heat 2.5g with 40ml of potassium hydroxide/ethanol TSl on a water-bath

under reflux for 30 minutes. Add 30ml of water, evaporate the ethanol,
acidify the hot mixture with 15ml of hydrochloric acid (~70g/l) TS, cool,
and shake with 50 ml of ether R. Wash the ether layer with three quanti-
ties, each of a mixture of 5 ml of sodium chloride (400 g/1) TS and 5 ml of
water, dry the ether layer over anhydrous sodium sulfate R, and filter.
Evaporate the filtrate and dry the residue under reduced pressure at room
temperature. Melt the residue, introduce it into capillary tubes, and keep
the tubes for 24 hours in a well-closed container. Melting temperature, not

lower than 53 °C.
452
Assays. Transfer about 0.4 g, accurately weighed, to a glass-stoppered separat-

ing funnel, and dissolve in 50 ml of dichloromethane R. Add 25ml of water and
shake vigorously for 1 minute. Allow the layers to separate. If an emulsion
is formed, add a few drops of glacial acetic acid R. Carry out the extraction
three more times using 25ml, 20ml, and 20ml of water. Combine the
dichloromethane extracts and use them in the assay for monoglycerides. Filter the
aqueous layers through a filter-paper moistened with water, wash the filter with
two quantities, each of 5 ml, of water, and dilute the combined filtrates and wash
liquids to 100 ml with water. Use this solution for the assay for free glycerol.
Free glycerol
Place 50ml of the prepared aqueous solution in a 500-ml ground-glass-
stoppered conical flask, add 25ml of periodic-acetic acid TS, and shake cau-
Allow to stand at a temperature between 25 and 30 °C for 30 minutes.

tiously.
Add 100 ml of water and 25ml of potassium iodide (80g/l) TS. Titrate with
sodium thiosulfate (0.1 mol/1) VS, using 1ml of starch TS as indicator added
towards the end of the titration. Repeat the procedure without the Glyceryl
monostearate being examined and make any necessary corrections.
Each ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to 2.3mg of glycerol.
Monoglycerides
Filter thecombined dichloromethane extracts through a plug of cotton-wool.
Wash the separating funnel and the filter with three quantities, each of 5ml, of
dichloromethane R, and dilute the filtrate to 100 ml with dichloromethane R.
Carry out the assay as described for free glycerol, using 50 ml of the
dichloromethane solution.
Each ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to 17.2 mg of mono-

glycerides, calculated as C 20 H 40 O 4 .
GLYCEROLUM
GLYCEROL
CH2 — OH
CH — OH
CH2 — OH
CsHaOa
453
Chemical name. Glycerol; 1,2,3-propanetriol; CAS Reg. No. 56-81-5.
Other name. Glycerin.
Description. A clear, colourless or almost colourless, syrupy liquid; odourless.
Miscibility. Miscible with water and ethanol (-750 g/1) TS; slightly miscible
with acetone R; practically immiscible with ether R.
Category. Solvent; humectant.
Storage. Glycerol should be kept in a tightly closed container.
Additional information. Glycerol is hygroscopic.
Requirements
Glycerol contains not less than 95 0 . % and not more than the equivalent of
101 0 % of
. C 3H 8O 3 ,
Identity tests
A. Impregnate a piece of filter-paper with alkaline potassio-mercuric iodide TS;
place it over a test-tube containing 1ml of Glycerol with 2g of potassium
hydrogen sulfate R and heat; the paper turns black.
B. Mix 2g with 10 ml of water and add 1 drop of phenolphthalein/ethanol TS;

the solution remains colourless. Add 1 drop of methyl red/ethanol TS; the
colour changes to yellow.
C. Mix 1 ml with 0.5ml of nitric acid (-1000 g/1) TS and superimpose 0.5ml of
potassium dichromate (100 g/1) TS; a blue ring is produced at the interface
of the two liquids. AUow to stand for 10 minutes; the blue colour does not
diffuse into the lower layer.
Refractive index. ttS = 1.470 - 1.475.
Relative density, dll = 1.258 - 1.263.
Chlorides. Use 5g in a mixture of 2ml of nitric acid (~130g/l) TS and 20ml

of water, and proceed as described under 2.2.1 Limit test for chlorides, using
1.0 ml of hydrochloric acid CITS; the chloride content is not more than lOpg/g.
454
Sulfates. Use 24 g and proceed as described under 2.2.2 Limit test for sulfates;
the sulfate content is not more than 20 pg/g.
Clarity and colour of solution. Mix 25 g with sufficient water to produce

50 ml; the solution is clear. Dilute 10 ml of this solution to 25 ml with water;
the solution is colourless.
Chlorinated compounds. Place about 5g, accurately weighed, in a dry

round-bottomed 100-ml flask, add 15ml of morpholine R, and connect to a suit-
able reflux condenser. Reflux gently for 3 hours. Rinse the condenser with
10 ml of water, adding the washings back into the flask, and cautiously acidify
with nitric acid (~1000g/l) TS. Transfer the solution to a suitable comparison
tube, add 0.5ml of silver nitrate (0.1 mol/1) VS, dilute with water to exactly
50ml, and mix thoroughly; the turbidity is not more pronounced than that of
a solution prepared similarly but without refluxing and to which 0.2ml of
hydrochloric acid (0.02 mol/1) VS has been added (30 pg of Cl/g).
Acidity. Dilute 25 g to 50ml with carbon-dioxide-free water R and add 0.5ml

of phenolphthalein/ethanol TS; the solution remains colourless. Titrate with
carbonate-free sodium hydroxide (0.1 mol/1) VS; not more than 0.2 ml is
required to obtain a pink colour. (Keep the solution for “Fatty acids and esters”.)
Fatty acids and esters. To the above solution, add 5ml of carbonate-free
sodium hydroxide (0.5 mol/1) VS, boil the mixture for 5 minutes, cool, add phe-
nolphthalein/ethanol TS, and with hydrochloric acid (0.5 mol/1) VS.
titrate
Repeat the procedure without the Glycerol being examined and make any nec-
essary corrections. Not more than 1.0ml of carbonate-free sodium hydroxide
(0.5 mol/1) VS is consumed.
Aldehydes and reducing substances. Transfer 5ml of Glycerol to a glass-

stoppered flask, mix with 10ml of water and 1ml of fuchsin/sulfurous acid
TS. Allow the mixture to stand in the dark for 1 hour; the colour of the solu-
tion is not more intense than that of a solution of potassium permanganate
(0.0002mol/l) VS.

Fischer method. Method A, using 1 .5 g of Glycerol; the water content is not
more than 20 mg/g.
Assay. Transfer about 0.4 g, accurately weighed, to a 600-ml beaker, dilute

with 50 ml of water, add bromothymol blue/ethanol TS, and acidify with sul-
furic acid (0.1 mol/1) VS to a green or greenish yellow colour. Neutralize with
sodium hydroxide (0.05 mol/1) VS to a definite blue endpoint, showing no green
tinge. Prepare a reagent blank containing 50ml of water, and neutralize in the
455
same manner. Pipette 50 ml of sodium metaperiodate TS into each beaker, swirl

gently to mix, cover with a watch-glass, and allow to stand for 30 minutes at
room temperature (not exceeding 35 °C). Dilute each solution with water to
about 300 ml and, using a pH-meter, titrate with sodium hydroxide (0.1 mol/1)
VS to pH 8.1 + 0.1 for Glycerol and pH 6.5 + 0.1 for the blank. Make any nec-
essary corrections for the blank.
Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to 9.210mg of

G,^Hg 03 .
GLYCEROLUM 85% m/m

GLYCEROL 85% m/m
Other name. Dilute glycerol.
Description. A clear, colourless or almost colourless, syrupy liquid; odourless.
Miscibility. Miscible with water and ethanol (-750 g/1) TS; slightly miscible
with acetone R; practically immiscible with ether R.
Storage. Glycerol 85% m/m should be kept in a tightly closed container.
Additional information. Glycerol 85% m/m is hygroscopic.
Requirements
Definition. Glycerol 85% m/m is a mixture of glycerol and water.
Glycerol 85% m/m contains not less than 83.5% m/m and not more than the
equivalent of 88.5% m/m of CsHgOg, calculated with reference to the anhy-
drous substance.
Identity tests
A. Impregnate a piece of filter-paper with alkaline potassio-mercuric iodide TS;
placeit over a test-tube containing 1ml of Glycerol 85% m/m with 2g of
potassium hydrogen sulfate R and heat; the paper turns black.
B. Mix 2g with 10 ml of water and add 1 drop of phenolphthalein/ethanol TS;

the solution remains colourless. Add 1 drop of methyl red/ethanol TS; the
colour changes to yellow.
456
C. Mix 1 ml with 0.5ml of nitric acid (~1000g/l) TS and superimpose 0.5ml of

potassium dichromate (lOOg/1) TS; a blue ring is produced at the interface
of the two liquids. Allow to stand for 10 minutes; the blue colour does not
diffuse into the lower layer.
Refractive index, nff = 1.449 - 1.455.
Relative density. 1^20 = 1.219 - 1.230.
metals content according to Method A; not more than 5 pg/g.
Chlorides. Use 5g in a mixture of 2ml of nitric acid (~130g/l) TS and 20ml

of water, and proceed as described under 2.2.1 Limit test for chlorides, using
1.0 ml of hydrochloric acid CITS; the chloride content is not more than lOpg/g.
Sulfates. Use 24 g and proceed as described under 2.2.2 Limit test for sulfates;
the sulfate content is not more than 20pg/g.
Clarity and colour of solution. Mix 25 g with sufficient water to produce

50 ml; the solution is clear. Dilute 10 ml of this solution to 25 ml with water;
the solution is colourless.
Chlorinated compounds. Place about 5g, accurately weighed, in a dry

round-bottomed 100-ml flask, add 15ml of morpholine R, and connect to a suit-
able reflux condenser. Reflux gently for 3 hours. Rinse the condenser with
10 ml of water, adding the washings back into the flask, and cautiously acidify
with nitric acid (-lOOOg/1) TS. Transfer the solution to a suitable comparison
tube, add 0.5ml of silver nitrate (0.1 mol/1) VS, dilute with water to exactly
50ml, and mix thoroughly; the turbidity is not more pronounced than that of
a solution prepared similarly, but without refluxing and to which 0.2 ml of
hydrochloric acid (0.02 mol/1) VS has been added (30 pg of Cl/g).
Acidity. Dilute 25 g to 50.0 ml with carbon-dioxide-free water R and add

0.5ml of phenolphthalein/ethanol TS; the solution remains colourless. Titrate
required to obtain a pink colour. (Keep the solution for “Fatty acids and esters’’.)
Fatty acids and esters. To the above solution, add 5 ml of carbonate-free

sodium hydroxide (0.5 mol/1) VS, boil the mixture for 5 minutes, cool, add
phenolphthalein/ethanol TS, and titrate with hydrochloric acid (0.5mol/l) VS.
Repeat the procedure without the Glycerol 85% ni/m being examined and make
any necessary corrections. Not more than 1.0 ml of carbonate-free sodium
hydroxide (0.5mol/l) VS is consumed.
457
Aldehydes and reducing substances. Transfer 5 ml of Glycerol 85% ni/m to

a glass-stoppered flask, mix with 10 ml of water and 1 ml of fuchsin/sulfurous
acid TS. Allow the mixture to stand in the dark for 1 hour; the colour of the
solution isnot more intense than that of a solution of potassium permanganate
(0.0002mol/l) VS.

Fischer method, Method A, using 0.2 g of Glycerol 85% m/m\ the water content
is not less than 0.12g/g and not more than 0.15g/g.
Assay. Transfer about 0.4 g, accurately weighed, to a 600-ml beaker, dilute

with 50 ml of water, add bromothymol blue/ethanol TS, and acidify with sul-
furic acid (0.1 mol/1) VS to a green or greenish yellow colour. Neutralize with
sodium hydroxide (0.05 mol/1) VS to a definite blue endpoint, showing no green
tinge. Prepare a reagent blank containing 50ml of water, and neutralize in the
same manner. Pipette 50 ml of sodium metaperiodate TS into each beaker, swirl
gently to mix, cover with a watch-glass, and allow to stand for 30 minutes at
room temperature (not exceeding 35 °G). Dilute each solution with water to
about 300 ml and, using a pH-meter, titrate with sodium hydroxide (0.1 mol/1)
VS to pH 8.1 + 0.1 for Glycerol 85% m/m and pH 6.5 ± 0.1 for the blank. Make
any necessary corrections for the blank.
Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to 9.210mg of

CsHaOs.
GRISEOFULVINUM
GRISEOFULVIN
Molecular formula. Ci/Hi/ClOe
Graphic formula.
458
Chemical name. 7-Chloro-2',4,6-trimethoxy-6'|3-methylspiro[benzo£uran-

2(3W),r-[2]cyclohexene]-3,4'-dione; (rS-fMK.s)-7-chloro-2',4,6-trimethoxy-6'-
methylspiro[benzofuran-2(3£7),r-[2]cyclohexene]-3,4'-dione; CAS Reg. No.
126-07-8.
Description. White to pale cream powder; almost odourless.
TS; freely soluble in tetrachloroethane R.
Category. Antifungal.
Storage. Griseofulvin should be kept in a well-closed container.
Additional information. The particles of Griseofulvin are generally up to

5 pm in maximum dimension, although occasionally larger particles may be
present that exceed 30 pm.
Requirements
Definition. Griseofulvin contains not less than 97.0% and not more than
102.0% of C17H17CIO6, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from griseofulvin RS or with the reference spectrum of
griseofulvin.

ethylmethylketone R and 1 volume of xylene R as the mobile phase. Apply
separately to the plate 10 pi of each of 2 solutions in chloroform R contain-
ing (A) 0.50 mg of the test substance per ml and (B) 0.50 mg of griseofulvin
RS per ml. After removing the plate from the chromatographic chamber,
C. Dissolve 5 mg in 1 ml of sulfuric acid (-1760 g/1) TS and add 5 mg of pow-
dered potassium dichromate R; a wine-red colour is produced.
459
Specific optical rotation. Use a 10 mg/ml solution in dimethylformamide R;

[a]g>"'= = +354 to+364°.
Particle size. In a mortar grind 10 mg with 10 drops of hydroxyethylceUulose

TS, add a further 3.50 ml of hydroxyethylceUulose TS and grind again. Trans-
fer adrop of the suspension to a suitable counting chamber 0.10 deep, place mm
a cover glass over it, and examine under a microscope 10 fields of vision of
0.04 mm^ area each, using a magnification of 600x; not more than 30 crystals
larger than 5 pm are visible in any field of vision.
Solution in dimethylformamide. A solution of 0.75g in 10 ml of dimethyl-

formamide R is clear and not more intensely coloured than standard colour
Matter soluble in light petroleum. Reflux l.Og with 40ml of light petro-
leum R for 10 minutes, cool and filter. Wash the flask 3 times with 10ml of
light petroleum R, filter, evaporate the combined filtrates on a water-bath, and
dry at 105 °C for 1 hour; the weight of the residue does not exceed 2.0 mg.
lOmg/g.
Acidity. Dissolve 0.25 g in 20 ml of neutralized ethanol TS and titrate with car-

bonate-free sodium hydroxide (0.02 mol/1) VS, phenolphthalein/ethanol TS
being used as indicator; not more than 1.0 ml is required to obtain the midpoint
Assay. Dissolve about O.lOg, accurately weighed, in sufficient dehydrated

ethanol R to produce 200ml and dilute 2ml of this solution to lOOml with
dehydrated ethanol R. Determine the absorbance of this solution in a 1-cm
layer at the maximum at about 291 nm and calculate the content of C17H17CIO6,
using the absorptivity value of 68.6 (£1°™ = 686).
GUMMI ARABICUM
ACACIA
Chemical name. Gum arabic; CAS Reg. No. 9000-01-5.
Description. Colourless or light yellowish brown, translucent or somewhat

opaque spheroidal tears or angular fragments with numerous fissures on the
460
surface; very brittle; fractured surfaces are glassy and occasionally iridescent;
odourless; tasteless and mucilaginous.
Solubility. Very slowly soluble in twice its mass of water, leaving only a very
small residue of vegetable particles; practically insoluble in ethanol (-750 g/1)
TS and ether R.
Category. Viscosity-increasing agent; emulsifying agent; suspending agent;

micro-encapsulating agent.
Storage. Acacia should be kept in a well-closed container, and stored in a cool

place.

purity of Acacia since it is of natural origin. It should be enzyme-free.
Requirements
Definition. Acacia is the air-hardened, gummy exudate from the stem and
branches oi Acacia Senegal (L.) Willdenow or other species ot Acacia of African
origin; it contains mainly polymers of salts of arabic acid.
Macroscopical examination. Spheroidal, oval or reniform pieces, the diam-

eter varying from 1-3 cm, white, yellowish white, yellow, or pale amber, some-
times with a pinkish tint, translucent or somewhat opaque, friable, frequently
with a cracked surface, easily broken into transparent angular fragments with
a glassy appearance and occasionally iridescent. Acacia also occurs as white to
yellowish white, thin flakes, powder, or fine granules.
Microscopical examination. The flakes appear as colourless striated frag-

ments. The powder presents colourless, bright, angular, irregular fragments
with only traces of starch or vegetable tissues visible. Stratified membrane is
not apparent. The granules appear as colourless, glassy, angular, irregular frag-
ments up to 100 pm in thickness, some of which exhibit parallel linear streaks.
Identity tests {Note: Powder the material before performing the tests.)
A. Dissolve Ig in 2 ml of water, add 2 ml of ethanol (-750 g/1) TS, and shake;

a white, gelatinous mucilage is formed which becomes fluid on adding
10 ml of water.
B. Dissolve 0.2 g in 10 ml of water and add 4 drops of lead subacetate TS; a

white, flocculent or curdy precipitate is instantaneously formed.
Starch and dextrin. Dissolve Ig in 10ml of water, boil and cool, then add
0.1ml of iodine (0.05 mol/1) VS; no blue or reddish brown colour develops.
461
Sucrose and fructose. Dissolve 0.3g in 5ml of water and add 0.1 g of resor-
cinol R and 2 ml of hydrochloric acid (-420 g/1) TS. Heat on a water-bath for 1
minute; no yellow or pink colour develops.
Tannin. Dissolve 1 g in 10ml of water and add 0.2ml of ferric chloride (65 g/1)
TS; a gelatinous precipitate is formed, but neither the precipitate nor the liquid
shows a dark blue colour.
Solubility in water and acidity. Dissolve 1 g in 2 ml of water; the solution
flows readily and is acid when tested with pH-indicator paper R.
Insoluble matter. To 5g, add 100ml of water and 15 ml of hydrochloric acid

(~70g/l) TS, and, while shaking frequently, boil gently for 15 minutes. Filter the
hot mixture through a tared sintered-glass crucible, wash the residue with hot
water, dry at 105 °C for 1 hour, and weigh; the residue weighs not more than
50 mg (1%).
0.15g/g.
HALOPERIDOLUM
HALOPERIDOL
Molecular formula. C21H23CIFNO2
Graphic formula.
Chemical name. 4-[4-(^-Chlorophenyl)-4-hydroxypiperidino]-4'-fluorobuty-

rophenone; 4-[4-(4-chlorophenyl)-4-hydroxy-l-piperidinyl]-l-(4-fluorophenyl)-
1-butanone; CAS Reg. No. 52-86-8.
Description. A white to faintly yellowish, amorphous or microcrystalline

powder; odourless.
462

(-750 g/1) TS and in 200 parts of ether R.
Storage. Haloperidol should be kept in a well-closed container, protected from

light.
Requirements
Definition. Haloperidol contains not less than 98.0% and not more than
101.0% of C21H23CIFNO2, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from haloperidol RS or with the reference spectrum of
haloperidol.
B. The absorption spectrum of a 15|j.g/ml solution in a mixture of 1 volume

of hydrochloric acid (1 mol/1) VS and 99 volumes of methanol R, when
observed between 230 nm and 350 nm, exhibits a maximum at about

245 nm; the absorbance of a 1-cm layer at this wavelength is between 0.49
and 0.53 (preferably use 2-cm cells for the measurement and calculate the
absorbance of a 1-cm layer).
C. Carry out the combustion as described under 2.4 Oxygen flask method,
using 20 mg of the test substance and a mixture of 3 ml of sodium hydrox-
ide (~80g/l) TS and 2 ml of water as the absorbing liquid. When the process
is complete, dilute to 10 ml with water; the resulting solution complies with
the following tests:
{a) Add 0.1 ml to a mixture of 0.1 ml of a freshly prepared sodium alizarin-

sulfonate (lg/1) TS and 0.1ml of zirconyl nitrate TS; the red colour of
the solution changes to clear yellow.
{b) Acidify 5 ml with sulfuric acid (-100 g/1) TS and boil gently for 2 minutes;
the solution yields reaction A described under 2.1 General identification
463

is preferable) and a mixture of 80 volumes of chloroform R, 10 volumes of
glacial acetic acid R and 10 volumes of methanol R as the mobile phase. Apply
separately to the plate 10 pi of each of 3 solutions in chloroform R containing
(A) 10 mg of the test substance per ml, (B) 0.050 mg of the test substance per
ml, and (C) 0.10 mg of the test substance per ml. After removing the plate from
the chromatographic chamber, allow it to dry in air, spray with potassium
iodobismuthate TS2, and examine the chromatogram in daylight. Any spot

than that obtained with solution B. Not more than one of any such spots is
more intense than the spot obtained with solution C.

R1 and titrate with perchloric acid (0.1 mol/1) VS, determining the endpoint
potentiometrically as described under 2.6 Non-aqueous titration, Method A.
C2,H23C1FN02.
Additional requirements for Hatop>eridol for parenteral use

HALOTHANUM
HALOTHANE
Molecular formula. C2HBrClF3
Graphic formula.
CICHCF,
I
Br
Chemical name. 2-Bromo-2-chloro-l,l,l-trifluoroethane; CAS Reg. No.

151-67-7.
464
.
Description. A colourless, mobile, heavy liquid; odour, characteristic, resem-

bling that of chloroform.
TS, ether R, and trichloroethylene R.
Storage. Halothane should be kept in a well-closed container, protected from

light, and stored at a temperature not exceeding 25 °C.
Additional information. Halothane is a noninflammable liquid. Halothane

contains not less than 0.08 mg/g and not more than 0.12mg/g of thymol, as a
stabilizer.
Requirements
Identity tests
To a test-tube, transfer 2ml of rert. -butanol R, 0.1ml of the test liquid, 1ml of
copper edetate TS, 0.5ml of ammonia (-260 g/1) TS and 2ml of hydrogen per-
oxide (-60 g/1) TS; this constitutes solution 1. Similarly, prepare a blank without
the test liquid; this constitutes solution 2. Place both tubes in a water-bath at
50 °C for 15 minutes, cool and add 0.3 ml of glacial acetic acid R.
A. To 1 ml of each of solutions 1 and 2 add 0.5 ml of a mixture of equal volumes
of sodium alizarinsulfonate (1 g/1) TS and zirconyl nitrate TS; a red colour

is produced in solution 2 and a yellow colour in solution 1
B. To 1 ml of each of solutions 1 and 2 add 1 ml of a mixture of 1.02 g of potas-
sium hydrogen phthalate R dissolved in 30 ml of sodium hydroxide

(0.1 mol/1) VS and diluted to 100ml with water (= buffer pH 5.2). Then add
{a) 1ml of phenol red/ethanol TS that has been previously diluted with an
equal volume of water and {b) 0.1ml of tosylchloramide sodium (15g/l)

TS; a yellow colour is produced in solution 2 and a bluish red colour in
solution 1.
C. To 2 ml of each of solutions1 and 2 add 0.5ml of sulfuric acid (-570 g/1) TS,
0.5ml of acetone R and 0.2 ml of potassium bromate (50 g/1) TS. Shake, then
place in a water-bath at 50 °C for 2 minutes. Cool, add 0.5ml of a mixture
of equal volumes of nitric acid (-lOOOg/1) TS and water, and 0.1 ml of silver
nitrate (40 g/1) TS; solution 2 remains clear and an opalescence is produced
in solution 1, which changes to a white precipitate after a few minutes.
Mass density, P 20 = 1.865-1. 875 g/ml.
465
Free halides. Shake 10ml with 20ml of carbon-dioxide-free water R for 3

minutes. To 5 ml of the aqueous layer add 5 ml of water, 1 drop of nitric acid
(~1000g/l) TS, and 0.2 ml of silver nitrate (40g/l) TS; no opalescence is pro-
duced. (Keep the remaining aqueous layer for the test of free halogens).
Free halogens. To 10ml of the aqueous layer obtained from the test for free
halides add 1ml of potassium iodide/starch TS; no blue colour is produced.
Acidity or alkalinity. Shake 20 ml with 20ml of carbon-dioxide-free water R

for 3 minutes. To the aqueous layer add a few drops of bromocresol
purple/ethanol TS; not more than 0.1ml of sodium hydroxide (0.01 mol/1) VS
or 0.6ml of hydrochloric acid (0.01 mol/1) VS is required to obtain the midpoint
of the indicator (grey).
Thymol. Use 3 dry 25-ml stoppered cylinders and place in the first one 0.5ml
of the test liquid, in the second one 0.5ml of thymol TS2, and in the third one
0.5 ml thymol TS3. To each of the 3 cylinders add 5 ml of carbon tetrachlo-
of
ride R and 5.0ml of titanium dioxide/sulfuric acid TS. Shake the cylinders vig-
orously for 30 seconds and allow to stand until the layers have separated. When
viewed transversally the yellowish-brown colour of the lower layer obtained
in the cylinder containing the test liquid is intermediate in intensity between
the colours of the corresponding layers in the other 2 cylinders (0.08-0.12mg/g
of thymol).
Related substances. Carry out the test as described under 1.14.5 Gas chro-
matography, using 3 solutions (1) trichlorotrifluoroethane TS serving as an
internal standard, (2) the test liquid, and (3) the test liquid containing 0.05 pi of
trichlorotrifluoroethane R per ml.
For the procedure use a glass column 2.75 m long and 5.0 mm in internal diame-
ter,the first 1.8 m of which are packed with an adequate quantity of an adsorbent
composed of 30 g of macrogol 400 R supported on 70 g of pink firebrick R, and the
remainder with an adequate quantity of an adsorbent composed of 30 g of
dinonyl phthalate R supported on 70 g of pink firebrick R. Maintain the column at
50 °C, use nitrogen R as the carrier gas and a flame ionization detector.
In the chromatogram obtained with solution 3, the peak area due to the
trichlorotrifluoroethane is greater than the total area of any other peaks except
that due to the halothane; in calculating the peak area due to the trichlorotri-
fluoroethane, allowance may
be necessary for any impurities having the same
retention time as revealed in the chromotogram obtained from solution 2.
466
HEPARINUM CALCICUM
HEPARIN CALCIUM
Chemical name. Heparin calcium; CAS Reg. No. 37270-89-6.
Solubility. Freely soluble in water.
Storage. Heparin calcium should be kept in a tightly closed container.
Labelling. The designation Heparin calcium for parenteral use indicates that the
enteral administration. The label should also state the name and quantity of any
added substances, and the source of the material (lung or mucosal). Expiry date.
Additional information. Heparin calcium may be prepared from the lungs of

oxen or the intestinal mucosae of oxen, pigs, or sheep. Attention should be paid
to minimize or eliminate microbiological contamination and substances low-
ering blood pressure. Heparin calcium is moderately hygroscopic.
Requirements
Definition. Heparin calcium is a preparation containing the calcium salt of a
sulfated glucosaminoglycan present in mammalian tissues. It has the charac-
teristic property of delaying the clotting of fresh blood.
Heparin calcium intended for the manufacture of a parenteral dosage form con-
tains not less than 150IU per mg, and Heparin calcium not intended for use
in the manufacture of a parenteral dosage form contains not less than 120IU
per mg, both calculated with reference to the dried substance.
Identity tests
A. Delays the clotting of fresh blood.
B. Specific optical rotation, use a 40mg/ml solution; [a]n = not less than
-t35°.
C. A 20mg/ml solution yields the reactions described under 2.1 General iden-
467
Calcium. Proceed with about 0.2 g, accurately weighed, as described under 2.5
Complexometric titrations for calcium. Each ml of disodium edetate (0.05 mol/1)
VS is equivalent to 2.004
mg of Ca; 95-115mg/g, calculated with reference to
the dried substance. (As an alternative, determine the content of calcium by
atomic absorption spectrophotometry under 1.8 Atomic spectrometry: emis-
sion and absorption.)
Nitrogen. Carry out Method A as described under 2.10 Determination of

nitrogen, using about 0.1 g, accurately weighed, and 5 ml of nitrogen-free sul-
furic acid (~1760g/l) TS. Each ml of sulfuric acid (0.05mol/l) VS is equivalent
to 1.401 mg of nitrogen; not more than 25mg/g, with reference to the dried
substance.
Protein and nucleotidic impurities. Measure the absorbance of a 1-cm layer

of a 4 mg/ml solution at a wavelength of 260 nm and 280 nm; at 260 nm not
greater than 0.20 and at 280 nm not greater than 0.15.
Clarity and colour of solution. A solution containing 5000 lU per ml is clear

Sulfated ash. Use 0.2 g; 0.32-0.40g/g, with reference to the dried substance.

exceeding 0.5 kPa or about 5mm of mercury) over phosphorus pentoxide R; it
loses not more than 0.080 g/g.
pH value. pH of lOmg/ml solution in carbon-dioxide-free water R, 5.5-8. 0.
Assay. The anticoagulant activity of heparin is determined in vitro using a bio-

logical assay to compare its ability to delay the clotting of recalcified citrated
sheep plasma with that of the reference substance. The following method is
suitable for carrying out the assay (other methods may also be applicable).
The onset of clottingdetermined either as a change in optical density (by

is
direct visual inspection, preferably using indirect illumination against a matt

black background, or by spectrophotometry, recording at a wavelength of
approximately 600 nm) or as a change in fluidity (by visual detection while
manually tilting the tube or by mechanical recording while stirring, taking care
to cause the minimum disturbance of the solution during the initial phase of
clotting). Use appropriate tubes according to the chosen technique.
468
Prepare a solution of Heparin calcium and a solution of heparin RS in sodium

chloride (9g/l) TS, each containing an accurately known number of lU of heparin
per ml. Using sodium chloride (9g/l) TS prepare from each solution a series of
dilutions in geometric progression such that the clotting time obtained with the
lowest concentration is not less than 1.5 times the blank recalcification time, and
the clotting time obtained with the highest concentration is such as to give a sat-
isfactory log dose-response curve, as determined in a preliminary test.
Place in an ice-bath 12 labelled tubes for each dilution: T,, T2, T3, etc. for
Heparin calcium and S], S2, S3, etc. for heparin RS. To each tube add 1.0ml of
thawed plasma substrate R and 1.0 ml of the appropriate dilution, either from
Heparin calcium or heparin RS, mixing each tube carefully, and not allowing
bubbles to form. (The detection technique employed may require the addition
of different volumes of plasma substrate, consequently the appropriate adjust-
ment of all tubes would be needed.) Transfer all the tubes to a water-bath at
37 °C, and allow to equilibrate for about 15 minutes. Add to each tube, mixing
after each addition, 1 ml of a dilution of cephalin TS and 1 ml of kaoHn sus-
pension TS freshly prepared just before use. (A suitable dilution of cephalin TS
is one that, under the conditions of the assay, gives a blank recalcification time
of not more than 60 seconds.) After exactly 2 minutes, add 1.0ml of calcium
chloride (3.7 g/1) TS. Record in seconds the interval between this addition and
the onset of clotting, determined according to the chosen technique. Similarly
determine the blank recalcification time at the beginning and at the end of the
procedure, using 1.0 ml of sodium chloride (9 g/1) TS in place of one of the
heparin dilutions; the two values for the blank should not differ significantly.
Repeat the procedure using fresh dilutions of the initial solutions and carrying
out the incubation in the reverse order (first tubes S, then tubes T).
Transform the clotting times to logarithms using the mean values for the dupli-
cate tubes and calculate the results by standard statistical methods.
Carry out not fewer than 3 independent assays. For each assay prepare fresh
solutions of Heparin calcium and heparin RS, and use a different, freshlythawed
portion of the stored plasma substrate R.
Calculate the potency of Heparin calcium by combining the results of the assays
by standard statistical methods. If the variance is significant (P = 0.01), due to
differences between assays, it is possible to obtain a combined estimate by cal-
culating the non-weighted mean of potency estimates.
The estimated potency is not less than 90% and not more than 111% of the
stated potency. The fiducial limits of error of the estimated potency (P = 0.95)
are not less than 80% and not more than 125% of the stated potency.
469
Additional requirements for Heparin calcium for

parenteral use
terial endotoxins; Heparin calcium intended for the manufacture of a parenteral
dosage form, without further appropriate procedure for the removal of bacte-
rial endotoxins, contains not more than 0.01 lU of endotoxin RS per lU of
heparin activity. The addition of divalent cations may be necessary in order to
fulfil the validation criteria.
HEPARINUM NATRICUM
HEPARIN SODIUM
Chemical name. Heparin sodium; CAS Reg. No. 9041-08-1.
Storage. Heparin sodium should be kept in a tightly closed container.
Labelling. The designation Heparin sodium for parenteral use indicates that
the substance complies with the additional requirements and may be used for
parenteral administration. The name and quantity of
label should also state the
any added substances, and the source of the material (lung or mucosal). Expiry
date.
Additional information. Heparin sodium may be prepared from the lungs of

oxen or the intestinal mucosae of oxen, pigs, or sheep. Attention should be paid
to minimize or eliminate microbiological contamination and substances low-
ering blood pressure. Heparin sodium is moderately hygroscopic.
Requirements
Definition. Heparin sodium is a preparation containing the sodium salt of a
sulfated glucosaminoglycan present in mammalian tissues. It has the charac-
teristic property of delaying the clotting of fresh blood.
Heparin sodium intended for the manufacture of a parenteral dosage form con-
tains not less than 150IU per mg, and Heparin sodium not intended for use
470
in themanufacture of a parenteral dosage form contains not less than 120IU

per mg, both calculated with reference to the dried substance.
Identity tests
A. Delays the clotting of fresh blood.
B. Specific optical rotation, use a 40mg/ml solution; [a]™ is not less than +35 °.
yields the characteristic reactions. If reaction B is to be used, prepare a
20mg/ml solution.
Sodium. Determine by atomic absorption spectrophotometry under 1.8

Atomic spectrometry: emission and absorption at a wavelength of 330.3 nm,
using a sodium hollow cathode lamp and a flame of suitable composition (e.g.
11 litres of air and 2 litres of acetylene per minute). Prepare a solution of 5 mg
in 10ml of hydrochloric acid (0.1 mol/1) VS containing 1.27 mg/ml of caesium
chloride R. As a reference solution use sodium standard (200 pg of Na per ml)
TS and use dilutions containing 25, 50, and 75 pg of Na per ml in the same
mixture of caesium chloride and hydrochloric acid as prepared above; 95-
125 mg of Na per g, calculated with reference to the dried substance.
Nitrogen. Carry out Method A as described under 2.10 Determination of

nitrogen, using about 0.1 g, accurately weighed, and 5 ml of nitrogen-free sul-
furic acid (-1760 g/1) TS. Each ml of sulfuric acid (0.05mol/l) VS is equivalent
to 1.401 mg of nitrogen; not more than 25mg/g, with reference to the dried
substance.
Protein and nucleotidic impurities. Measure the absorbance of a 1-cm layer

of a 4 mg/ml solution at a wavelength of 260 nm and 280 nm; at 260 nm not
greater than 0.20 and at 280 nm not greater than 0.15.
Clarity and colour of solution. A solution containing 5000 lU per ml is clear

Sulfated ash. Use 0.2g; 0.30-0.43 g/g, with reference to the dried substance.

exceeding 0.5 kPa or about 5mm of mercury) over phosphorus pentoxide R; it
loses not more than 0.080 g/g.
471
pH value. pH of lOmg/ml solution in carbon-dioxide-free water R, 5.5-8. 0.
Assay. The anticoagulant activity of heparin is determined in vitro using a bio-

logical assay to compare its ability to delay the clotting of recalcified citrated
sheep plasma with that of the reference substance. The following method is
suitable for carrying out the assay (other methods may also be applicable).
The onset of clottingdetermined either as a change in optical density (by

is
direct visual inspection, preferably using indirect illumination against a matt

black background, or by spectrophotometry, recording at a wavelength of
approximately 600 nm) or as a change in fluidity (by visual detection while
manually tilting the tube or by mechanical recording while stirring, taking care
to cause the minimum disturbance of the solution during the initial phase of
clotting). Use appropriate tubes according to the chosen technique.
Prepare a solution of Heparin sodium and a solution of heparin RS in sodium

chloride (9g/l) TS, each containing an accurately known number of lU of heparin
per ml. Using sodium chloride (9g/l) TS prepare from each solution a series of
dilutions in geometric progression such that the clotting time obtained with the
lowest concentration is not less than 1.5 times the blank recalcification time, and
the clotting time obtained with the highest concentration is such as to give a sat-
isfactory log dose-response curve, as determined in a preliminary test.
Place in an ice-bath 12 labelled tubes for each dilution: Ti, T 2 T3, etc. for
,
Heparin sodium and Si, S2, S3, etc. for heparin RS. To each tube add 1.0ml of
thawed plasma substrate R and 1.0 ml of the appropriate dilution, either from
Heparin sodium or heparin RS, mixing each tube carefully, and not allowing
bubbles to be formed. (The detection technique employed may require the
addition of different volumes of plasma substrate, consequently the appropri-
ate adjustment of all tubes would be needed.) Transfer all the tubes to a water-
bath at 37 °C, and allow to equilibrate for about 15 minutes. Add to each tube,
mixing after each addition, 1 ml of a dilution of cephalin TS and 1 ml of kaolin
suspension TS freshly prepared just before use. (A suitable dilution of cephalin
TS is one that, under the conditions of the assay, gives a blank recalcification
time of not more than 60 seconds.) After exactly 2 minutes, add 1.0 ml of
calcium chloride (3.7 g/1) TS. Record in seconds the interval between this addi-
tion and the onset of clotting, determined according to the chosen technique.
Similarly determine the blank recalcification time at the beginning and at the
end of the procedure, using 1.0 ml of sodium chloride (9 g/1) TS in place of one
of the heparin dilutions; the two values for the blank should not differ signifi-
cantly. Repeat the procedure using fresh dilutions of the initial solutions and
carrying out the incubation in the reverse order (first tubes S, then tubes T).
Transform the clotting times to logarithms using the mean values for the dupli-
cate tubes and calculate the results by standard statistical methods.
472
Carry out not fewer than 3 independent assays. For each assay prepare fresh
solutions of Heparin sodium and heparin RS, and use a different, freshlythawed
portion of the stored plasma substrate R.
Calculate the potency of Heparin sodium by combining the results of the assays
by standard statistical methods. If the variance is significant (P = 0.01), due to
differences between assays, it is possible to obtain a combined estimate by cal-
culating the non-weighted mean of potency estimates.
The estimated potency is not less than 90% and not more than 111% of the
stated potency. The fiducial limits of error of the estimated potency (P = 0.95)
are not less than 80% and not more than 125% of the stated potency.
Additional requirements for Hefârin sodium for fârenteral use

terial endotoxins; Heparin sodium intended for the manufacture of a parenteral
dosage form, without further appropriate procedure for the removal of bacte-
rial endotoxins, contains not more than 0.01 lU of endotoxin RS per lU of
heparin activity. The addition of divalent cations may be necessary in order to
fulfil the validation criteria.
HOMATROPINI HYDROBROMIDUM
HOMATROPINE HYDROBROMIDE
Molecular formula. Ci 6 H 2 iN 03 ,HBr
Graphic formula.
473
Chemical name. lafi,5af?-Tropan-3a-ol mandelate (ester) hydrobromide;

(±)-««iio-8-methyl-8-azabicyclo[3.2.1]oct-3-yl a-hydroxybenzeneacetate hydro-
bromide; CAS Reg. No. 51-56-9.
Solubility. Freely soluble in water; sparingly soluble in ethanol (-750 g/1) TS;
Category. Mydriatic.
Storage. Homatropine hydrobromide should be kept in a tightly closed con-

Requirements
Definition. Homatropine hydrobromide contains not less than 98.5% and not
more than 101.0% of CigH 2 iN 03 ,HBr, calculated with reference to the dried
substance.
Identity tests
A. Dissolve lOmg in 1ml of water, add ammonia (~100g/l) TS to render the
solution slightly alkaline, and shake with 5 ml of chloroform R. Evaporate
the chloroform layer to dryness on a water-bath and add 1 .5 ml of mercuric
chloride/ethanol TS to the residue; a yellow colour is produced, which turns
red on heating.
B. A 20 mg/ml solution yields reaction A described under 2.1 General identifi-

cation tests as characteristic of bromides.
15mg/g.
Foreign alkaloids. Dissolve 10 mg in 2 ml of water and add 0.25 ml of tannic

acid (50 g/1) TS; no precipitate is produced.
Related alkaloids. Dissolve 5 mg in 0.25 ml of fuming nitric acid R and evap-

orate to dryness on a water-bath. Allow to cool, add 0.1 ml of acetone R and
0.1 ml of a mixture of 1 volume of potassium hydroxide/ethanol (0.5 mol/1) VS
474
and 4 volumes of aldehyde-free ethanol (~750g/l) TS; no violet or reddish violet

colour is produced.
Assay. Dissolve about 0,3 g, accurately weighed, in 30ml of glacial acetic acid
is equivalent to 35.63
mg of C] 5H 2 iN 03 ,HBr.
HOMATROPINI METHYLBROMIDUM
HOMATROPINE METHYLBROMIDE
Br
Ci7H24BrN03
Chemical name. 3a-Hydroxy-8-methyl-laf/,5af/-tropanium bromide (±)-
mande late; (±)-e)«4/o-3- [(hydroxy phenylacetyl)oxy ]- 8,8- dime thyl-8-azonia-

bicyclo-[3.2.1]octane bromide; CAS Reg. No. 80-49-9.
Solubility. Very soluble in water; freely soluble in ethanol (~750g/l) TS; prac-
tically insoluble in ether R and acetone R.
Category. Mydriatic.
Storage. Homatropine methylbromide should be kept in a tightly closed con-

Additional information. Homatropine methylbromide darkens on exposure

to light.
475
Requirements
Homatropine methylbromide contains not less than 98 5 % and not more than
.
the equivalent of 101.0% of C] 7 H 24BrN 03 calculated with reference to the

,
dried substance.
Identity tests
A. Dissolve lOmg in 1ml of water, add ammonia (~100g/l) TS to render the
solution slightly alkaline, and shake with 5 ml of chloroform R. Evaporate
the chloroform layer to dryness on a water-bath and add 1 .5 ml of mercuric
chloride/ethanol TS to the residue; no yellow or red colour is produced (dis-
tinction from homatropine, atropine, and other solanaceous alkaloids).

Loss on drying. Dry to constant mass at 105°C; it loses not more than
lOmg/g.
6 volumes of 1 -propanol R, 3 volumes of water, 2 volumes of methanol R, and
1 volume of glacial acetic acid R as the mobile phase. Apply separately to the
plate 5 pi of each of two solutions in a mixture of 9 volumes of methanol R and

1 volume of water containing (A) 40
mg of Homatropine methylbromide per
ml and (B) 0.4 mg of Homatropine methylbromide per ml. After removing the
plate from the chromatographic chamber, allow it to dry in air, spray it first
with potassium iodobismuthate TS2 and then with sodium nitrite (50g/l) TS.
2.6 Non-aqueous titration. Method A.

Ci7H24BrN03.
476
HYDRALAZINI HYDRO CHLORIDUM

HYDRALAZINE HYDROCHLORIDE
Molecular formula. CbHsN 4,HC1
Graphic formula.
NHNHj
Chemical name. 1-Hydrazinophthalazine monohydrochloride; \{2H)-

phthalazinone hydrazone monohydrochloride; CAS Reg. No. 304-20-1.
Other name. Apressinum.
Solubility. Soluble in 25 parts of water; slightly soluble in ethanol (-750 g/1)

TS; very slighdy soluble in ether R.
Category. Antihypertensive drug.
Storage. Hydralazine hydrochloride should be kept in a well-closed container,

Additional information. Hydralazine hydrochloride melts at about 275 °C

with decomposition. Even in the absence of light, it is gradually degraded on
temperatures.
Requirements
Definition. Hydralazine hydrochloride contains not less than 98.0% and not
more than 101.0% of CbH 8N 4 ,HC 1 ,
substance.
Identity tests
A. The absorption spectrum of a lOpg/ml solution, when observed between
220 nm and 350 nm, exhibits maxima at 240 nm, 260 nm, 303 nm, and
477
315 nm; the absorbances of a 1-cm layer at these wavelenghts are about 0.58,
0.54, 0.27, and 0.21, respectively.
B. Dissolve 0.5g in a mixture of 100ml of water and 8ml of hydrochloric acid

(~70g/l) TS, add 20ml of sodium nitrite (lOg/1) TS, allow to stand for 10
minutes and filter. Wash the residue with water and dry at 105°C; melting
temperature, about 210°C.

Water-insoluble substances. Transfer 2.0g to a 250-ml conical flask, add

100 ml of water, and shake by mechanical means for 30 minutes. Filter the solu-
tion through a tared sintered glass crucible, rinse the flask, and wash any undis-
solved residue into the crucible. Wash the residue with three 10-ml portions of
water, dry at 105°C for 3 hours, cool and weigh; the residue weighs not more
than 10 mg (5mg/g).

exceeding O.bkPa or about 5mm of mercury) over phosphorus pentoxide R for
8 hours; it loses not more than 5.0mg/g.
chromatography, using silica gel R1 as the coating substance. For the mobile
phase, shake a mixture of 2 volumes of ethyl acetate R, 2 volumes of ammonia
(-260 g/1) TS and 8 volumes of hexane R, allow to separate and use the upper
layer. For the preparation of the test solution dissolve 0. 10 g of the test substance
in a mixture of 100 volumes of methanol R and 1 volume of hydrochloric acid
(-420 g/1) TS, and dilute to 20 ml with the same solvent mixture; to 2.0ml of this
solution add 1.0ml of salicylaldehyde TS, centrifuge and decant the supernatant
liquid (solution A). For the reference solution, dissolve 25.0 mg of hydrazine
sulfate R in 10ml 100ml using a mixture of 1 volume
of water and dilute to of
hydrochloric acid (~420g/l) TS and 100 volumes of methanol R; dilute 1.0ml to
100ml with the same solvent mixture. To 2.0ml of this solution add 1.0ml of
salicylaldehyde TS, centrifuge and decant the supernatant liquid (solution B).
Apply separately to the plate 40 pi of each of solutions A and B. After removing
the plate from the chromatographic chamber, allow it to dry in air and spray
with 4-dimethylaminobenzaldehyde TS6. Examine the chromatogram in ultra-
violet light (254 nm). Any spot obtained with solution A, other than the princi-
pal spot, is not more intense than that obtained with solution B.
of hydrochloric acid (-420 g/1) TS, cool to room temperature, add 5ml of chlo-
roform R, and titrate with potassium iodate (0.05 mol/1) VS, shaking continu-
478
ously, until the purple colour of iodine in the chloroform layer disappears.
The endpoint is reachedwhen the chloroform layer remains colourless for at
least 5 minutes. Each ml of potassium iodate (0.05 mol/1) VS is equivalent to
9.832 mg of CsH 8N 4 ,HCl.
HYDRARGYRI OXYCYANIDUM
MERCURIC OXYCYANIDE
Chemical name. CAS Reg. No. 73360-53-9.

TS.
Category. Antiseptic (for topical use).
Storage. Mercuric oxycyanide should be kept in a tightly closed container,

Additional information. Mercuric oxycyanide is slowly discoloured by light.

It might explode on triturating or mixing with other substances. The solution
should not be heated on an open flame.
Requirements
Definition. Mercuric oxycyanide is a mixture of approximately 1 part of
Hg(CN) 2 ,HgO and 2 parts of Hg(CN) 2 .
Mercuric oxycyanide contains not less than 14.5% and not more than 17.2%
of HgO, and not less than 82.5% and not more than 85.5% of Hg(CN) 2 .
Identity tests
A. Immerse a small piece of copper plate in a 0.05g/ml solution for some
minutes, remove, wash with water, and rub the plate with paper; a bright
silvery surface is produced.
B. To a 0.05 g/ml solution add potassium iodide (80g/l) TS; a yellow solution
is produced which on the addition of ammonia (-100 g/1) TS gives a reddish
brown precipitate.
C. To 1 ml of a 0.05 g/ml solution add 0.05 g of ferrous sulfate R and sufficient

sodium hydroxide (-80 g/1) TS to precipitate the iron as hydroxide. Boil the
479
mixture and acidify with hydrochloric acid (~70g/l) TS; a blue colour or pre-
cipitate is produced.
Chlorides. Dissolve 1.75g in 20ml of water, add 10ml of sodium hydroxide

(~80g/l) TS and 25ml of formaldehyde TS, and boil gendy for 10 minutes. Cool,
filter,wash the precipitate with water, combine the filtrate and washings, neu-
tralize with nitric acid (-130 g/1) TS and add sufficient water to produce 100 ml.
Proceed as described under 2.2.1 Limit test for chlorides, using 40ml of this
solution and 6ml of nitric acid (-130 g/1) TS. To prepare the standard opales-
cence mix 10ml of sodium hydroxide (-80 g/1) TS with 12.25ml of hydrochlo-
ric and boil the mixture gently for 10 minutes. Cool, filter, wash the
acid CITS,
with water, combine the filtrate and washings, neutralize with nitric acid
filter
(-130 g/1) TS, and add sufficient water to produce 100 ml. Proceed as described
under 2.2.1 Limit test for chlorides, using 40ml of this solution and 6ml of nitric
acid (-130 g/1) TS; the chloride content is not more than 0.35 mg/g.
Clarity of solution. A solution of 0.050 g in 10 ml of water is clear.
Sulfated ash. Carry out the ignition under a hood; not more than 2.5 mg/g.
Loss on drying. Dry to constant weight at 105°C; it loses not more than 10 mg/g.
Assay
For mercuric oxide. Dissolve about 0.5g, accurately weighed, in 50ml of
water, add 1 g of sodium chloride R and titrate with hydrochloric acid (0.1 mol/1)
VS using methyl orange/ethanol TS as indicator. Repeat the operation without
the substance being examined and make any necessary corrections. Each ml of
hydrochloric acid (0.1 mol/1) VS is equivalent to 10.83 mg of HgO. (Keep the
solution for the assay for mercuric cyanide).
For mercuric cyanide. Use the solution obtained from the assay for mercuric
oxide, add 3g of potassium iodide R, and continue to titrate with hydrochlo-
ric acid (0.1 mol/1) VS. Each ml of hydrochloric acid (0.1 mol/1) VS is equivalent
to 12.63mg of Hg(CN) 2 .
HYDROCHLOROTHIAZIDUM
HYDROCHLOROTHIAZIDE
Molecular formula. C7H8CIN3O4S2
480
Graphic formula.
0 0
\\//
Chemical name. 6-Chloro-3,4-dihydro-2H-l ,2,4-benzothiadiazine-7-sulfon-

amide 1,1-dioxide; CAS Reg. No. 58-93-5.

almost odourless.
Solubility. Very slightly soluble in water; practically insoluble in ether R;

soluble in 200 parts of ethanol (-750 g/1) TS and in 20 parts of acetone R.
Category. Diuretic.
Storage. Hydrochlorothiazide should be kept in a well-closed container.
Requirements
Definition. Hydrochlorothiazide contains not less than 98.0% and not more
than 102.0% of C7H8CIN3O4S2, calculated with reference to the dried substance.
Identity tests
infrared region.
spectrum obtained from hydrochlorothiazide RS or with the reference spec-
trum of hydrochlorothiazide.
B. Mix lOmg of the test substance and lOmg of disodium chromotropate R,

add 1ml of water and, cautiously, 5ml of sulfuric acid (~1760g/l) TS; a
purple colour is produced.
Free chlorides. For the preparation of the test solution shake 0.3 g with 20ml
of water and 10ml of nitric acid (~130g/l) TS for 5 minutes, and filter. Proceed
with the filtrate as described under 2.2.1 Limit test for chlorides; the chloride
481
lOmg/g.
Diazotizable substances. For the test solution place about 0.01 g, accurately
weighed, in a 50-ml volumetric flask, and dissolve in 10 ml of methanol R.
Dilute to volume with water and mix.
For the reference solution weigh 5.0 mg of 4-amino-6-chloro-l,3-

benzenedisulfonamide R, transfer to a 10-ml volumetric flask, and dissolve
in 1ml of methanol R. Dilute to volume with water and mix. Dilute 4 ml of
this solution with sufficient water to produce 100ml (=20pg/ml).
Transfer 5 ml of the test solution and of the reference solution to separate 50-
ml volumetric flasks, and 5 ml of water to a third 50-ml volumetric flask to serve
as a blank. To each flask add 1 ml of freshly prepared sodium nitrite (lOg/1) TS
and 5ml of hydrochloric acid (-70g/l) TS, and allow to stand for 5 minutes.
Add 2 ml of ammonium sulfamate (25g/l) TS, allow to stand for 5 minutes with
frequent swirling, then add 2 ml of freshly prepared disodium chromotropate
(lOg/1) TS and
10 ml of sodium acetate (150 g/1) TS. Dilute with water to volume
and mix. Measure the absorbance at the maximum at about 500 nm, against
the blank. The absorbance of the test solution does not exceed that of the ref-
erence solution (lOmg/g).
Assay. Dissolve about 0.3 g, accurately weighed, in 50ml of pyridine R, add 5

drops of azo violet TS and titrate quickly with sodium methoxide (0.1 mol/1)
VS to a deep blue endpoint, as described under 2.6 Non-aqueous titration.
Method B. Each ml of sodium methoxide (0.1 mol/1) VS is equivalent to
14.89mg of C7H8CIN3O4S2.
HYDROCORTISONI ACETAS
HYDROCORTISONE ACETATE
482
Graphic formula.
0
II
Chemical name. 21-(Acetyloxy)-lip,17-dihydroxypregn-4-ene-3,20-clione;

lip,17,21-trihydroxypregn-4-ene-3,20-dione 21-acetate; CAS Reg. No. 50-03-3.
Other name. Cortisol acetate.

Category. Adrenocortical steroid.
Storage. Hydrocortisone acetate should be kept in a well-closed container,

Additional information. Hydrocortisone acetate melts at about 220 °C with

decomposition.
Requirements
Definition. Hydrocortisone acetate contains not less than 97.0% and not more
than 102.0% of C23H32OS, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from hydrocortisone acetate RS or with the reference spec-
trum of hydrocortisone acetate.

483
=
hours and carry out the chromatography in the same direction as the
impregnation. As the mobile phase, use a mixture of 75 volumes of toluene
R and 25 volumes of chloroform R. Apply separately to the plate 2 pi of each
of 2 solutions in a mixture of 9 volumes of chloroform R and 1 volume of
methanol R containing (A) 2.5mg of the test substance per ml and (B)
2.5 mg of hydrocortisone acetate RS per ml. Develop the plate for a distance
of 15 cm. After plate from the chromatographic chamber,
removing the
allow it to dry in have evaporated, heat at 120 °C
air until the solvents
for 15 minutes, spray with sulfuric acid/ethanol TS, and then heat at 120 °C
for 10 minutes. Allow to cool, and examine the chromatogram in daylight
and in ultraviolet light (365 nm). The principal spot obtained with solution
A corresponds in position, appearance, and intensity with that obtained
with solution B.
Specific optical rotation. Use a lOmg/ml solution in dioxan R; [a]™ -1-157
to -tl68°.
lOmg/g.
95 volumes of dichloroethane R, 5 volumes of methanol R and 0.2 volumes of
allow it to dry in air until the solvents have evaporated; then heat at 105 °C for
10 minutes, allow to cool, and examine the chromatogram in ultraviolet light
Assay. Dissolve about 20 mg, accurately weighed, in sufficient ethanol

(-750 g/1) TS to produce 100ml; dilute 5.0ml of this solution to 100ml with the
same solvent. Measure the absorbance of a 1-cm layer of the diluted solution
at the maximum at about 242 nm. Calculate the amount of C23H32O6 in the sub-
stance being tested by comparison with hydrocortisone acetate RS, similarly
and concurrently examined. In an adequately calibrated spectrophotometer the
absorbance of the reference solution should be 0.40 + 0.02 (preferably use
484
HYDROCORTISONI NATRII SUCCINAS

HYDROCORTISONE SODIUM SUCCINATE
Molecular formula. C25H33NaOs
Graphic formula.
CHjOOClCHjljCOONa
CO
Chemical name. Cortisol 21-(sodium succinate); 21-(3-carboxy-l-

oxopropoxy)-llj3,17-dihydroxypregn-4-ene-3,20-dione monosodium salt; CAS
Reg, No. 125-04-2.
Description. A white or almost white, crystalline powder or amorphous solid;

odourless.
Solubility. Freely soluble in water; soluble in 34 parts of ethanol (-750 g/1) TS

and in 200 parts of dehydrated ethanol R; practically insoluble in ether R.
Storage. Hydrocortisone sodium succinate should be kept in a tightly closed

Additional information. Hydrocortisone sodium succinate is hygroscopic.

Requirements
Definition. Hydrocortisone sodium succinate contains not less than 97.0%
and not more than 103.0% of C 25 H 33NaOs, calculated with reference to the
dried substance.
485
=
Identity tests
spectrum obtained from hydrocortisone sodium succinate RS or with
the reference spectrum of hydrocortisone sodium succinate.

test as described
using R1 as the coating substance and a freshly prepared mixture
silica gel
of 3 volumes of 1 -butanol R, 1 volume of acetic anhydride R, and 1 volume
of water as the mobile phase. Apply separately to the plate 2[xl of each of
2 solutions in methanol R containing (A) 2.5 mg of the test substance per ml
and (B) 2.5 mg of hydrocortisone sodium succinate RS per ml. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air until
the solvents have evaporated, spray it with a mixture of 10 ml of sulfuric
acid (~1760g/l) TS and 90ml of ethanol (-750 g/1) TS, heat it at 120°C for
10 minutes, allow it to cool, and examine the chromatogram in ultraviolet
light (365 nm). The principal spot obtained with solution A corresponds in
position, appearance, and intensity with that obtained with solution B.
C. When tested for sodium as described under 2.1 General identification tests,

20mg/ml solution.
Specific optical rotation. Use a lOmg/ml solution in ethanol (~750g/l) TS

and calculate with reference to the dried substance; [a]™ -tl40° to -tl50°.
30mg/g.
to the plate 1 pi of each of 2 solutions in a mixture of equal volumes of chlo-
roform R and methanol R containing (A) 15 mg of the test substance per ml and
(B) 0.15 mg of the test substance per ml. After removing the plate from the chro-
then heat it at 105°C for 10 minutes; allow it to cool, spray it with blue tetra-
zolium/sodium hydroxide TS, and examine the chromatogram in daylight. Any
Sodium. Dissolve with gentle heating about 1 g, accurately weighed, in 75 ml

of glacial acetic acid R. Add 20ml of dioxan R, 0.15ml of crystal violet/acetic
486
acid TS, and titrate with perchloric acid VS. Each ml of perchloric
(0.1 mol/1)
VS is equivalent to 2.299mg of Na; the content is not less than
acid (0.1 mol/1)
46.0mg and not more than 48.4mg of Na per g, calculated with reference to

produce 100 ml; dilute 5 ml to 100 ml with water. Measure the absorbance of a
1-cm layer of the diluted solution at the maximum at about 248nm. Calculate
the amount of C 2 5 H 33 NaOs in the substance being tested by comparison with
,
hydrocortisone sodium succinate RS, similarly and concurrently examined. In

an adequately calibrated spectrophotometer the absorbance of the reference
HYDROCORTISONUM
HYDROCORTISONE
Graphic formula.
Chemical name. lip,17,21-Trihydroxypregn-4-ene-3,20-dione; CAS Reg. No.

50-23-7.
Other name. Cortisol.
Solubility. Very slightly soluble in water and ether R; sparingly soluble in

ethanol (-750 g/1) TS and acetone R.
487
Storage. Hydrocortisone should be kept in a well-closed container, protected

from light.
Additional information. Hydrocortisone melts at about 214 °C with

decomposition.
Requirements
Definition. Hydrocortisone contains not less than 97.0% and not more than
Identity tests

infrared region.
spectrum obtained from hydrocortisone RS or with the reference spectrum of
hydrocortisone.

nation. Use chloroform R as the mobile phase. Apply separately to the plate
2 pi of each of 2 solutions in a mixture of 9 volumes of chloroform R and 1
volume of methanol R containing (A) 2.5 mg of the test substance per ml,
and (B) 2.5 mg of hydrocortisone RS per ml. Develop the plate for a distance
of 15 cm. After removing the plate from the chromatographic chamber,
allow it to dry in air until the solvents have evaporated, heat at 120 °C for
15 minutes, spray with sulfuric acid/ethanol TS, and then heat at 120 °C for
in ultraviolet light (365 nm). The principal spot obtained with solution A cor-
solution B.
Specific optical rotation. Use a lOmg/ml solution in dioxan R; = -tl50

to -tl56°.
lOmg/g.
488
methanol R and 1.2 volumes of water as the mobile phase. Apply separately
evaporated, heat at 105°C for 10 minutes, allow to cool, and examine the chro-
solution B.

at the maximum at about 242 nm. Calculate the amount of C21H30O5 in the
substance being tested by comparison with hydrocortisone RS, similarly and
HYDROXOCOBALAMINI CHLORIDUM
HYDROXOCOBALAMIN CHLORIDE
HYDROXOCOBALAMINI SULFAS
HYDROXOCOBALAMIN SULFATE
Molecular formula. C62H90CIC0N13O15P; C124H1S0C02N26O34P2S.
Relative molecular mass. 1383 (hydroxocobalamin chloride); 2791 (hydrox-

ocobalamin sulfate).
489
Graphic formula for the base.
Chemical name. Cobinamide dihydroxide dihydrogen phosphate (ester),

mono(inner salt), 3'-ester with 5,6-dimethyl-l-a-D-ribofuranosylbenzimida-
zole monohydrochloride; cobinamide dihydroxide dihydrogen phosphate
(ester), mono(inner salt), 3'-ester with 5,6-dimethyl-l-a-D-ribofuranosyl-lf/-
benzimidazole monohydrochloride; Coa-[a-(5,6-dimethylbenzimidazolyl)]-

CoP-hydroxocobamide chloride; CAS Reg. No. 59461-30-2.
Cobinamide dihydroxide dihydrogen phosphate (ester), mono(inner salt), 3'-

ester with 5,6-dimethyl-l-a-D-ribo£uranosylbenzimidazole sulfate (salt) (2:1);
cobinamide dihydroxide dihydrogen phosphate (ester), mono(inner salt),
3'-ester with 5,6-dimethyl-l-a-D-ribofuranosyl-lW-benzimidazole sulfate (salt)
(2 :
1); 2(Coa-[a-(5,6-dimethylbenzimidazolyl)]-CoP-hydroxocobamide) sulfate
(salt) (2 :
1).
Solubility. Soluble in water.
Category. Antianaemia drug.
Storage. Hydroxocobalamin chloride or sulfate should be kept in a tightly

closed container, protected from light, and stored in a cool place.
stance is the chloride or the sulfate salt.
490
Additional information. Even in the absence of light, Hydroxocobalamin

chloride and Hydroxocobalamin sulfate are gradually degraded on exposure to
a humid atmosphere, the decomposition being faster at higher temperatures.
Requirements
Definition. Hydroxocobalamin chloride contains not less than 96.0% and not
more than 102.0% of C 62 H 9 oClCoN] 30 5 P, calculated with reference to the dried
]
substance; Hydroxocobalamin sulfate contains not less than 96.0% and not
more than 102.0% of Ci 24 H, 8 oCo 2 N 26034 P 2 S, calculated with reference to the
dried substance.
Identity tests
A. The absorption spectrumof a 40pg/ml solution in pH 4.5 acetate buffer TS,
274nm, 351 nm, and 525nm; the ratio of the absorbance of a 1-cm layer at
525 nm to that at 351 nm is about 0.34, and the ratio of the absorbance at
274 nm to that at 351 nm is about 0.80.
B. Heat cautiously about 2 mg in a porcelain crucible with a few drops of sul-
furic acid (~1760g/l) TS until a faintly bluish residue is produced. Cool, add
0.05 ml of water and then a few drops of a saturated solution of ammonium
thiocyanate R; a blue-green colour is produced.
C. Place about 2mg 2ml of water

in a 100-ml glass-stoppered flask, dissolve in
and add 5ml of phosphoric acid (-1440 g/1) TS. Insert in the flask a flat-
bottomed glass tube 1cm in diameter and 2 cm long, containing 1ml of
lithium carbonate/trinitrophenol TS. Close the flask and expose it for 4
hours to a bright light; the colour of the reagent in the glass tube remains
unchanged (distinction from cyanocobalamin).
D. For the chloride salt prepare a 20 mg/ml solution. It yields reaction B

described under 2.1 General identification tests as characteristic of chlorides.
For the sulfate salt prepare a 20mg/ml solution. It yields reaction A
described under 2.1 General identification tests as characteristic of sulfates.
or about 5 mm
of mercury) for 2 hours; the chloride salt loses between 80mg/g
and 120mg/g and the sulfate salt between 80mg/g and 160mg/g.
Other cobalamins. Carry out the test as described under 1.14.3 Column chro-
matography shaking 20g of diethylaminoethylcellulose R with200ml of sodium
hydroxide (0.5mol/l) VS, dilute with water to obtaina homogeneous suspension.
491
allow to and discard the supernatant liquid. Using a suitable filter, wash
settle,
with water washings are free from alkali, then transfer the adsorbent to
until the
a tube, length 22 cm, diameter 1,2 cm, and provided with a stopcock. Allow to
setde and tap the tube until the height of the adsorbent is about 14 cm. Wash with
water until the pH of the eluate is the same as that of the water.
Similarly prepare a second column, slurrying carboxymethylcellulose R with

hydrochloric acid (0.5 mol/1) VS, dilute with water, allow to and discard
settle,
the supernatant liquid. Using a suitable filter, wash with water

wash- until the
ings are free from acid, then transfer the adsorbent to a tube, length 22 cm,
diameter 1 .2 cm, and provided with a stopcock. Allow to settle and tap the tube
until the height of the adsorbent is about 10 cm. Wash with water until the pH
of the eluate is the same as that of the water.
Cover each column with a plug of glass wool, and allow to drain until only a
small amount of water remains above the adsorbents.
Place the column of diethylaminoethylcellulose above the other column so that

the effluent runs into the carboxymethylcellulose.
Weigh accurately about 0.05 g of the substance to be examined, dissolve it in

20 ml of water, and acidify with sufficient hydrochloric acid (~70g/l) TS to
obtain a pH of 4.0. Introduce this solution to the diethylaminoethylcellulose
column and allow it to run through both columns, rejecting the first colourless
eluate. Elute with water the pH of which has previously been adjusted to 4.0
with hydrochloric acid (~70g/l) TS, Collect the coloured eluate into a 50-ml
volumetric flask and adjust to volume with water. Measure the absorbance of
this solution in a 1-cm layer at the maximum at about 361 nm, and calculate
the content of other cobalamins in mg/g, using the absorptivity value of 20.7
(A!*m = 207); not more than 30 mg/g.
Acidic impurities. Elute the diethylaminoethylcellulose column from the

above test for other cobalamins with sodium chloride (lOg/1) TS, collecting
50ml of eluate. Measure the absorbance of this solution in a 1-cm layer at the
maximum between 351 nm and 361 nm, and calculate the content of acidic
impurities in mg/g, using the absorptivity value of 19,0 (Al°cm = 190); not more
than 30 mg/g.
Assay
Dissolve about 20 mg, accurately weighed, in sufficient acetate buffer, pH

4.5, TS produce 500 ml. Measure the absorbance of this solution in a 1-cm
to
layer at maximum at about 351 nm and calculate the content of
the
Ce;2 H 9 oClCoNi30 5 P Or Ci24H]8oCo2N26034P2S, using the absorptivity values of
,
19.0 or 18.8, respectively (Ajim = 190 or 188, respectively).
492
HYDROXOCOBALAMINUM
HYDROXOCOBALAMIN
Hydroxocobalamin anhydrous
Hydroxocobalamin hydrate
Molecular formula. C62H89CoNi30i5P (anhydrous); C62Hs9CoNi30i5P,H20

(monohydrate).
Relative molecular mass. 1346 (anhydrous); 1365 (monohydrate).
Graphic formula.
Chemical name. Cobinamide dihydroxide dihydrogen phosphate (ester),

zole; cobinamide dihydrogen phosphate (ester)-mono(inner salt), 3'-ester with
5, 6-dim ethyl- 1-a-D-ribofuranosyl-l /7-benzimidazole; Coa-[a-(5,6-dimethyl-
benzimidazolyl)]-CoP-hydroxocobamide; CAS Reg. No. 13422-51-0 (anhy-
drous). Cobinamide dihydroxide monohydrate, dihydrogen phosphate (ester),
zole; cobinamide dihydroxide monohydrate, dihydrogen phosphate (ester),
mono(inner salt) 3'-ester with 5,6-dimethyl-l-a-D-ribofuranosyl-l/7-
benzimidazole; Coa-[a-(5,6-dimethylbenzimidazolyl)]-CoP-hydroxocobamide
monohydrate; CAS Reg. No. 13422-52-1 (monohydrate).
Other names. Vitamin B] 2 a for anhydrous Hydroxocobalamin and Vitamin

Bi 2 b for Hydroxocobalamin hydrate.
493
Solubility. Sparingly soluble in water and ethanol (-750 g/1) TS; practically
insoluble in acetone R.
Category. Antianaemia drug.
Storage. Hydroxocobalamin should be kept in a tightly closed container, pro-

tected from light, and stored in a cool place.
stance is in the anhydrous or hydrated form.
Additional information. In aqueous solution, Hydroxocobalamin exists as

hydroxocobamide in equilibrium with the hydrated ionic form. The anhydrous
form of Hydroxocobalamin is very hygroscopic. Even in the absence of light,
it is gradually degraded on exposure to a humid atmosphere, the decomposi-
tion being faster at higher temperatures.
Requirements
Definition. Hydroxocobalamin contains not less than 96.0% and not more
than 102.0% of C(S2 H 89 CoNi 30 i5
P, calculated with reference to the dried
substance.
Identity tests
A. The absorption spectrumof a 40pg/ml solution in pH 4.5 acetate buffer TS,
274nm, 351 nm, and 525nm; the ratio of the absorbance of a 1-cm layer at
525 nm to that at 351 nm is about 0.34, and the ratio of the absorbance at
274 nm to that at 351 nm is about 0.80.
B. Heat cautiously about 2 mg in a porcelain crucible with a few drops of sul-

furic acid (~1760g/l) TS until a faintly bluish residue is produced. Cool, add
0.05 ml of water and then a few drops of a saturated solution of ammonium

thiocyanate R; a blue-green colour is produced.
C. Place about 2mg ml of water

in a 100-ml glass-stoppered flask, dissolve in 2
and add 5 ml of phosphoric acid (-1440 g/1) TS. Insert in the flask a flat-
bottomed glass tube 1cm in diameter and 2 cm long, containing 1ml of
lithium carbonate/trinitrophenol TS. Close the flask and expose it for 4
hours to a bright light; the colour of the reagent in the glass tube remains
unchanged (distinction from cyanocobalamin).
494
Loss on drying. Dry Hydroxocobalamin hydrate at 100 °C under reduced

pressure (not exceeding O.dkPa or about 5mm of mercury) for 2 hours; it loses
between 140mg/g and 180mg/g.
Other cobalamins. Carry out the test as described under 1.14.3 Column chro-
matography, shaking 20 g of diethylaminoethylcellulose R with 200 ml of
sodium hydroxide (0.5 mol/1) VS, dilute with water to obtain a homogeneous
suspension, allow to and discard the supernatant liquid. Using a suitable
settle,
filter, wash with water washings are free from alkali, then transfer the
until the
adsorbent to a tube, length 22 cm, diameter 1.2 cm, and provided with a stop-
cock. Allow to settle and tap the tube until the height of the adsorbent is about
14 cm. Wash with water until the pH of the eluate is the same as that of the
water.
Similarly prepare a second column, slurrying carboxymethylcellulose R with

hydrochloric acid (0.5mol/l) VS, dilute with water, allow to settle, and discard
the supernatant liquid. Using a suitable filter, wash with water until the wash-
ings are free from acid, then transfer the adsorbent to a tube, length 22 cm,
diameter 1.2 cm, and provided with a stopcock. Allow to settle and tap the tube
until the pH of the eluate is the same as that of the water.
Cover each column with a plug of glass wool and allow to drain until only a
small amount of water remains above the adsorbents.
Place the column of diethylaminoethylcellulose above the other column so that

the effluent runs into the carboxymethylcellulose.
Weigh accurately about 0.05 g of the substance to be examined, dissolve it in

20ml of water, and acidify with sufficient hydrochloric acid (~70g/l) TS to
obtain a pH of 4.0. Introduce this solution to the diethylaminoethylcellulose
column and allow run through both columns, rejecting the first colourless
it to
eluate. Elute with water, the pH of which has previously been adjusted to 4.0
with hydrochloric acid (~70g/l) TS. Collect the coloured eluate into a 50-ml
volumetric flask and adjust to volume with water. Measure the absorbance of
this solution in a 1-cm layer at the maximum at about 361 nm and calculate the
content of other cobalamins in mg/g, using the absorptivity value of 20.7
(AT™ = 207); not more than 30 mg/g.
Acidic impurities. Elute the diethylaminoethylcellulose column from the

above test for other cobalamins with sodium chloride (lOg/1) TS, collecting
50ml of eluate. Measure the absorbance of this solution in a 1-cm layer at the
maximum between 351 nm and 361 nm, and calculate the content of acidic
impurities in mg/g, using the absorptivity value of 19.0 = 190); not more
than 30 mg/g.
495
Assay
Dissolve about 20 mg, accurately weighed, in sufficient acetate buffer, pH 4,5,

TS to produce 500 ml. Measure the absorbance of this solution in a 1-cm layer
at the maximum at about 351 nm and calculate the content of C62H89CoNi30,5P,
using the absorptivity value of 19.5 = 195).
Additional requirements for Hydroxocohalamin for

p>arenteral use
Bacterial endotoxins. Carry out the test as described under 3,4 Test for bac-
HYDROXYETHYLCELLULOSUM
HYDRO XYETHYLCELLULOSE
Chemical name. Cellulose 2-hydroxyethyl ether; CAS Reg, No. 9004-62-0.
Description. A white or yellowish white powder or granules; odourless or

almost odourless.
Solubility. Soluble in hot and cold water forming a colloidal solution; practi-
cally insoluble in acetone R, ethanol (-750 g/1) TS, ether R, and toluene R.
Category. Stabilizer; suspending agent.
Storage. Hydroxyethylcellulose should be kept in a well-closed container.
Labelling. The designation on the container of hydroxyethylcellulose should

state its viscosity.
Additional information. Hydroxyethylcellulose may contain suitable anti-

caking agents. After drying, it is hygroscopic.
Requirements
Definition. Hydroxyethylcellulose is a partially substituted poly(hydrox-
yethyl) ether of cellulose.
496
Identity tests
A. Disperse 1 g of dried Hydroxyethylcellulose in 50 ml of carbon-dioxide-free
water R. After 10 minutes, dilute to 100ml with the same solvent and stir
until completely dissolved. While stirring, heat 10 ml on a water-bath (keep
the remaining solution for test B, for “pH value”, and for “Reducing sub-
stances”); no cloudiness appears above 50 °C and no precipitate is formed.
B. Place 1 ml of the above solution onto a glass plate and allow to evaporate;
a thin film is formed.
C. Dissolve 5mg in 1ml of water, add 1ml of phenol (50g/l) TS and 5ml of
sulfuric acid (~1760g/l) TS, shake carefully, and allow to cool; a red colour
develops.
lOOmg/g.
pH value. pH of the solution prepared in identity test A, 5. 5-8. 5.
Reducing substances. Add 5 ml of water to 5 ml of the solution prepared in

identity test A, thenadd 15 drops of sulfuric acid (0.5mol/l) VS and 1.5ml of
potassium permanganate (0.002 mol/1) VS. Heat the mixture to 50 °C for not
less than 5 minutes; the colour of the solution remains unchanged.
HYDROXYPROPYLCELLULOSUM
HYDROXYPROPYLCELLULOSE
Chemical name. Cellulose 2-hydroxypropyl ether; CAS Reg. No. 9004-64-2.
Description. A white or yellowish white powder; odourless.
Solubility. Soluble in cold water, ethanol (~750 g/1) TS, methanol R, and propy-
lene glycol R giving colloidal solutions; practically insoluble in hot water.
Category. Film-coating agent; tablet binder; granulating agent; viscosity-

increasing agent; suspending agent.
497
Storage. Hydroxypropylcellulose should be kept in a well-closed container.
Labelling. The designation on the container of Hydroxypropylcellulose

should state its viscosity.
Additional information. Hydroxypropylcellulose is hygroscopic after drying.
Requirements
Definition. Hydroxypropylcellulose is a cellulose having some of the hydroxyl
groups in the form of 2-hydroxypropyl ether.
Identity tests
A. While stirring, add Ig of dried Hydroxypropylcellulose to 50 ml of carbon-
dioxide-free water R heated to 90 °C. Allow to cool, dilute to 100 ml with

the same solvent, and stir until completely dissolved. While stirring, heat
10 ml on a water-bath (keep the remaining solution for test B and for “pH
value”); above 40 °C a cloudy solution or a flocculent precipitate is formed,
and on cooling the solution becomes clear.
C. Dissolve without heating 0.2g in 15ml of sulfuric acid (~1125g/l) TS. Pour
the solution with stirring into 100 ml of ice-water, and dilute to 250 ml with
ice-water. While cooling in ice-water, mix thoroughly in a test-tube 1 ml of
the prepared solution with 8ml of sulfuric acid (-1760 g/1) TS, added drop
by drop. Heat in a water-bath for exactly 3 minutes and immediately cool
in ice-water. While cold, add carefully 0.6ml of triketohydrindene/sodium
metabisulfite TS, mix well, and allow to stand at 25 °C; a pink colour is
immediately produced, which changes to violet within 100 minutes.
70mg/g.
pH value. pH of the solution prepared for identity test A, 5. 0-8. 5.
498
HYPROMELLOSUM
HYPROMELLOSE
Chemical name. Cellulose 2-hydroxypropyl methyl ether; CAS Reg. No.
9004-65-3.
Other name. Hydroxypropylmethylcellulose.
Description. A white or creamy white, fibrous or granular powder; odourless

or almost odourless.
Solubility. Soluble in cold water, forming a clear or viscous, colloidal solution;

practically insoluble in ethanol (-750 g/1) TS and ether R; soluble in mixtures of
methanol R and dichloromethane R.
Categoiy. Suspending agent; tablet binder; viscosity-increasing agent.
Storage. Hypromellose should be kept in a well-closed container.
Labelling. The designation on the container of Hypromellose should state its
viscosity.
Requirements
Definition. Hypromellose is a propylene glycol ether of methylcellulose.
Identity tests
A. Disperse 1 g of Hypromellose in 100 ml of water, allow the beaker to stand
until the dispersion becomes transparent and mucilaginous (about 5 hours),
swirl the beaker, stir until completely dissolved. To two 10-ml aliquots
and
(keep the remaining solution for test B and for “pH value”) add an equal
volume of sodium hydroxide (1 mol/1) VS or hydrochloric acid
either
(1 mol/1) VS; both mixtures remain unchanged.
C. Add Ig of powdered Hypromellose to 100ml of boiling water and stir the

mixture; a slurry is formed, but the powdered material does not dissolve.
Cool the slurry to 20 °C and stir; the resulting liquid is a clear or opalescent,
mucilaginous colloidal mixture.
under 2.2.3 Limit test for heavy metals. Procedure 3, adding 1 ml of hydroxy-
499
lamine hydrochloride (200 g/1) TS to the solution of the residue; determine the
heavy metals content according to Method A; not more than 10|tg/g.
pH value. pH of the solution prepared for identity test A, 5. 0-8.0.
IBUPROFENUM
IBUPROFEN
Graphic formula.
Chemical name. /^-Isobutylhydratropic acid; a-methyl-4-(2-methylpropyl)

benzeneacetic acid; 2-(/^-isobutylphenyl)propionic acid; CAS Reg. No. 15687-
27-1.
Description. Colourless crystals or a white, crystalline powder; odour,

characteristic.
Solubility. Practically insoluble in water; soluble in 1.5 parts of ethanol

(-750 g/1) TS, in 2 parts of ether R, and in 1.5 parts of acetone R.
Category. Analgesic; anti-inflammatory.
Storage. Ibuprofen should be kept in a well-closed container.
Requirements
Definition. Ibuprofen contains not less than 98.5% and not more than 100.5%
of CisHisOj, calculated with reference to the dried substance.
500
Identity tests

infrared region.
spectrum obtained from ibuprofen RS or with the reference spectrum of
ibuprofen.
B. The absorption spectrum of a 0.25 mg/ml solution in sodium hydroxide

(0.1 mol/1) VS, when 220nm and 350nm, is qualitatively
observed between
similar to that of a 0.25 mg/ml solution of ibuprofen
RS in sodium hydrox-
VS (maxima occur at about 264 nm and 273 nm, and a shoul-
ide (0.1 mol/1)
der at about 259 nm). The absorbances of the solutions at the respective
maxima do not differ from each other by more than 3%. The absorbance
of a 1-cm layer at the wavelengths of the main maxima at 264 nm and
273 nm are about 0.46 and 0.39, respectively (preferably use 2-cm cells for
the measurement and calculate the absorbances of 1-cm layers).

Related substances
A. Carry out the test as described under 1.14.5 Gas chromatography, using
a solution of the test substance prepared as follows: To O.lOg add dia-
zomethane TS until effervescence ceases and a persistent yellow colour is
produced. Remove the solvent in a current of nitrogen, warming gently if
necessary, and dissolve the residue in 2 ml of chloroform R. Use this solu-

tion in procedures A1 and A2.
In procedure A1 use a glass column 1.8 m long and 3.0 mm

in internal diam-
eterpacked with an adsorbent composed of 1 g of macrogol 20M R and 9g
of acid-washed, silanized kieselguhr R3, and maintained at 135 °C. Use
nitrogen R as the carrier gas and a flame ionization detector. In this system,
determine only those impurities with a relative retention time of less than
2.5, taking the retention time of ibuprofen as 1.0. The ratio of the total area
of the peaks due to these impurities to the area under the peak of ibupro-
501
fen does not exceed 0.010, and the corresponding ratio for the peak of any
individual impurity does not exceed 0,003.
In procedure A2 use a glass column 1.8 m long and 3.0 mm in internal diam-
eter packed with an adsorbent composed of 0.5 g of methyl silicone gum R.
0.2 g of cyanoethylmethyl silicone gum R and 9.3 g of acid-washed, silanized
kieselguhr R4 and maintained at 170 °C. Use nitrogen R as the carrier Gas
and a flame ionization detector. In this system, determine only those impu-
rities with a relative retention time of between 1.5 and 6.0, taking the reten-
tion time of ibuprofen as 1.0. The ratio of the total area of the peaks due to
these impurities to the area under the peak of ibuprofen does not exceed
0 010
. .
The sum of the ratios found in procedures A1 and A2 does not exceed 0,015.

1-hexane R, 5 volumes of ethyl acetate R and 1 volume of glacial acetic acid
R as the mobile phase. Apply separately to the plate 5 pi of each of 2 solu-
tions in chloroform R containing (A) lOOmg of the test substance per ml and
(B) 1 mg of the test substance per ml. After removing the plate from the
chromatographic chamber, allow it to dry in air, spray very lightly with a

lOmg/ml solution of potassium permanganate R in sulfuric acid (~100g/l)
TS, heat at 120 °C for 20 minutes, and examine the chromatogram in ultra-
violet light (365 nm). Any spot obtained with solution A, other than the prin-
Assay. Dissolve about 0.4 g, accurately weighed, in 100 ml of ethanol

(-750 g/1) TS previously neutralized to phenolphthalein/ethanol TS, and titrate
with carbonate-free sodium hydroxide (0.1 mol/1) VS, using phenolphthalein/
ethanol TS as indicator. Repeat the operation without the substance being
examined and make any necessary corrections. Each ml of carbonate-free
sodium hydroxide (0.1 mol/1) VS is equivalent to 20.63 mg of C13H18O2.
502
IDOXURIDINUM
IDOXURIDINE
HN
CH,OH
OH
C^HnlNsOs
Chemical name. 2'-Deoxy-5-iodouridine; CAS Reg. No. 54-42-2.
Solubility. Slightly soluble in water, ethanol (-750 g/1) TS and hydrochloric

acid (-70 g/1) TS; freely soluble in sodium hydroxide (-80 g/1) TS.
Category. Anti-infective agent.
Storage. Idoxuridine should be kept in a well-closed container, protected from

light.
Labelling. The designation Idoxuridine for sterile non-injectable use indicates

that the substance complies with the additional requirement and may be used
for sterile applications. Expiry date.
Additional information. Melting temperature, about 180 °C, with

decomposition.
Requirements
Idoxuridine contains not less than 98 0 . %
C 9 H IN 2 O 5
11 ,
Identity tests
503

infrared region.
spectrum obtained from idoxuridine RS or with the reference spectrum of
idoxuridine.
B. Dissolve about 2 mg in 50 ml of sodium hydroxide (0.01 mol/1) VS; the

absorption spectrum, when observed between 230 nm and 350 nm, exhibits
a maximum at about 279 nm.
C. See the test described below under “Related substances". The principal spot
obtained with solution D corresponds in position, appearance, and inten-
sity to that obtained with solution E.
D. Heat about 5 mg in a test-tube over an open flame; violet vapours are

evolved.
Specific optical rotation. Use a O.lOg/ml solution in sodium hydroxide (1
mol/1) VS and determine the rotation immediately after preparation. Calculate

with reference to the dried substance; [a]D°“‘^ = -1-28° to +32°.
Iodine and iodide. For solution (A) dissolve O.lOg in a mixture of 20ml of
water and 5 ml of sodium hydroxide (~80g/l) TS, and immediately add 5 ml of
sulfuric acid (-100 g/1) TS. Cool in an ice-bath, allow to stand for 10 minutes,
shaking occasionally, and filter. For solution (B) dissolve 0.11 Ig of potassium
iodide R in sufficient water to produce 1000ml. To 1.0ml of this solution add
19ml of water, 5ml of sodium hydroxide (~80g/l) TS, and 5ml of sulfuric acid
(-100 g/1) TS, mix, and filter. Transfer both filtrates from solutions A and B to
103 separate comparison tubes, to each add 10 ml of dichloromethane R and 3
drops of potassium iodate (0.05 mol/1) VS, shake for 30 seconds, and allow to
stand. The colour in the dichloromethane layer of solution A is not more
intense than that produced with solution B when compared as described under
1.11 Colour of liquids.
Solution in alkali. A solution of O.lOg in 10ml of sodium hydroxide (1 mol/1)

VS is clear and colourless.
Loss on drying. Dry about 1 g to constant mass at 60 °C under reduced pres-
sure (not exceeding 0.6kPa or about 5mm

of mercury) over phosphorus pen-
toxide R; it loses not more than lOmg/g.
5 volumes of 2-propanol R, 4 volumes of dichloromethane R, and 1 volume of
504
of each of 3 solutions in a mixture of 5 volumes of methanol R and 1 volume

of ammonia (-260 g/1) TS containing (A) 40 mg of Idoxuridine per ml, (B)
0.20 mg of Idoxuridine per ml, (C) 0.10 mg of Idoxuridine per ml, (D) 4 mg of
Idoxuridine per ml, and(E) 4 mg of idoxuridine RS per ml. After removing the
plate from the chromatographic chamber, dry it in a current of cold air and
repeat the development. After removing the plate following the second devel-
opment from the chromatographic chamber, dry it in a current of cold air, and
examine the chromatogram in ultraviolet light (254 nm).
intense than that obtained with solution B (0.5%). Not more than 3 such spots
are more intense than the spot obtained with solution C (0.25%).

mamide R and titrate with tetrabutylammonium hydroxide (0.1 mol/1) VS as
described under 2.6 Non-aqueous titration. Method B, determining the end-
point potentiometricaUy.

35.41 mg of Cc,HiiIN 205 .
Additional requirement for Sterile non-injectahle Idoxuridine

IMIPRAMINI HYDROCHLORIDUM
IMIPRAMINE HYDROCHLORIDE
Chemical name. 5-[3-(Dimethylamino)propyl]-10,ll-dihydro-5/f-dibenz[/?,/]

azepine monohydrochloride; 10,1 l-dihydro-A/,Af-dimethyl-5//-dibenz[/?,/]
azepine-5-propanamine monohydrochloride; CAS Reg. No. 113-52-0.
Other name. Imizine.
505
Description. A white or slightly yellowish, crystalline powder; odourless or

almost odourless.
Solubility. Freely soluble in water, ethanol (-750 g/1) TS; practically insoluble
in ether R.
Category. Psychotherapeutic drug.
Storage. Imipramine hydrochloride should be kept in a tightly closed con-

Additional information. Even in the absence of light, Imipramine hydrochlo-

ride is gradually degraded on exposure to a humid atmosphere, the decompo-
sition being faster at higher temperatures.
Requirements
Imipramine hydrochloride contains not less than 98 0 . %
and not more than the
of C, 9 H 24N 2 ,HC 1 calculated with reference to the dried
,
substance.
Identity tests
infrared region.
spectrum obtained from imipramine hydrochloride RS or with the reference
spectrum of imipramine hydrochloride.
B. Dissolve 2 mg in 2.0 ml of water and add 2 ml of nitric acid (-1000 g/1) TS;
an intense blue colour is produced.
C. Dissolve 0.05g in 3ml of water and add 0.05ml of quinhydrone/methanol

TS; no red colour develops within 15 minutes.
Clarity and colour of solution. Dissolve rapidly by shaking and triturating

with a glass rod 1 g in 10ml of carbon-dioxide-free water R; the solution is clear.
Immediately dilute half the solution with an equal volume of water; it is not
506
more intensely coloured than standard colour solution Yw3 when compared as
described under 1.11 Colour of liquids. (Keep the remaining solution for the
“pH value’’.)
5.0mg/g.
pH value. pH of the solution prepared for the “Clarity and colour of solution”
measured immediately after preparation, 3. 6-5.0.
5 volumes of hydrochloric acid (-250 g/1) TS, 5 volumes of water, 35 volumes
of glacial acetic acid R, and 55 volumes of ethyl acetate R as the mobile phase.
Apply separately to the plate 10 pi of each of three solutions in methanol R pre-
pared immediately before use containing (A) 25 mg of Imipramine hydrochlo-
ride per ml, (B) 0.05 mg of Imipramine hydrochloride per ml, and (C) 0.05 mg
of iminodibenzyl R per ml. After removing the plate from the chromatographic
chamber, allow it to dry for 5 minutes, spray with a solution of 0.5 g of potas-
sium dichromate R dissolved in 100 ml of a mixture of 4 volumes of water and
1 volume of sulfuric acid (~1760g/l) TS. Examine the chromatogram immedi-
ately in daylight.
Any spot obtained with solution A, other than the principal spot which should
be blue in colour and the spot corresponding to iminodibenzyl as obtained with
solution C, is not more intense than the spot obtained with solution B. Fur-
thermore, any spot obtained with solution A corresponding to iminodibenzyl

is not more intense than the spot obtained with solution C.
(0.1 mol/1) VS as described under 2.6 Non-aqueous titration. Method A.

C„H 24N ,HC2 1.
507
INDINAVIRI SULFAS
INDINAVIR SULFATE
H.SO,
C36H47N504,H204S
Chemical name. (2S)-l-[(2S,4R)-4-benzyl-2-hydroxy-5-[[(lS,2/^)-2-hydroxy-

2,3-dihydro-lfi-inden-l-yl]amino]-5-oxopentyl]-W-(l ,l-dimethylethyl)-4-
(pyridin-3-ylmethyl)piperazine-2-carboxamide sulfate; CAS Reg. No.
157810-81-6.
Solubility. Freely soluble in water, soluble in methanol.
Category. Antiretroviral (protease inhibitor).
Storage. Indinavir sulfate should be kept in a tightly closed container, pro-

tected from light.
Additional information. Indinavir sulfate occurs as the monoethanolate

which is hygroscopic. It converts to the hydrate upon loss of ethanol and expo-
sure to moist air.
Requirements
98.5% and not more than 101.0% of
Indinavir sulfate contains not less than
C36H47N504,H204S Calculated on anhydrous, ethanol free basis.
Identity tests
• Either tests A, B and D, or tests C and D may be applied.
508
phy, using silica gel R6 as the coating substance and a mixture of 8
volumes of dichloromethane R and 2 volumes of 2-propanol as the
5 mg of indinavir RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry exhaustively in a current of cool
air. Examine the chromatogram in ultraviolet light (254 nm).

volumes of dichloromethane R and 2 volumes of 2-propanol as the
5 mg of indinavir RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry exhaustively in a current of cool
air. Spray with vanillin/sulfuric acid TSl. Heat the plate for a few
minutes at 120 °C. Examine the chromatogram in daylight.

B. The absorption spectrum of a 0.1 00 mg/ml solution, when observed

between 220 nm and 280 nm, exhibits one maximum at about 260 nm; the
specific absorbance (AJ°(°J is between 56 and 65.
C. Dissolve 0.1 g in 10 ml of water, add 2 ml of sodium hydroxide (~80g/l) TS

and shake. Filter the resulting precipitate and wash with two 3-ml quanti-
ties of water. Dry the washed precipitate for one hour at 105 °C. Using the
dried precipitate thus obtained, carry out the examination as described
under "1.7 Spectrophotometry in the infrared region’’. The infrared absorp-
tion spectrum is concordant with the spectrum obtained from indinavir RS
or with the reference spectrum of indinavir.

Specific optical rotation. Use a lO.Omg/ml solution and calculate with ref-
erence to the anhydrous and ethanol free substance; = -1-27° to -1-31 °.
509

Fischer method, Method A, using 0.5 g of the substance; the water content is
Ethanol content. Determine by 1.14.5 Gas chromatography, using static

head-space injection. Use a fused-silica capillary or wide bore column 30 m long
and 0.32 mm or 0.53 mm
in internal diameter coated with macrogol 20 R M
(film thickness; 0.25 pm).
As detector use a flame ionization detector.
Use nitrogen forchromatography R or helium R as the carrier gas at an appro-

priate pressure and a split ratio 1 :5 with a linear velocity of about 35 cm/sec.
The following head-space injection conditions may be used:
Equilibration temperature (°C) 80

Equilibration time (min) 60
Transfer line temperature (°C) 85
Pressurization time (s) 30
Injection volume (ml) 1
Maintain the temperature of the column at 30 °C for 7 min, then raise the tem-
perature at a rate of 35 °C per min to 180°C and maintain for 10 min, main-
taining the temperature of the injection port at 140 °C and that of the flame
ionization detector at 250 °C.
Test solution. Dissolve 0.200 g of the test substance in purified water and dilute
to 20.0ml with the same solvent. Introduce 5.0ml of this solution and 1.0ml
of purified water into a headspace vial. Prepare two more vials.
Reference solutions. Add 0.200 g of ethanol R to purified water and dilute to

200.0ml with the same solvent. Transfer respectively 2.0 ml, 3.0 ml and 4.0ml
in separate headspace injection vials and bring the volume to 6.0ml with puri-
fied water.
510
Blank solution. Introduce 6.0 ml of purified water into a headspace vial.
Analyse the blank solution and then alternatively three times the test solution
and the three reference solutions.
The test is not valid unless the relative standard deviation on the areas of the
peaks obtained from the test solutions is not more than 5%.
Calculate the ethanol content by using the results obtained with the test solu-
tion and with the reference solutions; the ethanol content is not less than
raphy R (5 pm).
Use the following conditions for gradient elution:
Mobile phase A: 30 volumes of acetonitrile R, 5 volumes of phosphate buffer

pH 7.5 and 65 volumes of purified water.
Mobile phase B: 70 volumes of acetonitrile R, 5 volumes of phosphate buffer

Prepare the phosphate buffer pH 7.5 by dissolving l.Og of anhydrous disodium

hydrogen phosphate R in 50 ml of purified water, adjust the pH to 7.5 by adding
phosphoric acid (-105 g/1) TS and dilute it to 100 ml with purified water.
Time Mobile phase A Mobile phase B Comments

(min) (% v/v) (% v/v)
0-5 93 7 Isocratic
5-25 93 to 20 7 to 80 Linear gradient
30-35 20 to 93 80 to 7 Return to the initial conditions
35-45 93 7 Isocratic re-equilibration
Prepare the following solutions. For solution (1) use 2.0 mg of the test substance
per ml. For solution (2) dilute a suitable volume of solution (1) to obtain a con-
centration equivalent to 2pg of Indinavir sulfate per ml.
For the system suitability test: prepare solution (3) using 2 ml of solution (1)
and 2 ml of sulfuric acid (190 g/1), heat carefully in a boiling water bath for 10
minutes.
511
spectrophotometer set at a wavelength of 220 nm.
Maintain the column temperature at 40 °C.
Inject20 pi of solution (3). The test is not valid unless the resolution factor
between the two major peaks, with a retention time between 15 and 20min,
is less than 3.5. If necessary adjust the amount of acetonitrile in mobile
not
phase A, or adjust the gradient program.
Inject alternatively 20 pi each of solutions (1) and (2).
In the chromatograms obtained with solution (1), the area of any peak, other
than the principal peak, not greater than the area of the principal peak
is
obtained with solution %). The sum of the areas of all peaks, other than
(2) (0.1
the principal peak, is not greater than five times the area of the principal peak
obtained with solution (2) (0.5%). Disregard any peak with an area less than
0.5 times the area of the principal peak in the chromatogram obtained with
solution (2) (0.05%).
Assay. Dissolve 0.300 g, accurately weighed, in 50 ml of water and titrate with

sodium hydroxide (0.1 mol/1) VS, determine the end point potentiometrically.
C 36 H 47N 504 ,H 204 S; calculate with reference to the anhydrous and ethanol free
substance.
INDOMETACINUM
INDOMETACIN
Molecular formula. C19H16CINO4
Graphic formula.
512
Chemical name. l-(^-Chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic

acid; l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lf/-indole-3-acetic acid; CAS
Reg. No. 53-86-1.
Description. A white or a pale yellow, crystalline powder; odourless or almost

odourless.

TS and ether R.
Category. Analgesic; anti-inflammatory.
Storage. Indometacin should be kept in a well-closed container, protected

from light.
Additional information. Indometacin exhibits polymorphism. The poly-

morph specified in the monograph corresponds to the crystal form of
indometacin RS.
Requirements
Definition. Indometacin contains not less than 98.0% and not more than
101.0% of C19H16CINO4, calculated with reference to the dried substance.
Identity tests

infrared region. The infrared absorption spectrum obtained from the solid
state of the test substance is concordant with the spectrum obtained from
indometacin RS or with the indometacin (confirmation
reference spectrum of
of polymorphic form).
B. Dissolve 0.1 g in 100ml of water containing 0.5ml of sodium hydroxide

(1 mol/1) VS. To a 1 ml-portion add 1 ml of freshly prepared sodium nitrite
(lg/1) TS and allow to stand for 5 minutes. Add 0.5ml of sulfuric acid
(-1760 g/1) TS; a deep yellow colour is produced. To another 1-ml portion
add 1 ml of sodium TS, and allow to stand for 5 minutes. Add

nitrite (1 g/1)
0.5 ml of hydrochloric acid (-420 g/1) TS; a green colour is produced.
513
chromatography, using silica gel R2 as the coating substance and preparing the
slurry in sodium dihydrogen phosphate (45g/l) TS. As the mobile phase, use a
mixture of 7 volumes of ether R and 3 volumes of light petroleum R. Apply
separately to the plate 10 pi of each of 2 solutions in methanol R containing (A)
20mg of the test substance per ml and (B) O.lOmg of the test substance per ml.
Assay. Dissolve about 0.33 g, accurately weighed, in 75 ml of acetone R

through which nitrogen R free from carbon dioxide has previously been passed
for 15 minutes. Maintain a constant stream of nitrogen through the solution
and titrate with carbonate -free sodium hydroxide (0.1 mol/1) VS using phe-
nolphthalein/ethanol TS as indicator or determining the endpoint potentio-
metrically. Repeat the operation without the substance being examined and
make any necessary corrections. Each ml of carbonate-free sodium hydroxide
(0.1 mol/1) VS is equivalent to 35.78mg of C19H15CINO4.
514
Ji
INSULINUM
INSULIN
pGlu- Val-lle-Gly-H Pork Insulin

4 3 2 1
Chain A
Gin - Cys - Cys - Thr - Ser - He - Cys - Ser - Leu - Tyr - Gin - Leu - Glu - Asn - Tyr - Cys - Asn - OH
5 6 9 10 11 12 13 14 15 16 17 18 19 20 21
pGIn - His - Leu - Cys-Gly- Ser - His - Leu - Val - Glu - Ala - Leu - Tyr - Leu - Vat - Cys - Gly- Glu —
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
^ Asn - Val - Phe - H Chain B HO - Ala - Lys - Pro - Thr - Tyr - Phe - Phe - Gly - Arg—
3 2 1 30 29 28 27 26 25 24 23 22
-Glu - Val -Me- Gly -H Beef Insulin

4 3 2 1
-Gin - Cys - Cys - Ala - Ser - Val- Cys - Ser - Leu - Tyr - Gin - Leu - Glu - Asn - Tyr - Cys- Asn - OH
5 6 7v 8 9 10 11 12 13 14 15 16 17 18 19 20 21
-Gin - His - Leu - Cys - Gly - Ser - His - Leu - Val - Glu - Ala - Leu - Tyr - Leu - Val - Cys - Gly - Glu
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
- Asn - Val- Phe -H HO - Ala - Lys - Pro - Thr - Tyr - Phe - Phe - Gly - Arg -
3 2 1 30 29 28 27 26 25 24 23 22
C 256H 331 N 65 O 76S 6 (pork)

C 254 H 377N 65 O 75 S 6 (beef)
Definition. Insulin is the natural antidiabetic hormone obtained from pork or

beef pancreas and purified.
Chemical name. [Pork] Insulin; porcine insulin; CAS Reg. No. 12584-58-6.
[Beef] Insulin; bovine insulin; CAS Reg. No. 11070-73-8.
Solubility. Practically insoluble in water, ethanol (~750g/l) TS, and ether R;

dissolves in dilute solutions of mineral acids and alkali hydroxides.
Category. Antidiabetic agent.
Storage. Insulin should be kept in a tighdy closed container, protected from

light, and stored at a temperature not below -20 °C.
515
Labelling. The designation on the container should state the animal source of
the insulin; expiry date.
Additional information. Insulin is hygroscopic; it degrades in solutions of

alkali hydroxides. Attention should be paid to the microbiological quality since
Insulin is of natural origin.
The which represents a unique case among prepara-

quality of insulin,
tions in theWHO Model List of Essential Drugs, cannot be adequately
controlled by methods adopted for The International Pharmacopoeia.
Therefore, it is advised to refer to methods described in other pharma-
copoeias, such as those of China, Europe, Japan, UK, and USA.
lODUM
IODINE
Molecular formula. L
Chemical name. Iodine; CAS Reg. No. 7553-56-2.
Description. Heavy, greyish black plates or granules, having a metallic lustre.
Solubility. Very slightly soluble in water; soluble in ethanol (-750 g/1) TS;
freely soluble in carbon tetrachloride R, carbon disulfide R, and ether R.
Category. External antiseptic.
Storage. Iodine should be kept in a tightly closed container, preferably made

of glass and provided with a glass stopper.
Additional information. Iodine volatilizes slowly at room temperature

giving violet, irritant vapours.
Requirements
Definition. Iodine contains not less than 99.5% and not more than 100.5%
of I.
516
Identity tests
A. Dissolve 0.05g in 10ml of ethanol (~750g/l) TS; the colour of the solution
is reddish brown. Dissolve 0.05g in 10ml of carbon tetrachloride R; the
colour of the solution is violet.
B. To a saturated solution in water add starch TS; a blue colour is produced.

Boil the mixture for a short time; the solution loses its colour, but on cooling
the colour reappears.
Chlorides and bromides. Triturate 1.5g with 10ml of water, filter, wash the
filter and dilute the filtrate to 15 ml with water. To the solution add 0.5 g of zinc
R powder. When the solution has become decolourized, filter and wash the
filter with sufficient water to adjust the volume of the filtrate to 20 ml. To 5 ml
of the filtrate (keep the remaining filtrate for the test for cyanides) add 1.5ml
of ammonia (-260 g/1) TS and 3 ml of silver nitrate (40g/l) TS, filter, wash the
filter with sufficient water to adjust the volume of the filtrate to 10 ml, add
1.5ml of nitric acid (~1000g/l) TS and allow to stand for 1 minute. Any opales-
cence in the solution is not more intense than that obtained from a solution
simultaneously prepared by mixing 10.75ml of water, 0.25 ml of hydrochloric
acid (0.01 mol/1) VS, 0.2ml of nitric acid (~130g/l) TS, and 0.3ml of silver nitrate
(40 g/1) TS; this indicates that the content of chlorides and bromides is not more
than 0.25 mg/g.
Cyanides. To 5 ml of the filtrate obtained in the test for chlorides and bro-
mides add 0.2ml of ferrous sulfate (15g/l) TS and 1ml of sodium hydroxide
(-80 g/1) TS. Heat for a few minutes and acidify with hydrochloric acid (-70 g/1)
TS; no blue or green colour is produced.
Non-volatile residue. Place about 1 g, accurately weighed, in a porcelain dish

and heat on a water-bath until the iodine has volatilized. Dry the residue for 1
hour at 105 °C and weigh; not more than l.Omg/g.
Assay. Dissolve about 0.5 g of finely powdered test substance, accurately

weighed, in a solution of 1 g of potassium iodide R in 5 ml of water. Dilute with
water to about 50 ml, add 1 ml of hydrochloric acid (-70 g/1) TS and titrate with
sodium thiosulfate (0.1 mol/1) VS, using starch TS as indicator. Each ml of
sodium thiosulfate (0.1 mol/1) VS is equivalent to 12.69mg of I.
Additional requirement for Iodine for pârenteral use

517
lOHEXOLUM
lOHEXOL
C19H26I3N3O9
Chemical name. N,N'-Bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)-

acetamido]-2,4,6-triiodoisophthalamide; 5-[acetyl(2,3-dihydroxypropyl)amino]-
A/,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-l,3-benzenedicarboxamide; CAS
Reg. No. 66108-95-0.
Description. A white to greyish, amorphous powder; odourless.
Solubility. Very soluble in water and methanol R.
Storage. lohexol should be kept in a well-closed container, protected from

light.
Additional information. Melting range, 177-1 87 °C.
Requirements
lohexol contains not less than 98 5 .% and not more than the equivalent of
101 .5 % of C 19 H 26 I 3 N 3 O 9 calculated with reference to the anhydrous substance.
,
Identity tests

spectrum obtained from iohexol RS or with the reference spectrum of iohexol.
518
B. The absorption spectrum of a lOpg/ml when observed between

solution,
230 nm and 350 nm, exhibits a maximum at about 245 nm; the absorbance
of a 1-cm layer at the maximum wavelength is about 0.36.
D. Heat 0.05 g in a suitable crucible; violet vapours are evolved.
Aluminium. Place in three separatory funnels 2 ml of a freshly prepared alu-

minium standard (lOpg Al/ml) TS, 10ml of a solution containing 0.5g of
lohexol per ml, and 10 ml of water to serve as a blank. To each funnel add
5 ml of ammonium chloride buffer, pH 10.5, TS and dilute to 25ml with water.
To every funnel add 5 ml of 8-hydroxyquino line/ chloroform TS and shake for
2 minutes. Allow the phases to separate and measure the absorbance of the
extracts against the extracted blank solution at a maximum at about 395 nm;
the aluminium content is not more than 4pg/g.
Copper. Place in three separatory funnels 0.25ml of a freshly prepared copper

standard (lOpg/ml Cu) TS, 10 ml of a solution containing 0.5 g of lohexol per
ml, and 15ml of water To each funnel add 1 ml of ammo-
to serve as a blank.
nium pyrrolidinedithiocarbamate (lOg/1) TS and 5 ml of acetate buffer, pH 4.5,
TS and dilute to 25ml with water. To each funnel add 5 ml of isobutyl methyl
ketone R and shake for 2 minutes. Allow the phases to separate and measure
the absorbance of the extracts against the extracted blank solution at a
maximum at about 435 nm; the copper content is not more than 0.5pg/g.
Halides. Dissolve 5g in 20 ml of water and titrate with silver nitrate

(0.001 mol/1) VS, determining the endpoint potentiometrically, using silver/silver
chloride electrodes. Repeat the procedure without the lohexol being examined
and make any necessary corrections. Each ml of silver nitrate (0.001 mol/1) VS
is equivalent to 0.1269mg of 1; the content of halides, calculated as iodides,
does not exceed 20pg/g.
Colour of solution. Dissolve 6.47 g in sufficient water to produce 10 ml (apply

a correction for any presence of water). The concentration of the solution is
64.7%, equivalent to 300mg of 1 per ml. Filter through a MiUipore filter
0.22 pm, and measure the absorbance in a 1-cm cell using water as a blank at
about 400 nm, 420 nm, and 450 nm. The absorbance is not greater than 0.200,
0.050, and 0.025, respectively.

Fischer method. Method A, using 0.2 g; the water content is not more than
50mg/g.
519
50 volumes of 1-butanol R, 11 volumes of acetic acid (-300 g/1) TS, and 25
volumes of water as the mobile phase. Apply separately to the plate lOpl of
each of four solutions in methanol R containing (A) lOmg of lohexol per ml,
(B) 10 mg of iohexol RS per ml, (C) 20 mg of lohexol per ml, and (D) 40 pg of
lohexol per ml. After removing the plate from the chromatographic chamber,
(254 nm).
Any spot obtained with solution C, other than the principal spot, is not more
Primary aromatic amines

• The solutions must be protected from light throughout the procedure.
To about 0.2 g, accuratelyweighed, add 15 ml of water, mix to dissolve, and

allow to stand in an ice-bath for 5 minutes. Add 1.5ml of hydrochloric acid
(-250 g/1) TS and 2 ml of sodium nitrite (10 g/1) TS, mix, and return the flask to
the ice-bath for exactly 4 minutes. Add 1ml of sulfamic acid (50 g/1) TS and
again place the flask in the ice-bath for exactly 1 minute. Remove the flask from
the bath, add 0.5ml of freshly prepared N-(l-naphthyl)ethylenediamine
hydrochloride/propylene glycol TS, mix, and dilute to 25 ml with water. Within
20 minutes measure the absorbance of a 5-cm layer at the maximum at about
495nm against a solvent cell containing a solution prepared by treating 15ml
of water in a similar manner; the absorbance does not exceed 0.21.
Assay. Carry out the combustion as described under 2.4 Oxygen flask method
for iodine, using about 7.5 mg, accurately weighed. Titrate the liberated iodine
with sodium thiosulfate (0.01 mol/1) VS.
Each ml of sodium thiosulfate (0.01 ml/1) VS is equivalent to 0.4562 mg of

Cl9H26l3hl309-
IPECACUANHAE RADIX
IPECACUANHA ROOT
Description. Odour, slight; taste, bitter, nauseous and acrid.
Category. Expectorant; emetic.
Storage. Ipecacuanha root should be kept in a well-closed container, protected

from light.
520
Additional information. Even in the absence of light, the powder of

Ipecacuanha root is gradually degraded on exposure to a humid atmosphere,
Requirements
Definition. Ipecacuanha root consists of the dried rhizome and roots of
Cepha'ilis if>ecacuanha (Brotero) A. Richard (Earn. Rubiaceae) or of Cephaelis
acuminata Karsten, or of a mixture of both species. The principal alkaloids are
emetine and cephaeline.
Ipecacuanha root contains not less than 2.0% of total alkaloids, calculated as
emetine.
Macroscopic characteristics
Cephaelis ipecacuanha. Dark brick-red to very dark brown, somewhat tortuous
root, seldom more than 15 cm long or 6 mm thick; the root is closely annulated
externally, having rounded ridges completely encircling it; the fracture is short
in the bark and spKntery in the wood; a transversely cut surface shows a wide
greyish bark and a small uniformly dense wood. The rhizomes are short lengths
attached to roots; they are cylindrical, up to 2 mm
in diameter, finely wrinkled
longitudinally, and with pith occupying approximately one-sixth of the whole
diameter.
Cephaelis acuminata. In general it resembles the root of Cephaelis ipecacuanha,

but differs in the following particulars: often up to 9mm thick; external surface
greyish brown or reddish brown with transverse ridges at intervals of about
1-3 mm; the ridges are about 0.5-1 mm wide, extending about half-way round
the circumference and fading at the extremities into the general surface level.
Microscopic characteristics
Cephaelis ipecacuanha. A transverse section of the root shows a narrow, brown
cork layer of thin-walled polyhedral, tubular cells and a wide parenchymatous
zone of pheUoderm; the abundant starch, consisting of simple

latter contains
granules and compound granules of 2-8 components, the individual granules

being oval, rounded, or roughly hemispherical, and seldom more than 15 pm in
diameter; the phloem is present as a narrow unlignified zone; the xylem is dense,
consisting mainly of narrow tracheids intermixed with a smaller proportion of
vessels, both with numerous bordered pits in their lateral walls, the vessel
element having simple circular perforations; crystal cells, each containing a
bundle of raphides, 30-80 pm long, occur in the parenchymatous regions. A trans-
verse section through an internode of the rhizome shows several layers of thin-
walled cork, a somewhat collenchymatous cortex, a pericycle containing groups
of large, distinctly pitted sclereids, a narrow ring of phloem, and a wide ring of
xylem surrounding a pith composed of thin-walled, pitted, parenchymatous cells.
521
Cephaelis acuminata. Similar to Cephaelk ipecacuanha except that the individual

starch granules may be up to 22 pm in diameter.
Identity test
silica gel R1 as the coating substance and a mixture of 93 volumes of chloro-
form R, 6.5volumes of methanol R, and 0.5 volumes of ammonia (-260 g/1) TS
as the mobile phase, and allow the solvent front to ascend only 10 cm above
the line of application. Apply separately to the plate as bands, 20 mm
by 3 mm,
lOpl of each of the following solutions: for solution A, add to 0.1 g of finely
powdered test substance in a small test-tube, 0.05ml of ammonia (-260 g/1) TS
and 5 ml of chloroform R, stir vigorously with a glass rod, allow to stand for
30 minutes and filter; for solution B, dilute 1 ml of solution A to 25 ml with
chloroform R; for solution C, dissolve 5 mg of emetine hydrochloride RS and
6mg of cephaeline hydrochloride R in sufficient methanol R to produce 20 ml.
air until the odour of solvent is no longer detectable, spray it with a mixture
of 0.05g of iodine R in 10ml of chloroform R, and heat it at 60°C for 10

minutes. Examine the chromatogram first in daylight; a lemon-yellow zone
appears at about midpoint, corresponding to emetine, and below it a light
brown zone, corresponding to cephaeline. Then examine the chromatogram in
ultraviolet light (365 nm); the zone corresponding to emetine shows an intense
yellow fluorescence, and that corresponding to cephaeline, a light blue fluo-
rescence. The chromatogram obtained with solution A shows, in addition,
several very small zones due to secondary alkaloids. The chromatogram
obtained with solution B shows only 2 zones, which correspond to those
With Cephaelis ipecacuanha, the zone corresponding to cephaeline obtained

with solution A is much smaller than the corresponding zone obtained with
solution C.
With Cephaelis acuminata, the principal zones obtained with solution A corre-
spond in position, appearance, and intensity with those obtained with solution
C.
Ash. Carry out the procedure as described under 4.1 Determination of ash and
acid-insoluble ash; not more than 60mg/g.
Acid-insoluble ash. Carry out the procedure as described under 4.1 Deter-
mination of ash and acid-insoluble ash; not more than 30mg/g.
Foreign matter. Weigh about 200 g and spread it in a thin layer on a glass
plate. Detect the foreign matter by eye or with the use of a 6x lens, separate
it from the root, and weigh it; not more than lOmg/g.
522
Assay. Weigh accurately about 7.5 g of finely powdered test substance, trans-
fer it to a dry flask, add 100 ml of ether R, and shake for 5 minutes. Add 5 ml
of ammonia (-100 g/1) TS, shake frequently during 1 hour, add 5ml of water,
and shake vigorously; decant the ether layer into a dry flask, filtering through
a plug of adsorbent cotton. Wash the residue with two quantities, each of
25 ml of ether R, decanting each portion and filtering through the same plug of
adsorbent cotton. Remove most of the solvent from the combined ether
extracts by distillation and the remainder by gentle warming with a current of
air blown into the flask. Dissolve the residue in 5 ml of previously neutralized
ethanol (-710 g/1) TS by warming on a water-bath, add 15ml of hydrochloric
acid (0.1 mol/1) VS and titrate the excess of acid with sodium hydroxide
(0.1 mol/1) VS, using 0.5 ml of methyl red/methylthioninium chloride TS as indi-
cator. Each ml of hydrochloric acid (0.1 mol/1) VS is equivalent to 24.03mg of
total alkaloids, calculated as emetine.
ISONIAZIDUM
ISONIAZID
Molecular formula. CeHjNsO
Graphic formula.
Chemical name. 4-Pyridinecarboxylic acid hydrazide; CAS Reg. No. 54-85-3.
Other name. Isonicotinic acid hydrazide.
very slightly soluble in ether R.
Category. Tuberculostatic.
Storage. Isoniazid should be kept in a well-closed container, protected from

light.
523
Requirements
Definition. Isoniazid contains not less than 98.0% and not more than 101.0%
of C6H7N3O, calculated with reference to the dried substance.
Identity tests

the spectrum obtained from isoniazid RS or with the reference spectrum of
isoniazid.
B. Heat 0.05g with about 1 g of anhydrous sodium carbonate R; pyridine, per-

ceptible by its odour, is produced.
C. Dissolve 0.1 g in 2 ml of water and add 10 ml of a hot solution of vanillin

(lOg/1) TS, scratch the inside of the test-tube and allow to stand; a yellow
precipitate is obtained. Filter, recrystaUize from 5 ml of ethanol (-600 g/1) TS,
and dry at 105°C; melting temperature, about 227 °C.
Clarity and colour of solution. A solution of O.SOg in 10ml of water is clear

and colourless.
lOmg/g.
Free hydrazine. Carry out the test as described under 1.14.1 Thin-layer chro-
volumes of acetone R and 2 volumes of water as the mobile phase. Apply sep-
arately to the plate 10 pi of each of 2 solutions in a mixture of 1 volume of
acetone R and 1 volume of water containing (A) O.lOg of the test substance per
ml, and (B) 20 pg of hydrazine hydrate R per ml. After removing the plate from
the chromatographic chamber, allow it to dry in a current of air, spray with 4-
dimethylaminobenzaldehyde TS3, and examine the chromatogram in daylight.
524
The spot obtained with solution B is more intense than any spot, correspond-
ing in position and appearance, obtained with solution A.

produce 100ml. To 25.0ml of this solution add 100ml of water, 20ml of
hydrochloric acid (-250 g/1) TS, 0.2 g of potassium bromide R, and 3 drops of
methyl red/ethanol TS. Titrate with potassium bromate (0.0167 mol/1) VS,
adding the titrant drop by drop and shaking till the red colour disappears.
Repeat the operation without the substance being examined and make any nec-
essary corrections. Each ml of potassium bromate (0.0167 mol/1) VS is equiva-
lent to 3.429 mg of C^HyNsO.
ISOPRENALINI HYDRO CHLORIDUM

ISOPRENALINE HYDROCHLORIDE
Molecular formula. CnHi 7N 03 ,HCl
Graphic formula.
HO
Chemical name. 3,4-Dihydroxy-a-[(isopropylamino)methyl]benzyl alcohol

hydrochloride; 4-[l-hydroxy-2-[(l-methylethyl)amino]ethyl]-l,2-benzenediol
hydrochloride; a-[(isopropylamino)methyl]protocatechuyl alcohol hydrochlo-
ride; CAS Reg. No. 51-30-9.
Category. Bronchodilator.
Storage. Isoprenaline hydrochloride should be kept in a tightly closed con-

Additional information. Isoprenaline hydrochloride gradually darkens in

colour on exposure to air and light. Even in the absence of light, Isoprenaline
525
hydrochlorideis gradually degraded on exposure to a humid atmosphere, the
Requirements
Definition. Isoprenaline hydrochloride contains not less than 97.5% and not
more than 101.0% of C„Hi 7N 03 ,HCl, calculated with reference to the dried
substance.
Identity tests
A. The absorption spectrum of a 0.050mg/ml solution, when observed
between 240 nm and 350 nm, exhibits a maximum at about 280 nm; the
absorbance of a 1-cm layer at this wavelength is about 0.50.
B. Add 1ml of a l.Omg/ml solution to each of two flasks, one containing

10ml of buffer phthalate, pH 3.4, TS, the other containing 10ml of buffer
phosphate, pH 6.4, TS; other buffers having the same pH may also be used.
Add ml of iodine (0.1 mol/1) VS, allow to stand for 5 minutes and add 2
0.5
ml of sodium thiosulfate (0.1 mol/1) VS. In the solution of pH 3.4, a strong
red colour is produced; in the solution of pH 6.4, a strong red-violet colour
is produced (distinction from levarterenol).
C. A 0.05 g/ml solution yields reaction B, described under 2.1 General identifi-
D. Melting temperature, about 169°C with decomposition.


exceeding 0.6kPa or about 5mm of mercury) over phosphorus pentoxide R for
4 hours; it loses not more than lOmg/g.
Isoprenalone. The absorbance of a 1-cm layer of a l.Omg/ml solution in sul-

furic acid (0.005mol/l) VS at 310nm is not more than 0.2 (preferably use 2-cm
R1 with the aid of the minimum of heat, add 10 ml of mercuric acetate/acetic
acid TS, and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6
526
Non-aqueous titration. Method ml

A. Each of perchloric acid (0.1 mol/1) VS is
equivalent to 24.77mg of CnHi/NOgjHCl.
Additional requirements for Isop>renaline hydrochloride for

p'arenteral use
ISOPRENALINI SULFAS
ISOPRENALINE SULFATE
Molecular formula. (CnHi 7 N 03) 2 ,H 2 S 04 ,
2 H 20
Graphic formula.
HO
HO 2HjO
V_/ I
OH
Chemical name. 3,4-Dihydroxy-a-[(isopropylamino)methyl]benzyl alcohol

sulfate (2: 1) (salt) dihydrate; 4-[l-hydroxy-2-[(l-methylethyl)amino]ethyl]-l,2-
benzenediol sulfate (2: 1) (salt) dihydrate; a-[(isopropylamino)methyl]protocat-
echuyl alcohol sulfate (2:1) (salt) dihydrate: CAS Reg. No. 6700-39-6.
Category. Bronchodilator.
Storage. Isoprenaline sulfate should be kept in a tightly closed container, pro-

tected from light.
Additional information. Isoprenaline sulfate gradually darkens in colour on

exposure to air and light. Even in the absence of light, Isoprenaline sulfate is
527

Requirements
Definition. Isoprenaline sulfate contains not less than 98.0% and not more
than 101.0% of (Ci,Hi 7 N 03 ) 2 ,H 2 S 04 calculated with reference to the anhy-
,
drous substance.
Identity tests
A. The absorption spectrum of a 0.050mg/ml solution, when observed
between 240 nm and 350 nm, exhibits a maximum at about 280 nm; the
absorbance of a 1-cm layer at this wavelength is about 0.50.
B. Add 1ml of a l.Omg/ml solution to each of two flasks, one containing

10ml of buffer phthalate, pH 3.4, TS, the other containing 10ml of buffer
phosphate, pH 6.4, TS; other buffers having the same pH may also be used.
Add 0.5ml of iodine (0.1 mol/1) VS, allow to stand for 5 minutes and add
2 ml of sodium thiosulfate (0.1 mol/1) VS. In the solution of pH 3.4, a strong
red colour is produced; in the solution of pH 6.4, a strong red-violet colour
is produced (distinction from levarterenol).
C. A 0.05 g/ml solution yields reaction A, described under 2.1 General identi-
fication tests as characteristic of sulfates.
D. Dissolve 0.1 g in 10ml of ethanol (~750g/l) TS, boil for 2 minutes and filter.
Add 5 ml of ether R to the filtrate; a white, crystalline precipitate is formed;

melting temperature, about 162 °C (anhydrous isoprenaline sulfate).


Fischer method, Method A, using about 0.15g of the substance; not less than
pH value. pH of a lOmg/ml solution, 4. 0-5. 5.
Isoprenalone. The absorbance of a 1-cm layer of a l.Omg/ml solution in sul-

furic acid (0.005mol/l) VS at 310nm is not more than 0.2 (preferably use 2-cm
528

R1 and 40 ml of acetonitrile R, and titrate with perchloric acid (0.1 mol/1) VS as
described under 2.6 Non-aqueous titration. Method A. Each ml of perchloric
acid (0.1 mol/1) VS is equivalent to 52. 06 mg of (CnHi 7 N 03 ) 2 ,H 2 S 04 .
Additional requirements for Isop'renaline sulfate for

parenteral use
ISOSORBIDI DINITRAS DILUTUS

DILUTED ISOSORBIDE DINITRATE
C6H8N2O8
CAUTION: To permit safe handling, Isosorbide dinitrate is mixed with a suit-

able inert diluent, such as lactose or mannitol.
Appropriate precautions in handling undiluted Isosorbide dinitrate need to be

observed, since it can explode if subjected to percussion or excessive heat. Only
exceedingly small amounts should be isolated.
Chemical name. 1,4: 3,6-Dianhydrosorbitol 2,5-dinitrate; CAS Reg. No.

87-33-2.
Description. Undiluted Isosorbide dinitrate is a fine, white, crystalline

powder.
Solubility. Undiluted Isosorbide dinitrate is very slightly soluble in water;

sparingly soluble in ethanol (-750 g/1) TS; very soluble in acetone R; freely
529
The solubility of the diluted product depends on the diluent and its
concentration.
Category. Antianginal drug.
Storage. Diluted Isosorbide dinitrate should be kept in a tighdy closed con-

Labelling. The designation on the container should state the percentage

content of Isosorbide dinitrate, C 6 HSN O 8
2 .
Additional information. Diluted Isosorbide dinitrate may contain a suitable

stabilizer, such as up to 1% of ammonium phosphate.
Requirements
Diluted Isosorbide dinitrate contains not less than 95 0 % and not more than
.
105 0 % of the amount of C6H8N20a

. stated on the label. It usually contains
20-50% of Isosorbide dinitrate.
Identity tests
reference spectrum of isosorbide dinitrate. (Instructions for the preparation of the
spectrum will he given on the reference spectrum.)
B. Dissolve in a test-tube a quantity equivalent to lOmg of Isosorbide dinitrate

in a mixture of 1.0ml of water and about 2.0ml of sulfuric acid (~1760g/l)
TS and cool. Introduce slowly 3.0 ml of ferrous sulfate (15g/l) TS to form
two layers; a brown colour is formed at the interface of the two liquids.
C. Shake a quantity equivalent to 25mg of Isosorbide dinitrate with 10ml of

acetone R and filter. Evaporate the filtrate to dryness at a temperature not
exceeding 40 °C and dry the residue under reduced pressure (not exceeding
O.dkPa or about 5mm of mercury) over phosphorus pentoxide R for 16
hours; melting point of the residue, about 71 °C.
metals content according to Method B; not more than lOpg/g.

exceeding 0.6kPa or about 5mm of mercury) over phosphorus pentoxide R for
16 hours; it loses not more than lOmg/g.
530
Inorganic nitrates. Carry out the test as described under 1.14.1 Thin-layer
6 volumes of toluene R, 3 volumes of ethyl acetate R, and 1.5 volumes of glacial
acetic acid R as the mobile phase. Apply separately to the plate 5 ml of each of
the two following solutions. For solution (A) shake a quantity equivalent to
0.10 g of Isosorbide dinitrate in 5 ml of ethanol (-750 g/1) TS and filter. Solution
(B) must be freshly prepared by dissolving 10 mg of potassium nitrate R in 1 ml
of water and diluting to 100 ml with ethanol (-750 g/1) TS. After removing the
plate from the chromatographic chamber, dry it in a current of air, and spray
with freshly prepared potassium iodide/starch TSl. Expose the plate to ultra-
violet light for 15 minutes. Examine the chromatogram in daylight.
Any spot corresponding to the nitrate ion obtained with solution A is not more
intense than that obtained with solution B (0.5%, calculated as potassium
nitrate).
8 volumes of toluene R and 2 volumes of ethyl acetate R as the mobile phase.
Apply separately to the plate 20 (il of each of the following 2 solutions. For solu-
tion (A) shake a quantity equivalent to 0.20 g of Isosorbide dinitrate with 5 ml
of acetone R and filter. For solution (B) dilute 1 volume of solution A to 200
volumes with acetone R. After removing the plate from the chromatographic
chamber, dry it in a current of air, and spray with diphenylamine/sulfuric acid
TS. Examine the chromatogram in daylight.
intense than that obtained with solution B (0.5%).
Assay. Shake a quantity equivalent to about 25 mg of Isosorbide dinitrate,

accurately weighed, with 15 ml of glacial acetic acid R for 15 minutes. Dilute
with sufficient glacial acetic acid R to produce 25ml and filter. To 1.0ml of the
filtrate add 2ml of phenoldisulfonic acid TS, allow to stand for 15 minutes, add
50 ml of water, make alkaline with ammonia (-260 g/1) TS, cool, and add suffi-
cient water to produce 100ml (solution A). Prepare similarly a solution con-
taining 0,20 g of potassium nitrate R, previously dried at 105°C, in 5 ml of water
and add sufficient glacial acetic acid R to produce 25ml. To 5 ml of the result-
ing solution add sufficient glacial acetic acid R to produce 50ml. To 1.0ml of
this solution add 2 ml of phenoldisulfonic acid TS, allow to stand for 15
minutes, add 50 ml of water, make alkaline with ammonia (-260 g/1) TS, cool,
and add sufficient water to produce 100ml (solution B).
Measure the absorbance of a Tcm layer at the maximum at about 405 nm of

solution A against a solvent cell containing solution B, and calculate the content
of C 6H 8N 2 O 8 .
531
Each ml of the potassium nitrate solution is equivalent to 0.934 mg of

C6H8N2O5.
KALII CHLORIDUM
POTASSIUM CHLORIDE
Potassium chloride (non-injectable)
Potassium chloride for parenteral use
Molecular formula. KCl
Chemical name. Potassium chloride; CAS Reg. No. 7447-40-7.

(-750 g/1) TS.
Category. Ionic equilibration agent.
Storage. Potassium chloride should be kept in a well-closed container.
Labelling. The designation Potassium chloride for parenteral use indicates that
the substance complies with the additional requirement given at the end of this
monograph and may be used for parenteral administration or for other sterile
applications.
Requirements
Definition. Potassium chloride contains not less than 99.0% and not more
than 100.5% of KCl, calculated with reference to the dried substance.
Identity tests
A. To a 0.05 g/ml solution add 2 ml of sodium hydroxide (~80g/l) TS; it yields
the reaction described under 2.1 General identification tests as characteris-
tic of potassium.

532
Iron. Use l.Og; the solution complies with the 2.2.4 Limit test for iron; not
more than 40 pg/g.
Calcium and magnesium. To 20ml of a 10 mg/ml solution add 2 ml of

ammonia (~100g/l) TS, 2 ml of ammonium oxalate (25g/l) TS and 2 ml of dis-
odium hydrogen phosphate (40g/l) TS; no turbidity is produced within 5
minutes.
Barium. Dissolve 0.5g in 10ml of carbon-dioxide-free and ammonia-free

water R and add 1ml of sulfuric acid (-100 g/1) TS; no turbidity is produced
within 1 minute.

under 2.2.5 Limit test for arsenic; the arsenic content is not more than 3 pg/g.
Bromides. Dissolve 0.08 g in 10 ml of water, add 0.25 ml of sulfuric acid

(0.5mol/l) VS and 2 drops of tosylchloramide sodium (15g/l) TS and mix; allow
to stand for 2 minutes and extract with 5 ml of chloroform R. Prepare a refer-
ence solution by treating a mixture of 1.0ml of potassium bromide (0.119g/l)
TS and 9 ml of water in a similar manner. Transfer the extracts to matched
10-ml stoppered test-tubes of transparent glass, add to each tube 3ml of fuchsin
TS, shake vigorously and allow to separate. The colour of the chloroform layer
of the test solution when viewed transversely against a white background is
not more intense than that produced in the reference solution when compared
as described under 1.11 Colour of liquids.
Iodides. Moisten 5g by adding, drop by drop, a recendy prepared mixture

composed of 25 ml of starch TS, 2 ml of sulfuric acid (0.5 mol/1) VS, 2 ml of
sodium nitrite (10 g/1) TS, and 25 ml of water. Examine the mixture in daylight;
no particles show any trace of blue colour within 5 minutes.


and colourless.
lOmg/g.
Acidity or alkalinity. Dissolve 5.0 g in 50ml of carbon-dioxide-free water R,

and add 0.1ml of bromothymol blue/ethanol TS; not more than 0.2 ml of
533
sodium hydroxide (0.02 mol/1) VS or 0.2ml of hydrochloric acid (0.02 mol/1) VS

is required to obtain the midpoint of the indicator (green).

100ml. To 10.0ml add 50ml of water, 5ml of nitric acid (-130 g/1) TS, 25.0ml
of silver nitrate (0.1 mol/1) VS, and 2 ml of dibutyl phthalate R, shake the flask
and titrate with ammonium thiocyanate (0.1 mol/1) VS using 2.5ml of ferric
ammonium sulfate (45 g/1) TS as indicator. Each ml of silver nitrate (0.1 mol/1)
VS is equivalent to 7.455 mg of KCl.
Additional requirement for Potassium chloride for

parenteral use
Bacterial endotoxins. Carry out the test as described under 3.4 Test for
bacterial endotoxins; contains not more than 8.8IU of endotoxin RS per
miUiequivalence.
Sodium. Determine by atomic absorption spectrophotometry as described

under 1.8 Atomic spectrometry: emission and absorption, at a wavelength of
589 nm; use a standard solution of sodium chloride R, previously dried to con-
stant weight, dissolved in 1000ml of water to contain 508. 4mg of NaCl
(0.2 mg of Na per ml); the sodium content is not more than l.Omg/g.
Additional requirement for Potassium chloride for sterile use

KALII CITRAS
POTASSIUM CITRATE
Molecular formula. C 6 H 5 K.s 07 ,H 20
Graphic formula.
CH.COOK
HO — C — COOK
I
• H,0
I
CHjCOOK
534
Chemical name. Tripotassium monohydrate; tripotassium 2-hydroxy-

citrate
1,2,3-propanetricarboxylate monohydrate; CAS Reg. No. 6100-05-6
(mono-hydrate).
Description. Transparent crystals or a white, granular powder; odourless.
Solubility. Freely soluble in water; practically insoluble in ethanol (-750 g/1)

TS.
Category. Systemic alkalinizing substance; component of oral rehydration salt
mixtures.
Storage. Potassium citrate should be kept in a tightly closed container.
Additional information. Potassium citrate is deliquescent when exposed to

moist air.
Requirements
Definition. Potassium citrate contains not less than 99.0% and not more than
101.0% of C^HsKsO/, calculated with reference to the anhydrous substance.
Identity tests
A. To a O.lg/ml solution add 2 ml of sodium hydroxide (~80g/l) TS; it yields
tic of potassium.
B. A O.lg/ml solution yields reaction A described under 2.1 General identifi-

Sodium. Dissolve 1 g in 10 ml of water, add 6ml of potassium antimonate TS,

and allow to stand for 15 minutes; the solution is clear or any opalescence pro-
duced is not more pronounced than that of opalescence standard TS2.
Oxalates and tartrates. Dissolve 1 g in 4ml of hydrochloric acid (~70g/l) TS,

add 4ml of ethanol (~750g/l) TS and 1.0ml of calcium chloride (55 g/1) TS; the
solution remains unchanged within 1 hour.
Clarity and colour of solution. A solution of l.Og in 10 ml of carbon-dioxide-

free water R is clear and colourless.
535
Readily carbonizable substances. Dissolve 0.20 g in 10 ml of sulfuric acid

(~1760g/l) TS and heat on a water-bath at 80-90 °C for 1 hour; the solution is
not more intensely coloured than standard colour solutions Yw5 or Gnh when
compared as described under 1.11 Colour of liquids.

Fischer method, Method A, using about 0.5 g of the substance; stir and allow
it to remain in contact with the dehydrated methanol R for 15 minutes, stir
again for 1 minute, and then titrate; the water content is not less than 40mg/g

add 0.1 ml of phenolphthalein/ethanol TS; not more than 0.2 ml of hydrochlo-
ric acid (0.1 mol/1) VS or 0.2ml of sodium hydroxide (0.1 mol/1) VS is required
to change the colour of the solution.

Rl, heat to about 50 °C, allow to cool to room temperature, add 0.25ml of 1-
naphtholbenzein/acetic acid TS, and titrate with perchloric acid (0.1 mol/1) VS
until a green colour is obtained as described under 2.6 Non-aqueous titration.
Method A. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 10.21 mg
of C^H^KsO;.
KALII lODIDUM
POTASSIUM IODIDE
Molecular formula. KI
Chemical name. Potassium iodide; CAS Reg. No. 7681-11-0.
Description. Colourless crystals or a white, granular powder; odourless.
Solubility. Soluble in 0.7 part of water, in 17 parts of ethanol (-750 g/1) TS, in
2 parts of glycerol R, and in 75 parts of acetone R.
Category. Antifungal, expectorant.
Storage. Potassium iodide should be kept in a well-closed container, protected

from light.
536
Requirements
Definition. Potassium iodide contains not less than 99.0% and not more than
101.0% of KI, calculated with reference to the dried substance.
Identity tests
A. To a 0.05 g/ml solution add 2 ml of sodium hydroxide (~80g/l) TS; it yields
tic of potassium.
B. A 0.05 g/ml solution yields reaction B, described under 2.1 General identifi-
cation tests as characteristic of iodides.
Barium. Dissolve 0.5g in 10ml of carbon-dioxide-free and ammonia-free

water R and add 1ml of sulfuric acid (-100 g/1) TS; no turbidity is produced
within 1 minute.
lodates. Dissolve 0.5 gin 10 ml of carbon-dioxide-free water R, and add 2

drops of sulfuric acid (-100 g/1) TS, followed by 1 drop of starch TS; there is
no immediate production of a blue colour.
Nitrates, nitrites and ammonia. To a solution of l.Og in 5ml of water con-

tained in a test-tube of about 40 ml capacity, add 5 ml of sodium hydroxide
(~80g/l) TS and about 0.2 g of aluminium R wire. Insert a small plug of puri-
fied cotton wool in the upper part of the test-tube, and place a piece of moist-
ened red litmus paper R over the mouth of the tube. Heat the test-tube with
its contents in a water-bath for 15 minutes; no blue coloration of the paper is
discernible.
Sulfates. Dissolve 2.5 g in 20ml of water and proceed as described under 2.2.2
Thiosulfates. Dissolve l.Og in 10ml of carbon-dioxide-free water R and add

0.1 ml of starch TS; not more than 0.05 ml of iodine (0.01 mol/1) VS is required
to produce a blue colour.

and colourless.
lOmg/g.
537
Alkalinity. Dissolve l.Og in 10ml of carbon-dioxide-free water R, add 0.1ml

of sulfuric acid (0.05mol/l) VS, and 1 drop of phenolphthalein/ethanol TS; no
colour is produced.
Assay. Dissolve about 0.3g, accurately weighed, in 10ml of water. Add 10ml
of a mixture of 50 ml of starch TS and 1 drop of iodine/ethanol TS. Titrate with
silver nitrate (0.1 mol/1) VS until a pale yellow colour is produced. Each ml of
silver nitrate (0.1 mol/1) VS is equivalent to 16.60mg of Kl.
KAOLINUM
KAOLIN
Chemical name. Kaolin; CAS Reg. No. 1332-58-7.
Other name. Bolus alba.
Description. A white or greyish white, unctuous powder, free from gritty par-
ticles; odour, clay-like.
Solubility. Practically insoluble in water and in organic solvents; insoluble in

mineral acids and solutions of alkali hydroxides.
Category. Tablet and capsule diluent; suspending agent.
Storage. Kaolin should be kept in a well-closed container.
Labelling. The designation on the container of Kaolin should state whether it
is intended for internal use.

quality, since Kaolin is of mineral origin.
Requirements
Definition. Kaolin is a purified, natural hydrated aluminium silicate, the com-
position of which is variable.
Identity tests
A. Boil 0.5 g with 1 g of sodium hydroxide R and 5 ml of water for 5 minutes.
Dilute the mixture with sufficient water to produce 10 ml and filter. To 5 ml
(keep the remaining filtrate for test B) add 0.5 g of ammonium chloride R,
shake, and boil the solution; a white, gelatinous precipitate is produced.
538
B. To the filtrate from test A, add 1ml of hydrochloric acid (-420 g/1) TS; an
almost colourless, gelatinous precipitate is produced.
Acid-soluble substances. To 5g add 7.5ml of hydrochloric acid (~70g/l) TS

and 27.5 ml of water, and boilfor 5 minutes. Filter, wash the residue with water,
and dilute the combined filtrate and wash liquids with sufficient water to
produce 50 ml. To 10ml (keep the remaining solution for “Heavy metals”) add
1.5 ml of sulfuric acid (-100 g/1) TS, evaporate to dryness on a water-bath, ignite,
and weigh; the residue weighs not more than lOmg (lOmg/g).
Heavy metals. To 5 ml of the solution prepared above add 5 ml of water,

10ml of hydrochloric acid (~420g/l) TS, and 25ml of methylisobutylketone R.
Shake for 2 minutes, allow to separate, and evaporate the aqueous layer to
dryness on a water-bath. Dissolve the residue in 1ml of acetic acid (~300g/l)
TS and dilute to 40ml with water. Determine the heavy metals content as
described under 2.2.3 Limit test for heavy metals. Method A; not more than
50pg/g.
Iron. Triturate 2g in a mortar with 10ml of water and add 0.5g of sodium
salicylate R; not more than a slight red tint is produced.
Loss on ignition. Ignite to constant mass between 550 and 600 °C; it loses not
more than 150mg/g.
Acidity or alkalinity. To 1 g add 20 ml of carbon-dioxide-free water R, shake

for 2 minutes, and filter. To 10 ml of the filtrate add 0.1ml of phenolph-
thalein/ethanol TS; the solution remains colourless. Titrate with sodium
hydroxide (0.01 mol/1) VS; not more than 0.25 ml is required to obtain a pink
colour.
Swelling power. Triturate 2g with 2 ml of water; the mixture does not flow.
Adsorption capacity. Transfer 1 g to a 25-ml glass stoppered tube, add 10ml

of a solution containing 0.37g of methylthioninium chloride R in 100ml of
water, and shake vigorously for 2 minutes. Centrifuge and dilute 1 ml of the
supernatant solution to 100 ml with water; the colour produced is not darker
than that of a solution containing 3.0 mg of methylthioninium chloride R in
100 ml of water.
Additional requirement for Kaolin intended for internal use

Heavy metals. To 10ml of the solution prepared under “Acid-soluble sub-
stances”, add 10ml of water, 20ml of hydrochloric acid (-420 g/1) TS, and
25ml of methylisobutylketone R. Shake for 2 minutes, allow to separate, and
evaporate the aqueous layer to dryness on a water-bath. Dissolve the residue
in 1ml of acetic acid (-300 g/1) TS and dilute to 40ml with water. Determine
539
the heavy metals content as described under 2.2.3 Limit test for heavy metals,
Method A; not more than 25|J.g/g.
KETAMINI HYDROCHLORIDUM
KETAMINE HYDROCHLORIDE
• HCI
CH,
Ci3Hi6C1NO,HC1
Chemical name. (+)-2-(o-Chlorophenyl)-2-(methylamino)cyclohexanone hydro-

chloride; (+)-2-(2-chlorophenyl)-2-(methylamino)cyclohexanone hydrochloride;
CAS Reg. No. 1867-66-9.
Description. A white, crystalline powder; odour, characteristic.
Solubility. Freely soluble in water and methanol R; soluble in ethanol

Storage. Ketamine hydrochloride should be kept in a well-closed container.
Requirements
Ketamine hydrochloride contains not less than 98 5 % and not more than the
.
equivalent of 101 0 % of Ci 3 Hi 6 ClNO,HCl, calculated with reference to the

.
dried substance.
Identity tests

infrared region.
spectrum obtained from ketamine hydrochloride RS or with the reference
spectrum of ketamine hydrochloride.
540
B. The absorption spectrum of a 0.33 mg/ml solution in hydrochloric acid

maxima at about 269 nm and 276nm. The ratio of the absorbance at
269nm to that at 276nm is between 1.10 and 1.22.
C. Dissolve 1 g in 10ml of water and add 1 ml of sulfuric acid (~100g/l) TS and

1 ml of ammonium reineckate (lOg/1) TS; a light red precipitate is produced.
D. A O.lg/ml solution yields reaction B described under 2.1 General identifi-
Clarity and colour of solution. A solution of 2 g in 10 ml of water is clear

and colourless.
5-Omg/g.
49 volumes of cyclohexane R and 1 volume of isopropylamine R as the mobile
phase. Apply separately to the plate 2|xl of each of two solutions in methanol
R containing (A) 50 mg of Ketamine hydrochloride per ml and (B) 0.25 mg of
Ketamine hydrochloride per ml. Develop the plate for a distance of 10 cm. After
and spray the plate evenly with modified Dragendorff reagent TS, dry, spray
with hydrogen peroxide (~60g/l) TS, and examine the chromatogram in
daylight.
Assay. Dissolve about 0.5 g, accurately weighed, in 1ml of formic acid

(-1080 g/1) TS and add 70ml of a mixture of 6 volumes of acetic anhydride R
and 1 volume of glacial acetic acid Rl. Add 10 ml of mercuric acetate/acetic acid
TS and titrate with perchloric acid (0.1 mol/1) VS, determining the endpoint
541

Ci3Hi6C1NO,HC1.
Additional requirements for Ketamine hydrochloride for

prarenteral use
KETOCONAZOLUM
KETOCONAZOLE
C26H28CI2N4O4
Chemical name. +-as-l-Acetyl-4-[/?-[2-(2,4-dichlorophenyl)-2-(imidazol-l-

ylmethyl)-l,3-dioxolan-4-yl]methoxy]phenyl]piperazine; ds-l-acetyl-4-[4-[[2-
(2,4-dichlorophenyl)-2-(lh?-imidazol-l-ylmethyl)-l,3-dioxolan-4-yl]methoxy]
phenyl]piperazine; as-l-acetyl-4-[/2-[2-(2,4-dichlorophenyl)-2-(l//-imidazol-l-
ylmethyl)-l,3-dioxolan-4-yl]methoxy]phenyl]-piperazine; CAS Reg. No.
65277-42-1.
Solubility. Practically insoluble in water; freely soluble in dichloromethane R;

soluble in methanol R; sparingly soluble in ethanol (-750 g/1) TS.
542
Storage. Ketoconazole should be kept in a well-closed container, protected

from light.
Requirements
Ketoconazole contains not less than 99 0. % and not more than the equivalent
of 101 0 % of
. C 26H 28 CI2N 4 O 4 ,
Identity tests

infrared region.
spectrum obtained from ketoconazole RS or with the reference spectrum of
ketoconazole.
C. Place 1 ml of nitric acid (~1000g/l) TS in a porcelain dish and add 10 mg of

the substance; a clear orange-red solution is produced.
D. Place 30 mg in a porcelain dish, add 0.3 g of anhydrous sodium carbonate R,

and heat over an open flame for 10 minutes. Allow to cool, add 5 ml of nitric
acid (-130 g/1) TS to the residue, stir, and filter. To 1ml of the filtrate add
1ml of water. The solution yields reaction A described under 2.1 General
identification tests as characteristic of chlorides.
under 2,2,3 Limit test for heavy metals, Procedure 3; determine the heavy
metals content according to Method A; not more than 20mg/g.
Loss on drying. Dry to constant mass at 105 °C; it loses not more thanSmg/g.
4 volumes of dioxan R, 4 volumes of methanol R, and 2 volumes of ammo-
nium acetate TS as the mobile phase. Apply separately to the plate 5 pi of each
543
of 5 solutions in the mobile phase containing (A) 6 mg of Ketoconazole per ml,

(B) 6mg of ketoconazole RS per ml, for (C) prepare a mixture of 6 mg of each
of ketoconazole RS and econazole nitrate RS per ml, (D) 30 pg of ketoconazole
RS per ml, and 15pg of ketoconazole RS per ml. After removing the plate
(E)
from the chromatographic chamber, allow it to dry in a current of warm air for
15 minutes. Expose the plate to iodine vapours until the spots appear and
examine the chromatogram in daylight.
The test is not valid unless solution C shows two clearly separated spots. Any
than the principal spot obtained with solution D (0.5%) and only one such spot
is more intense than that obtained with solution E (0.25%).
Assay. Dissolve about 0.2g, accurately weighed, in 70ml of a mixture of 1

volume of glacial acetic acid R1 and 7 volumes of ethylmethyUcetone R, and
titration. Method A, determining the end-point potentiometrically.

C26H28CI2N4O4.
LACTOSUM
LACTOSE
Lactose, anhydrous
Lactose monohydrate
tt = 0 (anhydrous)
K = 1 (monohydrate)
C 12 H 22 O], (anhydrous)
C] 2 H 220 ii,H 20 (monohydrate)
544
=
Chemical name. Lactose; 4-0-|3-D-galactopyranosyl-D-glucose; CAS Reg.

No. 63-42-3.
Lactose monohydrate; 4-O-P-D-galactopyranosyl-D-glucose monohydrate;

CAS Reg. No. 64044-51-5.
Solubility. Freely but slowly soluble in water; very slightly soluble in ethanol
Storage. Lactose should be kept in a well-closed container.
Labelling. The designation on the container of Lactose should state whether

it is the monohydrate or the anhydrous form.

purity, since Lactose is of natural origin.
Requirements
Identity tests
A. Dissolve 0.1 g in 10 ml of water, add 3 ml of potassio-cupric tartrate TS, and
heat; a red precipitate is formed.
B. Dissolve 0.25g in5ml of water, add 5ml of ammonia (-260 g/1) TS, and heat
in a water-bath at 80 °C for 10 minutes; a red colour develops.
C. Dissolve 20 mg in 5 ml of water, add 0.2 ml of methylamine hydrochloride

(20 g/1) TS, and heat to boiling for 30 seconds. Add 0.2 ml of sodium hydrox-
ide (-200 g/1) TS; the initial yellow colour of the solution turns to red.
Specific optical rotation. Dissolve 10 g in 80 ml of water while heating to

50 °C. Allow to cool and add 0.2 ml of ammonia (-100 g/1) TS. After 30 minutes
dilute to 100 ml with water. Measure the rotation at 20 °C and calculate with
reference to the anhydrous substance; [a]™ -1-54.4 to -1-55.9°.
Heavy metals. Use l.Og for the preparation of the test solution, add 1ml of
hydrochloric acid (0.1 mol/1) VS and proceed as described under 2.2.3 Limit test
for heavy metals. Procedure 1; determine the heavy metals content according
to Method A; not more than 5 pg/g.
Starch. Dissolve 1 .5 g in 1 0 ml of boiling water. Cool, and add 1 drop of iodine

(0.05 mol/1) VS; no blue colour develops.
545
Clarity and colour of solution. A solution of 3 g in 10 ml of boiling water is
colourless or nearly colourless, clear, and odourless.
Ethanol-soluble substances. Add lOg of finely powdered Lactose to 40ml

of ethanol (~750g/l) TS and shake for 10 minutes. Filter, evaporate 10ml of the
filtrate to dryness, dry the residue at 100 °C for 10 minutes, and weigh; not
more than 20 mg.

Fischer method. Method A, using either:
— 2g of anhydrous Lactose; the water content is not more than lOmg/g; or
— monohydrate; the water content

0.5 g of Lactose is not less than 45mg/g
Acidity or alkalinity. Boil 6g with 25ml of carbon-dioxide-free water R, cool,

and add 0.3ml of phenolphthalein/ethanol TS; the solution remains colourless.
Titrate with carbonate-free sodium hydroxide (0.1 mol/1) VS; not more than
0.4ml is required to obtain the midpoint of the indicator (pink).
LEVAMISOLI HYDROCHLORIDUM
LEVAMISOLE HYDROCHLORIDE
C„Hi2N2S,HC1
Chemical name. (-)-2,3,5,6-Tetrahydro-6-phenylimidazo[2,l-l7]thiazole mono-

hydrochloride; (S)-2,3,5,6-tetrahydro-6-phenylimidazo[2,l-l’]thiazole monohy-
drochloride; CAS Reg. No. 16595-80-5.
Solubility. Freely soluble in water; soluble in ethanol (-750 g/1) TS; slightly
546
Category. Anthelminthic drug.
Storage. Levamisole hydrochloride should be kept in a well-closed container,

Requirements
Levamisole hydrochloride contains not less than 98 5 % and not more than
.
101 0 % of C]]Hi 2 N 2 S,HC 1 calculated with reference to the dried substance.

.
,
Identity tests

infrared region.
spectrum obtained from levamisole hydrochloride RS or with the reference
spiectrunt of levamisole hydrochloride.
C. Dissolve about 0.06g in 20 ml of water, add 2 ml of sodium hydroxide

(~80g/l) TS, boil for 10 minutes, and cool.Add a few drops of sodium
nitroprusside (45g/l) TS; a red colour is produced which fades on standing.
Specific optical rotation. Use a 0.050 g/ml solution in carbon-dioxide-free

water R and calculate with reference to the dried substance; [a]g’‘‘“ = 121.5° to
-128°.
Clarity and colour of solution. A solution of 0.50g in 10ml of carbondiox-

ide- free water R is clear and not more intensely coloured than standard colour
Ywl when compared as described under 1.11 Colour of liquids,
5-Omg/g.
547
60 volumes of toluene R, 40 volumes of acetone R, and 1 volume of ammonia
of 4 solutions in methanol R containing (A) 50 mg of Levamisole hydrochloride
per ml, (B) 5.0 mg of Levamisole hydrochloride per ml, (C) 0.25 mg of Lev-
amisole hydrochloride per ml, and (D) 5.0 mg of levamisole hydrochloride RS
per ml. After removing the plate from the chromatographic chamber, dry it at
105 °C for 15 minutes, and examine the chromatogram in ultraviolet light
(254 nm).
Expose the plate to iodine vapour in a tightly closed chamber for 15 minutes
and examine the chromatogram in daylight.
Any spot obtained with solution A, other than any spot with a very low Rf
value, is not more intense than that obtained with solution C (0.5%).
Assay. Dissolve about 0.2 g, accurately weighed, in 30ml of ethanol (~750g/l)

TS and add 5ml of hydrochloric acid (0.01 mol/1) VS. Titrate with sodium
hydroxide (0.1 mol/1) VS, determining the two inflection points potentiometri-
cally. Record the volume, in ml, of sodium hydroxide (0.1 mol/1) VS consumed
between the two inflection points.
Each ml of sodium hydroxide (O.lmol/1) VS is equivalent to 24.08mg of

C„Hi2N2S,HC1.
LEVODOPUM
LEVODOPA
Molecular formula. C 9 HnN 04
548
Graphic formula.
H
I
C COOH
I
NH^
Chemical name. (-)-3-(3,4-Dihydroxyphenyl)-L-alanine; 3-hydroxy-L-tyro-

sine; CAS Reg. No. 59-92-7.

Storage. Levodopa should be kept in a tightly closed container, protected from

light, and stored in a cool place.
Requirements
Definition. Levodopa contains not less than 98.5% and not more than 101.0%
of C 9 H NO
11 4, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from levodopa RS or with the reference spectrum of
levodopa.
C. To 5mg add 1ml of water, 1ml of pyridine R and 5mg of 4-nitrobenzoyl

chloride R, mix and allow to stand for 3 minutes; a violet colour is produced
which changes to pale yellow on boiling. While shaking, add 0.1ml of
sodium carbonate (200 g/1) TS; the violet colour reappears.
549
=
Specific optical rotation. Transfer about 0.5 g, accurately weighed, to a 25-

ml volumetric flask, dissolve it in 10ml of hydrochloric acid (1 mol/1) VS, add
5g of methenamine R, swirl the contents to dissolve the methenamine, dilute
with sufficient hydrochloric acid (1 mol/1) VS to produce 25 ml, mix, and allow
to stand in the dark at 25°C for 3 hours; [a]™ -160 to -167°.
Clarity and colour of solution. Dissolve 0.50 g in a mixture of 2 ml of

hydrochloric acid (~70g/l) TS and 8 ml of water; the solution is clear and not
more intensely coloured than standard colour solution Yw2 when compared as
described under 1.11 Colour of liquids.
lOmg/g.
chromatography, using cellulose R2 as the coating substance and a mixture of
50 volumes of 1 -butanol R, 25 volumes of glacial acetic acid R, and 25 volumes
of water as the mobile phase. Prepare a fresh solution of O.lOg of the test sub-
stance dissolved in 5 ml of anhydrous formic acid R and dilute to 10 ml with
methanol R; this constitutes solution A. Dilute 0.5ml of solution A to 100ml
with methanol R; this constitutes solution B. Separately prepare a fresh solu-
tion of O.lOg of levodopa RS dissolved in 5 ml of anhydrous formic acid R and
dilute to 10 ml with methanol R. Dilute 0.5 ml of this solution to 100 ml with
methanol R; this constitutes solution C. Dissolve 30 mg of tyrosine R in 1 ml of
anhydrous formic acid R and dilute to 100 ml with methanol R. Mix 1ml of
this solution with 1 ml of solution A; this constitutes solution D. Apply sepa-
rately to the plate 10 pi of each of solutions A, B, and C, and 20 pi of solution
D. Dry the plate in a stream of air before placing it in the chromatographic
chamber. Develop the plate, remove it from the chamber, allow it to dry in air,
spray with a freshly prepared solution containing 2 volumes of ferric chloride
(25g/l) TS and 1 volume of potassium ferricyanide (50g/l) TS, and examine the
chromatogram immediately in ultraviolet light (365 nm). Any spot obtained
obtained with solution B. The test is valid only if the chromatogram obtained
with solution D shows two distinctly separated spots.
Assay. Dissolve by heating about 0.18g, accurately weighed, in 5 ml of anhy-

drous formic acid R, add 25 ml of glacial acetic acid R1 and 25 ml of dioxan R,
550

alent to 19.72 mg of C 9H 11 NO 4.
LEVONORGESTRELUM
LEVONORGESTREL
Graphic formula.
Chemical name. (-)-13-Ethyl-17-hydroxy-18,19-dinor-17a-pregn-4-en-20-yn-

3-one; CAS Reg. No. 797-63-7.

TS and ether R.
Category. Contraceptive.
Storage. Levonorgestrel should be kept in a well-closed container, protected

from light.
Requirements
Definition. Levonorgestrel contains not less than 98.0% and not more than
102.0% of C 21 H 28 O 2 ,
Identity tests

551
spectrum obtained from levonorgestrel RS or with the reference spectrum of

levonorgestrel.
Specific optical rotation. Use a lOmg/ml solution in chloroform R; [a]™"*- =

-30.0 to -35.0°.
5-Omg/g.
Acidity or alkalinity. Dissolve O.lOg in 30ml of dehydrated ethanol R and

add 0.5ml of methyl red/ethanol TS; not more than 0.15ml of sodium hydrox-
ide (0.01 mol/1) VS is required to obtain a yellow colour and not more than
0.30ml of hydrochloric acid (0.01 mol/1) VS is required to obtain a red colour.
8 volumes of chloroform R and 2 volumes of acetone R as the mobile phase.
Apply separately to the plate 10 pi of each of 3 solutions in chloroform R con-
taining (A) lOmg of the test substance per ml, (B) O.lOmg of the test substance
per ml, and (C) O.lOmg of levonorgestrel RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air until the solvents have
evaporated, spray it with a mixture of 50ml of methanol R and lOml of sulfu-
ric acid (~1760g/l) TS, and examine the chromatogram in ultraviolet light
Ethynyl group. Dissolve about 0.2 g, accurately weighed, in about 40ml of

tetrahydrofuran R. Add 10ml of silver nitrate (lOOg/1) TS and titrate with
sodium hydroxide (0.1 mol/1) VS, determining the endpoint potentiometrically,
using a glass and a calomel electrode that contains potassium nitrate solution
as the electrolyte. Repeat the operation without the substance being examined
and make any necessary corrections. Each ml of sodium hydroxide (0.1 mol/1)
VS is equivalent to 2.503 mg of — C=CH; the content of ethynyl group is not
less than 78.1mg per g and not more than 81.8mg per g.

552
at about 241 nm. Calculate the amount of C 21 H 28 O 2 in the substance being

tested by comparison with levonorgestrel RS, similarly and concurrently exam-
erence solution should be 0.54 + 0.03.
LEVOTHYROXINUM NATRICUM
LEVOTHYROXINE SODIUM
Molecular formula. Ci 5 Hiol 4 NNa 04 (anhydrous); Ci 5 Hiol 4NNa 04 ,H 20
(mono-hydrate).
Graphic formula.
n =0 (anhydrous}
n = 1 (monohydrate)
Chemical name. Monosodium L-thyroxine; monosodium 0-(4-hydroxy-3,5-

diiodophenyl)-3,5-diiodo-L-tyrosine; 3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-
diiodophenyl]-L-alanine salt; CAS Reg. No. 55-03-8 (anhydrous);
monosodium
Monosodium L-thyroxine monohydrate; monosodium 0-(4-hydroxy-3,5-
diiodo-phenyl)-3,5-diiodo-L-tyrosine monohydrate; 3-[4-(4-hydroxy-3,5-
diiodophenoxy)-3,5-diiodophenyl]-L-alanine monosodium salt monohydrate;
CAS Reg. No. 31178-59-3 (monohydrate).
Other name. Thyroxine sodium.
Description. An almost white or slightly coloured powder, or a fine, slightly
coloured, crystalline powder; odourless.
TS; practically insoluble in acetone R and ether R. It dissolves in solutions of
alkali hydroxides.
Category. Thyroid hormone.
553
Storage. Levothyroxine sodium should be kept in a tightly closed container,

Additional information. Levothyroxine sodium may contain a variable quan-

tity of water of crystallization; anhydrous levothyroxine sodium is hygroscopic.
Requirements
Definition. Levothyroxine sodium contains not less than 97.0% and not
more than 101.0% of CisHioLNNaO^, calculated with reference to the dried
substance.
Identity tests
A. Dissolve 5mg in 2.0ml of nitric acid (-130 g/1) TS and warm the solution; a
brown to violet colour is produced. Cool, add 1.0ml of chloroform R and

shake; the colour of the chloroform layer turns violet.
B. Dissolve 5 mg in a mixture of 2.0 ml of ethanol (-750 g/1) TS and about

0.2 ml of hydrochloric acid (-70 g/1) TS, add about 0.25 ml of sodium nitrite
(10 g/1) TS and allow 2-3 minutes in

to stand for 15 minutes or heat for
a water-bath; a yellow solution is produced. Cool and add ammonia
(-100 g/1) TS to make the solution alkaline; the colour changes to red.
C. Examine the chromatograms obtained in the test for liothyronine (see

below). The principal spot in the chromatogram obtained with solution A
is similar in position, colour, and size to the principal spot in the chro-
matogram obtained with reference solution B.
D. When tested for sodium as described under 2.1 General identification tests,
yields the characteristic reactions. If reaction B is to be used, ignite 20 mg

and dissolve the residue in acetic acid (-60 g/1) TS.
Specific optical rotation. Dissolve 0.5 g in 22 ml of a gently boiling mixture

of 1 volume of hydrochloric acid (1 mol/1) VS and 4 volumes of ethanol
(-750 g/1) TS. Cool and dilute to 25ml with the same mixture of solvents.
Calculate with reference to the dried substance; [a]c = -1-16.0 to -1-20. 0°C.
Loss on drying. Dry to constant weight at 105°C; it loses not less than
Liothyronine. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using a plate coated with a mixture of 30 g of silica gel R3 and
60ml of a solution containing 0.75g of soluble starch R in 100ml of water. Use
the plate directly without heating. As the mobile phase use a mixture of 20
volumes of ammonia (-260 g/1) TS, 35 volumes of 2-propanol R, and 55 volumes
554
of ethyl acetate R. For the preparation of the test and reference solutions, make
up the following solvent mixture; to 5 volumes of ammonia (-260 g/1) TS add
70 volumes of methanol R and mix. For solution A, dissolve 0.10 g of the test
substance in the solvent mixture to produce 5 ml of concentrated solution, then
dilute 1.0ml of this solution with the same solvent mixture to produce 2.0ml
mg of levothyroxine sodium RS
of diluted solution. For solution B, dissolve 50
in the ammonia/methanol solvent mixture to produce 5 ml. For solution C, dis-
solve 5 mg of liothyronine RS in the ammonia/methanol solvent mixture to
produce 25 ml of concentrated solution, then dilute 1.0 ml of this solution with
the same solvent mixture to produce 2.0ml of diluted solution. For solution D,
mix 1.0 ml of the concentrated solution A with 1.0 ml of the concentrated solu-
tion C. Apply separately to the plate 5 pi of each of diluted solution A, solu-
tion B, diluted solution C, and solution D. After removing the plate from the
chromatographic chamber, allow it to dry in air, spray it with ferric chloride/fer-
ricyanide/arsenite TS, and examine the chromatogram in daylight. Any spot

corresponding to liothyronine in the chromatogram obtained with solution A
is not more intense than the spot in the chromatogram obtained with solution
C. The test is not valid unless the chromatogram obtained with solution D
shows two clearly separated spots.
Soluble halides. Shake lOmg with 10ml of water containing about 0.05 ml of
nitric acid (-130 g/1) TS for 5 minutes and filter. Dilute the filtrate to 10ml with
water and add 0.15ml of silver nitrate (40 g/1) TS; any opalescence produced is
not more intense than that of a solution simultaneously prepared by adding
0.15ml of silver nitrate (40g/l) TS and 0.10ml of hydrochloric acid (0.02 mol/1)
VS to 10ml of water (7mg/g as chlorides).
Assay. Carry out the combustion as described under 2.4 Oxygen flask method,
using about 25 mg of the test substance, accurately weighed, and 10 ml of
sodium hydroxide (10 g/1) TS as the absorbing liquid. When the process is com-
plete, proceed as described under the 4.2 Determination of iodine value. Each
ml of sodium thiosulfate (0.05 mol/1) VS is equivalent to 1.665mg of
C]5HioTNNa04.
LIDOCAINI HYDROCHLORIDUM
LIDOCAINE HYDROCHLORIDE
Molecular formula. Ci4H22N20,HCl,H20
555
Graphic formula.
CH,
/=( I
NHCCHjNICjHjlj- HCI-HjO
^ ^
\h.
Chemical name. 2-(Diethylamino)-2',6' acetoxylidide monohydrochloride

monohydrate; 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide monohy-
drochloride monohydrate; CAS Reg. No. 6108-05-0.
Solubility. Soluble in 0.7 part of water and in 1.5 parts of ethanol (-750 g/1)
Storage. Lidocaine hydrochloride should be kept in a tightly closed container,

Additional information. Lidocaine hydrochloride causes local numbness

after being placed on the tongue. Even in the absence of light, Lidocaine
Requirements
Definition. Lidocaine hydrochloride contains not less than 99.0% and not
more than 101.0% of Ci 4 H 22 N 20 ,HCl, calculated with reference to the anhy-
drous substance.
Identity tests
• Either tests A and C or tests B, C, and D may be applied.

reference spectrum of lidocaine hydrochloride.
B. Dissolve 0.15g in 10ml of water, make the solution alkaline with sodium
hydroxide (-80 g/1) TS, filter, and wash the precipitate with water. Dissolve
the precipitate in 1 ml of ethanol (-750g/l) TS, add 0.5ml of cobaltous chlo-
ride TS, and shake for 2 minutes; a bluish green precipitate is produced.
556
C. A 0.05 g/ml solution yields reaction B, described under 2.1 General identifi-
D. Dissolve 0.1 g in 10ml of water and add 10ml of trinitrophenol (7 g/1) TS.
Filter, wash the precipitate with water and dry at 105°C. Melting tempera-
ture, about 230 °C (picrate).

and colourless.

Primary aromatic amines. Dissolve O.lOg in 4 ml of hydrochloric acid

(~70g/l) TS using a 100-ml volumetric flask. Cool the solution in an ice-bath.
In a test-tube, dissolve 50 mg of sodium nitrite R in 10 ml of water and cool the
solution. To the flask in the ice-bath add half of the volume of the cooled
sodium nitrite solution. Allow to stand for 10 minutes, lift the flask from the
ice-bath, and add 1 g of urea R. Shake the flask frequently and when the evo-
lution of gas has ceased (about 15 minutes), add 2.5 ml of sodium hydroxide
(~80g/l) TS in which lOmg of thymol R has previously been dissolved. Add
5 ml of sodium hydroxide (-80 g/1) TS, allow to stand for 10 minutes, and dilute
to volume. Prepare a blank as described above, but without the substance being
examined. The test solution is not more intensely coloured than the blank solu-

Rl, add 10 ml of mercuric acetate/acetic acidTS, and titrate with perchloric acid
(0.1 mol/1) VS, as described under 2.6 Non-aqueous titration. Method A. Each
ml of perchloric acid (0.1 mol/1) VS is equivalent to 27.08 mg of Ci4H22N20,HCl.
Additional requirements for Lidocaine hydrochloride for

p>arenteral use
557
LIDOCAINUM
LIDO CAINE
Graphic formula.
CH3
Chemical name. 2-(Diethylamino)-2’,6’-acetoxylidide; 2-(diethylamino)-N-

(2,6-dimethylphenyl)acetamide; CAS Reg. No. 137-58-6.
Description. A white or slightly yellow, crystalline powder; odour, charac-

teristic.
Solubility. Practically insoluble in water; very soluble in ethanol (-750 g/1) TS;
freely soluble in benzene R and ether R.
Storage. Lidocaine should be kept in a tightly closed container, protected from

light.
Additional information. Lidocaine causes local numbness after being placed

on the tongue.
Requirements
Definition. Lidocaine contains not less than 99.0% and not more than 101.0%
of C] 4 H 22N 20 ,
Identity tests
558

infrared region.
spectrum obtained from lidocaine RS or with the reference spectrum of
lidocaine,
B. Dissolve 0.1 g in 1 ml of ethanol (-750 g/1) TS, add 0.5ml of cobaltous chlo-
ride TS, and shake for 2 minutes; a bluish green precipitate is produced.
C. Dissolve 0.1 g in 15ml of ethanol (~750g/l) TS and add 10ml of trinitro-

phenol (7 g/1) TS. Filter, wash the precipitate with water, and dry at 105 °C.
Melting temperature, about 230 °C (picrate).
a mixture of 4ml of hydrochloric acid (~70g/l) TS and 21ml of water, and
proceed as described under 2.2.3 Limit test for heavy metals, Procedure 1; deter-
mine the heavy metals content according to Method A; not more than 20pg/g.

20ml of water, filter if necessary, and proceed as described under 2.2.1 Limit
Sulfates. Dissolve 0.50 g in 5 ml of hydrochloric acid (-70 g/1) TS, and proceed
than 1 mg/g.
Loss on drying. Dry to constant weight over phosphorus pentoxide R at

ambient temperature; it loses not more than 5.0 mg/g.
Primary aromatic amines. Dissolve O.lOg in 4ml of hydrochloric acid

(-70 g/1) TS using a lOO-ml volumetric flask. Cool the solution in an ice-bath.
In a test-tube, dissolve 50 mg of sodium nitrite R in 10 ml of water and cool the
solution. To the flask in the ice-bath, add half of the volume of the cooled
sodium nitrite solution. Allow to stand for 10 minutes, lift the flask from the
ice-bath, and add 1 g of urea R. Shake the flask frequently and when the evo-
lution of gas has ceased (about 15 minutes), add 2.5 ml of sodium hydroxide
(-80g/l) TS in which lOmg of thymol R have previously been dissolved. Add
5 ml of sodium hydroxide (-80 g/1) TS, allow to stand for 10 minutes and dilute
to volume. Prepare a blank as described above, but without the substance being
examined. The test solution is not more intensely coloured than the blank solu-
559

alent to 23.43 mg of C 14 H 22 N 2 O.
LINDANUM
LINDANE
Molecular formula. CeHeCh
Graphic formula.
Cl
Chemical name. Y-l,2,3,4,5,6-Hexachlorocyclohexane; (la,2a,3p,4a,5a,6(3)-

1,2,3,4,5,6-hexachlorocyclohexane; CAS Reg. No. 58-89-9.
Other names. Gamma benzene hexachloride; gammahexachlorcyclohexane.
Description. A white, crystalline powder; odour, slight.
Solubility. Practically insoluble in water; soluble in dehydrated ethanol R and

ether R.
Category. Pediculicide; scabicide.
Storage. Lindane should be kept in a well-closed container.
Requirements
Definition. Lindane contains not less than 99.0% and not more than 100.5%
of CeHeCls, calculated with reference to the anhydrous substance.
560
Identity tests
infrared region.
spectrum obtained from lindane RS or with the reference spectrum of lindane.
B. To 1ml of a 5.0mg/ml solution add 3ml of ethanol (~750g/l) TS and 1ml

of potassium hydroxide/ethanol TS 1 and allow to stand for 10 minutes; the
mixture yields reaction B, described under 2.1 General identification tests as
characteristic of chlorides.
Congealing temperature. Not below 112.0°C.
Free chlorides. For the preparation of the test solution shake 1.2 g with 30ml
of water for 1 minute, and filter. To the filtrate add 10ml of nitric acid
(-130 g/1) TS and proceed as described under 2.2.1 Limit test for chlorides; the
chloride content is not more than 0.2mg/g.

is not more than S.Omg/g.
Acidity or alkalinity. Boil 1.5g with 30 ml of water for 1 minute and filter. To
10 ml of the filtrate add 2 drops of phenolphthalein/ethanol TS and titrate with
carbonate-free sodium hydroxide (0.01 mol/1) VS; not more than 0.2ml is
required to obtain a pink colour. Add 0.4ml of hydrochloric acid (0.01 mol/1)
VS and 5 drops of methyl red/ethanol TS; the colour changes to orange.
Assay. To about 0.4 g, accurately weighed, add 25 ml of ethanol (-750 g/1) TS

and warm on a water-bath until dissolved. Cool, add 10ml of potassium
hydroxide/ethanol (1 mol/1) VS, swirl gently, and allow to stand for 10 minutes.
Dilute to 150ml with water, neutralize with nitric acid (-130 g/1) TS, add 10ml
in excess of the acid, followed by 50ml of silver nitrate (0.1 mol/1) VS. Filter,
wash the residue with water, and titrate the combined filtrate and washings
with ammonium thiocyanate (0.1 mol/1) VS, using ferric ammonium sulfate
(45 g/1) TS as indicator. Repeat the operation without the substance being exam-
ined and make any necessary corrections. Each ml of silver nitrate (0.1 mol/1)
VS is equivalent to 9.693 mg of C^HeCh.
561
LITHII CARBONAS
LITHIUM CARBONATE
Molecular formula. Li 2 C 03
Chemical name. Dilithium carbonate; CAS Reg. No. 554-13-2.
Solubility. Soluble in 100 parts of water; less soluble in boiling water; very
Category. Antidepressant.
Storage. Lithium carbonate should be kept in a well-closed container.
Additional information. Lithium carbonate has a slightly alkaline taste.
Requirements
Definition. Lithium carbonate contains not less than 99.5% and notmore than
100.5% of LLCOs, calculated with reference to the dried substance.
Identity tests
A. Moisten a few crystals with hydrochloric acid (-420 g/1) TS and introduce
them on a platinum wire into the flame of a Bunsen burner; a carmine-red
colour is produced in the flame.
B. Dissolve 0.2 g in 5 ml of hydrochloric acid (-420 g/1) TS,

boil, add 2 ml of
sodium hydroxide (-80 g/1) TS, 5 ml of disodium hydrogen phosphate

(40 g/1) TS and boil; a white precipitate is produced.
C. To a small amount add hydrochloric acid (-70 g/1) TS; it effervesces and the
gas is colourless.Add a few drops of calcium hydroxide TS; immediately a
white precipitate is formed.
10 ml of acetic acid (-60 g/1) TS, adjust the pH to 3-4, dilute to 40 ml with water
and mix. Determine the heavy metals content as described under 2.2.3 Limit
test for heavy metals, according to Method A; not more than 20pg/g.
562
Arsenic. Use a solution of 5.0 g dissolved in a mixture of 15ml of brominated

hydrochloric acid AsTS and 45ml of water and remove the excess bromine
with a few drops of stannous chloride AsTS; proceed with the solution as
described under 2,2,5 Limit test for arsenic; the arsenic content is not more than
2 hg/g-
Calcium and magnesium. Dissolve 1.0 g in 30 ml of hydrochloric acid

(1 mol/1) VS and neutralize with ammonia (-100 g/1) TS, filter if necessary, and
divide into 2 equal portions. To one portion add 1 ml of ammonium oxalate

(25 g/1) TS; precipitate is produced when the mixture is allowed
no turbidity or
minutes (limit of calcium). To the second portion add 1 ml of dis-
to stand for 5
odium hydrogen phosphate (40 g/1) TS; no turbidity or precipitate is produced
when the mixture is allowed to stand for 5 minutes (limit of magnesium).
Chlorides. Dissolve 0.35 gin a mixture of 3ml of nitric acid (~130g/l) TS and
Sodium. Determine by atomic absorption spectrophotometry ‘T.8 Atomic

spectrometry: emission and absorption” at a wavelength of 589 nm; use a stan-
dard solution prepared by dissolving sodium chloride R, previously dried to
constant weight, in 1000 ml of water to give a solution containing 508.4 mg of
NaCl (0.2mg of Na per ml); the sodium content is not more than 2.0mg/g.
Sulfates. Dissolve 0.5 g in 20 ml of water, add 3 ml of hydrochloric acid

(-250 g/1) TS, and proceed as described under 2,2.2 Limit test for sulfates; the
sulfate content is not more than 1 mg/g.
5.0 mg/g.
Assay. Dissolve about 0.75 g, accurately weighed, in 100ml of water, add

50ml of hydrochloric acid (1 mol/1) VS, boil to remove carbon dioxide, cool,
and titrate the excess acid with sodium hydroxide (1 mol/1) VS, using methyl
orange/ethanol TS as indicator. Each ml of hydrochloric acid (1 mol/1) VS is
equivalent to 36.95 mg of LLCOs.
LOPERAMIDI HYDROCHLORIDUM
LOPERAMIDE HYDROCHLORIDE
Molecular formula. C29H33C1N202,HC1
563
Graphic formula.
Chemical name. 4-()^-Chlorophenyl)-4-hydroxy-Af,Af-dimethyl-a,a-diphenyl-

1-piperidinebutyramide monohydrochloride; 4-(4-chlorophenyl)-4-hydroxy-
N,N-dimethyl-a,a-diphenyl-l-piperidinebutanamide monohydrochloride;
CAS Reg. No. 34552-83-5.
Description. A white to slightly yellowish powder.
Solubility. Slightly soluble in water and in dilute acids; freely soluble in

methanol R.
Category. Antidiarrhoeal drug.
Storage. Loperamide hydrochloride should be kept in a well-closed container.
Requirements
Definition. Loperamide hydrochloride contains not less than 98.0% and not
more than 102.0% of C 29 H 33 C 1 N 202 ,HC 1 ,
substance.
Identity tests

spectrum obtained from loperamide hydrochloride RS or with the reference
spectrum of loperamide hydrochloride.
B. Transfer about 0.04g, accurately weighed, to a 100-ml volumetric flask, dis-

solve in about 50 ml of 2-propanol R, add 10 ml of hydrochloric acid
(0.1 mol/1) VS and volume with 2-propanol R. The absorption spec-
dilute to
trum of containing the same solvent
this solution against a solvent cell
mixture, when observed between 230 nm and 350 nm, is qualitatively
similar to that of a solution of loperamide hydrochloride RS concurrently
examined (maxima occur at about 253 nm, 259nm, 265nm, and 273nm).
564
The absorbances of the solutions at the respective maxima do not differ

from each other by more than 3%.
D. A lOmg/ml solution yields reaction A described under 2.1 General identifi-

Sulfated ash. Not more than 2,0mg/g.

or about 5mm of mercury) for 4 hours; it loses not more than 5.0mg/g.
85 volumes of chloroform R, 10 volumes of methanol R, and 5 volumes of
formic acid (~1080g/l) TS as the mobile phase. Apply separately to the plate
10 pi of each of 2 solutions in chloroform R containing (A) 10 mg of the test
substance per ml and (B) O.lOmg of the test substance per ml. After removing
the plate from the chromatographic chamber, allow it to dry in air and expose
it to iodine vapours. Examine the chromatogram in daylight. Any spot obtained

Rl, add 10ml of mercuric acetate/acetic acid TS and 0.15ml of 1-naphthol-
benzein/acetic acid TS as indicator, and titrate with perchloric acid (0.1 mol/1)
VS, as described under 2.6 Non-aqueous titration. Method A. Each ml of per-
chloric acid (0.1 mol/1) VS is equivalent to 51.35mg of C29H33C1N202,HC1.
MAGNESII HYDROXIDUM
MAGNESIUM HYDROXIDE
Molecular formula. Mg(OH)2
Chemical name. Magnesium hydroxide; CAS Reg. No. 1309-42-8.
Description. A white, fine, amorphous powder; odourless.
dilute acids.
565
Category. Antacid.
Storage. Magnesium hydroxide should be kept in a tightly closed container.
Requirements
Definition. Magnesium hydroxide contains not less than 95.0% and not more
than 100.5% of Mg(OH) 2 ,
Identity tests
A. Dissolve lOmg in 1.0ml of hydrochloric acid (~70g/l) TS, add 1.0ml of
ammonium chloride (lOOg/1) TS, 0.5ml of disodium hydrogen phosphate
(100 g/1) TS, and 1.0ml of ammonia (~100g/l) TS; a white, crystalline pre-
cipitate is formed, which is soluble in acetic acid (-300 g/1) TS.
B. Dissolve lOmg in 1.0ml of hydrochloric acid (~70g/l) TS, and add 2.0ml of
sodium hydroxide (-80 g/1) TS; a white, gelatinous precipitate is produced,
which is insoluble in an excess of sodium hydroxide (-80 g/1) TS. Add a few
drops of iodine TS; the precipitate turns dark brown.
Heavy metals. Dissolve l.Og in 15ml of hydrochloric acid (-250 g/1) TS and
shake with 25 ml of methylisobutylketone R for 2 minutes. Allow to stand, sep-
arate the layers,and evaporate the aqueous layer to dryness. Dissolve the
residue in15ml of water and proceed as described under 2,2.3 Limit test for
heavy metals, Procedure 1; determine the heavy metals content according to
Method A; not more than 30pg/g.
arsenic content is not more than 3[ig/g.
Calcium. Dissolve 5.0 g in a mixture of 50ml of acetic acid (-300 g/1) TS and
50 ml of water, boil for 2 minutes, cool, and dilute to 100 ml with acetic acid
(-120 g/1) TS. Filter, if necessary, through a previously ignited and tared porce-
lain or silica filter crucible of suitable porosity to give a clear filtrate. Dilute
1.3 ml of the to 150 ml with water (retain the filter for the test of
filtrate
To 0.20ml of ethanolic calcium standard

substances insoluble in acetic acid).
(lOOpg/ml Ca) TS add 0.8 ml of ammonium oxalate (50 g/1) TS. After 1 minute
add 1ml of acetic acid (-120g/l) TS and 15ml of the diluted filtrate prepared
above.
Prepare similarly a standard solution using a mixture of 10 ml of calcium stan-

dard (lOpg/ml Ca) TS and 5 ml of water.
After 15 minutes any opalescence produced in the test solution is not more
intense than that in the standard (15mg/g).
566
Iron. Dissolve 0.15g in 5ml TS and dilute to

of hydrochloric acid (~70g/l)
10ml with water. Proceed with 4.0ml of the resulting solution as described
under 2.2.4 Limit test for iron; not more than 700|ug/g.
Water-soluble substances. Mix 2.0g with 100ml of water and boil for 5
minutes. Filter while still hot, allow to cool, and dilute to 100ml with water.
Evaporate 50ml of the filtrate to dryness and dry at 105°C to constant weight;
the residue weighs not more than 20 mg.
Substances insoluble in acetic acid. Any residue remaining on the filter used
be examined in the limit test for calcium,
in the preparation of the solution to
when washed with water, dried and ignited at 600 °C, weighs not more than
5 mg.
Loss on ignition. Heat 0.5 g gradually to 900 °C and ignite to constant mass;
it loses not less than 0.300 g/g and not more than 0.325 g/g.
0.33 g/g.
Assay. Dissolve about 0.05 g, accurately weighed, in 2 ml of hydrochloric acid

(~70g/l) TS and proceed with the titration as described under 2.5 Complexo-
metric titrations for magnesium. Each ml of disodium edetate (0.05 mol/1) VS
is equivalent to 2.916mg of Mg(OH) 2 .
MAGNESII OXIDUM
MAGNESIUM OXIDE
Light magnesium oxide
Heavy magnesium oxide
Molecular formula. MgO
Chemical name. Magnesium oxide; CAS Reg. No. 1309-48-4.
Description. A
white powder; odourless. Light Magnesium oxide is very
bulky, whereas heavy Magnesium oxide is a dense powder.
dilute acids.
567
Category. Antacid.
Storage. Magnesium oxide should be kept in a tightly closed container.
The designation on the container should

Labelling. state whether it is light
Magnesium oxide or heavy Magnesium oxide.
Requirements
Definition. Magnesium oxide contains not less than 98.0% and not more than
100.5% of MgO, calculated with reference to the ignited substance.
Identity tests
A. Dissolve lOmg in 1.0ml of hydrochloric acid (~70g/l) TS, add 1.0ml of
ammonium chloride (lOOg/1) TS, 0.5ml of disodium hydrogen phosphate
(100 g/1) TS, and 1.0ml of ammonia (~100g/l) TS; a white, crystalline pre-
cipitate is formed, which is soluble in acetic acid (-300 g/1) TS.
B. Dissolve lOmg in 1.0ml of hydrochloric acid (~70g/l) TS, and add 2.0ml of
sodium hydroxide (-80 g/1) TS; a white, gelatinous precipitate is produced,
which is insoluble in an excess of sodium hydroxide (-80 g/1) TS. Add a few
drops of iodine TS; the precipitate turns dark brown.
Heavy metals. Dissolve 5.0g in a mixture of 70ml of acetic acid (~300g/l) TS

and 30 ml of water, boil for 2 minutes, cool, and dilute to 100ml with acetic
acid (-120 g/1) TS. Filter, if necessary, through a previously ignited and tared
porcelain or silica filter crucible of suitable porosity to give a clear filtrate. To
20ml of the filtrate (retain the filter for the test of substances insoluble in acetic
acid and the remaining filtrate for the limit test for calcium) add 15 ml of
hydrochloric acid (-250 g/1) TS and shake with 25 ml of methylisobutylketone
R for 2 minutes. Allow to stand, separate the layers, and evaporate the aqueous
layer to dryness. Dissolve the residue in 1ml of acetic acid (-300 g/1) TS and
dilute to 40 ml with water and mix. Determine the heavy metals content as
described under 2.2.3 Limit test for heavy metals, according to Method A; not
more than 40 pg/g.
arsenic content is not more than 3pg/g.
Barium. To O.lOg add a few drops of hydrochloric acid (-250g/l) TS and dis-
solve in 20ml of water. Add 0.10ml of sulfuric acid (-100 g/1) TS and shake; no
precipitate is produced within 10 minutes.
Calcium. Dilute 1.3 ml of the filtrate obtained in the test for heavy metals to
150 ml with water.
568
To 0.20 ml of ethanolic calcium standard (100 Jtg/ml Ca) TS add 0.8 ml of ammo-
nium oxalate (50g/l) TS. After 1 minute add 1ml of acetic acid (~120g/l) TS
and 15 ml of the diluted filtrate prepared above.
Prepare similarly a standard solution using a mixture of 10 ml of calcium stan-

dard (lOpg/ml Ca) TS and 5 ml of water. After 15 minutes, any opalescence pro-
duced in the test solution is not more intense than that in the standard (15mg/g).
Iron. Dissolve 0.15g in 5ml of hydrochloric acid (~70g/l) TS and dilute to

10ml with water. Proceed with 4.0ml of the resulting solution as described
under 2.2.4 Limit test for iron; not more than 500[xg/g.
Water-soluble substances. Mix 2.0g with 100ml of water and boil for 5
minutes. While still hot filter through a coarse sintered-glass filter, allow to cool,
and dilute to 100 ml with water. Evaporate 50 ml of the filtrate to dryness and
dry at 105 °C to constant weight; the residue weighs not more than 20 mg.
Substances insoluble in acetic acid. Any residue remaining on the filter used
examined in the test for heavy metals,
in the preparation of the solution to be
when washed with water, dried, and ignited at 600 °C, weighs not more than
5 mg.
Loss on ignition. Ignite l.Og at 900 °C to constant weight; it loses not more
than lOOmg/g.
Assay. Dissolve about 0.35 g, accurately weighed, in 20 ml of hydrochloric acid

(~70g/l) TS and proceed with the titration as described under 2.5 Complexo-
metric titrations for magnesium. Each ml of disodium edetate (0.05 mol/1) VS
is equivalent to 2.015mg of MgO.
MAGNESII STEARAS
MAGNESIUM STEARATE
{H3C [CHdie COsljMg
C36H7oMg04
Chemical name. Magnesium stearate; magnesium octadecanoate; CAS Reg.

No. 557-04-0.
Description. A white, very fine powder of low bulk density; unctuous and
readily adheres to the skin; odour, very faint, of stearic acid.
569
Solubility. Practically insoluble in water, ethanol (-750 g/1) TS, and ether R;
slightly soluble in hot ethanol (~750g/l) TS.
Category. Tablet and capsule lubricant; glidant; antiadherent.
Storage. Magnesium stearate should be kept in a well-closed container.
Requirements
Definition. Magnesium stearate consists mainly of magnesium stearate
(Ci 7 H C 02 Mg
35 )2
with variable proportions of magnesium palmitate
(Ci 5 H iC 02 Mg
3 )2
and magnesium oleate (Ci 7 H 33 C 02 ) 2 Mg.
Magnesium stearate contains not less than 3 8 % and not more than the equiv-
.
alent of 5 8 . % of Mg, calculated with reference to the dried substance.
Identity tests
A. To 5g add 50ml of ether R, 20 ml of nitric acid (-130 g/1) TS, and 20ml of
water. Heat under a reflux condenser until completely dissolved, and allow
to cool. Separate the aqueous layer, shake the ether layer with two quanti-
ties,each of 4 ml, of water, combine the aqueous solutions, wash with
15ml of ether R, and dilute to 50ml with water. (Retain this solution for
and for “Chlorides”.) Evaporate the ether layer to dryness and
identity test B
dry the residue at 105°C. The congealing point of the residue is not lower
than 53 °C. (Keep the residue for “Acid value of fatty acids”.)
B. To 1 ml of the above aqueous solution, add 1 ml of ammonia (-100 g/1) TS;
a white precipitate is formed which dissolves on the addition of 1 ml of

ammonium chloride (100 g/1) TS. Add 1 ml of disodium hydrogen phosphate
(40 g/1) TS; a white, crystalline precipitate is produced.
Chlorides. Proceed with 2 ml of the aqueous solution from identity test A as

described under 2.2.1 Limit test for chlorides; the chloride content is not more
than 0.25 mg/g.
60 mg/g.
Acidity or alkalinity. Boil Ig with 20 ml of carbon-dioxide-free water R for

1 minute under constant shaking, cool, and filter. To 10 ml of the filtrate add 2
drops of bromothymol blue/ethanol TS and titrate with either hydrochloric
570
acid (0.1 mol/1) VS or sodium hydroxide (0.1 mol/1) VS; not more than 0.05ml
of either titrant is required to obtain the midpoint of the indicator (green).
Acid value of fatty acids. Use 0.2 g of the residue obtained in identity test A
and dissolve in 25 ml of the prescribed mixture of solvents; 195-210.
Assay. With caution, heat gendy about 0.5 g, previously dried and accurately
weighed, and gradually ignite until a white residue is obtained. Cool, add
10ml of hydrochloric acid (~70g/l) TS, and warm on a water-bath for 10
minutes. Dilute with 25 ml of hot water, add sodium hydroxide (~80g/l) TS
until the solution becomes slightly turbid, and then add 10 ml of ammonium
chloride buffer, pH 10.0, TS. Proceed with the titration as described under 2.5
Complexometric titrations for magnesium.
Each ml of disodium edetate (0.05mol/l) VS is equivalent to 1.215mg of Mg.
MAGNESII SULFATIS HEPTAHYDRAS

MAGNESIUM SULFATE HEPTAHYDRATE
MgS04,7H20
Chemical name. Magnesium sulfate (1 :

1) heptahydrate; CAS Reg. No.
10034-99-8.
Other name. Epsom salt.
Description. Brilliant, colourless crystals or a white, crystalline powder;

odourless.

TS.
Category. Cathartic drug.
Storage. Magnesium sulfate heptahydrate should be kept in a well-closed

container.
Additional information. Magnesium sulfate heptahydrate effloresces in

warm, dry air.
571
g
Requirements
Magnesium sulfate heptahydrate contains not less than 99 0
. % and not more
than the equivalent of 100.5 % of MgS 04 ,
substance.
Identity tests
A. Dissolve 10 mg in 2 ml of water and add 1ml of ammonia (~100g/l) TS; a
white precipitate is produced which redissolves after adding 1 ml of ammo-
nium chloride (lOOg/1) TS. Then add 1 ml of disodium hydrogen phosphate
(40g/l) TS; a white, fine crystalline precipitate is formed.
B. A 20mg/ml solution yields reaction A described under 2.1 General identifi-

Arsenic. Use a solution of 5 g in 35 ml of water and proceed as described under

2.2.5 Limit test for arsenic; the arsenic content is not more than 2pg/g.

the chloride content is not more than 300pg/g.
Iron. Use 2.0 g; the solution complies with the 2.2.4 Limit test for iron; not
more than 20pg/g.
Clarity and colour of solution. A solution of Ig in 10ml of water is clear

and colourless.
Loss on drying. Dry 0.5 at 1 10-120 °C for 1 hour and then at 400 °C to con-
stant mass; it loses not less than 0.48 g/g and not more than 0.52 g/g.
Acidity or alkalinity. Dissolve l.Og in 10ml of water and add 0.05ml of

phenol red/ethanol TS; not more than 0.2 ml of hydrochloric acid (0.01 mol/1)
VS or sodium hydroxide (0.01 mol/1) VS is required to obtain the midpoint of
the indicator (pink).
Assay. Dissolve about 0.25 g, accurately weighed, in 100ml of water, and

proceed with the titration as described under 2.5 Complexometric titrations for
magnesium.
Each ml of disodium edetate (0.05mol/l) VS is equivalent to 6.018mg of MgS 04 .
572
Additional requirements for Magnesium sulfate heftahydrate

for parenteral use
MANNITOLUM
MANNITOL
Molecular formula. CfsHnOg
Graphic formula.
H H OH OH
I I I I
HOCH, C
1111
OH
C
OH
C
H
C
H
CH,OH
Chemical name. D-Mannitol; CAS Reg. No. 69-65-8.
Category. Diuretic.
Storage. Mannitol should be kept in a well-closed container.
Additional information. Mannitol has a sweet taste.
Requirements
Definition. Mannitol contains not less than 98.0% and not more than 102.0%
of C 6H 14 O 6 ,
Identity tests
A. Transfer about l.Og, accurately weighed, to a 100-ml volumetric flask, and
add 80 ml of ammonium molybdate (45 g/1) TS, previously filtered if neces-
573
=
sary. Add sulfuric acid (~50g/l) TS to volume and mix. Measure the optical
rotation and calculate the specific rotation as described under 1.4 Determi-
nation of optical rotation and specific rotation; [a]o -1-137 to -1-145°.
B. To 0.5 g add 2.5ml of acetyl chloride R, then add cautiously 0.5ml of pyri-
dine R. Warm the mixture until it becomes turbid, cool in ice, and collect
the precipitate on a sintered-glass filter. Recrystallize the precipitate several
times from ether R and dry at 60 °C for 1 hour; melting temperature, about
123 °C (mannitol hexaacetate).
metals content according to method A; not more than lOpg/g.


30ml of water and proceed as described under 2.2.1 Limit test for chlorides;
Sulfates. Dissolve 5.0 g in 40 ml of water and proceed as described under 2.2.2


5.0 mg/g.
Acidity. Dissolve 5.0 g in 50 ml of carbon-dioxide-free water R and titrate with

carbonate-free sodium hydroxide (0.02mol/l) VS, phenolphthalein/ethanol TS
being used as indicator; not more than 0.3 ml is required to obtain the midpoint
Sorbitol. Carry out the test as described under 1.14.1 Thin-layer chromatog-
raphy, using silica gel R1 as the coating substance and a mixture of 85 volumes
of 2-propanol R and 15 volumes of a 2 g/1 solution of boric acid R as the mobile
phase. Prepare a solution of l.Og of finely powdered test substance in 10ml of
ethanol (-750 g/1) TS, shake for 30 minutes and filter (solution A). Apply sepa-
rately to the plate 1 pi of test solution A and 2 pi of a 1 .Omg/ml solution of sor-
bitol R in water (B).
574
Develop the plate at room temperature, the process taking up to 5 hours. After
removing the plate from the chromatographic chamber, allow it to dry at
110°C for 5 minutes, cool, and spray with a 1 g/1 solution of potassium per-
manganate Rin sulfuric acid (0.5 mol/1) VS. Heat the plate at 110°C until brown
spots appear and examine the chromatogram in daylight. The spot obtained
with solution B is more intense than any spot, corresponding in position and
appearance, obtained with solution A.

100ml. Transfer 10ml to a stoppered flask, add 20.0ml of a 21.4 g/1 solution of
sodium metaperiodate R and 2 ml of sulfuric acid (-100 g/1) TS and heat on a
water-bath for 15 minutes. Cool, add 3g of sodium hydrogen carbonate R, 25
ml of sodium arsenite (0.05mol/l) VS, and 5ml of a 200 g/1 solution of potas-
sium iodide R; allow to stand for 15 minutes, and titrate with iodine (0.05 mol/1)
VS until the first trace of yellow colour appears. Repeat the procedure without
the test substance and determine the difference in volume of iodine (0.05 mol/1)
VS required for the titration. Each ml of iodine (0.05mol/l) VS is equivalent to
1.822 mg of CcHmOj.
Additional requirements for Mannitol for parenteral use

terial endotoxins; contains not more than 4IU of endotoxin RS per g for dosage
forms with a concentration of less than 100 g/1 of mannitol, and a limit of not
more than 2.5 lU of endotoxin RS per g for dosage forms with a concentration
of 100 g/1 or more of mannitol.
MEBENDAZOLUM
MEBENDAZOLE
Molecular formula. CieHisNsOa
575
Graphic formula.
H
NHCOOCH3
Chemical name. Methyl 5-benzoyl-2-benzimidazolecarbamate; methyl

(5-benzoyl- lff-benzimidazol-2-yl)carbamate; CAS Reg. No. 31431-39-7.
Description. A white to slightly yellow powder.
Solubility. Practically insoluble in water, dilute mineral acids, ethanol

(-750 g/1) TS and ether R; freely soluble in formic acid (~1080g/l) TS.
Category. Anthelmintic drug.
Storage. Mebendazole should be kept in a well-closed container, protected

from light.
Requirements
Definition. Mebendazole contains not less than 98.0% and not more than
102.0% of C16H13N3O3, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from mebendazole RS or with the reference spectrum of
mebendazole.
B. Shake 20 mg with 2.0 ml of sodium hydroxide (-80 g/1) TS, and heat the yel-
lowish coloured suspension until it dissolves; the solution is yellow. Add a
few drops of copper(ll) sulfate (160 g/1) TS; a greenish precipitate is pro-
duced. Add a few drops of ammonia (-100 g/1) TS; the colour turns to green-
ish blue.
C. Dissolve 20mg in 2ml of sulfuric acid (~1760g/l) TS; a yellow solution is
produced. Carefully dilute with 3 ml of water; the yellow colour disappears.
576
Then add 1.0ml of silver nitrate (40g/l) TS; a white precipitate is formed,
which does not dissolve in an excess of ammonia (~100g/l) TS,
Loss on drying. Dry at 105°C under reduced pressure (not exceeding 0.6kPa
chromatography, using silica gel R6 (a precoated plate from a commercial
source is suitable) as the coating substance and a mixture of 90 volumes of chlo-
roform R, 5 volumes of methanol R, and 5 volumes of formic acid (~1080g/l)
TS as the mobile phase. Apply separately to the plate 10 pi of each of 2 solu-
tions prepared as follows: (A) Dissolve 50 mg of the test substance in 1.0 ml of
formic acid (-1080 g/1) TS in a 10-ml volumetric flask, dilute to volume with
chloroform R, and mix; (B) Dilute 1.0 ml of solution A to 200 ml with a mixture
of 9 volumes of chloroform R and 1 volume of formic acid (-1080 g/1) TS. After
with solution A, other than the principal spot, is no larger or more intense than
the main spot obtained with solution B.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 29.53mg of CigHisNsOg.
MEDROXYPROGESTERONI ACETAS
MEDROXYPROGESTERONE ACETATE
577
Chemical name. 17-Hydroxy-6a-methylpregn-4-ene-3,20-dione acetate; 17-

(acetyloxy)-6a-methylpregn-4-ene-3,20-dione; CAS Reg, No. 71-58-9.

almost odourless.
Solubility. Practically insoluble in water; soluble in acetone R and dioxan R;

slightly soluble in ethanol (-750 g/1) TS, methanol R, and ether R.
Category. Progestogen.
Storage. Medroxyprogesterone acetate should be kept in a well-closed con-

Requirements
Medroxyprogesterone acetate contains not less than 97 0 % and not more than
.
the equivalent of 103 0 . %

of C 24 H 34 O 4 calculated with reference to the dried
,
substance.
Identity tests

spectrum obtained from medroxyprogesterone acetate RS or with the refer-
ence spectrum of medroxyprogesterone acetate.

preparing two plates and using kieselguhr R1 as the coating substance and
a mixture of 1 volume of propylene glycol R and 9 volumes of acetone R to
impregnate the plate, dipping it about 5 mm into the liquid. After the solvent
nation. Use a mixture of equal volumes of cyclohexane R and light petro-
leum R1 as the mobile phase. (Keep one plate for the “Related substances”.)
Apply separately to one plate 2 pi of each of three solutions in a mixture
of 9 volumes of chloroform R and 1 volume of methanol R containing (A)
2.5 mg of Medroxyprogesterone acetate per ml, (B) 2.5 mg of medrox-
yprogesterone acetate RS per ml, and (C) a mixture of equal volumes of solu-
tions A and B. Develop the plate for a distance of 15 cm. After removing the
578
vents have evaporated, heat at 120 °C for 15 minutes, and spray the hot
plate with sulfuric acid/ethanol TS. Heat at 120°C for a further 10 minutes,
allow to cool, and examine the chromatogram in daylight and in ultravio-
let light (365 nm).

ance, and intensity with that obtained with solution B. A single spot is
D, Use 20 mg; it yields the reaction described under 2.1 General identification
tests as characteristic of acetylated substances.
Specific optical rotation. Use a lOmg/ml solution in dioxan R; [a]o = -t-45
to -t51
Loss on drying. Dry at 105 °C for 3 hours; it loses not more than lOmg/g.
Related substances. To the plate kept from “Identity test B” and using the
same mobile phase, apply separately 5 pi of each of three solutions in chloro-
form R containing (A) 5 mg of Medroxyprogesterone acetate per ml, (B)
0.15mg of Medroxyprogesterone acetate per ml, and (C) 0.05mg of Medrox-
yprogesterone acetate per ml. After removing the plate from the chromato-
graphic chamber, allow it to dry in air until the solvents have evaporated, heat
at 120 °C for 30 minutes, and spray with 4-toluenesulfonic acid/ethanol TS.
Heat again at 120 °C for 10 minutes, expose the plate to iodine vapours for 10
minutes, and examine the chromatogram in daylight.
intense than that obtained with solution B. Not more than one such spot is
more intense than that obtained with solution C.
Assay. Dissolve about 0.1 g, accurately weighed, in sufficient ethanol (~750g/l)

TS to produce 100ml; dilute 1.0ml of this solution to 100ml with the same
solvent.
Measure the absorbance of the diluted solution in a 1-cm layer at the maximum
at about 241 nm and calculate the content of C 24 H 34 O 4 using the absorptivity
value of 42.6 (A{°((„ = 426).
579
Additional requirement for Medroxyfarogesterone acetate for

parenteral use
MEFLOQUINI HYDRO CHLORIDUM

MEFLOQUINE HYDROCHLORIDE
C,7H,6p6N20,HCl
Chemical name. DL-ef)’r/zra-a-2-piperidyl-2,8-bis(tri£luoromethyl)-4-quinoline-

methanol monohydrochloride; (l^*,S*)-(±)-a-2-piperidinyl-2,8-bis(trifluoromethyl)
-4-quinolinemethanol monohydrochloride; CAS Reg. No. 51773-92-3.
Description. A white to slightly yellow, crystalline powder.
Solubility. Very slightly soluble in water; freely soluble in methanol R; soluble

in ethanol (-750 g/1) TS; sparingly soluble in dichloromethane R.
Storage. Mefloquine hydrochloride should be kept in a tightly closed con-

Additional information. Mefloquine hydrochloride melts at about 260 °C,

with decomposition.
Requirements
Mefloquine hydrochloride contains not less than 99.0% and not more than the
equivalent of 101.0% of C] 7 H] 6 p 6 N 20 ,HCl, calculated with reference to the
anhydrous and solvent-free substance.
580
Identity tests
• Either tests A and E or tests B, C, D, and E may be applied.

infrared region.
spectrum obtained from mefloquine hydrochloride RS or with the reference
spectrum of mefloquine hydrochloride.
C. Transfer about 10 mg to a porcelain crucible, add 45 mg of magnesium oxide

R, and ignite until an almost white residue is obtained. Allow to cool, add
2.0ml of water, 0.05ml of phenolphthalein/ethanol TS, and about 1ml of
hydrochloric acid (~70g/l) TS. Filter, to the filtrate add a freshly prepared
mixture of 0.10ml of sodium alizarinsulfonate (1 g/1) TS and 0.10ml of zir-
conyl nitrate TS, mix, and allow to stand for 5 minutes. Prepare similarly a
reagent blank; a yellow colour is produced, whereas the reagent blank is
red.
D. To 20mg add about 0.2ml of sulfuric acid (~1760g/l) TS and view the
mixture under ultraviolet light (365 nm); a blue fluorescence is observed.
E. A 0.05g/ml solution yields reaction B described under 2.1 General identifi-

metals content according to Method A; not more than 20[ig/g.
Solution in methanol. A solution of 0.50 g in 10 ml of methanol R is clear

and not more intensely coloured than standard colour solution Ywl when

Fischer method. Method A, using l.Og of Mefloquine hydrochloride; the water
8 volumes of dichloromethane R, 1 volume of glacial acetic acid Rl, and 1
volume of methanol R as the mobile phase. Apply separately to the plate 5 pi
of each of 4 solutions in methanol R containing (A) 8 mg of Mefloquine
581
hydrochloride per ml, (B) l.dmg of Mefloquine hydrochloride per ml, (C)
1.6mg of mefloquine hydrochloride RS per ml, and (D) 0.04 mg of mefloquine
hydrochloride RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in a current of warm air for 15 minutes, and spray
with a freshly prepared mixture of 1 volume of sulfuric acid (~1760g/l) TS and
40 volumes of potassium iodoplatinate TS. Then spray again with hydrogen
peroxide (-330 g/1) TS and examine the chromatogram in daylight.
intense than that obtained with solution D (0.5%).
Ethanol, methanol, and acetone. Carry out the test as described under
1.14.5 Gas chromatography, using a stainless steel column (2m x 2.2mm)
packed with graphitized carbon (135-175 pm) (this may be obtained from a
commercial source) which is impregnated with a 0.05g/ml solution of macro-
gol 20M R. Maintain the column at 70 °C, the injection port at 200 °C, and the
detector at 250 °C. Use helium R as the carrier gas at a flow rate of 35 ml per
minute, and a flame ionization detector.
Use the following two solutions: (1) dissolve 1.0 g of Mefloquine hydrochloride
in 10ml of dimethylformamide R, and (2) prepare a mixture of 1.0 g of methanol
R, 1.0 g of dehydrated ethanol R, and l.Og of acetone R diluted to 100ml with
dimethylformamide R; further dilute 1.0 ml of this solution to 100ml with
dimethylformamide R.
Inject 1 pi each of solutions 1 and 2.
solutions 1 and 2, and calculate the total content of ethanol, methanol, and
acetone; the total content does not exceed 5mg/g.

Rl, add 5ml of mercuric acetate/acetic acid TS, and titrate with perchloric acid

CizH,T6N20,HC1.
582
MEGLUMINUM
MEGLUMINE
H H OH H
HO CH,
T
C
T
C
T
C
!
C CH, — NH CH,
i i i i
OH OH H OH
CzH.^NOs
Chemical name. l-Deoxy-l-(methylamino)-D-glucitol; CAS Reg. No.

6284-40-8.

almost odourless.
Category. Used in the preparation of meglumine amidotrizoate and meglu-

mine iotroxate as radiocontrast media.
Storage. Meglumine should be kept in a well-closed container.
Requirements
Meglumine contains not less than 99 0 % and not more than the equivalent
. of
100 5 % of C7H17NO5, calculated with reference to the dried substance.
.
Identity tests
A. To 5 ml of water add 0.5ml of paraldehyde R and 0.5 ml of sulfuric acid
(-190 g/1) TS. Shake and warm carefully until a cloudy solution appears, then
allow to cool for 15 minutes. Freshly prepare a solution containing 0.1 g of
sodium nitroprusside R per ml and to 0.2 ml add 1 ml of the above solution,
then add 50 mg of Meglumine and 2 ml of a solution of 50 mg of sodium
tetraborate R per ml; a blue colour develops slowly which becomes more
intense with time.
B. Dissolve 0.2 g in 2 ml of water, add 0.05 ml of methyl red/ethanol TS, and

neutralize with sulfuric acid (0.25 mol/1) VS. Add 1 ml of sodium hydroxide
(0.1 mol/1) VS and Ig of boric acid R; the solution becomes acidic.
583
Specific optical rotation. Use a O.lOg/ml solution; [a]o

"'^
= -15.7 to-17.3°C.
Reducing sugars. Dissolve 0.25 g in 5 ml of water, add 5 ml of potassio-cupric

tartrate TS and boil for 2 minutes; no red-brown precipitate is produced.

and colourless.
lOmg/g.
Assay. Dissolve about 0.5 g, accurately weighed, in 40 ml of water and titrate

with hydrochloric acid (0.1 mol/1) VS, using methyl red/ethanol TS as
indicator.
Each ml of hydrochloric acid (0.1 mol/1) VS is equivalent to 19.52 mg of

C7Hi,N05.
Additional requirement for Meglumine for faarenteral use

ing, per kg of the rabbit’s mass, a solution in sterile water R containing 0.6g of
Meglumine in not more than 5 ml.
Additional requirement for Meglumine for sterile use

584
MERCAPTOPURINUM
MERCAPTOPURINE
SH
C5H4N4S,H20
Chemical name. Purine-6-thiol monohydrate; l,7-dihydro-6H-purine-6-

thione monohydrate; CAS Reg. No. 6112-76-1.
Solubility. Practically insoluble in water and ether R; slightly soluble in ethanol

(-750 g/1) TS; dissolves in solutions of alkali hydroxides.
Storage. Mercaptopurine should be kept in a well-closed container, protected

from light.
Additional information. CAUTION: Mercaptopurine must be handled with

It melts at a temperature exceeding 308 °C with decomposition.
Requirements
Mercaptopurine contains not less than 97 0 %
and not more than the equiva-
.
lent of 102 0 . %
of C.5 H 4 N 4 S, calculated with reference to the anhydrous
substance.
Identity tests
R and dilute to 100ml with
A. Dissolve 20 mg in 5 ml of dimethyl sulfoxide
hydrochloric acid (0.1 mol/1) 5ml of this solution to 200 ml with
VS. Dilute
hydrochloric acid (0.1 The absorption spectrum of the diluted
mol/1) VS.
solution, when observed between 230 nm and 350 nm, exhibits a maximum
at about 325 nm.
585
B. Dissolve 20mg in 20ml of warm ethanol (~750g/l) TS and add 1ml of a

saturated solution of mercuric acetate R in ethanol (-750 g/1) TS; a white
precipitate is produced.
C. Dissolve 20mg in 20ml of warm ethanol (-750 g/1) TS and add 1ml of a
solution containing lOmg of lead acetate R per ml of ethanol (-750g/l) TS;
a yellow precipitate is produced.

Fischer method, Method A, using 0.15g; the water content is not less than
lOOmg/g and not more than 120mg/g.
Hypoxanthine. Carry out the test as described under 1.14.1 Thin-layer chro-
volumes of acetone R, 7 volumes of water, and 3 volumes of ammonia
two solutions containing (A) 50 mg of Mercaptopurine dissolved in 1ml of
dimethyl sulfoxide R and diluted to 10ml with methanol R, and (B) lOmg
of hypoxanthine R dissolved in 10ml of dimethyl sulfoxide R and diluted to
100 ml with methanol R. After removing the plate from the chromatographic
light (254 nm).

mamide R, add 5 drops of thymol blue/dimethylformamide TS, and titrate with
Non-aqueous titration. Method B.
Each ml of sodium methoxide (0.1 mol/1) VS is equivalent to 15.22 mg of

C5H4N4S.
586
DL-METHIONINUM
DL-METHIONINE
/CO,H
H 3^ ^"ir ‘
C"
/ and enantiomer
H 'nH^
CsHnNOjS
Chemical name. (i^,S)-2-Amino-4-(methylthio)butyric acid; CAS Reg. No. 59-

51-8.
Description. An almost white, crystalline powder or small flakes.
Solubility. Sparingly soluble in water; very slightly soluble in ethanol

(-750 g/1) TS. It dissolves in dilute acids and in dilute solutions of the alkali
hydroxides.
Category. Antidote.
Storage. DL-Methionine should be protected from light.
Additional information. Melting temperature, about 270 °C.
Requirements
DL-Methionine contains not less than 99 0 % and not more than 101 0 % of
. .
C 5 H 11 NO 2 S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from DL-methionine RS or with the reference spectrum of
DL-methionine.
C. Dissolve together about 0.1 g of DL-Methionine with 0.1 g of glycine R in

4.5ml of sodium hydroxide (-80 g/1) TS, add 1ml of 25 mg/ml solution of
587
sodium nitroprusside R, heat at 40 °C for 10 minutes, and allow to cool. Add

2 ml of a mixture of 1 volume of phosphoric acid (-1440 g/1) TS and 9
volumes of hydrochloric acid (-420 g/1) TS; a deep-red colour is produced.
D. Use a 0.050 g/ml solution in hydrochloric acid (1 mol/1) VS and measure the
angle of optical rotation as described under 1.4 Determination of optical
rotation and specific rotation; [a]o = -0.05° to -1-0.05°.
Chlorides. Dissolve 1.2g in a mixture of 5ml of nitric acid (-130g/l) TS and

Sulfates. Dissolve l.Og in 20ml of water for injections R by heating to 60 °C,

cool to 10°C, and filter. Proceed with the filtrate as described under 2.2.2 Limit
test for sulfates; the sulfate content is not more than 0.2mg/g.
Clarity and colour of solution. A solution of 0.20g in 10ml of carbondiox-

ide-free water R is clear and colourless.
pH value. pH of a 20 mg/ml solution in carbon-dioxide-free water R, 5.4-6. 1.
6 volumes of 2-butanol R, 2 volumes of glacial acetic acid R, and 2 volumes of
tions containing (A) 20 mg of DL-Methionine per ml, (B) 0.40 mg of DLMe-
thionine per ml, (C) 0.40mg of DL-methionine RS per ml, and (D) 0.040mg of
DL-methionine RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air, spray with triketohydrindene/butanol/acetic acid
TS and heat at 105°C for 15 minutes. Examine the chromatogram in daylight.
intense than that obtained with solution D (0.2%).
Assay. Dissolve about 0.14g, accurately weighed, in 3ml of anhydrous formic

acid R and add 30 ml of glacial acetic acid Rl. Without delay titrate with per-
chloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous titration.
Method A, determining the end-point potentiometrically.
588

CsHnNO^S.
METHOTREXATUM
METHOTREXATE
Graphic formula.
Chemical name. (+)-N-[/?-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]

benzoyl]-L-glutamic acid; N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methy-
lamino]benzoyl]-L-glutamic acid; CAS Reg. No. 59-05-2.
Description. A yellow to orange, crystalline powder.
Solubility. Practically insoluble in water, ethanol (-750 g/1) TS, dichloroe thane
R, and ether R; very soluble in diluted solutions of alkali hydroxides and
carbonates.
Storage. Methotrexate should be kept in a tightly closed container, protected

from light.
Additional information. Methotrexate is gradually affected by light.

CAUTION: Methotrexate must be handled with care, avoiding contact with
the skin and inhalation of airborne particles.
Requirements
Definition. Methotrexate contains not less than 96.0% and not more than
102.0% of C20H22N8O5, calculated with reference to the anhydrous substance.
589
Identity tests
infrared region.
spectrum obtained from methotrexate RS or with the reference spectrum of
methotrexate.
B. The absorption spectrum of a lO.Opg/ml solution in sodium hydroxide

maxima at about 258 nm, 303 nm, and 371 nm. The ratio of the absorbance
at 303 nm to that at 371 nm is between 2.8 and 3.3.
Specific optical rotation. Dissolve 0.25g in 12ml of sodium carbonate (lOg/1)

TS, dilute with water to 25 ml, and calculate the result with reference to the
anhydrous substance; [a]o = +19 to +24°.

Assay. Carry out the test as described under 1.14.4 High performance liquid
chromatography, using a column 10 cm long and 6 mm
in internal diameter
packed with silica gel, 5 pm in diameter, the surface of which has been modi-
fied with chemically bonded octadecylsilyl groups.
As the mobile phase, use a mixture of 8 volumes of acetonitrile R with 92

volumes of phosphate/citrate buffer pH 6.0, TS.
Prepare the following solutions in the above-mentioned mobile phase contain-

ing (A) 0.10 mg of the test substance per ml, (B) 0.10 mg of methotrexate RS per
ml, and (C) O.lOmg of methotrexate RS and O.lOmg of folic acid RS per ml for
the system suitability test.
Operate at room temperature with a flow rate of about 1.4 ml per minute. As
a detector use an ultraviolet spectrophotometer at a wavelength of about
303 nm, fitted with a low-volume flow cell (10 pi is suitable), and a suitable
recorder.
Make 6 replicate injections of solution C, each of 20 pi. The resolution factor

between methotrexate and folic acid should be not less than 5.0, with a rela-
tive standard deviation for the methotrexate peak of not more than 2.5%
(adjust the flow rate and the ratio of the mobile phase if it does not conform).
Inject 20 pi of each of solutions A and B. Measure the peak responses and

calculate the content in % of C20H22NSO5 using the following formula:
590
1 Q 0 (AiM 2 T)/{A 2JVI i), in which Ai and A 2

peak responses of the test sub-
are the
stance and the reference substance, respectively, and Mi and 2 are the con- M
centrations of the test solution and the reference solution, respectively, and T
corresponds to the degree of purity of methotrexate RS.
Additional requirement for Methotrexate for parenteral use

METHYLCELLULOSUM
METHYLCELLULO SE
Chemical name. Cellulose methyl ether; CAS Reg. No. 9004-67-5.
Description. A white, yellowish white, or greyish white powder or loose,

cotton-like, fibrous material; odourless.
Solubility. Practically insoluble in hot water, ethanol (-750 g/1) TS, ether R and
acetone R; soluble in glacial acetic acid R and in a mixture of equal volumes of
ethanol (-750 g/1) TS and chloroform R.
Category. Coating agent; viscosity-increasing agent; tablet binder.
Storage. Methylcellulose should be kept in a well-closed container.
Labelling. The designation on the container of Methylcellulose should state

its viscosity.
Additional information. Methylcellulose is hygroscopic after drying. In cold

water it swells and produces a clear to opalescent, viscous, colloidal suspension
which is neutral to litmus R.
Requirements
Definition. Methylcellulose is a methyl ether of cellulose.
Methylcellulose contains not less than 26 0 % and not more than

. the equiva-
lent of 32 0 % of methoxy (-OCH3) groups.
.
Identity tests
A. While stirring, add Ig of dried Methylcellulose to 50 ml of carbon-dioxide-
free water R heated to 90 °C. Allow to cool, dilute to 100ml with the same
591
solvent, and stir until completely dissolved. Heat 10ml in a water-bath while
stirring (keep the remaining solution for test B); above 50 °C a cloudy solu-
tion or a flocculent precipitate is formed, and on cooling the solution
becomes clear.
B. Place 1 ml of the above solution on a glass plate and allow to evaporate; a

thin film is formed.
C. Dissolve without heating 0.2g in 15ml of sulfuric acid (~1125g/l) TS. Pour
the solution with stirring into 100 ml of ice-water, and dilute to 250
ml with
ice-water. While cooling in ice-water, mix thoroughly in a test-tube 1 ml of
the prepared solution with 8ml of sulfuric acid (-1760 g/1) TS, added drop
by drop. Heat in a water-bath for exactly 3 minutes and immediately cool
in ice-water. While cold, carefully add 0.6ml of triketohydrindene/sodium
metabisulfite TS and mix well. Allow to stand at 25 °C; a pink colour is
immediately produced, which does not change to violet within 100 minutes.
lOOmg/g.
Assay. Carry out the assay as described under 2.9 Determination of methoxyl,
using about 0.05 g, previously dried and accurately weighed.

(-OCH3).
METHYLDOPUM
METHYLDOPA
Molecular formula. CjoHisNO^,! V2H2O
592
Graphic formula.
1^H,0
Chemical name. L-3-(3,4-Dihydroxyphenyl)-2-methylalanine sesquihydrate;

3-hydroxy-a-methyl-L-tyrosine sesquihydrate; CAS Reg. No. 41372-08-1.
Description. White to yellowish white, fine powder or lumps; odourless.
uble in ether R.
Category. Antihypertensive.
Storage. Methyldopa should be kept in a well-closed container, protected

from light.
Requirements
Definition. Methyldopa contains not less than 98.0% and not more than
101.0% of C]oHi 3 N 04 calculated with reference
,
Identity tests

infrared region.
spectrum obtained from methyldopa RS or with the reference spectrum of
methyldopa.

test as described
using cellulose R2 and a mixture of 50 volumes of
1 R and 25 volumes of water as
-butanol R, 25 volumes of glacial acetic acid
in hydrochloric acid (1 mol/1) VS containing (A) 10 mg of the test substance
per ml and (B) 10 mg of methyldopa RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry in a current of warm air,
spray with a freshly prepared solution composed of 2 volumes of ferric chlo-
ride (25 g/1) TS and 1 volume of potassium ferricyanide (50 g/1) TS, and
examine the chromatogram in daylight. The principal spot obtained with
593
=
C. To 5 mg add 1ml of water, 1ml of pyridine R, and 5 mg of 4-nitrobenzoyl

chloride R and heat to boiling. While shaking, add 0.1ml of sodium car-
bonate (200 g/1) TS; an orange or amber colour is produced.
Specific optical rotation. Use a 44 mg/ml solution in aluminium chloride TS

and calculate with reference to the anhydrous substance; [a]™ -25 to
-28°.

content is not less than lOOmg/g and not more than 130mg/g.
Acidity. Dissolve l.Og in 100ml of carbon-dioxide-free water R with the aid

of heat and titrate with sodium hydroxide (0.1 mol/1) VS, methyl red/ethanol
TS being used as indicator; not more than 0.5ml is required to obtain the mid-
3-O-Methyl derivative. Carry out the test as described under 1.14.1 Thin-
layer chromatography, using cellulose R2 as the coating substance and a
mixture of 65 volumes of 1 -butanol R, 15 volumes of glacial acetic acid R, and
25 volumes of water as the mobile phase. Apply separately to the plate (A)
lOpl of a lOmg/ml solution of the test substance dissolved in a mixture of 4
volumes of hydrochloric acid (-250 g/1) TS and 96 volumes of methanol R, (B)
10 pi of a 50pg/ml solution of (-)-3-(4-hydroxy-3-methoxyphenyl)-2-methy-
lalanine RS, and (C) 20 pi of a mixture of equal volumes of solutions A and B.
a current of warm air, and spray with a mixture of 5 volumes of a 0.05 g/ml
solution of sodium nitrite R and 45 volumes of a 3 mg/ml solution of 4-nitroani-
line R dissolved in a mixture of 80 volumes of hydrochloric acid (-420 g/1) TS
and 20 volumes of water. Dry in a current of warm air, spray with sodium car-
bonate (75 g/1) TS, and examine the chromatogram in daylight. The spot
obtained with solution B is more intense than any spot, corresponding in posi-
tion and appearance, obtained with solution A. The test is valid only if the
chromatogram obtained with solution C shows two distinctly separated spots.

Rl, add 20ml of dioxan R, and titrate with perchloric acid (0.1 mol/1) VS as
acid (0.1 mol/1) VS is equivalent to 21.12mg of C 10 H 13NO 4 .
594
METHYLIS HYDROXYBENZOAS
METHYL HYDROXYBENZOATE
CsHaOs
Chemical name. Methyl /^-hydroxybenzoate; methyl 4-hydroxybenzoate;

CAS Reg. No. 99-76-3.
Other name. Methylparaben.

almost odourless.
Solubility. Very slightly soluble in water; soluble in boiling water; freely

Storage. Methyl hydroxybenzoate should be kept in a well-closed container.
Additional information. Methyl hydroxybenzoate is normally used in com-

bination with other hydroxybenzoates.
Requirements
Methyl hydroxybenzoate contains not less than 99 0 % and not more than the
.
of CgHsOs, calculated with reference to the dried substance.
Identity tests
A. Complies with the test under “Melting range".
B. To 0.5g add 5ml of sodium hydroxide (-80 g/1) TS, and heat in a water-bath
for 5 minutes. After cooling, add 6ml of sulfuric acid (-190 g/1) TS, collect
the precipitate on a filter, wash thoroughly with a small amount of water,
595
Sulfated ash. l.Omg/g.

or 5mm of mercury) for 2 hours; it loses not more than 5.0mg/g.
Acidity. Dissolve 0.2 g in 5 ml of ethanol (-750 g/1) TS, add 5 ml of carbon-

Assay. Place about 80mg, accurately weighed, in a ground-glass-stoppered

acetic acid R. Cool in ice-water and add 10 ml of hydrochloric acid (-420 g/1)
30ml of potassium iodide (80 g/1) TS, close the flask, and mix. Titrate with
towards the end of the titration. Repeat the procedure without the Methyl
One volume of sodium thiosulfate (0.1 mol/1) VS corresponds to two volumes

(0.0333mol/l) VS is equivalent to 5.073mg of CaHsOj.
METHYLROSANILINII CHLORIDUM
METHYLROSANILINIUM CHLORIDE
CH, CK "
I I
C,5H3oC1N3
596
Chemical name. C.I. Basic violet 3; N-[4-[bis[4-(dimethylamino)phenyl]

methylene]-2,5-cyclohexadien-l-ylidene]-N-methyknethanaminium chloride;
CAS Reg. No. 548-62-9.
Other names. Crystal violet, gentian violet.
Description. A dark green powder or greenish, glistening pieces having a

metallic lustre; odourless or almost odourless.
Solubility. Sparingly soluble in water; soluble in ethanol (-750 g/1) TS and glyc-
erol R; practically insoluble in ether R.
Category. Anti-infective drug.
Storage. Methylrosanilinium chloride should be kept in a tightly closed con-

Requirements
Methylrosanilinium chloride contains not less than 96 0
. % and not more than
the equivalent of 101 . 0 % of C 25 H 30 CIN 3 ,
calculated with reference to the anhy-
drous substance.
Identity tests
A. See the test described below under “Related substances”. The principal spot
obtained with solution A corresponds to the spot with the lowest Revalue
of the three distinct spots obtained with solution B. A spot other than the
principal spot may be present on the chromatogram obtained with solution
A; this other spot corresponds to the spot with intermediate R;-value
B. Dissolve about 20 mg in 1 0 ml of water and add 5 drops of hydrochloric acid

(-420 g/1) TS. To 5 ml of this solution add tannic acid (50 g/1) TS, drop by
drop; a blue precipitate is produced (keep the remaining solution for test C).
C. To the remaining solution from test B add 0.5 g of zinc R powder, and warm
the mixture; the solution discolours rapidly. Place on a filter-paper 1 drop of
this solution adjacent to 1 drop of ammonia (-lOOg/1) TS; a blue colour is
produced at the zone of contact.
Ethanol-insoluble matter. Add 1.0 g to 50 ml of ethanol (-750 g/1) TS and boil

under reflux for 15 minutes. Filter through a tared filtering crucible, wash the
residue on the filter with hot ethanol (-750 g/1) TS until no violet colour appears
597
in the washings, and dry the crucible at 105 °C for 1 hour; the residue weighs
not more than lOmg (1.0%).

Fischer method, Method A, using 0.5 g of Methylrosanilinium chloride; the
water content is not more than 0.075g/g.
100 volumes of 1-butanol R, 5 volumes of ammonium chloride (20g/l) TS, and
0.5 volume of formic acid (~1080g/l) TS as the mobile phase. Apply separately
to the plate 5 pi of each of 2 solutions in methanol R containing (A) 1 mg of
Methylrosanilinium chloride per ml, and (B) 1 mg of methyl violet 2B R per ml;
also apply to the plate 10 pi of each of 4 solutions in methanol R containing (C)
lOmg of Methylrosanilinium chloride per ml, (D) 2.5mg of Methylrosanilin-
ium chloride per ml, (E) 0.05 mg of 4,4'-bis(dimethylamino) benzophenone R
per ml, and (F) 0.05 mg of Methylrosanilinium chloride per ml. After removing
the chromatogram in ultraviolet light (254 nm).
Any spot obtained with solution C corresponding to 4,4'-bis(dimethylamino)

benzophenone is not more intense than that obtained with solution E (0.5%).
Any spot obtained with solution D, other than the principal spot or any spot
due to 4,4'-bis(dimethylamino) benzophenone, is not more intense than that
obtained with solution F (2.0%).
Assay. Transfer about 0.4 g, accurately weighed, to a 300-ml conical flask, add
25ml of water and 10 ml of hydrochloric acid (-420 g/1) TS. Replace the air in
the flask with carbon dioxide R and maintain a stream of carbon dioxide R
through the flask during the determination. Add 50.0 ml of titanium trichloride
(0.1 mol/1) VS, heat to boiling, continuing to boil gently for 10 minutes, swirling
the liquid occasionally. Cool, add 5 ml of ammonium thiocyanate (10 g/1) TS,
and titrate with ferric ammonium sulfate (0.1 mol/1) VS until a faint red colour
is produced. Repeat the procedure without the Methylrosanilinium chloride
being examined and make any necessary corrections.
Each ml of titanium trichloride (0.1 mol/1) VS is equivalent to 20.40mg of

C,5H3oC1N3.
598
METHYLTESTO STERONUM
METHYLTESTOSTERONE
Graphic formula.
OH
Chemical name. 17|3-Hydroxy-17-methylandrost-4-en-3-one; CAS Reg. No.

58-18-4.
Description. Colourless or almost colourless crystals or a white or slightly

yellowish white, crystalline powder; odourless.

TS; sparingly soluble in ether R.
Category. Androgen.
Storage. Methyltestosterone should be kept in a well-closed container, pro-

tected from light.
Requirements
Definition. Methyltestosterone contains not less than 97.0% and not more
than 102.0% of C20H30O2, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from methyltestosterone RS or with the reference spec-
trum of methyltestosterone.
599

of propylene glycol R and 90 volumes of acetone R to impregnate the plate,
nation. As the mobile phase, use a mixture of 80 volumes of cyclohexane
R and 20 volumes of toluene R. Apply separately to the plate 2 pi of each
of 2 solutions in a mixture of 9 volumes of chloroform R and 1 volume of
methanol R containing (A) l.Omg of the test substance per ml and (B)
1.0 mg of methyltestosterone RS per ml. Develop the plate for a distance of
15 cm. After removing the plate from the chromatographic chamber, allow
it to dry in air until the solvents have evaporated, heat at 120 °C for 15
minutes, spray with 4-toluenesulfonic acid/ethanol TS, and then heat at
120 °C for 10 minutes. Allow to cool, and examine the chromatogram in
daylight and in ultraviolet light (365 nm). The principal spot obtained with
Specific optical rotation. Use a 10 mg/ml solution in ethanol (-750 g/1) TS;
[a]?T<= = -H78 to-t85°.
Solution in ethanol. A solution of 0.50g in 10ml of ethanol (-750 g/1) TS is

clear and not more intensely coloured than standard colour solution Yw2 when
lOmg/g.
90 volumes of chloroform R and 10 volumes of acetone R as the mobile phase.
Apply separately to the plate 5 pi of each of 2 solutions in ethanol (-750 g/1) TS
allow it to dry in air until the solvents have evaporated, and examine the chro-
solution B.

600
same Measure the absorbance of a 1-cm layer of the diluted solution

solvent.
at the maximum at about 242 nm. Calculate the amount of C 20 H 30 O 2 in the
substance being tested by comparison with methyltestosterone RS, similarly
METHYLTHIONINII CHLORIDUM
METHYLTHIONINIUM CHLORIDE
Molecular formula. CieHiaClNfsS (anhydrous); CMHi8ClNf3S,3H20 (trihydrate).
Relative molecular mass. 319.9 (anhydrous); 373.9 (trihydrate).
Graphic formula.
Cr . nHjO
n = 0 (anhydrous)
n = 3 (trihydrate)
Chemical name. C.I. Basic Blue 9; 3,7-bis(dimethylamino)phenothiazin-5-

ium chloride; CAS Reg. No. 61-73-4 (anhydrous).
C.I. Basic Blue 9 trihydrate; 3,7-bis(dimethylamino)phenothiazin-5-ium chlo-

ride trihydrate; CAS Reg. No. 7220-79-3 (trihydrate).
Other name. Methylene blue.
Description. Dark green crystals with a metallic lustre or a dark green, crys-
talline powder; odourless or almost odourless.
Category. Antidote.
Storage. Methylthioninium chloride should be kept in a tightly closed con-

Additional information. Methylthioninium chloride is hygroscopic.
601
Requirements
Definition. Methylthioninium chloride contains not less than 97.0% and
not more than 101.0% of CieHiaClNsS, calculated with reference to the dried
substance.
Identity tests
A. The absorption spectrum of a 5 pg/ml solution in hydrochloric acid (~70g/l)
TS, when observed between 230 nm and 800 nm, exhibits 4 maxima at
about 258 nm, 288 nm, 680 nm, and 745 nm.
B. Dissolve Img in 10 ml of water; a deep blue colour is produced. Add 2.0ml

of hydrochloric acid (~70g/l) TS and 0.25g of zinc R powder; the colour of
the solution is discharged; filter and expose the filtrate to the air; the blue
colour of the solution reappears.
C. Mix 0.05 g with 0.5 g of anhydrous sodium carbonate R in a porcelain cru-

cible. Carefully heat the mixture to a red glow for 10 minutes. Cool, dis-
solve the residue in 10ml of nitric acid (-130 g/1) TS and filter. The filtrate
teristic of chlorides.
Copper or zinc. Ignite 1.0 g in a porcelain crucible using as low a temperature

as practicable, until all of the carbon is oxidized. Cool the residue,add 15ml
of nitric acid (-130 g/1) TS and boil for 5 minutes. Separately prepare a refer-
ence solution by boiling a quantity of copper(II) sulfate R, equivalent to 200 pg
of Cu, with 15ml of nitric acid (-130 g/1) TS for 5 minutes. Filter separately the
cooled test and reference solutions, and wash any residue with 10 ml of water.
Combine filtrate and washings of the test solution and similarly combine
the
the and washings of the reference solution; add to each an excess of
filtrate
ammonia (~100g/l) TS and filter the solutions into 50-ml volumetric flasks.
Wash the precipitates with small portions of water, adding the washings to the
filtrates; dilute the contents of each flask with water to volume, mixing thor-
oughly. To 25 ml of each of the solutions add 10 ml of hydrogen sulfide TS; no

turbidity is produced within 5 minutes (absence of zinc) and any dark colour
produced in the test solution is not more intense than that of the reference
solution (the copper content is not more than 0.20 mg/g).
Iron. Mix 4g with 200 ml of water in a long-necked, round-bottomed flask,

add 15 ml of nitric acid (-1000 g/1) TS, bring gently to the boil and continue
boiling until the volume of liquid is reduced to about 20 ml. Allow to cool, add
10ml of sulfuric acid (~1760g/l) TS and mix. Heat to boiling and add small suc-
cessive quantities of nitric acid (-1000 g/1) TS, cooling before each addition,
until a colourless liquid is obtained. Heat until white fumes are evolved; if dark-
ening occurs at this stage continue the treatment with nitric acid (-1 000 g/1) TS,
finally heating until white fumes are again evolved. Allow the colourless liquid
602
to cool, add 25 ml of a saturated solution of ammonium oxalate R in water, and

boil until the slight froth completely subsides. Cool, dilute to 50ml with water;
5 ml of the diluted solution complies with the 2.2.4 Limit test for iron; not more
than O.lOmg/g.
Loss on drying. Dry to constant weight at 105°C; it loses not less than
Foreign dyes. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using as the coating substance a slurry prepared from silica gel R1
and a mixture of equal volumes of potassium dihydrogen phosphate (27.2 g/1)
TS and disodium hydrogen phosphate (28.4 g/1) TS. As the mobile phase, use
a mixture of 20 volumes of 1-propanol R, 4 volumes of anhydrous formic acid
R, and 1 volume of water. Apply to the plate 2 pi of a solution prepared by dis-
solving 25 mg of the test substance in sufficient methanol R to produce 10 ml.
an oven at 105°C. At an value of about 0.5, 3-4 spots appear, placed very
close to each other, the lowest spot being violet in colour and the others red,
the intensity of the colour increasing in ascending order of the spots. No other
spot is detected.
Assay. Transfer about 0.3 g, accurately weighed, to a 100-ml volumetric flask,

dissolve in 30 ml of water by warming on a water-bath, and allow the solution
to cool. While shaking, add 50.0ml of potassium dichromate (0.0167 mol/1) VS,
dilute to volume with water, and mix. Repeat the shaking intermittently for
10 minutes, and filter; discard the first 20ml of the filtrate. Transfer 50.0ml of
the filtrate to a glass-stoppered flask, add 40 ml of sulfuric acid (-190 g/1) TS and
1 g of potassium iodide R, mix, and allow the closed flask to stand in the dark
for 5 minutes. Add 100 ml of water and titrate with sodium thiosulfate
(0.1 mol/1) VS, using starch TS as indicator, until a blue-green colour is obtained.
Repeat the operation without the substance being examined and make any
ml of potassium dichromate (0.0167 mol/1) VS is
necessary corrections. Each
equivalent to 10.66mg of CigHiBClNsS.
Additional requirements for Methylthioninium chloride for

parenteral use
bacterial endotoxins; contains not more than 2.5 lU of endotoxin RS per mg.
603
j
METOCLOPRAMIDI HYDRO CHLORIDUM

METOCLOPRAMIDE HYDROCHLORIDE
Molecular formula. Ci 4 H 22 ClN 302 ,HCl,H 20
Graphic formula.
HCHj CHj N (C^ I
HCI
'OCH,
Chemical name. 4-Amino-5-chloro-W-[2-(diethylamino)ethyl]-o-anisamide

monohydrochloride monohydrate; 4-amino-5-chloro-N-[2-(diethylamino)-
ethyl]-2-methoxybenzamide monohydrochloride monohydrate; CAS Reg.
No. 54143-57-6 (monohydrate).

almost odourless.
Solubility. Very soluble in water; freely soluble in ethanol (-750 g/1) TS;
Category. Antiemetic drug.
Storage. Metoclopramide hydrochloride should be kept in a well-closed

Requirements
Definition. Metoclopramide hydrochloride contains not less than 98.0% and
not more than 101.0% of Ci 4 H 22 ClN 302 ,HCl, calculated with reference to the
Identity tests

spectrum obtained from metoclopramide hydrochloride RS or with the ref-
erence spectrum of metoclopramide hydrochloride.

604
maxima about 273 nm and 309 nm; the absorbances of a 1-cm layer
at at
these wavelengths are about 0.79 and 0.69, respectively.
C. Dissolve 0.05 g in 5 ml of water and add 5 ml of 4-dimethylaminobenzalde-

hyde TS5; a yellow-orange colour is produced.

Clarity and colour of solution. A solution of 1 .0 g in 1 0 ml of carbon-dioxide-

free water R is clear and not more intensely coloured than standard colour

pH value. pH of a O.lOg/ml solution in carbon-dioxide-free water R, 4. 5-6. 5.
of 95 volumes of 1 -butanol R and 5 volumes of ammonia (-260 g/1) TS as the
mobile phase. Apply separately to the plate 5 pi of each of 2 solutions in
methanol R containing (A) 50 mg of the test substance per ml and (B) 0.50 mg

R, add 10 ml of mercuric acetate/acetic acid TS and titrate with perchloric acid
2.6 Non-aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/1) VS
is equivalent to 33.63 mg of Ci 4 H 22 ClN 302 ,HCl.
Additional requirements for Metoclopwamide hydrochloride for

fârenteral use
metoclopramide.
605
METRIFONATUM
METRIFONATE
Molecular formula. C4H8CI3O4P
Graphic formula.
Cl H
— C — C — PO
I
I
Cl
I I
'ÔCH,^
Cl OH
Chemical name. Dimethyl (2,2,2-trichloro-l-hydroxyethyl)phosphonate;

CAS Reg. No. 52-68-6.
Description. A white or yellowish white, crystalline powder.
Solubility. Sparingly soluble in water; very soluble in ethanol (-750 g/1) TS and
acetone R.
Category. Antischistosomal drug.
Storage. Metrifonate should be kept in a tightly closed container, protected

from light.
Additional information. Even in the absence of light, Metrifonate is gradu-

faster at higher temperatures. CAUTION: Metrifonate must be handled with
Requirements
Definition. Metrifonate contains not less than 95.0% and not more than
101.0% of C4H8CI3O4P, calculated with reference to the anhydrous substance.
Identity tests
A. In a porcelain crucible mix 0.1 g with 0.2 g of a mixture composed of equal
parts of R and anhydrous sodium carbonate R, heat it care-
potassium nitrate
fully to a red glow for 3 minutes, cool the residue, and dissolve it in a
mixture of 7 ml of water and 3 ml of nitric acid (-1000 g/1) TS. Keep 2 ml of
this solution for test B. To 5ml add 2ml of ammonium molybdate (95 g/1)
TS and heat to boiling; a yellow, crystalline precipitate is produced.
606
B. To 2ml of the solution kept in test A add 1ml of silver nitrate (0.1 mol/1)
VS; a white precipitate is produced, which is soluble in 3 ml of ammonia
(~100g/l) TS.
Congealing temperature. Melt 6.2 g in a water-bath at 85 °C, add 2g of

calcium sulfate R, heat again to 80 °C, and proceed as described under 1.2.2
Congealing point; not below 73.5 °C.
Acetone-insoluble matter. Shake l.Og with 20ml of acetone R for 15

minutes, filter, wash the filter with 10ml of acetone R and dry it at 105 °C for
1 hour; the content of insoluble matter is not more than 5.0mg/g.

pH value. pH of a 5.0 mg/ml solution, 2. 0-3. 5.
Assay. Dissolve about l.Og, accurately weighed, in 90ml of methanol R. Add

10ml of monoethanolamine R and allow the solution to stand at 20 °C ± 0.5
°C for 1 hour. Then cool it in an ice-bath for 10 minutes, add 20ml of nitric
acid (-1000 g/1) TS, and titrate at 20°C with silver nitrate (O.lmol/1) VS, deter-
mining the endpoint potentiometrically using a platinum electrode and a
calomel reference electrode. Each ml of silver nitrate (0.1 mol/1) VS is equiva-
lent to 25.74 mg of C 4H 8 CI 3 O 4 P.
METRONIDAZOLI BENZOAS
METRONIDAZOLE BENZOATE
ON
o
o
CH3
C13H13N304
Chemical name. 2-(2-Methyl-5-nitro-llT-imidazol-l-yl)ethyl benzoate; 2-

methyl-5-nitro-lH-imidazole-l-ethanol benzoate; CAS Reg. No. 13182-89-3.
607
Description. A white or slightly yellowish, crystalline powder.
Solubility. Practically insoluble in water; freely soluble in dichloromethane R;

soluble in acetone R; slighdy soluble in ethanol (-750 g/1) TS; very slightly
soluble in ether R.
Storage. Metronidazole benzoate should be kept in a well-closed container,

Requirements
Metronidazole benzoate contains not less than 98 5 and not more than
. %
101 0 . %
of C 13 H 13N 3 O 4 calculated with reference to the dried substance.
,
Identity tests

infrared region.
spectrum obtained from metronidazole benzoate RS or with the reference
spectrum of metronidazole benzoate.
C. To about lOmg add lOmg of zinc R powder, 1ml of water, and about
0.3 ml of hydrochloric acid (-420 g/1) TS. Heat on a water-bath for 5 minutes
and cool. The solution yields the reaction described for the identification of
primary aromatic amines under 2.1 General identification tests, producing
a red precipitate.
pH value. pH of a 20mg/ml suspension in carbon-dioxide-free water R,

5.0-7.0.
608
chromatography, using silica gel R2 as the coating substance. Heat to activate
the plate at 110°C for 1 hour and cool before use. As the mobile phase, use
ethyl acetate R. Apply separately to the plate 10 pi of each of 8 solutions in
acetone R containing (A) 20mg of Metronidazole benzoate per ml, (B) 2.0mg
of Metronidazole benzoate per ml, (C) 2.0 mg of metronidazole benzoate RS
per ml, (D) O.lOmg of Metronidazole benzoate per ml, (E) 0.040mg of Metron-
idazole benzoate per ml, (F) O.lOmg of metronidazole RS per ml, (G) O.lOmg
of 2-methyl-5-nitroimidazole R per ml, and for solution (H) dissolve 10 mg of
metronidazole RS and 10 mg of 2-methyl-5-nitroimidazole R in sufficient
acetoneR to produce 50 ml. After removing the plate from the chromatographic
light (254 nm).
Any spot corresponding to metronidazole or to 2-methyl-5-nitroimidazole

obtained with solution A is not more intense than the corresponding spot
obtained with solutions F and G (0.5%). Any spot obtained with solution A,
other than the principal spot and the spots corresponding to metronidazole and
to 2-methyl-5-nitroimidazole, is not more intense than that obtained with solu-
tion D (0.5%), and not more than one such spot is more intense than that
obtained with solution E (0.2%). The test is not valid unless the chromatogram
obtained with solution H shows two clearly separated principal spots.


G13H13N3O4.
METRONIDAZOLUM
METRONIDAZOLE
Molecular formula. G6H9N3O3
Graphic formula.
609
Chemical name. 2-Methyl-5-nitroimidazole-l-ethanol; 2-methyl-5-nitro-lH-

imidazole-l-ethanol; CAS Reg. No. 443-48-1.
Description. A white or pale yellow, crystalline powder; odourless or almost

odourless.
Solubility. Sparingly soluble in water; slightly soluble in ethanol (-750 g/1) TS,
and ether R.
Category. Antitrichomonal; antiamoebic.
Storage. Metronidazole should be kept in a well-closed container, protected

from light.
Additional information. Metronidazole is stable in air, but darkens on expo-

sure to light.
Requirements
Definition. Metronidazole contains not less than 99.0% and not more than
101.0% of C^HgNsOg, calculated with reference to the dried substance.
Identity tests
infrared region.
spectrum obtained from metronidazole RS or with the reference spectrum of
metronidazole.
B. To lOmg add lOmg of zinc R powder, 1ml of water, and 0.25ml of

hydrochloric acid (-250 g/1) TS and heat in a water-bath for 5 minutes; cool
in ice, add 0.5 ml of sodium nitrite (100 g/1) TS, and remove the excess nitrite
with sufficient sulfamic acid (50 g/1) TS. Add 0.5ml of the resulting solution
to a mixture of 0.5ml of 2-naphthol TSl and 2 ml of sodium hydroxide
(-80 g/1) TS; an orange-red colour is produced.
5-Omg/g.
9 volumes of chloroform R and 1 volume of diethylamine R as the mobile
610
phase. Apply separately to the plate 5 pi of each of 2 solutions in acetone R

containing (A) 20 mg of the test substance per ml (warm slightly if necessary
to dissolve the substance)and (B) 0.10 mg of the test substance per ml. After

Rl, add 3 drops of 1-naphtholbenzein/acetic acid TS as indicator and titrate
with perchloric acid (0.1 mol/1) VS, as described under 2.6 Non-aqueous titra-
tion, Method A. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to
17.12mg of C 6 H 9 N 3 O 3 .
Additional requirements for Metronidazole for parenteral use

MICONAZOLI NITRAS
MICONAZOLE NITRATE
Molecular formula. C] 8 Hi 4 Cl 4 N 20 ,HN 03
Graphic formula.
Chemical name. l-[2,4-Dichloro-P-[(2,4-dichlorobenzyl)oxy]-phenethyl]imida-

zole mononitrate; l-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]-
ethyl]-lH-imidazole mononitrate; CAS Reg. No. 22832-87-7.

almost odourless.
611
Solubility. Very slightly soluble in water and ether R; soluble in 140 parts of
ethanol (-750 g/1) TS.
Storage. Miconazole nitrate should be kept in a well-closed container, pro-

tected from light.
Additional information. Miconazole nitrate melts at about 182 °C with

decomposition.
Requirements
Definition. Miconazole nitrate contains not less than 98.5% and not more
than 101.5% of CisHi 4 Cl 4 N 20 ,HN 03 calculated with reference to the dried
,
substance.
Identity tests

spectrum obtained from miconazole nitrate RS or with the reference spectrum
of miconazole nitrate.
B. The absorption spectrum of a 0.40 mg/ml solution in a mixture of 9 volumes

of methanol R and 1 volume of hydrochloric acid (0.1 mol/1) VS, when
observed between 230 nm and 350 nm, exhibits maxima at about 264 nm,
272 nm, and 280 nm; the absorbances of a 1-cm layer at these wavelengths
are about 0.40, 0.58, and 0.48, respectively.
C. Shake lOmg with 5ml of water and cool in an ice-bath. Keeping the sus-
pension cool throughout, add 0.4 ml of potassium chloride (100 g/1) TS,
0.1ml of diphenylamine/sulfuric acid TS, and, drop by drop with shaking,
5 ml of sulfuric acid (-1760 g/1) TS; an intense blue colour is produced.

of mercury); it loses not more than 5.0mg/g.
60 volumes of hexane R, 30 volumes of chloroform R, 10 volumes of methanol
R, and 1 volume of ammonia (-260 g/1) TS as the mobile phase. Apply sepa-
612
lately to the plate 50 pi of each of 2 solutions in a mixture of equal volumes of

chloroform R and methanol R containing (A) 10 mg of the test substance per
ml and 25 pg of the test substance per ml. After removing the plate from
(B)
the chromatographic chamber, allow it to dry in air, spray it with iodine/chlo-
roform TS, and examine the chromatogram in daylight. Any spot obtained with
with solution B.

R1 and titrate with perchloric acid (0.1 mol/1) VS, determining the endpoint
CisHi4Cl4N20,HN03.
MORPHINI HYDROCHLORIDUM
MORPHINE HYDROCHLORIDE
Molecular formula. Ci 7 Hi 9 N 03 ,HCl, 3 H 20
Graphic formula.
Chemical name. 7,8-Didehydro-4,5a-epoxy-17-methylmorphinan-3,6a-diol

hydrochloride (1:1) (salt) trihydrate; CAS Reg. No. 6055-06-7.
Description. Colourless, needle-like crystals or a white, crystalline powder;

odourless.

Category. Analgesic.
613
Storage. Morphine hydrochloride should be kept in a tightly closed container,

Additional information. Even in the absence of light, Morphine hydrochlo-

Requirements
Definition. Morphine hydrochloride contains not less than 98.0% and not
more than 101.0% of Ci 7 Hi 9N 03 ,HCl, calculated with reference to the dried
substance.
Identity tests
A. To 5 ml of a 1 mg/ml solution add 3 drops of a freshly prepared potassium
ferricyanide (10 g/1) TS and 1 drop of ferric chloride (25 g/1) TS; a bluish green
colour is produced.
B. To 5 ml of a 1 mg/ml solution add 1ml of hydrogen peroxide (-60 g/1) TS,

1 ml of ammonia (-100 g/1) TS, and 1 drop of copper (II) sulfate (80 g/1) TS;
a transient red colour is produced.
C. To about 1 mg
add 0.5ml of sulfuric acid (~1760g/l) TS containing 1 drop
of formaldehyde TS; a purple colour is produced, which changes quickly to
violet.
D. Dissolve a few mg in 5 ml of water, add 1 drop of hydrochloric acid

(-70 g/1) TS and 1 ml of potassium iodobismuthate TS2; an orange or orange-
red precipitate is produced immediately.
E. A 20 mg/ml solution yields reaction B, described under 2.1 General identi-

fication tests as characteristic of chlorides.
Specific optical rotation. Use a 20 mg/ml solution and calculate with refer-
ence to the dried substance; [a]c = -109 to -115 °.
Acidity. Dissolve 0.2 g in 10 ml of carbon-dioxide-free water R, and titrate with

sodium hydroxide (0.02 mol/1) VS, using methyl red/ethanol TS as indicator;
not more than 0.2 ml is required to obtain the midpoint of the indicator
(orange).
614
Meconate. Dissolve 0.2 g in 5 ml of water, add 5 ml of hydrochloric acid

(~70g/l) TS and a few drops of ferric chloride (25g/l) TS; no red colour is
produced.
Related alkaloids. Transfer about 0.5 g, accurately weighed, to a separator,

add 15ml of water, 2ml of sodium hydroxide (lOg/1) TS, and 10ml of chloro-
form R. Shake, allow to separate and transfer the chloroform layer to another
separator. Repeat the extraction with further quantities of chloroform R, each
of 10 ml. Wash the combined chloroform solutions with 4 ml of sodium hydrox-
ide (lOg/1) TS and then twice with water, using 5 ml each time. Separate the
chloroform layer and evaporate it carefully to dryness on a water-bath. Add to
the residue thus obtained 10ml of sulfuric acid (0.01 mol/1) VS, heat until dis-
solved, cool, add 1 drop of methyl red/ethanol TS, and titrate the excess acid
with sodium hydroxide (0.02mol/l) VS; not less than 8.75ml is required to
obtain the midpoint of the indicator (orange).
Noscapine. Dissolve 0.05 g in 2 ml of sulfuric acid (-1760 g/1) TS and heat the
solution on a water-bath; no violet colour is produced.
ml of perchloric acid (0.1 mol/1) VS is equivalent to 32.18mg of Ci 7 Hi 9 N 03 ,HCl.
Additional requirement for Morp'hine hydrochloride for

parenteral use
MORPHINI SULFAS
MORPHINE SULFATE
Molecular formula. (Ci 7 Hi 9 N 03 )2
,H 2 S 04 5 H 20
,
615
Graphic formula.
Chemical name. 7,8-Didehydro-4,5a-epoxy-17-methylmorphinan-3,6a-diol

sulfate (2:1) (salt) pentahydrate; CAS Reg. No. 6211-15-0.
Description. White, feathery needles of a white, crystalline powder or cubical

masses; odourless.

Category. Analgesic.
Storage. Morphine sulfate should be kept in a tightly closed container, pro-

tected from light.
Additional information. Morphine sulfate loses water of hydration on expo-

sure to air and darkens in colour on prolonged exposure to light.
Requirements
Definition. Morphine sulfate contains not less than 98.0% and not more
than 101.0% of (Ci 7 Hi 9 N 03) 2 ,H 2 S 04 calculated with reference to the dried
,
substance.
Identity tests
A. To 5 ml of a 1 mg/ml solution add 3 drops of a freshly prepared potassium
ferricyanide (10 g/1) TS and 1 drop of ferric chloride (25 g/1) TS; a bluish green
colour is produced.
B. To 5ml of a 1 mg/ml solution add 1ml of hydrogen peroxide (-60 g/1) TS,
1ml of ammonia (-lOOg/1) TS and 1 drop of copper (II) sulfate (80 g/1) TS;
a transient red colour is produced.
616
C. To about 1 mg
add 0.5ml of sulfuric acid (~1760g/l) TS containing 1 drop
of formaldehyde TS; a purple colour is produced, which changes quickly to
violet.

ence to the dried substance; = -106 to -110
Loss on drying. Dry at 145°C for 1 hour; it loses not less than 90mg/g and
Acidity. Dissolve 0.2 g in 10 ml of carbon-dioxide-free water R, and titrate with

sodium hydroxide (0.02 mol/1) VS, using methyl red/ethanol TS as indicator;
not more than 0.2 ml is required to obtain the midpoint of the indicator
(orange).
Meconate. Dissolve 0.2 g in 5 ml of water, add 5 ml of hydrochloric acid

(~70g/l) TS and a few drops of ferric chloride (25g/l) TS; no red colour is
produced.
Related alkaloids. Transfer about 0.5 g, accurately weighed, to a separator,

add 15ml of water, 2ml of sodium hydroxide (lOg/1) TS, and 10ml of chloro-
form R. Shake, allow to separate, and transfer the chloroform layer to another
separator. Repeat the extraction with further quantities of chloroform R, each
of 10 ml. Wash the combined chloroform solutions with 4 ml of sodium hydrox-
ide (lOg/1) TS and twice with water, using 5ml each time. Separate the chlo-
roform layer and evaporate it carefully to dryness on a water-bath. Add to the
10ml of sulfuric acid (0.01 mol/1) VS, heat until dissolved,
residue thus obtained
cool,add 1 drop of methyl red/ethanol TS, and titrate the excess acid with
sodium hydroxide (0.02 mol/1) VS; not less than 8.75 ml is required to obtain
the midpoint of the indicator (orange).
Noscapine. Dissolve 0.05g in 2ml of sulfuric acid (-1760 g/1) TS and heat the
solution on a water-bath; no violet colour is produced.
(Ci7Hi,N03)2,H2S04.
617
Additional requirements for Morphine sulfate for

parenteral use
NALOXONI HYDROCHLORIDUM
NALOXONE HYDROCHLORIDE
Naloxone hydrochloride, anhydrous
Naloxone hydrochloride, dihydrate
Molecular formula. Ci9H2iN04,HCl (anhydrous); Ci9H2iN04,HCl,2H20

(dihydrate).
Graphic formula.
Chemical name. (-)-17-Allyl-4,5a-epoxy-3,14-dihydroxymorphinan-6-one

hydrochloride; 4,5î-epoxy-3,14-dihydroxy-17-(2-propenyl)morphinan-6-one
hydrochloride; (-)-12-aUyl-7,7a,8,9-tetrahydro-3,7a-dihydroxy-4a/i-8,9c-imino-
ethanophenanthro[4,5-^’cT|furan-5(6T?)‘Cme hydrochloride; CAS Reg. No.
357-08-4 (anhydrous).
(-)-17-Allyl-4,5a-epoxy-3,14-dihydroxymorphinan-6-one hydrochloride dihy-

drate; 4,5a-epoxy-3,14-dihydroxy-17-(2-propenyl)morphinan-6-one hydroch-
loride dihydrate; (-)-12-allyl-7,7a,8,9-tetrahydro-3,7a-dihydroxy-4aH-8,9c-
imino-ethanophenanthro[4,5-l?cT]furan-5(6H)-one hydrochloride dihydrate;
CAS Reg. No. 51481-60-8 (dihydrate).
618
Solubility. Soluble in water; slightly soluble in ethanol (-750 g/1) TS; practi-
Category. Narcotic antagonist.
Storage. Naloxone hydrochloride should be kept in a tightly closed container,

Labelling. The designation on the container of Naloxone hydrochloride

should state whether the substance is in the anhydrous form or is the
dihydrate.
Additional information. Even in the absence of light, Naloxone hydrochlo-

sition being faster at higher temperatures. It melts at about 177 °C.
Requirements
Definition. Naloxone hydrochloride contains not less than 98.0% and not
more than 102.0% of C] 9H 2 iN 04 ,HCl, calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from naloxone hydrochloride RS or with the reference
spectrum of naloxone hydrochloride.
B. Dissolve 0.05 g in 5 ml of hydrochloric acid (0.1 mol/1) VS and add 0.3 ml of

ferric chloride (25 g/1) TS; a purplish blue colour is produced.
C. A 0.05g/ml solution yields reaction A described under 2.1 General identifi-

Specific optical rotation. Use a 25 mg/ml solution and calculate with refer-
ence to the dried substance; [aj^ = -170 to -181 °.
Loss on drying. Dry to constant weight at 105 °C; anhydrous Naloxone

hydrochloride loses not more than 5.0mg/g. Naloxone hydrochloride dihy-
drate loses not more than llOmg/g.
619
chromatography, using silica gel R1 as the coating substance, and as the mobile
phase prepare the following solution: shake 100 ml of 1 -butanol R with 60 ml
of ammonia (~17g/l) TS, discard the lower layer, and mix 95 volumes of the
upper layer with 5 volumes of methanol R. Dry the plate in a current of air.
Apply separately to the plate 5 pi of each of the two following solutions. For
solution (A) dissolve 40 mg of Naloxone hydrochloride in 2 ml of water and
dilute to 5 ml with methanol R. For solution (B) dilute 0.5ml of solution A to
100 ml with methanol R. Develop the chromatogram protected from light. After
spray with ferric chloride/potassium ferricyanide TS, and examine the chro-
intense than that obtained with solution B (0.5%). Disregard any spot remain-
ing at the point of application.
Chlorides content. Dissolve about 0.3 g, accurately weighed, in 50 ml of

methanol R. Add 5ml of glacial acetic acid R and 0.1ml of eosin Y (5g/l) TS,
and titrate with silver nitrate (0.1 mol/1) VS until a pink colour is produced. Each
ml of silver nitrate (0.1 mol/1) VS is equivalent to 3.545mg of Cl; the content of
chlorides is not less than 95.4 mg/g and not more than 99.4 mg/g, calculated
Rl, add 10ml of acetic anhydride R, 10ml of mercuric acetate/acetic acid TS,
alent to 36.38 mg of Ci 9 H 2 iN 04 ,HCl.
Additional requirements for Naloxone hydrochloride for

p>arenteral use
terial endotoxins; contains not more than 500 lU of endotoxin RS per mg.
620
NATRII AMIDOTRIZOAS
SODIUM AMIDOTRIZOATE
CH.
CiiH8l3N2Na04
Chemical name. Monosodium 3,5-diacetamido-2,4,6-triiodobenzoate; mono-

sodium 3,5-bis(acetylamino)-2,4,6-triiodobenzoate; CAS Reg. No. 737-31-5.
Other names. Sodium diatrizoate; diatrizoate sodium.
Description. A white powder; odourless or almost odourless.
practically insoluble in acetone R and ether R.
Storage. Sodium amidotrizoate should be kept in a well-closed container, pro-

tected from light.
Requirements
Sodium amidotrizoate contains not less than 98 0 % and not more than the
.
equivalent of 102 0 % of C]iHal 3 N 2 Na 04 calculated with reference to the anhy-

.
,
drous substance.
Identity tests
• Either tests A and E or tests B, C, D, and E may be applied.

infrared region.
spectrum obtained from sodium amidotrizoate RS or with the reference spec-
trum of sodium amidotrizoate.
621

using silica gel R4 as the coating substance and 20 volumes of chloroform
R, 10 volumes of methanol R, and 2 volumes of ammonia (-260 g/1) TS as

the mobile phase. Apply separately to the plate 10 pi of each of two solu-
tions in a mixture of 80 mg of sodium hydroxide R in 100 ml of methanol R
containing (A) 1 mg of Sodium amidotrizoate per ml and (B) 1 mg of sodium
amidotrizoate RS per ml. After removing the plate from the chromato-
graphic chamber, allow it to dry in air, and examine the chromatogram in
ultraviolet light (254 nm).

C. Place 20 mg into a flask, add 5 ml of sodium hydroxide (-80 g/1) TS, and boil
gently under a reflux condenser for 10 minutes. Cool, add 5ml of hydrochlo-
ric acid (-70 g/1) TS, and cool in ice for 5 minutes. Add 4 ml of sodium nitrite
(10 g/1) TS, cool in ice for 5 minutes, add 0.3 g of sulfamic acid R, swirl gently
until effervescence ceases, and add 2 ml of N-(l-naphthyl)ethylenediamine
hydrochloride (5 g/1) TS; an orange-red colour is produced.
D. Heat 0.1 g in a suitable crucible; violet vapours are evolved.
E. When tested for Sodium as described under 2.1 General identification tests,

20mg/ml solution.
Iodides. Dissolve 0.8 g in 10ml of water, add drop by drop nitric acid
(-130 g/1) TS until complete precipitation is obtained, then add an excess of
3 ml. Filter, and wash the precipitate with 5 ml of water; to the filtrate add 1 ml
of hydrogen peroxide (-330 g/1) TS and 1ml of chloroform R, and shake. To

serve as a reference solution, treat similarly 2 ml of iodide standard (20 pg I/ml)
TS with 3 ml of nitric acid (-130 g/1) TS and sufficient water to equal the volume
of the solution to be tested. Any red-violet colour in the chloroform layer is
not darker than that obtained from the reference solution.

and colourless.

Fischer method. Method A, using 0.4 g; the water content is not more than
lOOmg/g.
622
Primary aromatic amines. Place about 1 g, accurately weighed, in a 50-ml

volumetric flask,and add 5ml of water, 10ml of sodium hydroxide (0.1 mol/1)
VS, and 25ml of dimethyl sulfoxide R. Stopper the flask,mix the contents by
gentle swirling, and allow to stand in an ice-bath, protected from light, for 5
minutes. Keeping the flask in the ice-bath add slowly 2 ml of hydrochloric acid
(-250 g/1) TS, mix, and allow to stand for 5 minutes. Add 1.5ml of sodium
nitrite (35 g/1) TS, mix, and allow to stand for 5 minutes. Add 2 ml of sulfamic
acid (50 g/1) TS, mix, and again allow to stand for 5 minutes. Add 2 ml of N-{\-
naphthyl)ethylenediamine hydrochloride/Tpropanol TS and mix. Remove the
flask from the ice-bath and allow to stand in water at 22-25 °C for 10 minutes
with occasional gentle shaking. Dilute to 50 ml with dimethyl sulfoxide R and
mix.
Within 5 minutes after the addition of the last reagent measure the absorbance
at about 470nm, against a solvent cell containing the reagents prepared in a
similar manner. The absorbance does not exceed 0.40.
Assay. To about 0.3 g, accurately weighed, add 30 ml of sodium hydroxide

(50 g/1) TS and 0.5 g of zinc R powder. Boil under a reflux condenser for 1 hour.
Cool the flask to room temperature, rinse the condenser with 20ml of water,
and filter. Thoroughly rinse the flask and add the rinse liquid to the filter. To
the filtrate add 5 ml of glacial acetic acid R and 1ml of tetrabromophenolph-
thalein ethyl ester TS and titrate with silver nitrate (0.05 mol/1) VS until the
yellow precipitate formed just turns green.
Each ml of silver nitrate (0.05mol/l) VS is equivalent to lO.bOmg of

CiiH8l,dSl2Na04.
NATRII CALCII EDETAS

SODIUM CALCIUM EDETATE
Molecular formula. CioHi2CaN2Na20s,2H20
623
Graphic formula.
NaOOCCHj CHjCOONa
N
• 2HjO
ooc
Chemical name. Disodium [(ethylenedinitrilo)tetraacetato]calciate(2-) dihy-

drate; (OC-6-21)-disodium [[N,N'-l,2-ethanediylbis[N-(carboxymethyl)glyci-
nato]](4-)-N, N',0, O', 0”0‘''']calciate(2-) dihydrate; calcium chelate of the
disodium salt of ethylenediamine-Ai,N,Ai',N'-tetraacetic acid dihydrate; CAS
Reg. No. 6766-87-6 (dihydrate).
Description. A white or creamy white powder; almost odourless.
Solubility. Soluble in 2 parts of water; very slightly soluble in ethanol

Category. Antidote, chelating agent for metals, mainly lead.
Storage. Sodium calcium edetate should be kept in a tightly closed container.
Additional information. Even in the absence of light. Sodium calcium

edetate is gradually degraded on exposure to a humid atmosphere, the decom-
position being faster at higher temperatures.
Requirements
Definition. Sodium calcium edetate contains not less than 97.0% and not
more than 102.0% of CioHi 2 CaN 2 Na 208 calculated with reference to the anhy-
,
drous substance.
Identity tests
A. Dissolve 2.0 g in 25 ml of water, add 2.0 ml of lead nitrate (100 g/1) TS, shake
and add 6ml of potassium iodide (80 g/1) TS; no yellow precipitate can be
observed (keep the solution for test C).
B. To 0.15ml of ferric chloride (25g/l) TS add 0.15ml of ammonium thio-

cyanate (75 g/1) TS; to this deep red-coloured solution add about 0.05 g of
the test substance; the deep red colour changes to yellow.
C. To the solution prepared in test A, add ammonia (-100 g/1) TS, drop by drop,
until an alkaline reaction is obtained with pH-indicator paper R. Add 5 ml
of ammonium oxalate (25 g/1) TS; a white precipitate is produced (distinc-
tion from disodium edetate).
624

quantity and dissolve the residue in acetic acid (~60g/l) TS.
Cyanide. Dissolve l.Og in 50ml of water, add 0.1 g of potassium iodide R,

4ml of ammonia (-100 g/1) TS, and 0.10ml of silver nitrate (0.01 mol/1) VS, and
allow to stand for 1 minute; the opalescence produced is not less than that
of a standard solution containing 1ml of nitric acid (~130g/l) TS, 1.0ml of
hydrochloric acid (O.OOOlmol/1) VS, 9ml of water, and 0.10ml of silver nitrate
(0.1 mol/1) VS, this solution having also been allowed to stand for 1 minute.
Disodium edetate. Dissolve 5.0 g in 20 ml of water, add 10ml of ammonia

buffer TS2 and 0.2 g of mordant black 11 indicator mixture R, and titrate with
magnesium chloride (0.1 mol/1) VS. Not more than 1.5 ml of magnesium chlo-
ride (0.1 mol/1) VS is required.
Iron. Ignite 0.5 g and dissolve the residue in 40 ml of water. Treat the solution
as described under 2.2.4 Limit test for iron; not more than 80pg/g.

Assay. Dissolve about 0.5g, accurately weighed, in 90ml of water, add 7g of

methenamine R and 5 ml of hydrochloric acid (-70 g/1) TS. Titrate with lead
nitrate (0.05 mol/1) VS, using xylenol orange indicator mixture R. Each ml of
lead nitrate (0.05mol/l) VS is equivalent to 18.71 mg of C]oHi2CaN2Na20g.
Additional requirements for Sodium calcium edetate for

f'arenteral use
sodium edetate.
625
NATRIICHLORIDUM
SODIUM CHLORIDE
Molecular formula. NaCl
Chemical name. Sodium chloride; CAS Reg. No. 7647-14-5.
Category. Ionic equilibration agent.
Storage. Sodium chloride should be kept in a well-closed container.
Additional information. Sodium chloride has a saline taste.
Requirements
Definition. Sodium chloride contains not less than 99.0% and not more than
100.5% of NaCl, calculated with reference to the dried substance.
Identity tests
A. When tested for sodium as described under 2.1 General identification tests,

20mg/ml solution.


under 2.2.5 Limit test for arsenic; not more than 4pg/g.
Barium. Dissolve 4g in 20 ml of water, filter if necessary, divide the solution

into 2 portions and place them in two separate matched tubes. To 1 portion
add 2 ml of sulfuric acid (-100 g/1) TS and to the other, 2 ml of water; the solu-
tions remain equally clear for not less than 30 minutes when viewed down the
vertical axis of the tube in diffused light against a black background.
626
Calcium and magnesium. To 20ml solution add 2ml each of

of a lOmg/ml
ammonia (~100g/l) TS, ammonium oxalate (25g/l) TS, and disodium hydrogen
phosphate (40g/l) TS; no turbidity is produced within 5 minutes.
Iodides and bromides. Digest 2g of finely powdered test substance for 3

hours with 25 ml of warm ethanol (-750 g/1) TS, cool, and filter. Evaporate the
dryness, dissolve the residue in 5 ml of water, add 1 ml of chloroform
filtrate to
R, and cautiously add, drop by drop, with constant agitation, 5 drops of chlo-
rine TS, previously diluted with twice their volume of water; the chloroform
does not acquire a violet, yellow or orange colour.
Iron and sodium ferrocyanides. To 0.5g add 5 drops of sulfuric acid

(-100 g/1) TS and carefully heat until dry. Dissolve the residue in 6ml of water,
add 1ml of hydrochloric acid (-70g/l) TS, 1 drop of hydrogen peroxide
(-60 g/1) TS, and 2 ml of ammonium thiocyanate (-75 g/1) TS; this constitutes
solution A. Prepare in a similar manner solution B using 0.40 ml of iron stan-
dard FeTS instead of the residue obtained from the test substance. Solution A
is not more intensely coloured than solution B when compared as described
under 1.11 Colour of liquids (16pg/g of total Fe).

Clarity and colour of solution. A solution of 1.0 g in 10 ml of carbon-dioxide-

lOmg/g.

add 2 drops of bromothymol blue/ethanol TS; not more than 0.1 ml of sodium
hydroxide (0.02 mol/1) VS or 0.2 ml of hydrochloric acid (0.02 mol/1) VS is
required to attain the midpoint of the indicator (green).

with silver nitrate (0.1 mol/1) VS, using potassium chromate (100 g/1) TS as indi-
cator. Each ml of silver nitrate (0.1 mol/1) VS is equivalent to 5.844 mg of NaCl.
Additional requirements for Sodium chloride for

parenteral use
terial endotoxins; contains not more than 5.0 lU of endotoxin RS per g.
627
NATRII CITRAS
SODIUM CITRATE
Sodium citrate, anhydrous
Sodium citrate, dihydrate
Molecular formula. CgHsNasO/ (anhydrous); C 6 HsNa 307 2 H 20 ,
(dihydrate).
Graphic formula.
CH,— COONa
HO —C
I
COONa . nHÔ
I
CHj— COONa
n 7= 0 (anhydrous)
n 2 (dihydratel
Chemical name. Trisodium citrate; trisodium 2-hydroxy- 1,2, 3-propanetricar-

boxylate; CAS Reg. No. 68-04-2 (anhydrous).
Trisodium citrate dihydrate; trisodium 2-hydroxy-l,2,3-propanetricarboxylate

dihydrate; CAS Reg. No. 6132-04-3 (dihydrate).
Solubility. Freely soluble in water and very soluble in boiling water; practi-
cally insoluble in ethanol (~750g/l) TS and ether R.
Category. Systemic alkalinizing agent; component of oral rehydration salt

mixtures.
Storage. Sodium citrate should be kept in a tightly closed container.
Labelling. The designation on the container of Sodium citrate should state

whether the substance is the dihydrate or is in the anhydrous form.
Additional information. Sodium citrate is slightly deliquescent in moist air.
Requirements
Definition. Sodium citrate contains not less than 99.0% and not more than
101.0% of CfiHsNaaOj, calculated with reference to the anhydrous substance.
628
Identity tests

20mg/ml solution.
B. A 20mg/ml solution yields reaction B described under 2.1 General identifi-

Oxalates. Dissolve 0.5 g in 4 ml of water, add 3 ml of hydrochloric acid

(-420 g/1) TS and 1 g of granulated zinc R, and heat on a water-bath for 1 minute.
Allow to stand for 2 minutes, decant the liquid into a test-tube containing
0.25 ml of phenylhydrazine hydrochloride (10 g/1) TS, and heat to boiling. Cool
rapidly, transfer to a graduated cylinder, and add an equal volume of hydrochlo-
ric acid (-420 g/1) TS, followed by 0.25ml of potassium ferricyanide (50 g/1) TS.
Shake and allow to stand for 30 minutes; any pink colour produced is not more
intense than that of a similarly treated solution containing 4 ml of oxalic acid
(0.05 g/1) TS.


Fischer method. Method A. For the anhydrous form use about 1 g of the sub-
stance; the water content is not more than lOmg/g. For the dihydrate use about
0.3 g of the substance; the water content is not less than O.lOg/g and not more
than 0.13g/g.

add 0.1 ml of phenolphthalein/ethanol TS; not more than 0.2 ml of hydrochlo-
ric acid (0.1 mol/1) VS or 0.2ml of sodium hydroxide (0.1 mol/1) VS is required
to change the colour of the solution.

Rl, heat to about 50 °C, allow to cool to room temperature, add 0.25ml of 1-
naphtholbenzein/acetic acid TS, and titrate with perchloric acid (0.1 mol/1) VS
until a green colour is obtained as described under 2.6 Non-aqueous titration.
of CfsH^NasO/.
629
NATRII CROMOGLICAS
SODIUM CROMOGLICATE
Molecular formula. C 23 Hi 4 Na 20 i,
Graphic formula.
Chemical name. Disodium 5,5'-[(2-hydroxytrimethylene)dioxy]bis[4-oxo-

4H-l-benzopyran-2-carboxylate]; disodium 5,5'-[(2-hydroxy-l,3-propanediyl)
bis(oxy)]-bis[4-oxo-4H-l-benzopyran-2-carboxylate]; CAS Reg. No. 15826-37-6.
Solubility. Freely soluble in water; slightly soluble in methanol R; very slightly

soluble in ethanol (-750 g/1) TS; practically insoluble in ether R.
Storage. Sodium cromoglicate should be kept in a tightly closed container,

Additional information. Sodium cromoglicate is hygroscopic.
Requirements
Definition. Sodium cromoglicate contains not less than 98.0% and not more
than 101.0% of C 23 Hi 4Na 20 n, calculated with reference to the dried substance.
Identity tests

630
spectrum obtained from sodium cromoglicate RS or with the reference spec-

trum of sodium cromoglicate,
B. See the test described below under “Related substances”. The spot obtained
with solution B corresponds in position, appearance, and intensity with that
C. Dissolve 5 mg in 0.5 ml of methanol R and add 3 ml of 4-aminoantipyrine

TSl, and allow to stand for 5 minutes; an intense yellow colour is produced.

20mg/ml solution.
Oxalates. Dissolve O.lOg in 20ml of water, add 5.0ml of iron salicylate TS

and sufficient water to produce 50ml; the absorbance at about 480 nm is not
less than that of a solution containing 0.35 mg of oxalic acid R prepared in a
similar manner.
exceeding O.dkPa or about 5mm of mercury); it loses not more than lOOmg/g.
Acidity or alkalinity. Dissolve l.Og in 25ml of carbon-dioxide-free water R

and add 0.1ml of bromothymol blue/ethanol TS; not more than 0.25ml of
sodium hydroxide (0.1 moI/1) VS or 0.25ml of hydrochloric acid (0.1 mol/1) VS
is required to obtain the midpoint of the indicator (green).
chromatography, using silica gel R4 as the coating substance, omitting the
heating, and allowing the plate to stand overnight at room temperature. As
the mobile phase use a mixture of 9 volumes of chloroform R, 9 volumes of
methanol R, and 2 volumes of glacial acetic acid R. Apply separately to the plate
10 pi of each of 3 solutions in a mixture of 1 volume of acetone R, 4 volumes
of tetrahydrofuran R (which has been freed from stabilizer by passage through
a column of suitable alumina), and 6 volumes of water, containing (A) 20 mg
of the test substance per ml, (B) 0.10 mg of the test substance per ml, and (C)
0.10 mg of sodium cromoglicate RS per ml. After removing the plate from the
chromatographic chamber, allow it to dry in air, and examine the chro-
moving ahead of the principal spot, is not more intense than that obtained with
solution B.
631
Assay. Dissolve about 0.18g, accurately weighed, in a mixture of 25ml of

propylene glycol R and 5 ml of 2-propanol R, warming slightly, cool and add
30ml of dioxan R. Titrate with perchloric acid/dioxan (0.1 mol/1) VS, deter-
mining the endpoint potentiometrically as described under 2.6 Non-aqueous
titration. Method A. Each ml of perchloric acid/dioxan (0.1 mol/1) VS is equiv-
alent to 25.62 mg of C23Hi4Na20i,.
NATRII FLUORIDUM
SODIUM FLUORIDE
Molecular formula. NaF
Chemical name. Sodium fluoride; CAS Reg. No. 7681-49-4.
Description. A white powder; odourless.
Solubility. Soluble in water; practically insoluble in ethanol (-750 g/1) TS.
Category. Mineral salt.
Storage. Sodium fluoride should be kept in a well-closed container.
Requirements
Definition. Sodium fluoride contains not less than 98.0% and not more than
101.0% of NaF, calculated with reference to the dried substance.
Identity tests

20mg/ml solution.
B. Place O.lOg in a lead or platinum crucible, add about 1ml of sulfuric acid
(-1760 g/1) TS, cover the crucible with a piece of clear polished glass, and
heat on a water-bath for 15 minutes. Remove the glass cover, rinse with
water and wipe dry; the surface of the glass is etched.
C. Add a few mg of the test substance to a mixture of O.lOml of freshly pre-

pared sodium alizarinsulfonate (10 g/1) TS and O.lOml of zirconyl nitrate TS;
a red colour develops,which changes to yellow.
632
lOmg/g.
Acidity or alkalinity. Dissolve l.Og in 20ml of water in a platinum dish, add

and dissolve into this solution 3 g of potassium nitrate R, and cool to 0 °C. Add
0.15 ml of phenolphthalein/ethanolTS; not more than 2.0 ml of sodium hydrox-
ide (0.05mol/l) VS or 1.0ml of sulfuric acid (0.05mol/l) VS is required to obtain
the midpoint of the indicator (pink). (Keep the solution for the test of
fluorosili cates.)
FluorosUicates. Heat to boiling the solution obtained in the test for acidity or
alkalinity and titrate while hot with sodium hydroxide (0.05 mol/1) VS until a
permanent pink colour is produced; not more than 1.5ml of sodium hydroxide
(0.05 mol/1) VS are required.
Assay. Dissolve about 30 mg, accurately weighed, in 1.0 ml of water. Add cau-
tiously 15 ml of acetic anhydride R and boil gently for 2 minutes, placing a small
funnel on the flask to serve as a reflux condenser. Cool and titrate with per-
Method A. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 4.199mg
of NaF.
NATRII HYDRO GENOCARBONAS

SODIUM HYDROGEN CARBONATE
Molecular formula. NaHCOs
Chemical name. Monosodium carbonate; CAS Reg. No. 144-55-8.
Other name. Sodium bicarbonate.
Solubility. Soluble in water; practically insoluble in ethanol (-750 g/1) TS.
Category. Systemic alkalinizing agent; antacid.
633
Storage. Sodium hydrogen carbonate should be kept in well-closed

containers.
Additional information. Sodium hydrogen carbonate has a saline and

slightly alkaline taste. It is stable in air, but slowly decomposes in moist air.
Requirements
Definition. Sodium hydrogen carbonate contains not less than 99.0% and
not more than 101.0% of NaHCOg, calculated with reference to the dried
substance.
Identity tests

20mg/ml solution.
B. To a small amount add hydrochloric acid (~70g/l) TS; it effervesces and the
gas is colourless.Add a few drops of calcium hydroxide TS; immediately a
white precipitate is formed.
C. To a 20 mg/ml solution add at room temperature a few drops of magnesium

sulfate (50g/l) TS; no precipitate is formed. Boil the mixture; a white pre-
cipitate is formed.
Ammonium. Heat l.Og in a test-tube; the gas evolved does not turn moist-
ened red litmus paper R blue.
Arsenic. Use a solution of 3.3 g in 35ml of water and proceed as described

Calcium. Boil a 20 mg/ml solution for 5 minutes; the solution is clear or at

most very slightly opalescent.
Carbonates. pH of a 50 mg/ml solution in carbon-dioxide-free water R, not

more than 8.6.
Chlorides. Dissolve mixture of 2 ml of nitric acid (-130 g/1) TS and

1.7 g in a
634
Sulfates. Dissolve 2.5 g in 40ml of water, add 1ml of hydrochloric acid

(-250 g/1) TS, and proceed as described under 2.2,2 Limit test for sulfates; the
sulfate content is not more than 0.2mg/g.
Clarity and colour of solution. A solution of 0.50g in 10ml of carbon-


with hydrochloric acid (0.2 mol/1) VS, using 3 drops of methyl orange/ethanol
TS as indicator. Each ml of hydrochloric acid (0.2 mol/1) VS is equivalent to
16,80mg ofNaHCOj.
Additional requirements for Sodium hydrogen carbonate for

parenteral use
bacterial endotoxins; contains not more than 5.0 lU of endotoxin RS per
miUiequivalence.
NATRII HYDROXYDUM
SODIUM HYDROXIDE
NaOH
Chemical name. Sodium hydroxide; sodium hydroxide (Na(OH)); CAS Reg.

No. 1310-73-2.
Description. White or almost white, fused masses, sticks, pellets, or flakes;

they are hard and brittle, showing a crystalline fracture.
Solubility. Very soluble in water and ethanol (~750g/l) TS.
Category. Alkalinizing agent; used in the preparation of sodium lactate

solution.
Storage. Sodium hydroxide should be kept in a tightly closed container.
635
Additional information. CAUTION-. Sodium hydroxide must be handled

with care, avoiding contact with the skin. It is very deliquescent, strongly alka-
line, and corrosive. It rapidly absorbs carbon dioxide.
Requirements
Sodium hydroxide contains not less than 97 5 % of total alkali,
. calculated as
NaOH, and not more than the equivalent of 2 5 % of Na 2 C 03 . .
Identity tests

20mg/ml solution.
B. A solution is strongly alkaline.

Aluminium, iron, and matter insoluble in hydrochloric acid. Boil 5 g with

70ml of hydrochloric acid (~70g/l) TS. Cool, make alkaline with ammonia
(~100g/l) TS, boil, filter, and wash with a mixture of equal volumes of water
and ammonium nitrate (50g/l) TS. Ignite the residue to constant mass; not more
than 5 mg.
Potassium. Dissolve 0.25 g in 5 ml of water, acidify with acetic acid (~60g/l)

TS, and add 5 drops of sodium cobaltinitrite (lOOg/1) TS; no precipitate is
formed.
Chlorides. Dissolve 0.35g in a mixture of 2ml of nitric acid (-130 g/1) TS and

(~70g/l) TS, and proceed as described under 2.2.2 Limit test for sulfates; the
sulfate content is not more than 1.2 mg/g.
Assay. Dissolve about 2g, accurately weighed, in 80 ml of carbon-dioxide-free

water R. Add 0.3ml of phenolphthalein/ethanol TS and titrate with hydrochlo-
ric acid (1 mol/1) VS until the red colour of the indicator disappears. Note the
consumption of acid. Add 0.3ml of methyl orange/ethanol TS to the solution
636
and continue the titration with hydrochloric acid (1 mol/1) VS until a perma-
nent red colour is obtained.
Each ml of hydrochloric acid (1 mol/1) VS used in the second titration is equiv-

alent to lOb.Omg of Na 2 C 03 Each ml of hydrochloric acid
. (1 mol/1) VS used in
the combined titrations is equivalent to 40.00mg of total alkali, calculated as
NaOH.
NATRII NITRIS
SODIUM NITRITE
Molecular formula. NaNOj
Chemical name. Nitrous acid, sodium salt; CAS Reg. No. 7632-00-0.
Description. A white to slightly yellow, granular powder, or white or almost

white, opaque, fused masses or sticks; odourless.
Solubility. Freely soluble in water; sparingly soluble in ethanol (~750g/l) TS.
Category. Vasodilator.
Storage. Sodium nitrite should be kept in a well-closed container, protected

from light.
Additional information. Sodium nitrite is deliquescent in air. Even in the

Requirements
Definition. Sodium nitrite contains not less than 98.0% and not more than
100.5% of NaNOj, calculated with reference to the dried substance.
Identity tests
A. When tested for sodium as described under 2.1 General identification tests
yields the characteristic reactions.If reaction B is to be used, prepare a
O.lOg/ml solution acidified with acetic acid (-300 g/1) TS.
B. Dissolve O.lOg in 1.0ml of water and add 1.0ml of ferrous sulfate(~15g/l)

TS; after a few minutes a deep brown colour is produced.
637
C. Dissolve 0.20g in 1.0ml of water and add 1.0ml of sulfuric acid (~100g/l)
TS; brownish red fumes are evolved. Add a few drops of starch/iodide TS;
a blue colour is produced.
Heavy metals. Dissolve l.Og in 10ml of hydrochloric acid (~70g/l) TS, evap-
orate to dryness on a water-bath and until the odour of hydrochloric acid is no
longer perceptible. Proceed with the residue as described under 2.2.3 Limit test
for heavy metals, Procedure 1; determine the heavy metals content according
to Method A; not more than 20|ig/g.
Chlorides. For the preparation of the test solution, boil 2.5g in a mixture of
3 ml of water and 2 ml of nitric acid (~1000g/l) TS, cool, and proceed as
described under 2.2.1 Limit test for chlorides; the chloride content is not more
than 0.1 mg/g.
Loss on drying. Dry at ambient temperature over silica gel, desiccant, R for
4 hours; it loses not more than S.Omg/g.
Acidity or alkalinity. To a solution of 0.5g in 10ml of water add 0.1ml of

sodium hydroxide (0.01 mol/1) VS and 0.25ml of phenol red/ethanol TS; a red
colour is produced. Add 0.3 ml of hydrochloric acid (0.01 mol/1) VS; the colour
changes to yellow.

100ml. To 10.0ml of this solution add 20.0ml of potassium permanganate
(0.02 mol/1) VS and 10ml of sulfuric acid (~1760g/l) TS. AUow to stand for 10
minutes, add 0.5 g of potassium iodide R, swirl the flask, and titrate the liber-
ated iodine with sodium thiosulfate (0.1 mol/1) VS, using starch TS as indicator.
Each ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to 3.450 mg of NaNOj.
Additional requirements for Sodium nitrite for parenteral use

NATRII NITROPRUSSIDUM
SODIUM NITROPRUSSIDE
Molecular formula. Na2[Fe(CN)5N0],2H20
638
Graphic formula.
’
2
• 2Na* • 2HjO
Chemical name. Disodium pentacyanonitrosylferrate(2-) dihydrate; dis-

odium (OC-6-22)-pentakis(cyano-C)nitrosylferrate(2-) dihydrate; CAS Reg.
No. 13755-38-9 (dihydrate).
Other names. Sodium nitroferricyanide; sodium nitroprussiate.
Description. Reddish brown crystals or powder; odourless or almost

odourless.
Solubility. Freely soluble in water; slightly soluble in ethanol (-750 g/1) TS,
Category. Antihypertensive drug.
Storage. Sodium nitroprusside should be kept in a tightly closed container,

Requirements
Definition. Sodium nitroprusside contains not less than 99.0% and not more
than 100.5% of Na 2 [Fe(CN)sNO], calculated with reference to the anhydrous
substance.
Identity tests
of a 1-cm layer at this wavelength is about 1.10.
B. When tested for sodium under

2.1 General identification tests
as described
yields the characteristic reactions.If reaction B is to be used, prepare a
O.lOg/ml solution acidified with acetic acid (-300 g/1) TS.
C. Dissolve 5mg in 2.0ml of water, add 0,1ml of acetone R and 0.5ml of

sodium hydroxide (-80 g/1) TS; an orange colour is produced. Add about
2 ml of acetic acid (-300 g/1) TS; the colour changes to purple.
639

30ml of water, and proceed as described under 2,2,1 Limit test for chlorides;

Ferricyanide. Dissolve 0.50g in 20ml of ammonium acetate buffer, pH 4.62,

TS, and divide the solution into two equal portions, A and B. To portion B add
1 ml of ferricyanide standard (50pg/ml) TS, then add to both portions 5 ml of
ferrous ammonium sulfate (lg/1) TS and dilute to 50 ml with water. Prepare a
blank solution by dissolving 0.25 g of the substance to be examined in 10 ml of
ammonium acetate buffer, pH 4.62, TS, and diluting to 50 ml with water. Allow
to stand for hour and measure the absorbance of the solutions at the
1
maximum about 720nm. The absorbance of portion A when measured

at
against the blank is not greater than the absorbance of portion B when mea-
sured against portion A (0.2 mg/g).
Ferrocyanide. Dissolve 2.0g in 40ml of water and divide the solution into
two equal portions, A and B. To portion B add 2 ml of ferrocyanide standard
(lOOpg/ml) TS, thenadd to both portions 0.2ml of ferric chloride (50g/l) TS and
dilute to 50 ml with water. Allow to stand for 5 minutes. Prepare a blank solution
by dissolving l.Og of the substance to be examined in sufficient water to produce
50 ml. Measure the absorbance of the solutions at the maximum at about 695 nm.
The absorbance of portion A when measured against the blank is not greater than
the absorbance of portion B when measured against portion A (0.2 mg/g).
Insoluble matter. Dissolve 10. Og in 50ml of water, heat the solution on a

water-bath for 30 minutes, filter, wash the insoluble matter with water, and dry
to constant weight at 105°C; the content of insoluble matter is not more than
0.1 mg/g.

is not less than 90 mg/g and not more than 150 mg/g.
Assay. Dissolve about 0.35g, accurately weighed, in 100ml of water. Add

0.1ml of sulfuric acid (-100 g/1) TS and 20ml of ethanol (~750g/l) TS. Titrate
with silver nitrate (0.1 mol/1) VS, determining the endpoint potentiometrically
using a silver/silver chloride electrode system. Each ml of silver nitrate
(0.1 mol/1) VS is equivalent to 13.10mg of Na 2 [Fe(CN)sNO].
640
NATRII SALICYLAS
SODIUM SALICYLATE
Molecular formula. C/HsNaOs
Graphic formula.
Chemical name. Sodium 2-hydroxybenzoate; CAS Reg. No. 54-21-7.
Description. Small, colourless crystals or shiny flakes, or a white, crystalline

powder; odourless or almost odourless.
Solubility. Freely soluble in water and ethanol (-750 g/1) TS; practically
Category. Analgesic; antiphlogistic.
Storage. Sodium salicylate should be kept in a well-closed container, protected

from light.
Additional information. Sodium salicylate is discoloured on exposure to

light.
Requirements
Definition. Sodium salicylate contains not less than 99.0% and not more than
101.0% of C/HsNaOa, calculated with reference to the dried substance.
Identity tests

20mg/ml solution.
B. A 0.05 g/ml solution yields the reaction described under 2.1 General identi-
fication tests as characteristic of salicylates.
Heavy metals. For the preparation of the test solution use 2.0 g dissolved in
45ml of water, add 5ml of hydrochloric acid (~70g/l) TS, and filter. Dilute
25ml of the filtrate to40ml with water and mix; determine the heavy metals
641
content as described under 2.2.3 Limit test for heavy metals, according to
Method A; not more than 20|J.g/g.
Chlorides. Dissolve 1.25 g in a mixture of 5 ml of water and 5 ml of ethanol

(~710g/l) TS. Add 1 ml of nitric acid (~1000g/l) TS, filter, and proceed with the
filtrate as described under 2.2.1 Limit test for chlorides; the chloride content is
Sulfates. Dissolve 0.85 g in 20 ml of water, add 1ml of hydrochloric acid

(-250 g/1) TS, and filter. Proceed with the filtrate as described under 2.2.2 Limit
test for sulfates; the sulfate content is not more than 0.6mg/g.
Sulfites and thiosulfates. Dissolve l.Og in 20ml of water, add 1ml

of hydrochloric acid (-250 g/1) TS, and filter. Titrate the filtrate with iodine
(0.05 mol/1) VS; not more than 0.15ml of titrant is required to produce a yellow
colour.
Clarity and colour of solution. A freshly prepared solution of l.Og in 10ml

of water is clear and not more intensely coloured than standard colour solution
Rdl when compared as described under 1.11 Colour of liquids.
S.Omg/g.
Acidity. Dissolve 2.0g in 50ml of carbon-dioxide-free water R and add 10

drops of phenol red/ethanol TS; the solution is yellow. Titrate with sodium
hydroxide (0.1 mol/1) VS; not more than 0.2 ml is required to produce a red
colour.
aqueous titration, Method A. Each nil of perchloric acid (0.1 mol/1) VS is equiv-
alent to 16.01 mg of C HsNa 03
7 .
NATRII STIBOGLUCONAS
SODIUM STIBOGLUCONATE
Chemical name. D-Gluconic acid cyclic ester with antimonic acid (H 8 Sb 209)
(2 :
1), trisodium salt, nonahydrate; 2,4:2',4'-0-(oxydistibylidyne)bis-D-gluconic
acid, Sb,Sb'- dioxide, trisodium salt, nonahydrate; CAS Reg. No. 16037-91-5.
Description. A colourless, mostly amorphous powder; odourless.
642
Solubility. Very soluble in water; practically insoluble in ethanol (-750 g/1) TS

and ether R.
Category. Antileishmaniasis drug.
Storage. Sodium stibogluconate should be kept in a well-closed container.
Requirements
Definition. Sodium stibogluconate is a pentavalent antimony compound of
indefinite composition. It has been represented by the formula C 6H 9Na 209Sb,
but it usually contains less than 2 atoms of sodium for each atom of antimony.
Sodium stibogluconate contains not less than 30.0% and not more than 34.0%
of total antimony, calculated with reference to the dried substance.
Identity tests
A. Heat a small quantity of the test substance; it chars without melting. Dis-
solve the residue in acetic acid (-60 g/1) TS; it yields reaction B, characteris-
tic of sodium as described under 2.1 General identification tests.
B. Dissolve 0.5g in 10 ml of water and add a few drops of hydrogen sulfide

TS; an orange precipitate is produced, which dissolves in sodium hydrox-
ide (-80 g/1) TS.
C. A lOmg/ml solution is dextrorotatory.
Chlorides. Dissolve 2.5 g in a mixture of 50 ml of water, 2 ml of nitric acid

(-130 g/1) TS, and 75 ml of acetate buffer, pH 5.0, TS. Titrate with silver nitrate
(0.1 mol/1) VS, determining the endpoint potentiometrically; not more than
3.0 ml of silver nitrate (0.1 mol/1) VS are required.
Colour and pH value. Dissolve 0.3 g in 10ml of water and heat the solution
in an autoclave under reduced pressure (about 70kPa) for 30 minutes; the solu-
tion is colourless or almost colourless and has a pH between 5.0 and 5.6.
Trivalent antimony. Dissolve 2.0 g in 30 ml of water, add 15 ml of hydrochlo-

ric acid (-250 g/1) TS, and titrate with potassium bromate (0.00833 mol/1) VS
using methyl/orange TS as indicator; not more than 1.3 ml of potassium

bromate (0.00833 mol/1) VS are required.
643
Assay. Place about 0.25 g, accurately weighed, in a 300-ml long-necked flask,

add 10ml of nitric acid (~1000g/l) TS and 5ml of sulfuric acid (~1760g/l) TS,
and heat cautiously over a small flame, keeping the liquid in motion by rotat-
ing the flask; remove the flask from the flame at the onset of the first vigorous
reaction until this subsides. Continue to heat until white fumes are evolved and
allow the liquid to cool. Add 1ml of nitric acid (-1000 g/1) TS and again heat
until white fumes are evolved. Add 1 g of ammonium sulfate R, again heat to
the point of fuming, cool thoroughly, and add 1 g of tartaric acid R and 60 ml
of water. To the clear, almost colourless solution add 1 g of potassium iodide R
and boil the solution gently for about 5 minutes or until free iodine is expelled,
the liquid becoming pure yellow in colour. Cool, add sodium hydroxide
(-400 g/1) TS until just alkaline (about 15ml), cool again, acidify with sulfuric
acid (-100 g/1) TS until just acid, add an excess of sodium hydrogen carbonate
R, and titrate with iodine (0.02 mol/1) VS, using starch TS as indicator if
necessary. Each ml of iodine (0.02 mol/1) VS is equivalent to 2.435 mg of total
antimony.
NATRII SULFAS
SODIUM SULFATE
Molecular formula. Na2SO4,10H2O
Chemical name. Disodium sulfate decahydrate; sulfuric acid disodium salt,
decahydrate; CAS Reg. No. 7727-73-3 (decahydrate).
Other name. Glauber's salt.
Description. Colourless crystals or a white powder; odourless.

TS.
Category. Laxative.
Storage. Sodium sulfate should be kept in a well-closed container.
Additional information. Sodium sulfate partially dissolves in its own water

of crystallization at about 33 °C.
644
Requirements
Definition. Sodium sulfate contains not less than 99.0% and not more than
100.5% of Na 2 S 04 calculated with reference
,
Identity tests

0.05 g/ml solution.

Ammonium salts. Transfer 1 g to a test-tube, add about 0.3g of potassium

hydroxide R and heat the mixture; a moistened red litmus paper R placed in
the evolved vapours does not turn blue.
Arsenic. Use a solutionof 5g in 35 ml of water and proceed as described under

2.2.5 Limit test for arsenic; the arsenic content is not more than 2pg/g.
Calcium. To two separate comparison tubes transfer 0.2 ml of ethanolic

calcium standard (lOOpg/mlCa) TS, add 1.5ml of ammonium oxalate (25g/l)
TS, allow to stand for 1 minute, and then add 1 ml of acetic acid (-120 g/1) TS.
To one tube add a solution of the substance to be examined containing 0.5 g in

15ml of water, and to the second tube add 10ml of calcium standard (lOpg/ml
Ga) TS and 5 ml of water. Observe any opalescence produced after 15 minutes;
the opalescence in the first tube is not more intense than that in the second
tube (200pg/g).

Iron. Using l.Og prepare a solution in 40 ml of water and proceed as described

under 2.2.4 Limit test for iron; not more than 40pg/g.
Magnesium. Dissolve 0.5g in 10ml of water, add 1 ml of glycerol R, 0.15ml

of titan yellow TS, 0.2ml of ammonium oxalate (50 g/1) TS, and 5 ml of sodium
hydroxide (-80 g/1) TS, and shake; any pink colour produced is not more intense
than that of a similarly treated mixture of 5 ml of magnesium standard (lOpg/ml
Mg) TS and 5 ml of water.
645

Reducing substances. Dissolve 0.5g in 5ml of water, add 1 ml of sulfuric acid

(-100 g/1) TS and 0.20ml of potassium permanganate (0.002mol/l) VS. Allow
to stand for 15 minutes; no discoloration is observed.
Loss on drying. Dry at 30 °C for 1 hour and then to constant weight at

130 °C; it loses not less than 0.52 g/g and not more than 0.57 g/g.
Acidity or alkalinity. Dissolve 0.5g in 10ml of carbon-dioxide-free water R

and add 0.1ml of bromothymol blue/ethanol TS; not more than 0.5ml of car-
bonate-free sodium hydroxide (0.01 mol/1) VS or 0.5ml of hydrochloric acid
(0.01 mol/1) VS is required to obtain the midpoint of the indicator (green).
Assay. Dissolve about 0.25 g, accurately weighed, in 250ml of water, add

10 ml of hydrochloric acid (-70 g/1) TS, heat to boiling and add a sufficient quan-
tity of barium chloride (50 g/1) TS. Heat on a water-bath for 30 minutes, stir-
ring occasionally. Collect the precipitate, wash, dry and ignite at 600 °C. Each
g of residue is equivalent to 0.608 g of Na 2 S 04 .
NATRII SULFAS ANHYDRICUS

SODIUM SULFATE, ANHYDROUS
Molecular formula. Na 2 S 04
Chemical name. Disodium sulfate; sulfuric acid disodium salt, anhydrous;

CAS Reg. No. 7757-82-6 (anhydrous).

TS.
Category. Laxative.
Storage. Anhydrous sodium sulfate should be kept in a well-closed container.
Additional information. Anhydrous sodium sulfate is hygroscopic.
646
Requirements
Definition. Anhydrous sodium sulfate contains not less than 99.0% and not
more than 100.5% of Na 2 S 04 ,
Identity tests

20mg/ml solution.

Ammonium salts. Transfer 0.5 g to a test-tube, add about 0.3 g of potassium

hydroxide R and heat the mixture; a moistened red litmus paper R placed in
the evolved vapours does not turn blue.

Calcium. To two separate comparison tubes transfer 0.2 ml of ethanolic

calcium standard (lOOpg/mlCa) TS, add 1.5ml of ammonium oxalate (25g/l)
1 minute, and then add 1 ml of acetic acid (-120 g/1) TS.
TS, allow to stand for
To one tube add a solution of the substance to be examined containing 0.22 g
in 15ml of water, and to the second tube add 10ml of calcium standard
(10 pg/mlGa) TS and 5ml of water. Observe any opalescence produced after 15
minutes; the opalescence in the first tube is not more intense than that in the
second tube (450pg/g).

Iron. Using 0.44 g prepare a solution in 40 ml of water and proceed as

described under 2.2.4 Limit test for iron; not more than 90pg/g.
Magnesium. Dissolve 0.22g in 10ml of water, add 1 ml of glycerol R, 0.15ml

of titan yellow TS, 0.25ml of ammonium oxalate (50 g/1) TS, and 5 ml of sodium
hydroxide (-80 g/1) TS, and shake; any pink colour produced is not more intense
than that of a similarly treated mixture of 5 ml of magnesium standard (lOpg/ml
Mg) TS and 5 ml of water.
647

Reducing substances. Dissolve 0.25 g in 5 ml of water, add 1ml of sulfuric

acid (-100 g/1) TS and 0.20 ml of potassium permanganate (0.002 mol/1) VS.
Allow to stand for 15 minutes; no discoloration is observed.
50mg/g.
Acidity or alkalinity. Dissolve 0.22 g in 10ml of carbon-dioxide-free water R

and add 0.1ml of bromothymol blue/ethanol TS; not more than 0.5ml of car-
bonate-free sodium hydroxide (0.01 mol/1) VS or 0.5ml of hydrochloric acid
Assay. Dissolve about 0.1 g, accurately weighed, in 250ml of water, add 10ml
of hydrochloric acid (-70 g/1) TS, heat to boiling, and add a sufficient quantity
of barium chloride (50 g/1) TS. Heat on a water-bath for 30 minutes, stirring
occasionally. Collect the precipitate, wash, dry and ignite at 600 °C. Each g of
residue is equivalent to 0.608 g of Na 2 S 04 .
NATRII THIOSULFAS
SODIUM THIOSULFATE
Molecular formula. Na 2 S 203 5 H 20 ,
Chemical name. Disodium thiosulfate pentahydrate; disodium thiosulfate

(Na 2 S 20 s) pentahydrate; thiosulfuric acid (H 2 S 2 O 3), disodium salt, pentahydrate;
CAS Reg. No. 10102-17-7 (pentahydrate).
Description. Transparent, colourless crystals; odourless.
Solubility. Very soluble in water; practically insoluble in ethanol (-750 g/1) TS.
Category. Antidote.
Storage. Sodium thiosulfate should be kept in a tightly closed container.
Additional information. Sodium thiosulfate effloresces in dry air at temper-

atures exceeding 33 °C and dissolves in its water of crystallization at about
49 °C.
648
Requirements
Definition. Sodium thiosulfate contains not less than 99.0% and not more
than 101.0% of Na 2 S 203 5 H 20
,
.
Identity tests
A. Dissolve l.Og in 10ml of water (use this solution also for tests B and C). To
2 ml add 1.0ml of iodine TS; the colour of the solution is discharged. Add
0.25 ml of barium chloride (50g/l) TS; the solution remains clear.
B. To 2 ml A add 1.0 ml of hydrochloric acid

of the solution prepared in test
(~70g/l) TS; an odour of sulfur dioxide is perceptible and a precipitate of
sulfur is produced (proceed with caution).
C. To 2 ml of the solution prepared in test A add 2 ml of silver nitrate (0.1 mol/1)

VS; a white precipitate is formed, but quickly becomes yellowish, then
black.
D. When tested for sodium as described under 2.1 General identification

tests yields the characteristic reactions. If reaction B is to be used, prepare
a O.lOg/ml solution.
Heavy metals. Dissolve l.Og in 10 ml of water, add slowly 5 ml of hydrochlo-

ric acid (~70g/l) TS, and evaporate to dryness on a water-bath. Add 15ml of
water to the residue, boil gently for 2 minutes and filter. Heat the filtrate to
boiling, add sufficient bromine TSl to the hot filtrate to produce a clear solu-
tion and provide a slight excess of bromine. Boil the solution to expel the excess
bromine. Cool, add 0.05ml of phenolphthalein/ethanol TS and neutralize with
sodium hydroxide (1 mol/1) VS. Adjust the pH to 3-4 with acetic acid (~60g/l)
TS, dilute to 40ml with water, and determine the heavy metals content as
described under 2.2.3 Limit test for heavy metals, according to Method A; not
more than 20 pg/g.
Chlorides. Dissolve 1.20 g in a mixture of 20ml of water and 2 ml of nitric

acid (-130g/1) TS, boil gently for 3-4 minutes, cool, filter, and proceed with the
as described under 2.2.1 Limit test for chlorides; the chloride content is
filtrate
Sulfates and sulfites. Dissolve 0.25g in 10ml of water, add 5ml of iodine TS,
and gradually more iodine TS, drop by drop, until a very faint persistent yellow
colour is produced. Proceed as described under 2.2.2 Limit test for sulfates; the
sulfate content is not more than 2mg/g.
Sulfides. Dissolve Ig in 10ml of water and add 0.05ml of sodium nitroprus-

side (45 g/1) TS; the solution does not become violet.
649
Clarity and colour. A solution of 1.0 g in 10ml of carbon-dioxide-free water

R is clear and colourless.
pH value. pH of a O.lOg/ml solution in carbon-dioxide-free water R, 6, 0-8, 4.

with iodine (0.05 mol/1) VS using starch TS as indicator, added towards the end
of the titration. Each ml of iodine (0.05 mol/1) VS is equivalent to 24.82 mg of
Na2S203,5H20.
Additional requirements for Sodium thiosulfate for

parenteral use
NATRII VALPROAS
SODIUM VALPROATE
Molecular formula. CsH]5Na02
Graphic formula.
CH3CH2CHjCHCOjNa
I
CHjCHjCHj
Chemical name. Sodium 2-propylvalerate; sodium 2-propylpentanoate; CAS

Reg. No. 1069-66-5.

almost odourless.
Storage. Sodium valproate should be kept in a well-closed container.
Additional information. Sodium valproate is deliquescent.
650
Requirements
Definition. Sodium valproate contains not less than 98.0% and not more than
101.0% of C Hi 5 Na 02
8 ,
Identity tests
A. Dissolve 0.5g in 5ml of water, add 5ml of chloroform R and 1ml of
hydrochloric acid (~70g/l) TS, shake vigorously for 1 minute, allow to sep-
arate, dry the lower layer with anhydrous sodium sulfate R, filter, and evap-
orate to dryness. Carry out the examination of a thin film of the residue as
described under 1.7 Spectrophotometry in the infrared region. The infrared
absorption spectrum is concordant with the spectrum obtained from val-
proic acid RS or with the reference sfrectrum of valproic acid.
B. Dissolve 0.5 g in 5 ml of water and add 1 ml of cobalt(Il) nitrate (lOOg/1) TS;

a purple precipitate is produced, which is soluble in carbon tetrachloride R.
C. A 20 mg/ml solution yields reaction B, described under 2.1 General identi-

fication tests as characteristic of sodium.


Iron. Using 0.2 g prepare a solution in 40 ml of water and proceed as described

under 2.2.4 Limit test for iron; not more than 50pg/g.
Sulfates. Dissolve 2.5g in 20ml of water and proceed as described under 2.2.2
Clarity and colour of solution. The opalescence of a solution of 2.0g in

10ml of carbon-dioxide-free water R is not more intense than that of opales-
cence standard TS2 and the solution is colourless.
20 mg/g.
Acidity or alkalinity. Dissolve 2.0 g in 20ml of carbon-dioxide-free water R

and add 0.1 ml of phenolphthalein/ethanol TS; not more than 1.5ml of sodium
651
hydroxide VS or 1.5ml of hydrochloric acid

(0.1 mol/1) (0.1 mol/1) VS is required
to obtain the midpoint of the indicator (pink).
Related substances. Carry out the test as described under 1.14.5 Gas chro-
matography using 3 solutions:
Solution 1. 0.20 mg of octanoic acid R (internal standard) per ml of

dichlorome thane R.
Solution 2. Dissolve 0.50g of the substance being examined in 10ml of water,

acidify with sulfuric acid (~190g/l) TS, and shake with 3 quantities, each of
20ml, of dichloromethane R. Wash the combined dichloromethane extracts
with 10 ml of water, shake with anhydrous sodium sulfate R, filter and evap-
orate the filtrate at a temperature not exceeding 30 °C to a volume of about
10ml, using a rotary evaporator.
examined in 10 ml of a mixture
Solution 3. Dissolve 0.50 g of the substance being
of 2.0mgof octanoic acid R in 10ml of sodium hydroxide (0.1 mol/1) VS,
acidify with sulfuric acid (~190g/l) TS, and shake with 3 quantities, each of
20ml, of dichloromethane R. Wash the combined dichloromethane extracts
with 10 ml of water, shake with anhydrous sodium sulfate R, filter and evap-
orate the filtrate at a temperature not exceeding 30 °C to a volume of about
10ml, using a rotary evaporator.
For the procedure use a glass column, 1.5m long and 0.4 cm in internal diam-
eter, packed with an adequate quantity of an adsorbent composed of 15 g of a
phase consisting of an ester of macrogol 20 M
and terephthalic acid, together
with Ig of phosphoric acid (-1440 g/1) TS supported on 84g of acid-washed,
silanized diatomaceous support R (150-180 pm). Maintain the column at
170 °C, use nitrogen R as the carrier gas and a flame ionization detector. In the
chromatogram obtained with solution 3, the total area of all the peaks, exclud-
ing the main peak and those due to the solvent and the internal standard, is
not greater than the area of the peak due to the internal standard.

with perchloric acid (0.1 mol/1) VS, as described under 2.6 Non-aqueous titra-
tion, Method A. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to
16.62mg of CaHisNaOj.
652
NELFINAVIRI MESILAS
NELFINAVIR MESILATE
C32H45N30,S,CH403S
Chemical name. (3S,4aS,8aS)-N-(l,l-dimethylethyl)-2-[(2R,3R)-2-hydroxy-3-

[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylsulfanyl)butyl]decahydroiso-
quinoline-3-carboxamide methanesulfonate;CAS reg. No. 159989-65-8.
Solubility. Practically insoluble in water and soluble in methanol R.
Category. Antiretroviral (Protease Inhibitor).
Storage. Nelfinavir mesilate should be kept in a tightly closed container, pro-

tected from light.
Additional information. Nelfinavir mesilate is hygroscopic.
Requirements
Definition. Nelfinavir mesilate contains not less than 98.5% and not more
than 101.0% of C 32 H 45 N 304 S,CH 403 S, calculated with reference to the dried
substance.

653
Identity tests
• Either tests A and B or test C may be applied.
5 mg of nelfinavir mesilate RS per ml. After removing the plate from
the chromatographic chamber, allow it to dry exhaustively in a current
of cool air. Examine the chromatogram in ultraviolet light (254 nm).

volumes of dichloromethane R, 20 volumes of acetonitrile R, 10
5 mg of nelfinavir mesilate RS per ml. After removing the plate from
the chromatographic chamber, allow it to dry exhaustively in a current
of cool air. Spray with basic potassium permanganate (~5g/l) TS.

B. The absorption spectrum of a 40pg/ml solution in methanol R, when

253 nm; the specific absorbance (Ar^) is 124 to 136.

infrared region.
spectrum obtained from nelfinavir mesilate RS or with the reference spectrum
of nelfinavir mesilate.
Specific optical rotation. Use a 10.0 mg/ml solution in methanol R and cal-
culate with reference to the dried substance; = -105° to -125°.
654
Heavy metals. Use l.Og in 30 ml of methanol R for the preparation of the test
solution as described under 2.2.3 Limit test for heavy metals, Procedure 2; deter-
mine the heavy metals content according to Method A; not more than 20pg/g.
30mg/g.
raphy R (5 pm).
Mobile phase A: 27 volumes of acetonitrile R, 20 volumes of methanol R, 28

volumes of phosphate buffer pH 3.4 and 25 volumes of purified water.
Mobile phase B: 41 volumes of acetonitrile R, 31 volumes of methanol R and

28 volumes of phosphate buffer pH 3.4.
Prepare the phosphate buffer pH 3.4 by dissolving 4.88 g of anhydrous sodium

dihydrogen phosphate R in 800 ml of purified water, adjust the pH to 3.4 by
adding phosphoric acid (~105g/l) TS and dilute it to 1000ml with purified
water.

(min) (% v/v) (% v/v)

75-80 0 to 100 100 to 0 Return to the initial conditions
Prepare the following solutions using mobile phase A as diluent. For solution
(1) use 2.0mg of the test substance per ml. For solution (2) dilute a suitable
volume of solution (1) to obtain a concentration equivalent to lO.Opg of Nelfi-
navir mesilate per ml. For solution (3) use 100 pg of methanesulfonic acid R
per ml.
and 5 ml of sulfuric acid (475 g/1), heat carefully in a boiling water bath for
30 minutes.
655
Maintain the column at 35 °C.
Inject 20 pi of solution (4). The test is not valid unless the resolution factor
between the principal peak (retention time = about 27 minutes) and the peak
with a retention time relative to the principal peak of about 0.2 is not less than
15. The test is also not valid unless the resolution factor between the last two
peaks out of three peaks, which increase during the decomposition process, is
not less than 4.0. The ratio of the retention times of these two peaks relative
to the principal about 1.8 and 1.9 respectively. If necessary adjust the
peak is
amount of acetonitrile R in both mobile phases A and B, or adjust the gradient

program.
Inject 20 pi of solution (3).
than the principal peak, is not greater than that in the chromatogram obtained
with solution (2) (0.5%). The sum peaks, other than the prin-
of the areas of all
cipal peak, not greater than twice the area of the principal peak in the chro-
is
matogram obtained with solution (2) (1.0%). Disregard any peak with an area
less than 0.1 times the area of the principal peak in the chromatogram obtained
with solution (2) (0.05%) and any peak due to methanesulfonic acid, corre-
sponding to the principal peak in the chromatogram obtained with solution (3).
Assay. Dissolve about 0.50 g, accurately weighed, in 50ml of methanol R and

titrate with sodium hydroxide (0.1 mol/1) VS, determine the end point poten-
tiometrically. Perform a blank determination and make the necessary correc-

tion. Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to 66.39mg of
C32H,5N30,S.CH403S.
NEOMYCINI SULFAS
NEOMYCIN SULFATE
Chemical name. Neomycin sulfate; CAS Reg. No. 1405-10-3.
Description. A white or yellowish white powder; odourless or almost

odourless.
656
TS; practically insoluble in acetone R and ether R.
Storage. Neomycin sulfate should be kept in a tightly closed container and

Additional information. Neomycin sulfate is hygroscopic. Even in the

the decomposition being faster at higher temperatures. An aqueous solution is
dextrorotatory.
Requirements
Definition. Neomycin sulfate is a mixture of sulfate salts of substances pro-
duced by the growth of Streptoniyces fradiae, the main components of which are
neomycin B and its stereoisomer neomycin C.
Neomycin sulfate contains not less than 600 International Units of neomycin
per mg, calculated with reference to the dried substance.
Identity tests
using silica gel R3 as the coating substance and freshly prepared ammonium
acetate (40 g/1) TS as the mobile phase. Apply separately to the plate 1 pi of
each of 2 solutions containing (A) 20 mg of the test substance per ml, and
(B) 20 mg of neomycin B sulfate RS per ml. After removing the plate from
the chromatographic chamber, allow it to dry in air for 10 minutes, heat at
105 °C for 1 hour, and spray with triketohydrindene/butanol TS. Heat it
again at 105°C for 5 minutes and examine the chromatogram in daylight.

The with solution A corresponds in position and
principal red spot obtained
appearance with that obtained with solution B.
B. Dissolve lOmg in 5ml of water, add 0.1 ml of pyridine R and 2ml of trike-
tohydrindene hydrate (1 g/1) TS, and heat on a water-bath at a temperature
between 65 and 70°C for 10 minutes; a deep violet colour is produced.

657

Neamine. Carry out the test as described under 1.14.1 Thin-layer chro-
matography, using a plate prepared as follows: Mix 0.3 g of carbomer R with
240 ml of water and allow to stand for 1 hour with occasional moderate
shaking. Adjust the pH to 7 by slowly adding, with continuous shaking, sodium
hydroxide (~80g/l) TS, then add 30 g of silica gel R3. Coat the plate with a layer
of 0.75mm thickness, heat the plate at 110°C for 1 hour, allow to cool, and
use immediately. As the mobile phase, use potassium dihydrogen phosphate
(100 g/1) TS. Apply separately to the plate 10 pi of each of 2 solutions contain-
ing (A) 2.5mg of the test substance per ml and (B) 0.05mg of neamine
hydrochloric RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in a current of warm air, spray it with triketohy-
drindene/stannous chloride TS, and heat it at 110°C for 15 minutes. Examine
the chromatogram in daylight. Any spot obtained with solution A corre-
sponding to neamine is not more intense than that obtained with solution B.
Content of sulfates. Dissolve 1 g in 200 ml of water, add 3 ml of hydrochlo-

ric g/1) TS, heat to boiling, and add 25ml of hot barium chloride
acid (-420
Heat on a water-bath for 4 hours with stirring, collect the precipi-
(50 g/1) TS.
tate, wash with water, dry, ignite, and weigh. Each g of residue is equivalent
to 411. 6mg of sulfates; the sulfate content is not less than 250mg/g and not
more than 310mg/g.
antibiotics, using either {a) Bacillus pumilus (NCTC 8241; ATCC 14884) as the
test organism, culture medium Cml with a final pH of 8.0-8. 1, sterile phos-
phate buffer pH 8.0 TSl or TS2, an appropriate concentration of neomycin
39 °C; or {b) Staphylococcus aureus (ATCC 29737) as the test organism, culture
medium Cml with a final pH of 7. 8-8.0, sterile phosphate buffer pH 8.0 TSl
or TS2, an appropriate concentration of neomycin (usually between 2 and
20 lU per ml), and an incubation temperature of 35-39 °C; or (c) Staphylococcus
epiderniidis (ATCC 12228) as the test organism, culture medium Cml with a
final pH of 8.0-8. 1, sterile phosphate buffer pH 8.0 TSl or TS2, an appropriate

concentration of neomycin (usually between 0.5 and 21U per ml), and an incu-
bation temperature of 35-39 °C. The precision of the assay is such that the fidu-
cial limits of error of the estimated potency (? = 0.95) are not less than 95%
error of the estimated potency (P = 0.95) is not less than 600 lU of neomycin
per mg, calculated with reference to the dried substance.
658
NEOSTIGMINI BROMIDUM
NEOSTIGMINE BROMIDE
Molecular formula. Ci2Hi9BrN202
Graphic formula.
Chemical name. (m-Hydroxyphenyl)trimethylammonium bromide dimethyl-

carbamate; 3-[[(dimethylamino)carbonyl]oxy]-N,N,N-trimethylbenzenaminium
bromide; CAS Reg. No. 114-80-7.
tically insoluble in ether R.
Category. Cholinergic.
Storage. Neostigmine bromide should be kept in a tighdy closed container,

Requirements
Definition. Neostigmine bromide contains not less than 98.0% and not more
than 101.0% of Ci 2 Hi 9 BrN 202 calculated with reference to the dried substance.
,
Identity tests
A. Heat 0.05 g with 0.4 g of potassium hydroxide R and 2 ml of ethanol
(-750 g/1) TS on a water-bath for 3 minutes. Replace the evaporated ethanol,
cool, and add 2 ml of water and 2 ml of diazobenzenedisulfonic acid TS; a
red colour is produced.
659
B. To a solution of 0.1 g in 5ml of water add 15ml of trinitrophenol (7 g/1) TS,

wash the precipitate with water, and dry at 105 °C; melting temperature,
about 185°C (picrate).

Sulfates. Dissolve 2.5g in 40 ml of water and proceed as described under2.2.2

20mg/g.
Acidity. Dissolve 0.2 g in 20 ml of carbon-dioxide-free water R and titrate to
pH 7.0 with sodium hydroxide (0.02 mol/1) VS; not more than 0.1 ml is required.
3 -Hydroxyphenyltrimethylammonium bromide. The absorbance of a 1-

cm layer of a freshly prepared 5.0mg/ml solution in sodium carbonate (lOg/1)
TS at 294 nm is not more than 0.2 (preferably use 2-cm cells for the measure-
ment and calculate the absorbance of a 1-cm layer).

Rl, add 5 ml of acetic anhydride R and 10 ml of mercuric acetate/acetic acid TS.
Titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous
30.32mg of Ci2Hi9BrN202.
NEOSTIGMINI METILSULFAS
NEOSTIGMINE METILSULFATE
Graphic formula.
660
Chemical name. (w-Hydroxyphenyl)trimethylammonium methyl sulfate

dimethylcarbamate; 3-[[(dimethylamino)carbonyl]oxy]-N,AfN-trimethylben-
zenaminium methyl sulfate; CAS Reg. No. 51-60-5.
Solubility. Very soluble in water; freely soluble in ethanol (-750 g/1) TS.
Storage. Neostigmine metilsulfate should be kept in a tightly closed container,

Requirements
Definition. Neostigmine metilsulfate contains not less than 98.0% and not
more than 100.5% of Ci3H22N20gS, calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from neostigmine metilsulfate RS or with the reference
spectrum of neostigmine metilsulfate.
B. See the test described under “Related substances”. The spot obtained with
solution B corresponds in position, appearance, and intensity with that
C. Heat 0.05 g with 0.4 g of potassium hydroxide R and 2 ml of ethanol

(-750 g/1) TS on a water-bath for 3 minutes. Adjust to the original volume
with ethanol (-750 g/1) TS, cool, and add 2 ml of water and 2 ml of dia-
zobenzenedisulfonic acid TS; a red colour is produced.
D. Mix 20 mg with 0.5 g of sodium carbonate R and heat to fusion in a small

mass with 10 ml of water until it has disintegrated,
crucible; boil the fused
then filter. Add 0.2ml of bromine
TSl to the filtrate, heat to boiling, acidify
with hydrochloric acid (-70 g/1) TS, and expel the excess of bromine by
boiling; the resulting solution yields reaction A described under 2.1 General
identification tests as characteristic of sulfates.
Melting range. 144-149°C, after drying at 105°C for 3 hours.
661
Chlorides. Dissolve 0.20g in 10ml of water, add 1ml of nitric acid (~130g/l)
TS and 1ml of silver nitrate (40g/l) TS; no immediate appearance of opales-
cence is observed.
Sulfates. Dissolve 0.20g in 10ml of water, add 1.5ml of hydrochloric acid

(~70g/l) TS and 1ml of barium chloride (50g/l) TS; no ready appearance of
turbidity is produced.

and not more intensely coloured than standard colour solution Bnl when com-
lOmg/g.
Acidity. Dissolve 0.20g in 10ml of water and add 0.1ml of phenolph-

thalein/ethanol TS; the test solution is colourless. Add 0.30ml of sodium
hydroxide (0.01 mol/1) VS; the solution turns red.
67 volumes of water, 30 volumes of methanol R, and 3 volumes of diethylamine
R as the mobile phase (a certain type of precoated plate may not be suitable
with this mobile phase). Apply separately to the plate 10 pi of each of 3 solu-
tions containing (A) 20 mg of the test substance per ml, (B) 0.10 mg of the test
substance per ml, and (C) 0.10 mg of neostigmine metilsulfate RS per ml. After
removing the plate from the chromatographic chamber allow it to dry in a
current of warm air, spray it with 4-nitroaniline TS2 and then with sodium
hydroxide (0.1 mol/1) VS. Dry the plate again in a current of warm air, spray it
with potassium iodobismuthate TS2, and examine the chromatogram in day-
Assay. Dissolve about 0.15g, accurately weighed, in 20ml of water, transfer

to an ammonia semi-micro distillation apparatus, and add 25ml of sodium
hydroxide (-400 g/1) TS. Pass a current of steam through the mixture and collect
the distillate in 50 ml of sulfuric acid (0.01 mol/1) VS until the total volume
reaches about 200 ml. Titrate the excess of acid with sodium hydroxide
(0.02 mol/1) VS using methyl red/ethanol TS as indicator. Repeat the operation
without the substance being tested; the difference between the titrations rep-
resents the amount of acid required to neutralize the dimethylamine formed
from the neostigmine. Each ml of sulfuric acid (0.01 mol/1) VS is equivalent to
6.688mg of C13H22N2O6S.
662
Additional requirement for Neostigmine metilsulfate for

parenteral use
NEVIRAPINUM
NEVIRAPINE
Nevira|)ine, anhydrous
Nevirapine heminydrate
n = 0 (anhydrous)
n = '/2 (hemihydrate)
Molecular formula. C 15 H 14N 4 O (anhydrous); Ci 5 Hi 4N 40 ,-jH 20 (hemihydrate)
Relative molecular mass. 266.3 (anhydrous); 275.3 (hemihydrate)
Chemical name. ll-cyclopropyl-4-methyl-5,ll-dihydro-6H-dipyrido[3,2-l?:2',

CAS Reg. No. 129618-40-2. (anhydrous) 11-cyclo-
3'-e][l,4]diazepin-6-one;
propyl-4-methyl-5,l l-dihydro-6ff-dipyrido[3,2-lr:2',3'-e][l,4]diazepin-6-one
hemihydrate
Description. A white to almost white powder.
Solubility. Practically insoluble in water, sparingly to slightly soluble in

dichlorome thane R, slightly soluble in methanol R.
Category. Antiretroviral (Non-Nucleoside Reverse Transcriptase Inhibitor).
Storage. Nevirapine should be kept in a well closed container.
stance is the hemihydrate or is in the anhydrous form.
663
Requirements
Nevirapine contains not less than 98 0 . %
C15H14N4O, calculated with reference to the anhydrous substance.
Identity test
volumes of dichloromethane R, 10 volumes of methanol R and 3
volumes of glacial acetic acid R as the mobile phase. Apply separately
to the plate 5 |il of each of 2 solutions in methanol containing (A) 5 mg
of the test substance per ml and (B) 5 mg of anhydrous nevirapine RS
per ml. After removing the plate from the chromatographic chamber,
allow it to dry exhaustively in air or in a current of cool air. Examine

A.2, Carry out the test as described under 1.14.1 Thin-layer chromatogra-
volumes of dichloromethane R, 10 volumes of methanol R and 3
volumes of glacial acetic acid R as the mobile phase. Apply separately
to the plate 5 pi of each of 2 solutions in methanol containing (A) 5 mg
of the test substance per ml and (B) 5 mg of anhydrous nevirapine RS
per ml. After removing the plate from the chromatographic chamber,
allow it to dry exhaustively in air or in a current of cool air. Dip the
plate in basic potassium permanganate (~lg/l) TS. Examine the chro-


283 nm with a specific absorbance (Alcm), calculated with reference to the
anhydrous substance, between 251 and 285.

infrared region. For the anhydrous substance, the infrared absorption spec-
trum is concordant with the spectrum obtained from anhydrous nevirapine
RS or with the reference spectrum of anhydrous nevirapine For the hemihy-
664
drate, the infrared absorption spectrum shows a characteristic sharp

absorbance at about 3503 cm"’; after heating the test substance for one hour
at 140°C and cooling, the infrared absorption spectrum is concordant with
the spectrum obtained from anhydrous nevirapine RS or with the reference
spectrum of anhydrous nevirapine.

Fischer method, Method A. For the anhydrous form, use about 1 g of the test
substance; the water content is not more than 2 mg/g. For the hemihydrate, use
about 0.30 g of the test substance; the water content is not less than 31 mg/g
and not more than 39 mg/g.
Related substances
Prepare fresh solutions and perform the tests without delay
matography, using a stainless steel column (15 cm x 4.6mm), packed with hexa-
decylamidylsilyl silica gel for chromatography (5|tm).
The mobile phase consists of a filtered and degassed mixture of 20 volumes of

acetonitrile R and 80 volumes of a 3.6g/l solution of ammonium dihydrogen
phosphate R adjusted to pH 5.0 using ammonia (-260 g/1) TS.
Prepare the following solutions. For solution (1) dissolve 24 mg of the test sub-
stance in 4 ml add 80ml of the mobile phase and sonicate.
of acetonitrile R,
Dilute to 100.0ml with the mobile phase. For solution (2) dilute 5.0ml of solu-
tion (1) to 50.0 ml with the mobile phase. Then dilute 5.0ml of this solution to
50.0ml with the same solvent. For solution (3) dissolve 6mg of nevirapine
impurity B RS in a mixture of 25 ml of acetonitrile R and 55 ml of mobile phase,
sonicate for 30min and dilute to 100.0ml with the mobile phase. Then mix
6.0 ml of this solution with 3.0 ml of solution (1) and dilute to 50.0 ml with the
mobile phase. For solution (4) dissolve 12 mg of anhydrous nevirapine RS in
2 ml of acetonitrile R and 40ml of the mobile phase and sonicate. Dilute to
50.0ml with the mobile phase. Then dilute 3.0ml of this solution to 25.0ml
with the mobile phase. For solution (5) Dilute 3.0 ml of solution (1) to 25.0ml
with the mobile phase.
Inject separately 50 pi of solution (2) in replicate injections in the chromato-

graphic system. The relative standard deviation for peak areas of nevirapine in
665
replicate injections of solution (2) is not more than 5.0%. The test is not valid
unless the column efficiency determined for nevirapine using solution (2) is not
less than 10000. The peak symmetry factor of nevirapine should be between
0.8 and 1.2.
Inject 50 pi of solution (3). The test is not valid unless the resolution between
nevirapine and nevirapine impurity B RS is not less than 5.
Inject separately 50 pi each of solution (1) and of mobile phase in the chro-
matographic system and record the chromatograms for 6 times the retention
time of nevirapine. Examine the mobile phase chromatogram for any extrane-
ous peaks and disregard the corresponding peaks observed in the chro-
matogram obtained with solution (1).
In the chromatogram obtained with solution (1), the following impurity peaks
with reference to nevirapine (reten-
are eluted at the following relative retention
tion time about 7.6min): impurity B about 0.7; impurity A about 1.5; impurity
C about 2.8.
In the chromatogram obtained with solution (1) the area of any individual peak
corresponding to impurity A or C is not greater than 0.2 times the area of the
principal peak in the chromatogram obtained with solution (2) (0.2%) and the
area of any individual peak corresponding to impurity B, when multiplied by
a correction factor of 0.77, is not greater than 0.2 times the area of the princi-
pal peak in the chromatogram obtained with solution (2) (0.2%). The area of
any other impurity peak is not greater than 0.1 times the area of the principal
peak in the chromatogram obtained with solution (2) (0.1%). The sum of the
areas of all peaks, other than the principal peak, is not greater than 0.6 times
the area of the principal peak in the chromatogram obtained with solution (2)
(0.6%). Disregard any peak with an area less than 0.05 times the area of the
principal peak in the chromatogram obtained with solution (2) (0.05%).
Assay
matography, using the conditions given under Related substances.
Inject alternatively 25 pi of solutions (4) and (5).
Measure the areas of the peaks responses obtained in the chromatogram from
solutions (4) and (5), and calculate the percentage content of C, 5 Hi 4 N 40 with
reference to the anhydrous substance.
Impurities
The following list of known and potential impurities that have been shown to
be controlled by the tests in this monograph is given for information.
666
A. R = C2H5: ll-ethyl-4-methyl-5,ll-dihydro-6/f-dipyrido[3,2-^:2',3'-e][l,4]
diazepin-6-one,
B. R= H: 4-metiiyl-5,ll-dihydro-6H-dipyrido[3, 2-^:2', 3'-c][l,4]diazepin-6-one,
C. R = CH2-CH2-CH3: 4-methyl-ll-propyl-5,ll-dihydro-6H-dipyrido[3,2-/7:2',
3'-e] [1 ,4] diazepin-6-one.
NICLOSAMIDUM
NICLOSAMIDE
Niclosamide, anhydrous
Niclosamide monohydrate
Molecular formula. C13H8CI2N2O4 (anhydrous); Ci3HaCl2N204,H20 (mono-

hydrate)
Relative molecular mass. 327.1 (anhydrous); 345.1 (monohydrate)
Graphic formula.
OH
n = 0 (anhydrous)
n = 1 (monohydrate)
Chemical name. 2',5-Dichloro-4'-nitrosalicylanilide; 5-chloro-N-(2-chloro-4-

nitrophenyl)-2-hydroxybenzamide; CAS Reg. No. 50-65-7 (anhydrous).
monohydrate;
2',5-Dichloro-4'-nitrosalicylanilide 5-chloro-W-(2-chloro-4-
nitrophenyl)-2-hydroxybenzamide monohydrate; CAS Reg. No. 73360-56-2
(monohydrate).
667
Description. A cream-coloured, crystalline powder; odourless.

(-750 g/1) TS; slightly soluble in ether R, and acetone R.
Category. Taeniacide.
Storage. Niclosamide should be kept in a tightly closed container.
Labelling. The designation on the container of Niclosamide should state

whether the substance is the monohydrate or is in the anhydrous form.
Additional information. Anhydrous Niclosamide is hygroscopic.
Requirements
Definition. Niclosamide contains not less than 98.0% and not more than
100.5% of C 13 H 8 CI N 2 O 4
2 ,
Identity tests

reference spectrum of a relevant form of niclosamide.
B. Dissolve Img in 2 ml of dimethylformamide R and add 2 drops of potas-

sium hydroxide/ethanol TSl; a strong red colour is produced.
C. Dissolve 0.1 g in 1ml of acetic anhydride R and boil for 10 minutes. Cool
and add 10 ml of water. Collect the precipitate on a filter, wash with water,
recrystaUize from ethanol (~750g/l) TS, and dry at 105°C; melting temper-
ature, about 178 °C (acetyl-derivative).
Loss on drying. Dry to constant weight at 105 °C. Anhydrous Niclosamide

more than 5.0mg/g. Niclosamide monohydrate loses not less than
loses not
Acidity or alkalinity. Boil 0.8 g in 40ml of water for 1 minute and filter. To
10ml of the filtrate add 2 drops of phenolphthalein/ethanol TS and 0.2ml of
carbonate-free sodium hydroxide (0.01 mol/1) VS; a red colour is produced. Add
5 drops of methyl red/ethanol TS and 0.4ml of hydrochloric acid (0.01 mol/1)
VS; the colour of the solution changes from red to orange.
668
2-ChIoro-4-nitroaniline. Boil g with 20ml of methanol R for 2 minutes,

0.1
cool, add sufficient hydrochloric acid (1 mol/1) VS to produce 50ml, and filter.
To 10ml of the add 1.0ml of sodium nitrite (3g/l) TS and allow to stand
filtrate
for 10 minutes; add 1ml of ammonium sulfamate (25g/l) TS, shake, allow
to stand for 10 minutes, and add 1ml of N-(l-naphthyl)ethylenediamine
hydrochloride (5g/l) TS. Treat similarly 10 pg of 2-chloro-4-nitroaniline R. The
colour produced in the test solution is not more intense than that of the refer-
ence solution when compared as described under 1.11 Colour of liquids.
5-Chlorosalicylic acid. Boil 0.5g with 10ml of water for 2 minutes, cool,
filter, and add to the filtrate a few drops of ferric chloride (25g/l) TS; no red or
violet colour is produced.

mamide R and titrate with tetrabutylammonium hydroxide (0.1 mol/1) VS
(0.1 mol/1) VS is equivalent to 32.71 mg of C13H8CI2N2O4.
NICOTINAMIDUM
NICOTINAMIDE
Molecular formula. C 6 H 6N 2 O
Graphic formula.
Chemical name. 3-Pyridinecarboxamide; 3-pyridinecarboxylic acid amide;

CAS Reg. No. 98-92-0.

almost odourless.
Solubility. Soluble in 1 part of water and 2 parts of ethanol (-750 g/1) TS;
Category. Vitamin.
669
Storage. Nicotinamide should be kept in a well-closed container.
Requirements
Definition. Nicotinamide contains not less than 99.0% and not more than
101.0% of C 6H 6N 2 O, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from nicotinamide RS or with the reference spectrum of
nicotinamide.
B. Dissolve lOmg in 10ml of water. To 2ml add 2ml of thiocyanate reagent,

obtained by adding, drop by drop, ammonium thiocyanate (0.1 mol/1) VS to
bromine TSl until the yellow coloration disappears. Then add 3 ml of aniline
(25g/l) TS and shake; a yellow colour is produced.
C. Boil gently 0.1 g with 1ml of sodium hydroxide (~80g/l) TS in a test-tube;

ammonia, perceptible by its odour, is evolved.
Clarity and colour of solution. A solution of 2.5 g in 10ml of water is clear

and not more intensely coloured than standard colour solution Yw2 when

reduced pressure (not exceeding O.bkPa or about 5 mm
of mercury) over silica
gel, desiccant, R or phosphorus pentoxide R; it loses not more than 5.0mg/g.
48 volumes of chloroform R, 10 volumes of water and 45 volumes of dehy-
drated ethanol R as the mobile phase. Apply separately to the plate 5 pi of each
670
of 2 solutions in a mixture of equalvolumes of ethanol (-750 g/1) TS and water

containing (A) 0.12 g of the test substance per ml and (B) 0.30 mg of the test

chloric acid (0.1 mol/1) VS is equivalent to 12.21 mg of C 6 H 5 N 2 O.
NIFEDIPINUM
NIFEDIPINE
Ci/HiaNtOe
Chemical name. l,4-Dihydro-2,6-dimethyl-4-(o-nitrophenyl)-3,5-pyridinedi-

carboxylate dimethyl ester; l,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-
pyridinedicarboxylic acid dimethyl ester; CAS Reg. No. 21829-25-4.
Solubility. Nifedipine is practically insoluble in water; freely soluble in

acetone R; sparingly soluble in dehydrated ethanol R.
Category. Cardiovascular drug; calcium-channel blocking agent.
Storage. Nifedipine should be kept in a tightly closed container, protected

from light.
Additional information. CAUTION: Nifedipine decomposes on exposure to

daylight, artificial light of certain wavelengths, and ultraviolet light.
671
Requirements
Nifedipine contains not less than 98 0 % and not more than 102 0 %
. . of
CijHisNaOe, calculated with reference to the dried substance.
Note-. Throughout the monograph perform the tests and the assay in the dark
or under a suitable fluorescent light, using low-actinic glassware.
Identity tests

infrared region.
spectrum obtained from nifedipine RS or with the reference spectrum of
nifedipine.
B. See the test described below under “Related substances, Test B”. The prin-
cipal spot obtained with solution A corresponds in position, appearance, and
intensity with that obtained with solution B.
C. Using gende heat dissolve 25mg in 10ml of a mixture of 5 volumes of

ethanol (~750g/l) TS, 3.5 volumes of water, and 1.5 volumes of hydrochlo-
ric acid (-420 g/1) TS. Add 0.5 g of granulated zinc R and allow to stand for
filter. To the filtrate add 5 ml of sodium
5 minutes, swirling occasionally, and
TS and allow to stand for 2 minutes. Add 2.0ml of ammo-
nitrite (lOg/1)
nium sulfamate (50 g/1) TS, shake vigorously but carefully, and add 2.0ml
of N-(l-naphthyl)ethylenediamine hydrochloride (5 g/1) TS; an intense red
colour is produced which does not fade within 5 minutes.
Sulfated ash. Use an ignition temperature of 600 °C; not more than l.Omg/g.
Related substances
matography, using a column (15cm x 4.6mm) packed with
stainless steel
particles of silica gel, the surface ofwhich has been modified with chemi-
cally bonded As the mobile phase, use a
octadecylsilyl groups (5-10 pm).
mixture of 55 volumes of water, 36 volumes of methanol R, and 9 volumes
of acetonitrile R.
672
Prepare the following solutions in methanol R: for solution (A) dissolve

0.20 g of Nifedipine in 20ml of methanol R and dilute to 50 ml with the
mobile phase; solution (B) 0.4 mg of dimethyl 2,6-dimethyl-4-(2-
nitrophenyl) pyridine-3, 5-dicarboxylate RS per ml; solution (C) 0.4 mg
of dimethyl 2, 6-dimethyl-4-(2-nitrosophenyl)pyridine-3, 5-dicarboxylate RS
120 per ml; and for solution (D) mix 1.0ml each of solutions B and C and
0.10ml of solution A, dilute to 20ml with the mobile phase, then dilute
2.0 ml of the resulting solution to 10 ml with the mobile phase.
Operate with a flow rate of 1.0 ml per minute. As a detector use an ultravi-
olet spectrophotometer set at a wavelength of about 235 nm; the use of an
electronic integrator is advisable.
Inject20 pi of solution D. The peaks are eluted in the following order;

dimethyl 2, 6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylate; dimethyl
2,6-dimethyl-4-(2-nitrosophenyl)pyridine-3,5-dicarboxylate; and nifedipine.
The retention time of nifedipine is about 15.5 minutes.
The test is not valid unless, in the chromatogram obtained with solution D:
— the resolution between the peaks corresponding to dimethyl 2,6-

dimethyl-4-(2-nitrophenyl)pyridine-3, 5-dicarboxylate and dimethyl 2,6-
dimethyl-4-(2-nitrosophenyl)pyridine-3,5-dicarboxylate is greater than
1.5; and
— the resolution between the peaks corresponding to dimethyl 2,6-

dimethyl-4-(2-nitrosophenyl)pyridine-3,5-dicarboxylate and nifedipine
is greater than 1.5.
Adjust the sensitivity of the system so that the height of the peak corre-
sponding to dimethyl 2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxy-
late is not less than 20% of the full scale of the recorder.
each of solutions A and D. Record the chromatogram

Inject alternately 20 pi
for twice the retention time of nifedipine.

from solutions A and D, and calculate the content of the related substances
as a percentage. In the chromatogram obtained with solution A, none of
the peaks, other than the principal peak and the peaks corresponding
to dimethyl 2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylate and
dimethyl 2, 6-dime thyl-4-(2-nitrosophenyl)pyridine-3, 5-dicarboxylate, has
an area greater than that of the peak corresponding to nifedipine in the
chromatogram obtained with solution D (0.1%). The areas of the peaks
corresponding to dimethyl 2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,
5-dicarboxylate and dimethyl 2,6-dimethyl-4-(2-nitrosophenyl)pyridine-3,5-
673
dicarboxylate are not greater than the corresponding peaks in the chro-
matogram obtained with solution D (0.1%). The total amount of related
substances does not exceed 0.3%. Disregard any peak with an area less than
10% of the area of the peak corresponding to nifedipine in the chro-
matogram obtained with solution D (0.01%).
B. Carry out the test as described under 1.14.1 Thin-layer chromatography, in

an unsaturated chamber, using silica gel R6 as the coating substance and a
mixture of 6 volumes of cyclohexane R and 4 volumes of ethyl acetate R as
in methanol R containing (A) 1.0 pg of Nifedipine per ml, (B) 1.0 mg of
nifedipine RS per ml, and (C) 10 pg of Nifedipine per ml. After removing the
plate from the chromatographic chamber, allow it to dry in air, and examine
Assay. Dissolve about 0.13g, accurately weighed, in a mixture of 25 ml of tert-

butanol R and 25 ml of perchloric acid TS, and titrate with ceric ammonium
sulfate (0.1 mol/1) VS, using 0.1 ml of ferroinTS as indicator until the pink colour
is discharged, titrating slowly towards the end-point. Repeat the procedure
without the Nifedipine being examined and make any necessary corrections.
Each ml of ceric ammonium sulfate (0.1 mol/1) VS is equivalent to 17.32 mg of

C17H18N2O5.
NIFURTIMOXUM
NIFURTIMOX
Graphic formula.
674
Chemical name. 3-Me thyl-4- [(5-nitrofurfurylidene)amino] thiomorpholine

1,1-dioxide; 3-methyl-N-[(5-nitro-2-furanyl)methylene]-4-thiomorpholinamine
1,1-dioxide; CAS Reg. No. 23256-30-6.
Description. A yellow to orange-yellow, crystalline powder; odourless.
Solubility. Practically insoluble water and ether

in R; freely soluble in
dimethylformamide R; sparingly soluble in dioxan R.
Category. Antitrypanosomal drug.
Storage. Nifurtimox should be kept in a well-closed container.
Requirements
Definition. Nifurtimox contains not less than 98.0% and not more than
102.0% of C10H13N3O5S, calculated with reference to the dried substance.
Identity tests

infrared region.
trum obtained from nifurtimox RS or with the reference spectrum of nifurtimox.
C. Dissolve 20mg in a mixture of 2.0ml of dimethylformamide R and 2.0ml

of water. Add 0.25ml of copper(ll) sulfate (160g/l) TS and 4 drops of pyri-
dine R; a dark green colouris produced. Add 5 ml of chloroform R and shake;
the green colour remains in the aqueous layer.
Loss on drying. Dry to constant weight at 105°C; not more than 5.0mg/g.
from a commercial source is suitable) and a mixture of equal volumes of ethyl
acetate R and hexane R as the mobile phase. Apply separately to the plate the
following 3 solutions in acetone R; (A) 10 pi containing 10 mg of the test sub-
675
stance per ml, (B) 2 pi containing 1.0 mg of the test substance per ml, and (C)
2 pi containing 1.0 mg of nifurtimox RS per ml. Allow the spots to dry before
placing the plate into an unsaturated chromatographic chamber and develop
the plate for a distance of 10 cm. After removing the plate from the chromato-
graphic chamber, allow it to dry in air, and examine the chromatogram in ultra-
violet light (254 nm). Any spot obtained with solution A, other than the
Assay. Dissolve about 0.1 g, accurately weighed, in 20ml of dimethylfor-

mamide R and 80 ml of ethanol (-750 g/1) TS. Add 5 ml of hydrochloric acid
(-250 g/1) TS and start to bubble carbon dioxide R into the solution, keeping it
up throughout the determination. After 3 minutes add 30.0ml of titanium
trichloride (0.1 mol/1) VS and 3 ml of potassium thiocyanate (200 g/1) TS. Allow
to stand for 15 minutes and titrate the excess titanium trichloride with ferric
ammonium sulfate (0.1 mol/1) VS. Each ml of titanium trichloride (0.1 mol/1) VS
is equivalent to 4.788mg of C 10 H 13 N 3 O 5 S.
NIRIDAZOLUM
NIRIDAZOLE
Molecular formula. C1SH6N4O3S
Graphic formula.
Chemical name. l-(5-Nitro-2-thiazolyl)-2-imidazolidinone; CAS Reg. No.

61-57-4.
Description. A yellow, crystalline powder; odourless or almost odourless.
Solubility. Practically insoluble in water and ether R; soluble in dimethylfor-

mamide R and pyridine R; slightly soluble in ethanol (-750 g/1) TS and acetone
R.
676
Storage. Niridazole should be kept in a tightly closed container.
Requirements
Definition. Niridazole contains not less than 97.0% and not more than
103.0% of C 6H 6N 4 O 3 S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from niridazole RS or with the reference spectrum of
niridazole.
C. Dissolve 20mg in 2.0ml of acetone R, add 2.0ml of sodium hydroxide

(~80g/l) TS, shake, and allow to stand for 10 minutes; a dark red colour is
produced in the lower layer.
metals content according to Method A; not more than 20[ig/g.
Sulfates. Add
0.5 g of the test substance to a mixture of 2.0ml of hydrochlo-
ric TS and 8.0 ml of water, heat to boiling, cool to room temper-
acid (~70g/l)
ature, and dilute the filtrate to 10 ml with water; the solutionis clear. Add
filter,
1.0 ml of barium chloride (0.5 mol/1) VS and again heat to boiling.
Similarly prepare a comparison solution containing 0.10 ml of sulfuric acid

(0.01 mol/1) VS, 2.0ml of hydrochloric acid (~70g/l) TS, 8.0ml of water, 1.0ml
of barium chloride (0.5 mol/1) VS, and heat to boiling.
Allow both solutions to stand for 1 hour; the opalescence in the solution pre-
pared from the test substance is not more intense than that produced in the
comparison solution.
677
Solution in dimethylformamide. A solution of O.lOg in 10ml of dimethyl-

formamide R is clear.
Sulfated ash. Not more than 3.0mg/g,
or about 5 mm of mercury) for 3 hours; it loses not more than 5.0mg/g.
from a commercial source is suitable) and a mixture of 12 volumes of toluene
R and 8 volumes of acetone R as the mobile phase. Apply separately to the
plate 5 pi of each of 4 solutions in pyridine R containing (A) lOmg of the test
substance per ml (heat slightly to dissolve, if necessary), (B) 10 mg of niridazole
RS per ml (heat slightly to dissolve, if necessary), (C) 20 pg of 2-amino-5-nitroth-
iazole RS per ml, and (D) 40 pg of niridazole-chlorethylcarboxamide RS per ml.
a current of air; examine the chromatogram immediately in ultraviolet light
(365 nm) and again after 15 minutes’ irradiation. Any spot obtained with solu-
with solution C viewed immediately and not more intense than that obtained
with solution D viewed after 15 minutes.
Assay. Dissolve about 40 mg, accurately weighed, in 4 ml of dimethylfor-

mamide R and dilute with sufficient dehydrated ethanol R to produce 200 ml;
dilute 5.0ml of this solution to 100ml with the same solvent. Measure the
absorbance of a 1-cm layer of the diluted solution at the maximum at about
359 nm. Calculate the amount of C6H6N4O3S in the substance being tested by
comparison with niridazole RS, similarly and concurrently examined. In an ade-
quately calibrated spectrophotometer the absorbance of a lOpg/ml solution of
niridazole RS should be 0.70 ± 0.03.
NITRAZEPAMUM
NITRAZEPAM
Molecular formula. CisHnNsOs
678
Graphic formula.
Chemical name. l,3-Dihydro-7-nitro-5-phenyl-2H-l,4-benzodiazepin-2-one;

CAS Reg. No. 146-22-5.
Description. A yellow, crystalline powder; odourless or almost odourless.

TS; very slighdy soluble in ether R.
Category. Sedative; hypnotic.
Storage. Nitrazepam should be kept in a well-closed container, protected from

light.
Requirements
Definition. Nitrazepam contains not less than 98.5% and not more than
Identity tests
A. NOTE: Carry out the following operations in subdued light. Prepare a
0.005 mg/ml solution in a mixture of 1 volume of hydrochloric acid (1 mol/1)
VS and 9 volumes of methanol R and examine immediately. The absorption
spectrum, when observed between 230 nm and 350 nm, exhibits a
maximum only at about 280 nm; the absorbance of a 1-cm layer at this
wavelength is about 0.45.
B. Dissolve lOmg in 1ml of methanol R, warming if necessary, and add

0.05ml of sodium hydroxide (-80 g/1) TS; an intense yellow colour is
produced.
C. Dissolve 20 mg in a mixture of 5 ml of hydrochloric acid (-420 g/1) TS and

10ml of water, boil for 5 minutes, cool, and add 2ml of sodium nitrite
(1 g/1) TS. Allow to stand for 1 minute, add 1ml of sulfamic acid (5 g/1) TS,
mix, allow to stand for 1 minute, and add 1 ml of N-(l-naphthyl)ethylene-
diamine hydrochloride (1 g/1) TS; a red colour is produced.
679
under 2.2,3 Limit test for heavy metals, Procedure 3; determine the heavy
Related substances. Carry out the test in subdued light as described under
1.14.1 Thin-layer chromatography, using silica gel R4 as the coating substance
and a mixture of 85 volumes of nitromethane R and 15 volumes of ethyl acetate
R as the mobile phase. Apply separately to the plate 10 pi of each of 2 freshly
prepared solutions in a mixture of equal volumes of chloroform R and methanol
R containing (A) 25 mg of the test substance per ml and (B) 25 pg of the test
substance per ml. Develop the plate for a distance of 12 cm. After removing the
with solution B.
Assay. Dissolve about 0.25 g, accurately weighed, in 30ml of acetic anhydride

R, and titrate with perchloric acid (0.1 mol/1) VS, determining the endpoint
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 28. 13 mg of C 15 H 11 N 3 O 3 .
NITROFURANTOINUM
NITROFURANTOIN
Nitrofurantoin, anhydrous
Nitrofurantoin monohydrate
Molecular formula. C8H6N4O5 (anhydrous); CsH5N405,H20 (monohydrate).
680
Graphic formula.
NH
CH=N — N nHjO
\\
n =0 (anhydrous)
n=1 (monohydrate)
Chemical name. l-[(5-Nitrofurfurylidene)amino]hydantoin; l-[[(5-nitro-2-

£uranyl)methylene]amino]-2,4-imidazolidinedione; CAS Reg. No. 67-20-9
(anhydrous).
l-[(5-Nitrofurfurylidene)amino]hydantoin monohydrate; l-[[(5-nitro-2-

furanyl)-methylene]amino]-2,4-imidazolidinedione monohydrate; CAS Reg.
Other name. Furadoninum.
Description. Lemon-yellow crystals or a yellow, crystalline powder; odour-


(-750 g/1) TS; soluble in dimethylformamide R.
Storage. Nitrofurantoin should be kept in a well-closed container, protected

from light, and stored at a temperature not exceeding 25 °C.
Labelling. The designation on the container of Nitrofurantoin should state

whether the substance is the monohydrate or is in the anhydrous form.
Additional information. Nitrofurantoin melts at about 271 °C with decom-

position. Nitrofurantoin and its solutions are discoloured by alkali and by expo-
sure to light and are decomposed upon contact with metals other than stainless
steel and aluminium.
Requirements
Definition. Nitrofurantoin contains not less than 98.0% and not more than
Identity tests
681

infrared region. For Nitrofurantoin monohydrate, the substance must be
previously dried to constant weight at 105 °C. The infrared absorption spec-
trum is concordant with the spectrum obtained from nitrofurantoin RS or
with the reference spectrum of nitrofurantoin.
B. Carry out the following operations in subdued light.
Prepare a blank solution by dissolving 3.6 g of sodium acetate R in 20 ml of

water, adding 0.3 ml of glacial acetic acid R, and diluting with sufficient
water to produce 200ml. Dissolve 0.12g in 50ml of dimethylformamide R
and add sufficient water to produce 1000 ml. Dilute 5 ml of this solution to
100 ml with the blank solution prepared above. Measure the absorption
spectrum of the resulting solution against 10ml of the blank solution to
which 0.05ml of dimethylformamide R has been added. When observed
between 220 nm and 400 nm, the absorption spectrum exhibits maxima at
about 266nm and 367 nm. The absorbances of a 1-cm layer at these wave-
lengths are about 0.32 and 0.46, respectively. The ratio of the absorbance at
367 nm to that at 266 nm is between 1.36 and 1.42.
C. Dissolve 0.2 g in a mixture of 10ml of dimethylformamide R and 1 drop of

glacial acetic acid R. To 0.5ml of this solution add 2ml of water, 0.15ml of
copper(II) sulfate (80g/l) TS, and 0.2 ml of pyridine R; shake with 3 ml
of chloroform R and allow the layers to separate; a green colour is produced
in the chloroform layer.
D. Dissolve 5mg in 5ml of sodium hydroxide (0.1 mol/1) VS; a deep yellow
solution is produced, which becomes deep orange-red on standing.
Loss on drying. Dry to constant weight at 105 °C; anhydrous Nitrofurantoin

loses not more than lOmg/g. Nitrofurantoin monohydrate loses not less than
50mg/g and not more than 71 mg/g.
90 volumes of nitromethane R and 10 volumes of methanol R as the mobile
phase. Apply separately to the plate lOpl of each of 2 solutions: (A) dissolve
0.25 g of the test substance in a minimum volume of dimethylformamide R and
dilute to 10 ml with acetone R; (B) dilute 1ml of solution A to 100 ml with
acetone R. After removing the plate from the chromatographic chamber, allow
it to dry in air and heat it at 105 °C for 5 minutes. Spray the warm plate with
phenylhydrazine/hydrochloric acid TS, heat it again at 105 °C for 10 minutes,

682
Assay. Dissolve about 0,4 g, accurately weighed, in a mixture of 10ml of

dimethylformamide R and 10 ml of dioxan R, add 0.10ml of thymol blue/
dimethylformamide TS and titrate with lithium methoxide (0.1 mol/1) VS to a
dark green endpoint, as described under 2.6 Non-aqueous titration, Method B.
Each ml of lithium methoxide (0.1 mol/1) VS is equivalent to 23.82 mg of
CsH^NÔs,
NONOXINOLUM 9
NONOXINOL 9
C9H,9C6H4(0CH2CH2)„0H
{Average value of n = 9, with a possible range of
Chemical name. Polyethylene glycol mono(/<’-nonylphenyl) ether; a-{4-

nonylphenyl)-co-hydroxypoly-(oxy-l,2-ethanediyl); CAS Reg. No. 26027-38-3,
Description. A clear, colourless to light yellow, viscous liquid.
Solubility. Miscible with water, ethanol (-750 g/1) TS and olive oil R.
Category. Adjunctive contraceptive agent.
Storage. Nonoxinol 9 should be kept in a tightly closed container.
Additional information. Nonoxinol 9 should be kept away from oxidizing

agents.
Requirements
Definition. Nonoxinol 9 is an anhydrous liquid mixture containing mainly
monononylphenyl ethers of macrogols.
The content isnot less than 95 0 % and not more than 105 0 % of Nonoxinol
. .
9, calculated with reference to the anhydrous substance.
683
Identity tests
reference spectrum of nonoxinol 9.
B. See the test described below under “Assay”. The retention time of the major
peak in the chromatogram obtained with solution A corresponds to that in
the chromatogram obtained with solution B.

Macrogol. Transfer about 10 g, accurately weighed, to a 250-ml beaker. Add

100 ml of ethyl acetate R and allow to dissolve using a magnetic stirrer. Trans-
fer to a 500-ml separatory funnel fitted with a glass stopper with the aid of
100 ml of sodium chloride (300 g/1) TS. Insert the stopper and shake vigorously
for 1 minute. Remove the stopper carefully. Immerse a thermometer into the
mixture and place the funnel so that it is partially immersed in a water-bath
maintained at 50 °C. Swirl the funnel gently while letting the internal temper-
ature rise to between 40 and 45 °C. Once this is reached remove the funnel
from the bath immediately, dry the outside surface, and drain the aqueous layer
into another 500-ml separatory funnel. Extract the organic layer with 100ml
of sodium chloride (300 g/1) TS a second time, combining the two aqueous
extracts. Discard the organic layer. Wash the combined aqueous layers with
100 ml of ethyl acetate R and separate the aqueous layer into another 500-ml
separatory funnel. Discard the organic layer. Extract the aqueous layer with two
successive 100-ml portions of dichlorome thane R, filtering the organic layers
through a folded filter paper (e.g. grade Whatman 2V) and combining them in
a 250-ml beaker. Evaporate to dryness on a water-bath and continue heating
until the odour of dichloromethane is no longer perceptible. Allow the beaker
to cool. Add 25ml of acetone R and allow the residue to dissolve using a mag-
netic stirrer. Filter into a tared 250-ml beaker, rinsing with two 25-ml quanti-
ties of acetone R. Evaporate to dryness on a water-bath. Dry under reduced
pressure (not exceeding 0.6kPa or about 5 mmof mercury) at 60 °C for 1 hour.

Allow the beaker to cool, and weigh; not more than 16mg/g.
Cloudiness of solution. Transfer l.Og to a 250-ml beaker, add 99g of water,

and mix to dissolve. Pour about 30 ml of the solution into a 70-ml test-tube.
Place the test-tube into a water-bath and stir the contents constantly with a
thermometer until the solution becomes cloudy, then immediately remove the
test-tube from the bath, so that the temperature does not rise further by more
than 2°C, and continue stirring. The temperature at which the solution
684
becomes and when the

sufficiently clear entire thermometer bulb is clearly
visible is between 52 and 56°C.
Ethylene oxide and dioxan. Carry out the test as described under 1,14.5 Gas
chromatography, with the apparatus equipped with an injection system for
the performance of head-space chromatography. Use a capillary glass or quartz
column (30 m x 0.32 mm), the inner surface of which is coated with a thick
layer of polydimethylsiloxane R (1.0 pm). Maintain the temperature of the
column at 50 °C for 5 minutes. Increase the temperature at a rate of 5°C per
minute to ISO^C, and then increase the temperature again at a rate of 30 °C
per minute to 230 °C, and maintain it at this point for 5 minutes. Maintain the
temperature of the injection port at 150 °C and that of the detector at 250 °C.
Use helium R or nitrogen R as the carrier gas with a linear velocity of about
20 cm per second and a split ratio of 1 :20; use a flame-ionization detector.
Prepare the following solutions; for solution (A) weigh l.Og of Nonoxinol 9,
add 1.0ml of water, mix to obtain a homogeneous solution, and allow to stand
at 70°C for weigh l.Og of Nonoxinol 9, add 0.5ml
45 minutes; for solution (B)
of ethylene oxide TS and 0.5ml of dioxan TS, mix to obtain a homogeneous

solution, and allow to stand at 70 °C for 45 minutes; for solution (C) add to
0.5ml of ethylene oxide TS 0.10ml of a freshly prepared lOmg/1 solution of
acetaldehyde R and 0.1 0 ml of dioxan TS, mix to obtain a homogeneous solu-
tion, and allow to stand at 70 °C for 45 minutes.
Inject1.0ml of the gaseous phase of solution C. Adjust the sensitivity of the

system so that the heights of the peaks corresponding to ethylene oxide and
acetaldehyde in the chromatogram obtained are at least 15% of the full scale
of the recorder. The test is not valid unless the resolution between the peaks
corresponding to acetaldehyde and ethylene oxide is at least 2.0 and the peak
of ethylene oxide is detected with a signal-to-noise ratio of at least 5.
Inject separately 1.0ml each of the gaseous phases of solutions A and B.
solutions A and B. The mean areas of the ethylene oxide and dioxan peaks
obtained with solution A are not greater than half the mean area of the corre-
sponding peak obtained with solution B (1 mg/g of ethylene oxide and 50 mg/g
of dioxan).
Assay. Determine by 1.14.4 High performance liquid chromatography, using

a stainless steel column (25 cm x 4 mm) packed with particles of porous silica,
the surface of which has been modified with chemically bonded dihydrox-
ypropane groups (diol) (10 pm). As mobile phase A, use a mixture of 2 volumes
of ethyl acetate R and 8 volumes of hexane R. As mobile phase B, use a mixture
of 2.5 volumes of methanol R and 97.5 volumes of ethyl acetate R.
685
Prepare the following solutions in mobile phase A: solution (A) 2.0 mg of

Nonoxinol 9 per ml; and solution (B) 2,0 mg of nonoxinol 9 RS per ml.
Operate with a flow rate of 1,0ml per minute. As a detector use an ultraviolet
Use the following gradient elution system:
Time (minutes) Mobile phase A Mobile phase B Comment

(% v/v) (% v/v)
0-2 100 0 equilibration

2-10 100 ^ 84 0 ^ 16 linear gradient
10-20 84 ^ 70 16 ^ 30 linear gradient
20-30 70 ^ 62 30 ^ 38 linear gradient
30-40 62 ^ 57 38 ^ 43 linear gradient
40-50 57 -4 54 43 -4 46 linear gradient
50-70 54 ^ 50 46 ^ 50 linear gradient
70-75 50 ^ 50 50 ^ 50 isocratic
75-76 50 ^ 100 50 ^0 re-equilibration
Inject 100 pieach of solutions A and B. The nonoxinol oligomers elute as sharp
distinct peaks, and their areas should be included in the peak response for
nonoxinol 9.
solutions A and B. The sum of the areas of any peaks corresponding to
nonoxynols with chain lengths k < 4 or k > 16 is not greater than 1.0% of the
sum of the areas of the peaks corresponding to nonoxynols with chain lengths
tt= 4 to = 16. Calculate the content of Nonoxinol 9 as a percentage, with ref-
erence to the anhydrous substance.
NORETHISTERONI ACETAS
NORETHISTERONE ACETATE
686
Graphic formula.
0
II
Chemical name. 17-Hydroxy-19-nor-17a-pregn-4-en-20-yn-3-one acetate;

17-(acetyloxy)-19-nor-17a-pregn-4-en-20-yn-3-one; 17a-ethynyl-17-hydrox-
yestr-4-en-3-one acetate; CAS Reg, No. 51-98-9.
Solubility. Practically insoluble in water; soluble in 12.5 parts of ethanol

(-750 g/1) TS and in 4 parts of acetone R; sparingly soluble in ether R.
Category. Progestational steroid.
Storage. Norethisterone acetate should be kept in a well-closed container, pro-

tected from light.
Requirements
Definition. Norethisterone acetate contains not less than 97.0% and not more
than 103.0% of C22H28O3, calculated with reference to the dried substance.
Identity tests
infrared region.
spectrum obtained from norethisterone acetate RS or with the reference spec-
trum of norethisterone acetate.
B. See the test described under “Related substances”. The principal spot
C. Heat 0.1 g with 2ml of potassium hydroxide/ethanol (0.5mol/l) VS in a

water-bath for 5 minutes. Cool, add 2 ml of sulfuric acid (-700 g/1) TS and
boil gently for 1 minute; ethyl acetate, perceptible by its odour (proceed
with caution) is produced.
687
=
Specific optical rotation. Use a 20 mg/ml solution in dioxan R; [a]™ -32

to -38 °
5.0mg/g.
equal volumes of toluene R and ethyl acetate R as the mobile phase. Apply sep-
arately to the plate 10 pi, in two portions of 5 pi, of each of 3 solutions in chlo-
roform R containing (A) lOmg of the test substance per ml, (B) O.lOmg of the
test substance per ml, and (C) O.lOmg of norethisterone acetate RS per ml. After
until the solvents have evaporated, and spray with sulfuric acid/ethanol TS.
Heat the plate to 105 °C for 15 minutes, allow to cool, and examine the chro-
matogram in daylight. Any spot obtained with solution A, other than the prin-

(-750 g/1) TS to produce 100ml; dilute 10.0ml of this solution to 100ml with
substance being tested by comparison with norethisterone acetate RS, similarly
NORETHISTERONI ENANTAS
NORETHISTERONE ENANTATE
o
C27H3803
688
=
Chemical names. 17-Hydroxy-19-nor-17a-pregn-4-en-20-yn-3-one hep-

tanoate; 17-[(l-oxoheptyl)oxy]-19-nor-17a-pregn-4-en-20-yn-3-one; CAS Reg.
No. 3836-23-5.
Other name. Norethindrone enantate.
Description. A white to creamy white, crystalline powder; odourless.
Solubility. Practically insoluble in water; freely soluble in acetone R, methanol

R, dehydrated ethanol R, dioxan R, and ether R; slightly soluble in light petro-
leum R.
Category. Contraceptive.
Storage. Norethisterone enantate should be kept in a tightly closed container,

Requirements
Norethisterone enantate contains not less than 96 0 . % and not more than the
equivalent of 104 0 %
. of C 27H 38 O 3 ,
substance.
Identity tests
infrared region.
spectrum obtained from norethisterone enantate RS or with the reference
sjaectrum of norethisterone enantate.
B. The absorption spectrum of a 13.5pg/ml solution in methanol R, when

observed between 210nm and 290nm, exhibits a maximum at about
240 nm.
C. Dissolve 1 mg in 1 ml of dehydrated ethanol R and add 0.5ml of sulfuric acid

(-1760 g/1) TS; a violet solution is produced with a red fluorescence.
Specific optical rotation. Use a 20 mg/ml solution in chloroform R; [a]o

-10.0 to -15.0°.
Solution in chloroform. A solution of 0.2 g in 10ml of chloroform R is clear

and almost colourless.
689
Loss on drying. Dry over silica gel, desiccant, R at ambient temperature for
4 hours; it loses not more than 5,0mg/g.
2 volumes of cyclohexane R and 1 volume of ethyl acetate R as the mobile
phase. Apply separately to the plate 5 pi of each of two solutions in chloroform
R containing (A) 20mg of Norethisterone enantate per ml and (B) 0.1 mg of
Norethisterone enantate per ml. After removing the plate from the chromato-
graphic chamber, allow it to dry in air and examine the chromatogram in ultra-
violet light (254nm). Spray the plate with antimony trichloride TS, heat at
110°C for 15 minutes, and examine the chromatogram in ultraviolet light
(365 nm).
Free enantic acid. Dissolve 0.3g in 10ml of neutralized ethanol (-750 g/1) TS
using bromothymol blue/ethanol TS as indicator. Titrate the solution quickly
with sodium hydroxide (0.01 mol/1) VS to a light blue endpoint; not more than
0.3ml (corresponding to 1.3mg/g of enantic acid).
Assay. Dissolve about 13.5 mg, accurately weighed, in sufficient methanol R

to produce 100ml; dilute 10ml of this solution to 100ml with the same solvent.
Measure the absorbance of the diluted solution in a 1-cm layer at the maximum
at about 240 nm and calculate the content of C27H3s03 using the absorptivity
value of 42.8 (Al"! = 428).
Additional requirement for Norethisterone enantate for

prarenteral use
NORETHISTERONUM
NORETHISTERONE
690
Graphic formula.
C=CH
Chemical name. 17-Hydroxy-19-nor-17a-pregn-4-en-20-yn-3-one; 17 a-

ethynyl-17-hydroxyestr-4-en-3-one; CAS Reg. No. 68-22-4.

(-750 g/1) TS, and in 80 parts of acetone R.
Storage. Norethisterone should be kept in a weU-closed container, protected

from light.
Requirements
Definition. Norethisterone contains not less than 97.0% and not more than
103.0% of C 20 H 26 O 2 ,
Identity tests
infrared region.
spectrum obtained from norethisterone RS or with the reference spectrum of
norethisterone.
Specific optical rotation. Use a lOmg/ml solution in chloroform R; [otjo”

'"
=
-23 to -27°.
5.0mg/g.
691
,
95 volumes of chloroform R and 5 volumes of methanol R as the mobile phase.
Apply separately to the plate 10 pi in two portions of 5 pi, of each of 3 solu-
tions in chloroform R containing (A) 10 mg of the test substance per ml, (B)
O.lOmg of the test substance per ml, and (C) O.lOmg of norethisterone RS
itto dry in air until the solvents have evaporated, and spray with sulfuric
acid/ethanol TS. Heat the plate to 105°C for 15 minutes, allow to cool, and
examine the chromatogram in daylight. Any spot obtained with solution A,
solution B.

(-750 g/1) TS to produce 100ml; dilute 10.0ml of this solution to 100ml with
substance being tested by comparison with norethisterone RS, similarly and
NOSCAPINI HYDROCHLORIDUM
NOSCAPINE HYDROCHLORIDE
Molecular formula. C22H23N07,HC1,H20
Graphic formula.
Chemical name. Narcotine hydrochloride monohydrate; (51^)-5-[(lS)-6,7-

dimethoxyphthalidyl]-5,6,7,8-te trahy d ro -4 me thoxy- 6-methyl 1
- -
3-dioxolo[4,5-g]isoquinoline hydrochloride monohydrate; [S-(R*,S*)]-6,7-
692
dime thoxy-3 -(5, 6,7,8- tetrahydro-4-me thoxy-6-methyl- l,3-dioxolo[4, 5-g]iso-

quinolin-5-yl)-l(3f7)-isobenzofuranone hydrochloride monohydrate; CAS Reg.
No. 912-60-7 (anhydrous).
Solubility. Freely soluble in water and ethanol (-750 g/1) TS; practically insol-
uble in ether R.
Storage. Noscapine hydrochloride should be kept in a well-closed container,

Additional information. Noscapine hydrochloride is hygroscopic.
Requirements
Definition. Noscapine hydrochloride contains not less than 98.5% and not
more than 101.0% of C 22 H 23 N 07 ,HC 1
,
substance.
Identity tests
A. Dissolve 0.1 g in 10ml of water, make the solution alkaline with ammonia
(-100 g/1) TS, and shake it with 10ml of chloroform R. Separate the chloro-
form layer, wash it with 5 ml of water, and filter. Evaporate the filtrate
almost to dryness on a water-bath, add 1 ml of dehydrated ethanol R, and
evaporate to dryness. Carry out the examination using the dried residue as
absorption spectrum is concordant with the spectrum obtained from
noscapine RS or with the reference spectrum of noscapine.
B. Dilute 5ml of ammonia (-35 g/1)TS to 100ml with methanol R; then dilute
further 1 ml of this solution to 100 ml with methanol R. The absorption spec-
trum of a 0.050mg/ml solution in the prepared ammonia/methanol mixture,
291 nm and 310nm and a minimum at about 263 nm. The ratio of the
absorbance at 310nm to that at 291 nm is between 1.2 and 1.3.
C. Place about 0.1 g on a porcelain dish, add drop by drop sulfuric acid
(~1760g/l) TS and mix; a greenish yellow colour is obtained, which turns
red and finally violet on heating.
693
=


(0.01 mol/1) VS and calculate with reference to the dried substance; [ajg*
-t38.5 to -t44.0°.
65mg/g.
of 45 volumes of acetone R, 45 volumes of toluene R, 7 volumes of ethanol
(-750 g/1) TS, and 3 volumes of ammonia (-260 g/1) TS as the mobile phase.
TS containing (A) 20 mg of the test substance per ml, and (B) 0.20 mg of the
chamber, allow it to dry in air, spray it with iodine (0.1 mol/1) VS, and examine
the chromatogram in daylight. Any spot obtained with solution A, other than
the principal spot, is not more intense than that obtained with solution B.
Assay. Dissolve about 0.5g, accurately weighed, in 50ml of a mixture of 7

volumes of acetic anhydride R and 3 volumes of glacial acetic acid Rl, add
10ml of mercuric acetate/acetic acid TS and titrate with perchloric acid
ml of perchloric acid (0.1 mol/1) VS is equivalent to 44.99mg of C22H23N07,HC1.
NOSCAPINUM
NOSCAPINE
694
Graphic formula.
Chemical name. Narcotine; (5i?)-5-[(lS-6,7-dimethoxyphthalidyl]-5, 6,7,8-

tetrahydro-4-methoxy-6-methyl-l,3-dioxolo[4,5-g]isoquinoline; [S-(R*,S’*)]-6,7-
dimethoxy-3-(5, 6,7, 8-tetrahydro-4-methoxy-6-methyl-l, 3-diox olo[4,5-g]isoqui
nolin-5-yl)-l(3/i)-isobenzo£uranone; CAS Reg. No. 128-62-1.

almost odourless.
Solubility. Practically insoluble in water; sparingly soluble in boiling water;

slightly soluble in ethanol (-750 g/1) TS and ether R.
Storage. Noscapine should be kept in a well-closed container.
Requirements
Definition. Noscapine contains not less than 98.5% and not more than
Identity tests

infrared region.
spectrum obtained from noscapine RS or with the reference spectrum of
noscapine.
B. The absorption spectrum of a 0.050 mg/ml solution in methanol R, when

and 310nm and a minimum at about 263 nm. The ratio of the absorbance
at 310nm to that at 291 nm is about 1.2.
695
C. To lOmg add about 0.5ml of sulfuric acid (~1760g/l) TS and mix; a greenish
yellow solution is formed, which becomes red and finally violet on heating.

(0.1 mol/1) VS; [a]g>"^ = +42 to +48°.
lOmg/g.
of 45 volumes of acetone R, 45 volumes of toluene R, 7 volumes of ethanol
(-750 g/1) TS, and 3 volumes of ammonia (-260 g/1) TS as the mobile phase.
TS containing (A) 20 mg of the test substance per ml and (B) 0.20 mg of the test
allow it to dry in air, spray it with iodine (0.1 mol/1) VS, and examine the chro-
Rl, warming gently, and titrate with perchloric acid (0.1 mol/1) VS as described
(0.1 mol/1) VS is equivalent to 41.34mg of C 22 H 23NO 7.
NYSTATINUM
NYSTATIN
Chemical name. Nystatin; CAS Reg. No. 1400-61-9.
Description. A yellow to light brown powder.

(-750 g/1) TS and methanol R; practically insoluble in ether R.
Storage. Nystatin should be kept in a tightly closed container, protected from

lightand stored at a temperature not exceeding 5°C.
696
Additional information. Nystatin is hygroscopic. Even in the absence of

light, it is gradually degraded on exposure to a humid atmosphere, the decom-
position being faster at higher temperatures. The potency of Nystatin may fall
at a rate of 1% per month when it is stored under the conditions mentioned
above.
Requirements
Definition. Nystatin is a mixture of substances produced by the growth of
certain strains of Strep’toniyces noursei, the main component of which is nystatin
A,.
Nystatin contains not less than 4400 International Units per mg, calculated with
Identity tests
A. Dissolve O.lOg in a mixture of 50ml of methanol R and 5ml of glacial acetic
acid R, add sufficient methanol R to produce 100 ml, and dilute 1ml to
100 ml with methanol R. The absorption spectrum of the resulting solution,
when observed between 240 nm and 350 nm, exhibits three maxima at
about 291 nm, 305nm, and 319nm; the ratio of the absorbance of a Tcm
layer at 291 nm to that at 305 nm is between 0.61 and 0.73, and the ratio of
the absorbance at 319nm to that at 305 nm is between 0.83 and 0.96.
B. Shake 30 mg with 5 ml of water for 2 minutes, add 2 ml of a solution pre-

pared by dissolving 0.1 g of pyrogallol R in 100ml of decolorized fuchsin
TS, heat on a water-bath until a dark pink colour is produced, cool, and
allow to stand for 1 hour; the pink colour is retained.

pH value. Shake 0.3g with 10ml of carbon-dioxide-free water R; pH of the

suspension, 6.5-8. 0.
antibiotics, using Petri dishes or rectangular trays filled to a depth of 1-2 mm
with culture medium Cm3 having a final pH of 6.0-6.2 and inoculated with
Saccharomyces cerevisiae (NCYC ATCC
9763) as the test organism, adding an
87;
appropriate concentration of nystatin (usually between 25 and 300 lU per ml)
and incubating at a temperature of 29-33 °C. Prepare the solutions of the test
substance by dissolving 75 mg in sufficient dimethylformamide R to produce
50ml and dilute 10ml to 200 ml with sterile phosphate buffer pH 6.0, TS3.
Protect the solutions from light throughout the assay. The precision of the assay
is such that the fiducial limits of error of the estimated potency (P = 0.95)
697
are not less than 95% and not more than 105% of the estimated potency.
The upper fiducial limit of error of the estimated potency (P = 0.95) is not less
than 4400 lU of nystatin per mg, calculated with reference to the dried
substance.
OLEUM ARACHIDIS
ARACHIS OIL
Chemical name. Peanut oil; CAS Reg. No. 8002-03-7.
Other name. Peanut oil.
Description. A yellowish, clear, and viscous liquid; odourless, or with a very

faint nut-like odour.
Miscibility. Very slightly miscible with ethanol (-750 g/1) TS; miscible with
ether R, and light petroleum R.
Category. Oleaginous vehicle; solvent.
Storage. Arachis oil should be kept in a well-filled and weU-closed container,

protected from light, and stored in a cool place.
Additional information. The quality of the material described is not suitable

for parenteral administration.
Requirements
Definition. Arachis oil is the refined fixed oil obtained from the seed kernels
of Arachis hypogaea L.
Identity test
To 0.5ml add 10ml of potassium hydroxide/ethanol TSl and, while shaking
intermittently, heat in a water-bath at 80 °C for 15 minutes. Allow to stand for
1 hour; a cloudy, gelatinous mixture is formed which adheres to the walls of
the tube.
Refractive index, no = 1.468-1.472.
Relative density. di“ = 0.912-0.920.
698
Iodine value. 83-103.
Unsaponifiable matter. Not more than 15mg/g.
Peroxide value. Not more than 5.0.
OXAMNIQUINUM
OXAMNIQUINE
Molecular formula. Ci 4 H 2 iN 303
Graphic formula.
Chemical name. l,2,3,4-Tetrahydro-2-[(isopropylamino)methyl]-7-nitro-6-

quinolinemethanol; l,2,3,4-tetrahydro-2-[[(l-methylethyl)amino]methyl]-7-
nitro-6-quinolinemethanol; CAS Reg. No. 21738-42-1.
Description. A yellow-orange, crystalline powder.
Solubility. Sparingly soluble in water, soluble in methanol R and acetone R.
Category. Antischistosomal.
Storage. Oxamniquine should be kept in a well-closed container.
Requirements
Definition. Oxamniquine contains not less than 97.0% and not more than
103.0% of Ci,|H 2 iN 303 calculated
,
Identity tests
699

infrared region.
spectrum obtained from Oxamniquine RS or with the reference spectrum of
oxamniquine.
B. To lOmg add lOmg of zinc R powder, 1.0ml of water, and about 0.5ml of
hydrochloric acid (-250 g/1) TS. Heat in a water-bath for 5 minutes, cool in
ice,add 1ml of sodium nitrite (100 g/1) TS, and remove the excess nitrite by
adding sufficient sulfamic acid (50 g/1) TS. Add 0.5ml of the resulting solu-
tion to a mixture of 0.5ml of 2-naphthol TSl and 2.0 ml of sodium hydrox-
ide (~80g/l) TS; an orange-red colour is produced.
Iron. Ignite l.Og; cool and dissolve the residue in 5ml of hydrochloric acid
(-250 g/1) FeTS and 30 ml of water. Treat the solution as described under 2.2.4
Limit test for iron; the iron content is not more than 50p,g/g.

pH value. Shake 0.1 g with 10ml of carbon-dioxide-free water R; pH of the

suspension, 8.0-10.0.
from a commercial source is suitable) and 20 volumes of chloroform R, 10
volumes of hexane R, 2 volumes of 2-propanol R, and 0.5 volume of iso-
propylamine R as the mobile phase. Apply separately to the plate 10 pi of each
of 2 solutions in a mixture of equal volumes of chloroform R and methanol R
allow it to dry in air for 10 minutes until the solvents have evaporated. Examine
the chromatogram in ultraviolet light first at 254 nm then at 365 nm. Any spot
Rl, previously neutralized to oracet blue R/acetic acid TS. Titrate with per-
700
chloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous titration,

of C14H21N3O3.
OXYGENIUM
OXYGEN
O2
Chemical name. Oxygen; CAS Reg. No. 7782-44-7.
Solubility. One volume dissolves in about 32 volumes of water and in about

7 volumes of ethanol (~750g/l) TS, both at a pressure of 101.3kPa and 20°C.
Category. Gas for inhalation.
Storage. Oxygen should be kept as compressed gas or liquid at cryogenic tem-

perature, in appropriate containers complying with the safety regulations of the
national authority. Valves or taps should not be lubricated with oil or grease.
Labelling. An ISO standard* requires that cylinders containing oxygen

intended for medical use should bear the name of the contents in legible and
permanent characters and, preferably, also the molecular formula O 2.
Additional information. In the analysis of medicinal gases certain tests are

not intended for hospital pharmacists. They are solely applicable by laborato-
ries equipped with the specialized apparatus.
Requirements
Oxygen contains not less than 99.5% v/v of O 2 .
• Oxygen labelled as having been produced by the air-licjuefaction process may be

exempted from the recjuirements of the tests for carbon monoxide and carbon dioxide.
’
International Standard 3>2. Gas cylinders for medical use - marking for identification of content. Interna-
tional Organization for Standardization, Switzerland, 1977.
701
Identity tests
A. Place a glowing splinter of wood into the test gas; the splinter bursts into
flame.
B. Shake with alkaline pyrogallol TS; the test gas is absorbed and the solution
becomes dark brown (distinction from Dinitrogen oxide).
• Note-. For the following tests deliver the test gas at a rate of 4 litres per hour.
Carbon monoxide
A. The apparatus (Fig. 7) consists of the following parts connected in series:
—a U-tube (Ul) containing desiccant silica gel R impregnated with

chromium trioxide R;
— a wash bottle (FI) containing 100 ml of potassium hydroxide (-400 g/1)

TS;
— a U-tube (U2) containing pellets of potassium hydroxide R;
— a U-tube (U3) containing phosphorus pentoxide R dispersed on previ-

ously granulated, fused pumice;
—a U-tube (U4) containing 30 g of recrystallized iodine pentoxide R in

granules, previously dried at 200 °C and kept at a temperature of 120 °C
(T) during the test. The iodine pentoxide is packed in the tube in 1-cm
columns separated by 1-cm columns of glass wool to give an effective
length of 5 cm;
— a reaction tube (F2) containing 2.0 ml of potassium iodide (160 g/1) TS

and 0.15ml of starch TS.
Flush the apparatus with 5.0 litres of argon R. If necessary, discharge the blue
colour in tube F2 containing potassium iodide (160 g/1) TS by adding a suffi-
cient volume of freshly prepared sodium thiosulfate (0.002 mol/1) VS. Con-
tinue flushing with argon R until not more than 0.045ml of sodium
thiosulfate (0.002 mol/1) VS is litres of argon
required after the passage of 5.0
R. Pass 7.5 litres of the test gas from the container through the apparatus.
Flush the last traces of liberated iodine into the reaction tube by passing 1.0
litre of argon R through the apparatus. Titrate the liberated iodine with
sodium thiosulfate (0.002 mol/1) VS. Repeat the procedure using 7.5 litres of
argon R.
702
Figure 7. Apparatus for the determination of carbon monoxide in medicinal gases

Measurements in mm.
B. Determine the content using a carbon monoxide detector tube. Pass the
The gas supply is connected to a pressure regulator and needle valve. Connect
the flexible tubing fitted with a Y-piece to the valve and adjust the flow of the
test gas to purge the tubing to an appropriate flow. Fit the carbon monoxide
detector tube to the metering pump following the manufacturer's instructions.
Connect the open end of the tube to the short leg of the tubing and operate
the pump sufficiently to pass a suitable volume of the test gas through the
tube. Read the value corresponding to the length of the coloured layer or the
intensity of the colour on the graduated scale; not more than 5pl/l.
Note-. For the following tests - “Carbon dioxide”, “Oxidizing substances”, and
“Acidity and alkalinity” - pass the test gas through the appropriate reagent con-
tained in a hermetically closed flat-bottomed glass cylinder (with dimensions
such that 50 ml of liquid reaches a height of 12-14 cm) that is fitted with (a) a
delivery tube terminated by a capillary 1 mm
in internal diameter and placed
within 2mm of the bottom of the cylinder; and (b) an outlet tube.
Prepare the reference solutions in identical cylinders.
Carbon dioxide
703
A. Pass 1.0 litre of the test gas through 50ml of a clear solution of barium
hydroxide (0.15mol/l) VS. Similarly prepare a reference solution by adding

1.0ml of a 1.1 mg/ml solution of sodium hydrogen carbonate R in carbon-
dioxidefree water R to 50ml of barium hydroxide (0.15mol/l) VS.
Any turbidity in the solution after the passage of the test gas is not more
intense than that of the reference solution (300pl/l).
B. Determine the content using a carbon dioxide detector tube. Pass the

Fit the carbon dioxide detector tube to the metering pump following the
leg of the tubing and operate the pump sufficiently to pass a suitable volume
of the test gas through the tube. Read the value corresponding to the length
of the coloured layer or the intensity of the colour on the graduated scale;
not more than 300 pl/1.
Oxidizing substances. To two cylinders add 50ml of freshly prepared potas-

sium iodide/starch TSl and about 0.2ml of glacial acetic acid R. Protect the
cylinders from light Pass 5.0 litres of the test gas into one of the solutions and
compare the colour produced.
The solutions in both cylinders remain colourless.
Water
A. The apparatus consists either of an electrolytic hygrometer as described

below, an appropriate humidity detector tube, or a capacity hygrometer.
The measuring cell consists of a thin film of phosphoric anhydride placed

between two coiled platinum wires that act as electrodes. The water vapour
in the test gas is absorbed by the phosphoric anhydride to form phosphoric
acid, which acts as an electrical conductor.
Before introducing the test gas into the device, allow the gas to stabilize at
room temperature and make sure that the temperature is constant through-
out the apparatus. Apply a continuous voltage across the electrodes to
produce electrolysis of the water and regeneration of phosphoric anhydride.
Measure the resulting electrical current, which is proportional to the water
704
content in the test gas. (This is a self-calibrating system that obeys Faraday’s
law.)
Calculate the content of water; not more than 60 |.tg/l.
B. Determine the content using a water vapour detector tube. Pass the required
volume of the test gas through the tube, the calibration of which is verified
according to the manufacturer’s instructions.

Connect the with a Y-piece to the valve and adjust the
flexible tubing fitted
flow of the test gas to purge the tubing to an appropriate flow.Fit the water
vapour detector tube to the metering pump following the manufacturer’s
instructions. Connect the open end of the tube to the short leg of the tubing
and operate the pump sufficiently to pass a suitable volume of the test gas
through the tube. Read the value corresponding to the length of the coloured
layer or the intensity of the colour on the graduated scale; not more than
60pl/l.
Acidity and alkalinity. Pass 2.0 litres of the test gas through a mixture of
0.10ml of hydrochloric acid (0.01 mol/1) VS and 50ml of carbon-dioxide-free
water R.
For reference solution 'I, use 50ml of carbon-dioxide-free water R. For reference
solution 2, use a mixture of 0.20ml of hydrochloric acid (0.01 mol/1) VS and
50 ml of carbon-dioxide-free water R.
To each solution add 0.1 ml of methyl red/ethanol TS; the intensity of the colour
in the solution of the test gas is between those of reference solutions 1 and 2.
Assay
A. For the determination use a 25-ml capacity gas burette (Fig. 8) in the form
of a chamber with at its upper end, a tube graduated in 0.2% between 95

and 100, and isolated at each end by a tap with a conical barrel. The lower
tap is joined to a tube with an olive-shaped nozzle and is used to introduce
the test gas into the apparatus. A cylindrical funnel above the upper tap is
used to introduce the absorbent solution. Wash the burette with water and
dry. Open the two taps. Connect the nozzle to the container of the test gas
and set the flow rate to 1 litre per minute. Flush the burette by passing the
gas through it for 1 minute. Close the upper tap of the burette and imme-
diately afterwards the lower tap. Rapidly disconnect the burette from the
container of the test gas, and give a half turn to the upper tap to eliminate
any excess pressure in the burette. Keeping the burette vertical, fill the funnel
705
Figure8. Burette used for the assay of oxygen Measurements in mm.

with a freshly prepared mixture of 21 ml of potassium hydroxide (~560g/l)

TS and 130ml of sodium dithionite (200g/l) TS. Open the upper tap slowly.
The solution absorbs the oxygen and enters the burette. Allow to stand for
10 minutes without shaking.
706
Read the level of the liquid meniscus on the graduated part of the burette;
the figure represents the content of oxygen as a percentage in v/v.
B. Oxygen in medicinal gases can also be determined using a paramagnetic

analyser, which measures electronically the molecule’s interaction with
magnetic fields.
Carry out the method according to the instrument manufacturer’s instructions.
OXYTETRACYCLINI DIHYDRAS
OXYTETRACYCLINE DIHYDRATE
Oxytetracycline dihydrate (non-injectable)
Oxytetracycline dihydrate, sterile
Molecular formula. C22H24N209,2H20
Graphic formula.
Chemical name. (46,4aR,5S,5al?,65',12aS)-4-(Dimethylamino)-l,4,4a,5,5a,6,

ll,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl-l,ll-dioxo-2-naph-
thacenecarboxamide dihydrate; [4S-(4a,4aa,5a,5aa,6P,12aa)]-4-(dimethy-
lamino)-l,4,4a,5,5a,6,ll,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl
-l,ll-dioxo-2naphthacenecarboxamide dihydrate; CAS Reg. No. 6153-64-6
(dihydrate).
Description. A pale yellow, crystalline powder; odourless.

(-750 g/1) TS; freely soluble in dilute acids and alkalis.
Storage. Oxytetracycline dihydrate should be kept in a tightly closed con-

707
Labelling. The designation sterile Oxytetracycline dihydrate indicates that the

substance complies with the additional requirements for sterile Oxytetracycline
dihydrate and may be used for parenteral administration or for other sterile
applications.
Additional infonnation. Oxytetracycline dihydrate darkens on exposure to

strong sunlight. It deteriorates in solutions having a pH below 2 and is rapidly
destroyed by alkali hydroxide solutions.
Requirements
Definition. Oxytetracycline dihydrate contains not less than 920 International
Units of Oxytetracycline per mg, calculated with reference to the anhydrous
substance.
Identity tests
using a kieselguhr coating prepared as follows: To 25 g of kieselguhr R1 add
50ml of a mixture of 2.5 ml of glycerol R and 47.5 ml of disodium edetate
the plates with this mixture and allow them to dry at room temperature for
about 70-90 minutes, or until sufficiently dry to give a satisfactory separa-
tion. As the mobile phase, take 200ml of a mixture of 2 volumes of ethyl
acetate R, 2 volumes of chloroform R, and 1 volume of acetone R, shake
with 25ml of disodium edetate (0.1 mol/1) VS previously adjusted to pH 7
with ammonia (-100 g/1) TS, allow to settle, and use the lower layer. Apply
(A) 0.50 mg of the test substance per ml, (B) 0.50 mg of Oxytetracycline dihy-
drate RS per ml, and (C) a mixture of 0.50 mg of chlortetracycline hydrochlo-
ride RS per ml, 0.50 mg of Oxytetracycline dihydrate RS per ml, and
0.50 mg of tetracycline hydrochloride RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air, expose it to the
vapour of ammonia (-260 g/1) TS, and examine the chromatogram in ultra-
violet light (365 nm). The principal spot obtained with solution A corre-
sponds in position, appearance, and intensity with that obtained with
B. To about Img add 2ml of sulfuric acid (-1760g/l) TS; a deep red colour is
produced, which changes to yellow on the addition of 0.1 ml of water.
Specific optical rotation. Dissolve 0.25 g in sufficient hydrochloric acid

(0.1 mol/1) VS to produce 25 ml, and allow to stand for 60 minutes. Measure the
rotation and calculate with reference to the anhydrous substance; [a]§’"‘- =
-203 to -216°.
708

Absorption in the ultraviolet region. The absorption spectrum of a 20 pg/ml

solution in hydrochloric acid (0.1 mol/1) VS, when observed between 230 nm and
400 nm, exhibits 2 maxima at about 268 nm and 353 nm. The absorbance of a
1-cm layer at 353 nm is not less than 0.54 and not more than 0.58.
Light-absorbing impurities. Prepare a 2.0mg/ml solution in a mixture of 1

volume of hydrochloric acid (1 mol/1) VS and 99 volumes of methanol R and
measure within 1 hour of preparation the absorbance of a 1-cm layer at
430 nm; the absorbance does not exceed 0.25. Prepare a 10 mg/ml solution in a
mixture of 1 volume of hydrochloric acid (1 mol/1) VS and 99 volumes of
methanol R, and measure within 1 hour of preparation the absorbance of a
1-cm layer at 490 nm; the absorbance does not exceed 0.20.
antibiotics, using either {a) Bacillus p’umilus (NCTC 8241 or ATCC 14884) as the
test organism, culture medium Cml with a final pH of 63-6.6, sterile phos-
phate buffer, pH 4.5 TS, an appropriate concentration of oxytetracycline
39 °C, or (b) Bacillus cereus (ATCC 11778) as the test organism, culture medium
Cml with a final pH of 5. 9-6.0, sterile phosphate buffer, pH 4.5 TS, an appro-
priate concentration of oxytetracycline (usually between 0.5 and 2IU per ml),
and an incubation temperature of 30-33 °C. The precision of the assay is such
that the fiducial limits of error of the estimated potency (P = 0.95) are not less
than 95% and not more than 105% of the estimated potency. The upper fidu-
cial limit of error of the estimated potency (P = 0.95) is not less than 900 lU of
oxytetracycline per mg, calculated with reference to the anhydrous substance.
Additional Requirements for Oxytetracycline Dihydrate for

sterile use
oxytetracycline.

membrane filtration test procedure and using a diluting fluid containing 1 ml of
macrogol /^^-isooctylphenyl ether R per litre of peptone (1 g/1) TS. Use the sam-
pling plan described under 3.2.1 Test for sterility of non-injectable preparations.
709
OXYTETRACYCLINI HYDROCHLORIDUM
OXYTETRACYCLINE HYDROCHLORIDE
Oxytetracycline hydrochloride (non -injectable)
Oxytetracycline hydrochloride, sterile
Molecular formula. C22H24N209,HC1
Graphic formula.
• HCI
Chemical name. (46’,4ajR,5S,5al?,6S,12a6’)-4-(Dimethylamino)l,4,4a,5,5a,6,

1 l,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl-l,l l-dioxo-2-naph-
thacenecarboxamide monohydrochloride; [4S-(4a,4aa,5a,5aa,6P,12aa)]-4-
(dimethylamino)-l,4,4a,5,5a,6,l l,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-
6-methyl- 1,11 -dioxo-2-naphthacenecarboxamide monohydrochloride; CAS
Reg. No. 2058-46-0.
Storage. Oxytetracycline hydrochloride should be kept in a tightly closed con-

Labelling. The designation sterile Oxytetracycline hydrochloride indicates

that the substance complies with the additional requirements for sterile Oxyte-
tracycline hydrochloride and may be used for parenteral administration or for
other sterile applications.
Additional information. Oxytetracycline hydrochloride is hygroscopic. Even

in the absence of light, it is gradually degraded on exposure to a humid atmos-
phere, the decomposition being faster at higher temperatures. Dissolved in
water it becomes turbid on standing owing to the separation of the base caused
710
through partial hydrolysis of the hydrochloride. It deteriorates in solutions of

pH below 2, and is rapidly destroyed by alkali hydroxide solutions.
Requirements
Definition. Oxytetracycline hydrochloride contains not less than 870 Interna-
tional Units of Oxytetracycline per mg, calculated with reference to the anhy-
drous substance.
Identity tests
using a kieselguhr coating prepared as follows: To 25g of kieselguhr R1 add
50ml of a mixture of 2.5ml of glycerol R and 47.5ml of disodium edetate
the plates with this mixture, and allow them to dry at room temperature
for about 70-90 minutes, or until sufficiently dry to give a satisfactory sep-
aration.As the mobile phase, take 200 ml of a mixture of 2 volumes of ethyl
acetate R, 2 volumes of chloroform R, and 1 volume of acetone R, shake
with 25ml of disodium edetate (0.1 mol/1) VS previously adjusted to pH 7
with ammonia (~100g/l) TS, allow to settle, and use the lower layer. Apply
(A) 0.50 mg of the test substance per ml, (B) 0.50 mg of Oxytetracycline
hydrochloride RS per ml, and (C) a mixture of 0.50 mg of chlortetracycline
hydrochloride RS per ml, 0.50 mg of Oxytetracycline hydrochloride RS per
ml, and 0.50 mg of tetracycline hydrochloride RS per ml. After removing the
plate from the chromatographic chamber, allow it to dry in air, expose it to
the vapour of ammonia (-260 g/1) TS, and examine the chromatogram in
ultraviolet light (365 nm). The principal spot obtained with solution A cor-
B. To about Img add 2ml of sulfuric acid (~1760g/l) TS; a deep red colour is
produced, which changes to yellow on the addition of 0.1 ml of water.
C. A 0.05 g/ml solution yields reaction B described under 2.1 General identifi-
Specific optical rotation. Dissolve 0.25 g in sufficient hydrochloric acid

(0.1 mol/1) VS to produce 25ml and allow to stand for 60 minutes. Measure the
rotation and calculate with reference to the anhydrous substance; [ajn'’"'^ =
-188 to -200°.
711

Absorption in the ultraviolet region. The absorption spectrum of a

20pg/ml solution in hydrochloric acid (0.1 mol/1) VS, when observed between
230 nm and 400 nm, exhibits 2 maxima at about 268 nm and 353 nm. The
absorbance of a 1-cm layer at 353 nm is not less than 0.54 and not more than
0.58.
Light-absorbing impurities. Prepare a 2.0mg/ml solution in a mixture of 1

volume of hydrochloric acid (1 mol/1) VS and 99 volumes of methanol R and
measure within 1 hour of preparation the absorbance of a 1-cm layer at
430 nm; the absorbance does not exceed 0.50.
Prepare a 10 mg/ml solution in a mixture of 1 volume of hydrochloric acid

(1 mol/1) VS and 99 volumes of methanol R and measure within 1 hour of pre-
paration the absorbance of a 1-cm layer at 490 nm; the absorbance does not
exceed 0.20.
antibiotics, using either {a) Bacillus pumilus (NCTC 8241 or ATCC 14884) as the
test organism, culture medium Cml with a final pH of 6.5-6.6, sterile phos-
phate buffer, pH 4.5 TS, an appropriate concentration of oxytetracycline
39 °C, or {b) Bacillus cereus (ATCC 11778) as the test organism, culture medium
Cml with a final pH of 5. 9-6.0, sterile phosphate pH
an appro-
buffer, 4.5 TS,
priate concentration of oxytetracycline (usually between
and 2IU), and an
0.5
incubation temperature of 30-33 °C. The precision of the assay is such that the
fiducial limits of error of the estimated potency (P = 0.95) are not less than 95%
error of the estimated potency (P = 0.95) is not less than 870 lU of oxytetracy-
cline per mg, calculated with reference to the anhydrous substance.
Additional Requirements for Oxytetracycline Hydrochloride

for sterile use
oxytetracycline.

membrane nitration test procedure. Use the sampling plan described under
712
1
SELECTED WHO PUBLICATIONS OF RELATED INTEREST
Basic tests for drugs:

pharmaceutical substances, medicinal plant materials
and dosage forms
1998 (94 pages)
Basic tests for pharmaceutical dosage forms

1991 (134 pages)
Quality Assurance of Pharmaceuticals: a compendium of guidelines

and related materials
Volume 1: 1997 (244 pages)
Volume 2: good manufacturing practices and inspection. 2004 (236 pages)
WHO Expert Committee on Specifications for

Pharmaceutical Preparations
Fortieth report
WHO Technical Report Series, No. 937,
2006 (461 pages)
International nonproprietary names (INN) for

pharmaceutical substances
Cumulative list no 1 .
2004 (available in CD-ROM format only)
The use of essential medicines

Report of the WHO Expert Committee
(including the 13th Model List of Essential Medicines)
2004 (133 pages)
WHO Expert Committee on Biological Standardization

Fifty-fourth report
2005 (160 pages)
Further information on these or other Wt-iO publications can be obtained from

WFfO Press, World Health Organization, 1211 Geneva 27, Switzerland.
Attention is also drawn to the WHO Medicines web site
(http://www.who.int/medicines)
*
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The World Health Organization was established in 1948 as a specialized agency of the United
Nations serving as the directing and coordinating authority for international health matters and
public health, One of WHO's constitutional functions is to provide objective and reliable information
and advice in the field of human health, a responsibility that it fulfils in part through its extensive
programme of publications.
The Organization seeks through its publications to support national health strategies and address
the most pressing public health concerns of populations around the world. To respond to the needs
of Member States at all levels of development, WHO publishes practical manuals, handbooks and
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books are closely tied to the Organization's priority activities, encompassing disease prevention
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To ensure the widest possible availability of authoritative information and guidance on health
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throughout the world, WHO's books contribute to achieving the Organization's principal objective
- the attainment by all people of the highest possible level of health.
The International
Pharmacopoeia
FOURTH EDITION
Pharmacop’oea Internationalis
Editio Quarta
Volume 2
World Health Organization

Geneva
2006
WHO Library Cataloguing-in-Publication Data
The International pharmacopoeia = Pharmacopoea internationalis. - 4'"’’
ed.
2 V.
1. Pharmacopoeias 2. Pharmaceutical preparations - standards 3. Pharmaceutical preparations -

analysis 4. Dosage forms - standards LWorld Health Organization.
ISBN 92 4 155301 X (LC/NLM classification: QV 738.1)
© World Health Organization 2006
World Health Organization can be obtained from WHO

All rights reserved. Publications of the
Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791
3264; fax: +41 22 791 4857; email: bookorders@who.inO Requests for permission to reproduce or
.
translate WHO
publications - whether for sale or for noncommercial distribution — should be
addressed to WHO Press, at the above address (fax: +41 22 791 4806; email: permissions@who.int).
The designations employed and the presentation of the material in this publication do not imply
the expression of any opinion whatsoever on the part of the World Health Organization concern-
ing the legal status of any country, territory, city or area or of its authorities, or concerning the
delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines
for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products does not imply that they
are endorsed or recommended by World Health Organization in preference to others of a
the
similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary
products are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization to verify the infor-
mation contained in this publication. However, the published material is being distributed without
warranty of any kind, either express or implied. The responsibility for the interpretation and use
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damages arising from its use.
Printed in Singapore
Contents
Preface iv
History ix
Acknowledgements xiii
General Notices 1
Monographs 21
Pharmaceutical substances (A to O)
Monographs 713
Pharmaceutical substances (P to Z)
Dosage forms 945
General monographs 945
Capsules 947
Ophthalmic preparations 951
Parenteral preparations 956
Suppositories 960
Tablets 963
Topical semi-solid dosage forms 969
Specific monographs 975
Radiopharmaceuticals 1113
Methods of Analysis 1137
Reagents, test solutions and volumetric solutions 1281
Supplementary information 1439
Index 1467
Monographs
Pharmaceutical
substances
PAPAVERINI HYDRO CHLORIDUM

PAPAVERINE HYDROCHLORIDE
Molecular formula. C 2 oH 2 iN 04 ,HCl
Graphic formula.
• HCI
Chemical name. 6,7-Dimethoxy-l-veratrylisoquinoline hydrochloride; 1-
[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline hydrochloride;
CAS Reg. No. 61-25-6.
Solubility. Sparingly soluble in water; soluble in 120 parts of ethanol (-750 g/1)
Category. Spasmolytic.
Storage. Papaverine hydrochloride should be kept in a well-closed container,

Requirements
Definition. Papaverine hydrochloride contains not less than 98.5% and not
more than 101.0% of C oH 2 iN 04 ,HCl,
substance.
Identity tests

infrared region.
spectrum obtained from papaverine hydrochloride RS or with the reference
spectrum of papaverine hydrochloride.
713
B. Treat about 10 mg with 3 ml of acetic anhydride R, add cautiously 3 drops

of sulfuric acid (~1760g/l) TS and heat on a water-bath for 3-4 minutes; a
yellow colour with a green fluorescence is produced.

D. Dissolve 20 mg in 10ml of water, add drop by drop ammonia (~100g/l) TS,

and set aside. Filter, wash the precipitate with water, and dry at 105 °C;
melting temperature, about 146°C (papaverine base).

dioxide-free water R is clear and not more intensely coloured than standard
colour solution Gn2 when compared as described under 1.11 Golour of liquids.
Loss on drying. Dry to constant weight at 105°G; it loses not more than
lOmg/g.
Related substances. Garry out the test as described under 1.14.1 Thin-layer
7 volumes of toluene R, 2 volumes of ethyl acetate R, and 1 volume of diethy-
solutions in a mixture of 4 volumes of hydrochloric acid (0.01 mol/1) VS and 1
volume of ethanol (-750 g/1) TS containing (A) 0.050g of the test substance per
ml and (B) 0.50 mg of codeine R per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in a current of warm air until the odour
of diethylamine is no longer perceptible, and examine the chromatogram in
ultraviolet light (254 nm). Any spot obtained with solution A, other than the

ml of perchloric acid (0.1 mol/1) VS is equivalent to 37.59mg of G 2 oH 2 iN 04 ,HCl.
714
PARACETAMOLUM
PARACETAMOL
Graphic formula.
HO NHCOCH3
^
Chemical name. 4'-Hydroxyacetanilide; N-(4-hydroxyphenyl)acetamide;

CAS Reg, No. 103-90-2,
Other name. Acetaminophen.
Solubility. Sparingly soluble in water; freely soluble in ethanol (-750 g/1) TS

and acetone R; practically insoluble in ether R.
Category. Analgesic; antipyretic.
Storage. Paracetamol should be kept in a tightly closed container, protected

from light.
Requirements
Definition. Paracetamol contains not less than 98.5% and not more than
Identity tests
A. Dissolve 0.05g in 100ml of methanol R. To 1 ml of this solution add 0.5ml
of hydrochloric acid (0.1 mol/1) VS and dilute to 100ml with methanol R.
Protect the solution from light and immediately measure the absorbance of
a 1-cm layer at the maximum wavelength of about 249nm; about 0.88.
B. Dissolve 0.1 g in 10ml of water and add 0.05ml of ferric chloride (25 g/1) TS;
a violet-blue colour is produced.
C. Boil 0.1 g with 1ml of hydrochloric acid (~70g/l) TS for 3 minutes, add
10ml of water and cool; no precipitate is formed. Add 0.05ml of potassium
715
dichromate (0.0167mol/l) VS; a violet colour, which does not turn to red
(distinction from phenacetin) is slowly produced.
Heavy metals. Use l.Og and a mixture of 85 volumes of acetone R and 15

volumes of water for the preparation of the test solution as described under
2.2.3 Limit test for heavy metals, Procedure 2; determine the heavy metals
content according to Method A; not more than lOpg/g.
5-Omg/g.
4-Aminophenol. Dissolve 0.5g in a mixture of equal volumes of methanol R

and water and dilute to 10 ml with this solvent mixture. Add 0.2 ml of alkaline
sodium nitroprusside TS, mix, and allow to stand for 30 minutes. Prepare sim-
ilarly a reference solution containing 0.5 g of 4-aminophenol-free paracetamol
R and 0.5ml of a solution containing 0.050 mg/ml of 4-aminophenol R in the
same solvent mixture. The colour of the test solution is not more intense than
that of the reference solution (0.05 mg/ g).
of 65 volumes of chloroform R, 25 volumes of acetone R, and 10 volumes of
toluene R as the mobile phase. Allow the solvent front to ascend 14 cm above
the line of application, using an unlined chromatographic chamber. Prepare the
following 4 test solutions: For solution (A) transfer l.Og of finely powdered sub-
stance to be examined to a glass-stoppered tube, add 5 ml of ether R, and shake
mechanically for 30 minutes. Centrifuge the tube until a clear supernatant liquid
is obtained and separate this from the solid. For solution (B) dilute 1 ml of solu-
tion A to 10 ml with ethanol (-750 g/1) TS. For solution (C) dissolve 25 mg of 4-
chloroacetanilide R in 50 ml of ethanol (-750 g/1) TS. For solution (D) dissolve
0.25 g of 4-chloroacetanilide R and
0.1 g of the substance to be examined in suf-
ficient ethanol (-750 g/1) TS
produce 100ml. Apply separately to the plate
to
200 pi of solution A and 40 pi of each of the remaining 3
solutions. After remov-
ing the plate from the chromatographic chamber, allow it to dry in a current
of warm air and examine the chromatogram in ultraviolet light (254 nm). Any
spot due to 4-chloroacetanilide obtained with solution A is not more intense
than the corresponding spot obtained with solution C. Any spot obtained with
solution B, other than the principal spot and the spot corresponding to 4-
chloroacetanilide, is not more intense than the spot obtained with solution C.
The test is valid only if the chromatogram obtained with solution D shows two
distincdy separated spots corresponding to 4-chloroacetanilide and the sub-
stance being examined, the latter having a lower Revalue.
716
Assay. Transfer about 0.25g, accurately weighed, to a flask, add 10ml of

hydrochloric acid (~70g/l) TS and boil under a reflux condenser for 1 hour.
Wash the condenser with 30 ml of water, add 1 g of potassium bromide R to
the combined solution, and proceed as described under 2.7 Nitrite titration,
titrating with sodium nitrite (0.1 mol/1) VS. Each ml of sodium nitrite (0.1 mol/1)
VS is equivalent to 15.12mg of C 8H 9NO 2 .
PARAFFINUM ALBUM
WHITE, SOFT PARAFFIN
PARAFFINUM FLAVUM
YELLOW SOFT PARAFFIN
Chemical name. White and yellow petrolatum.
Other names. White petrolatum, yellow petrolatum; vaselinum album,

vaselinum flavum.
Description. A white or a pale yellow to yellow, soft, unctuous mass; odourless.
ether R, and in most fixed and volatile oils.
Category. Ointment base.
Storage. White and yellow soft paraffins should be kept in a weU-closed container.
Additional information. In a thin layer or when melted, both paraffins show

a slight fluorescence. Melting point, within 38-60 °C.
Requirements
Definition. White and yellow soft paraffins are purified mixtures of semi-solid
hydrocarbons obtained from petroleum. White soft paraffin is bleached. To
prevent oil separation, soft paraffins may contain a suitable stabilizer.
Identity tests
A. Melt 2g until a homogeneous mass is obtained and immediately add 2 ml
of water and 0.2ml of iodine (0.1 mol/1) VS. Heat; as soon as two liquid
phases are obtained, shake and cool; the upper solid phase should have a
pinkish violet colour.
717
B. Heat a small quantity of either White soft paraffin or Yellow soft paraffin
and ignite; a luminous flame is observed and a deposit of carbon is formed.
Alkalinity. To 35 g add 100 ml of boiling water, cover the beaker, and, while
stirring, heat to boiling for 5 minutes. Allow the phases to separate, transfer
the aqueous layer to a suitable dish, and wash the paraffin with two portions,
each of 50 ml, of boiling water which are added to the dish. Add 1 drop of phe-
nolphthalein/ethanol TS and boil; the colour does not change to pink. (Keep
this solution for “Acidity”.)
Acidity. To the above solution, add 0.1ml of methyl orange/ethanol TS; the
colour does not change to red or pink.
Organic acids. To 20 g add 100 ml of a mixture of equal volumes of neutral-

ized ethanolTS and water, mix thoroughly, and heat to boiling. Add 1 ml of
phenolphthalein/ethanol TS and titrate rapidly with carbonate-free sodium
hydroxide (0.1 mol/1) VS to a sharp pink endpoint, the colour change being
observed in the ethanol-water layer; not more than 0.4ml of carbonate-free
sodium hydroxide (0.1 mol/1) VS is required.
Fixed oils, fats, and rosin. Digest lOg with 50ml of sodium hydroxide (-200 g/1)
TS at 100 °C for 30 minutes. Separate the aqueous layer and acidify with sulfuric
acid (-570 g/1) TS; the remaining phase does not show any oil or solid matter.
Ultraviolet absorption. Dissolve 50mg in 100ml of 2,2,4-trimethylpentane

R. Measure the absorbance of a 1-cm layer at about 290 nm. White soft paraf-
fin does not exceed 0.5; yellow soft paraffin does not exceed 0.75.
PARAFFINUM DURUM
HARD PARAFFIN
Chemical name. Paraffin wax; paraffin waxes and hydrocarbon waxes; CAS
Reg. No. 8002-74-2.
Description. A colourless or white, slightly unctuous mass showing a crys-

talline structure; odourless.
Solubility. Practically insoluble in water and ethanol (-750g/l) TS; freely

soluble in ether R.
Category. Ointment base; viscosity-increasing agent.
718
Storage. Hard paraffin should be kept in a well-closed container.
Additional information. Congealing point, within 47-65 °C.
Requirements
Definition. Hard paraffin is a purified mixture of solid hydrocarbons obtained
from petroleum.
Identity tests
A. Melt 2g until a homogeneous mass is obtained and immediately add 2 ml of
water and 0.2ml of iodine (0.1 mol/1) VS. Heat; as soon as two liquid phases
are obtained, shake and cool; the upper solid phase has a pinkish violet colour.
B. Heat a small quantity of Hard paraffin and ignite; a luminous flame is
observed and a deposit of carbon is formed.
Acidity or alkalinity. Boil 5g with 10ml of ethanol (~710g/l) TS previously

neutralized to litmus TS, cool, and add a few drops of litmus TS; the solution
is neutral (violet).
PAROMOMYCINI SULFAS
PAROMOMYCIN SULFATE
Molecular formula. C23H45N50i4,xH2S04
Graphic formula.
719
Chemical name. 0-2,6-Diamino-2,6-dideoxy-|3-L-idopyranosyl-(1^3)-0-P-

D-ribofuranosyl-(1^5)-0-[2-amino-2-deoxy-a-D-glucopyranosyl-(1^4)]-
2-deoxystreptamine sulfate (salt); 0-2-amino-2-deoxy-a-D-glucopyranosyl-
(1—>4)-0-[0-2,6-diamino-2,6-dideoxy-P-L-idopyranosyl-(l—>3)-P-D-ribofura-
nosyl-(l—>5)]-2-deoxy-D-streptamine sulfate (salt); CAS Reg. No. 1263-89-4.
Description. A creamy white to light yellow powder; odourless or almost

odourless.
Solubility. Very soluble in water; practically insoluble in ethanol (-750 g/1) TS

and ether R.
Storage. Paromomycin sulfate should be kept in a tightly closed container,

Additional information. Paromomycin sulfate is very hygroscopic. Even in

the absence of light, it is gradually degraded on exposure to a humid atmos-
Requirements
Definition. Paromomycin sulfate contains not less than 675 International
Units of paromomycin per mg, calculated with reference to the dried substance.
Identity tests
using silica gel R3 as the coating substance and a freshly prepared ammo-
nium acetate (40 g/1) TS as the mobile phase. Apply separately to the plate
1 |il of each of 2 solutions containing (A) 20 mg of the test substance per ml
and (B) 20 mg of paromomycin sulfate RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air for 10 minutes,
heat it at 105°C for 1 hour, and spray it with triketohydrindene/butanol TS.
Heat it again at 105 °C for 5 minutes and examine the chromatogram in day-
light. The principal red spot obtained with solution A corresponds in posi-
tion and appearance with that obtained with solution B.

ence to the dried substance; = -tSO to -1-55°.
720

pH value. pH of a 30mg/ml solution in carbon-dioxide-free water R, 5, 0-7, 5.
antibiotics, using either {a) Bacillus subtilis (NCTC 10400) as the test organism,
culture medium Cml with a final pH of 8.0, sterile phosphate buffer pH 7.8,
TS, an appropriate concentration of paromomycin (usually between 1 and 4IU
per ml), and an incubation temperature of 37-39 °C; or {b) Bacillus subtilis (ATCC
6633) as the test organism, culture medium Cml with a final pH of 7.8, sterile
phosphate buffer pH 8.0, TSl or TS2, an appropriate concentration of paro-
momycin (usually between 2 and 8IU per ml), and an incubation temperature
of 36-38 °C. The precision of the assay is such that the fiducial limits of error
of the estimated potency (? = 0.95) are not less than 95% and not more than
105% of the estimated potency. The upper fiducial limit of error of the esti-
mated potency (? = 0.95) is not less than 675 ID of paromomycin per mg, cal-
culated with reference to the dried substance.
PENICILLAMINUM
PENICILLAMINE
Molecular formula. C.sHnNOjS
Graphic formula.
CH3 H
I
I
HS — C---C— COOH
I I
CH3 NHj
Chemical name. 3-Mercapto-D-valine; 3,3-dimethyl-D-cysteine; CAS Reg.

No. 52-67-5.
Description. A white or almost white, crystalline powder; odour,

characteristic.
practically insoluble in chloroform R and ether R.
Category. Antidote.
721
Storage. Penicillamine should be kept in a tightly closed container, protected

from light.
Additional information. Even in the absence of light, Penicillamine is grad-

Requirements
Definition. Penicillamine contains not less than 95.0% and not more than
100.5% of C5H11NO2S, calculated with reference to the dried substance.
Identity tests
A. Dissolve 20 mg in 4 ml of water, add 2 ml of phosphotungstic acid TS and
allow to stand for a few minutes; a deep blue colour is produced.
B. Dissolve 20 mg in 5 ml of water, add 0.05ml of sodium hydroxide (-200 g/1)

TS and 20 mg of triketohydrindene hydrate R; an intense blue or violet-blue
colour is produced immediately.
Specific optical rotation. Use a 50 mg/ml solution in sodium hydroxide

(1 mol/1) VS; [a]g>’= = -58 to -68 °.

Mercury
• The operations described below must be carried out in subdued light.
Transfer about 0.5 g, accurately weighed, to a 650-ml long-necked flask con-

taining a few glass beads, incline the flask at an angle of about 45°, and add
2.5ml of nitric acid (-1000 g/1) TS through a small funnel placed in the mouth
of the flask.Allow the mixture to stand at room temperature until nitrous oxide
fumes are evolved and the vigorous reaction subsides (5-30 minutes). Add
2.5 ml of sulfuric acid (-1760 g/1) TS through the funnel and heat, gently at first
and then to the production of fumes of sulfur trioxide. Cool, then add cau-
tiously 2.5ml of nitric acid (-1000 g/1) TS, heat again to the production of sulfur
trioxide fumes, and cool. Repeat this treatment once more, then add 50ml of
water, rinsing the funnel, and collecting the rinsings in the flask. Remove the
funnel, boil the solution down to approximately half its volume (about 25 ml),
and cool to room temperature. Transfer to a 250-ml separating funnel with the
722
aid of water and dilute with water to 50 ml. Add 1 ml of disodium edetate
(20g/l) TS and 1ml of glacial acetic acid R and extract with small portions of
chloroform R until the last chloroform extract remains colourless. Discard the
chloroform extract and add 50ml of sulfuric acid (0.125mol/l) VS, 90ml of
water, and 10 ml of hydroxylamine hydrochloride (200 g/1) TS. Add dithizone
standard TS, in portions of 0.3-0. 5ml, from a 10-ml burette. After each addi-
tion, shake the mixture well, allow the chloroform layer to separate, and discard
it. Continue until an addition of dithizone standard TS remains green after
shaking.
From the volume of the dithizone standard TS used, calculate the amount of
mercury present in the test substance. It contains not more than 20 pg of Hg
per g.
of sodium hydroxide (1 mol/1) VS and 0.2 ml of dithizone TS and titrate with
mercuric nitrate (0.02 mol/1) VS. Each ml of mercuric nitrate (0.02 mol/1) VS is
equivalent to 5.968mg of C 5H 11 NO 2 S.
PENTAMIDINI ISETIONAS
PENTAMIDINE ISETIONATE
Molecular formula. Ci9H24N402,2C2H604S
Graphic formula.
HN CHjOH
O - (CHj)^ - 0 2 I
CHj -SO 3 H
Chemical name. 4,4'-(Pentamethylenedioxy)dibenzamidine bis(2-hydrox-

yethanesulfonate); 4,4'-[l,5-pentanediylbis(oxy)]bis[benzenecarboximidamide]-
bis(2-hydroxyethanesulfonate); CAS Reg. No. 140-64-7.

TS; practically insoluble in chloroform R and ether R.
Category. Antitrypanosomal drug; antileishmaniasis drug.
723
Storage. Pentamidine isetionate should be kept in a well-closed container.
Additional information. Pentamidine isetionate is hygroscopic.
Requirements
Definition. Pentamidine isetionate contains not less than 98.5% and not more
than 102.5% of C,9H24N402,2C2H604S, calculated with reference to the dried
substance.
Identity tests
B. To add 5 ml of water and heat to 80 °C to dissolve. Add 10 ml of sodium

0.5 g
hydroxide (-50g/l) TS, cool in ice, and filter. To 2 ml of the nitrate add
0.2 ml of nitric acid (-1000 g/1) TS followed by 0.2 ml of ceric ammonium
nitrate TS; a red-orange colour is produced.
Loss on drying. Dry to constant weight at 105°C; it loses not more than40mg/g.
chromatography, using silica gel R6, activated at 105 °C for 1 hour, as the
coating substance (a precoated plate from a commercial source is suitable), and
as the mobile phase the upper layer obtained by shaking together 10 volumes
of water, 8 volumes of 1 -butanol R, and 2 volumes of glacial acetic acid R. Apply
separately to the plate 10 |il of each of 2 solutions in methanol R containing (A)
50mg of the test substance per ml and (B) 0.25mg of the test substance per ml.

gen, using about 0.4 g of the test substance, accurately weighed, and 9 ml of
nitrogen-free sulfuric acid (~1760g/l) TS. Each ml of sulfuric acid (0.05mol/l)
VS is equivalent to 14.82 mg of Ci9H24N402,2C2Hs04S.
724
PENTAMIDINI MESILAS
PENTAMIDINE MESILATE
Molecular formula. Ci9H24N402,2CH403S
Graphic formula.
Chemical name. 4,4'-(Pentamethylenedioxy)dibenzamidine dimethanesul-

fonate; 4,4'-[l,5-pentanediylbis(oxy)]bis[benzenecarboximidamide] dimethane-
sulfonate; CAS Reg. No. 6823-79-6.
Description. A white or light pink, granular powder; almost odourless.
Solubility. Slighdy soluble in water and ethanol (-750 g/1) TS; practically insol-
uble in ether R, acetone R, and chloroform R.
Category. Antitrypanosomal drug; antileishmaniasis drug.
Storage. Pentamidine mesilate should be kept in a well-closed container.
Requirements
Definition. Pentamidine mesilate contains not less than 98.5% and not more
than 102.5% of Ci9H24N402,2CH403S, calculated with reference to the dried
substance.

Identity tests
A. To 0.5 g add 5 ml of water and heat to 80 °C to dissolve. Add 10 ml of sodium
hydroxide (-50 g/1) TS, cool in ice, and filter. To 2 ml of the filtrate add
0.2 ml of nitric acid (-1000 g/1) TS followed by 0.2 ml of ceric ammonium
nitrate TS; a yellow colour is produced.
725
g
B. Heat 0.5 with 1ml of sodium hydroxide (~400g/l) TS in a test-tube;

ammonia, perceptible by its odour, is evolved.
C. Dissolve 1 g in 10ml of water at 80°C, add 10ml of sodium hydroxide

(~50g/l) TS, cool in ice, filter (keep the filtrate for test D), wash with 10ml
of water, and dry the precipitate at 105°C; melting temperature, about
188 °C.
D. Transfer 10 ml of the filtrate obtained in test C to a platinum crucible, add
2.5ml of hydrogen peroxide (~60g/l) TS, mix with the help of a glass rod
and evaporate to dryness on a water-bath. Dissolve the residue in 1 ml of
water, add 1 ml of glacial acetic acid R and again evaporate to dryness on a
water-bath, then ignite until free from carbon. Cool, mix the residue with
5ml of water and filter, if necessary. Neutralize with hydrochloric acid
(~70g/l) TS and add an excess of 3ml. Boil for 30 seconds, cool and proceed
with reaction A described under 2.1 General identification tests as charac-
teristic of sulfates.
15mg/g.
pH value. pH of a 0.05 g/ml solution prepared in warm water and then cooled,
4.5-6.S.
chromatography, using silica gel R6, activated at 105 °C for 1 hour, as the
separately to the plate 10 pi of each of 2 solutions in methanol R containing (A)
50mg of the test substance per ml (warm, if necessary) and (B) 0.25mg of the
chamber, allow it to dry in air and examine the chromatogram in ultraviolet

acid (~1760g/l) TS. Each ml of sulfuric acid (0.05mol/l) VS is equivalent to
13.32mg of Ci9H24N402,2CH403S.
726
PETHIDINI HYDRO CHLORIDUM

PETHIDINE HYDROCHLORIDE
Molecular formula. Ci5H2iN02,HCl
Graphic formula.
Chemical name. Ethyl l-methyl-4-phenylisonipecotate hydrochloride; ethyl

l-methyl-4-phenyl-4-piperidinecarboxylate hydrochloride; CAS Reg. No.
50-13-5.
Other name. Meperidine hydrochloride.
Solubility. Very soluble in water; soluble in ethanol (~750g/l) TS; practically

Category. Narcotic analgesic.
Storage. Pethidine hydrochloride should be kept in a well-closed container,

Additional information. Even in the absence of light, Pethidine hydrochlo-

Requirements
Definition. Pethidine hydrochloride contains not less than 98.0% and not
more than 101.0% of C] 5 H 2 iN 02 ,HCl, calculated with reference to the dried
substance.
Identity tests
A. Dissolve 5 mg in 0.5ml of water, add about 0.1ml of formaldehyde TS and
2 ml of sulfuric acid (-1760 g/1) TS; an orange-red colour is produced.
727
B. Dissolve O.lg in 5ml of ethanol (~750g/l) TS and add 15ml of trinitrophe-

nol (7 g/1) TS; a yellow, crystalline precipitate is produced. Collect the pre-
cipitate on a filter, wash with water, and dry at 105 °C; melting temperature,
about 190 °C (picrate), (Keep a portion of the picrate for test C.)
C. Mix 5 mg of the picrate obtained in test B with 5 mg of the substance being

examined; melting temperature, not less than 20 °C lower than that of the
picrate alone.

Melting range. After drying to constant weight at 105 °C, 187-190 °C.

and colourless.
chromatography, using kieselguhr R1 as the coating substance and a mixture of
1 volume of 2-phenoxyethanol R and 9 volumes of acetone R to impregnate the
plate, dipping it about 5mm into the liquid. After the solvent has reached a
height of a least 15 cm, remove the plate from the chromatographic chamber and
allow the acetone to evaporate. Use the impregnated plate immediately, carry-
ing out the chromatography in the same direction as the impregnation. Prepare
the mobile phase by shaking together 1 volume of diethylamine R, 100 volumes
of light petroleum Rl, and 8 volumes of 2-phenoxyethanol R, allow to settle, and
use the supernatant liquid. For the preparation of the test solutions, dissolve
0.10 g in 5 ml of water, add 0.5ml of sodium hydroxide (-400 g/1) TS, 2 ml of
ether R, and shake. Allow the layers to separate and use the upper layer as solu-
tion A. Dilute 0.5ml of solution A to 50ml with ether R and use as solution B.
Apply separately to the plate 5 pi of each of solutions A and B. After removing
the plate from the chromatographic chamber, allow it to dry in air for 10
minutes, return it to the chamber, and allow the solvent mixture to ascend a
second time 12 cm above the line of application. Remove the plate, allow it to
dry in air for 10 minutes, and spray it with dichlorofluorescein TS. Allow it to
stand for 5 minutes and examine the chromatogram in daylight; red to orange
spots on a white to almost white background are obtained. Then examine the
chromatogram in ultraviolet light (365 nm). Any spot obtained with solution A,
solution B.
728

(0.1 mol/1) VS as described under 2.6 Non-aqueous titration, Method A. Each
ml of perchloric acid (0.1 mol/1) VS is equivalent to 28.38mg of Ci5H2iN02,HCl.
Additional requirements for Pethidine hydrochloride for

parenteral use
PHENOBARBITALUM
PHENOBARBITAL
Graphic formula.
Chemical name. 5-Ethyl-5-phenylbarbituric acid; 5-ethyl-5-phenyl-2,4,6-

(l/i3/i5//)-pyrimidinetrione; CAS Reg. No. 50-06-6.
Solubility. Soluble in about 1100 parts of water, in about 10 parts of ethanol

(-750 g/1) TS and in about 15 parts of ether R.
Category. Hypnotic; sedative; anticonvulsant.
Storage. Phenobarbital should be kept in a well-closed container.
Additional information. Phenobarbital may exhibit polymorphism.
729
Requirements
Definition. Phenobarbital contains not less than 98.0% and not more than
Identity tests
• Either test A or tests B, C, and D may be applied.

reference spectrum of phenobarbital.
B. Dissolve 20 mg in 5 ml of ethanol (-750 g/1) TS, add 1 drop of cobaltous chlo-
ride TS and 1 drop of ammonia (-100 g/1) TS; a violet colour is produced.
C. Shake for 3 minutes 0.1 g with 4 ml of sodium hydroxide (0.1 mol/1) VS and
1 ml of water. Filter and to 2 ml of the filtrate add 4 drops of mercuric chlo-
ride (65 g/1) TS; a white precipitate is formed, which dissolves on the addi-
tion of 5 ml of ammonia (-100 g/1) TS.
D. Dissolve 0.1 g in 2ml of sulfuric acid (-1760g/l) TS, add about lOmg of
sodium nitrite R, and warm on a water-bath for 10 minutes; an orange-
yellow colour with a brownish sheen is produced.
Solution in alkali. Dissolve l.Og in 4.0ml of sodium hydroxide (-80g/l) TS

and add 6.0ml of water; the solution is clear and colourless.
Acidity. Boil l.Og with 50ml of water for 2 minutes, adjust the volume to
50ml, and filter. To 10ml of the filtrate add 0.15ml of methyl red/ethanol TS;
not more than 0.1 ml of sodium hydroxide (0.1 mol/1) VS is required to obtain
the midpoint of the indicator (orange).
Phenylbarbituric acid. Boil l.Og with 5 ml of ethanol (-750 g/1) TS for 3

minutes under a reflux condenser; a clear solution is produced.
Neutral and basic impurities. Dissolve l.Og in a mixture of 5 ml of sodium

hydroxide (-80 g/1) TS and 10 ml of water and shake for 1 minute with 25 ml
of ether R. Wash the ethereal layer 3 times, each time with 5 ml of water, evap-
orate the ether, and dry the residue at 105 °C for 1 hour; the residue weighs
not more than 3.0 mg.
730
80 volumes of chloroform R, 15 volumes of ethanol (-750 g/1) TS, and 1 volume
of ammonia (-260 g/1) TS as the mobile phase. Apply separately to the plate
lOpl of each of 2 solutions in ethanol (-750g/l) TS containing (A) lOmg of the
testsubstance per ml and (B) 0.20 mg of the test substance per ml. After remov-
ing the platefrom the chromatographic chamber, allow it to dry in air, and

mamide R, add 2 drops of thymolphthalein/dimethylformamide TS and titrate
with sodium methoxide (0.1 mol/1) VS to a blue endpoint, as described under
2.6 Non-aqueous titration. Method B. Each ml of sodium methoxide (0.1 mol/1)
VS is equivalent to 23.22 mg of C H 12 N 2 O 3
12 .
PHENOBARBITALUM NATRICUM
PHENOBARBITAL SODIUM
Molecular formula. Ci 2 HnN 2 Na 03
Graphic formula.
Chemical name. Sodium 5-ethyl-5-phenylbarbiturate; 5-ethyl-5-phenyl-

2,4,6-(lH',3/f,5E/)-pyrimidinetrione monosodium salt; CAS Reg. No. 57-30-7.
Description. White, crystalline granules or a white, crystalline powder;

odourless.
Solubility. Freely soluble in water; soluble in ethanol (-750 g/1) TS; practically
Category. Hypnotic; sedative; anticonvulsant.
731
Storage. Phenobarbital sodium should be kept in a tightly closed container,

Additional information. Phenobarbital sodium is hygroscopic. Even in the

absence of light, Phenobarbital sodium is gradually degraded on exposure to a
Requirements
Definition. Phenobarbital sodium contains not less than 98.0% and not
more than 101.0% of Ci 2 HnN 2 Na 03 ,
substance.
Identity tests

reference spectrum of phenobarbital sodium.
B. Dissolve 0.2 g in 10ml of water and add 2 ml of hydrochloric acid (~70g/l)

TS; a white, crystalline precipitate is produced. Wash the precipitate with
water until free from chlorides, and dry at 105 °C; melting temperature,
about 175°C (phenobarbital). Keep the precipitate for test C.
C. Dissolve 20 mg of the precipitate obtained from test B in 5 ml of ethanol

(-750 g/1) TS, add 1 drop of cobaltous chloride TS and 1 drop of ammonia
(~100g/l) TS; a violet colour is produced.

20 mg/ml solution.

free water R remains clear and colourless during 15 minutes.
70mg/g.
pH value. pH of a O.lOg/ml solution in carbon-dioxide-free water R, 9.0-10.8.
Neutral and basic impurities. Dissolve l.Og in a mixture of 5 ml of sodium

hydroxide (-80 g/1) TS and 10 ml of water, and shake for 1 minute with 25 ml
of ether R. Wash the ethereal layer 3 times, each time with 5 ml of water, evap-
orate the ether, and dry the residue at 105°C for 1 hour; the residue weighs
not more than 3.0 mg.
732
80 volumes of chloroform R, 15 volumes of ethanol (-750 g/1) TS and 5 volumes
lOpl of each of 2 solutions in ethanol (-750g/l) TS containing (A) lOmg of the
testsubstance per ml and (B) 0.20 mg of the test substance per ml. After remov-
ing the platefrom the chromatographic chamber, allow it to dry in air, and
of hydrochloric acid (2 mol/1) VS, and extract with 50ml of ether R and then
with successive quantities, each of 25ml of ether R, until complete extraction
has been effected. Wash the combined extracts twice with water, using 5 ml
each time. Add the ether extract to the main ether extract, evaporate to low
bulk, add 2 ml of dehydrated ethanol R, evaporate to dryness, and dry the
residue to constant weight at 105 °C. Each g of residue is equivalent to 1.095g
of C] 2 Hi]N 2 Na 03 .
PHENOXYMETHYLPENICILLINUM
PHENOXYMETHYLPENICILLIN
Molecular formula. C](iHi 8 N 205 S
Graphic formula.
Chemical name. (2S,5R,6/^)-3,3-Dimethyl-7-oxo-6-(2-phenoxyacetamido)-4-

thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid; [2S-(2a,5a,6|3)]-3,3-
dimethyl-7-oxo-6 [(phenoxyacetyl)amino]-4-thia-l-azabicyclo [3.2.0]heptane-
2-carboxylic acid; CAS Reg. No. 87-08-1.
Description. A white, fine crystalline powder.
733
=
Solubility. Soluble in 1700 parts of water and in 7 parts of ethanol (~750g/l)

TS.
Category. Antibiotic,
Storage. Phenoxymethylpenicillin should be kept in a tightly closed container,

Additional information. Even in the absence of light, Phenoxymethylpeni-

cillin is gradually degraded on exposure to a humid atmosphere, the decom-
position being faster at higher temperatures.
Requirements
Definition. Phenoxymethylpenicillin contains not less than 95.0% and not
more than 102.0% of C16H18N2O5S, calculated with reference to the anydrous
substance.
Identity tests

spectrum obtained from phenoxymethylpenicillin RS or with the reference
spectrum of phenoxymethylpenicillin.

1 minute in a water-bath; the solution remains colourless.
C. Place 2 mg in a test-tube, add 1 drop of water and 2 ml of formaldehyde/sul-

furic acid TS and mix; the solution is red. Immerse the test-tube for 1 minute
in a water-bath; a red-brown colour is produced.
Specific optical rotation. Use a 10 mg/ml solution in 1 -butanol R and calcu-

late with reference to the anhydrous substance; [a]™ -1-I86 to -1-200°,

Fischer method, Method B, using about 0.3 g of the substance; not more than
15mg/g.
pH value. pH of a 5.0mg/ml suspension in water, 2. 4-4.0.
^-Hydroxyphenoxymethylpenicillin. Dissolve about 0.1 g, accurately

weighed, in sufficient sodium hydroxide (0.1 mol/1) VS to produce 100ml.
734
Monographs for pharmaceutical substances
Measure the absorbance of 1-cm layer at the maximum at about 306nm; not
a
more than 0.36 (preferably use 2-cm cells for the measurement and calculate
the absorbance of a 1-cm layer).
Ultraviolet absorbance range. Dilute 20ml of the solution obtained in the

test for ;^-hydroxyphenoxymethylpenicillin to 100 ml with sodium hydroxide
(0.1 mol/1) VS. Measure the absorbance of a 1-cm layer at the maximum at
about 274nm; not less than 0.56 and not more than 0.62.
Assay. Dissolve about 50 mg, accurately weighed, in a mixture of 0.6ml of

sodium hydrogen carbonate (40g/l) TS and 10 ml of water and dilute with suf-
ficient water to produce 1000ml. Transfer two 2.0-ml aliquots of this solution
into separate stoppered tubes. To one tube add 10.0ml of imidazole/mercuric
chloride TS, mix, stopper the tube, and place it in a water-bath at 60 °C for
exactly 25 minutes. Cool the tube rapidly to 20 °C (solution A). To the second
tube add 10.0ml of water and mix (solution B).

tion B.
B, calculate the amount of C16H1SN2O5S in the substance being examined by
comparison with phenoxymethylpenicillin potassium RS similarly and concur-
rently examined, but omitting the addition of sodium hydrogen carbonate
(40g/l) TS; each mg of phenoxymethylpenicillin potassium RS (C16H17KN2O5S)
is equivalent to 0.902 mg of phenoxymethylpenicillin (C16H18N2O5S). In an ade-
quately calibrated spectrophotometer the absorbance of the reference solution

should be 0.63 + 0.03.
PHENOXYMETHYLPENICILLINUM CALCICUM
PHENOXYMETHYLPENICILLIN CALCIUM
Molecular formula. (Ci(iHi7N205S)2Ca,2H20 or C32H34CaN40]oS2,2H20
735
Graphic formula.
Chemical name. Calcium bis[(2.S’,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenoxyac-

etamido)-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate] dihydrate; calcium
bis[[2S-(2a,5a,6P)]3,3-dimethyl-7-oxo-6-[(phenoxyacetyl)amino]-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylate] dihydrate; CAS Reg. No. 73368-74-8.
Description. A white, fine crystalline powder; odourless or with a faint char-

acteristic odour.
Solubility. Slowly soluble in 120 parts of water.
Storage. Phenoxymethylpenicillin calcium should be kept in a well-closed

container.

cillin calcium is gradually degraded on exposure to a humid atmosphere, the
Requirements
Definition. Phenoxymethylpenicillin calcium contains not less than 95.0%
and not more than 102.0% of (Ci 6 Hi 7 N 205 S) 2 Ca, calculated with reference to
the anhydrous substance.
Identity tests

spectrum obtained from phenoxymethylpenicillin calcium RS or with
the reference spectrum of phenoxymethylpenicillin calcium.

(~1760g/l) TS and mix; the solution is colourless. Immerse the test-tube for
736

D. Ignite a small quantity, dissolve the residue in hydrochloric acid (~70g/l) TS,
and make the solution alkaline by the addition of ammonia (~100g/l) TS;
the solution yields the reactions described under 2.1 General identification
tests as characteristic of calcium.

Fischer method. Method A, using about 0.2 g of the substance; not more than
50mg/g.
/^-Hydroxyphenoxymethylpenicillin. Dissolve about 0.11 g, accurately

Measure the absorbance of a 1-cm layer at the maximum at about 306 nm; not
more than 0.36 (use preferably 2-cm cells for the measurement and calculate

test for /^-hydroxyphenoxymethylpenicillin to 100 ml with sodium hydroxide

produce 1000ml. Transfer two 2.0-ml aliquots of this solution into separate
mix, stopper the tube, and place it in a water-bath at 60 °C for exactly 25
minutes. Cool the tube rapidly to 20 °C (solution A). To the second tube add
10.0ml of water and mix (solution B).
B, calculate the amount of (Ci 6Hi 7N 205 S) 2 Ca in the substance being tested by
comparison with phenoxymethylpenicillin potassium RS, similarly and con-
currently examined, taking into account that each mg of phenoxymethylpeni-
cillin potassium RS (C, 6 Hi 7 KN 205 S) is equivalent to 0.951 mg of
phenoxymethylpenicillin calcium (Ci^Hi 7 N 205 S) 2 Ca. In an adequately cali-
brated spectrophotometer the absorbance of the reference solution should be
0.63 ± 0.03.
737
PHENOXYMETHYLPENICILLINUM KALICUM
PHENOXYMETHYLPENICILLIN POTASSIUM
Molecular formula. C16H17KN2O5S
Graphic formula.
Chemical name. Potassium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenoxyac-

etamido)-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate; potassium [2S-
(2a,5a,6P)]-3,3-dimethyl-7-oxo-6- [(phenoxyacetyl)amino]-4-thia-l-azabicyclo-
[3,2.0]heptane-2-carboxylate; CAS Reg. No, 132-98-9.

Solubility. Soluble in about 1.5 parts of water; practically insoluble in ether R.
Storage. Phenoxymethylpenicillin potassium should be kept in a tightly closed

container; protected from light.

cillin potassium is gradually degraded on exposure to a humid atmosphere, the
Requirements
Definition. Phenoxymethylpenicillin potassium contains not less than 95.0%
and not more than 102.0% of C16H17KN2O5S, calculated with reference to the
dried substance.
Identity tests
738

spectrum obtained from phenoxymethylpenicillin potassium RS or with
the reference spectrum of phenoxymethylpenicillin potassium.


D. Ignite a small quantity, dissolve the residue in water and filter. To the fil-
trate, add 2 ml of sodium hydroxide (-80 g/1) TS; it yields the reaction
described under 2.1 General identification tests as characteristic of

potassium.
ence to the dried substance; = -1-215 to -1-235°.
Clarity of solution. A solution of 0.2 gin 10 ml of water is not more than

slightly opalescent.
15mg/g.
p-Hydroxyphenoxymethylpenicillin. Dissolve about 0.11 g, accurately

Measure the absorbance of a Tcm layer at the maximum at about 306 nm; not
more than 0.36 (preferably use 2-cm cells for the measurement and calculate

test for /^’-hydroxyphenoxymethylpenicillin to 100 ml with sodium hydroxide

mix, stopper the tube and place it in a water-bath at 60 °C for exactly 25
10.0ml of Water and mix (solution B).
739

tion B.

the difference
B, calculate theamount of CigHi 7 KN 205 S in the substance being tested by
comparison with phenoxymethylpenicillin potassium RS, similarly and
PHENYLHYDRARGYRI NITRAS
PHENYLMERCURIC NITRATE
Ci2HiiHg2N04
Chemical name. Nitratophenylmercury; (nitrato-O)phenylmercury; CAS

Reg. No. 55-68-5.
Description. White, lustrous plates or a white, crystalline powder; odourless.
TS; soluble in glycerol R and in fixed oils.
Storage. Phenylmercuric nitrate should be kept in a tightly closed container,

Additional information. Phenylmercuric nitrate is affected by light. It melts

at about 188°C with decomposition.
Requirements
Definition. Phenylmercuric nitrate is a mixture of phenylmercuric nitrate and
phenylmercuric hydroxide.
Phenylmercuric nitrate contains not less than 98 0

. % and not more than the
equivalent of 101 0 . % of Ci 2 HnHg 2 N 04 ,
substance.
740
Identity tests
A. To 10 ml of a saturated solution add 2 drops of sodium sulfide TS; a white
precipitate is produced. Boil the mixture and allow to stand; the precipitate
becomes black.
B. Heat 0.5g with 0.5g of zinc R powder, 0.5g of reduced iron R, and 5ml of
sodium hydroxide (-200 g/1) TS. Place a piece of moistened red litmus paper
R over the vapours; the colour of the paper changes to blue.
C. To 10ml of a saturated solution add 1ml of hydrochloric acid (~70g/l) TS

and heat to boiling; a white precipitate is produced. Filter, and to 5 ml of the
filtrate add 2 ml of ferrous sulfate (15 g/1) TS; it yields reaction A described
under 2.1 General identification tests as characteristic of nitrates.
Mercuric salts and heavy metals. Heat 0.1 g with 15 ml of water, cool, filter,
and add 0.1ml of sodium sulfide TS to the filtrate; a precipitate is produced,
the colour of which remains unchanged.
Residue on ignition. Not more than 5.0mg/g.
lOmg/g.
Acidity. To a 0.2 mg/ml solution add 3 drops of bromocresol green/ethanol TS;

the solution is neutral (green).
Assay. Transfer about 0.2 g, accurately weighed, to a conical flask, and dis-
solve in 90 ml of water and 10 ml of nitric acid (-1000 g/1) TS. Add 2 ml of ferric
ammonium sulfate (45 g/1) TS and titrate with ammonium thiocyanate
(0.05 mol/1) VS.
Each ml of ammonium thiocyanate (0.05mol/l) VS is equivalent to 0.01586g of

C]2HiiHg2N04.
PHENYTOINUM
PHENYTOIN
741
Graphic formula.
Chemical name. 5,5-Diphenylhydantoin; 5,5-diphenyl-2,4-imidazolidine-

dione; CAS Reg. No. 57-41-0.

Storage. Phenytoin should be kept in a tightly closed container, protected

from light.
Requirements
Definition. Phenytoin contains not less than 98.0% and not more than
101.0% of C] 5 H, 2 N 202 calculated with reference
,
Identity tests

infrared region.
trum obtained from phenytoin RS or with the reference spectrum of phenytoin.
B. Dissolve 20 mg in 2 ml of ammonia (-100 g/1) TS and add 5 ml of silver

nitrate (40 g/1) TS; a white precipitate is produced.
C. Dissolve 5 mg in 1 ml of boiling ethanol (-750 g/1) TS, add 2 ml of water, 2

drops of pyridine R, and 2 drops of copper (II) sulfate (80g/I) TS; allow to
cool; a blue-violet, crystalline precipitate is produced.
Heavy metals. Use 1.0 g for the preparation of the test solution, as described
742
lOmg/g.
Acidity or alkalinity. Shake 2 g for 1 minute with 40 ml of carbon-dioxide-

free water R, and filter. To 10 ml of the filtrate add 2 drops of phenolph-
thalein/ethanol TS; no colour is produced. Add 0.15 ml of carbonate-free
sodium hydroxide (0.01 mol/1) VS; a pink colour is produced. Add 0.3 ml of
hydrochloric acid (0.01 mol/1) VS and 5 drops of methyl red/ethanol TS; a red
or orange colour is produced.

mamide R, add 2 drops of thymol blue/dimethylformamide TS and titrate with
Non-aqueous titration. Method B. Each ml of sodium methoxide (0.1 mol/1) VS
is equivalent to 25.23 mg of C 15 H N 2 O
12 2.
PHENYTOINUM NATRICUM
PHENYTOIN SODIUM
Molecular formula. Ci5H,]N2Na02
Graphic formula.
Chemical name. 5,5-Diphenylhydantoin monosodium salt; 5,5-diphenyl-2,4-

monosodium salt; CAS Reg. No. 630-93-3.
imidazolidinedione
Solubility. Soluble in water, giving a slighdy turbid solution owing to partial

hydrolysis; soluble in ethanol (-750 g/1) TS; practically insoluble in ether R.
743
Storage. Phenytoin sodium should be kept in a tightly closed container.
Additional information. Phenytoin sodium is somewhat hygroscopic and on

exposure to air gradually absorbs carbon dioxide.
Requirements
Definition. Phenytoin sodium contains not less than 98.5% and not more than
101.0% of C] 5 HiiN 2 Na 02 calculated with reference
,
Identity tests
• Either tests A and D or tests B, C, D, and E may be applied.
A. Shake 0.1 g with 20ml of water, acidify with hydrochloric acid (~70g/l) TS,
and extract with chloroform R; wash the chloroform extract with water
and evaporate to dryness. Carry out the examination with the residue as
described under 1 .7 Spectrophotometry in the infrared region. The infrared
absorption spectrum is concordant with the spectrum obtained from pheny-
toin RS or with the reference spectrum of phenytoin.
B. Dissolve 0.1 g in a mixture of 1 ml of pyridine R and 9 ml of water, add 1 ml

of copper (II) sulfate/pyridine TS, and allow to stand for 10 minutes; a blue
C. Dissolve lOmg in 1ml of water, add 1 drop of ammonia (-100 g/1) TS and
heat until boiling begins. Add 1 drop of copper (II) sulfate/ammonia TS and
shake; a pink, crystalline precipitate is formed.

20mg/ml solution.
E. Shake 0.1 g with 20ml of water, acidify with hydrochloric acid (~70g/l) TS,
and extract with chloroform R; wash the chloroform extract with water and
evaporate to dryness. Melting temperature of the residue, about 295 °C
(phenytoin).
Heavy metals. To l.Og add 24ml of water and 6ml of hydrochloric acid
(~70g/l) TS; heat the mixture until boiling begins. Filter, cool, and filter again
through a suitable sintered glass filter. Dilute to 40 ml with water, mix, and
determine the content of heavy metals as described under 2.2.3 Limit test for
heavy metals, according to Method A; not more than lOpg/g.
744
Solution in alkali. To 20 mg add 8.0 ml of carbon-dioxide-free water R and

then add gradually 2.0ml of carbonate-free sodium hydroxide (0.1 mol/1) VS;
the solution is clear and not more intensely coloured than standard colour solu-
30mg/g.

Rl, add 3 drops of 1-naphtholbenzein/acetic acid TS as indicator and titrate
27.43mg of Ci 5 HnN 2 Na 02 .
Additional requirements for Phenytoin sodium for

parenteral use
PHYSOSTIGMINI SALICYLAS
PHYSOSTIGMINE SALICYLATE
Molecular formula. Ci 5 H 2 iN 302 ,C 7 H 503 or C 22 H 27N 3 O 5
Graphic formula.
COOH
Chemical name. Physostigmine monosalicylate; (3aS-ds)-l,2,3,3a,8,8a-

hexahydro-l,3a,8-trimethylpyrrolo[2,3-l']indol-5-ol, methylcarbamate (ester),
mono-(2-hydroxybenzoate); CAS Reg. No. 57-64-7.
Other name. Eserine salicylate.
745
=
Description. Colourless crystals; odourless.
Solubility. Sparingly soluble in water; soluble in ethanol (-750 g/1) TS; slightly
soluble in ether R.
Category. Anticholinesterase; miotic.
Storage. Physostigmine salicylate should be kept in a tightly closed container,

protected from light, and preferably in quantities not exceeding 1 g.
Additional information. Physostigmine salicylate is very poisonous. It

acquires a red tint when exposed to air or light. All tests should be performed
on freshly prepared solutions.
Requirements
Definition. Physostigmine salicylate contains not less than 98.0% and not
more than 101.0% of Ci 5 H 2 iN 302 ,C 7 Hg 03 ,
calculated with reference to the
dried substance.
Identity tests
A. To a 10 mg/ml solution add a few drops of sodium hydroxide (-80 g/1) TS;
a white precipitate is produced, which gradually turns pink. It dissolves in
an excess of the reagent to give a red solution.
B. Warm lOmg with a few drops of ammonia (-100 g/1) TS; an orange solu-
tion is produced. Evaporatethis solution and dissolve the residue in ethanol
(-750 g/1) TS. To the resulting blue solution add a few drops of acetic acid
(-300 g/1) TS; the colour is intensified. Dilute with water; a red fluorescence
appears.
C. To a 10 mg/ml solution add a few drops of ferric chloride (25 g/1) TS; a violet
colour, which remains after the addition of ethanol (-750 g/1) TS, appears.
Specific optical rotation. Use a lOmg/ml solution; [a]™ -90 to -94°.
Clarity and colour of solution. A solution of 0.10 gin 10 ml of water is clear

and colourless.
lOmg/g.
746
Eseridine. To 5 ml of a 10 mg/ml solution add 5 drops of hydrochloric acid

(~70g/l) TS, 1.5ml of potassium iodate (0.01 mol/1) VS and 1ml of chloroform
R. Shake for 1 minute; no violet colour develops in the chloroform layer.
Assay. Dissolve about 0.35 g, accurately weighed, in 30 ml of a mixture of

equal volumes of chloroform R and glacial acetic acid R1 Titrate with perchloric .
acid (0.1 mol/1) VS, determining the endpoint potentiometrically as described

(0.1 mol/1) VS is equivalent to 41.35mg of Ci 5 H 2 iN 302 ,C 7 H 503 .
PHYTOMENADIONUM
PHYTOMENADIONE
Graphic formula.
H H
Chemical name. Phylloquinone; [/^-[/^*,/^*-(£)]]-2-methyl-3-(3,7,ll,15-tetram-

ethyl-2-hexadecenyl)-l,4-naphthalenedione; 2-methyl-3-phytyl- 1,4-naphtho-
quinone; CAS Reg. No. 84-80-0.
Other names. Phytonadione; Vitamin Ki.
Description. A clear, yellow to amber-coloured, very viscous liquid; odour-

Miscibility. Practically immiscible with water; sparingly miscible with ethanol

(-750 g/1) TS; freely miscible with ether R.
747
Storage. Phytomenadione should be kept in a tightly closed container, pro-

tected from light.
Requirements
Definition. Phytomenadione contains not less than 97.0% and not more than
102.0% of C31H46O2.
Identity tests
A. The absorption spectrum of a lOpg/ml solution in 2,2,4-trimethylpentane
R, when observed between 230 nm and 350 nm, exhibits 4 maxima at about
243 nm, 249 nm, 261 nm, and 270 nm. The absorbances at those wavelengths
using 1-cm cells are about 0.40, 0.42, 0.38, and 0.39, respectively. The spec-
trum also exhibits minima at about 246 nm, 254 nm, and 266 nm. The ratio
of the absorbance at the minimum of about 254 nm to that at the maximum
of about 249nm is between 0.70 and 0.75.
B. The absorption spectrum of a 0.10 mg/ml solution in 2,2,4-trimethylpentane

R, when observed between 230 nm and 350 nm, exhibits a maximum at
about 327 nm and a minimum at about 285 nm. The absorbance of a Tcm
layer at the maximum is about 0.70, and at the minimum about 0.22.
C. Mix about 0.05 g of the test liquid with 5 ml of ethanol (~750g/l) TS and
add 1.0ml of potassium hydroxide/ethanol TSl; a green colour is produced.
Allow to stand for 15 minutes; the colour of the solution turns to red-brown.
Refractive index. «d = 1.525 - 1.529.
Acidity or alkalinity. Dissolve 1 g in 20ml of dehydrated ethanol R; the solu-

tion is neutral to litmus paper R.
Related substances. Carry out, in subdued light, the test described under
and a mixture of 80 volumes of cyclohexane R, 20 volumes of ether R, and 1
volume of methanol R as the mobile phase. Apply separately to the plate 10 pi
of each of 2 solutions in 2,2,4-trimethylpentane R containing (A) 5 mg of the
ml and (B) 0.05 mg of menadione R per ml. After removing
test substance per
from the chromatographic chamber, allow it to dry in air and examine
the plate
with solution B.
Assay
Carry out the following operations in subdued light.
748
Dissolve about 0.1 g, accurately weighed, in sufficient 2,2,4-trimethylpentane

R to produce 100 ml. Dilute 10ml 100ml with 2,2,4-trimethylpentane R and
to
further dilute 10 ml of this solution to 100 ml with the same solvent. Measure
the absorbance of this solution in a 1 -cm layer at the maximum at about
249 nm and calculate the content of C 31 H 4 SO 2 using the absorptivity value

,
of 42 (AiL = 420).
Additional requirements for Phytomenadione for

p'arenteral use
PILOCARPINI HYDRO CHLORIDUM

PILOCARPINE HYDROCHLORIDE
Molecular formula. CnHi6N202,HCl
Graphic formula.
Chemical name. Pilocarpine monohydrochloride; (3S-c/s)-3-ethyldihydro-

4-[(l-methyl-l/f-imidazol-5-yl)methyl]-2(3/i)-furanone monohydrochloride;
CAS Reg. No. 54-71-7.

almost odourless.
Solubility. Very soluble in water; freely soluble in ethanol (-750 g/1) TS;
Category. Parasympathomimetic; miotic.
Storage. Pilocarpine hydrochloride should be kept in a tighdy closed con-

749
Additional information. Pilocarpine hydrochloride is very poisonous; it is

hygroscopic and is affected by light. Even in the absence of light, Pilocarpine
Requirements
Definition. Pilocarpine hydrochloride contains not less than 98.5% and not
more than 101.0% of C]iH, 6 N 202 ,HCl, calculated with reference to the dried
substance.
Identity tests
A. Dissolve 10 mg in 5 ml of water, add 2 drops of sulfuric acid (~100g/l) TS,
1ml of hydrogen peroxide (~60g/l) TS, 1ml of toluene R, and 1 drop of
potassium dichromate (lOOg/1) TS, and shake well; the toluene layer
acquires a violet colour, whereas the aqueous layer remains yellow.
B. A 0.05 g/ml solution yields reaction B described under 2.1 General identifi-
ence to the dried substance; [a]o = -1-89 to -1-93°.
Nitrates. Dissolve 0.05 g in 5 ml of water and carefully add the solution to

5 ml of a 1 mg/ml solution of diphenylamine R in sulfuric acid (~1760g/l) TS,
ensuring that the liquids do not mix; no blue colour is produced at the inter-

and colourless.
20mg/g.
pH value. pH of a 5.0mg/ml solution, 3. 8-5. 2.
Related alkaloids. Carry out the tests as described under 1.14.1 Thin-layer
25 volumes of chloroform R, 20 volumes of acetone R, and 0.4 volumes of
of each of 2 solutions containing (A) 50 mg of the test substance per ml and (B)
750
1.0 mg of the test substance per ml. After removing the plate from the chro-
ml of perchloric acid (0.1 mol/1) VS is equivalent to 24.47 mg of C,]H]6N202,HC1.
Additional requirement for Pilocarpnne hydrochloride for

sterile use
PILOCARPINI NITRAS
PILOCARPINE NITRATE
Molecular formula. CnHi 6N 202 ,HN 03
Graphic formula.
Chemical name. Pilocarpine mononitrate; (3S-c(s)-3-ethyldihydro-4-[(l-

methyl-lf/-imidazol-5-yl)methyl]-2(3H)-furanone mononitrate; CAS Reg. No.
148-72-1.
Category. Parasympathomimetic; miotic.
Storage. Pilocarpine nitrate should be kept in a tightly closed container, pro-

tected from light.
751
Additional information. Pilocarpine nitrate is very poisonous; it is affected

by light. Even in the absence of light, Pilocarpine nitrate is gradually degraded
on exposure to a humid atmosphere, the decomposition being faster at higher
temperatures.
Requirements
Definition. Pilocarpine nitrate contains not less than 98.5% and not more than
101.0% of C]iHi 6N 2 C> 2 ,HN 03 calculated with reference to the dried substance.
,
Identity tests
A. Dissolve lOmg in 5ml of water, add 2 drops of sulfuric acid (~100g/l) TS,
1ml of hydrogen peroxide (~60g/l) TS, 1ml of toluene R, and 1 drop of
potassium dichromate (lOOg/1) TS, and shake well; the toluene layer
acquires a violet colour, whereas the aqueous layer remains yellow.
B. To 2 ml of a 0.05 g/ml solution add 2 ml of ferrous sulfate (15g/l) TS; it yields

of nitrates.
ence to the dried substance; [a]o = -1-80 to -1-83°.


and colourless.
20 mg/g.
pH value. pH of a 5.0 mg/ml solution in water, 3. 5-4. 5.
25 volumes of chloroform R, 20 volumes of acetone R, and 0.4 volume of
of each of 2 solutions containing (A) 50 mg of the test substance per ml and (B)
1.0 mg of the test substance per ml. After removing the plate from the chro-
752

Assay. Dissolve about 0.55 g, accurately weighed, in 30 ml o£ glacial acetic acid

aqueous titration. Method A. Each ml o£ perchloric acid (0.1 mol/1) VS is equiv-
alent to 27.13 mg o£ CnH] 6N 202 ,HN 03 .
Additional requirement for Pilocarpine nitrate for sterile use

Complies with 3.2.1 Test lor sterility o£ non-injectable preparations.
PIPERAZINI ADIPAS
PIPERAZINE ADIPATE
Molecular formula. C 4 HioN 2 ,C 6 Hio 04 or C 10 H 20N 2 O 4
Graphic formula.
CH,-CH,-COOH
'
I
CHj-CHj— COOH
Chemical name. Piperazine hexanedioate (1:1); hexahydro-l,4-diazine

adipate (1:1); CAS Reg. No. 142-88-1.
Solubility. Soluble in water; practically insoluble in ethanol (-750 g/1) TS and

ether R.
Storage. Piperazine adipate should be kept in a well-closed container.
753
Requirements
Definition. Piperazine adipate contains not less than 98.0% and notmore than
101.0% of C 4 HioN 2 ,C 6 Hio 04 calculated with reference to the dried substance.
,
Identity tests
A. Dissolve 0.1 g in 5ml of water, add 0.5g of sodium hydrogen carbonate R,
0.5ml of freshly prepared potassium ferricyanide (50g/l) TS, and 0.1ml
of mercury R. Shake vigorously for 1 minute, and allow to stand for 20
minutes; a reddish colour slowly develops.
B. Dissolve 0.5g in 10ml of water and add 5ml of hydrochloric acid (~250g/l)
TS. Extract 3 times with ether R, using 10ml each time, and keep the
aqueous layer for test C. Evaporate the ether extracts to dryness and dry at
105 °C: melting temperature, about 152 °C (adipic acid).
C. Cautiously heat the aqueous layer obtained from test B to eliminate any dis-
solved ether. Cool and add 0.5 g of sodium nitrite R. Heat to boiling and
cool in ice for 15 minutes, stirring if necessary to induce crystallization.
Filter, wash with 10ml of ice-water and dry the precipitate at 105 °C;
melting temperature, about 158 °C (N,N'-dinitrosopiperazine).
5.0mg/g.
Primary amines. For the preparation of the test solution dissolve 0.25 g in suf-
ficient water to produce 50ml. Transfer 0.5ml of this solution to a test-tube.
Separately transfer to a second test-tube 0.5ml of a solution containing lOpg/ml
of ethylenediamine R to serve as a reference solution. To both tubes add
0.5 ml of ethanol (-750 g/1) TS, 1 ml of diethoxytetrahydrofuran/acetic acid TS,
heat on a water-bath at 80 °C for 30 minutes, cool in ice, and add 3ml of 4-
dimethylaminobenzaldehyde TS4. Measure the absorbance at about 570 nm,
7-10 minutes after the addition of the last reagent, against a solvent cell con-
taining the reagents prepared in a similar manner. The absorbance of the test
solution is not more intense than that of the reference solution.
Assay. Dissolve about 0.20 g, accurately weighed, in 3.5ml of sulfuric acid

(0.5mol/l) VS and 10ml of water; add 100ml of trinitrophenol (7 g/1) TS, heat
754
on a water-bath for 15 minutes, and allow to stand for 1 hour. Filter, wash the
residue with successive quantities of trinitrophenol (7g/l) TS, using 10ml each
time, until the washings are free from sulfates. Finally, wash with dehydrated
ethanol R, and dry the residue to constant weight at 105°C. Each g of residue
isequivalent to 426.8 mg of C 4 Fl]oN 2 ,C 6 Hio 04 .
PIPERAZINI CITRAS
PIPERAZINE CITRATE
Molecular formula. (C 4 HioN 2) 3 2 C 6 Hs 07 ,
or C 24H 46N 6 O 14 (anhydrous)
Graphic formula.
CHjCOOH
• HO-C-COOH
I
CH COOH
Chemical name. Piperazine 2-hydroxy-l,2,3-propanetricarboxylate (3:2);

hexahydro-l,4-diazine citrate (3:2); CAS Reg. No. 144-29-6 (anhydrous).
Description. A fine, white, granular powder; almost odourless.
Solubility. Soluble in 1.5 parts of water; practically insoluble in ethanol

Storage. Piperazine citrate should be kept in a well-closed container, protected

from light.
Additional information. Piperazine citrate contains a variable amount of

water of crystallization.
Requirements
Definition. Piperazine citrate contains not less than 98.0% and not more than
101.0% of (C4 HioN 2)3,2C6H807, Calculated with reference to the anhydrous
substance.
755
Identity tests
A. Dissolve 0.1 g in 5ml of water, add 0.5g of sodium hydrogen carbonate R,
0.5ml of freshly prepared potassium ferricyanide (50g/l) TS, and 0.1ml of
mercury R. Shake vigorously for 1 minute, and allow to stand for 20
minutes; a reddish colour slowly develops.

C. Melting temperature, after drying at 105 °C, about 185 °C.
D. Dissolve 0.2 g in 5 ml of hydrochloric acid (~70g/l) TS, and add 0.5 g of

sodium nitrite R. Cool in ice for 15 minutes, stir if necessary to induce crys-
tallization, filter, wash with 10 ml of ice-water, and dry the precipitate at
105 °C; melting temperature, about 158 °C (iV,N'-dinitrosopiperazine).

content is not less than O.lOg/g and not more than 0.14g/g.
Primary amines. For the preparation of the test solution dissolve 0.25 g in suf-
ficient water to produce 50ml. Transfer 0.5ml of this solution to a test-tube.
Separately transfer to a second test-tube 0.5 ml of a solution containing lOpg/ml
of ethylenediamine R to serve as a reference solution. To both tubes add
0.5ml of ethanol (-750 g/1) TS, 1 ml of diethoxytetrahydrofuran/acetic acid TS,
heat on a water-bath at 80 °C for 30 minutes, cool in ice, and add 3ml of 4-
dimethylaminobenzaldehyde TS4. Measure the absorbance at about 570 nm,
7-10 minutes after the addition of the last reagent, against a solvent cell con-
taining the reagents prepared in a similar manner. The absorbance of the test
solution is not more intense than that of the reference solution.
Assay. Dissolve about 0.20 g, accurately weighed, in 3.5 ml of sulfuric acid

(0.5mol/l) VS and 10ml of water, add 100ml of trinitrophenol (7 g/1) TS, heat
on a water-bath for 15 minutes, and allow to stand for 1 hour. Filter, wash the
residue with successive quantities of trinitrophenol (7 g/1) TS, using 10ml each
time, until the washings are free from sulfates. Finally, wash with dehydrated
ethanol R, and dry the residue to constant weight at 105°C. Each g of residue
is equivalent to 393.5 mg of (C 4 HioN 2 ) 3 2 C(;Ha 07
,
.
756
FIX LITHANTHRACIS
COAL TAR
Chemical name. Coal tar; CAS Reg. No. 8007-45-2.
Description. Brown-black or black, viscous liquid; odour, characteristic and

strong resembling naphthalene.
Solubility. Slightly soluble in water; very slightly or partially soluble in ethanol

(-750 g/1) TS, ether R and hexane R.
Category. Keratoplastic agent.
Storage. Coal tar should be kept in a tightly closed container.
Additional information. Coal tar bums in air with a luminous sooty flame.
On exposure to air it hardens. Even in the absence of light, it is gradually
Requirements
Definition. Coal tar is a by-product usually obtained during the destructive
distillation of coal. It is a complex and undefined mixture of a great number of
chemical compounds. The product is available in various compositions.
Identity test
Carefully add 0.5g to 10ml of light petroleum R1 and allow to stand for 30
minutes; the supernatant liquid has a blue fluorescence in daylight and a more
intense fluorescence when viewed in ultraviolet light (365 nm).
PODOPHYLLI RESINA
PODOPHYLLUM RESIN
Chemical name. Podophyllum resin; CAS Reg. No. 8050-60-0.
Other name. Podophyllinum.
Description. Light brown to greenish yellow or brownish grey masses or an

amorphous powder.
757
Solubility. Practically insoluble in cold water; partially soluble in hot water,

and ether R; soluble in ethanol (~750g/l) TS.
Storage. Podophyllum resin should be kept in a tightly closed container, pro-

tected from light, and preferably stored at a temperature between 2 and 15 °C.
Labelling. The designation on the container should state the botanical source.
Additional information. CAUTION: Podophyllum resin must be handled

with care, avoiding contact with the skin and mucous membranes and inhala-
tion of airborne particles.
On exposure to light or to temperatures above 25 °C, it becomes darker in colour.
Requirements
Definition. Podophyllum resin is a mixture of resins obtained from the rhi-
zomes and roots of Podophyllum hexandrum Royle (P. emodi Wall.) or Podophyl-
lum peltatum L. after percolation with ethanol and precipitation from water or
very dilute acids.
Podophyllum resin contains not less than 40 0 % and not more than
. the equiv-
alent of 52 5 %
. of Podophyllum toxin (a and [3 peltatum), calculated with ref-
erence to the dried substance.
Identity tests
A. Dissolve 10 mg in 2 ml of ethanol (-750 g/1) TS and add 1 drop of ferric chlo-
ride (25g/l) TS; a deep, dark green colour is produced and the solution
appears black in reflected light.
B. Add 0.4 g, finely powdered, to 3 ml of ethanol (-535 g/1) TS, then add 0.5ml
of potassium hydroxide (1 mol/1) VS, shake gently, and allow to stand; the
resin of P. hexandrum produces a stiff jelly, whereas the resin of P. peltatum
does not gelatinize.
C. To two separate tubes add a few mg of Podophyllum resin, add potassium

hydroxide (1 mol/1) VS to one tube and sodium hydroxide (1 mol/1) VS to
the other, and allow to dissolve; a yellow solution is produced in both tubes
which becomes darker on standing. Add a few drops of hydrochloric acid

(-250 g/1) TS; a resin precipitates.
Matter insoluble in ethanol. Shake 1 g, finely powdered, with 20 ml of

ethanol (-750 g/1) TS for 5 minutes. Filter through a sintered-glass crucible
758
— R
(approx, porosity 40 pm), wash the filter with ethanol (-750 g/1) TS, and dry at
105 °C; the residue weighs not more than 25 mg.
Matter insoluble in ammonia. Shake powdered, with 30ml of

0.5 g, finely
ammonia (-100 g/1) TS for 30 minutes at about 20 °C.
Filter through a sintered-
glass crucible (approx, porosity 40 pm) and wash

filter with 30 ml the flask and
of water, the time taken for filtering and washing being not more than 10
minutes. Dry the filter with the residue to constant mass at 105 °C; the residue
from the resin of P. hexandrum weighs not less than 0.18 g and not more than
0.30 g, whereas the residue from the resin of P. peltatum weighs not more than
50 mg.
Loss on drying. Dry to constant mass at 1 05 °C; it loses not more than 50 mg/g.
Assay. Transfer about 0.45 g, finely powdered and accurately weighed, to a

glass-stoppered 50-ml flask, add 15 ml of chloroform R, and shake for 30
minutes. Filter, then transfer 10ml of the filtrate to a tared 100-ml flask, previ-
ously dried to constant mass at 70°C and containing 80 ml of light petroleum
R. Filter the precipitate through a tared sintered-glass crucible (approx, poros-
ity 40 pm), previously dried to constant mass at 70 °C, and wash the flask and
the filter with 20 ml of light petroleum R. Dry both of them at 70 °C for 1 hour,
cool, and weigh. Determine the mass of the residue, taking into account the
tare of the filter and the flask.
Each g of residue is equivalent to 0.30 g of Podophyllum resin.
POLYSORBATA 20, 60, 80

POLYSORBATES 20, 60, 80
HO— [CH2 — CHj— 0 ]„,^ JO— CH2 — CH2L — OH
H
O C — — CH2
[O CHîy — OH
H2C — — CH2—
[O CHjJ^ —
[Sum of w, X, y and z is 20 ;
R is H3C [CH2 ]-)q““C 02 ““ ,

H3C [^^2] 16 "
" CO2 )
or
H H
H3C — [CHJ, C
I
= C
I
[CHjh — CO2 1
759
Chemical names. Polysorbate 20: Polyoxyethylene 20 sorbitan monolaurate;

sorbitan monododecanoate, poly(oxy-l,2-ethanediyl) derivatives; CAS Reg.
No. 9005-64-5.
Polysorbate 60: Polyoxyethylene 20 sorbitan monostearate; sorbitan monooc-

tadecanoate, poly(oxy-l,2-ethanediyl) derivatives; CAS Reg. No. 9005-67-8.
Polysorbate 80: Polyoxyethylene 20 sorbitan monooleate; sorbitan mono[(Z)-9-

octadecenoate], poly(oxy-l,2-ethanediyl) derivatives; CAS Reg. No. 9005-65-6.
Description. Polysorbates 20 and 80 are yellowish or yellowish brown, oily

liquids. Polysorbate 60 is a gelatinous mass, and a clear liquid above 25 °C.
Solubility. Miscible with water, ethanol (~750g/l) TS, methanol R, and ethyl
acetate R; practically insoluble in fatty oils and in liquid paraffin R.
Category. Nonionic surfactant.
Storage. Polysorbates should be kept in tightly closed containers, protected

from light.
Additional information. Relative densities of Polysorbates 20 and 60 dfo =

about 1.10; Polysorbate 80 dfo = about 1.08.
Requirements
Definition. Polysorbates are mixtures of partial fatty acid esters of sorbitol and
its anhydrides, copolymerized with approximately 20 moles of ethylene oxide
for each mole of sorbitol and sorbitol anhydride.
In Polysorbate 20 the fatty acid is lauric acid which may contain other fatty
acids. In Polysorbate 60 the fatty acid is which may contain other
stearic acid
fatty acids, especially palmitic acid. In Polysorbate 80 the fatty acid is oleic acid.
Identity tests
A. A mixture of 6 volumes of Polysorbates and 4 volumes of water yields a
gelatinous mass at room temperature as well as at lower temperatures.
B. Dissolve O.lg in 5ml of chloroform R, and add O.lg of potassium thio-

cyanate R and 0.1 g of cobalt(II) nitrate R. Stir with a glass rod; the solution
becomes blue.
760
Hydroxyl value. Use Method B.
Polysorbate 20: 96-108.
Iodine value.
Polysorbates 20 and 60: not more than 5.0,
Saponification value.
Reducing impurities. Dissolve 2g in 25ml of hot water and add 25ml of sul-
furic acid (~100g/l) TS and 0.1 ml of ferroin TS. Titrate with ceric ammonium
nitrate (0.01 mol/1) VS, shaking continuously, until the colour change from red
to greenish blue persists for 30 seconds. Repeat the procedure without the
Polysorbates being examined and make any necessary corrections. Consump-
tion of ceric ammonium nitrate (0.01 mol/1) VS:
Polysorbates 20 and 60: not more than 2.0ml
Polysorbate 80: not more than 5.0ml.
Sulfated ash. Use 2.0g; not more than 2.5mg/g.

Fischer method, Method A, using 1 g; the water content is not more than
30mg/g.
761
POVIDONUM
POVIDONE
(CsH9NO)k
Chemical name. l-Vinyl-2-pyrrolidinone polymer; l-ethenyl-2-pyrrolidinone

homopolymer; CAS Reg. No. 9003-39-8.
Other name. Povidone, polyvinylpyrrolidone.
Description. A white to creamy white powder; odourless or almost odourless.
Solubility. Soluble in water, and ethanol (-750 g/1) TS; practically insoluble in
ether R.
Category. Tablet binder; tablet coating; viscosity-increasing agent.
Storage. Povidone should be kept in a tighdy closed container.
Labelling. The designation on the container of Povidone should state its
viscosity.
Additional information. Povidone is hygroscopic. It is not necessarily suit-
able for use as a plasma extender.
Requirements
Definition. Povidone consists of linear polymers of l-vinyl-2-pyrrolidinone
groups, the degree of polymerization of which results in polymers of various
molecular masses ranging from about 10000 to 700000.
Povidone contains not less than 11 5 . %

and not more than the equivalent of
12 8
. %
of nitrogen N, calculated with reference to the anhydrous substance.
Identity tests
A. Dissolve 0.5g in 5ml of water and add 10ml of hydrochloric acid (1 mol/1)
VS and 2 ml of potassium dichromate (100 g/1) TS; an orange-yellow pre-
cipitate is formed.
762
B. Dissolve 0.1 g in 1ml of water and add 0.2 ml of 4-dimethylaminoben-

zaldehyde TS6 and 0.1ml of sulfuric acid (-1760 g/1) TS; a pink colour is
produced.

Fischer method, Method A, using 0.5 g; the water content is not more than
50mg/g.
Aldehydes. To lOg add 180ml of sulfuric acid (-440 g/1) TS and boil under a
reflux condenser for 45 minutes. Cool, reassemble the apparatus, distil, and
collect lOOml of distillate in a flask that is placed in an ice-bath and contains
20ml of hydroxylamine hydrochloride (70 g/1) TS previously adjusted to pH 3.1.
Titrate the distillate with sodium hydroxide (0.1 mol/1) VS to pH 3.1. Repeat
the procedure without the Povidone being examined and make any necessary
corrections. Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to
4.405 mg of aldehyde, calculated as acetaldehyde. Not more than 4.6ml of
sodium hydroxide (0.1 mol/1) VS is required (2.0mg/g).
Vinylpyrrolidinone. Dissolve 10 g in 80 ml of water, add 1 g of sodium acetate

R and titrate with iodine (0.05 mol/1) VS until a permanent colour is obtained.
Add an excess of 3.0 ml of iodine (0.05mol/l) VS, allow to stand for 10 minutes,
and titrate the excess iodine with sodium thiosulfate (0.1 mol/1) VS, adding
starch TS towards the end of the titration. Repeat the procedure without the
Povidone being examined and make any necessary corrections. Not more than
3.6ml of iodine (0.05mol/l) VS is required (2.0mg/g).

acid (-1760g/l) TS. Carefully add 1 ml of hydrogen peroxide (-330 g/1) TS down
the wall of the flask, and heat. Repeat this addition three to six times, and con-
tinue to heat until a slightly green and clear solution is produced. Proceed with
the distillation as described. Repeat the procedure without the Povidone being
examined and make any necessary corrections.
Each ml of sulfuric acid (0.05 mol/1) VS is equivalent to 1.401 mg of N.
763
PRAZIQUANTELUM
PRAZIQUANTEL
Graphic formula.
Chemical name. 2-(Cyclohexylcarbonyl)-l,2,3,6,7, llb-hexahydro-4ff-

pyrazino-[2,l-«]isoquinolin-4-one; CAS Reg. No. 55268-74-1.

Solubility. Very slightly soluble in water; freely soluble in ethanol (-750 g/1)
TS.
Storage. Praziquantel should be kept in a well-closed container, protected

from light.
Requirements
Definition. Praziquantel contains not less than 98.5% and not more than
Identity tests

infrared region.
spectrum obtained from praziquantel RS or with the reference spectrum of
praziquantel.
764
B. See the test below under “Related substances”. The principal spot obtained
Loss on drying. Dry at 50 °C under reduced pressure (not exceeding O.bkPa

volumes of toluene R and 15 volumes of methanol R as the mobile phase. Use
an unlined chamber. Apply separately to the plate, in a current of nitrogen R,
10 pi of each of 2 solutions in chloroform R containing (A) 50 mg of the test sub-
stance per ml and (B) 50 mg of praziquantel RS per ml; further apply 2 pi of each
of 2 solutions in chloroform R containing (C) 0.5 mg of praziquantel RS per ml
and (D) 1.0 mg of praziquantel RS per ml. Allow the mobile phase to ascend
7 cm. After removing the plate from the chromatographic chamber, allow it to
dry in a current of warm air, chamber with iodine vapours
place the plate in a
and allow to stand for 20 minutes. Examine the chromatogram immediately in
daylight. Any spot obtained with solution A, other than the principal spot, is not
more intense than that obtained with solution C, except one spot above the
main spot which is not more intense than that obtained with solution D.
Assay. Dissolve about 0.04 g, accurately weighed, in sufficient ethanol

(-750 g/1) TS to produce 100ml. Measure the absorbance of a 1-cm layer at the
maximum at about 265 nm. Calculate the amount of C]9H24N202 in the sub-
stance being tested by comparison with praziquantel RS, similarly and con-
currently examined.
PREDNISOLONI ACETAS
PREDNISOLONE ACETATE
765
Graphic formula.
Chemical name. ll(3,17,21-Trihydroxypregna-l,4-diene-3,20-dione 21-acetate;

21-(acetyloxy)-lip,17-dihydroxypregna-l,4-diene-3,20-dione; CAS Reg. No.
52-21-1.

TS, and acetone R.
Storage. Prednisolone acetate should be kept in a well-closed container, pro-

tected from light.
Additional information. Prednisolone acetate has a melting temperature of

about 235 °C with decomposition.
Requirements
Definition. Prednisolone acetate contains not less than 96.0% and not more
than 104.0% of C 23 H 30 O 5 ,
Identity tests

infrared region.
spectrum obtained from prednisolone acetate RS or with the reference spec-
trum of prednisolone acetate.

766
allow it to stand at room temperature until the solvents have completely

evaporated. Use the impregnated plate within 2 hours, carrying out the
chromatography in the same direction as the impregnation. Use 75 volumes
of toluene R and 25 volumes of chloroform R as the mobile phase. Apply
separately to the plate 2 pi of each of 2 solutions in a mixture of 9 volumes
of chloroform R and 1 volume of methanol R containing (A) 2.5 mg of the
test substance per ml and (B) 2.5 mg of prednisolone acetate RS per ml.
Develop the plate for a distance of 15 cm. After removing the plate from the
chromatographic chamber, allow it to dry in air until the solvents have evap-
orated, heat it at 120 °C for 15 minutes, spray it with a mixture of 20ml of
sulfuric acid (~190g/l) TS and 80ml of ethanol (-750 g/1) TS, and then heat
it at 120 °C for 10 minutes. Allow it to cool and examine the chromatogram
in daylight and in ultraviolet light (365 nm). The principal spot obtained with
C. To 0.05g add 2ml of ethanol (~750g/l) TS and 2ml of sulfuric acid

(-700 g/1) TS and boil gently for 1 minute; ethyl acetate, perceptible by its
odour (proceed with caution), is produced.
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; = -1-112

to -H 119°.
lOmg/g.
allow it to dry in air until the solvents have evaporated, then heat it at 105°C
for 10 minutes. Allow it to cool, spray it with blue tetrazolium/sodium hydrox-
ide TS, and examine the chromatogram in daylight. Any spot obtained with
with solution B.

produce 100 ml; dilute 5.0 ml of this solution to 100 ml with the same solvent.
Measure the absorbance of a Tcm layer of the diluted solution at the maximum
at about 243 nm. Calculate the amount of C 23 H 3 oO<; in the substance being
tested by comparison with prednisolone acetate RS, similarly and concurrently
examined. In an adequately calibrated spectrophotometer the absorbance of
the reference solution should be 0.37 + 0.02.
767
PREDNISOLONI NATRII PHOSPHAS

PREDNISOLONE SODIUM PHOSPHATE
o
II
^y'ONa
ONa
C2iH27Na20sP
Chemical name. lip,17,21-Trihydroxypregna-l,4-diene-3,20-dione 21-(dis-

odium phosphate); (IIP) 1 l,17-dihydroxy-21-(phosphonooxy)-pregna-l,4-
diene-3,20-dione disodium salt; CAS Reg. No. 125-02-0.
Description. A white to light yellow, crystalline powder or granules.
Solubility. Freely soluble in water; soluble in methanol R; very slightly soluble

in ethanol (-750 g/1) TS and acetone R.
Category. Corticosteroid.
Storage. Prednisolone sodium phosphate should be kept in a tightly closed

Labelling. The designation Prednisolone sodium phosphate for sterile nonin-

jectable use indicates that the substance complies with the additional require-
ment and may be used for sterile applications. Expiry date.
Additional information. Prednisolone sodium phosphate is hygroscopic.
Requirements
Prednisolone sodium phosphate contains not less than 96 0 . % and not more
than 103 0. % of C2iH27Na208P, calculated with reference to the anhydrous
substance.
Identity tests
• Either tests A, D, and E or tests B, C, D, and E may be applied.
768
=

spectrum obtained from prednisolone sodium phosphate RS or with the ref-
erence spectrum of prednisolone sodium phosphate.

using silica gel R1 and a freshly prepared mixture
of 3 volumes of 1 volume of acetic anhydride R, and 1 volume
-butanol R, 1
of water as the mobile phase. Apply separately to the plate 2|tl of each of
4 solutions in methanol R containing (A) 2.5mg of Prednisolone sodium
phosphate per ml, (B) 2.5 mg of prednisolone sodium phosphate RS per ml,
(C) a mixture of equal volumes of solutions A and B, and (D) equal volumes
of solution A and a solution of 2.5mg of dexamethasone sodium phosphate
RS per ml of methanol R. After removing the plate from the chromato-
graphic chamber, allow it to dry in air until the solvents have evaporated,
spray with a mixture of 10ml of sulfuric acid (~1760g/l) TS and 90ml of
ethanol (-750 g/1) TS, heat at 120 °C for 10 minutes, allow to cool, and

ance, and intensity with that obtained with solution B. The principal spot
obtained with solution C appears as a single compact spot, whereas the
chromatogram obtained with solution D shows two spots which may not
be completely separated.
C. To about 2 mg add 2 ml of sulfuric acid (-1760 g/1) TS and shake to dissolve;

an intense red colour is produced within 5 minutes. Examine under ultravi-
olet light (365 nm); a reddish brown fluorescence is observed. Add the solu-
tion to 10 ml of water and mix; the colour fades and a greenish yellow
fluorescence is produced.

20mg/ml solution.
E. To 1 ml of a 20mg/ml solution add 3ml of nitric acid (-130 g/1) TS; it yields
of orthophosphates.
ence to the anhydrous substance; [a]™ -i-95° to -i-102°.

free water R is clear and not more intensely coloured than standard colour Bnl
when compared as described under 1.11 Colour of liquids.
769

Fischer method, Method A, using about 0.2 g of the substance; not more than
0.080g/g.
Inorganic phosphates. Dissolve 0.050g in sufficient water to produce 100 ml.

To 10 ml add 5ml of ammonium molybdate/vanadate TS, mix and allow to
stand for 5 minutes; any yellow colour produced is not more intense than that
of a reference solution prepared similarly using 10 ml of phosphate standard
(5pg/ml) TS.
Free prednisolone and other related substances

matography, using a stainless steel column (15cm x 4.6mm) packed with
cally bonded octadecylsilyl groups (5 pm). As the mobile phase, use a
mixture prepared as follows: weigh 1.36g of potassium dihydrogen phos-
phate R and 0.60 g of hexylamine R, transfer to a 250-ml conical flask, mix,
and allow to stand for 10 minutes, and then dissolve in 185ml of water. Add
65 ml of acetonitrile R, mix, and filter.
Prepare the following solutions in the mobile phase: solution (A) 2.5 mg of
Prednisolone sodium phosphate per ml; solution (B) 2.5 mg of prednisolone
sodium phosphate RS and 2.5mg of prednisolone RS per ml, dilute 1.0ml
of this solution to 25ml with the mobile phase; and for solution (C) dilute
1.0 ml of solution A to 50 ml with the mobile phase.
Operate with a flow rate of 1.0 ml per minute. As a detector use an ultra-
violetspectrophotometer set at a wavelength of 254 nm.
Inject 20 pi of solution B. Adjust the sensitivity of the system so that the

heights of the principal peaks in the chromatogram obtained with solution
B are 70-90% of the full scale of the recorder. The retention times for pred-
nisolone sodium phosphate are about 6.5 minutes, and for prednisolone
about 8.5 minutes. The test is not valid unless the resolution between the
peaks corresponding to prednisolone sodium phosphate and prednisolone
is not less than 4.5.
Inject alternately 20 pi each of solutions A and C. Continue the chro-

matography for 3 times the retention time of the principal peak.
770

from solutions A and C, and calculate the contents of free prednisolone and
other related substances as a percentage. In the chromatogram obtained
from solution A, the area of any peak, other than the principal peak, is not
greater than that of the principal peak obtained with solution C (2.0%), and
not more than one such peak has an area greater than half the area of the
principal peak obtained with solution C (1.0%). The sum of the areas of all
the peaks, other than the principal peak, is not greater than 1.5 times the
area of the principal peak obtained with solution C (3.0%). Disregard any
peak due to the solvent and any peak with an area less than 0.025 times the
area of the principal peak obtained with solution C.

using silica gel R1 as the coating substance and methanol R as the mobile
phase. Apply separately to the plate 2 pi of each of 2 solutions in methanol
R containing (A) 10 mg of Prednisolone sodium phosphate per ml, and (B)
0.20 mg of prednisolone RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in air for 5 minutes, spray with a solu-
tion of 3g of zinc chloride R in 10 ml of methanol R, heat at about 125°C
for 1 hour, and examine the chromatogram in ultraviolet light (365 nm).
intense than that obtained with solution B (2.0%).

100 ml. Dilute 5 ml to 250 ml with water and measure the absorbance of this
solution in a 1-cm layer at the maximum at about 247 nm.
Calculate the percentage content of C2iH27Na20sP using the absorptivity value
of 31.2 (A!L = 312).
Additional requirement for sterile non-injectahle Prednisolone

sodium p>ltosp>hate
PREDNISOLONUM
PREDNISOLONE
771
Graphic formula.
Chemical name. lip,17,21-Trihydroxypregna-l,4-diene-3,20-dione; CAS

Reg. No. 50-24-8.
Solubility. Soluble in 1300 parts of water and in 30 parts of dehydrated ethanol

R; soluble in methanol R and dioxan R.
Storage. Prednisolone should be kept in a tighdy closed container, protected

from light.
Additional information. Prednisolone is hygroscopic; it has a melting tem-

perature of about 230 °C with decomposition.
Requirements
Definition. Prednisolone contains not less than 97.0% and not more than
Identity tests

infrared region.
spectrum obtained from prednisolone RS or with the reference spectrum of
prednisolone.

has reached a height of at least 16 cm, remove the plate from the chro-
772
=

nation. Use chloroform R as the mobile phase. Apply separately to the plate
2 [tl R and 1
of each of 2 solutions in a mixture of 9 volumes of chloroform
volume of methanol R containing (A) 2.5 mg of the test substance per ml
and (B) 2.5mg of prednisolone RS per ml. Develop the plate for a distance
of 15 cm. After removing the plate from the chromatographic chamber,
allow it to dry in air until the solvents have evaporated, heat at 120 °C for
15 minutes, spray with sulfuric acid/ethanol TS, and then heat at 120 °C for
in ultraviolet light (365 nm). The principal spot obtained with solution A
corresponds in position, appearance, and intensity with that obtained with
solution B.
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; [a]™ -t-96
to -H 103°.
lOmg/g.
evaporated and heat at 105 °C for 10 minutes; cool, and examine the chro-
matogram in ultraviolet light (254 nm). Any spot obtained with solution A,
tion B.

at the maximum at about 242 nm. Calculate the amount of C21H28O5 in the
substance being tested by comparison with prednisolone RS, similarly and
773
PRIMAQUINI DIPHOSPHAS
PRIMAQUINE DIPHOSPHATE
Molecular formula. Ci 5 H 2 iN 30 ,
2 H 3 P 04 .
Graphic formula.
Chemical name. 8-[(4-Amino-l-methylbutyl)amino]-6-methoxyquinoline

phosphate (1:2); N''-(6-methoxy-8-quinolinyl)-l,4-pentanediamine phosphate
(1:2); CAS Reg. No. 63-45-6.
Description. An orange-red, crystalline powder; odourless or almost

odourless.
Solubility. Soluble in water; practically insoluble in ethanol (-750 g/1) TS, and
ether R.
Storage. Primaquine diphosphate should be kept in a well-closed container,

Requirements
Definition. Primaquine diphosphate contains not less than 98.0% and not
more than 102.0% of C] 5 H 2 iN 30 ,
2 H 3 P 04 calculated with reference to the dried
,
substance.
Identity tests

infrared region.
spectrum obtained from primaquine diphosphate RS or with the reference
spectrum of primaquine diphospate.
774
B. Dissolve lOmg in 5ml of water and add 1ml of ceric ammonium

sulfate/nitric acid TS; a deep violet colour is immediately produced (dis-
tinction from chloroquine).
C. To 1 ml of a 20mg/ml solution add 3ml of nitric acid (-130 g/1) TS; it yields
of orthophosphates.
lOmg/g.
3 volumes of dimethylamine/ethanolTS, 4 volumes of acetone Rand 5 volumes
of chloroform R as the mobile phase. To 5 ml of a solution containing 20 mg of
the test substance per ml add 5 ml of chloroform R and 0.5ml of ammonia
(-35 g/1) TS and shake. Separate the chloroform layer, filter and apply 5p.l of
this solution to the plate. After removing the plate from the chromatographic
light (365 nm). Only a single fluorescent spot is obtained.
0.9 g, accurately weighed, and 50ml of hydrochloric acid (-70 g/1) TS. Each ml
of sodium nitrite (0.1 mol/1) VS is equivalent to 45.53 mg of Ci 5 H 2 iN 30 2 H 3 P 04 ,
.
PROBENECIDUM
PROBENECID
Molecular formula. C13H19NO4S
Graphic formula.
(CHjCH^CHjljNSOj ^— COOH
—
Chemical name. /?-(Dipropylsulfamoyl)benzoic acid; 4-[(dipropylamino)sul-
fonyl]benzoic acid; CAS Reg. No. 57-66-9.
775

(-750 g/1) TS and in 12 parts of acetone R; soluble in dilute solutions of alkali
hydroxides.
Storage. Probenecid should be kept in a well-closed container.
Requirements
Definition. Probenecid contains not less than 98.0% and not more than
101.0% of C13H19NO4S, calculated with reference to the dried substance.
Identity tests

spectrum obtained from probenecid RS or with the reference spectrum of
probenecid.
obtained with solution A (first application) corresponds in position, appear-
ance and intensity with that obtained with solution B.
5-Omg/g.
Acidity. Heat 2.0g with 100ml of carbon-dioxide-free water R on a water-bath

for 30 minutes, cool and filter. Add 0.15ml of phenolphthalein/ethanol TS to the
filtrate and titrate with carbonate-free sodium hydroxide (0.1 mol/1) VS; not more
than 0.5 ml of the titrant is required to obtain the midpoint of the indicator.
776
of 15 volumes of 1-propanol R and 3 volumes of ammonia (~17g/l) TS as the

mobile phase. Prepare the 3 following solutions in a mixture of 1 volume of
ammonia (~17g/l) TS and 9 volumes of ethanol (~750g/l) TS containing (A)
lOmg of the test substance per ml, (B) lOmg of probenecid RS per ml, and (C)
0.050 mg of the test substance per ml. Apply to the plate 1 pi of each of solu-
tions A and B. Apply separately 20 pi of each of solutions A and C. After remov-
with solution A (second application), other than the principal spot, is not more
intense than that obtained with solution C.
Assay. Dissolve about l.Og, accurately weighed, in 50ml of neutralized

ethanol TS and titrate with carbonate-free sodium hydroxide (0.1 mol/1) VS,
using bromothymol blue/ethanol TS as indicator. Repeat the operation without
the substance being examined and make any necessary corrections. Each ml of
C, 3 H«N 04 S.
PROCAINAMIDI HYDRO CHLORIDUM

PROCAINAMIDE HYDROCHLORIDE
Molecular formula. Ci 3 H 2 iN 30 ,HCl
Graphic formula.
o
II
HjN CNHICHjljNICjHjlj HCI
Chemical name. /7-Amino-N-[2-(diethylamino)ethyl]benzamide monohy-

drochloride; 4-amino-N-[2-(diethylamino)ethyl]benzamide monohydrochlo-
Description. A white to yellowish white, crystalline powder; odourless.
Solubility. Very soluble in water; freely soluble in ethanol (-750 g/1) TS; very
Category. Antiarrhythmic.
777
Storage. Procainamide hydrochloride should be kept in a tighdy closed con-

Additional information. Procainamide hydrochloride is hygroscopic. Even in

the absence of light, Procainamide hydrochloride is gradually degraded on
temperatures.
Requirements
Definition. Procainamide hydrochloride contains not less than 98.0% and not
substance.
Identity tests
A. Dissolve 1 g in 10 ml of water, add 10 ml of sodium hydroxide (-200 g/1) TS,
and extract with 10 ml of chloroform R. To the extract add 10 ml of toluene

R, dry over anhydrous sodium sulfate R, and filter. Mix the filtrate with
5 ml of anhydrous pyridine R, add 1 ml of benzoyl chloride R drop by drop,
heat on a water-bath for 30 minutes, and pour into a mixture of 50ml of
water and 50 ml of sodium hydroxide (-200 g/1) TS. Extract with 10 ml of
ether R, wash the extract with 20ml of water, dilute with 30ml of ether R,
and allow to crystallize. Recrystallize from ethanol (-375 g/1) TS; melting
temperature, about 185 °C (benzoyl procainamide).
B. Dissolve 0.1 g in 2ml of water and add 2ml of potassium ferrocyanide

(45 g/1) TS. Add a few drops of hydrochloric acid (-70g/l) TS to acidify
slightly and heat; a light green precipitate is produced.
metals content according to Method A; not more than 20p,g/g.
Loss on drying. Dry to constant weight at 105°C: it loses not more than
3-Omg/g.
778
4 volumes of 1 -butanol R, 1 volume of glacial acetic acid R, and 2 volumes of
tions in ethanol (~750g/l) TS containing (A) 50mg of the test substance per ml
and (B) 0.25 mg of the test substance per ml. After removing the plate from the
chromatographic chamber, allow it dry in air, and examine the chromatogram

R and 15ml of glacial acetic acid Rl. Heat the solution until boiling. Add 20ml
of dioxan R and 20 ml of mercuric acetate/acetic acid TS and titrate with per-
of Ci 3 H 2 iN 30 ,HC 1 .
Additional requirements for Procainamide hydrochloride for

parenteral use
PROCAINI BENZYLPENICILLINUM
PROCAINE BENZYLPENICILLIN
Procaine ben^lpenicillin (non-injectable)
Proccdne benzylpenicillin, sterile
Molecular formula. CmHi 8N 204 S,Ci 3 H 2 oN 202 ,H 20
Graphic formula.
779
Chemical name. 2-(Diethylamino)ethyl /^-aminobenzoate compound with

(2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-l-azabicyclo-
[3,2.0]heptane-2-carboxylic acid (1 :
1) monohydrate; 2-(diethylamino)ethyl
4-aminobenzoate compound with [2S-(2a,5a,6|3)]-3,3-dimethyl-7-oxo-6-
[(phenyl-acetyl)amino]-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid
(1:1) monohydrate; CAS Reg. No. 6130-64-9 (monohydrate).
Other names. Penicillin G procaine, Procaine penicillin.

almost odourless.
Solubility. Slightly soluble in water; soluble in ethanol (-750 g/1) TS.
Storage. Procaine benzylpenicillin should be kept in a tightly closed container

and protected from light.
Labelling. The designation sterile Procaine benzylpenicillin indicates that the

substance complies with the additional requirements for sterile Procaine ben-
zylpenicillin and may be used for parenteral administration or for other sterile
applications.
Additional information. Solutions of Procaine benzylpenicillin are dextroro-

tatory. It is readily decomposed by acids, alkalis, and oxidizing agents. Even in
Requirements
Definition. Procaine benzylpenicillin contains not less than 96.0% and not
more than 100.5% of total penicillins calculated as Ci 5HigN 204 S,Ci 3H 2 oN 202
and not less than 38.5% and not more than 41.5% of C]3H2oN202, both calcu-
lated with reference to the anhydrous substance.
Identity tests
A. To 2 mg add 0.05ml of water followed by 2 ml of sulfuric acid
in a test-tube
(-1760 g/1) TS and mix; the solution is almost colourless. Immerse the test-
tube for minute in a water-bath; the solution remains almost colourless.

1
Place 2 mg in a second test-tube, add 1 drop of water and 2 ml of formalde-

hyde/sulfuric acid TS and mix; the solution is almost colourless, but after a
few minutes the colour changes to yellow-brown. Immerse the test-tube for
1 minute in a water-bath; a reddish brown colour is produced.
780
B. Dissolve lOmg 10ml of water and add 0.5ml of neutral red/ethanol TS.
in
Add sufficient sodium hydroxide (0.01 mol/1) VS to give a permanent orange
colour and then add 1.0 ml of penicillinase TS; the colour changes rapidly
to red.
aromatic amines under 2.1 General identification tests, producing a bright,
orange-red precipitate.

pH value. pH of a saturated solution containing about 3.0 g in 10 ml of carbon-

dioxide-free water R, 5. 0-7. 5.
Assay
For total penicillins. Dissolve about 0.045 g, accurately weighed, in sufficient
water to produce 1000 ml. Transfer two 2.0-ml aliquots of this solution into
separate stoppered tubes. To one tube add 10.0ml of imidazole/mercuric chlo-
ride TS, mix, stopper the tube and place in a water-bath at 60 °C for exactly 25
10.0ml of water and mix (solution B).

about314nm against a solvent cell containing a mixture of 2.0ml of water and
tion B.

the difference
B, calculate the amount of Ci6HisN204S,C 13 H 20N 2 O 2 in the substance being
tested by comparison with 0.050 g of benzylpenicillin sodium RS similarly
and concurrently examined, taking into account that each mg of benzyl-
penicillin sodium RS (Ci 6 Hi 7 N 2 Na 04 S) is equivalent to 1.601 mg of
Ci 6 Hi 8 N 204 S,Ci 3 H 2 oN 202 In 3 n adequately calibrated spectrophotometer the
.
For procaine. Dissolve about 0.5 g, accurately weighed, in 10 ml of water, add

5 ml of sodium carbonate (75g/l) TS and extract with four successive quantities,
each of 25ml of chloroform R, filter the chloroform extracts, and evaporate to
a small volume on a water-bath. Add 20.0ml of hydrochloric acid (0.1 mol/1)
VS and distil off the remaining chloroform. Cool, add 0.25ml of methyl
red/ethanol TS and titrate with sodium hydroxide (0.1 mol/1) VS. Each ml of
hydrochloric acid (0.1 mol/1) VS is equivalent to 23.63 mg of C 13H 20N 2 O 2 .
781
The International Pharniacoiaoeia
Additional requirements for Procaine Benzytf'enicittin for

sterile use
benzylpenicillin.

the membrane procedure with added penicillinase TS or the direct
filtration test
test procedure. Use the sampling plan described under 3.2.1 Test for sterility of
non-injectable preparations.
PROCAINI HYDROCHLORIDUM
PROCAINE HYDROCHLORIDE
Molecular formula. Ci3H2oN202,HCl
Graphic formula.
0
HjN
^ ^
COICHjljNICjHgij . HCI
Chemical name. 2-(Diethylamino)ethyl p-aminobenzoate monohydrochlo-

ride; 2-(diethylamino)ethyl 4-aminobenzoate monohydrochloride; CAS Reg.
No. 51-05-8.
Solubility. Soluble in 1 part of water and in 25 parts of ethanol (-750 g/1) TS;
Storage. Procaine hydrochloride should be kept in a tightly closed container,

Additional information. Procaine hydrochloride causes local numbness after

being placed on the tongue. Even in the absence of light. Procaine hydrochlo-
782
Requirements
Definition. Procaine hydrochloride contains not less than 99.0% and not
more than 101.0% of Ci 3 H 2 oN 202 ,HCl, calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from procaine hydrochloride RS or with the reference
spectrum of procaine hydrochloride.
B. About 0.05 g yields the reaction described for the identification of primary
aromatic amines under 2.1 General identification tests, producing a vivid red
precipitate.
C. Dissolve 0.05g in 5ml of water, add 5 drops of sulfuric acid (~100g/l) TS

and 2 drops of potassium permanganate (0.02 mol/1) VS; the violet colour
produced disappears quickly.
metals content according to Method A; not more than 20|o.g/g.

lOmg/g.
80 volumes of dibutyl ether R, 16 volumes of hexane R, and 4 volumes of glacial
acetic acid R as the mobile phase. Apply separately to the plate 5 pi of each of
2 solutions containing (A) 0.10 g of the test substance per ml and (B) 0.050 mg
783
of 4-aminobenzoic acid R per ml. After removing the plate from the chro-
matographic chamber, allow it to dry at 105 °C for 10 minutes, and examine
with solution B. The principal spot remains at the point of application.
Assay. Carry out the assay as described under 2.7 Nitrite titration; dissolve
about 0.5 g, accurately weighed, in 50 ml of hydrochloric acid (~70g/l) TS,
add 0.1 g of potassium bromide R, and titrate with sodium nitrite (0.1 mol/1)
VS. Each ml of sodium nitrite (0.1 mol/1) VS is equivalent to 27.28mg of
C,3H2„N202,HC1.
PROCARBAZINI HYDROCHLORIDUM
PROCARBAZINE HYDROCHLORIDE
Molecular formula. Ci 2 Hi 9 N 30 ,HCl
Graphic formula.
(CHjjjCHNHC
II
—C A CHjNHNHCHj HCI
Chemical name. N-lsopropyl-a-(2-methylhydrazino)-/^-toluamide monohy-

drochloride; N-(l-methylethyl)-4-[(2-methylhydrazino)methyl]benzamide mono-
hydrochloride; CAS Reg. No. 366-70-1.
Description. A white to yellowish, crystalline powder.
Solubility. Soluble in water and methanol R; sparingly soluble in ethanol

Storage. Procarbazine hydrochloride should be kept in a tightly closed con-

Additional information. Procarbazine hydrochloride melts at about 223 °C

with decomposition. Even in the absence of light. Procarbazine hydrochloride
being faster at higher temperatures. CAUTION: Procarbazine hydrochloride
784
must be handled with care, avoiding contact with the skin and inhalation of
airborne particles. Wear rubber gloves while handling this substance.
Requirements
Definition. Procarbazine hydrochloride contains not less than 98.5% and not
more than 100.5% of Ci 2 Hi 9 N 30 ,HCl, calculated with reference to the dried
substance.
Identity tests
infrared region.
spectrum obtained from procarbazine hydrochloride RS or with the refer-
ence spectrum of procarbazine hydrochloride.
B. A O.lOg/ml solution yields reaction B described under 2.1 General identifi-

5-Omg/g.
Assay. Dissolve about 0.125 g, accurately weighed, in a mixture of 5 ml of

formic acid (~1080g/l) TS and 20ml of glacial acetic acid Rl, add 5 ml of mer-
curic acetate/acetic acid TS, and titrate with perchloric acid (0.1 mol/1) VS as
described under 2.6 Non-aqueous titration, Method A. Each ml of perchloric
acid (0.1 mol/1) VS is equivalent to 25.78mg of Ci 2 Hi 9 N 30 ,HCl.
PROGESTERONUM
PROGESTERONE
785
Graphic formula.
COCH,
—H
Chemical name. Pregn-4-ene-3,20-dione; CAS Reg. No. 57-83-0.
Description. Colourless crystals or a white to slightly yellowish white, crys-


(-750 g/1) TS.
Storage. Progesterone should be kept in a well-closed container, protected

from light.
Additional information. Progesterone may exist in 2 polymorphic forms, one

of which melts at about 130 °C, the other at about 121 °C.
Requirements
Definition. Progesterone contains not less than 97.0% and not more than
Identity tests

spectrum obtained from progesterone RS or with the reference spectrum of
progesterone. If the spectrum obtained from the solid state of the test sub-
stance is not concordant with the spectrum obtained from the reference sub-
stance, compare the spectra using solutions in chloroform R containing
30mg/ml and a path length of 0.2mm.

of propylene glycol R and 90 volumes of acetone R to impregnate the plate.
786
dipping about 5
it mm beneath the surface of the liquid. After the solvent
has reached the height of at least 16 cm, remove the plate from the chro-
nation. As the mobile phase, use a mixture of 50 volumes of cyclohexane
R and 50 volumes of light petroleum R. Apply separately to the plate 5 pi
of each of 2 solutions in a mixture of 9 volumes of chloroform R and 1
volume of methanol R containing (A) 1.0 mg of the test substance per ml
and (B) 1.0 mg of progesterone RS per ml. Develop the plate for a distance
of 15 cm. After removing the plate from the chromatographic chamber allow
it to dry in air until the solvents have evaporated, heat at 120 °C for 15
minutes, spray with 4-toluenesulfonic acid/ethanol TS, and then heat at

120 °C for 10 minutes. Allow to cool and examine the chromatogram in day-
light and in ultraviolet light (365 nm). The principal spot obtained with solu-
tion A corresponds in position, appearance, and intensity with that obtained
with solution B.

[a]g>"^ = -Hl86to-)-196°.
5.0mg/g.
2 parts of chloroform R and 1 part of ethyl acetate R as the mobile phase. Apply
separately to the plate 10 pi of each of 2 solutions in a mixture of 1 volume of
ethanol (-750g/l) TS and 1 volume of chloroform R containing (A) lOmg of the
test substance per ml and (B) O.lOmg of the test substance per ml. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air until the
solvents have evaporated, and examine the chromatogram in ultraviolet light

at about 240 nm. Calculate the amount of C21H30O2 in the substance being
tested by comparison with progesterone RS, similarly and concurrently exam-
erence solution should be 0.54 ± 0.03.
787
PROGUANILI HYDRO CHLORIDUM

PROGUANIL HYDROCHLORIDE
C„Hi6C1N5,HC1
Cl
CH, NH NH
,
HCI
H,C
Relative molecular mass. 290.2.
Chemical name. l-(/^-Chlorophenyl)-5-isopropylbiguanide hydrochloride;

CAS Reg. No. 637-32-1.
Solubility. Slightly soluble in water, more soluble in hot water; sparingly

soluble in ethanol (-750 g/1) TS.
Storage. Proguanil hydrochloride should be kept in a well-closed container,

Requirements
Proguanil hydrochloride contains not less than 99 0 % and not more than .
101 0 % of CiiHi 6 C 1 N 5 ,HC 1 calculated with reference to the dried substance.

.
,
Identity tests

infrared region. The is concordant with the
infrared absorption spectrum
spectrum obtained from proguanil hydrochloride RS or with the reference
spectrum of proguanil hydrochloride.
B. Dissolve about 0.1 g in 10ml of water, add 5 drops of potassium ferro-

cyanide (45g/l) TS; a white precipitate is produced. Add 10-15 drops of
nitric acid (-130 g/1) TS; the precipitate dissolves.
C. Dissolve about 0.1 g in 10ml of water, add 3 drops of copper(ll) sulfate

(160 g/1) TS and 1.0ml of ammonia (-100 g/1) TS, shake, add 5ml of toluene
R, and shake again; a violet colour is produced in the toluene layer.
788

Acidity or alkalinity. To 35 ml of water maintained at a temperature of about

65 °C add 0.20 ml of methyl red/methylthioninium chloride TS, neutralize with
sodium hydroxide (0.01 mol/1) VS or hydrochloric acid (0.01 mol/1) VS, add
0.4 g of Proguanil hydrochloride, and stir until dissolved; the resulting solution
is not acidic and requires for neutralization not more than 0.2 ml of hydrochlo-
ric acid (0.01 mol/1) VS.
Chloraniline. For solution A, dissolve 0.10 g in 1ml of hydrochloric acid

(~70g/l) TS, and add sufficient water to produce 20ml. Cool to 5°C, add 1ml
of sodium nitrite (35g/l) TS, allow to stand at 5°C for 5 minutes, add 2 ml of
ammonium sulfamate (50g/l) TS, shake, and allow to stand for 10 minutes. Add
2ml of freshly prepared W-(l-naphthyl)ethylenediamine hydrochloride (1 g/1)
TS, dilute to 50ml with water, and allow to stand for 30 minutes. For solution
B, treat similarly 20 ml of a solution containing 1.25 pg of chloraniline R per ml.
Any magenta colour produced in solution A is not more intense than that
produced in solution B (250pg/g).
5.0 mm) packed with particles of silica gel, the surface of which has been mod-
ified with chemically bonded octadecylsilyl groups (5 pm). As the mobile phase,
use a solution prepared as follows: dissolve 1.88g of sodium hexanesulfonate

R in 1000 ml of a mixture of 12 volumes of methanol R, 8 volumes of water,
and 0.1 volume of glacial acetic acid R.
Prepare the following solutions in the mobile phase: solution (A) 1.0 pg of
Proguanil hydrochloride per ml; and solution (B) O.lOmg of Proguanil
hydrochloride per ml.
solutions A and B, and calculate the content of the related substances as a per-
centage. The sum of the areas of any peaks, other than the principal peak, in
the chromatogram obtained from solution B is not greater than that of the prin-
cipal peak obtained with solution A (1%).
789
(0.1 mol/1) VS as described under 2.6 Non-aqueous titration, Method A, deter-
mining the end-point potentiometrically.

C„Hi5C1N5,HC1.
PROMETHAZINI HYDROCHLORIDUM
PROMETHAZINE HYDROCHLORIDE
Molecular formula. Ci7H2oN2S,HCl
Graphic formula.
Chemical name. 10-[2-(Dimethylamino)propyl]phenothiazine monohydrochlo-

ride; A/A/a-trimethyl-10f/-phenothiazine-10-ethanamine monohydrochloride;
CAS Reg. No. 58-33-3.
Other name. Diprazinum.
Description. A white or faintly yellowish, crystalline powder; odourless or

almost odourless.
Category. Antiemetic drug.
Storage. Promethazine hydrochloride should be kept in a tightly closed con-

Additional information. On prolonged exposure to air. Promethazine

hydrochloride slowly oxidizes and acquires a blue colour. Even in the absence
of light, it is gradually degraded on exposure to a humid atmosphere, the
790
Requirements
Definition. Promethazine hydrochloride contains not less than 98.5% and not
more than 101.0% of Ci 7 H 2 oN 2 S,HCl, calculated with reference to the dried
substance.
Identity tests
A. Dissolve 20 mg in 5 ml of water and add 0.05 g of lead(IV) oxide R; no red
coloration is observed in the supernatant liquid but it slowly turns bluish.
B. Dissolve 0.25 g in 25 ml of water and add slowly, with 25 ml of trini- stirring,
trophenol TS. Allow the mixture to stand for 10 minutes, collect the
(7g/l)
precipitate and wash with a small quantity of water; melting temperature
after recrystallization from ethanol (~750g/l) TS and drying, about 160°C
with decomposition (picrate).
C. Dissolve 0.05 g in 5 ml of water and add 2 ml of nitric acid (~1000g/l) TS; a

dark red colour is produced, which turns yellowish on standing. The solu-
tion yields reaction A described under 2.1 General identification tests as
characteristic of chlorides.
Solution in chloroform. A solution of l.Og in 10ml of chloroform R is clear

and not more intensely coloured than standard colour solution Ywl when com-
5-Omg/g.
pH value. pH of a O.lOg/ml solution in carbon-dioxide-free water, 3. 5-5.0.
Related impurities. Carry out, in subdued light, the test as described under
and a mixture of 85 volumes of hexane R, 10 volumes of acetone R, and 5
volumes of diethylamine R as the mobile phase. Apply separately to the plate
10 pi of each of 2 freshly prepared solutions in a mixture of 95 volumes of
methanol R and 5 volumes of diethylamine R containing (A) 20 mg of the test
with solution B.
Assay. Dissolve about 0.4 g, accurately weighed, in 200 ml of acetone R, add

10ml of mercuric acetate/acetic acid TS and 3ml of methyl orange/acetone TS,
791
alent to 32.09 mg of Ci 7 H 2 oN 2 S,HCl.
Additional requirements for Promethazine hydrochloride for

prarenteral use
2-PROPANOLUM
2-PROPANOL
OH
CsHaO
Chemical name. Isopropyl alcohol; 2-propanol; CAS Reg. No. 67-63-0.
Description. A colourless, clear, and mobile liquid; odour, characteristic.
Miscibility. Miscible with water, ethanol (~750g/l) TS, and ether R.
Storage. 2-Propanol should be kept in a tightly closed container, in a cool place.
Additional information. 2-Propanol is volatile and flammable. Boiling range,

81-83°C.
Requirements
Identity tests
A. Mix 1 ml with 9 ml of water. To 1 ml of this solution add 2 ml of mercuric
sulfate TS and heat to boiling; a white to yellowish white precipitate is
produced.
792
B. Heat gently 1 ml with 3 ml of potassium dichromate (lOOg/1) TS and 1 ml of

sulfuric acid (-1760 g/1) TS; acetone, perceptible by its odour, is evolved.
Refractive index. tiS = 1.376 - 1.378.
Relative density, dfo = 0.783 - 0.787.
Nonvolatile residue. Evaporate 50 ml to dryness on a water-bath, heat at

105 °C for 1 hour, and weigh; not more than 2.5 mg (0.005%).
Acidity. To 50ml add 100ml of carbon-dioxide-free water R and 2 drops of

phenolphthalein/ethanol TS, and titrate with carbonate-free sodium hydroxide
(0.02 mol/1) VS to a pink colour that persists for 30 seconds; not more than
0.7 ml of carbonate-free sodium hydroxide (0.02 mol/1) VS is required.
Aldehydes and ketones. Transfer 25ml to a comparison tube, add 25ml of

water and 50 ml of hydroxylamine hydrochloride TS, mix, and allow to stand
for 5 minutes. Titrate with sodium hydroxide (0.1 mol/1) VS until the colour
matches that of 50 ml of hydroxylamine hydrochloride TS placed in a similar
tube and viewed down the vertical axis; not more than 2.0ml of sodium
hydroxide (0.1 mol/1) VS is required.
PROPRANOLOLI HYDROCHLORIDUM
PROPRANOLOL HYDROCHLORIDE
Molecular formula. Ci6H2iN02,HCl
Graphic formula.
OH
I
OCHjCHCH2NHCH(CH3)j
I ll I
•
HCI
Chemical name. (+)-l-(lsopropylamino)-3-(l-naphthyloxy)-2-propanol hydro-

chloride; (±)-l-[(l-methylethyl)amino]-3-(l-naphthalenyloxy)-2-propanol hydro-
chloride; CAS Reg. No. 318-98-9.
793
Solubility. Soluble in water and in ethanol (-750 g/1) TS; practically insoluble
in ether R.
Category. Antiadrenergic,
Storage. Propranolol hydrochloride should be kept in a well-closed container,

Requirements
Definition. Propranolol hydrochloride contains not less than 98.0% and not
substance.
Identity tests
infrared region.
spectrum obtained from propranolol hydrochloride RS or with the reference
spectrum of propranolol hydrochloride.

20 pg/ml solution in methanol R of propranolol hydrochloride RS (maxima
occur at about 290 nm, 306 nm, and 319nm). The absorbances of the solu-
tions at their respective maxima do not differ from each other by more than
3%. The absorbances of a 1-cm layer at those wavelengths are about 0.42,
0.25 and 0.15 (preferably use 2-cm cells for the measurement and calculate
the absorbances of 1-cm layers).

Specific optical rotation. Use a O.lOg/ml solution; the substance is optically

inactive.

5.0mg/g.
794
140 volumes of dichloroethane R, 60 volumes of methanol R, 2.5 volumes of
water, and 2.5 volumes of anhydrous formic acid R as the mobile phase. Apply
(A) 10 mg of the test substance per ml and (B) 0.050 mg of the test substance
per ml. Develop the plate for a distance of 10 cm. After removing the plate from
matogram in ultraviolet light (254 nm). Any spot obtained with solution A,
tion B.
Rl, and add 10ml of mercuric acetate/acetic acid TS, warming slightly if nec-
essary to effect solution. Cool and titrate with perchloric acid (0.1 mol/1) VS as
acid (0.1 mol/1) VS is equivalent to 29.58 mg of C]gH2iN02,HCl.
PROPYLENEGLYCOLUM
PROPYLENE GLYCOL
OH
C3H8O2
Chemical name. 1,2-Propanediol; CAS Reg. No. 57-55-6.
Description. A colourless, clear, and viscous liquid; odourless.
Miscibility. Miscible with water, ethanol (~750g/l) TS, and chloroform R.
Storage. Propylene glycol should be kept in a tightly closed container.
Additional information. Propylene glycol is hygroscopic. Boiling range

185-189 °C.
795
Requirements
Identity test
Dissolve 0.1 ml in sufficient water to produce 100ml, dilute 1 ml to 10ml, and
place 0.5ml of this solution in a test-tube. Cool in ice, add 5 ml of a cooled

mixture of 10ml of water and 90ml of sulfuric acid (-1760 g/1) TS, heat on a
water-bath at 70°C for 10 minutes, and cool again. Add 0.2ml of triketohy-
drindene/sodium metabisulfite TS; a violet colour slowly appears.
Refractive index, = 1.431 - 1.433.
Relative density, dfo = 1.035 - 1.040.
Heavy metals. Use 4 ml for the preparation of the test solution as described
Clarity and colour of solution. Propylene glycol should be clear and colour-
less.
Sulfated ash. Use 50 g; the residue weighs not more than 5mg (O.lmg/g).

Fischer method. Method A, using 5g; the water content is not more than
2-Omg/g.
Acidity. To 10ml add 40ml of water and 0.1ml of bromothymol blue/ethanol

TS; the solution is greenish yellow. Titrate with sodium hydroxide (0.1 mol/1)
VS; not more than 0.05 ml is required to obtain the midpoint of the indicator
(blue).
Oxidizing substances. To 10 ml add 5 ml of water, 2 ml of potassium iodide

(80 g/1) TS, and 2 ml of sulfuric acid (-100 g/1) TS, and allow to stand in a stop-
pered flask protected from light for 15 minutes. Titrate with sodium thiosul-
fate (0.05 mol/1) VS, using starch TS as indicator; not more than 0.2 ml is
required.
Reducing substances. To 1 ml add 1 ml of ammonia (-100 g/1) TS and heat in
a water-bath at 60 °C for 5 minutes; the solution is yellow. Without delay add

0.15ml of silver nitrate (0.1 mol/1) VS and allow to stand for 5 minutes; the
colour and aspect of the solution remain unchanged.
796
PROPYLIODONUM
PROPYLIODONE
o
C10H1J2NO3
Chemical name. Propyl 3,5-diiodo-4-oxo-l(4H)-pyridineacetate; CAS Reg.

No. 587-61-1.

almost odourless.
Storage. Propyliodone should be kept in a tightly closed container, protected

from light.
Requirements
Propyliodone contains not less than 99 0 . %
and not more than the equivalent
of 101 0 % of CioHul 2 N 03 calculated with reference to the dried substance.
.
,
Identity tests
A. The absorption spectrum of a 20pg/ml solution in dehydrated ethanol R,
239nm and 281 nm; the absorbances of a 1-cm layer at those wavelengths
are about 0.64 and 0.52, respectively.
B. Heat 0.1 g with a few drops of sulfuric acid (~1760g/l) TS in a suitable

crucible; violet vapours are evolved.
797
under 2.2,3 Limit test for heavy metals, Procedure 3; determine the heavy
metals content according to Method A; not more than 20p,g/g.
Halides. Shake 2.4g with 30ml of water for 15 minutes and filter. To 10ml of
the filtrate add 1 ml of nitric acid (-130 g/1) TS, 2 ml of sodium nitrite (lg/1) TS,
and 2 ml of chloroform R, shake well, and centrifuge. To serve as a reference
solution, treat similarly 2ml of iodide standard (20 pg I/ml) TS with 8 ml of
water. The content of halides, expressed as iodides, does not produce a solu-
tion with any red-violet colour more intense than that of the reference
solution.
Loss on drying. Dry to constant mass at 105°C; it loses not more than
5.0mg/g.
Acidity. Dissolve 1 g in 40 ml of hot 1 -propanol R, previously neutralized using

phenolphthalein/ethanol TS, cool, and allow to stand for 15 minutes with fre-
quent shaking. Filter, wash the residue with neutralized 1 -propanol R, filter
again, and combine the filtrate and the wash liquids. Titrate with sodium
hydroxide (0.05 mol/1) VS, using phenolphthalein/ethanol TS as indicator, until
a pink colour persists for 15 seconds; not more than 0.15ml of sodium hydrox-
ide (0.05 mol/1) VS is required.
Assay. Carry out the combustion as described under 2.4 Oxygen flask method
for iodine, using about 15 mg, accurately weighed. Titrate the liberated iodine
with sodium thiosulfate (0.02 mol/1) VS.

C10FI11I2NO3.
Additional requirement for Propyliodone for paarenteral use

798
PROPYLIS HYDROXYBENZOAS
PROPYL HYDROXYBENZOATE
OH
C10H12O3
Chemical name. Propyl /^-hydroxybenzoate; propyl 4-hydroxybenzoate;

CAS Reg. No. 94-13-3.
Other name. Propylparaben.

with a faintly aromatic odour.
Solubility. Very slightly soluble in water; slightly soluble in boiling water;

freely soluble in ethanol (-750 g/1) TS and ether R.
Storage. Propyl hydroxybenzoate should be kept in a well-closed container.
Additional information. Propyl hydroxybenzoate is normally used in com-

bination with other hydroxybenzoates.
Requirements
Propyl hydroxybenzoate contains not less than 99 0 % and not more than the
.
equivalent of 101.0% of C 10H 12 O 3 ,

substance.
Identity tests
A. Complies with the test under “Melting range”.
B. To 0.5 g add 5 ml of sodium hydroxide (-80 g/1) TS and heat in a water-bath

for 5 minutes. After cooling, add 6 ml of sulfuric acid (-190 g/1) TS, collect
799
the precipitate on a filter, wash thoroughly with a small amount of water,


or 5mm of mercury) for 2 hours; it loses not more than 5.0mg/g.
Acidity. Dissolve 0.2g in 5ml of ethanol (-750 g/1) TS, add 5ml of carbon-
Assay. Place about 80mg, accurately weighed, in a ground-glass-stoppered


acetic acid R. Cool in ice-water and add 10 ml of hydrochloric acid (-420 g/1)
30ml of potassium iodide (80 g/1) TS, close the flask and mix. Titrate with
towards the end of the titration. Repeat the procedure without the Propyl
One volume ofsodium thiosulfate (0.1 mol/1) VS corresponds to two volumes

(0.0333mol/l) VS is equivalent to 6.007mg of C 10 H 12 O 3 .
PROPYLTHIOURACILUM
PROPYLTHIOURACIL
Molecular formula. C7HioN20S
Graphic formula.
800
Chemical name. 6-Propyl-2-thiouracil; 2,3-dihydro-6-propyl-2-thioxo-4(lf/)-

pyrimidinone; 6-propyl-2-thio-2,4(l/7,3H)-pyrimidinedione; CAS Reg. No,
51-52-5.
Description. Colourless or pale cream-coloured crystals or a white or cream-

coloured, crystalline powder; odourless.

Category. Antithyroid substance.
Storage. Propylthiouracil should be kept in a well-closed container, protected

from light.
Additional information. Propylthiouracil has a bitter taste.
Requirements
Definition. Propylthiouracil contains not less than 98.0% and not more than
100.5% of C/H]oN 20S, calculated with reference to the dried substance.
Identity tests

reference spectrum of propylthiouracil.
B. Dissolve 0.05 g in 5 ml of boiling water and to the hot solution add a freshly
prepared solution of 20 mg of hydroxylamine hydrochloride R and 0.04 g of
anhydrous sodium carbonate R in 5 ml of water, to which 0.4ml of sodium
nitroprusside (45 g/1) TS has been added; a greenish blue colour is produced.
C. To 25mg add bromine TSl, drop by drop, until the substance is completely
dissolved. Warm until the colour is discharged, cool, and add 10 ml of
barium hydroxide (15 g/1) TS; a white precipitate is produced (distinction
from thiouracil, which yields a white precipitate that turns purple within 1
minute).
801
5-Omg/g.
Thiourea. Boil 0.50g with 50ml of water under a reflux condenser until dis-
solved and dilute 5 ml of the hot solution to 50 ml with water. Place 10 ml of
this solution in a test-tube and add to it 1 ml of thiourea (0.1 g/1) TS. Cool the
remainder of the hot solution, filter, and place 10 ml of the filtrate in a second
test-tube, to serve as a reference. To each tube add 0.5 g of sodium acetate R
and 5ml of silver nitrate (0.1 mol/1) VS and heat in a water-bath for 5 minutes.
The colour produced in the test solution, when viewed transversely against a
white background, is not more intense than that of the reference solution when
Assay. Transfer about 0.3 g, accurately weighed, to a 500-ml flask and add
30 ml of water. Add from a burette about 30 ml of sodium hydroxide (0.1 mol/1)
VS, heat to boiling, and shake the flask until solution is complete. Wash down
any particles on the wall of the flask with a small volume of water, then add
about 50ml of silver nitrate (0.1 mol/1) VS while mixing, and boil gently for 5
minutes. Add 1-2 ml of bromothymol blue/ethanol TS, and continue to titrate
with sodium hydroxide (0.1 mol/1) VS until a permanent blue-green colour is
produced. Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to 8.51 mg
of C 7 H 10N 2 OS.
PROTAMINI SULFAS
PROTAMINE SULFATE
Chemical name. Protamine sulfate; CAS Reg. No. 9009-65-8.
Description. A white or almost white powder; hygroscopic.
Solubility. Soluble in water; practically insoluble in ethanol (-750 g/1) TS and

ether R.
Category. Drug affecting blood coagulation.
Storage. Protamine sulfate should be kept in a tightly closed and tamper-

evident container.
Labelling. The designation Protamine sulfate for parenteral use indicates that
the substance complies with the additional requirements and may be used for
parenteral administration. Expiry date.
802
Additional information. Protamine sulfate binds with heparin in solution,

inhibiting its anticoagulant activity. It is prepared in conditions designed to min-
imize the risk of microbial contamination.
Requirements
Definition. Protamine sulfate is a mixture of sulfates of purified proteins
extracted from the sperm or roe of fish usually belonging to the family Clupi-
dae and Salmonidae.
The quantity of 1 mg of Protamine sulfate precipitates not less than lOOIU of

heparin sodium activity, calculated with reference to the dried substance.
Identity tests
A. Use a lOmg/ml solution in hydrochloric acid (0.1 mol/1) VS. Measure
the optical rotation and calculate with reference to the dried substance;
[a]g>"^ = 65° to -85°.
B. Dissolve 0.1 gin 5 ml of water, add 4.5 ml of water, 1.0 ml of sodium hydrox-
ide (~80g/l) TS, and 2.0ml of 1-naphthol TSl. Cool the mixture to 5°C and
add 0.5 ml of sodium hypobromite TS; an intense red colour is produced.
C. Dissolve 0.04 g in 2 ml of water and heat in a water-bath at 60 °C. Add

0.1ml of mercuric sulfate TS and mix; no precipitate is formed. Cool the
mixture in an ice-bath; a precipitate is formed.

Sulfates. Transfer 0.15g to a beaker and dissolve in 15ml of water. Add 5ml
of hydrochloric acid (~70g/l) TS, heat to boiling and slowly add to the boiling
solution 10ml of barium chloride (100 g/1) TS, Cover the beaker and heat in a
water-bath for 1 hour. Filter, and wash the precipitate several times with small
quantities of hot water. Dry and ignite the residue at 600 °C to constant mass.
Each g of residue is equivalent to 0.412 g of sulfates (SO4), calculated with ref-
erence to the dried substance; 0.16-0.24g/g.
Clarity and colour of solution. A solution of 0.50 g in 10 ml of water is
not more opalescent than opalescence standard TS2 and not more intensely
803
Loss on drying. Dry at 105 °C for 3 hours; it loses not more than 0.050g/g.
Light absorbance. Dissolve O.OSOg in 5ml of water and measure the

absorbance of a 1-cm layer at a wavelength between 260 nm and 280 nm; not
greater than 0.1.
Nitrogen. Proceed as described under 2.10 Determination of nitrogen, Method

B, using 10 mg of Protamine sulfate; the content of nitrogen is not less than
0.23g/g and not more than 0.27 g/g, calculated with reference to the dried
substance.
Assay. Prepare the following solutions: for solution (A) dissolve 15.0 mg of
Protamine sulfate in sufficient water to produce 100 ml; for solution (B) dilute
2.0 ml of solution A to 3.0 ml with water; for solution (C) dilute 1.0 ml of solu-
tion A to 3.0 ml with water.
As titrant use a solution of heparin RS in water containing about 170IU/ml.

Titrate each of solutions A, B, and C in duplicate and carry out 3 independent
assays. Measure accurately 1.5 ml of one of the solutions and introduce it to a
cell of a suitable spectrophotometer set at 420 nm. Add small volumes of the
titrant until a sharp change in transmittance is observed and note the volume
of titrant added.
For each individual titration, calculate the number of International Units of

heparin in the volume of titrant added, per mg of Protamine sulfate. Average the
18 values and test the linearity of the response using the usual statistical methods.
The assay isnot valid unless the relative standard deviations calculated for the
results obtained with each solution are less than 5% of the average result.
Additional requirements for Protamine sulfate for

p'arenteral use
PROTIONAMIDUM
PROTIONAMIDE
804
Graphic formula.
CHjCHjCH3
CSNHj
Chemical name. 2-Propylthioisonicotinamide; 2-propyl-4-pyridinecarboth-

ioamide; CAS Reg. No. 14222-60-7.
Description. Yellow crystals or a crystalline powder; odourless or almost

odourless.

methanol R; slightly soluble in ether R.
Storage. Protionamide should be kept in a well-closed container, protected

from light.
Requirements
Definition. Protionamide contains not less than 98.0% and not more than
101.0% of C9H12N2S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from protionamide RS or with the reference spectrum of
protionamide.
B. The absorption spectrum of a lOpg/ml solution in ethanol (-750 g/1) TS,

C. Heat 0.1 g with 5ml of hydrochloric acid (1 mol/1) VS; the vapours evolved
blacken lead acetate paper R.
805
5.0mg/g.
Acidity. Dissolve 2.0 g in 20ml of warm methanol R, add 20 ml of water, cool,

shake until crystallization occurs, and titrate with sodium hydroxide (0.1 mol/1)
VS, using cresol red/ethanol TS as indicator; not more than 0.2 ml is required
to obtain the midpoint of the indicator (orange).
Apply separately to the plate 5 pi of each of 2 solutions in methanol R con-
taining (A) 50 mg of the test substance per ml and (B) 0.25 mg of the test sub-
stance per ml. After removing the plate from the chromatographic chamber

alent to 18.03 mg of C9H12N2S.
PYRANTELI EMBONAS
PYRANTEL EMBONATE
Molecular formula. CnHi4N2S,C23Hi60(;
Graphic formula.
HOOC OH HO COOH
CHj
806
Chemical name. (£)- 1,4,5, 6-Tetrahydro-l -methyl-2-[2-(2-thienyl) vinyljpyrimi-

dine compound with 4,4'-methylenebis[3-hydroxy-2-naphthoate] (1:1); (£)-
l,4,5,6-tetrahydro-l-methyl-2-[2-(2-thienyl)ethenyl]pyrimidine 4,4'-methylene-
bis[3-hydroxy-2-naphthalenecarboxylate] (1:1); CAS Reg. No. 22204-24-6.
Other name. Pyrantel pamoate.
Solubility. Practically insoluble in water and methanol R; soluble in dimethyl

sulfoxide R; slightly soluble in dimethylformamide R.
Storage. Pyrantel embonate should be kept in a well-closed container, pro-

tected from light.
Requirements
Definition. Pyrantel embonate contains not less than 97.0% and not more
than 103.0% of C,]H]4N2S,C23H,605, calculated with reference to the dried
substance.
Identity tests

spectrum obtained from pyrantel embonate RS or with the reference spectrum
of pyrantel embonate.

and 300 nm. The ratio of the absorbance at 288 nm to that at 300 nm is about
1 . 0 .
C. Dissolve 5 mg in 1.0 ml of hydrochloric acid (~70g/l) TS and add 1.0ml of

formaldehyde/sulfuric acid TS; a purple colour is produced.
D. Melting temperature, above 250 °C with decomposition.

807
20 volumes of ethyl acetate R, 5 volumes of methanol R, and 1.5 volumes of
diethylamine R as the mobile phase. Apply separately to the plate 100 pi
of each of 2 solutions in a mixture of 5 volumes of chloroform R, 5 volumes of
methanol R, and 0.5 volumes of ammonia (-260 g/1) TS containing (A) 20 mg
of the test substance per ml and (B) 0.20 mg of the test substance per ml. After
current of air for 10 minutes and examine the chromatogram in ultraviolet light
Assay
• Perform the assay in subdued light and without any prolonged interrup-
tions, preferably using low-actinic glassware.
Transfer about O.lOg, accurately weighed, to a 200-ml volumetric flask, dis-

solve in a mixture of 10ml of dioxan R and 10ml of ammonia (-lOOg/1) TS,
and dilute to volume with perchloric acid (-140 g/1) TS. Filter, discard the first
10ml of the and transfer 5ml of the subsequent filtrate to a 50-ml vol-
filtrate,
umetric flask. TS and mix.

Dilute to volume with perchloric acid (~140g/l)
Transfer 25ml 250-ml separating funnel, add 100 ml of chloroform R, and
to a
shake well. Drain off the chloroform layer into a second separating funnel.
Repeat the extraction of the aqueous phase with a second 100-ml portion of
chloroform R, and combine the chloroform extracts into the same separating
funnel. Add 40ml of hydrochloric acid (0.05 mol/1) VS to the combined chlo-
roform extracts and shake well. Drain off the chloroform phase into a third
separating funnel and extract with a further 40-ml portion of hydrochloric acid
(0.05 mol/1) VS, discarding the chloroform phase. Combine the aqueous phases
in a 100-ml volumetric flask, rinse the separating funnel, draining into the vol-
umetric flask, and dilute to volume with hydrochloric acid (0.05 mol/1) VS.
Measure the absorbance of a Tcm layer of this solution at the maximum at
about 31 1 nm against a solvent cell containing hydrochloric acid (0.05 mol/1) VS.
amount of CnH]4N2S,C23Hi605 in the substance being tested by
Calculate the
comparison with pyrantel embonate RS, similarly and concurrently examined.
PYRAZINAMIDUM
PYRAZINAMIDE
808
Graphic formula.
CONHj
Chemical name. Pyrazinecarboxamide; CAS Reg. No. 98-96-4.
Solubility. Sparingly soluble in water; slightly soluble in ethanol (-750 g/1) TS.
Category. Antituberculosis drug.
Storage. Pyrazinamide should be kept in a well-closed container.
Requirements
Definition. Pyrazinamide contains not less than 98.5% and not more than
101.0% of C5H5N3O, calculated with reference to the anhydrous substance.
Identity tests

infrared region.
spectrum obtained from pyrazinamide RS or with the reference spectrum of
pyrazinamide.
B. The absorption spectrum of a lOpg/ml solution, when observed between

230 nm and 350 nm, exhibits a maximum at about 268 nm and another
smaller one at 310nm; the absorbance of a Tcm layer at 268 nm is about
0 . 66 .
C. Dissolve O.lg in 10ml of water and add 1ml of ferrous sulfate (15g/l) TS;
an orange-red colour develops turning to blue on the addition of 1 ml of
sodium hydroxide (-80 g/1) TS.
D. Boil 20 mg with 5 ml of sodium hydroxide (-200 g/1) TS; ammonia, percep-

tible by its odour, is evolved.
809

Ammonia. To 20 ml of carbon-dioxide-free water R add 1.0 ml of formalde-

hyde TS and 0.05 ml of phenolphthalein/ethanol TS, neutralize with carbon-
ate-free sodium hydroxide (0.1 mol/1) VS, if necessary, add 0.50g of the test
substance, and dissolve cautiously while heating. Titrate with carbonate-free
sodium hydroxide (0.1 mol/1) VS until a pink colour is obtained; not more than
0.50ml of carbonate-free sodium hydroxide (0.1 mol/1) VS is required.
Assay. Dissolve about 0.07 g, accurately weighed, in 15ml of chloroform R

and 5ml of acetic anhydride R, add 0.15ml of Sudan red TS as indicator, and

12.31 mg of C5H5N3O.
PYRIDOSTIGMINI BROMIDUM
PYRIDOSTIGMINE BROMIDE
Molecular formula. C9Hi3BrN202
Graphic formula.
Chemical name. 3-Hydroxy-l-methylpyridinium bromide dimethylcarba-

mate; 3-[[(dimethylamino)carbonyl]oxy]-l-methylpyridinium bromide; CAS
Reg. No. 101-26-8.
810
Description. A white or almost white, crystalline powder; odour, agreeable,

characteristic.
Solubility. Soluble in less than 1 part of water and ethanol (~750g/l) TS; prac-
Storage. Pyridostigmine bromide should be kept in a well-closed container,

Additional information. Pyridostigmine bromide is deliquescent.
Requirements
Definition. Pyridostigmine bromide contains not less than 98.5% and not
more than 101.0% of C 9 Hi 3BrN 202 ,
substance.
Identity tests
A. The absorption spectrum of a 25[xg/ml solution, when observed between
of a 1-cm layer at this wavelength is about 0.46 (preferably use 2-cm cells
for the measurement and calculate the absorbance of a 1-cm layer).
C. To 0.1 g add 0.6ml of sodium hydroxide (~80g/l) TS; an orange colour is
produced. Warm the solution; the colour changes to yellow and the vapours
evolved turn moistened red litmus paper blue.


811
67 volumes of water, 30 volumes of methanol R, and 3 volumes of diethylamine

R as the mobile phase. Apply separately to the plate 10 pi of each of 3 solu-
tions containing (A) 20 mg of the test substance per ml, (B) 0.10 mg of the test
substance per ml, and (C) O.lOmg of pyridostigmine bromide RS per ml. After
current of warm air, spray it with 4-nitroaniline TS2 and then with sodium
hydroxide (0.1 mol/1) VS. Dry the plate again in a current of warm air, spray it
with potassium iodobismuthate TS2 and examine the chromatogram in day-
Rl, add 10 ml of mercuric acetate/acetic acid TS and 2 drops of quinaldine
red/ethanol TS as indicator, and titrate with perchloric acid (0.1 mol/1) VS as
acid (0.1 mol/1) VS is equivalent to 26.11 mg of C9Hi3BrN202.
Additional requirements for Pyridostigmine bromide for

parenteral use
PYRIDOXINI HYDROCHLORIDUM
PYRIDOXINE HYDROCHLORIDE
Molecular formula. C 8 HuN 03 ,HCl
Graphic formula.
Chemical name. 5-Hydroxy-6-methyl-3,4-pyridinedimethanol hydrochlo-

ride; 3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridine hydrochloride; CAS
Reg. No. 58-56-0.
812
Category. Vitamin.
Storage. Pyridoxine hydrochloride should be kept in a tightly closed container,

Additional information. Even in the absence of light, Pyridoxine hydrochlo-

Requirements
Definition. Pyridoxine hydrochloride contains not less than 98.5% and not more
than 101.0% of C 8 HnN 03,HCl, calculated with reference to the dried substance.
Identity tests
VS,
at
length is about 0,43 (preferably use 2-cm cells for the measurement and cal-
B. The absorption spectrum of a 0.5 mg/ml solution in phosphate buffer pH

6.9, TS, when observed between 230 nm and 350 nm, exhibits maxima at
about 254 nm and 324 nm; the absorbances of a 1-cm layer at the maximum
wavelengths are about 0.18 and 0.35, respectively (preferably use 2-cm cells
for the measurement and calculate the absorbances of 1-cm layers).
C. In each of two test-tubes A and B, place 1ml of a 0.1 mg/ml solution and
2ml of sodium acetate (150g/l) TS. To tube A add 1 ml of water and to tube
B 1 ml of boric acid (50 g/1) TS and mix. Cool both tubes to about 20 °C and
rapidly add to each tube 1 ml of 2,6-dichloroquinone chlorimide/ethanol TS;
a blue colour is produced in tube A, whereas in tube B no blue colour is
observed.
813

and colourless.
Sulfated ash. Not more than l.Omg/g,

reduced pressure (not exceeding 0.6 kPa or about 5 mm of mercury) over silica
gel, desiccant, R; it loses not more than 5.0mg/g.
ml of perchloric acid (0.1 mol/1) VS is equivalent to 20.56mg of C 8 HnN 03 ,HCl.
PYRIMETHAMINUM
PYRIMETHAMINE
Molecular formula. C12H13CIN4
Graphic formula.
Chemical name. 2,4-Diamino-5-(/^-chlorophenyl)-6-ethylpyrimidine; 5-(4-

chlorophenyl)-6-ethyl-2,4-pyrimidinediamine; CAS Reg. No. 58-14-0.

TS and acetone R.
Storage. Pyrimethamine should be kept in a well-closed container, protected

from light.
814
Requirements
Definition. Pyrimethamine contains not less than 99.0% and not more than
101.0% of Ci 2 Ht 3 ClN 4 calculated with reference
,
Identity tests

infrared region.
spectrum obtained with pyrimethamine RS or with the reference spectrum of
pyrimethamine.

(0.005mol/l) VS, when observed between 230nm and 350nm, exhibits a
of a 1-cm layer at the maximum is about 0.48 and at the minimum about
0.45.
C. To add 1 ml of a mixture of equal volumes of glacial acetic acid R and

0.5 g
acetic anhydride R and heat under a reflux condenser for 30 minutes. Pour
the warm mixture into 25 ml of water; a white, crystalline precipitate is pro-
duced. Collect the precipitate on a filter, wash with water, recrystallize from
4ml of ethanol (~750g/l) TS mixed with 6ml of water, and dry at 105°C;
melting temperature, about 172 °C (2,4-diacetylpyrimethamine).
D. Ignite 0.1 g with 0.5g of anhydrous sodium carbonate R, extract the residue
with water, and filter. Neutralize with nitric acid (~130g/l) TS; it yields reac-
tion A described under 2.1 General identification tests as characteristic of
chlorides.
5-Omg/g.
Acidity or alkalinity. Boil 0.3 g with 15 ml of water, cool and filter. Add
0.25ml of methyl red/ethanol TS to the filtrate; a yellow colour is observed.
Not more than 0.1 ml of hydrochloric acid (0.05 mol/1) VS is required to change
the colour of the solution to red.
Rl, add 0.20ml of quinaldine red/ethanol TS as indicator, and titrate with per-
815

of Ci2H,3C1N4.
QUINIDINI SULFAS
QUINIDINE SULFATE
Molecular formula. (C2oH24N202)2,H2S04,2H20
Graphic formula.
H
CH=CH,
• HjSO^- 2HjO
CH,0
Chemical name. Quinidine sulfate (2 1) (salt), dihydrate; (9S)-6'-methoxycin-

:
chonan-9-ol sulfate (2 :
1) (salt), monohydrate; CAS Reg. No. 6591-63-5 (dihydrate).
Description. Colourless, needle-like crystals or a white, crystalline powder;

odourless.
Solubility. Sparingly soluble in water; soluble in ethanol (-750 g/1) TS; practi-
cally insoluble in ether R and acetone R.
Category. Antidysrhythmic drug.
Storage. Quinidine sulfate should be kept in a well-closed container, protected

from light.
Additional information. Quinidine sulfate has a very bitter taste. It darkens

in colour on exposure to light.
Requirements
Definition. Quinidine sulfate contains not less than 99.0% and not more than
101.0% of total alkaloids, calculated as (C 2 oH 24 N 2 Q 2) 2 ,H 2 SQ 4 and with reference
816
Identity tests
A. Dissolve 0.10 g in 10 ml of water; the solution produces a slight blue fluo-
rescence (keep the remaining solution for testB). To 1.0ml add a few drops
of sulfuric acid (-100 g/1) TS, and dilute with water to 5ml; a vivid blue flu-
orescence is produced.
B. To 1.0ml of the solution prepared for test A, add 4ml of water, 0.15ml of
bromine TSl, and 1.0ml of ammonia (-100 g/1) TS; an emerald-green colour
is produced.
C. Dissolve 0.05 g in 5 ml of hot water, cool, add 1 ml of silver nitrate (40 g/1)
TS, and stir with a glass rod; after a few minutes a white precipitate is pro-
duced, which is soluble in nitric acid (-130 g/1) TS.


(0.1 mol/1) VS and calculate with reference to the dried substance; [a]™ ”‘^ = -t275
to-t290°.
Clarity and colour of solution. Dissolve 0.20g in 10ml of hydrochloric acid

(0.1 mol/1) VS; the solution is clear and not more intensely coloured than stan-
dard colour solution Yw2 when compared as described under 1.11 Colour of
liquids.
Related cinchona alkaloids. Carry out the test as described under 1.14.1
Thin-layer chromatography, using silica gel R1 as the coating substance and a
mixture of 20 volumes of toluene R, 12 volumes of ether R, and 5 volumes of
of 4 solutions in methanol R containing (A) 10 mg of the test substance per ml,
(B) 0.25 mg of quinine R per ml, (C) 0.25 mg of cinchonine R per ml, and (D)
lOmg of the test substance dissolved in 1 ml of solution C. After removing the

plate from the chromatographic chamber, allow it to dry in a current of air for
15 minutes and repeat the development. Heat the plate at 105 °C for 30
minutes, allow it to cool, spray it with potassium iodoplatinate TS, and
tion B or solution C. Disregard any spot obtained with solution A immediately
817
below the principal spot. The test is valid only if the chromatogram obtained
Dihydroquinidine. Dissolve about 0.2g, accurately weighed, in 20ml of

water.Add 0.5g of potassium bromide R, 15ml of hydrochloric acid (~70g/l)
TS, and 0.1ml of methyl red/ethanol TS. Titrate with potassium bromate
(0.0167mol/l) VS until a yellow colour is produced. Add 0.5g of potassium
iodide R 200 ml of water, stopper the flask, and allow to stand in the dark
in
for 5 minutes. Titrate the iodine liberatedby excess potassium bromate in the
solution with sodium thiosulfate (0.1 mol/1) VS, adding 2 ml of starch TS when
the solution has reached a light yellow colour. Each ml of potassium bromate
(0.0167mol/l) VS is equivalent to 18.67 mg of (C2oH24N202)2,H2S04, calculated
with reference to the dried substance. Express the results of both the above
determination and the assay in percentages. The difference between the two
is not more than 15%.
Assay. Dissolve about 0.20 g, accurately weighed, in 10 ml of chloroform R

and 20 ml of acetic anhydride R, and titrate with perchloric acid (0.1 mol/1) VS,
alent to 24.90 mg of (C2 qH24N202)2,H2S04.
QUININI BISULFAS
QUININE BISULFATE
Molecular formula. C2 oH24N202,H2S04,7H20
Graphic formula.
Chemical name. Quinine sulfate (1:1) (salt), heptahydrate; (8aS,9R)-6'-

methoxycinchonan-9-ol sulfate (1 1) (salt), heptahydrate; (8S,9/^)-9-hydroxy-
:
818
6'-methoxycinchonan sulfate (1 :
1) (salt), heptahydrate; CAS Reg. No. 6183-68-
2 (heptahydrate).
Solubility. Freely soluble in water; sparingly soluble in ethanol (~750g/l) TS.
Storage. Quinine bisulfate should be kept in a well-closed container, protected

from light.
Additional information. Quinine bisulfate effloresces in dry air. Even in the

Requirements
Definition. Quinine bisulfate contains not less than 98.5% and not more than
101.5% of total alkaloids, calculated as C 2 oH 24N 2 Q 2 ,H 2 SQ 4 and with reference
Identity tests
A. Dissolve 5 mg in 10 ml of water and add 0.05 ml of sulfuric acid (~100g/l)
TS; a strong blue fluorescence is produced (keep the solution for test B).
B. To the solution prepared for test A add 0.15ml of bromine TSl and 1.0ml
of ammonia (~100g/l) TS; an emerald-green colour is produced.
C. Dissolve 0.05 g in 5 ml of water and add 1 ml of silver nitrate (40g/l) TS; no

white precipitate is produced.


(0.1 mol/1) VS and calculate with reference to the dried substance; =
-208 to -216°.

liquids.
819

or about 5 mm
of mercury) for 18 hours; it loses not less than 190mg/g and not
more than 240 mg/g.
diethylamine R as the mobile phase. Apply separately to the plate 4|tl of each
(B) 0.25 mg of quinine R per ml, (C) 0.25 mg of cinchonidine R per ml, and (D)
lOmg of the test substance dissolved in 1ml of solution C. After removing

the plate from the chromatographic chamber, allow it to dry in a current of air
for 15 minutes and repeat the development. Heat the plate at 105°C for 30
minutes, allow it to cool, spray it with potassium iodoplatinate TS, and
tion B or solution C. Disregard any spot obtained with solution A immediately
below the principal spot. The test is valid only if the chromatogram obtained
Dihydroquinine. Dissolve about 0.2 g, accurately weighed, in 20 ml of water.

Add 0.5 g of potassium bromide R, 15 ml of hydrochloric acid (~70g/l) TS, and
0.1 ml of methyl red/ethanol TS. Titrate with potassium bromate (0.0167 mol/1)
VS until a yellow colour is produced. Add 0.5 g of potassium iodide R in
200 ml of water, stopper the flask, and allow to stand in the dark for 5 minutes.
Titrate the iodine liberated by excess potassium bromate in the solution with
sodium thiosulfate (0.1 mol/1) VS, adding 2 ml of starch TS when the solution
has reached a light yellow colour. Each ml of potassium bromate (0.0167 mol/1)
VS is equivalent to 21. 13 mg of C2oH24N202,H2S04, calculated with reference to
the dried substance. Express the results of both the above determination and
the assay in percentages. The difference between the two is not more than
10 %.
Assay. Dissolve about 0.45 g, accurately weighed, in 15 ml of water. Add

25 ml of sodium hydroxide (0.1 mol/1) VS and extract with 3 quantities, each of
25 ml of chloroform R. Wash the combined chloroform extracts with 20 ml of
water. Dry the chloroform extracts with anhydrous sodium sulfate R, evapo-
rate to dryness under reduced pressure, and dissolve the residue in 50 ml of
glacial acetic acid Rl. Titrate with perchloric acid (0.1 mol/1) VS as described
(0.1 mol/1) VS is equivalent to 21.13mg of C2oH24N202,H2S04.
820
QUININI DIHYDROCHLORIDUM
QUININE DIHYDROCHLORIDE
Molecular formula. C 2 oH 24 N 202 2 HCl
,
Graphic formula.
Chemical name. (8aS,9R)-6'-Methoxycinchonan-9-ol dihydrochloride; (8S,

9R)-9-hydroxy-6'-methoxycinchonan dihydrochloride; CAS Reg. No. 60-93-5.
Solubility. Very soluble in water; soluble in ethanol (~750g/l) TS; practically

Storage. Quinine dihydrochloride should be kept in a well-closed container,

Additional information. Quinine dihydrochloride turns yellow on exposure

to light. Even in the absence of light, it is gradually degraded on exposure to a
Requirements
Definition. Quinine dihydrochloride contains not less than 99.0% and not
more than 101.0% of total alkaloids, calculated as C oH 24N 2 Q
2 2, 2 HCl and with
Identity tests
A. Dissolve 5mg in 10ml of water and add 0.05ml of sulfuric acid (~100g/l)
TS; a strong blue fluorescence is produced (keep the solution for test B).
821
B. To the solution prepared for test A add 0.15ml of bromine TSl and 1.0ml
of ammonia (~100g/l) TS; an emerald-green colour is produced.
C. Dissolve 0.05g in 5ml of water and add 1ml of silver nitrate (40g/l) TS; a


-223 to -229°.

liquids.
30mg/g.
(B) 0.25 mg of quinine R per ml, (C) 0.25 mg of cinchonidine R per ml, and (D)
lOmg of the test substance dissolved in 1 ml of solution C. After removing the

plate from the chromatographic chamber, allow it to dry in a current of air for
15 minutes and repeat the development. Heat the plate at 105 °C for 30
minutes, allow to cool, spray with potassium iodoplatinate TS and examine the
principal spot, is not more intense than that obtained with solution B or solu-
tion C. Disregard any spot obtained with solution A immediately below the
principal spot. The test is valid only if the chromatogram obtained with solu-
tion D shows two distinctly separated spots.

Add 0.5 g of potassium bromide R, 15 ml of hydrochloric acid (~70g/l) TS, and
TS until a yellow colour is produced. Add 0.5 g of potassium iodide R in
822
Titrate the iodine liberatedby excess potassium bromate in the solution with
has reached a lightyellow colour. Each ml of potassium bromate (0.0167 mol/1)
VS is equivalent to 19.87 mg of C2oH24N202,2HCl, calculated with reference to
the dried substance. Express the results of both the above determination and
the assay in percentages. The difference between the two is not more than
10 %.
Rl, add 20ml of acetic anhydride R and 10ml of mercuric acetate/acetic acid
TS, and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-
alent to 19.87 mg of C2 oH24N202,2HC1.
Additional requirement for Quinine dihydrochloride for

parenteral use
QUININI HYDRO CHLORIDUM

QUININE HYDROCHLORIDE
Molecular formula. C2 oH24N202,HC1,2H20
Graphic formula.
Chemical name. (8a,9i^)-6'-Methoxycinchonan-9-ol monohydrochloride (salt)
dihydrate; (8a,9R)-9-hydroxy-6'-methoxycinchonan hydrochloride (1 :

1) (salt)
dihydrate; CAS Reg. No. 6119-47-7.
Description. Silky, colourless crystals, often grouped in clusters; odourless.
823
Solubility. Soluble in water; freely soluble in ethanol (~750g/l) TS; very

Storage. Quinine hydrochloride should be kept in a well-closed container, pro-

tected from light.
Additional information. Quinine hydrochloride has a very bitter taste.
Requirements
Definition. Quinine hydrochloride contains not less than 98.5% and not more
than 101.0% of total alkaloids, calculated as C 2 oH 24N 2 Q 2 ,HCl and with refer-
Identity tests
A. Dissolve 0.1 g in 2.5ml of water; the solution is not fluorescent. Dilute
0.5 ml of this solution to 100 ml with water and add 2 drops of sulfuric
acid (~100g/l) TS; a strong blue fluorescence is produced.
B. To 5 ml of a 1 mg/ml solution add 2-3 drops of bromine TSl and 5 drops of

ammonia (~100g/l) TS; an emerald-green colour is produced.
C. A lOmg/ml solution yields reaction B described under 2.1 General identifi-


-240 to -258°.
Barium. To 15ml of a 0.3g/ml solution add 1 ml of sulfuric acid (~100g/l) TS;

the solution remains clear for not less than 15 minutes.
Sulfates. Dissolve 0.5 g in 20ml of water and proceed as described under 2.2.2
Limit test for sulfates; the sulfate content is not more than 1 mg/g.
Clarity and colour of solution. A solution of O.lOg in 10ml of water is clear

and colourless.
60 mg/g and not more than 100 mg/g.
824
and (B) 0.25 mg of cinchonidine R per ml. After removing the plate from the
chromatographic chamber, heat it at 105 °C for 30 minutes, allow it to cool,
spray with potassium iodoplatinate TS, and examine the plate in daylight. Any

Add 0.5g of potassium bromide R, 15ml of hydrochloric acid (~70g/l) TS and
Titrate the iodine liberated by excess potassium bromate in the solution
with sodium thiosulfate (0.1 mol/1) VS, adding 2 ml of starch TS when the solu-
tion has reached a light yellow coloration. Each ml of potassium bromate
(0.0167mol/l) VS is equivalent to 18.04 mg of C2oH24N202,HCl. Express the
results of both the above determination and the assay in percentages, calcu-
lated with reference to the dried substance. The difference between the two is
not more than 10%.

alent to 18.04 mg of C 2 oH 24N 202 ,HC 1.
QUININI SULFAS
QUININE SULFATE
Molecular formula. (C2oH24N202)2,H2S04,2H20
825
Graphic formula.
Chemical name. (8a,9i^)-6'-Methoxycinchonan-9-ol sulfate (2:1) (salt) dihy-

drate; (8a,9R)-9-hydroxy-6'-methoxycinchonan sulfate (2 ;
1) (salt) dihydrate;
CAS Reg. No. 6591-63-5.
Description. Colourless, needle-like crystals; odourless.
Solubility. Slighdy soluble in water, ethanol (-750 g/1) TS, and ether R.
Storage. Quinine sulfate should be kept in a well-closed container, protected

from light.
Additional information. Quinine sulfate has a very bitter taste.
Requirements
Definition. Quinine sulfate contains not less than 99.0% and not more than
101.0% of total alkaloids, calculated as (C 2 oH 24 N 202) 2 ,H 2 SQ 4 and with reference
Identity tests
A. Dissolve 5 mg in 10 ml of water and add 1 drop of sulfuric acid (-100 g/1)
TS; a strong blue fluorescence is produced.
B. To 5 ml of a 1 mg/ml solution add 2-3 drops of bromine TSl and 5 drops of

ammonia (-100 g/1) TS; an emerald-green colour is produced.

Specific optical rotation. Use a 30mg/ml solution in sulfuric acid (~100g/l)

TS and calculate with reference to the dried substance; [a]§’ = -240 to -250°.
826
Clarity and colour of solution. Dissolve 20 mg in 5 ml of hydrochloric acid

(0.1 mol/1) VS and add sufficient water to produce 10ml. This solution is clear
pH value. pH of a lOmg/ml suspension in carbon-dioxide-free water R,

5.7-6.6.

and (B) 0.25 mg of cinchonidine R per ml. After removing the plate from the
chromatographic chamber, heat it at 105 °C for 30 minutes, allow it to cool,
spray with potassium iodoplatinate TS and examine the chromatogram in day-

Add 0.5g of potassium bromide R, 15ml of hydrochloric acid (~70g/l) TS and
Titrate the iodine liberated by excess potassium bromate in the solution
with sodium thiosulfate (0.1 mol/1) VS, adding 2 ml of starch TS when the solu-
tion has reached a light yellow coloration. Each ml of potassium bromate
(0.0167 mol/1) VS is equivalent to 24.90mg of (C2 qH 24N202)2,H2S04. Express the
results of both the above determination and the assay in percentages, calcu-
lated with reference to the dried substance. The difference between the two is
not more than 10%.

chloric acid (0.1 mol/1) VS is equivalent to 24.90 mg of (C2oH24N202)2,H2S04.
827
RESERPINUM
RESERPINE
Graphic formula.
Chemical name. Methyl 18|3-hydroxy-ll,17a-dimethoxy-3P,20a-yohimban-

16P-carboxylate 3,4,5-trimethoxybenzoate (ester); methyl ll,17a-dimethoxy-
18P-[(3,4,5-trimethoxybenzoyl)oxy]-3P,20a-yohimban-16P-carboxylate; CAS
Reg. No. 50-55-5.
Description. Small, white to pale beige crystals or a white to pale beige, crys-
Solubility. Practically insoluble in water; soluble in 90 parts of acetone R; very

slightly soluble in methanol R, ethanol (~750g/l) TS, and ether R.
Category. Neuroleptic; hypotensive.
Storage. Reserpine should be kept in a well-closed container, protected from

light.
Additional information. Reserpine darkens slowly on exposure to light, but

more rapidly in solution.
Requirements
Definition. Reserpine contains not less than 98.0% and not more than 102.0%
of C 33 H 40N 2 O 9 ,
828
=
Identity tests

infrared region.
spectrum obtained from reserpine RS or with the reference spectrum of
reserpine.
B. To Img add 0.2ml of a freshly prepared lOg/1 solution of vanillin R in

hydrochloric acid (-250 g/1) TS; a pink colour is produced in about 2 minutes.
C. Mix 0.5mg with 5mg of 4-dimethylaminobenzaldehyde R and 0.2ml of

glacial acetic acid R and add 0.2ml of sulfuric acid (-1760 g/1) TS; a green
colour is produced. Add 1 ml of glacial acetic acid R; the colour changes to
red.
Specific optical rotation. Use a lOmg/ml solution in chloroform R; [a]™

-113 to -127°.

Oxidation products. Measure the absorbance of a 1-cm layer of a 0.2 mg/ml

solution in glacial acetic acid R at the maximum at about 388 nm; not greater
than 0.10 (preferably use 2 -cm cells for the measurement and calculate the
absorbance of a 1 -cm layer).
Assay
• The solutions must be protected from air and light throughout the assay.
Moisten about 25mg, accurately weighed, with 2 ml of ethanol (-750 g/1) TS,
add 2ml of sulfuric acid (0.25mol/l) VS and 10ml of ethanol (-750g/l) TS, and
warm gently to effect solution. Cool, dilute to 100.0 ml with ethanol (-750 g/1)
TS, and dilute 5.0 ml of this solution to 50.0ml with the same solvent. Trans-
fer 10.0 ml of the solution to a boiling tube, add 2 0 ml of sulfuric acid .
(0.25 mol/1) VS and 2.0 ml of freshly prepared sodium nitrite (3 g/1) TS, mix, and
heat in a water-bath at 55 °C for 30 minutes. Cool, add 1.0ml of freshly pre-
pared sulfamic acid (50 g/1) TS, and dilute to 25.0 ml with ethanol (-750 g/1) TS.
Measure the absorbance of a 1-cm layer at the maximum at about 390 nm,
against a solvent cell containing a solution prepared by treating a further
10.0ml of the solution in the same manner but omitting the sodium nitrite. Cal-
culate theamount of C 33 H 40N 2 O 9 in the substance being tested by comparison
with reserpine RS, similarly and concurrently examined. In an adequately cal-
829
ibrated spectrophotometer the absorbance of the reference solution should be

0.42 ± 0.01 (preferably use 2-cm cells for the measurement and calculate the
absorbance of a 1-cm layer).
Additional requirements for Reserpnne for parenteral use

RETINOLUM DENSATUM OLEOSUM

RETINOL CONCENTRATE, OILY FORM
Chemical name. 3,7 Dimethyl-9-(2,6,6-trimethyl-l-cyclohexen-Tyl)-2,4,6,8-

nonatetraen-l-ol; CAS Reg. No. 68-26-8.
Other name. Vitamin A concentrate (oily form).
Description. A yellow to brownish yellow, oily liquid.
Solubility. Practically insoluble in water; soluble or partly soluble in dehy-

drated ethanol R; miscible with organic solvents.
Category. Vitamin.
Storage. The oily form of Retinol concentrate should be kept in a well-closed

and well-filled container, protected from light, and stored at a temperature
between 8 and 15 °C. Once the container has been opened its contents should
be used as soon as possible; any part of the contents not used at once should
be protected by an atmosphere of inert gas.
Labelling. The designation on the container should state the name of the ester
or esters, whether any additional agents are added and their quantities, as well
as the method of solubilizing the liquid if partial crystallization has occurred.
830
Additional information. Even in the absence of light, the oily form of Retinol
concentrate is gradually degraded on exposure to a humid atmosphere, the
Partial crystallization may occur in concentrated solutions and upon

refrigeration.
Requirements
Definition. The oily form of Retinol concentrate consists of an ester or a
mixture of esters (acetate, propionate, or palmitate) of retinol (C20H30O), usually
prepared by synthesis. It may be diluted in a suitable vegetable oil. It
may contain suitable antimicrobial agents and stabilizing agents such as

antioxidants.
The declared content of retinol is not less than 500000IU/g. Retinol concen-
trate contains not less than 95.0% and not more than 110.0% of the amount
of C20H30O stated on the label.
Identity tests
cyclohexane R and 2 volumes of ether R as the mobile phase. Apply sepa-
rately to the plate 2 pi of each of 4 solutions in cyclohexane R containing
(A) 2 mg of Retinol per ml, (B) 2 mg of retinol acetate RS per ml, (C) 2 mg of
retinol propionate RS per ml, and (D) 2 mg of retinol palmitate RS per ml.
in air, and spray with antimony trichloride TS. Examine the chromatogram
in daylight.
The with solution A corresponds to one or more of

the spots obtained with solutions B, C, and D.
B. Dissolve a small drop in about 1ml of dichlorome thane R and add 5ml of
antimony trichloride TS; a blue colour is immediately produced which turns
gradually to violet-red.
Peroxides. For solution (A) dissolve 0.30 g in 25ml of a mixture of 4 volumes

of methanol R and 6 volumes of toluene R. For solution (B) prepare a solution
containing 0.27 g of ferric chloride R per ml, and add 1 .0 ml to 99 ml of a mixture
of 4 volumes of methanol R and 6 volumes of toluene R. Dilute 2.0 ml to
100ml with the same solvent mixture.
831
Place in 2 separate test-tubes in the following order, mixing after each addition,
3 ml of a solution containing 18 mg of ammonium thiocyanate R per ml, 10 ml
of methanol 0.3ml of ferrous sulfate/hydrochloric acid TS, and 15ml of
R,
toluene R. Then add 1,0ml of solution A into one tube and 1.0 ml of solution
B into the other, shake, and allow to stand for 5 minutes. The colour produced
with solution A is not more intense than that produced with solution B.
Assay
Carry out the assay as rapidly as possible, avoiding exposure to actinic light
and oxidizing agents, and maintaining whenever possible an atmosphere of
nitrogen above the solution.
The spectrophotometric measurements should be made at 20-25 °C. Before

each series of measurements, check the wavelength scale of the spectropho-
tometer as well as the absorbance scale. The cells filled with 2-propanol R must
not differ from each other in absorbance by more than 0.002 at each of the fol-
lowing wavelengths: 300 nm, 325 nm, 350 nm, and 370nm.
Carry out each determination in duplicate, using separately weighed amounts

of Retinol concentrate. Prepare a dilution series containing 25-lOOmg of Retinol
concentrate in 5 ml of K-pentane R and dilute with 2-propanol R to a presumed
concentration of 10-15 lU per ml. Verify that the absorption maximum of the
solution to be examined, measured against a solvent cell containing 2-propanol
between 325 nm and 327 nm. Measure the absorbances at 300 nm,
R, lies
326nm, 350 nm, and 370nm. Repeat the readings at each wavelength and take
the mean values. Calculate the ratio A/A326 for each wavelength. If the ratios
do not exceed 0.592 at 300nm, 0.537 350 nm, and 0.142 at 370nm, calculate
at
the content of retinol in International Units per gram from the expression: A 326
X V X 1900/100 w, where A 326 is the absorbance at 326 nm, V is the total volume
used for the dilution to give 10-15 lU per ml, m is the mass of Retinol concen-
trate in g, and 1900 is the factor to convert the specific absorbances of ester of
retinol into lU per g.
RIBOFLAVINUM
RIBOFLAVIN
832
H
Graphic formula.
CHjOH
HO —C —H
HO — C —
HO — C — H
I
Chemical name. 7,8-Dimethyl-10-(D-nl7o-2,3,4,5-tetrahydroxypentyl)isoal-

loxazine; CAS Reg. No. 83-88-5.
Description. A yellow to orange-yellow, crystalline powder; odourless or

almost odourless.

(-750 g/1) TS, ether R, and acetone R.
Category. Vitamin.
Storage. Riboflavin should be kept in a tightly closed container, protected

from light.
Additional information. Riboflavin has a bitter taste. Solutions of Riboflavin,

especially in dilute solutions of alkalis, deteriorate rapidly when exposed to
light.
Requirements
Definition. Riboflavin contains not less than 98.0% and not more than
Identity tests
infrared region.
spectrum obtained from riboflavin RS or with the reference spectrum of
riboflavin.
B. Dissolve Img in 100ml of water. The solution has a pale greenish yellow
colour by transmitted light and an intense yellowish green fluorescence
833
by reflected light. The addition of mineral acids or alkalis destroys the

fluorescence.
15mg/g.
Lumiflavin. Shake 25 mg with 10 ml of ethanol- free chloroform R for 5

minutes and Measure the absorbance of the filtrate in a 1-cm layer at the
filter.
maximum about 440 nm against a solvent cell containing ethanol-free chlo-

at
roform R; the absorbance does not exceed 0.025 (preferably use 2-cm cells for
the measurement and calculate the absorbance of a 1-cm layer).
Assay
• Carry out the operations in subdued light.
To about 0.075 g, accurately weighed, add 5 ml of water to ensure that the sub-
stance is completely wetted. Then add 5 ml of sodium hydroxide (~80g/l) TS.
As soon as this is completely dissolved, add 100 ml of water and 2.5 ml of glacial
acetic acid R,and sufficient water to produce 1000 ml. To 10 ml of this solu-
tion, add 1 ml of sodium acetate (50 g/1) TS and sufficient water to produce
50ml. Measure the absorbance of a 1-cm layer of the diluted solution at the
maximum at about 444 nm. Calculate the amount of C17H20N4O6 in the sub-
stance being tested by comparison with riboflavin RS, similarly and concur-
rently examined.
RIFAMPICINUM
RIFAMPICIN
834
Graphic formula.
OH
Chemical name. {2S,\2ZMEA6S,\7S,\?,R,19R,2QR,21S,22S,2?,S,2AE)-5,6,9,

17,19,21-Hexahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-[N-(4-
m ethyl- 1 - pipe razinyl) form imidoyl] -2, 7 -(epoxy pentad eca[l ,11,13]-
trienimino)naphtho[2,l-J’]-furan-l,l l-(2ff-dione, 21-acetate; 3-[[(4-methyl-l-
piperazinyl)imino]methyl]rifamycin; CAS Reg. No. 13292-46-1.
Other name. Rifampin.
Description. A brick red to red-brown, crystalline powder; odourless or

almost odourless.
Solubility. Very slightly soluble in water; freely soluble in chloroform R;

soluble in methanol R; slightly soluble in acetone R, ethanol (-750 g/1) TS, and
ether R.
Category. Antileprosy drug; antituberculosis drug.
Storage. Rifampicin should be kept in a tightly closed container, protected

from light and stored at a temperature not exceeding 15 °C, or in an atmos-
phere of nitrogen at a temperature not exceeding 30 °C.
Requirements
Definition. Rifampicin contains not less than 97.0% and not more than
Identity tests
infrared region.
spectrum obtained from rifampicin RS or with the reference spectrum of
rifampicin.
835
B. Dissolve50mg in 50ml of methanol R and dilute 1ml of this solution to

50ml with phosphate buffer, pH 7.4, TS. The absorption spectrum of
4 maxima at about 237nm, 254 nm, 334 nm, and 475nm; the ratio of the
absorbance of a 1-cm layer at the maximum at about 334 nm to that at the
maximum at about 475nm is about 1.75.
C. Suspend 25 mg in 25ml of water, shake for 5 minutes and filter. To 5 ml of

the nitrate add 1 ml of ammonium persulfate/phosphate buffer TS and shake
for a few minutes; the colour turns from orange-yellow to violet-red without
the formation of a precipitate.
Heavy metals. Place l.Og in a silica crucible and mix it with 4 ml of magne-
sium Heat cautiously to ignition and continue heating
sulfate/sulfuric acid TS.
until a white or at most greyish residue is obtained. Ignite at a temperature not
exceeding 800 °C, allow to cool, and moisten the residue with a few drops of
sulfuric acid (~100g/l) TS. Evaporate, ignite again, and allow to cool. Next, dis-
solve the residue in hydrochloric acid (-70g/l) TS, add, drop by drop, a solu-
tion of ammonia (-100 g/1) PbTS, until the pH of the solution is between 8 and
8.5, then add, also drop by drop, acetic acid (-60 g/1) PbTS to adjust the pH to
3-4, filter, dilute with water to 40 ml, and mix. Determine the heavy metals
content as described under 2.2.3 Limit test for heavy metals, according to
Method A; not more than 20pg/g.

pH value. Shake O.lOg with 10ml of carbon-dioxide-free water R; pH of the

suspension, 4.5-6.5.
chromatography, using silica gel R1 as the coating substance and preparing the
slurry with phosphate/citrate buffer pH 6.0, TS. As the mobile phase use a
mixture of 85 volumes of chloroform R and 15 volumes of methanol R. Apply
(A) 20mg of the test substance per ml, (B) O.lOmg of 3-formylrifamycin SV RS
per ml, (C) 0.30 mg of rifampicin quinone RS per ml, and (D) 0.20 mg of the
chamber, allow it to dry in air and examine the chromatogram in daylight. Any
coloured spots obtained with solution A, other than the principal spot, are not
more intense than the corresponding spots obtained with solutions B and C.
Any other spots obtained with solution A are not more intense than that
obtained with solution D.
836
Assay. Dissolve about O.lOg, accurately weighed, in sufficient methanol R to

produce 100 ml. Dilute 2 ml of this solution to 100 ml with phosphate buffer,
pH 7.4, TS. Measure the absorbance of the resulting solution in a 1-cm layer at
the maximum at about 475nm, using as the blank phosphate buffer, pH 7.4,
TS. Calculate the content of C 43H 58N 4 O 12 ,
using the absorptivity value of 18.7
(AIL = 187).
RITONAVIRUM
RITONAVIR
C37H48N6O5S2
Chemical name, thiazol-5-ylmethyl [(lS,2S,4S)-l-benzyl-2-hydroxy-4-[[(2S)-

3-methyl-2-[[methyl[[2-(l-methylethyl)thiazol-4-yl]methyl]
carbamoyl]amino]butanoyl]amino]-5-phenylpentyl]carbamate; CAS Reg.
NO. 155213-67-5.
Solubility. Practically insoluble in water, freely soluble in methanol R, spar-

ingly soluble in acetone R and very slighdy soluble in acetonitrile R.
Storage. Ritonavir should be kept in a well-closed container, protected from

light.
Additional information. Ritonavir may exhibit polymorphism.
837
Requirements
Ritonavir contains not less than 98 5 % and not more than 101 0 %
. . of
C37H48N6O5S2, calculated with reference to the dried substance.
Identity tests
A.l. Carry out the test as described under 1.14.1 Thin-layer chromatography,
dichlorome thane R, 20 volumes of acetonitrile R, 10 volumes of methanol R
and 3 volumes of ammonia (-260 g/1) TS as the mobile phase. Apply separately
to the plate 1 0 |il of each of 2 solutions in methanol containing (A) 5 mg of the
test substance per ml and (B) 5 mg of ritonavir RS per ml. After removing the
plate from the chromatographic chamber, allow it to dry exhaustively in a
current of cool air. Examine the chromatogram in ultraviolet light (254 nm).

phy, using silica gel R5 and a mixture of 67 volumes
of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol
R and 3 volumes of ammonia (-260 g/1) TS mobile phase. Apply sep-
as the
arately to the plate 10 pi of each of 2 solutions in methanol containing (A)
5 mg of the test substance per ml and (B) 5 mg of ritonavir RS per ml. After
removing the plate from the chromatographic chamber, allow it to dry
exhaustively in a current of cool air. Spray with basic potassium perman-
ganate (5 g/1) TS. Examine the chromatogram in daylight.
The principal spot obtainedwith solution A corresponds in position, appear-


observed between 220 nm and 280 nm, exhibits one maximum at about
240 nm; the specific absorbance (Al*m) is 116 to 128.

spectrum obtained from ritonavir RS or with the reference spectrum of riton-
avir. Ifthe spectra obtained in the solid-state show differences, dissolve the
test substance and the reference substance separately in a minimal amount
of methanol R, crystallise by adding just enough water drop by drop, filter
and dry for about one hour and record the spectra again.
838
Specific optical rotation. Use a 20.0mg/ml solution in methanol R; [a]o =

+7° to +10°.
mine the heavy metals content according to method A; not more than 20pg/g.
Loss on drying. Dry for 2 hours at 105 °C; it loses not more than 5mg/g.
raphy R (5 pm).
Mobile phase A: 35 volumes of acetonitrile R, 28 volumes sodium phosphate

buffer pH 4.0 and 37 volumes of purified water.
Mobile phase B: 70 volumes of acetonitrile R, 28 volumes sodium phosphate

buffer pH 4.0 and 2 volumes of purified water.
Prepare the sodium phosphate buffer pH 4.0 by dissolving 7.8 g of sodium dihy-
drogen phosphate dihydrate R and 1.88g of sodium hexanesulfonate R in
800 ml of purified water, adjust the pH to 4.0 by adding phosphoric acid
(-105 g/1) TS and dilute to 1000 ml with purified water.

(min) (% v/v) (% v/v)
(1) use 0.5 mg of the test substance per ml. For solution (2) dilute a suitable
volume of solution (1) to obtain a concentration equivalent to 0.5 pg of riton-
avir per ml.
and 1 ml of sulfuric acid (475g/l), heat in a boiling water bath for 20 minutes.
839
Inject 20|il of solution (3). The test is not valid unless the resolution between
the principal peak (retention time about 22 minutes) and the peak with a rela-
tive retention of about 0.8 is not less than 3.5. The test is also not valid unless
the resolution between the principal peak and the peak with a relative reten-
tion of about 1.5 is not less than 9.0. If necessary adjust the amount of ace-
tonitrile in both mobile phases A and B, or adjust the gradient programme.
In the chromatogram obtained with solution the area of any peak, other
(1),
than the principal peak, is not greater than three times the area of the princi-
pal peak obtained with solution (2) (0.3%). In the chromatogram obtained with
solution (1), the areas of not more than two peaks, other than the principal
peak, are greater than twice the area of the principal peak obtained with solu-
tion (2) (0.2%) and the areas of not more than four such peaks are greater than
the area of the principal peak obtained with solution (2) (0.1%). The sum of
the areas of all peaks, other than the principal peak, is not greater than ten times
the area of the principal peak obtained with solution (2) (1.0%). Disregard any
peak with an area less than 0.5 times the area of the principal peak in the chro-
matogram obtained with solution (2) (0.05%).
Assay. Dissolve 0.25 g, accurately weighed, in 30ml of glacial acetic acid R1

and titrate with perchloric acid (0.1 mol/1) VS, determine the end point poten-
tiometrically as described under 2.6 Non-aqueous titration Method A. Each ml
of perchloric acid (0.1 mol/1) VS is equivalent to 36.05 mg of C 37 H 4aN(;05 S 2 .
SACCHARINUM NATRICUM
SACCHARIN SODIUM
Saccharin sodium, anhydrous
Saccharin sodium, dihydrate
840
n = 0 (anhydrous)
n = 2 (dihydrate)
C7H4NNa03S (anhydrous)
C7H4NNa03S,2H20 (dihydrate)
Chemical name. l,2-Benzisothiazolin-3-one 1,1-dioxide, sodium salt; 1,2-

benzisothiazol-3(2f/)-one 1,1-dioxide, sodium salt; CAS Reg. No. 128-44-9
(anhydrous).
l,2-Benzisothiazolin-3-one 1,1-dioxide, sodium salt, dihydrate; 1,2-benzisoth-

iazol-3(2H)-one 1,1-dioxide, sodium salt, dihydrate; CAS Reg. No. 6155-57-3
(dihydrate).
Other name. Saccharimidum natricum.

almost odourless.
Category. Sweetening agent.
Storage. Saccharin sodium should be kept in a well-closed container.
Additional information. Saccharin sodium effloresces slowly in air and loses

about half of its content of water of crystallization. It has a very sweet taste,
even in very dilute solutions.
Requirements
Saccharin sodium contains not less than 98 0 % and not more than the equiv-
.
alent of 101 0 % of C7H4NNa03S, calculated with reference to the anhydrous

.
substance.
Identity tests
A. To 20mg add 0.04g of resorcinol R and 0.5ml of sulfuric acid (-1760 g/1) TS,
and heat gently until a dark green colour is observed. Allow to cool and add
10ml of water and 10ml of sodium hydroxide (~80g/l) TS; a fluorescent
green solution is produced.
B. Ignite 1 g and proceed with the residue as follows;
841
— Dissolve half of the residue in acetic acid (~60g/l) TS. When tested for
sodium as described under 2.1 General identification tests it yields reac-
tion B.
— Dissolve the remaining residue in hydrochloric acid (~70g/l) TS. It yields

reaction A described under 2.1 General identification tests as character-
istic of sulfates.
Arsenic. Transfer 3.3g to a crucible containing 3.3g of anhydrous sodium car-

bonate R. Moisten with a small quantity of water, evaporate to dryness on a
water-bath, and ignite to 550 °C until all black particles have disappeared. Cool,
dissolve the residue in 5 ml of hydrochloric acid (-250 g/1) AsTS, and proceed
as described under 2.2.5 Limit test for arsenic; the arsenic content is not more
than 3pg/g.

Fischer method. Method A, using 1 g of Saccharin sodium dihydrate; the water
Free acid or alkali. Dissolve 1 g in 10 ml of carbon-dioxide-free water R, add

5 ml of sulfuric acid (0.005 mol/1) VS, boil, cool, and titrate with sodium hydrox-
ide (0.01 mol/1) VS using phenolphthalein/ethanol TS as indicator; 4.5-5.5ml
are required to obtain a pink colour.
100 volumes of chloroform R, 50 volumes of methanol R, and 10 volumes of
ammonia (-260 g/1) TS as the mobile phase. Apply separately to the plate 5|xl
of each of the following four solutions: For solution (A) dissolve 2.6g of Sac-
charin sodium in 10ml of sodium hydrogen carbonate (lOOg/1) TS, add 12.5g
of diatomaceous support R as a filter-aid, and mix well. Transfer to a chro-
matographic tube, 250 mm
in length and with a diameter of 25mm, fitted at
the lower end with a sintered-glass disc and a stopcock. Pack the contents of
the tube by tapping on a padded surface and tamping firmly from the top. Elute
with dichloromethane R at a rate of 50 ml in 30 minutes. Evaporate the eluate
to dryness and dissolve the residue in 4 ml of acetone R. For solutions (B) dis-
solve 50 pg of toluene-2-sulfonamide RS per ml of acetone R, (C) 5 mg of Sac-
charin sodium per ml of methanol R, and (D) 50 pg of 4-sulfamoylbenzoic acid
R per ml of acetone R. After removing the plate from the chromatographic
chamber, dry in a current of warm air, heat at 105°C for 5 minutes, and spray
the hot plate with sodium hypochlorite TSl. Dry in a current of cold air until
a sprayed area of the plate below the line of application gives at most a faint
842
blue colour with 0.05 ml of a solution of 5 mg of potassium iodide R in 1 ml of

starch TS containing 1 % glacial acetic acid R. Avoid prolonged exposure to cold
air. Spray the plate again with the same mixture. Examine the chromatogram
in daylight.
Any spot obtained with solution A corresponding to toluene-2-sulfonamide is
not more intense than that obtained with solution B. Any spot obtained with
solution C corresponding to 4-sulfamoylbenzoic acid is not more intense than
that obtained with solution D.
aqueous titration, Method A.

C 7 H 4NNa 03 S.
SALBUTAMOLI SULFAS
SALBUTAMOL SULFATE
Molecular formula. (Ci 3 H 2 iN 03 ) 2 ,H 2 S 04
Graphic formula.
HOCHj
Chemical name. a'-[(tcrt-Butylamino)methyl]-4-hydroxy-m-xylene-a, a'-diol

sulfate (2:1) (salt); a’-[[(l,l-dimethylethyl)amino]methyl]-4-hydroxy-l,3-ben-
zenedimethanol sulfate (2:1) (salt); CAS Reg. No. 51022-70-9.
Solubility. Soluble in 4 parts of water; slightly soluble in ethanol (-750 g/1) TS,
and ether R.
843
Storage. Salbutamol sulfate should be kept in a well-closed container, pro-

tected from light.
Additional information. Even in the absence of light, Salbutamol sulfate is

Requirements
Definition. Salbutamol sulfate contains not less than 98.0% and not more
than 101.0% of Ci 3 H 2 iN 03 ,V2 H 2 S 04 calculated with reference to the dried
,
substance.
Identity tests
• Either tests A and D or tests B, C and D may be appHed.

spectrum obtained from salbutamol sulfate RS or with the reference spectrum
of salbutamol sulfate.

maximum atabout 276nm; the absorbance of a 1-cm layer at this wave-
C. Dissolve O.OSg in 5ml of water and add 0.1 ml of ferric chloride (25g/l) TS;
a reddish violet colour is produced. Add O.OSg of sodium hydrogen car-
bonate R; a fleshy precipitate is produced with an evolution of gas. Add a

few drops of sulfuric acid (-1760 g/1) TS; the solution becomes colourless.

4 volumes of ammonia (-260 g/1) TS, 16 volumes of water, 30 volumes of 2-
propanol R, and 50 volumes of ethyl acetate R as the mobile phase. Apply sep-
arately to the plate 5 pi of each of 2 solutions containing (A) 20 mg of the test
substance per ml and (B) O.lOmg of the test substance per ml. After removing
844
the platefrom the chromatographic chamber, allow it to dry in air until the sol-
vents have evaporated. Place the plate for a few minutes in an atmosphere sat-
urated with diethylamine R, spray it with diazotized sulfanilic acid TS, and
tion B.
R1 and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-
alent to 57.67 mg of Ci 3 H 2 iN 03 ,V2 H 2 S 04 .
Additional requirement for Salbutamot sulfate for

parenteral use
SALBUTAMOLUM
SALBUTAMOL
Molecular formula. Ci 3 H 2 iN 03
Graphic formula.
Chemical name. a'-[(tert-Butylamino)methyl]-4-hydroxy-w-xylene-a, a'-diol;

a‘- [[(1,1 -dimethylethyl)amino]methyl] -4-hydroxy- 1,3-benzene dimethanol;
CAS Reg. No. 18559-94-9.
Solubility. Soluble in 70 parts of water; soluble in ethanol (~750g/l) TS;

slighdy soluble in ether R.
845
Storage. Salbutamol should be kept in a well-closed container, protected from

light.
Requirements
Definition. Salbutamol contains not less than 98.0% and not more than
Identity tests

infrared region.
spectrum obtained from salbutamol RS or with the reference spectrum of
salbutamol.

maximum only at about 276nm; the absorbance of a 1-cm layer at this
wavelength is about 0.56.
C. Dissolve 0.05g in 5ml of water and add 0.1 ml of ferric chloride (25g/l) TS;
a reddish violet colour is produced. Add 0.05 g of sodium hydrogen car-
bonate R; a fleshy precipitate is produced with an evolution of gas. Add a
few drops of sulfuric acid (~1760g/l) TS; the solution becomes colourless.

4 volumes of ammonia (-260 g/1) TS, 16 volumes of water, 30 volumes of 2-
propanol R, and 50 volumes of ethyl acetate R as the mobile phase. Apply sep-
arately to the plate 5 |il of each of 2 solutions in methanol R containing (A)
20mg of the test substance per ml and (B) O.lOmg of the test substance per ml.
air until the solvents have evaporated. Place the plate for a few minutes in an
atmosphere saturated with diethylamine R, spray it with diazotized sulfanilic

acid TS, and examine the chromatogram in daylight. Any spot obtained with
with solution B.
846
R1 and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-
alent to 23.93 mg of C13H21NO3.
SAQUINAVIRI MESILAS
SAQUINAVIR MESILATE
C38H50N6O5.CH4O3S
Chemical name. (2S’)-N'-[(lS,21?)-l-benzyl-3-[(3S,4aS,8aS)-3-[(l,l-dimethylethyl)

carbamoyl] octahydroisoquinolin-2(lH)-yl]-2-hydroxypropyl] -2- [(quinolin-2-
ylcarbonyl)amino]butanediamide methanesulfonate;CAS Reg. No. 149845-06-7.
Solubility. Very slightly soluble in water and sparingly soluble in methanol R.
Storage. Saquinavir mesilate should be kept in a tightly-closed container, pro-

tected from light.
Additional information. Saquinavir mesilate is slightly hygroscopic.
Requirements
Definition. Saquinavir mesilate contains not less than 98.5% and not more
than 101 0 . % of C38H50N6O5.CH4O3S calculated with reference to the dried
substance.
847

Identity tests
volumes of dichloromethane R and 2 volumes of 2-propanol R as the
mobile phase. Apply separately to the plate 5 pi of each of the follow-
ing 2 solutions in methanol R (A) 5 mg of the test substance per ml and
(B) 5 mg of saquinavir mesilate RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry exhaustively in air

or in a current of cool air. Examine the chromatogram in ultraviolet
light (254 nm).

mobile phase. Apply separately to the plate 5 pi of each of the follow-
ing 2 solutions in methanol R (A) 5 mg of the test substance per ml and
(B) 5 mg of saquinavir mesilate RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry exhaustively in air

or in a current of cool air. Dip the plate in basic potassium perman-
ganate (~lg/l) TS. Examine the chromatogram in daylight.


239 nm; the specific absorbance (Aj*,^) is 580 to 640.

infrared region.
spectrum obtained from saquinavir mesilate RS or with the reference spec-
trum of saquinavir mesilate.
848
late with reference to the dried substance; [«]d = -33 to -39°.
Heavy metals. Use 0.5 g in 30ml of methanol R for the preparation of the test
mine the heavy metals content according to Method A; not more than 20 pg/g.
Loss on drying. Dry for 5 hours at 105 °C; it loses not more than lOmg/g.
raphy R (5 pm).
Mobile phase A: 50 volumes of a mixture of 5 parts of acetonitrile R and 2 parts

of methanol R, 15 volumes of phosphate buffer pH 3.4 and 35 volumes of
purified water.


adding phosphoric acid (-105 g/1) TS and dilute to 1000 ml with purified water.

(min) (%) (%)

Prepare the following solutions using mobile phase A as diluent For solution
volume of solution (1) to obtain a concentration equivalent to 0.5 pg per ml.
and 5ml of sulfuric acid (475g/l), heat carefully in a boiling water-bath for 30
minutes.
849
the peak due to saquinavir (retention time about 21 minutes) and the peak of
similar size with a relative retention of about 0.45 is not less than 14. The test
is also not valid unless the resolution between two smaller peaks of similar size,
eluted after the saquinavir peak and which increase during decomposition, is
not less than 2.0. The relative retention of these two peaks is about 1.8 and
1.9, respectively. If necessary, adjust the amount of acetonitrile in both mobile
phases A and B, or adjust the gradient programme.
than the principal peak, is not greater than twice the area of the principal peak
obtained with solution (2) (0.2%) and the area of not more than one such peak
is greater than the area of the principal peak obtained with solution 2 (0.1%).
The sum of the areas of all peaks, other than the principal peak, is not greater
than five times the area of the principal peak obtained with solution (2) (0.5%).
Disregard any peak with an area less than 0.5 times the area of the principal
peak in the chromatogram obtained with solution (2) (0.05%).
Assay. Dissolve about 0.500 g, accurately weighed, in 70 ml of methanol R and

titrate with sodium hydroxide (0.1 mol/1) VS determining the end-point poten-
tiometrically. Perform a blank determination and make the necessary correc-

tion. Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to 76.70mg of
C38H50N6O5.CH4O3S; calculate with reference to the dried substance.
850
SAQUINAVIRUM
SAQUINAVIR
CasHsoNeOs
Chemical name. (2S)-Ai'-[(lS,27?)-l-benzyl-3-[(3S,4ah',8aS)-3-[(l,l-dimethylethyl)

carbamoyl]octahydroisoquinolin-2(lH)-yl]-2-hydroxypropyl]-2-[(quinolin-2-
ylcarbonyl)amino]butanediamide; CAS Reg. NO. 127779-20-8.
Solubility. Practically insoluble in water and soluble in methanol.
Storage. Saquinavir should be kept at 2-8 °C in a tightly closed container, pro-

tected from light.
Additional information. Saquinavir is slightly hygroscopic.
Requirements
Saquinavir contains not less than 98 5 . % and not more than 101 0 . % of
CasHsoNeOs, calculated with reference to the dried substance.
Identity tests
851

in methanol R containing (A) 5 mg of the test substance per ml and (B)
5 mg of saquinavir RS per ml. After removing the plate from the chro-
of cool air. Examine the chromatogram in ultraviolet light (254 nm).

in methanol R containing (A) 5 mg of the test substance per ml and (B)
5 mg of saquinavir RS per ml. After removing the plate from the chro-
of cool air. Dip the plate in dilute basic potassium permanganate (1 g/1)
TS. Examine the chromatogram in daylight.


238 nm; the specific absorbance (Al”™) is 670 to 730.

infrared region.
spectrum obtained from saquinavir RS or with the reference spectrum of
saquinavir.
Specific optical rotation. Use a 5.0 mg/ml solution in methanol R; =

-50° to -56°.
solution as described under 2.2.3 Limit test for heavy metals. Procedure 2; deter-
mine the heavy metals content according to Method A; not more than lOpg/g.
Loss on drying. Dry for 5 hours at 105 °C; it loses not more than 20mg/g.
852

raphy R (5 pm).
Mobile phase A: 50 volumes of a mixture of 5 parts of acetonitrile R and 2 parts

methanol R, 15 volumes of phosphate buffer pH 3.4 and 35 volumes of puri-
fied water.


adding phosphoric acid (-105 g/1) TS and dilute to 1000 ml with purified water.

(min) (% v/v) (% v/v)

volume of solution (1) to obtain a concentration equivalent to 0.5 pg of
saquinavir per ml.
and 5ml of sulfuric acid (475g/l), heat carefully in a boiling water-bath for 30
minutes.
the peak due to saquinavir (retention time about 21 minutes) and the peak of
similar size with a relative retention of about 0.45 is not less than 14. The test
is also not valid unless the resolution between two smaller peaks of similar size,
eluted after the saquinavir peak and which increase during decomposition, is
not less than 2.0. The relative retention of these two peaks is about 1.8 and
853
1.9, respectively. If necessary, adjust the amount of acetonitrile in both mobile

phases A and B, or adjust the gradient programme.
Inject alternatively 20 pi each of solutions (1) and (2),
In the chromatogram obtained with solution (1), the area of any peak, other
than the principal peak, is not greater than twice the area of the principal peak
obtained with solution (2) (0.2%) and the area of not more than one such peak
is greater than the area of the principal peak obtained with solution (2) (0.1%).
The sum of the areas of all peaks, other than the principal peak, is not greater
than five times the area of the principal peak obtained with solution (2) (0.5%).
Disregard any peak with an arealess than 0.5 times the area of the principal
peak in the chromatogram obtained with solution (2) (0.05%).
Assay. Dissolve 0.300 g, accurately weighed, in 50 ml of glacial acetic acid R1

and titrate with perchloric acid (0.1 mol/1) VS, determine the end point poten-
tiometrically as described under 2.6 Non-aqueous titration method A. Each ml
of perchloric acid (0.1 mol/1) VS is equivalent to 33.54 mg of CsaHsoNÔs; cal-
culate with reference to the dried substance.
SELENII DISULFIDUM
SELENIUM DISULFIDE
SeSa
Chemical name. Selenium sulfide; CAS Reg. No. 7488-56-4.
Description. A bright orange to reddish brown powder.
Solubility. Selenium disulfide is practically insoluble in water and organic

solvents.
Storage. Selenium disulfide should be kept in a well-closed container.
Requirements
Selenium disulfide contains not less than 52.0% and not more than 55.5% of
Se.
854
Identity tests
A. Gently boil 0.05 g with 5 ml of nitric acid (~1 000 g/1) TS for 30 minutes, dilute
to 50 ml with water, and filter. To 5 ml of the filtrate add 1 0 ml of water and
5g of urea R, boil, cool, and add 2.0ml of potassium iodide (80 g/1) TS; a
yellow to orange colour is produced which darkens rapidly on standing.
(Keep this solution for test B.)
B. Allow the coloured solution obtained in test A to stand for 10 minutes, and
filter through kieselguhr Rl. The filtrate yields the reactions described under
2.1 General identification tests as characteristic of sulfates.
Soluble selenium compounds. For solution A, use lOg of Selenium disul-

fide, add 100 ml of water, mix well, allow to stand for 1 hour with frequent
shaking, and filter. For solution B, use a solution of selenious acid R containing
5pg of selenium per ml. To 10ml of each of solutions A and B, add 2 ml of a
solution containing about 1 ml of formic acid (-1080g/l) TS in 10ml of water,
and dilute both solutions to 50 ml with water. If necessary, adjust the pH to 2.5
± 0.5 with the diluted formic acid as prepared above. Then add 2.0 ml of freshly
prepared 3,3'-diaminobenzidine tetrahydrochloride (5 g/1) TS, allow to stand for
45 minutes, and adjust thepH to 6.5 ± 0.5 with ammonia (-100 g/1) TS. Shake
both solutions for 1 minute with 10 ml of toluene R, and allow to separate.
Measure the absorbances of a 1-cm layer of the toluene layers at 420 nm against
a solvent cell containing the same reagents treated as described above. The
absorbance of solution A is not more than that of solution B (5 pg of Se per g).
Assay. To about weighed, add 25 ml of fuming nitric acid R,

0.1 g, accurately
heat on a water-bath for 1 hour, cool, and dilute to 100 ml with water. To
25ml of this solution add 50 ml of water and 5g of urea R, and heat to boiling.
Cool, add 7 ml of potassium iodide (80g/l) TS, 3ml of starch TS, and titrate
immediately with sodium thiosulfate (0.1 mol/1) VS. Repeat the procedure
without the Selenium disulfide being examined and make any necessary
corrections.
Each ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to 1.974mg of Se.
855
SENNAE FOLIUM
SENNA LEAF
Alexandrian Senna leaf
Tinnevelly Senna leaf
Description. Odour, slight; taste, first mucilaginous and sweet, then slighdy bitter.
Storage. Senna leaf should be kept protected from light and moisture.
Additional information. Even in the absence of light. Senna leaf is gradually

Requirements
Definition. Senna leaf consists of the dried leaflets of Cassia senna L., known
as Alexandrian or Khartoum Senna (C. acutifolia Defile) or Tinnevelly Senna
(C. angustifolia Vahl), or a mixture of both species.
Senna leaf contains not less than 2.5% of hydroxyanthracene derivatives, cal-
culated as sennoside B.
Identity test
To about 0.5 g of the powdered leaf add 10 ml of sodium hydroxide/ethanol TS
and on a water-bath, dilute with 10 ml of water, and filter. Acidify the
boil
with hydrochloric acid (~70g/l) TS and extract with 10ml of ether R.
filtrate
Separate the ether layer and shake it with 5 ml of ammonia (~100g/l) TS; a
yellowish red colour is produced in the ammonia layer.
Macroscopic examination
Alexandrian Senna leaf. Pale greyish green, thin, fragile leaflets; lanceolate,
mucronate; length, 20-40 mm; width, 5-15mm, the maximum width being at
a point slightly below the centre; lamina, slightly undulant; both surfaces
covered with fine, short trichomes; pinnate venation, slightly prominent midrib
with lateral veins leaving the midrib at an angle of about 60° and anastomos-
ing to form a ridge parallel to the margin.
Tinnevelly Senna leaf Yellowish green leaflets; elongated and lanceolate; length,
25-50 mm; width at the centre, 7-20 mm; lamina, flat; both surfaces are smooth
with a very small number of trichomes, and marked with impressed transverse
or oblique lines.
856
Microscopic examination. Epidermis with polygonal cells containing

mucilage; unicellular thick-walled trichomes, length, up to 260 pm, slightly
curved at the base, warty; paracytic stomata on both surfaces; under the epi-
dermal cells a single row of palisade layer; cluster crystals of calcium oxalate
distributed throughout the lacunose tissue; on the adaxial surface of the leaf,
sclerenchymatous fibres and a gutter-shaped group of similar fibres on the
abaxial side containing prismatic crystals of calcium oxalate.
Water-soluble extractive. Shake 5g with a mixture of 0.5 ml of chloroform

R and 200ml of water, filter, evaporate 20ml of the filtrate and weigh; the
residue is not less than 300mg/g.
Stems and foreign matter. Weigh about 200 g and spread it in a thin layer.
Detect the stems and the foreign matter by eye or with the use of a 6x lens;
separate and weigh individually the stems and the foreign matter; not more
than 20mg/g of stems and not more than lOmg/g of foreign matter.
Assay. Place 0.15g of the powdered leaf in a 100-ml flask. Add 30.0ml of
water, mix, weigh and place in a water-bath at 80-90 °C. Heat under a reflux
condenser for 15 minutes. Allow to cool, weigh, and adjust to the original mass
with water. Centrifuge and transfer 20.0 ml of the supernatant liquid to a
150-ml separating funnel. Add 0.1ml of hydrochloric acid (~70g/l) TS and shake
with 3 quantities, each of 15 ml, of chloroform R. Allow to separate and discard
the chloroform layer. Add O.lOg of sodium hydrogen carbonate R and shake
for 3 minutes. Centrifuge and transfer 10.0 ml of the supernatant liquid to a
lOO-ml round-bottomed flask with a ground-glass neck. Add 20ml of ferric
chloride (65g/l) TS and mix. Heat for 20 minutes under a reflux condenser in a
water-bath with the water level above that of the liquid in the flask; add 1 ml
of hydrochloric acid (-420 g/1) TS and heat for a further 20 minutes, with fre-
quent shaking, to dissolve the precipitate. Cool, transfer the mixture to a sep-
arating funnel and shake with 3 quantities, each of 25ml, of ether R previously
used to rinse the flask. Combine the ether layers and wash with 2 quantities,
each of 15 ml, of water. Transfer the ether layers to a volumetric flask and dilute
to 100ml with ether R. Evaporate 10.0ml carefully to dryness and dissolve the
residue in 10.0ml of a solution containing 5 mg of magnesium acetate R per ml
of methanol R. Measure the absorbance of this solution in a 1-cm layer at the
maximum at about 515 nm against a solvent cell containing methanol R. Cal-
culate in % the amount of hydroxyanthracene derivatives as sennoside B with
an absorptivity value of 24.0 = 240) as follows: 25 A/W, where A is the
absorbance at 515 nm and Wis the mass of the material examined in g.
857
SENNAE FRUCTUS
SENNA FRUIT
Alexandrian Senna firuit
Tinnevelly Senna firuit
Description. Odour, slight; taste, first mucilaginous and sweet, then slightly
bitter.
Storage. Senna fruit should be kept protected from light and moisture.
Additional information. Even in the absence of light, Senna fruit is gradu-

Requirements
Definition. Alexandrian Senna fruit is the dried ripe fruit of Cassia senna L.,
(C. acutifolia Defile) and Tinnevelly Senna fruit is the dried ripe fruit of Cassia
angustifolia Vahl.
Alexandrian Senna fruit contains not less than 3.4% of hydroxyanthracene

derivatives and Tinnevelly Senna fruit contains notless than 2.2% of hydrox-
yanthracene derivatives, both calculated as sennoside B.
Identity test
Cut powder about 0.5g, add 10ml of sodium
the fruit into small pieces or
hydroxide/ethanol TS and
boil on a water-bath, dilute with 10 ml of water and
filter. Acidify the with hydrochloric acid (~70g/l) TS and extract with
filtrate
10ml of ether R. Separate the ether layer and shake it with 5ml of ammonia
(~100g/l) TS; a pink to red colour is produced in the ammonia layer.
Macroscopic examination. Leaflike, flat and thin pods, yellowish green to

yellowish brown with a dark brown central area, oblong or reniform.
Alexandrian Senna fruit. Pale to greyish green; length, about 40-50 mm; width,
20-25 mm; stylar point at one end, containing 6-7 obovate green to pale brown
seeds, with longitudinal prominent ridges on the testa.
Tinnevelly Senna fruit. Brown to greyish black; length, about 35-60 mm; width,
14-1 8 mm; stylar point at one end, containing up to 10 obovate green to pale
brown seeds, with indefinite transverse ridges.
858
Microscopic examination. Epicarp with very thick cuticularized isodiamet-

rical cells, occasional anomocytic or faaracytic stomata, and very few unicellular
and warty trichomes; hypodermis with collenchymatous cells; mesocarp with

parenchymatous tissue containing a layer of calcium oxalate prisms; endocarp
consisting of thick-walled fibre, mostly perpendicular to the longitudinal axis
of the fruit, but the inner fibres running at an oblique angle or parallel to the
longitudinal axis. Seeds, subepidermal layer of palisade cells with thick outer
walls; the endosperm has polyhedral cells with mucilaginous walls.
Water-soluble extractive. Shake 5g with a mixture of 0.5 ml of chloroform

R and 200ml of water, filter, evaporate 20ml of the filtrate and weigh; the
residue is not less than 250mg/g.
Foreign matter. Weigh about 200 g and spread it in a thin layer. Detect the
foreign matter by eye or with the use of a 6x lens, separate and weigh; not
more than lOmg/g.
Assay. Place 0.15g of the powdered fruit in a 100-ml flask. Add 30.0ml of
water, mix, weigh, and place in a water-bath at 80-90 °C. Heat under a reflux
condenser for 15 minutes. Allow to cool, weigh, and adjust to the original mass
with water. Centrifuge and transfer 20.0 ml of the supernatant liquid to a 150-
ml separating funnel. Add 0.1ml of hydrochloric acid (~70g/l) TS and shake
with 3 quantities, each of 15 ml, of chloroform R. AUow to separate and discard
the chloroform layer. Add O.lOg of sodium hydrogen carbonate R and shake
for 3 minutes. Centrifuge and transfer 10.0 ml of the supernatant liquid to a
100-ml round-bottomed flask with a ground-glass neck. Add 20ml of ferric
chloride (65g/l) TS and mix. Heat for 20 minutes under a reflux condenser in a
water-bath with the water level above that of the liquid in the flask; add 1 ml
of hydrochloric acid (7.420 g/1) TS and heat for a further 20 minutes, with fre-
quent shaking, to dissolve the precipitate. Cool, transfer the mixture to a sep-
arating funnel and shake with 3 quantities, each of 25ml, of ether R previously
used to rinse the flask. Combine the ether layers and wash with 2 quantities,
each of 15 ml, of water. Transfer the ether layers to a volumetric flask and dilute
to 100ml with ether R. Evaporate 10.0ml carefully to dryness and dissolve the
residue in 10.0ml of a solution containing 5 mg of magnesium acetate R per ml
of methanol R. Measure the absorbance of this solution in a 1-cm layer at the
maximum at about 515 nm against a solvent cell containing methanol R. Cal-
culate in % the amount of hydroxyanthracene derivatives as sennoside B with
an absorptivity value of 24.0 = 240) as follows: \.25A/W, where A is the
absorbance at 515 nm and W is the mass of the material examined in g.
859
SPECTINOMYCINI HYDROCHLORIDUM
SPECTINOMYCIN HYDROCHLORIDE
Molecular formula. Ci4H24N207,2HCl,5H20
Graphic formula.
• 2HCI • 5HjO
Chemical name. (2i?,4ai?,5ai?,6S,7S,8i?,9S,9al?,10aS)-Decahydro-4a,7,9-trihy-

droxy-2-methyl-6,8-bis(methylamino)-4/f-pyrano[2,3-i’][l,4]benzodioxin-4-
one dihydrocjfiloride pentahydrate; [2R-(2a,4aP,5aP,6P,7P,8[3,9a,9aa,10aP)]-
decahydro-4a,7,9-trihydroxy-2-methyl-6,8-bis(methylamino)-4/f-pyrano[2,3-
1?][1,4] benzodioxin-4-one dihydrochloride pentahydrate; CAS Reg. No. 22189-
32-8 (pentahydrate).

TS, chloroform R and ether R.
Storage. Spectinomycin hydrochloride should be kept in a well-closed

container.
Requirements
Definition. Spectinomycin hydrochloride contains not less than 600 pg of
C14H24N2O7 per mg, calculated with reference to the anhydrous substance.

for histamine-like substances (vasodepressor substances), using 1 ml per kg
of body mass of a solution in saline TS containing 25 mg of the substance

to be examined per ml.
860
Identity tests
spectrum obtained from Spectinomycin hydrochloride RS or with the refer-
ence spectrum of Spectinomycin hydrochloride.
B. A 20 mg/ml solution yields reaction B described under 2.1 General identifi-

Specific optical rotation. Use a O.lOg/ml solution, and calculate with refer-
ence to the anhydrous substance; [ajo’"'" = -1-15 to -1-21 °.

content is not less than 0.16g/g and not more than 0.20g/g.
pH value. pH of a O.lOg/ml solution, 3. 8-5. 6.
10 volumes of 1-propanol R, 8 volumes of water, 1 volume of glacial acetic acid
R, and 1 volume of pyridine R as the mobile phase. Apply separately to the
plate 10 pi of each of 2 solutions containing (A) 20 mg of the test substance per
ml and (B) 0.20 ml of the test substance per ml. After removing the plate from
the chromatographic chamber, allow it spray it with potassium
to dry in air,
permanganate (25g/l) TS, allow it to stand for 2-3 minutes, and examine the

membrane filtration test procedure.
Assay. Carry out the assay described under 1.14.5 Gas chromatography. As
an internal standard use a solution containing 2 mg of triphenylantimony R per
ml of dimethylformamide R. Use the following 2 solutions: (1) to about 30 mg
of spectinomycin hydrochloride RS, accurately weighed, add 10.0 ml of the
internal standard and 1.0ml of hexamethyldisilazane R, and shake intermit-
tently for 1 hour; and (2) to 30 mg of the substance being examined add
10.0ml of the internal standard and 1.0ml of hexamethyldisilazane R, and shake
intermittently for 1 hour. For the procedure use a flame ionization detector and
a glass column 1.3m long and 0.4 cm in internal diameter packed with an ade-
quate quantity of an adsorbent composed of 5 g of phenyl/methylpolysiloxane
R supported on 95 g of acid-washed and base-washed, silanized kieselguhr R4.
Maintain the column and the detector at about 215 °C and 270 °C, respectively.
861
and the injection part at about 265 °C. Use dry helium R as the carrier gas at a
flow rate of about 90 ml per minute. Prepare chromatogram A and B injecting
separately about 2.5 pi of each of solutions 1 and 2. Measure the area of the
major peak in each chromatogram, and calculate the content in pg of
C] 4 H 24 N 207 per mg in the substance being tested.
Additional requirement for Sfaectinomycin hydrochloride for

prarenteral use
spectinomycin.
SPIRONOLACTONUM
SPIRONOLACTONE
Molecular formula. C24H32O4S
Graphic formula.
Chemical name. 17-Hydroxy-7a-mercapto-3-oxo-17a-pregn-4-ene-21-car-

boxylic acid y-lactone acetate; 7a-(acetylthio)-17-hydroxy-3-oxo-17a-pregn-4-
ene-21 -carboxylic acid y-lactone; CAS Reg. No. 52-01-7.
Description. A light yellowish white to light yellowish brown powder;

odourless or with a faint characteristic odour.
Solubility. Practically insoluble in water; soluble in ethanol (~750g/l) TS.
862
Category. Diuretic.
Storage. Spironolactone should be kept in a well-closed container, protected

from light.
Additional information. Spironolactone may show preliminary melting at

about 135 °C, followed by resolidification.
Requirements
Definition. Spironolactone contains not less than 97.0% and not more than
101.5% of C24H32O4S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from Spironolactone RS or with the reference spectrum of
Spironolactone.

C. Shake 10 mg with 2 ml of sulfuric acid (-700 g/1) TS; an orange solution with
an intense yellowish green fluorescence is produced. Heat the solution
gently; the colour changes to deep red and hydrogen sulfide, which black-
ens lead acetate paper R, is evolved. Pour the solution into water; a green-
ish yellow, opalescent solution is produced.
Specific optical rotation. Use a lOmg/ml solution in chloroform R: [otlo”''" =

-33.0 to -37.0°.
Chromium. Place 0.20 g with 1 g of potassium carbonate R and 0.3 g of potas-

sium nitrate R into a platinum crucible; mix, heat gently until fused, and ignite
at 600-650 °C until the carbon is removed. Cool, dissolve the residue as com-
pletely as possible in 10 ml of water using gentle heat, filter, and dilute with suf-
ficient water to produce 20ml. To 10 ml add 0.5 g of urea R and acidify with
sulfuric acid (-190 g/1) TS, dilute to 20 ml with water, and add 0.5 ml of diphenyl-
carbazide TS. The colour produced is not deeper than that of a solution obtained
by adding 1 ml of sulfuric acid (-190 g/1) TS to 0.50 ml of a freshly prepared solu-
tion containing 2.83 mg of potassium dichromate R in 100ml of water, then
diluting to 20 ml with water and adding 0.5ml of diphenylcarbazide TS.
863
5-Omg/g.
from a commercial source is suitable) and butyl acetate R as the mobile phase.
Apply separately to the plate 5 pi of each of 2 solutions in chloroform R con-
taining (A) 20 mg of the test substance per ml and (B) 0.20 mg of the test sub-
stance per ml. After removing the plate from the chromatographic chamber,
allow it to dry at room temperature and develop the plate a second time allow-
ing the solvent to ascend 15 cm above the line of application. Remove the plate,
allow the solvent to evaporate at room temperature, spray it with sulfuric
acid/methanol TS, and heat at 105°C for 10 minutes. Examine the chro-
Mercapto compounds. Shake 2.0 g with 20ml of water, filter, and titrate
10ml of the filtrate with iodine (0.005mol/l) VS, using starch TS as indicator.
Repeat the operation without the test substance and make any necessary cor-
rections. Not more than 0.1ml of iodine (0.005mol/l) VS is required.

produce 100 ml and dilute 10 ml of this solution to 100 ml with methanol R.
about 238 nm, and calculate the content of C 24 H 32 O 4 S, using the absorptivity
value of 47 (A!°cm = 470).
STIBII NATRII TARTRAS

ANTIMONY SODIUM TARTRATE
Molecular formula. C 4 H 4Na 07 Sb
864
H
Graphic formula.
,0 — c=o
Na^ -Sb'— 0 — C—
\q — C— I
o — c=o
1
Chemical name. Sodium aqua[tartrato(4 )-0',0^,0^]antimoniate(III); CAS

Reg. No. 34521-09-0.
Description. Colourless, transparent scales or an almost white powder;

odourless.

(-750 g/1) TS.
Storage. Antimony sodium tartrate should be kept in a tightly closed con-

Additional information. Antimony sodium tartrate is hygroscopic. Even in

Requirements
Definition.Antimony sodium tartrate contains not less than 98.0% and not
more than 101.0% of C 4 H 4NaO/Sb, calculated with reference to the dried
substance.
Identity tests

lOmg/ml solution.
B. Dissolve 0.05g in 1.0ml of hydrochloric acid (~70g/l) TS and add 1.0ml of

hydrogen sulfide TS; an orange-red precipitate is produced, which is soluble
in ammonium sulfide TS and in sodium hydroxide (-80 g/1) TS.
C. Dissolve 20 mg in 0.2ml of water; it yields reaction B described under 2.1

General identification tests, as characteristic of tartrates.
865
Arsenic. Dissolve 1.3g in 10ml of water and add 16ml of stannated

hydrochloric acid (-250 g/1) AsTS in a flask, connect to a condenser, and distil
20 ml; wash the flask and condenser, return the distillate and washings to the
flask, add 0.05ml of stannous chloride AsTS, and redistil 16ml; proceed with
the distillate as described under 2.2.5 Limit test for arsenic; the arsenic content
is not more than 8pg/g.

and colourless.
60mg/g.
Acidity or alkalinity. Dissolve 1.0 g in 50 ml of water and add a few drops of

bromocresol green/ethanol TS; not more than 2 ml of either hydrochloric acid
(0.01 mol/1) VS or sodium hydroxide (0.01 mol/1) VS are required to obtain the
midpoint of the indicator (green) indicative of pH 4.5.
Assay. Dissolve about 0.5 g, accurately weighed, in 50ml of water, add 5g of

potassium sodium tartrate R and 2g of sodium tetraborate R. Titrate with
iodine (0.05 mol/1) VS using starch TS as indicator, added towards the end of
the titration. Each ml of iodine (0.05mol/l) VS is equivalent to 15.44mg of
C 4 H 4Na 07 Sb.
STREPTOMYCINI SULFAS
STREPTOMYCIN SULFATE
Streptomycin sulfate (non-injectable)
Streptomycin sulfate, sterile
Molecular formula. (C 2 iH 39 N 70 i 2) 2 , 3 H 2 S 04
866
Graphic formula.
Chemical name. 0-2-Deoxy-2-(methylamino)-a-L-glucopyranosyl-(l—>2)-0-

5-deoxy-3-C-formyl-a-L-lyxofuranosyl-(l—>4)-N,N'-bis(aminoiminomethyl)-
D-streptamine sulfate (2:3) (salt);CAS Reg. No. 3810-74-0.
Description. A white or almost white powder; odourless or with a slight

odour.
Solubility. Very soluble in water; practically insoluble in ethanol (-750 g/1) TS,
and ether R.
Storage. Streptomycin sulfate should be kept in a well-closed container and

protected from moisture.
Labelling. The designation sterile Streptomycin sulfate indicates that the sub-
stance complies with the additional requirements for sterile Streptomycin
sulfate and may be used for parenteral administration or for other sterile
applications.
Additional information. Streptomycin sulfate is hygroscopic, but it is stable

in air and on exposure to light.
867
Requirements
Definition. Streptomycin sulfate contains not less than 90.0% of
(C2 iH 39N70 2)2,3H2S04 and not less than 720 International Units per
i mg, both

for histamine-like substances (vasodepressor substances) using, per kg of
body weight, a solution containing 3 mg of streptomycin base in 1ml of
saline TS.
Identity tests
A. Dissolve 20 mg in 5 ml of water and boil for a few minutes with 10 drops of
sodium hydroxide (1 mol/1) VS; add 3 drops of hydrochloric acid (-250 g/1) TS
and 1 ml of ferric chloride (25 g/1) TS; an intense violet colour is produced.
B. Dissolve 0.1 g in 2ml of water, add 1ml of 1-naphthol TSl and 2ml of a
mixture of equal volumes of sodium hypochlorite (-40 g/1) TS and water; a
red colour is produced.


pared as described in 1.11 Colour of liquids.

Methanol. Transfer 0.2 g, accurately weighed, to a flask, dissolve in 5 ml of

water and add 0.05ml of sulfuric acid (0.05mol/l) VS; connect the flask to a dis-
tillation apparatus, distil and collect about 2.5ml of distillate in a 10-ml test-
tube. Transfer the distillate to a conical flask, rinsing the test-tube twice
with water, using 1ml each time, and add 25 ml of potassium dichromate
(0.0167mol/l) VS. Cautiously add 10ml of sulfuric acid (-1760 g/1) TS and heat
the resulting solution for 30 minutes on a water-bath; cool and dilute to about
500ml with water. Add 12.5ml of potassium iodide (80g/l) TS, allow to stand
for 5 minutes, and then titrate with sodium thiosulfate (0.1 mol/1) VS, using
starch TS as indicator, added towards the end of the titration, the endpoint
being reached when the dark blue colour turns pale green. Repeat the opera-
868
tion without the substance being tested; the difference between the volumes
used for the two titrations represents the amount of sodium thiosulfate
(0.1 mol/1) VS, equivalent to the methanol present. Each ml of sodium thiosul-
fate (0.1 mol/1) VS is equivalent to 0.534 mg of CH4O; the methanol content is
not more than 40mg/g, calculated as CH4O.
Assay
For streptomycin sulfate. Dissolve about O.lOg, accurately weighed, in
sufficient water to produce 100ml. To 5 ml add 5 ml of sodium hydroxide
(0.2 mol/1) VS and heat in a water-bath for exactly 10 minutes. Cool in ice for
exactly 5 minutes, add 3 ml of ferric ammonium sulfate TS2 and sufficient water
to produce 25ml, and mix. Exactly 20 minutes after the addition of the ferric
ammonium sulfate TS2 measure the absorbance of a 1-cm layer at the
maximum at about 525 nm, against a solvent cell containing a solution prepared
in the same manner but omitting the substance being examined. Calculate the
content of (C2iH39N70i2)2,3H2S04 in the substance, using the absorptivity value
of 1.18 (E!L = 11.8).
For potency. Carry out the assay as described under 3.1 Microbiological assay
of antibiotics, using either {a) Bacillus subtilis (NCTC 8236, or ATCC 11774) as
the test organism, culture medium Cml with a final pH of 7. 9-8.0, sterile phos-
phate buffer pH 8.0, TSl or TS2, an appropriate concentration of streptomycin
(usually between 5 and 20 lU), and an incubation temperature of 36-39 °C, or
(ATCC 6633) as the test organism, culture medium Cml with
{b) Bacillus subtilis
a final pH of 8. 0-8.1, sterile phosphate buffer pH 8.0, TSl or TS2, an appro-

priate concentration of streptomycin (usually between 3 and 15 lU), and an
incubation temperature of 35-37 °C. The precision of the assay is such that the
fiducial limits of error of the estimated potency (? = 0.95) are not less than 95%
error of the estimated potency (? = 0.95) is not less than 720 lU per mg, calcu-
lated with reference to the dried substance.
Additional Requirements for Streptomycin Sulfate for

sterile use
streptomycin.

membrane filtration test procedure. Use the sampling plan described under
869
SULFACETAMIDUM
SULFACETAMIDE
Molecular formula. CbHioNjOjS
Graphic formula.
Chemical name. iV-Sulfanilylacetamide; W-[(4-aminophenyl)sulfonyl]

acetamide; CAS Reg. No. 144-80-9.
Solubility. Very slighdy soluble in water; freely soluble in acetone R and

methanol R; soluble in ethanol (-750 g/1) TS.
Storage. Sulfacetamide should be kept in a well-closed container, protected

from light.
Requirements
Definition. Sulfacetamide contains not less than 99.0% and not more than
101.0% of CsHioNzOaS, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from Sulfacetamide RS or with the reference spectrum of
Sulfacetamide.
870
C. Dissolve O.lOg in 5ml of ethanol (~750g/l) TS, add about 0.2ml of sulfuric
acid (-1760 g/1) TS and heat; ethyl acetate, perceptible by its odour (proceed
Heavy metals. For the preparation of the test solution use 1.0 g dissolved in
a mixture of 10ml of water and 1.0ml of acetic acid (-300 g/1) TS, heat until
dissolved, cool, and filter. Dilute to 40ml with water and determine the heavy
metals content as described under 2.2.3 Limit test for heavy metals, according
to Method A; not more than 20|t.g/g.
Solution in alkali. Dissolve 0.5g in 10ml of sodium hydroxide (1 mol/1) VS;

the solution is clear or any opalescence produced is not more pronounced than
that of opalescence standard TS2, and the solution is not more intensely
Solution in acid. A solution of 1.0 g in 10 ml of hydrochloric acid (1 mol/1) VS

is clear or any opalescence produced is not more pronounced than that of
opalescence standard TS2.
5-Omg/g.
20 volumes of chloroform R, 2 volumes of methanol R, and 1 volume of
dimethylformamide R as the mobile phase. Apply separately to the plate 10 pi
of each of 3 solutions in a mixture of 9 volumes of ethanol (-750 g/1) TS and 1
volume of ammonia (-260 g/1) TS containing (A) 2.5 mg of the test substance
per ml, (B) 2.5mg of sulfacetamide RS per ml, and (C) 12.5 pg of sulfanilamide
RS per ml. After removing the plate from the chromatographic chamber, allow
it to dry in air until the solvents have evaporated. Spray the dried plate with
sulfuric acid/ethanol TS, heat it at 105°C for 30 minutes, and immediately

expose it to nitrous fumes in a closed chamber for 15 minutes (the nitrous fumes
may be generated by adding sulfuric acid (-700 g/1) TS drop by drop to a solu-
tion containing lOg of sodium nitrite R and 3g of potassium iodide R in
100 ml). Place the plate in a current of warm air for 15 minutes and spray it
with N-(Tnaphthyl)ethylenediamine hydrochloride/ethanol TS. If necessary,
allow it to dry before repeating the spraying and examine the chromatogram
is not more intense than that obtained with solution C.
871
0.5 g, accurately weighed, dissolved in a mixture of 20 ml of hydrochloric acid
(-250 g/1) TS and 50ml of water, and titrate with sodium nitrite (0.1 mol/1) VS.
Each ml of sodium nitrite (0.1 mol/1) VS is equivalent to 21. 42 mg of
CaH.oNÔsS.
SULFACETAMIDUM NATRICUM
SULFACETAMIDE SODIUM
Molecular formula. C8H9N2Na0sS,H20
Graphic formula.
Chemical name. N-Sulfanilylacetamide monosodium salt monohydrate; N-

[(4-aminophenyl)sulfonyl]acetamide monosodium salt monohydrate; CAS Reg.
Other name. Sulfacylum-natrium.
Description. A white or yellowish white, crystalline powder; odourless.
Solubility. Soluble in 1.5 parts of water; slightly soluble in ethanol (-750 g/1)
Storage. Sulfacetamide sodium should be kept in a well-closed container, pro-

tected from light.
Additional information. Even in the absence of light. Sulfacetamide sodium

Requirements
Definition. Sulfacetamide sodium contains not less than 99.0% and not more
than 101.0% of CsH 9N 2Na 03 S, calculated with reference to the anhydrous
substance.
872
Identity tests
A. Dissolve 1 g in 10ml of water and add 2ml of acetic acid (-300 g/1) TS; a
white precipitate is produced. Collect the precipitate on a filter, wash it with
cold water, and dry it at 105°C. Carry out the examination with the dried
residue as described under 1.7 Spectrophotometry in the infrared region.
The infrared absorption spectrum is concordant with the spectrum obtained
from Sulfacetamide RS or with the reference spectrum of sulfacetamide. Keep
the remaining precipitate for tests B and C.
B. Melting temperature of the precipitate obtained in test A, about 183°C

(sulfacetamide).
C. Dissolve 0.1 g of the precipitate obtained in test in 5ml of ethanol A

(-750 g/1) TS, add about 0.2 ml of sulfuric acid (-1760 g/1) TS, and heat;
ethyl acetate, perceptible by its odour (proceed with caution), is produced.
yields the characteristic reactions. If reaction B is to be used, ignite 0.5 g and

dissolve the residue in acetic acid (-60 g/1) TS.
Clarity and colour of solution. A solutionof 0.5g in 10 ml of carbon-dioxide-

free water R is clear and not more intensely coloured than standard colour solu-
tion Ywl when compared as described under 1.11 Colour of liquids.

pH value. pH of a 0.05 g/ml solution in carbon-dioxide-free water R, 8. 0-9. 5.
from a commercial source is suitable) and a mixture of 50 volumes of 1 -butanol
R, 25 volumes of dehydrated ethanol R, 25 volumes of water, and 10 volumes
5 pi of each of 3 solutions containing (A) O.lOg of the test substance per ml, (B)
0.50 mg of sulfanilamide RS per ml, and (C) 0.25 mg of sulfanilamide RS per ml.
air untilthe solvents have evaporated. Spray the plate with 4-dimethylamino-
benzaldehyde TS5. Any spot obtained with solution A, other than the princi-
pal spot, is not more intense than that obtained with solution B. Not more than
one of any such spots is more intense than the spot obtained with solution C.
873
0.5 g, accurately weighed, dissolved in a mixture of 50 ml of water and 20ml
of hydrochloric acid (~70g/l) TS, and titrate with sodium nitrite (0.1 mol/1) VS.
CaH,N 2 Na 03 S.
SULFADIAZINUM ARGENTUM
SULFADIAZINE SILVER
Ag
CioH 9AgN 402 S
Chemical name. N'-2-Pyrimidinylsulfanilamide monosilver(l+) salt; 4-
amino-Al-2-pyrimidinylbenzenesulfonamide monosilver(l+) salt; CAS Reg. No.

22199-08-2.
Solubility. Sulfadiazine silver is practically insoluble in water and ethanol

(-750 g/1) TS; slightly soluble in acetone R and ether R; soluble in ammonia
(-260 g/1) TS.
Storage. Sulfadiazine silver should be kept in a well-closed container, pro-

tected from light.
Requirements
Sulfadiazine silver contains not less than 98 0 % and not more than 102 0 %
. .
of C]oHtAgN 402 S, calculated with reference to the dried substance.
Identity tests
874
Prepare the following residue to be used in tests A, B, and C: dissolve 0.5 g in

5 ml of nitric acid (~1000g/l) TS, add 20 ml of water and 20ml of sodium chlo-
ride (400 g/1) TS, mix, and filter. Neutralize the filtrate with sodium hydroxide
(~80g/l) TS using phenolphthaleinyethanol TS as indicator, and add 2,0ml of
acetic acid (-60 g/1) TS; a white precipitate is produced. Filter, wash the pre-
cipitate on the filter with water, and dry it at 105°C for 1 hour.
A. Carry out the examination with the residue as described under 1.7 Spec-
trophotometry in the infrared region. The infrared absorption spectrum is
concordant with the spectrum obtained from sulfadiazine RS or with the
reference spectrum of sulfadiazine.
B. About lOmg of the residue yields the reaction described for the identifica-
tion of primary aromatic amines under 2.1 General identification tests, pro-
ducing an orange-red precipitate.
C. Dissolve about 0.1 g of the residue in 3ml of water, add 3ml of sodium
hydroxide (50 g/1) TS, shake, and filter. To a portion of the filtrate add 1 drop
of copper(II) sulfate (160 g/1) TS; a yellowish green precipitate is produced
that on standing turns to brownish red.
D. To about 0.1 g add 20ml of water, 2ml of nitric acid (-130 g/1) TS, and
mix; a curdy, white precipitate is produced which is soluble in ammonia
(-100 g/1) TS.
5-Omg/g.
pH value. Heat l.Og with 50ml of carbon-dioxide-free water R to 70 °C for

5 minutes, cool rapidly, and filter. pH of the filtrate, 5.5-7. 0.
7 volumes of dichloromethane R, 4 volumes of methanol R, and 1 volume of
ammonia (-260 g/1) TS as the mobile phase. {Note-. Mix the dichloromethane
and methanol before adding the ammonia.) Apply separately to the plate 10 pi
of each of the following 2 solutions. For solution (A) dissolve 50 mg of Sulfa-
diazine silver in 3.0ml of ammonia (-260 g/1) TS and dilute with sufficient
methanol R to produce 10 ml. For solution (B) dilute 1.0 ml of solution A
with a mixture containing 4 volumes of methanol R and 1 volume of ammonia
(-260 g/1) TS to produce 100 ml. Allow the spots to dry before development.
in air, and examine the chromatogram in ultraviolet light (254 nm).
Any spot obtained with solution A, other than the principal spot, is not larger
and more intense than that obtained with solution B (1.0%),
875
Assay. Transfer into a stoppered flask about 0.5 g, accurately weighed, and dis-
solve in 8ml of nitric acid (~130g/l) TS. Add 50ml of water and titrate with
ammonium thiocyanate (0.1 mol/1) VS, using ferric ammonium sulfate (45g/l)
TS as indicator. Repeat the procedure without the Sulfadiazine silver being
examined and make any necessary corrections.
Each mi of ammonium thiocyanate (0.1 mol/1) VS is equivalent to 35.71 mg of

C]oH 9AgN 402 S.
SULFADIMIDINUM
SULFADIMIDINE
Graphic formula.
Chemical name. N'-(4,6-Dimethyl-2-pyrimidinyl)sulfanilamide; 4-amino-A/-

(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide; CAS Reg. No. 57-68-1.
Other names. Sulfadimezinum, sulfamethazine.
Description. A white or creamy white, crystalline powder; odourless or

almost odourless.
TS; soluble in acetone R; practically insoluble in ether R.
Storage. Sulfadimidine should be kept in a well-closed container, protected

from light.
Requirements
Definition. Sulfadimidine contains not less than 99.0% and not more than
876
Identity tests

infrared region.
spectrum obtained from sulfadimidine RS or with the reference spectrum of
sulfadimidine.
aromatic amines under 2.1 General identification tests, producing an orange
precipitate.
C. Boil gently 0.5 g with 1 ml of sulfuric acid (-700 g/1) TS until a precipitate is
formed (about 2 minutes). Cool, add 15ml of sodium hydroxide (-80 g/1) TS
and extract the mixture with 20 ml of ether R. Filter the ether extract through
a layer of anhydrous sodium sulfate R and evaporate to dryness on a water-
bath. Melting temperature, about 153 °C (2-amino-4,6-dimethylpyrimidine).
5.0mg/g.
Acidity. Heat 2.0g with 100ml of carbon-dioxide-free water R at about 70°C

for 5 minutes, cool and filter. Titrate 25 ml of the filtrate with sodium hydrox-
ide (0.1 mol/1) VS, using bromothymol blue/ethanol TS as indicator; not more
than 0.2ml is required to obtain the midpoint of the indicator (green).
phase. Apply separately to the plate 10 pi of each of 2 solutions in acetone R
containing (A) lOmg of the test substance per ml and (B) 0.050mg of sulfanil-
amide RS per ml. After removing the plate from the chromatographic chamber,
heat it at 105°C for 10 minutes and spray it with 4-dimethylaminobenzalde-
hyde TS3. Any spot obtained with solution A, other than the principal spot, is
0.5 g, accurately weighed, dissolved in 50 ml of hydrochloric acid (-70 g/1) TS,
877
(0.1 mol/1) VS is equivalent to 27.83mg of C12H14N4O2S.
SULFADIMIDINUM NATRICUM
SULFADIMIDINE SODIUM
Molecular formula. Ci2Hi3N4Na02S
Graphic formula.
Chemical name. W'-(4,6-Dimethyl-2-pyrimidinyl)sulfanilamide monosodium

salt; 4-amino-Ai-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide monosodium
salt; CAS Reg. No. 1981-58-4.

almost odourless.
Solubility. Soluble in 2.5 parts of water and in 60 parts of ethanol (-750 g/1)
TS.
Storage. Sulfadimidine sodium should be kept in a well-closed container, pro-

tected from light.
Additional information. Even in the absence of light, Sulfadimidine sodium

Requirements
Definition. Sulfadimidine sodium contains not less than 98.0% and not
more than 101.0% of C]2Hi3N4Na02S, calculated with reference to the dried
substance.
878
Identity tests
A. Dissolve 0.1 g in 10ml of water,

with hydrochloric acid (~70g/l) TS,
acidify
filter, and dry the residue at 105°C. Carry out the examination with the
dried residue as described under 1.7 Spectrophotometry in the infrared

region. The infrared absorption spectrum is concordant with the spectrum
obtained from sulfadimidine RS or with the reference spectrum of
sulfadimidine.
B. Dissolve 0.1 g in 10ml of water, add 2ml of acetic acid (-300 g/1) TS, sepa-
rate the precipitate by filtration, and wash with cold water. It yields the
reaction described for the identification of primary aromatic amines under
2.1 General identification tests, producing a bright orange-red precipitate.
C. Boil gently 0.5g with 1.5ml of sulfuric acid (-700 g/1) TS until a precipitate
is formed (about 2 minutes). Cool, add 15ml of sodium hydroxide (-80 g/1)
TS and extract the mixture with 20 ml of ether R. Filter the ether extract
through a layer of anhydrous sodium sulfate R and evaporate to dryness
on a water-bath. Melting temperature, about 153 °C (2-amino-4,6-
dimethylpyrimidine).
D. When tested for sodium as described under 2.1 General identification tests
quantity and dissolve the residue in acetic acid (-60 g/1) TS.
Clarity and colour of solution. A solution of 3.3 g in 1 0 ml of carbon-dioxide-

free water R is clear and not more intensely coloured than standard colour solu-
20mg/g.
pH value. pH of a O.lOg/ml solution in carbon-dioxide-free water R,

10 0 - 11 0
. . .
phase. Apply separately to the plate lOpl of each of 2 solutions in a mixture of
9 volumes of ethanol (-750 g/1) TS and 1 volume of ammonia (-260 g/1) TS con-
taining (A) 10 mg of the test substance per ml and (B) 0.050 mg of sulfanilamide
879
RS per ml (for the preparation of solution A, first dissolve the test substance in
a little ammonia (-260 g/1) TS, add
9 volumes of ethanol (-750 g/1), and then
dilute to volume with the ethanol/ammonia mixture). After
the required
removing the plate from the chromatographic chamber, heat it at 105 °C for
10 minutes and spray it with 4-dimethylaminobenzaldehyde TS3. Any spot
0.5 g, accurately weighed, dissolved in a mixture of 75ml of water and 10ml
of hydrochloric acid (-420g/l) TS, and titrate with sodium nitrite (0.1 mol/1)
Ci2hli3N4Na02S.
SULFADOXINUM
SULFADOXINE
Graphic formula.
Chemical name. N*-(5,6-Dimethoxy-4-pyrimidinyl)sulfanilamide; 4-amino-

N-(5,6-dimethoxy-4-pyrimidinyl)benzenesulfonamide; CAS Reg. No. 2447-57-
6 .
TS and in methanol R; practically insoluble in ether R.
Storage. Sulfadoxine should be kept in a well-closed container, protected from

light.
880
Requirements
Definition. Sulfadoxine contains not less than 99.0% and not more than
Identity tests

infrared region.
spectrum obtained from sulfadoxine RS or with the reference spectrum of
sulfadoxine.
aromatic amines under 2.1 General identification tests, producing an orange-
red precipitate.
C. Dissolve 50 mg in 3 ml of sodium hydroxide (0.1 mol/1) VS, heating slightly.

Cool and add 1.0ml of copper(ll) sulfate (80g/l) TS; a greenish yellow pre-
cipitate is produced, the colour of which changes to blue (distinction from
certain other sulfonamides).
Chlorides. For the preparation of the test solution, boil 2.5 g with 30 ml of
water, cool and filter. Add 10ml of nitric acid (-130 g/1) TS to the filtrate and
proceed as described under 2.2.1 Limit test for chlorides; the chloride content
Sulfates. For the preparation of the test solution, boil 2.5 g with 40 ml of water,
cool and filter. Proceed with the nitrate as described under 2.2.2 Limit test for
sulfates; the sulfate content is not more than 0.2 mg/g.
Clarity and colour of solution. A solution of 0.50g in 10ml of hydrochlo-

ric acid (-70g/1) TS is clear and not more intensely coloured than standard
colour solution Yw3 when compared as described under 1.11 Colour of liquids.
5.0 mg/g.
881
Acidity. Heat 1.0 g with 50ml of carbon-dioxide-free water R at about 70°C

for 5 minutes, cool quickly to 20 °C, and filter; titrate 25 ml of the filtrate to pH
7.0 with sodium hydroxide (0.1 mol/1) VS; not more than 0.25ml is required.
phase. Apply separately to the plate lOpl of each of 2 solutions in a mixture of
9 volumes of ethanol (~750g/l) TS and 1 volume of ammonia (-260 g/1) TS con-
taining (A) 10 mg of the test substance per ml and (B) 0.050 mg of sulfanilamide
RS per ml. After removing the plate from the chromatographic chamber, heat
it at 105°C for 10 minutes and spray it with 4-dimethylaminobenzaldehyde
TS3. Any spot obtained with solution A, other than the principal spot, is not
0.5 g, accurately weighed, dissolved in a mixture of 75ml of water and 10ml
of hydrochloric acid (-250 g/1) TS, and titrate with sodium nitrite (0.1 mol/1)
C12H14N4O4S.
SULFAMETHOXAZOLUM
SULFAMETHOXAZOLE
Molecular formula. CjoHnNsOsS
Graphic formula.
Chemical name. N'-(5-Methyl-3-isoxazolyl)sulfanilamide: 4-amino-N-

(5-methyl-3-isoxazolyl)benzenesulfonamide; CAS Reg. No. 723-46-6.
Description. A white or yellowish white, crystalline powder; odourless.

(-750 g/1) TS and in 3 parts of acetone R.
882
Storage. Sulfamethoxazole should be kept in a well-closed container, pro-

tected from light.
Requirements
Definition. Sulfamethoxazole contains not less than 99.0% and not more than
101.0% of CioHiiNsOjS, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from sulfamethoxazole RS or with the reference spectrum
of sulfamethoxazole.
B. Dissolve 5 mg in 0.5ml of sodium hydroxide (~80g/l) TS and add 5 ml of

water. Add 0.1 g of phenol R, heat to boiling, cool, and add 1 ml of sodium
hypochlorite (~40g/l) TS; a golden yellow colour is immediately produced
and is persistent.
aromatic amines under 2.1 General identification tests, producing a red-
orange precipitate.
Solution in alkali. Dissolve 0.40 g in a mixture of 8.0 ml of water and 2.0ml

of sodium hydroxide (1 mol/1) VS; the solution is clear.
5.0mg/g.
Acidity. Heat l.Og with 50ml of carbon-dioxide-free water R at about 70°C

for 5 minutes, cool quickly to 20 °C, and filter; titrate 25ml of the filtrate, phenol-
phthalein/ethanol TS being used as indicator; not more than 0.35 ml of sodium
hydroxide (0.1 mol/1) VS is required to obtain the midpoint of the indicator.
883
per ml and (B) 12.5 pg of sulfanilamide RS per ml. After removing the plate from
evaporated. Spray the dried plate with sulfuric acid/ethanol TS, heat at 105°C
for 30 minutes, and immediately expose to nitrous fumes in a closed chamber
for 15 minutes (the nitrous fumes may be generated by adding sulfuric acid
(-700 g/1) TS drop by drop to a solution containing lOg of sodium nitrite R and
3g of potassium iodide R in 100 ml). Place the plate in a current of warm air
for 15 minutes and spray with iV-(l-naphthyl)ethylenediamine hydrochlo-
ride/ethanol TS. If necessary, allow to dry before repeating the spraying and
tion B.
0.5 g, accurately weighed, dissolved in a mixture of 40 ml of water and 20ml
of glacial acetic acid R; add 15 ml of hydrochloric acid (-70 g/1) TS and titrate
with sodium nitrite (0.1 mol/1) VS. Each ml of sodium nitrite (0.1 mol/1) VS is
equivalent to 25.33mg of C 10 H N 3 O 3 S.
11
Additional requirement for Sulfamethoxazole for

parenteral use
SULFAMETHOXYPYRIDAZINUM
SULFAMETHOXYPYRIDAZINE
Molecular formula. CiiHi 2 N 40 3 S ,
Graphic formula.
884
Chemical name. N'-(6-Methoxy-3-pyridazinyl)sulfanilamide; 4-amino-N-(6-

methoxy-3-pyridazinyl)benzenesulfonamide; CAS Reg. No. 80-35-3.
Description. A white or yellowish white, crystalline powder; odourless or

almost odourless.

(-750 g/1) TS; soluble in acetone R.
Storage. Sulfamethoxypyridazine should be kept in a well-closed container,

Additional information. Sulfamethoxypyridazine becomes gradually

coloured on exposure to light.
Requirements
Definition. Sulfamethoxypyridazine contains not less than 99.0% and not
more than 101.0% of CnH] 2 N 403 S, calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from sulfamethoxypyridazine RS or with the reference
spectrum of sulfamethoxypyridazine.
aromatic amines under 2.1 General identification tests, producing a bright
C. To 20mg add 10ml of sulfuric acid (-100 g/1) TS, mix to dissolve and care-
fully add 0.1ml of potassium bromate (50 g/1) TS; a yellow colour that
changes to amber is produced and a brown precipitate is gradually formed.
885
5-Omg/g.
Acidity. Heat l.Og with 50ml of carbon-dioxide-free water R at about 70°C

for 5 minutes, cool quickly to 20 °C, and filter; 25 ml of the filtrate requires for
titration to pH 7.0 not more than 0.35ml of sodium hydroxide (0.1 mol/1) VS.
per ml and (B) 12.5 pg of sulfanilamide RS per ml. After removing the plate from
evaporated. Spray the dried plate with sulfuric acid/ethanol TS, heat at 105°C
for 30 minutes, and immediately expose to nitrous fumes in a closed chamber
for 15 minutes (the nitrous fumes may be generated by adding sulfuric acid
(-700 g/1) TS drop by drop to a solution containing lOg of sodium nitrite R and
3g of potassium iodide R in 100 ml). Place the plate in a current of warm air
for 15 minutes and spray with W-(l-naphthyl)ethylenediamine hydrochlo-
ride/ethanol TS. If necessary, allow to dry before repeating the spraying and
tion B.
0.5 g, accurately weighed, dissolved in 50ml of hydrochloric acid (~70g/l) TS,
(0.1 mol/1) VS is equivalent to 28.03mg of Ci,H] 2 N 403 S.
SULFASALAZINUM
SULFASALAZINE
886
Graphic formula.
Chemical name. 5-[[/^’-(2-Pyridylsul£amoyl)phenyl]azo]salicylic acid; 2-
hydroxy-5-[[4-[(2-pyridinylamino)sul£onyl]phenyl]azo]benzoic acid; CAS Reg.

No. 599-79-1.
Other name. Salazosul£apyridine.
Description. A bright yellow to brownish yellow powder; odourless.
Solubility. Practically insoluble in water, and ether R; very slightly soluble in

ethanol (-750 g/1) TS; soluble in alkali hydroxides.
Storage. Sul£asalazine should be kept in a tightly closed container, protected

£rom light.
Additional information. Sulfasalazine melts at about 255 °C with

decomposition.
Requirements
Definition. Sulfasalazine contains not less than 93.0% and not more than
103.0% of C] 8 HhN 405 S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from sulfasalazine RS or with the reference sp’ectrum of
sulfasalazine.
B. The absorption spectrum of the solution as prepared in the assay below,

when observed between 230 nm and 600 nm, exhibits maxima and minima
at the same wavelengths as does the absorption spectrum of a solution of
sulfasalazine RS prepared in a similar manner.
887
C. Dissolve O.lOg in a mixture of 1.0ml of ethanol (-750 g/1) TS and 4ml of

hydrochloric acid (-70 g/1) TS, then add 0.20 g of zinc R powder. Heat on a
water-bath for 5 minutes and filter. To 1.0ml of the nitrate add 0.1ml of
ferric chloride (25 g/1) TS; a red colour is produced. To 1.0 ml of the filtrate
add 1.0ml of sodium nitrite (lOg/1) TS and allow to stand for 1 minute. Then
add 2.0ml of sodium hydroxide (-80 g/1) TS and 0.1ml of 2-naphthol TSl;
a strong red colour is produced.
Chlorides. Digest 2.0g with 100ml of water at 70°C for 5 minutes. Cool
immediately to room temperature and filter. To 25ml of the filtrate (keep the
remaining filtrate for the limit test for sulfates) add 1 ml of nitric acid
(-1000 g/1) TS and allow to stand for 5 minutes. Filter through a fine-texture
filter-paper and proceed with the filtrate as described under 2.2.1 Limit test for
chlorides; the chloride content is not more than 0.14mg/g.
Sulfates. To 25 ml of the filtrate retained from the limit test for chlorides add
1.5 ml of hydrochloric acid (2 mol/1) VS and allow to stand for 5 minutes. Filter
through a fine-texture filter-paper and proceed with the filtrate as described
under 2.2.2 Limit test for sulfates; the sulfate content is not more than 0.4mg/g.
10 mg/g.
Assay. Carry out the test as described under 1.14.1 Thin-layer chromatogra-
phy using silica gelR2 as the coating substance and a mixture of 4 volumes of
chloroform R, 1 volume of 1 -butanol R, 1 volume of acetone R, and 1 volume
of formic acid (-1080 g/1) TS as the mobile phase, allowing the chamber to equi-
librate for 18 hours. Apply separately to the plate 10 pi of each of 2 solutions

in dimethylformamide R containing (A) 12 mg of the test substance per ml, (B)
12 mg of sulfasalazine RS per ml, and (C) 10 pi of dimethylformamide R to serve
as a blank. Dry the spotted plate thoroughly at room temperature to remove
traces of dimethylformamide and develop the plate over a distance of 15 cm.
After removing the plate from the chromatographic chamber, mark the solvent
front and allow the solvent to evaporate. Locate the spots by examination of
the chromatogram in ultraviolet light (254 nm) and mark the sulfasalazine spots
at an 1?^ value of about 0.6, excluding an adjacent impurity band. Remove quan-
titatively and separately the silica gel mixture containing each of the 3 spots
from solutions A, B and C, transferring them to separate glass-stoppered cen-
trifuge tubes. (NOTE: The dimethylformamide R employed in the next step
should be obtained from a recently opened bottle and should contain less than
888
10 mg of water per Dimethylformamide dried over alkali pellets is not accept-

g.
able.) Add 10.0 ml of dimethylformamide R to each tube, insert the stopper,

shake for 10 minutes, and centrifuge until clear. Measure the absorbance of the
solutions in a 1-cm layer at the maximum at about 406 nm against a solvent
cell containing the blank obtained from solution C. Calculate the amount of
C18H14N4O5S in the substance being tested (solution A) by comparison with sul-

fasalazine RS (solution B).
SURAMINUM NATRICUM
SURAMIN SODIUM
Molecular formula. C5iH34N6Na(;023S6
Graphic formula.
CO CO
NHCONH
Chemical name. Hexasodium 8,8'-[ureylenebis[m-phenylenecarbonylim-

ino(4-methyl-w-phenylene)carbonylimino]]di-l,3,5-naphthalenetrisulfonate;
hexasodium 8,8'-[carbonylbis[imino-3,l-phenylenecarbonylimino(4-methyl-
3,l-phenylene)carbonylimino]]bis[l,3,5-naphthalenetrisulfonate]; CAS Reg.
No. 129-46-4.
Other name. Nagananinum.
Description. A white, pinkish white or cream-coloured, crystalline powder;

odourless.
Solubility. Very soluble in water; slightly soluble in ethanol (-750 g/1) TS; prac-
Category. Antifilarial drug; antitrypanosomal drug.
Storage. Suramin sodium should be kept in a tightly closed container, pro-

tected from light, and stored in a cool place.
Additional information. Suramin sodium is very hygroscopic and discolours

on exposure to light.
Requirements
Definition. Suramin sodium contains not less than 96.0% and not more than
100.5% of C5iH34N6Na5023S6, calculated with reference to the anhydrous
substance.

Therapeutic potency. For therapeutic potency, the sample is tested in

mice infected with a strain of Trypanosoma equiperdum, or other suitable
species of trypanosome sensitive to suramin sodium. The test may be
carried out as follows: Inoculate at least 10 mice with the trypanosomes.
After forty-eight hours examine the blood of each mouse microscopically
and estimate the number of trypanosomes per ml in the blood of each
mouse. The number should lie between 1000 and 20000. The estimate may
be made by examining a thin film of the blood in the form of a cover-slip
preparation, and counting the trypanosomes in at least 10 microscopic fields
with an area of 0.12mm^. The presence of 1 to 20 trypanosomes in each of
two fields corresponds approximately to a content of 1000 to 20000 try-
panosomes per ml.
Inject into 10 of the infected mice, intravenously, 0.16ml of a 50 mg/1 solu-

tion of the sample in freshly distilled water per g of body mass. Examine
the blood of each mouse microscopically, using a microscope with a 4-mm
objective, on the first and third days after the injection. If no trypanosomes
are found in the blood of 5 or more mice when 20 fields are examined on
the third day, the sample passes the test. If trypanosomes are found under
these conditions in the blood of more than 5 mice, repeat the test. The
sample passes the test if no trypanosomes are found under these conditions
in theblood of not less than 50% of the total number of mice treated.
Identity tests
A. Dissolve 20 mg in 2.0ml of water, add 1.0 ml of hydrochloric acid (~70g/l) TS,
and heat on a water-bath for 5 minutes. Cool, add 0.25ml of sodium nitrite
(lOg/1) TS, allow to stand for 1 minute, then add 1.0ml of sodium hydroxide
(~80g/l) TS and 0.15ml of 2-naphthol TSl; a red colour is produced.
890
B. In a porcelain crucible, mix 20 mg with O.lOg of anhydrous sodium car-

bonate R and heat until the gas evolution has ceased. Cool, dissolve the
residue in 4ml of hydrochloric acid (~70g/l) TS, and filter. To 2.0ml of the
nitrate add 0.25 ml of barium chloride (50g/l) TS; a white precipitate is
produced.
C. Dissolve 0.05 g in 2.0ml of water and add 0.25 ml of glacial acetic acid R; it
yields reaction B characteristic of sodium as described under 2.1 General

identification tests.
Chlorides. Dissolve 0.5g in 10ml of water, add 5ml of nitric acid (~130g/l)
TS, 5ml of silver nitrate (0.1 mol/1) VS, and 3 ml of nitrobenzene R, and shake
vigorously. Titrate the excess of silver nitrate with ammonium thiocyanate
(0.1 mol/1) VS, using 2ml of ferric ammonium sulfate (45g/l) TS as indicator;
not less than 3.6ml of ammonium thiocyanate (0.1 mol/1) VS are required.

(-250 g/1) TS, and proceed as described under 2.2.2 Limit test for sulfates; the
sulfate content is not more than 1 mg/g.
Clarity of solution. A solution of 0.50 g in 10ml of carbon-dioxide-free water

R is clear.

content is not more than O.lOg/g.
Free amines. Dissolve 5 g in 30 ml of water and add 30 ml of hydrochloric acid

(1 mol/1) VS. Titrate at a temperature between 15 and 20 °C with sodium nitrite
(0.1 mol/1) VS, stirring vigorously, until a blue colour is obtained on

starch/iodide paper R. The end-point of the titration is reached when the blue
colour is reproduced after the titrated solution has been allowed to stand for 1
minute. The titration can also be performed electrometricaUy. Repeat the oper-
ation without the substance to be examined. The difference in volume between
the two titrations does not exceed 0.4 ml.
Assay. To about 0.5g, accurately weighed, add 12ml of sulfuric acid (~700g/l)
TS, and boil under a reflux condenser for 1 hour; cool, and dilute to about
100 ml with water. Add Ig of potassium bromide R and titrate at a tempera-
ture between 15 and 20 °C with sodium nitrite (0.1 mol/1) VS, stirring vigor-
891
ously until a blue colour is obtained on starch/iodide paper R. The end-point

of the titration is reached when the blue colour is reproduced after the titrated
solution has been allowed to stand for 1 minute. The titration can also be per-
formed electrometrically. Repeat the operation without the substance to be
examined. Each ml of sodium nitrite (0.1 mol/1) VS is equivalent to 23.82 mg of
C5lH34N5Nfa6023S6.
SUXAMETHONII CHLORIDUM
SUXAMETHONIUM CHLORIDE
Molecular formula. Ci 4 H 3 oCl 2 N 204 2 H 20 ,
Graphic formula.
COOCHjCHjN*(CH3)j
(CH,)3 2cr> 2HjO
COOCHjCHjNhCHjjj
Chemical name. Choline chloride, succinate (2:1), dihydrate; 2,2'-[(l,4-dioxo-

l,4-butanediyl)bis(oxy)]bis[N,N,N-trimethylethanaminium] dichloride, dihy-
drate; 2,2'-succinyldioxybis(ethyltrimethylammonium) dichloride, dihydrate;
CAS Reg. No. 6101-15-1 (dihydrate).
Other name. Succinylcholine chloride.

almost odourless.
Solubility. Soluble in 1 pan of water; slightly soluble in ethanol (-750 g/1) TS;
Categoiy. Muscle relaxant.
Storage. Suxamethonium chloride should be kept in a tightly closed container,

Additional information. Suxamethonium chloride is hygroscopic. Even in

892
Requirements
Definition. Suxamethonium chloride contains not less than 98.0% and not
more than 101.0% of C14H30CI2N2O4, calculated with reference to the anhy-
drous substance.
Identity tests
A. Dissolve 25 mg in 1ml of water, add 0.1ml of cobalt(II) chloride (5g/l) TS
and 0.1 ml of potassium ferrocyanide (45g/l) TS; an emerald green colour is
produced.
B. Dissolve 0.05 g in 10 ml of water, add 10 ml of sulfuric acid (~100g/l) TS and

30ml of ammonium reineckate (lOg/1) TS; a pink precipitate is produced.
Allow to stand for 30 minutes, filter and wash with water, then with ethanol
(-750 g/1) TS and with ether R. Dry the residue at 80 °C; melting tempera-
ture, about 183 °C.


and colourless.

Fischer method, Method A, using about 0.15g of the substance; not less than
80mg/g and not more than lOOmg/g.
chromatography, using cellulose R2 as the coating substance and a mixture of
4 volumes of 1 -butanol R, 1 volume of acetic acid (-300 g/1) TS, and 5 volumes
of water as the mobile phase. Apply separately to the plate 10 pi of each of 2
solutions containing (A) S.Omg of the test substance per ml and (B) O.lOmg of
the test substance per ml. Develop the plate for 2V2 hours. After removing the
plate from the chromatographic chamber, dry it at 90 °C for 15 minutes, allow
it to cool, spray it with potassium iodoplatinate TS2 and examine the chro-
893

alent to 18.07 mg of CMH 30 CI N O
2 2 4.
Additional requirements for Suxamethonium chloride for

fcârenteral use
terial endotoxins; contains not more than 2.01U of endotoxin RS per mg.
TALCUM
TALC
Chemical name. Talc; talc [Mg 3 H 2 (Si 03 ) 4 ]; CAS Reg. No. 14807-96-6.
Description. A white or almost white, very fine, homogeneous, unctuous

powder; odourless.
Solubility. Practically insoluble in water, dilute solutions of acids and alkalis,

ethanol (-750 g/1) TS, and ether R.
Category. Tablet and capsule lubricant; glidant; diluent.
Storage. Talc should be kept in a well-closed container.
Additional information. Talc adheres readily to the skin. Attention should be

paid to its microbiological quality since it is of mineral origin.
Requirements
Definition. Talc is a powdered, natural hydrate of magnesium silicate that
may contain variable proportions of aluminium and iron.
Identity tests
A. Transfer 0.5 g to a metal crucible, add Ig of potassium nitrate R and 3g of
anhydrous sodium carbonate R, mix, and heat until melted. Allow to cool,
add 20ml of boiling water, mix, and filter. Wash the filter with 50ml of
water. Take up the residue with a mixture of 0.5ml of hydrochloric acid
(-420 g/1) TS and 5 ml of water and filter. To the filtrate add 1 ml of ammonia
(-260 g/1) TS and 1ml of ammonium chloride (100 g/1) TS and filter. To this
filtrate add 1 ml of disodium hydrogen phosphate (100 g/1) TS; a white, crys-
talline precipitate is produced.
894
B. Using a copper wire mix 0.1 g with lOmg of sodium fluoride R placed in a
lead or platinum crucible, and add a few drops of sulfuric acid (~1760g/l)
TS Cover the crucible with a thin, transparent, plastic
to obtain a thin slurry.
plate from which a drop of water is suspended and warm gently; a white
ring is produced around the drop of water within a short time.
Microscopic examination. Irregular plates, length up to 50 pm, are observed.

Talc is free from microscopic asbestos fibres. A 1 mg/ml solution of methylth-
ioninium chloride R in ethanol (~750g/l) TS does not notably stain the particles.
Arsenic and heavy metals. Transfer 10 g add 50 ml of

to a 250-ml flask,
hydrochloric acid (0.5mol/l) VS, attach a reflux condenser, and heat on a water-
bath for 30 minutes. Cool, transfer the mixture to a beaker, and allow the undis-
solved material to settle. Decant the supernatant liquid through a thick, strong
filter-paper of medium grade into a 100-ml volumetric flask, retaining as much
as possible of the undissolved material in the beaker. Wash the beaker with
three 10-ml portions of hot water, decanting each washing through the same
filter. Finally, wash the filter with 15 ml of hot water, cool the filtrate and dilute
to volume, and mix. Use this solution for the following tests:
— Use 10ml and proceed as described under 2.2.5 Limit test for arsenic; the
— Use 5ml and proceed as described under 2.2.3 Limit test for heavy metals.
Procedure 1; determine the heavy metals content according to Method A;
Carbonates. Suspend 0.25g in 40ml of sulfuric acid (~100g/l) TS; no effer-

vescence is observed.
Acid-soluble substances. Digest l.Og with 20ml of hydrochloric acid

(~70g/l) TS 50 °C for 15 minutes, cool, add sufficient water to produce
at
50 ml, and the filtrate is not clear, centrifuge it.) To 25 ml of the clear
filter. (If
solution (keep the remaining solution for “Iron”), add 1ml of sulfuric acid
(~100g/l) TS, and evaporate to dryness. Ignite the residue at 800 + 25 °C and
weigh; not more than 20mg/g.
Iron. Acidify 10ml of the filtrate obtained in the test above for “Acid-soluble
substances” with hydrochloric acid (~70g/l) TS and add 1 ml of potassium fer-
rocyanide (45g/l) TS; no blue colour is observed.
Reaction and soluble substances. Boil 10 g with 50 ml of water for 30

minutes, adding water from time to time to maintain approximately the orig-
inal volume, and filter; the filtrate is neutral to litmus paper R. Evaporate 20 ml
of the filtrate to dryness and dry the residue at 105°C for 1 hour; the mass of
the residue does not exceed 4 mg (1 mg/g).
895
Loss on ignition. Ignite l.Og at 1000°C to constant mass; it loses not more
than 65mg/g.
TAMOXIFENI CITRAS
TAMOXIFEN CITRATE
CH— CO^H
C— COjH
CH— CO^H
C26H2,N0,C,H307
Chemical name. (Z)-2-[/^-(l, 2 -Diphenyl-1 -butenyI)phenoxy]-A/',Al-dimethy-

lethylamine citrate (1:1); (Z)-2-[4-(l, 2-diphenyl- 1 -butenyl)phenoxy] -N,N-
dimethylethanamine 2-hydroxy-l,2,3-propanetricarboxylate (1:1); CAS Reg.
No. 54965-24-1.
Solubility. Slightly soluble in water and acetone R; soluble in methanol R.
Category. Antiestrogen.
Storage. Tamoxifen citrate should be kept in a well-closed container, protected

from light.
Requirements
Tamoxifen citrate contains not less than 99 0 % and not more than the equiv-
.
alent of 101 0 % of C 26 H 29 N 0 ,C 6 Hb07 calculated with reference to the dried

.
,
substance.
Identity tests
896

spectrum obtained from tamoxifen citrate RS or with the reference spectrum
of tamoxifen citrate.

using silica gelR4 as the coating substance and a mixture of 9 volumes of
toluene R and volume of triethylamine R as the mobile phase. Apply sep-
1
arately to the plate 5 pi of each of two solutions in methanol R containing

(A) 10 mg of Tamoxifen citrate per ml and (B) 10 mg of tamoxifen citrate RS
it to dry in air, and examine the chromatogram in ultraviolet light (254 nm).
The with solution A corresponds

principal spot obtained in position, appear-
ance, and intensity with that obtained with solution B
C. To lOmg add 4ml of pyridine R and 2ml of acetic anhydride R, and shake;
a yellow colour is immediately produced. Heat on a water-bath for 2
minutes; a light pink to red colour is produced.
5.0mg/g.
£-isomer and related substances. Carry out the test as described under
1.14.4 High performance liquid chromatography, using a stainless steel column
(20 cm X 5 mm) packed with particles of silica gel, the surface of which has been
modified with chemically bonded octadecylsilyl groups (5 pm). As the mobile
phase, use a mixture of 400 volumes of acetonitrile R and 600 volumes of water
containing 0.9 g/1 of sodium dihydrogen phosphate R and 4.8 g/1 of N,N-
dimethyloctylamine R adjusted to pH 3.0 with phosphoric acid (-105 g/1) TS.
Prepare the following solutions in the mobile phase: for solution (A) use 1.0 mg
of Tamoxifen citrate per ml; for solution (B) use 1.0 mg of tamoxifen citrate E-
isomer RS per ml; for solution (C) dilute 1 volume of solution A to 100 volumes
with the mobile phase; and for solution (D) dilute 1 volume of solution B to
100 volumes with the mobile phase.
897
spectrophotometer set at a wavelength of about 240 nm, the instrument being
fitted with a low volume flow cell (10 pi).
Equilibrate the column with the mobile phase at a flow rate of 1.0 ml per minute
for about 30 minutes.
Make three replicate injections of 10 pi each of solution D, adjusting the sensi-

tivity of the system so that the height of the peak is not less than 40% of the
full scale of the recorder.
Inject alternately 10 pi each of solutions A, C, and D. The test is not valid unless
the peak obtained with solution D elutes prior to that for solution C, and with
solution A there is baseline separation between all the peaks.
solutions A, C, and D, and calculate the content of the related substances as a
percentage. Calculate the content of £-isomer comparing the peaks obtained
with solutions A and D; not more than lOmg/g. In the chromatogram obtained
with solution A, the area of any peak, other than the peak due to the £-isomer
obtained with solution D, is not greater than half that of the peak obtained
with solution C (0.5%). Furthermore, the sum of the areas is not greater than
the peak obtained with solution C (1%).
Assay. Dissolve about 1 g, accurately weighed, in 150 ml of glacial acetic acid

Rl, add 0.25ml of 1-naphtholbenzein/acetic acid TS, and titrate with perchlo-
ric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous titration. Method A.

C,,H2<,NO,C6HsO„
TESTOSTERONI ENANTAS
TESTOSTERONE ENANTATE
898
Graphic formula.
I5CH3
Chemical name. Testosterone, heptanoate; 17(3-[(l-oxoheptyl)oxy]androst-4-

en-3-one; CAS Reg. No. 315-37-7.

almost odourless.
Solubility. Practically insoluble in water; very soluble in ethanol (-750 g/1) TS,
ether R, and acetone R.
Category. Androgen.
Storage. Testosterone enantate should be kept in a tightly closed container,

protected from light, and stored at a temperature not exceeding 15 °C.
Additional information. Testosterone enantate melts at about 37 °C.
Requirements
Definition. Testosterone enantate contains not less than 97.0% and not more
than 103.0% of C 26H 40 O 3, calculated with reference to the dried substance.
Identity tests

spectrum obtained from testosterone enantate RS or with the reference sfrec-
truni of testosterone enantate.
C. Suspend 5 mg in 2.0ml of a mixture prepared by previously cooling 2

volumes of sulfuric acid (-1760 g/1) TS and 1 volume of ethanol (-750g/l)
TS, then place it in a water-bath; a greenish yellow fluorescence develops
899
that changes to orange, whereas the walls of the tube take on a dichroic
blue colour, changing to red below a certain depth.
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; [a]o = +77

to +83°.

Free heptanoic acid. Dissolve 0.5 g, accurately weighed, in 10 ml of ethanol

(-750 g/1) TS, previously neutralized to bromothymol blue/ethanol TS, and
titrate immediately with sodium hydroxide (0.01 mol/1) VS, using bromothy-
mol blue/ethanol TS as indicator; not more than 0.6ml of sodium hydroxide
92 volumes of dichloroethane R, 8 volumes of methanol R, and 0.5 volume of
containing (A) 20 mg of the test substance per ml; (B) 0.20 mg of the test sub-
stance per ml; (C) 1.0 mg of the test substance per ml and (D) 1.0 mg of testos-
terone enantate RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air and heat it at 110 °C for 10 minutes. Spray the
hot plate with sulfuric acid/ethanol TS, again heat it at 110°C for 10 minutes,
Assay. Dissolve about 20 mg, accurately weighed, in dehydrated ethanol R to

at about 241 nm. Calculate the amount of C26H40O3 in the substance being
tested by comparison with testosterone enantate RS, similarly and concurrently
examined. In an adequately calibrated spectrophotometer the absorbance of a
lOpg/ml solution of testosterone enantate RS in dehydrated ethanol R should
be 0.42 + 0.02.
Additional requirements for Testosterone enantate for

parenteral use
900
TESTOSTERONI PROPIONAS
TESTOSTERONE PROPIONATE
Graphic formula.
OCOCHjCHj
Chemical name. 17P-(l-Oxopropoxy)androst-4-en-3-one; IZP-hydroxyan-

drost-4-en-3-one propionate; CAS Reg. No. 57-85-2.
Description. Colourless or slightly yellowish crystals or a white or slightly

yellowish powder; odourless.

and ether R; soluble in vegetable oils.
Category. Androgen.
Storage. Testosterone propionate should be kept in a well-closed container,

Requirements
Definition. Testosterone propionate contains not less than 97.0% and not
more than 102.0% of C22H32O3, calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from testosterone propionate RS or with the reference
sp’ectrum of testosterone propionate.
901
=
Specific optical rotation. Use a 10 mg/ml solution in dioxan R; [a]™ +81

to +91
Solution in ethanol. A solution of 0.50 gin 10 ml of ethanol (-750 g/1) TS is

clear and not more intensely coloured than standard colour solution Yw2 when
Loss on diying. Dry to constant weight at 105 °C; it loses not more than 5.0 mg/g.
containing (A) 20 mg of the test substance per ml; (B) 0.20 mg of the test sub-
stance per ml; (C) 1.0 mg of the test substance per ml and (D) 1.0 mg of testos-
terone propionate RS per ml. After removing the plate from the
chromatographic chamber, allow it to dry in air and heat at 110 °C for 10
minutes. Spray the hot plate with sulfuric acid/ethanol TS, again heat it at
110°C for 10 minutes, and examine the chromatogram in ultraviolet light

at the maximum at about 241 nm. Calculate the amount of C22H32O3 in the sub-
stance being tested by comparison with testosterone propionate RS, similarly
TETRACAINI HYDRO CHLORIDUM

TETRACAINE HYDROCHLORIDE
Molecular formula. Ci 5 H 24N 202 ,HCl
902
Graphic formula.
CH3(CHjl3 NH — —COOCHjCH3N(CH3)j . HCI
Chemical name. 2-(Dimethylamino)ethyl ;?-(butylamino)benzoate monohy-

drochloride; 2-(dimethylamino)ethyl 4-(butylamino)benzoate monohydrochlo-
ride; CAS Reg, No. 136-47-0,
Other names. Amethocaine hydrochloride; dicainum.
Solubility. Soluble in about 8 parts of water; soluble in ethanol (-750 g/1) TS;
Storage. Tetracaine hydrochloride should be kept in a tightly closed container,

Additional information. Tetracaine hydrochloride is hygroscopic; it has a

slightly bitter taste and causes local numbness after being placed on the tongue.
Tetracaine hydrochloride melts at about 148 °C or may exist in either of the
two polymorphic forms, one of which melts at 134 °C and the other at 139 °C.
Mixtures of the forms melt within the range 134-147 °C.
Requirements
Definition. Tetracaine hydrochloride contains not less than 98.0% and not
more than 101.0% of Ci 5 H 24N 202 ,HCl, calculated with reference to the dried
substance.
Identity tests
A. Dissolve 0.2g in 10ml of water and add 1ml of ammonium thiocyanate
(75 g/1) TS. Collect the precipitate on a filter, recrystallize from water, and
dry it at 80 °C for 2 hours; melting temperature, about 131 °C.


903
lOmg/g.
80 volumes of dibutyl ether R, 16 volumes of hexane R, and 4 volumes of glacial
acetic acid R as the mobile phase. Place the plate in the chromatographic
chamber, dipping it about 5 mm
into the liquid. After the solvent has reached
a height of about 12 cm, remove the plate from the chromatographic chamber
and dry it for a few minutes in a current of warm air. Allow it to cool and apply
separately to the plate 5 pi of each of 2 solutions containing (A) O.lOg of the
test substance perml and (B) 0.050 mg of 4-aminobenzoic acid R per ml. Allow
the solvent front to ascend 10cm above the line of application. After removing
the platefrom the chromatographic chamber, dry it at 105°C for 10 minutes
obtained with solution B. The principal spot remains on the baseline.
0.5 g, accurately weighed, dissolved in a mixture of 50ml of water and 5 ml of
hydrochloric acid (~420g/l) TS, and titrate with sodium nitrite (0.1 mol/1) VS.
Each ml of sodium nitrite (0.1 mol/1) VS is equivalent to 30.08mg of
Ci 5 H 24 N 202 ,HC 1 .
Additional requirements for Tetracaine hydrochloride for

sterile use
TETRACYCLINI HYDROCHLORIDUM
TETRACYCLINE HYDROCHLORIDE
Tetracycline hydrochloride (non-injectable)
Tetracycline hydrochloride, sterile
Molecular formula. C 22 H 24N 20 s,HCl
904
Graphic formula.
- HCI
I2
Chemical name. (4S,4aS,5aS,6S,12a.S’)-4-Dimethylamino-l,4,4a,5,5a,6,ll,

12a-octahydro-3,6,10,12,l 2a -pentahydroxy-6-m ethyl- l,ll-dioxo-2-
naphthacenecarboxamide monohydrochloride; [45-(4a,4aa,5aa,6P, 12aa)]-4-
(dimethylamino)-l,4,4a,5,5a,6,ll,12a-octahydro-3,6,10,12,12a-pentahydroxy-
6-methyl- 1,11 -dioxo-2-naphthacenecarboxamide mono hydrochloride; CAS
Reg, No. 64-75-5.
TS; practically insoluble in acetone R and ether R.
Storage. Tetracycline hydrochloride should be kept in a tightly closed con-

Labelling. The designation sterile Tetracycline hydrochloride indicates that

the substance complies with the additional requirements for sterile Tetracycline
hydrochloride and may be used for parenteral administration or for other sterile
preparations.
Additional information. Tetracycline hydrochloride decomposes rapidly in

solutions below pH 2, and less rapidly in solutions above pH 7. Even in the
absence of light. Tetracycline hydrochloride is gradually degraded on exposure
to a humid atmosphere, the decomposition being faster at higher temperatures.
Requirements
Definition. Tetracycline hydrochloride contains when tested according to
assayA not less than 96.0% and not more than 102.0% of C22H24N208,HC1,
and when tested according to assay B not less than 950 International Units per
mg, both calculated with reference to the dried substance.
Identity tests
A. Carry out the test as described under 1.14.1 Thin-layer chromatography, but
using an unlined chamber and a cellulose coating prepared as follows: To
905
0.275 g of carbomer R add 120 ml of water, let the mixture stand for 1 hour
while shaking it from time to time; then add gradually while stirring a suf-
ficientvolume of sodium hydroxide (~80g/l) TS to adjust to pH 7.0. To this

mixture add 30 g of cellulose R1 and a sufficient quantity of water (usually
60-80 ml) to obtain a coating substance of suitable consistency. Coat the
plates with a layer 0.4mm thick, and allow them to dry at room tempera-
ture. The plates thus coated are used after a suitable treatment both for the
identity test and the test of “related substances”. For the identity test spray
the plate with phosphate/citrate buffer pH 4.5, TS, until traces of moisture
appear. Dry the plate at 50 °C for 30 minutes.
Prepare the following solutions immediately before use while protected

from bright light: Dissolve 5.0 mg of the test substance, 5.0 mg of chlorte-
tracycline hydrochloride RS, 5.0 mg of oxy tetracycline hydrochloride RS,
and 5.0 mg of tetracycline hydrochloride RS in sufficient methanol R to
produce 10ml; this constitutes solution A. Dissolve 5.0 mg of chlortetracy-
cline hydrochloride RS and 5.0 mg of oxy tetracycline hydrochloride RS in
sufficient methanol R to produce 10ml; this constitutes solution B. Dissolve
5.0 mg of chlortetracycline hydrochloride RS, 5.0 mg of oxytetracycline
hydrochloride RS, and 5.0 mg of tetracycline hydrochloride RS in sufficient
methanol R to produce 10ml; this constitutes solution C.
Apply separately to the plate 1 pi of each of solutions A, B and C, and spray

it very finely and uniformly with trimethylpyridine (50g/l) TS until traces
of humidity appear (about 8 ml).
Pour the mobile phase consisting of a mixture of 60 volumes of ethyl acetate

R, 30 volumes of acetone R, and 6 volumes of water in the unlined chro-
matographic chamber. Place the plate in the chamber in such a manner that
it is not in contact with the mobile phase. Allow the plate to become impreg-
nated with the vapours for 1 hour. Then dip the plate into the mobile phase
and allow the chromatogram to develop to a distance of 15 cm. After remov-
ing the plate from the chromatographic chamber, allow it to dry in air,
expose it to the vapour of ammonia (-260 g/1) TS, and examine the chro-
matogram immediately in ultraviolet light (365 nm). Three principal clearly
separated spots are obtained with solution A corresponding in position,
appearance, and intensity with those obtained with solution C, two of
which correspond with the spots obtained with solution B.
B. To about 1 mg add 2 ml of sulfuric acid (-1760 g/1) TS; a red-violet colour is
produced which on the addition of 0.1ml of water changes to yellow.
906
Specific optical rotation. Use a lOmg/ml solution in hydrochloric acid

(0.01 mol/1) VS and calculate with reference to the dried substance; [a]o =
-239 to -258°.

chromatography, using a plate as prepared under the identity test A. To a suf-
ficient volume of disodium edetate (0.1 mol/1) VS add sodium hydroxide
(~80g/l) TS to adjust to pH 7.0, and use this solution to spray the plate
uniformly until traces of moisture appear. Dry the plate at 50 °C for 30 minutes.
Prepare the following solutions immediately before use, protecting them from
bright light: Dissolve O.lOg of the test substance in sufficient methanol R
to produce 10 ml; this constitutes solution A. Dilute 2.5 ml of solution A to
10.0ml with methanol R; this constitutes solution B. Dissolve 5.0mg of
4-epianhydrotetracycline hydrochloride RS insufficient methanol R to produce
20ml; this constitutes solution K. Dilute 2ml of solution K to 10ml with
methanol R; this constitutes solution C. Dissolve 5.0 mg of 4-epitetracycline
hydrochloride RS in sufficient methanol R to produce 8 ml; this constitutes solu-
tion L. Dilute 2 ml of solution L to 10ml with methanol R; this constitutes solu-
tion D. Dissolve 5.0 mg of anhydrotetracycline hydrochloride RS in sufficient
methanol R to produce 20 ml; this constitutes solution M. Dilute 2 ml of solu-
tion M to 10ml with methanol R; this constitutes solution E. Dissolve 20mg
of chlortetracycline hydrochloride RS in sufficient methanol R to produce
20ml; this constitutes solution N. Dilute 2 ml of solution N to 10 ml with
methanol R; this constitutes solution E Dissolve 10 mg of tetracycline
hydrochloride RS in sufficient methanol R to produce 20ml; this constitutes
solution P. Mix together 0.5ml of each of the following solutions K, L, M, N
and P; this constitutes solution G.
Apply separately to the plate 1 pi of each of solutions A, B, C, D, E, F, and G,

and spray it very finely and uniformly with trimethylpyridine (50g/l) TS until
traces of humidity appear (about 8 ml).
As the mobile phase, use a mixture of 60 volumes of ethyl acetate R, 30 volumes

of acetone R, and 6 volumes of water. Allow the chromatogram to develop to
a distance of 15 cm. After removing the plate from the chromatographic
chamber, allow it to dry in air, expose it to the vapour of ammonia (-260 g/1)
TS, and examine the chromatogram immediately in ultraviolet light (365 nm).
The spot corresponding to 4-epitetracycline hydrochloride obtained with solu-
tion B is not more intense than that obtained with solution D (5% of 4-epite-
tracycline hydrochloride). The spots corresponding to 4-epianhydrotetracycline
907
hydrochloride, anhydrotetracycline hydrochloride, and chlortetracycline

hydrochloride obtained with solution A are not more intense than those
obtained with solution C (0.5% of 4-epianhydrotetracycline hydrochloride),
solution E (0.5% of anhydrotetracycline hydrochloride), and solution F (2% of
chlortetracycline hydrochloride). The test is not valid unless the chromatogram
obtained with solution G shows 5 clearly separated spots.
Anhydroderivatives. Dissolve about 0.2 g, accurately weighed, in sufficient

hydrochloric acid (0.02mol/l) VS to produce 50ml. Place 10.0ml in a separator,
add 10 ml of chloroform R and 10 ml of citrate buffer, pH 5.4 TS, and shake for
2 minutes. Separate the chloroform layer and measure the absorbance at 437 nm
against a solvent cell containing chloroform R; not more than 0.18 (preferably
use 2-cm cells for the measurement and calculate the absorbance of a 1 -cm layer).
Assay
A. Dissolve about 0.25 g, accurately weighed and previously dried at 60 °C
under reduced pressure, in 5 ml of formic acid (~1080g/l) TS and 10ml of
add 10ml of dioxan R, 5ml of mercuric acetate/acetic
glacial acetic acid Rl,
acid TS,and titrate with perchloric acid (0.1 mol/1) VS as described under
2.6 Non-aqueous titration. Method A. Each ml of perchloric acid (0.1 mol/1)
VS is equivalent to 48.09 mg of C22H24N208,HC1.
B. Carry out the assay as described under 3.1 Microbiological assay of antibi-
otics, using either (a) Bacillus pumilus (NCTC 8241 or ATCC 14884) as the
test organism, culture medium Cml with a final pH of 6. 5-6. 6, sterile phos-
phate buffer, pH 4.5 TS, an appropriate concentration of tetracycline (usually
between and 20 lU), and an incubation temperature of 37-39 °C, or {b)
2
Bacillus cereus (ATCC 11778) as the test organism, culture medium Cml with
a final pH of 5. 9-6.0, sterile phosphate buffer, pH 4. STS, an appropriate con-
centration of tetracycline (usually between 0.5 and 2IU), and an incubation
temperature of 30-33 °C. The precision of the assay is such that the fiducial
limits of error of the estimated potency [P = 0.95) are not less than 95% and
not more than 105% of the estimated potency. The upper fiducial limit of
error of the estimated potency (P = 0.95) is not less than 950 lU per mg, cal-
culated with reference to the dried substance.
Additional requirements for Tetracycline hydrochloride for

sterile use
Sterility. Complies with the 3.2.2 Sterility testing of antibiotics, applying the
membrane filtration test procedure. Use the sampling plan described under
908
THIAMINI HYDROBROMIDUM
THIAMINE HYDROBROMIDE
Thiamine hydrobromide, anhydrous
Thiamine hydrobromide hemihydrate
Molecular formula. Ci2Hi7BrN40S,HBr (anhydrous); Ci2Hi7BrN40S,HBr,

-iH20 (hemihydrate).
Graphic formula.
n =0 (anhydrous)
n =1/2 (hemihydrate)
Chemical name. Thiamine bromide, monohydrobromide; 3-[(4-amino-2-

methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium
bromide, monohydrobromide; CAS Reg. No. 4234-86-0 (anhydrous).
Thiamine bromide, monohydrobromide, hemihydrate; 3-[(4-amino-2-methyl-

5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium bromide,
mono-hydrobromide, hemihydrate; CAS Reg. No. 62084-87-1 (hemihydrate).
Description. A white to yellowish white, crystalline powder; odour, slight

and characteristic.
Solubility. Freely soluble in water and in methanol R; sparingly soluble in

ethanol (-750 g/1) TS; practically insoluble in ether R.
Category. Component of vitamin B.
Storage. Thiamine hydrobromide should be kept in a tightly closed, non-

metallic container, protected from light.
Requirements
Definition. Thiamine hydrobromide contains not less than 98.0% and not
more than 101.0% of Ci2Hi7BrN40S,HBr, calculated with reference to the dried
substance.
909
Identity tests
A. Dissolve lOmg in 1 ml of water, add 1 ml of sodium hydroxide (~80g/l) TS,
0.5ml of potassium ferricyanide (lOg/1) TS; the solution remains pale
yellow. Shake with5ml of 2-butanol R and allow to stand for 5-10 minutes;
in bright daylight or in ultraviolet light (365 nm) the 2-butanol layer shows
a blue fluorescence.
B. Spread a small quantity of the powder on a watch-glass; the odour is slight

and characteristic, resembling that of yeast.


and colourless.
Loss on drying. Dry to constant weight at 105 °C. Anhydrous thiamine

hydrobromide loses not more than 5.0mg/g. Thiamine hydrobromide hemi-
hydrate loses not more than 25mg/g.
pH value. pH of a 0.06g/ml solution, 2. 7-3. 4.

acid Rl, add 10ml of mercuric acetate/acetic acid TS, and titrate with per-
of Ci 2 H, 7 BrN 40 S,HBr.
THIAMINI HYDROCHLORIDUM
THIAMINE HYDROCHLORIDE
Molecular formula. Ci 2 Hi 7 ClN 40 S,HCl
910
Graphic formula.
Cl~* HCI
Chemical name. Thiamine chloride, hydrochloride; 3-[(4-amino-2-methyl-5-

pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium chloride, mono-
hydrochloride; CAS Reg. No. 67-03-8.
Description. Colourless crystals or a white or yellowish white, crystalline

powder; odour, slight and characteristic.
Solubility. Soluble in 1 part of water and in 100 parts of ethanol (-750 g/1) TS;
practically insoluble in acetone R and ether R.
Storage. Thiamine hydrochloride should be kept in a tightly closed, non-

Additional information. Even in the absence of light, Thiamine hydrochlo-

sition being faster at higher temperatures. When exposed to air, the anhydrous
product rapidly absorbs about 4g of water per lOOg. Melting temperature,
about 248 °C with some decomposition. In solution at pH 4.0 or less, it loses
its activity only very slowly. Neutral and alkaline solutions deteriorate rapidly,
especially in contact with air.
Requirements
Definition. Thiamine hydrochloride contains not less than 98.0% and not
more than 101.0% of Ci 2 H] 7 ClN 40 S,HCl, calculated with reference to the dried
substance.
Identity tests
A. Dissolve 10 mg in1 ml of water, add 1 ml of sodium hydroxide (-80 g/1) TS
and 0.5ml of potassium ferricyanide (lOg/1) TS; the solution remains pale
yellow.
Shake with 5 ml of 2-butanol R and allow to stand for 5-10 minutes; in bright
daylight or in ultraviolet light (365 nm) the 2-butanol layer shows a blue
fluorescence.
911



50mg/g.
pH value. pH of a 25 mg/ml solution, 2.7-3.3.

Rl, add 10 ml of mercuric acetate/acetic acid TS, and titrate with perchloric
acid (0.1 mol/1) VS as described under 2.6 Non-aqueous titration. Method A.
Ci 2 Hi 7 C 1 N 40 S,HC 1 .
THIAMINI MONONITRAS
THIAMINE MONONITRATE
Graphic formula.
912
Chemical name. Thiamine nitrate (salt); 3-[(4-amino-2-methyl-5-pyrimidinyl)

methyl]-5-(2-hydroxyethyl)-4-methylthiazolium nitrate (salt); CAS Reg. No.
532-43-4.
Description. Colourless crystals or a white, crystalline powder; odour, slight

and characteristic.
Solubility. Sparingly soluble in water; very slightly soluble in ethanol

(-750 g/1) TS.
Storage. Thiamine mononitrate should be kept in a tightly closed, non-

Additional information. Even in the absence of light, Thiamine mononitrate

Requirements
Definition. Thiamine mononitrate contains not less than 98.0% and not more
than 101.0% of C12H17N5O4S, calculated with reference to the dried substance.
Identity tests
A. Dissolve 10 mg in 1 ml of water, add 1 ml of sodium hydroxide (-80 g/1) TS
and 0.5ml of potassium ferricyanide (lOg/1) TS; the solution remains pale
yellow. Shake with 5ml of 2-butanol R and allow to stand for 5-10 minutes;
in bright daylight or in ultraviolet light (365 nm) the 2-butanol layer shows

C. To 2 ml of a 0.05 g/ml solution add 2 ml of ferrous sulfate (15 g/1) TS; it yields
of nitrates.

913
lOmg/g.
Rl, add 0.15ml of 1-naphtholbenzein/acetic acid TS, and titrate with perchlo-
ric acid (0.1 mol/1) VS to a green end-point, as described under 2.6 Non-aqueous

16.37mg of C12H17N5O4S.
THIOACETAZONUM
THIOACETAZONE
Molecular formula. C10H12N4OS
Graphic formula.
CH =N - NH - CS -NHj
NH- CO — CH3
Chemical name. 4'-Formylacetanilide 4'-(thiosemicarbazone); W-[4-[[(amino-

thioxomethyl)hydrazono]methyl]phenyl]acetamide; CAS Reg. No. 104-06-3.
Description. Pale yellow crystals or a yellow, crystalline powder; almost

odourless.
TS and methanol R; soluble in 10 parts of dimethylformamide R.
Storage. Thioacetazone should be kept in a tightly closed container, protected

from light.
914
Requirements
Definition. Thioacetazone contains not less than 98.0% and not more than
102.0% of C10H12N4OS, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from thioacetazone RS or with the reference spectrum of
thioacetazone.
B. The absorption spectrum of a 3.0pg/ml solution in dehydrated ethanol R,

C. Dissolve lOmg by heating in 1ml of sodium hydroxide (5mol/l) VS, add

0.25ml of lead acetate (80g/l) TS and boil for 1 minute; a black precipitate
is produced.
D. About lOmg yields the reaction described for the identification of primary
aromatic amines under 2.1 General identification tests, producing a red
colour.
5-Omg/g.
chromatography, using silica gel R4 as the coating substance and ethyl acetate
R as the mobile phase. Apply separately to the plate lOpl of each of 2 solu-
tions in a mixture of 9 volumes of methanol R and 1 volume of water
containing (A) 2.0mg of the test substance per ml, and (B) 4.0 pg of
/^-acetamidobenzalazine RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in air, spray it evenly with nitric acid
(-130 g/1) TS, and examine the chromatogram in ultraviolet light (365 nm). Any
915
Thiosemicarbazide. Finely powder the test substance and to about 2 g, accu-

rately weighed, add sufficient water to produce 50 ml. Allow to stand for at
least 1 hour, shaking occasionally. Filter, reject the first few ml of the filtrate,
and transfer 25 ml of the filtrate to a 250-ml conical flask. Acidify with sul-
furic acid (-100 g/1) TS and titrate with ceric sulfate (0.1 mol/1) VS, using o-
phenanthrolineTS as indicator, to a blue end-point that persists for 1 minute.
Not more than 0.8ml of ceric sulfate (0.1 mol/1) VS is required (not more than
l.Omg/g).
Assay. Dissolve about 0.1 g, accurately weighed, in 60ml of methanol R

by warming to 60 °C on a water-bath. Slowly add 20 ml of hot silver
nitrate/methanol TS, maintain the temperature at 60 °C until the formed pre-
cipitate congeals and settles, leaving a clear supernatant liquor. Cool and filter
through a dried and weighed sintered-glass crucible and wash the precipitate
with methanol R until the washings are free from silver. Dry to constant weight
at 105 °C, cool and weigh. Each g of precipitate is equivalent to 460. 6mg of
C,oHi 2 N 40 S.
THIOPENTALUM NATRICUM
THIOPENTAL SODIUM
C„Hi7N2Na02S
Chemical name. Sodium 5-ethyl-5-(l-methylbutyl)-2-thiobarbiturate; 5-

ethyldihydro-5-(l-methylbutyl)-2-thioxo-4,6(l//,5//)-pyrimidinedione
monosodium salt; CAS Reg. No. 71-73-8.
Description. A yellowish white powder; odour, characteristic.
Solubility. Freely soluble in water and ethanol (-750 g/1) TS; practically
916
Storage. Thiopental sodium should be kept in a tightly closed container, pro-

tected from light.
Additional information. Thiopental sodium is hygroscopic. Even in the

Requirements
Thiopental sodium contains not less than 97 0 . % and not more than the
equivalent of 102 0 %. of CnHi 7N 2 Na 02 S, calculated with reference to the
dried substance.
Identity tests
A. Place about 0.5g in a separatory funnel, add 10ml of water and acidify with
hydrochloric acid (~70g/l) TS. Shake with 20ml of ether R, separate the
ether layer, wash with 10 ml of water, dry over anhydrous sodium sulfate
R, and filter. Evaporate the filtrate to dryness over a water-bath and dry the
residue at 100-105°C. Carry out the examination as described under 1.7
is concordant with the spectrum obtained from thiopental RS or with the
reference spectrum of thiopental.
B. See the test described below under “Related substances”. Apply 10 |il of each
of solutions B and C and develop the chromatogram for a dis-
to the plate
tance of 18 cm. The with solution B corresponds in
position and intensity with that obtained with solution C.
C. Fuse 0.2 g with 1 g of sodium hydroxide R in a test-tube until the glass glows
red; the melt turns red-brown and vapours are evolved. Insert moistened
pH-indicator paper R into the vapours; its coloration is changed to an alka-
line range.Cool and add 5 ml of water to the melt, mix well, and filter.
Acidify the filtrate with sulfuric acid (-100 g/1) TS and heat gently; the
vapours evolved turn a strip of lead nitrate paper R to brown and then to
black.

20mg/ml solution.
917

and not more intensely coloured than standard colour solution Gn5 when com-
chromatography, using silica gel R4 as the coating substance and the lower layer
of a mixture of 5 volumes of ammonia (-260 g/1) TS, 15 volumes of ethanol
(-750 g/1) TS, and 80 volumes of chloroform R as the mobile phase. Apply
separately to the plate 20 pi of each of three solutions containing (A) 10 mg of
Thiopental sodium per ml (disregard any slight residue), for solution (B) dilute
1 ml of solution A to 10ml, for solution (C) dissolve 85mg of thiopental RS in
10ml of sodium hydroxide (~80g/l) TS and dilute with sufficient water to

produce 100 ml, and for solution (D) dilute 0.5 ml of solution A to 100 ml. After
removing the plate from the chromatographic chamber, examine the chro-
matogram immediately in ultraviolet light.
intense than that obtained with solution D (0.5%). Disregard any spot at the
point of application.
of sulfuric acid (~100g/l) TS, and extract with four 10-ml quantities of chloro-
form R. Filter the combined chloroform extracts, evaporate the filtrate to
dryness on a water-bath, and dissolve the residue in 30 ml of dimethylfor-
mamide R previously neutralized with lithium methoxide (0.1 mol/1) VS. Titrate
immediately with lithium methoxide (0.1 mol/1) VS, using 0.1ml of thymol
blue/methanol TS as indicator, until a blue colour is obtained. Protect the solu-
tion from atmospheric carbon dioxide during the titration.
Each ml of lithium methoxide (0.1 mol/1) VS is equivalent to 26.43 mg of

C]iHi7N2Na02S.
TIABENDAZOLUM
TIABEND AZOLE
918
Graphic formula.
Chemical name. 2-(4-Thiazolyl)benzimidazole; 2-(4-thiazolyl)-lfi-benzimi-

dazole; CAS Reg. No. 148-79-8.
Description. A white to almost white powder; odourless or almost odourless.

Storage. Tiabendazole should be kept in a well-closed container.
Requirements
Definition. Tiabendazole contains not less than 98.0% and not more than
101.0% of C10H7N3S, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from tiabendazole RS or with the reference spectrum of
tiabendazole.
B. The absorption spectrum of a 4.0pg/ml solution in hydrochloric acid

VS, when observed between 230 nm and 350 nm, exhibits maxima
(0.1 mol/1)
atabout 243 nm and 302 nm; the absorbances of a 1-cm layer at these
maxima are about 0.23 and 0.49, respectively.
C. Dissolve 5mg in 5ml of hydrochloric acid (0.1 mol/1) VS, add 3mg of 1,4-
phenylenediamine dihydrochloride R, and shake until dissolved. Add 0.1 g
of zinc R powder, mix, allow to stand for 2 minutes, and add 10ml of ferric
ammonium sulfate (45 g/1) TS; a deep blue or blue-violet colour is produced.
919
5-Omg/g,
50 volumes of toluene R, 20 volumes of glacial acetic acid R, 8 volumes of
acetone R, and 2 volumes of water as the mobile phase. Apply separately to
the plate 10 pi of each of 2 solutions in methanol R containing (A) 10 mg of the
test substance per ml and (B) 0.15 mg of the test substance per ml. After remov-
Assay. Dissolve about 0.16g, accurately weighed, in 30 ml of glacial acetic

acid R1 and titrate with perchloric acid (0.1 mol/1) VS as described under 2.6
Non-aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/1) VS is
equivalent to 20.13g of C10H7N3S.
TIMOLOLI MALEAS
TIMOLOL MALEATE
,C(CH3>3
Ci3H24N403S,C4H404
Chemical name. (-)-(S)-l-(Ktf-Butylamino)-3-[(4-morpholino-l,2,5-thiadia-

zol-3-yl)oxy]-2-propanol maleate (1:1) (salt); (S')-l-[(l,l-dimethylethyl)amino]-
3-[[4-(4-morpholinyl)-l,2,5-thiadiazol-3-yl]oxy]-2-propanol (Z)-2-butenedioate
(1:1) (salt); CAS Reg. No. 26921-17-5.
Description. A white or almost white powder; odourless or almost odourless.
Solubility. Soluble in water, methanol R, and ethanol (-750 g/1) TS; practically
Category. Antiglaucoma drug.
920
Storage. Timolol maleate should be kept in a well-closed container, protected

from light.
Requirements
Timolol maleate contains not less than 98 0 . %
and not more than the equiva-
lent of 101 0 . %
of Ci 3H24N403S,C4H404, Calculated with reference to the dried
substance.
Identity tests

infrared region.
spectrum obtained from timolol maleate RS or with the reference spectrum of
timolol maleate.
B. The absorption spectrum of a 25pg/ml solution in sulfuric acid (0.05 mol/1)

VS, when observed between 230nm and 350 nm, exhibits a maximum at
about 295 nm; the absorbance of a 1-cm layer at this wavelength is about
0.52.
C. Dissolve 0.2 g in 3 ml of water, add 2 ml of sodium hydroxide (-200 g/1) TS,

and shake with three quantities, each of 3 ml, of ether R. Warm the aqueous
layer in a water-bath for 10 minutes, add 2 ml of bromine TSl, boil, and
cool. Add 0.2ml of this solution to lOmg of resorcinol R dissolved in 3ml
of sulfuric acid (-1760 g/1) TS, and heat in a water-bath for 15 minutes; a
bluish black colour is produced.

(1 mol/1) VS; [a]g>’= = -11.7° to -12.5°.

and colourless.
exceeding 0.6kPa or 5 mm of mercury); it loses not more than 5.0mg/g.
from a commercial source is suitable) and a mixture of 80 volumes of
921
dichlorome thane R, 20 volumes of methanol R, and 1 volume of ammonia

of three solutions in methanol R containing (A) 50 mg of Timolol maleate per
ml, (B) 0.2 mg of Timolol maleate per ml, and (C) 0.1 mg of Timolol maleate
to dry in and examine the chromatogram in ultraviolet light (254 nm). Then
air,
expose the plate to iodine vapours for 2 hours and examine the chromatogram
in daylight.
Using both methods of visualization, any spot obtained with solution A, other
than the principal spot, is not more intense than that obtained with solution B,
and not more than two such spots are more intense than that obtained with
solution C.

Rl, add 3 drops of 1-naphtholbenzein/acetic acid TS as indicator, and titrate
with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-aqueous titra-
tion, Method A.

C]3H24N403S,C4H404.
Additional requirement for Timolol maleate for sterile use

TOLBUTAMIDUM
TOLBUTAMIDE
Graphic formula.
CH3 —y==\y— S02NHCNH(CH3>3CH3

(1
II
Chemical name. l-Butyl-3-(/3-tolylsulfonyl)urea; W-[(butylamino)carbonyl]-4-

methylbenzenesulfonamide; CAS Reg. No. 64-77-7.
922

almost odourless.

(-750 g/1) TS; soluble in acetone R.
Category. Antidiabetic.
Storage. Tolbutamide should be kept in a tightly closed container, protected

from light.
Additional information. Even in the absence of light. Tolbutamide is gradu-

Requirements
Definition. Tolbutamide contains not less than 99.0% and not more than
Identity tests

infrared region.
spectrum obtained from tolbutamide RS or with the reference spiectruni of
tolbutamide.
B. Boil 0.2gwith 8ml of sulfuric acid (-700 g/1) TS under a reflux condenser
for 30 minutes.Cool and filter. Keep the precipitate for test C. Make the fil-
by adding sodium hydroxide (-300 g/1) TS, and carry
trate strongly alkaline
out a steam distillation for 30 minutes. Collect the distillate in 30 ml of
hydrochloric acid (0.1 mol/1) VS. To 2ml of the solution containing the
distillateadd 0.2g of sodium acetate R and 10ml of sodium tetraborate
(lOg/1) TS. Cool the mixture for 10 minutes in an ice-bath, add 1ml of 4-
nitroaniline TSl and 2.7 ml of sodium nitrite (100 g/1) TS and allow to stand
for 30 minutes. Add 2.5ml of sodium hydroxide (-80 g/1) TS drop by drop;
after a few minutes an intense red colour develops.
C. Wash the precipitate obtained in test B with 4ml of water and dry at
105 °C; melting temperature, about 136°C.
923
5.0mg/g.
15 volumes of 2-propanol R, 3 volumes of cyclohexane R, 1 volume of ammonia
(-260 g/1) TS and 1 volume of water as the mobile phase. Apply separately to
the plate 5|il of each of 2 solutions in acetone R containing (A) lOmg of the
test substance per ml and (B) 0.050 mg of 4-toluenesulfonamide R per ml. After
removing the platefrom the chromatographic chamber, allow it to dry in a
current of warm air, heat at 110 °C for 10 minutes, spray the hot plate with
sodium hypochlorite TSl and dry in a current of cold air until a sprayed area
of the plate below the line of application gives at most a very faint blue colour
with 1 drop of potassium iodide/starch TS; avoid prolonged exposure to cold
air. Spray the plate with potassium iodide/starch TS, allow it to stand for 5
minutes, and examine the chromatogram in daylight. Any spot obtained with
with solution B.
Assay. Dissolve about 0.55 g, accurately weighed, in 30 ml of ethanol

(-750 g/1) TS previously neutralized to phenolphthalein/ethanol TS, add 20ml
of carbon-dioxide-free water R, and titrate with carbonate-free sodium
hydroxide VS, using phenolphthalein/ethanol TS as indicator. Repeat
(0.1 mol/1)
the operation without the substance being examined and make any necessary
corrections. Each ml of carbonate-free sodium hydroxide (0.1 mol/1) VS is
equivalent to 27.04 mg of C 12 H 18N 2 O 3 S.
TRIHEXYPHENIDYLI HYDROCHLORIDUM
TRIHEXYPHENIDYL HYDROCHLORIDE
Molecular formula. C2oH3iNO,HCl
924
Graphic formula.
Chemical name. a-Cyclohexyl-a-phenyl-l-piperidinepropanol hydrochlo-

Other names. Benzhexol hydrochloride; cyclodolum.
Description. A white or off-white, crystalline powder; odourless or almost

odourless.
Solubility. Slightly soluble in water; soluble in ethanol (-750 g/1) TS, and
methanol R.
Categoiy. Anticholinergic; antiparkinsonism drug.
Storage. Trihexyphenidyl hydrochloride should be kept in a tightly closed

container.
Requirements
Definition. Trihexyphenidyl hydrochloride contains not less than 98.0% and
not more than 101.0% of C oH 3 iNO,HCl,
substance.
Identity tests

spectrum obtained from trihexyphenidyl hydrochloride RS or with the ref-
erence sfrectrum of trihexyphenidyl hydrochloride.
B. Dissolve 0.5g in 5 ml of warm methanol R and add sufficient sodium

hydroxide (-80 g/1) TS to make the solution alkaline to litmus paper R; a
white precipitate is formed. Collect the precipitate, wash with a small
portion of water, recrystallize from methanol R, and dry under reduced
pressure (not exceeding 0.6kPa or about 5mm of mercury) over silica gel,
desiccant, R for 2 hours; melting temperature, about 1 15 °C (trihexyphenidyl
base).
925
5.0mg/g.
pH value. Dissolve 1 .0
g in 1 00 ml of carbon-dioxide-free water R by warming,
then cool; pH of this solution, 5. 0-6.0.
Piperidylpropiophenone. Dissolve O.lOg in a mixture of 40ml of water and

1 ml of hydrochloric acid (1 mol/1) VS with the aid of heat, cool, and add suffi-
cient water to produce 100ml. Measure the absorbance of a 1-cm layer at the
maximum at about 247 nm; not more than 0.5.
(0.1 mol/1) VS, as described under 2.6 Non-aqueous titration. Method A. Each
ml of perchloric acid (0.1 mol/1) VS is equivalent to 33.79mg of C 2 oH 3 iNO,HCl.
TRIMETHADIONUM
TRIMETHADIONE
Molecular formula. C 6 H 9 NO 3
Graphic formula.
Chemical name. 3,5,5-Trimethyl-2,4-oxazolidinedione; CAS Reg. No.

127-48-0.
Description. Colourless, granular crystals; odour, slightly camphoraceous.
Solubility. Soluble in water; freely soluble in ethanol (-750 g/1) TS, and ether
R.
926
Storage. Trimethadione should be kept in a well-closed container, and stored

in a cool place.
Requirements
Definition. Trimethadione contains not less than 98.0% and not more than
101.0% of CsHgNOg, calculated with reference to the dried substance.
Identity tests

infrared region.
spectrum obtained from trimethadione RS or with the reference spectrum of
trimethadione.
B. To 5ml of a 20mg/ml solution add 2ml of barium hydroxide (15g/l) TS; a

precipitate is immediately produced.
C. Heat 0.5g with 10ml of sodium hydroxide (~80g/l) TS on a water-bath

for 30 minutes, evaporate to low bulk, cool in ice, and cautiously add
hydrochloric acid (~70g/l) TS until acid to litmus paper R. To 0.5 ml add 2
drops of ferric chloride (25g/l) TS; a deep yellow colour is produced. Retain
the remainder of the solution for test D.
D. Extract the remainder of the solution obtained in test C 3 times with ether
R, using 10 ml each time; evaporate the combined ether extracts on a water-
bath for 30 minutes and scratch the irmer surface of the container to induce
crystallization; melting temperature, about 80 °C (a-hydroxyisobutyric
acid).
Melting range. 45-47 °C, determined without previous drying.
Assay. Carry out the assay as described under 1.14.5 Gas chromatography. As
an internal standard use 2-phenylethanol TS. Use the following 3 solutions: (1)
to O.lOg of trimethadione RS add 5ml of 2-phenylethanol TS and sufficient
methanol R to produce 10 ml, (2) dissolve 0.20 g of the substance being
examined in sufficient methanol R to produce 10ml, and (3) to 0.20 g of the
substance being examined add 5 ml of 2-phenylethanol TS and sufficient
methanol R to produce 10 ml. For the procedure use a glass column 1.5 m long
927
and 0.4 cm in internal diameter packed with an adequate quantity of an adsor-

bent composed of lOg of diethylene glycol succinate R supported on 90g of
acid-washed, silanized kieselguhr R4. Maintain the column at 105 °C, use nitro-
gen R as the and a flame ionization detector. Prepare chromatograms
carrier gas
A, B, and C from
solutions 1, 2 and 3, respectively. Measure the appropriate
peak areas chromatograms A, B, and C, and calculate the content of
in
CSH 9NO 3 using the data obtained from chromatograms A and C, introducing
,
if necessary the correction resulting from chromatogram B.
TRIMETHOPRIMUM
TRIMETHOPRIM
Graphic formula.
Chemical name. 2,4-Diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine; 5-

[(3,4,5-trimethoxyphenyl)methyl]-2,4-pyrimidinediamine; CAS Reg. No.
738-70-5.
Description. A white, crystalline powder; odourless or almost odourless.
Solubility. Sparingly soluble in water; soluble in methanol R; practically insol-

uble in ether R.
Storage. Trimethoprim should be kept in a well-closed container.
Requirements
Definition. Trimethoprim contains not less than 98.5% and not more than
101 0 . % of C 14 H 18N 4 O 3 ,
928
Identity tests
infrared region.
spectrum obtained from trimethoprim RS or with the reference spectrum of
trimethoprim.
B. Dissolve 25 mg in 5 ml of sulfuric acid (0.005 mol/1) VS, heat if necessary,

and add 2 ml of a mixture of 1.6g of potassium permanganate R dissolved
in sufficient sodium hydroxide (0.1 mol/1) VS to produce 100ml. Heat to
boiling and add to the hot solution 0.4ml of formaldehyde TS. Mix, add
1 ml of sulfuric acid (0.5 mol/1) VS, mix, and again heat to boiling. Cool to
room temperature and filter. To the filtrate add 2 ml of chloroform R and

shake the flask vigorously; a green fluorescence is produced in the chloro-
form layer when examined in ultraviolet light (365 nm).
lOmg/g.
pH value. Shake 0.20 g with 20ml of carbon-dioxide-free water R for 1 minute

and filter; pH of the filtrate, 7. 5-8. 5.
Related substances
• The operations must be performed in a well-ventilated hood.
silica gel R2 as the coating substance and a mixture of 85 volumes of ethyl
acetate R, 10 volumes of methanol R, 5 volumes of water, and 2 volumes of
anhydrous formic acid R as the mobile phase; an unlined chromatographic
chamber should be used and the solvent front allowed to ascend 17 cm above
the line of application. Apply separately to the plate 5 pi of each of 2 solutions
in a mixture of 5 volumes of chloroform R, 4.5 volumes of methanol R, and
1 volume of water containing (A) 40 mg of the test substance per ml and (B)
0.080 mg of the test substance per ml. Pour the mobile phase into the chamber
and insert the plate immediately so as to avoid prior saturation of the chamber.
a stream of cold air for 5 minutes, and examine the chromatogram in ultravio-
let light (254 nm). Place the plate in a closed chamber containing chlorine, pro-
duced by mixing equal volumes of a 15mg/ml solution of potassium
permanganate R and hydrochloric acid (~70g/l) TS, placed at the bottom of the
chamber, and allow to stand for 20 minutes. Remove the plate from the
chamber and drive off the chlorine in a current of cold air until the area below
the line of application does not give any blue colour on the addition of 0.05ml
929
of starch/iodide TS. Spray the plate with starch/iodide TS, and examine the
Rl, and titrate with perchloric acid (0.1 mol/1) VS, as described under 2.6
equivalent to 29.03mg of C 14 H 18N 4 O 3 .
Additional requirement for Trimethojarim for parenteral use

TROPICAMIDUM
TROPICAMIDE
o
and enantiomer
Cl7hl2ohl202
Chemical name. Ai-Ethyl-2-phenyl-A/-(4-pyridylmethyl)hydracrylamide; N-

ethyl-P-(hydroxymethyl)-N-(4-pyridinylmethyl)-benzeneacetamide; CAS Reg.
No. 1508-75-4.
Solubility. Slightly soluble in water; freely soluble in dichloromethane R and

Categoiy. Mydriatic.
Storage. Tropicamide should be kept in a tightly closed container, protected

from light.
Labelling. The designation Tropicamide for sterile non-injectable use indicates

that the substance complies with the additional requirement and may be used
for sterile applications. Expiry date.
930
Requirements
Tropicamide contains not less than 99 0 . %
C 17H 20N 2 O 2 calculated with reference to the dried substance.
,
Identity tests

infrared region.
spectrum obtained from tropicamide RS or with the reference spectrum of
tropicamide.
B. The absorption spectrum of a 0.04 mg/ml solution in hydrochloric acid

C. Dissolve 5 mg in 3 ml of a mixture of 9 ml of acetic anhydride R, 1ml of

and O.lOg of citric acid R. Heat on a water-bath
acetic acid (~300g/l) TS, for
5-10 minutes: a reddish yellow colour is produced.

or about 5 mm
of mercury) for 4 hours; it loses not more than 5.0mg/g.
Tropic acid. To lO.Omg add 5mg ofsodium tetraborate R and 0.35ml of a

freshly prepared solution containing g of dimethylaminobenzaldehyde R in
1 .0
10ml of a mixture of 9 volumes of TS and 1 volume of

sulfuric acid (~1760g/l)
water. Heat on a water-bath for 3 minutes. Cool in ice-water and add 5 ml of
acetic anhydride R; no violet-red colour develops (0.05%).
95 volumes of dichloromethane R, 5 volumes of methanol R, and 0.5 volume
10 pi of each of 3 solutions in dichloromethane R containing (A) 20 mg of Tropi-
camide per ml, (B) 0.10 mg of Tropicamide per ml, and (C) 40 pg of Tropicamide
to dry in air, and examine the chromatogram in ultraviolet light (254 nm).
931
intense than that obtained with solution B (0.5%). Not more than one such
spot is more intense than that obtained with solution C (0.2%).
Rl, and titrate with perchloric acid (0.1 mol/1) VS, using 1-naphtholbenzein/
acetic acid TS as indicator until the colour changes from orange to green as
described under 2.6 Non-aqueous titration, Method A.

C17H20N2O2.
Additional requirement for sterile non-injectable Tropncamide

TUBOCURARINI CHLORIDUM
TUBOCURARINE CHLORIDE
Molecular formula. C37H4iClN206,HCl,5H20
Graphic formula.
Chemical name. (-f-)-Tubocurarine chloride hydrochloride, pentahydrate;

7',12'-dihydroxy-6,6'-dimethoxy-2,2',2'-trimethyltubocuraranium chloride hy-
drochloride pentahydrate; CAS Reg. No. 6989-98-6 (pentahydrate).
Description. A white to yellowish white, crystalline powder; odourless.
Solubility. Soluble in 20 parts of water and 30 parts of ethanol (~750g/l) TS;

practically insoluble in acetone R, and ether R.
932
Storage. Tubocurarine chloride should be kept in a tightly closed container.
Additional information. Tubocurarine chloride melts at about 270 °C with

decomposition.
Requirements
Definition. Tubocurarine chloride contains not less than 98.0% and not more
than 102.0% of C 37H 4 iClN 20 (;,HCl, calculated with reference to the dried
substance.
Identity tests
A. Dissolve 10 mg in 1 ml of water and add 1 ml of mercuric nitrate TS; a cherry
red colour is slowly produced.
B. Dissolve lOmg in 1 ml of water and add 0.1 ml of ferric chloride (25g/l) TS;
a green colour is produced, which becomes brown on warming on a
water-bath.

Specific optical rotation. Use a 10 mg/ml solution, which has been allowed
to stand for 3 hours, and calculate with reference to the dried substance;
[a]g>"= = -t210to-t220°.
Chloroform-soluble substances. Dissolve 0.25 g in 150ml of water, add

5 ml of a saturated solution of sodium hydrogen carbonate R, and extract with
3 quantities, each of 20ml, of chloroform R. Wash the combined chloroform
extracts with 10 ml of water, filter the chloroform solution into a beaker, wash
the filter with 2 successive quantities, each of 5ml, of chloroform R, and add
the washings to the filtrate. Evaporate the combined filtrate and washings on
a water-bath and dry the residue at 105 °C for 1 hour; the weight of the residue
is not more than 5mg (2.0%). Add 10ml of water to the residue; the residue
does not dissolve. Then add 1 ml of hydrochloric acid (~70g/l) TS; the residue
dissolves.

exceeding O.dkPa or about 5 mm
of mercury); it loses not less than 90mg/g and
933
R1 by warming on a water-bath, cool, and add 60 ml of acetic anhydride R and
10ml of mercuric acetate/acetic acid TS. Titrate with perchloric acid (0.1 mol/1)
VS, determining the end-point potentiometrically as described under 2.6
equivalent to 34.08 mg of C37H4]C1N20(;,HC1.
VERAPAMILI HYDROCHLORIDUM
VERAPAMIL HYDROCHLORIDE
Molecular formula. C27H38N204,HC1
Graphic formula.
CHICHjjj
Chemical name. 5-[(3,4-Dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxy-

phenyl)-2-isopropylvaleronitrile hydrochloride; a-[3-[[2-(3,4-dimethoxyphenyl)-
ethyl]methylamino]propyl]-3,4-dimethoxy-a-(l-methylethyl)benzeneacetonitrile
monohydrochloride; CAS Reg. No. 152-11-4.

almost odourless.
TS.
Storage. Verapamil hydrochloride should be kept in a well-closed container,

934
Requirements
Definition. Verapamil hydrochloride contains not less than 99,0% and not
more than 101.0% of C 27H 38 N 204 ,HC 1, calculated with reference to the dried
substance.
Identity tests
• Either tests A and D or tests B, C, D and E may be applied.

infrared region.
spectrum obtained from verapamil hydrochloride RS or with the reference
spectrum of verapamil hydrochloride.

maxima at about 229nm and 278nm; the absorbances of a 1-cm layer at
these maxima are about 0.63 and 0.24, respectively.
C. Dissolve 20mg 2.5ml of water, add 0.5ml of sulfuric acid (~570g/l) TS

in
and 0.2ml of potassium permanganate (lOg/1) TS; a violet precipitate is pro-
duced, which quickly dissolves to produce a very pale yellow solution.

E. Melting temperature, about 143 °C.
Clarity and colour of solution. A solution of 0.50 g in 10 ml of

carbon-dioxide-free water R is clear and colourless.
Readily carbonizable substances. Dissolve O.lOg in 5ml of sulfuric acid

(~1760g/l) TS. After 5 minutes the solution is clear and not more intensely
5-Omg/g.
pH value. pH of a 0.05 g/ml solution in carbon-dioxide-free water R, 4. 5-6. 5.
Related substances
using silica gel R5 as the coating substance (a precoated plate from a corn-
935
mercial source is suitable) and a mixture of 85 volumes of cyclohexane R
and 15 volumes of diethylamine R as the mobile phase. Apply separately to

the plate 10 pi of each of 3 solutions in chloroform R containing (A) 50 mg
of the test substance per ml, (B) 25 pg of the test substance per ml, and (C)
50 pg of the test substance per ml. After removing the plate from the chro-
matographic chamber, allow it to dry at room temperature for 10 minutes
and develop the plate a second time. Remove the plate, heat it at 110°C for
1 hour, allow it to cool, and spray it with a solution prepared by dissolving
5g of ferric chloride R and 2g of iodine R in 50 ml of tartaric acid (200 g/1)

TS, applying a total of 15-20ml of the reagent. Examine the plate immedi-
ately in daylight disregarding any spot on the line of application. Up to 3
secondary spots may be more intense than the spot obtained with solution
B but they must be less intense than the spot obtained with solution C. Any
other secondary spots in the chromatogram obtained with solution A must
be less intense than the spot obtained with solution B.
B. Carry out test A once more using a mixture of 70 volumes of toluene R, 20

volumes of methanol R, 5 volumes of acetone R, and 5 volumes of glacial
acetic acid R as the mobile phase. The result is the same as that with test
A.
Assay. Dissolve about 0.5 g, accurately weighed, in 30ml of glacial acetic

acid Rl, add 10ml of mercuric acetate/acetic acid TS followed by 0.15ml of 1-
naphtholbenzein/acetic acid TS as indicator, and titrate with perchloric acid
ml of perchloric acid (0.1 mol/1) VS is equivalent to 49.11 mg of C27H38N204,HC1.
Additional requirements for Verapamil hydrochloride for

parenteral use
936
VINBLASTINI SULFAS
VINBLASTINE SULFATE
0CH3
C46H5sN409,H2S04
Chemical name. Vincaleukoblastine sulfate (1 :

1) (salt); CAS Reg. No.
143-67-9.
Description. A white to slightly yellow, amorphous or crystalline powder.
Solubility. Freely soluble in water; very slightly soluble in ethanol (~750g/l)

Storage. Vinblastine sulfate should be kept in a tightly closed container, pro-

Additional information. CAUTION: Vinblastine sulfate must be handled

with care, avoiding contact with the skin and inhalation of airborne particles.
It is very hygroscopic and unstable. Before the bottle is opened, it should be
allowed to come to room temperature in a desiccator.
Requirements
Vinblastine sulfate contains not less than 96 0 % and not more than the equiv-
.
alent of 101 0 % of C46H5 sN 409,H2S04, Calculated with reference to the dried

.
substance.
937
=
Identity tests

spectrum obtained from vinblastine sulfate RS or with the reference spectrum
of vinblastine sulfate.
B. See the test described below under “Related alkaloids”. The principal spot
C. To 1 mg add 0.2 ml of vanillin/hydrochloric acid TS and allow to stand for

1 minute; a pink colour is produced (distinction from vincristine sulfate).

Specific optical rotation. Use a 20 mg/ml solution in methanol R and calcu-

latewith reference to the dried substance; [a]™ -20 to -35°.
Clarity of solution. A solution of 30mg in 10ml of water is clear.
Loss on drying. Dry 60 °C under reduced pressure (not exceeding 0.6kPa

at
or 5mm of mercury) for 16 hours; it loses not more than 170mg/g.
80 volumes of toluene R, 40 volumes of chloroform R, and 6 volumes of diethy-
lamine R as the mobile phase. Apply separately to the plate 5 |xl of each of three
solutions in methanol R containing (A) 10 mg of Vinblastine sulfate per ml, (B)
0.2 mg of vincristine sulfate RS per ml, and (C) 10 mg of vinblastine sulfate RS
to dry in air, and examine the chromatogram in ultraviolet light (254 nm).

produce 500 ml.

about 267 nm. Calculate the content of C46H5aN409,H2S04, using the absorp-
tivity value of 18.5 (A)"™ = 185).
938
VINCRISTINI SULFAS
VINCRISTINE SULFATE
Molecular formula. C46H56N40io,H2S04
Graphic formula.
Chemical name. Leurocristine sulfate (1 :

1) (salt); 22-oxovincaleukoblastine
sulfate (1 :
1) (salt); CAS Reg. No. 2068-78-2.
Description. A white to slightly yellow, amorphous or crystalline powder;

odourless.
Storage. Vincristine sulfate should be kept in a tightly closed container, pro-

Additional information. Vincristine sulfate is hygroscopic and very toxic.

CAUTION: Vincristine sulfate must be handled with care, avoiding contact
939
Requirements
Definition. Vincristine sulfate contains not less than 95.0% and not more than
105.0% of C46H56N40 io,H 2S04, Calculated with reference to the dried substance.
Identity tests

The infrared absorption spectrum of the substance dried
infrared region.
under reduced pressure for 16 hours at 40 °C is concordant with the spec-
trum obtained from vincristine sulfate RS similarly prepared or with the ref-
erence spectrum of vincristine sulfate.
C. To about 1 mg add 0.2 ml of vanillin/hydrochloric acid TS and allow to stand

for approximately 1 minute; an orange colour is produced (distinction from
vinblastine sulfate).


pH value. pH of l.Og/ml solution, 3. 5-4. 5.
40 volumes of toluene R, 20 volumes of chloroform R, and 3 volumes of diethy-
solutions in methanol R containing (A) 10 mg of the test substance per ml, (B)
0.20 mg of the test substance per ml, and (C) 10 mg of vincristine sulfate RS per

produce 500 ml. Measure the absorbance of this solution in a 1-cm layer at the
maximum at about 297 nm and calculate the content of C46H56N40io,H2S04,
using the absorptivity value of 17.7 (Al’™ = 177).
940
WARFARINUM NATRICUM
WARFARIN SODIUM
Molecular formula. Ci 9 Hi 5 Na 04 Ci9H]5Na04,C3H80,H20
;
(hyclate).
Relative molecular mass. 330.3; 408.4 (hyclate).
Graphic formula.
ONa CHjCOCHj
n = 0
n = 1
Chemical name. 3-(a-Acetonylbenzyl)-4-hydroxycoumarin sodium salt; 4-

sodium salt; CAS
hydroxy-3-(3-oxo-l-phenylbutyl)-2ih-l-benzopyran-2-one
Reg. No. 129-06-6.
3-(a-Acetonylbenzyl)-4-hydroxycoumarin sodium salt compound with 2-
propanol monohydrate; 4-hydroxy-3-(3-oxo-l-phenylbutyl)-2H-benzopyran-2-

one sodium salt 2-propanol monohydrate.
Description. A white, amorphous or crystalline powder; odourless.
Solubility. Soluble in less than 1 part of water and in ethanol (-750 g/1) TS;
Storage. Warfarin sodium should be kept in a well-closed container, protected

from light.
Labelling. The designation on the container of Warfarin sodium should state

whether the substance is in the amorphous or the crystalline, clathrate form.
Additional information. Warfarin sodium is discoloured by light. Even in the

941
Requirements
Definition. Warfarin sodium contains not less than 98.0% and not more than
102.0% of Ci 9 Hi 5 Na 04 calculated with reference
,
to the anhydrous and 2-
propanol-free substance.
Identity tests
A. Dissolve 0.1 g in 25 ml of water, add 0.1 ml of hydrochloric acid (~70g/l) TS,
collect the precipitate on a filter (keep the filtrate for test C), wash with
water, and dry the residue at 105°C. Melting temperature, about 162°C
(warfarin). (Keep the residue for test B.)
B. Carry out the examination of the residue obtained in test A as described

under 1.7 Spectrophotometry in the infrared region. The infrared absorp-
tion spectrum is concordant with the spectrum obtained from warfarin RS
or with the reference spectrum of warfarin.
C. The filtrate obtained in test A yields reaction B described under 2.1 General
identification tests as characteristic of sodium.
D. Dissolve 1 g in 10 ml of water, add 5 ml of nitric acid (~1000g/l) TS, and filter.

To the filtrate add 2 ml of potassium dichromate (0.0167 mol/1) VS and shake
for 5 minutes; only the clathrate yields a light greenish blue solution.
Clarity of solution. The opalescence of a solution of 0.50 g in 10 ml of carbon-

dioxide-free water R is not more intense than that of opalescence standard TS2.

5 volumes of chloroform R, 5 volumes of cyclohexane R, and 2 volumes of
glacial acetic acid R as the mobile phase. Apply separately to the plate 20 pi of
each of 2 solutions in acetone R containing (A) 20 mg of the test substance per
the chromatographic chamber, allow it to dry in air and immediately examine
with solution B.
Absorbance in alkaline solution. Dissolve 1.25 g, accurately weighed, in

10ml of sodium hydroxide (50g/l) TS, filter through a membrane filter, and,
942
within 15 minutes, measure the absorbance of a 1-cm layer at the maximum

atabout 385 nm against a solvent cell containing sodium hydroxide (50g/l) TS;
not more than 0.3.
2-Propanol content. Dissolve about 0.8g, accurately weighed, in 25.0ml of

water. Add 25.0ml of sulfuric acid (0.125mol/l) VS while swirling, filter, and
transfer 10 ml of the clear filtrate to a 250-ml flask containing 40 ml of water.
Add some boiling chips, then 30 ml of potassium dichromate TS3, and connect
the flask to a condenser by means of a 75° connecting tube. Distil 60ml, col-
lecting the distillate in 20 ml of sodium hydroxide (~80g/l) TS contained in a
250-ml iodine flask immersed in an ice-bath. Add, with swirling, 20.0 ml of
iodine (0.1 mol/1) VS, insert the stopper in the flask, and allow to stand for 30
minutes. Add 5 ml of hydrochloric acid (~420g/l) TS through the well in the
flask, rinse the well and the neck of the flask with water, swirl to mix, remove
the stopper, and titrate the excess iodine with sodium thiosulfate (0.1 mol/1) VS,
adding 3 ml of starch TS towards the end of the titration. Each ml of iodine
(0.1 mol/1) VS is equivalent to 1.001 mg of 2-propanol. The amorphous sub-
stance contains not more than 3 mg per g and the crystalline clathrate contains
between 43 and 83 mg of 2-propanol per g.
Assay. Dissolve about 0.1 g, accurately weighed, in sufficient sodium hydrox-

ide (0.01 mol/1) VS to produce 100ml and dilute 10ml to 1000ml with sodium
hydroxide (0.01 mol/1) VS. Measure the absorbance of a 1-cm layer of the
diluted solution at the maximum at about 308 nm. Calculate the amount of
Ci 9 Hi 5 Na 04 in the substance being tested by comparison with warfarin RS,
similarly and concurrently examined, taking into account that each mg of war-
farin RS is equivalent to 1.071 mg of CigHisNaO^. In an adequately calibrated
spectrophotometer the absorbance of the reference solution should be 0.47 + 0.03.
ZINCI OXYDUM
ZINC OXIDE
Molecular formula. ZnO
Chemical name. Zinc oxide; CAS Reg. No. 1314-13-2.
Description. A white or faintly yellowish white, very fine, amorphous

powder, free from grittiness; odourless.
hydrochloric acid (-70 g/1) TS.
943
Category. Mild astringent used topically as a protective.
Storage. Zinc oxide should be kept in a well-closed container.
Additional information. Zinc oxide gradually absorbs carbon dioxide from

the air.
Requirements
Definition. Zinc oxide contains not less than 99.0% and not more than
100.5% of ZnO, calculated with reference to the freshly ignited substance.
Identity tests
A. Heat strongly a small amount of the substance; it assumes a yellow colour,
which disappears on cooling.
B. Dissolve20mg in 2.0ml of hydrochloric acid (~70g/l) TS, add 0.15ml of

potassium ferrocyanide (45g/l) TS; a greenish white precipitate is formed.
Arsenic. Use a solution of 1.6g in 35ml of hydrochloric acid (~70g/l) TS and

Carbonates and acid-insoluble substances. Mix 2.0g with 10ml of water,

add 30ml of sulfuric acid (~100g/l) TS, and heat on a water-bath with constant
stirring; no effervescence occurs and the resulting solution is clear and colourless.
Iron. Dissolve 0.20 g in 5 ml of hydrochloric acid (-250 g/1) TS and 30 ml of

water. Treat the solution as described under 2.2.4 Limit test for iron; not more
than 200pg/g.
Lead. Add 2 g to 20 ml of water, stir well, add 5 ml of glacial acetic acid R, and
warm on a water-bath until solution is effected. Then add 0.25 ml of potassium
chromate (100 g/1) TS; no turbidity or precipitate is produced.
Loss on ignition. Ignite 1.0 g at 500 °C to constant weight; it loses not more
than lOmg/g.
Alkalinity. Mix Ig with 10 ml of hot water, add 0.1ml of phenolphthalein/

ethanol TS and filter; if the filtrate is red, not more than 0.3 ml of hydrochloric
acid (0.1 mol/1) VS is required to discharge the colour.
Assay. Dissolve about 0.15g, accurately weighed, in 10ml of acetic acid

(-120 g/1) TS and proceed with the titration as described under 2.5 Com-
plexometric titrations for zinc. Each ml of disodium edetate (0.05mol/l) VS is
equivalent to 4. 069 mg of ZnO.
944
Monographs
Dosage Forms:
General monograph
CopyrigfiJed i
Monographs for dosage forms
Dosage Forms: General monographs

CAPSULES
Definition
Capsules are solid dosage forms with hard or soft shells. They are of various
shapes and sizes, and contain a single dose of one or more active ingredients.
They are intended for oral administration, but preparations for alternative appli-
cations, such as vaginal or rectal, are also available in this presentation. These
preparations may require a special formulation, method of manufacture, or
form of presentation, appropriate to their particular use. For this reason they
may not comply with certain sections of this monograph. Starch capsules (often
known as cachets) are not described in this monograph.
The different categories of capsules that exist include hard, soft, and mod-
ified-release capsules. Their surfaces may bear symbols or other markings. They
should be sufficiently robust to withstand handling, including packaging,
storage, and transportation, without cracking or breaking. They should be pack-
aged and stored in a manner that protects them from microbial contamination.
Capsule shells are made of gelatin or other substances, the consistency of
which may be modified by the addition of substances such as glycerol and sor-
bitol. Preservatives may also be necessary. The shell should disintegrate in the
presence of digestive fluids so that the contents are released. The contents of
capsules may be solid, liquid, or of a paste-like consistency. Capsule shells and
contents may contain excipients such as diluents, solvents, surface-active sub-
stances, opaque fillers, antimicrobial agents, sweeteners, colouring matter,
flavouring substances, disintegrating agents, glidants, lubricants, and substances
capable of modifying the behaviour of the active ingredient(s) in the gastroin-
testinal tract. The contents should not cause deterioration of the shell.
When excipients are used, it is necessary to ensure that they do not
adversely affect the stability, dissolution rate, bioavailability, safety, or efficacy
must be no incompatibility between any of
of the active ingredient(s); there
the components of the dosage form.
Visual inspection
Unpack and inspect at least 20 capsules. They should be smooth and undam-
aged. Evidence of physical instability is demonstrated by gross changes in
physical appearance, including hardening or softening, cracking, swelling,
mottling, or discoloration of the shell.
Unifonnity of mass
Capsules comply with the test for 5.2 Uniformity of mass for single-dose prepa-
rations, unless otherwise specified in the individual monograph.
947
Uniformity of content
A requirement for compliance with the test for 5.1 Uniformity of content for
single-dose preparations is specified in certain individual monographs where
the active ingredient is 5% or less of the total formulation. In such cases the
test for 5.2 Uniformity of mass for single-dose preparations is not required.
Dissolution test
Where a requirement for the “Dissolution test” is specified in the individual
monograph, compliance with 5.3 Disintegration test for tablets and capsules is
not required.
Labelling
Every pharmaceutical preparation must comply with the labelling requirements
established under Good Manufacturing Practice.
The label should include:
(1) the name of the pharmaceutical product;

(2) the name(s) of the active ingredient(s); INNs should be used wherever
possible;
(3) the amount of the active ingredient(s) in each capsule and the number
of capsules in the container;
(4) the batch (lot) number assigned by the manufacturer;
(5) the expiry date and, when required, the date of manufacture;
(6) any special storage conditions or handling precautions that may be
necessary;
(7) directions for use, warnings, and precautions that may be necessary; and
(8) the name and address of the manufacturer or the person responsible for
placing the product on the market.
Storage
Capsules should be kept in well-closed containers. They should be protected
from light, excessive moisture, or dryness, and should not be subjected to tem-
peratures above 30 °C. Additional special packaging, storage, and transporta-
tion recommendations are specified in the individual monograph.
Requirements for specific types of capsules
Hard cap>sules
Definition
Hard capsules have shells consisting of two prefabricated cylindrical sections
that fit together. One end rounded and closed, and the other
of each section is
is open. The contents of hard capsules form (powder or

are usually in solid
granules); in certain cases the contents may be in the form of encapsulated
powders or micropeUets.
948
Manufacture
The manufacturing and filling processes for hard capsules should meet the
requirements of Good Manufacturing Practice, especially with regard to cross-
contamination. The following information is intended to provide very broad
guidelines concerning the main steps to be followed during production, indi-
cating those that are the most important.
The particle size of the active ingredient(s) is of primary significance in
determining the rate and extent of dissolution, the bioavailability, and the uni-
formity of a drug product, especially for substances of low solubility in aqueous
media. In order to obtain a suitable formulation, it is usually necessary to mix
the active ingredient(s) with a number of excipients. It is essential that such

mixing is carried out in a manner that ensures homogeneity. Sometimes, the
physical characteristics of the mixture allow it to be directly filled into the shell,
but it may filling. Normally the
occasionally be necessary to granulate before
granulate needs to be mixed with lubricants and/or disintegrating agents. The
use of excessive amounts of lubricants should be avoided since these may dele-
teriously affect the capsules.
A uniform mass of the capsule mixture is volume trically fed into the nar-
rower lower section of the shell body which is then closed by slipping the larger
section or cap over it. The security of the closure may be ensured by suitable
means.
Throughout manufacturing, certain procedures should be validated and
monitored by carrying out appropriate in-process controls. These should be
designed to guarantee the effectiveness of each stage of production. In-process
controls during hard capsule production should include the particle size of the
active ingredient(s), the homogeneity and moisture content of the mixture and/
or granulate (as well as of the shells), the size of granules, the flow of the final
mixture, and the uniformity of mass, capsule size, integrity of the seals, and
disintegration or dissolution rate (e.g. for modified-release capsules) of the
finished dosage form.
Disintegration test
Hard capsules comply with 5.3 Disintegration test for tablets and capsules.
Use water as the immersion fluid unless hydrochloric acid (0.1 mol/1) VS is
specified in the individual monograph. Operate the apparatus for 30 minutes
and examine the state of the capsules.
If capsules float, use a disc as described under 5.4 Disintegration test for
suppositories.
Soft cap'sutes
Definition
Soft capsules have thicker shells than hard capsules, and preservatives are
usually added. The shells are of one piece and various shapes. Partial migration
of the contents into the shell may occur (and vice versa) depending on the
nature of the materials used and the product in question.
949
Manufacture
The manufacturing processes for soft capsules should meet the requirements
of Good Manufacturing Practice. The following information is intended to
provide very broad guidelines concerning the main steps to be followed during
production, indicating those that are the most important.

mixing is manner that ensures homogeneity. Soft gelatin cap-
carried out in a
sules are usually formed, filled, and sealed in one operation. However, shells
for extemporaneous use are sometimes prefabricated. Liquids may be incor-
porated directly. Solids are usually dissolved or dispersed in a suitable
excipient(s) to give a solution or dispersion of thick consistency.
controls during soft capsule production should include the particle size of the
active ingredient(s), the homogeneity of the mixture, the viscosity of the con-
tents, and the uniformity of mass, capsule size, integrity of the seals, and dis-
integration or dissolution rate (e.g. for modified-release capsules) of the finished
dosage form.
Disintegration test
Soft capsules comply with 5.3 Disintegration test for tablets and capsules, using
water as the immersion fluid unless hydrochloric acid (0.1 mol/1) VS is speci-
fied in the individual monograph. Operate the apparatus for 30 minutes and
examine the state of the capsules.
Modified-retease capsules
Definition
Modified-release capsules are hard or soft capsules in which the contents or the
shell or both contain additives or are prepared by special procedures such as
micro-encapsulation which, separately or together, are designed to modify the
rate of release of the active ingredient(s) in the gastrointestinal tract.
Extended-release capsules
Definition
Extended-release capsules are designed to slow the rate of release of the active
ingredient(s) in the gastrointestinal tract.
All requirements for these specialized dosage forms are given in the indi-
vidual monographs.
950
Delayed-retease capsules (enteric capsules)

Definition
Delayed-release capsules are hard or soft capsules prepared in such a manner
that either the shell or the contents resist the action of gastric fluid but release
the active ingredient(s) in the presence of intestinal fluid.
Manufacture
The statements given under either hard or soft capsules apply, as appropriate
to delayed-release capsules.
Disintegration test
Delayed-release capsules comply with 5.3 Disintegration test for tablets and
capsules, using hydrochloric acid (0.1 mol/1) VS as the immersion fluid. Operate
the apparatus for 2 hours, unless otherwise specified in the individual mono-
graph (but in any case for not less than 1 hour), and examine the state of the
capsules. No capsule should show signs of disintegration or rupture permitting
the contents to escape. Replace the acid by phosphate buffer solution, pH 6.8,
TS with added pancreatin R where specified in the individual monograph.
Operate the apparatus for 60 minutes and examine the state of the capsules.
OPHTHALMIC PREPARATIONS
Definition
Ophthalmic preparations (eye preparations) are sterile, liquid, semi-solid, or
solid preparations that may contain one or more active pharmaceutical ingre-
dient! s) intended for application to the conjunctiva, the conjunctival sac or the
eyelids.
The choice of base and any excipients used for the preparation of oph-
thalmic preparations must be proven through product development studies not
to affect adversely either the stability of the final product or the availability of
the active ingredients at the site of action. The addition of colouring agents is
not recommended.
Unless the active ingredient itself has antimicrobial activity, ophthalmic
preparations supplied as multidose preparations may include a suitable antimi-
crobial agent. The antimicrobial activity should remain effective throughout the
entire period of use.
The different categories of ophthalmic preparations include drops consist-
ing of emulsions, solutions or suspensions, and ointments.
Manufacture
The manufacturing processes should meet the requirements of Good Manu-
facturing Practices, especially with regard to cross-contamination. The follow-
951
ing information is intended to provide very broad guidelines concerning the

main steps to be followed during production, indicating those that are the most
important.
controls during production of ophthalmic preparations should include moni-
toring environmental conditions (especially with respect to particulate and
microbial contamination), pyrogens (use of a limulus amoebocyte lysate (LAL)
test may be advantageous), pH and clarity of solution, and integrity of con-
tainer (absence of leakage, etc.). Appropriate limits should be set for the parti-
cle size of the active ingredient(s).
It is essential that ophthalmic preparations are sterile. An aseptic manufac-
turing process is usuallyemployed when the dosage form does not allow
routine sterilization methods to be used.'
Packaging must be adequate to protect ophthalmic preparations from light,
moisture, microbial contamination, and damage due to handling and

transportation.
Visual inspection
Inspect the ointments, aqueous or oily solution, suspensions, or emulsions.
Evidence of physical and/or chemical instability is demonstrated by notice-
able changes in colour and odour.
Sterility
Ophthalmic preparations comply with 3.2 Test for sterility.
Particle size
Ophthalmic preparations containing dispersed solid particles comply with the
following test.
Take a quantity of the preparation (shake the container gently if necessary)

corresponding to at least 10 pg of solid active ingredient and place in a count-
ing cell or spread in a thin layer on a slide. Firmly apply a cover-glass and scan
the whole area of the sample under a microscope.^
For each 10 pg of solid active substance not more than 20 particles should
have a maximum dimension greater than 25 pm and not more than two of these
particles should have a maximum dimension greater than 50 pm. None of the
particles should have a maximum dimension greater than 90 pm.
^
5.8 Methods of sterilization.
^
For practical reasons, the whole sample is first scanned at low magnification (e.g, x50) and
particles >25 pm are identified. The larger particles can then be measured at a higher magnifica-
tion (e.g. x200-x500).
952
Containers
The materials for containers and closures should not adversely affect the quality
of the preparation or allow diffusion of any kind into or across the material of
the container into the preparation. The container should be fitted with a closure
that minimizes microbial contamination and a device that reveals whether the
container has ever been opened.
Labelling
established by Good Manufacturing Practices.

(2) the name(s) of the active ingredient(s); International Nonproprietary
Names (INN) should be used wherever possible;
(3) the concentration(s) of the active ingredient(s) and the amount or the
volume of preparation in the container;
(5) the expiry date, the utilization period, and, when required, the date of
manufacture;
necessary;
(7) if applicable, the period of use after opening the container;
(8) directions for use, warnings and precautions that may be necessary;
placing the product on the market;
(10) if applicable, the name(s) and concentration(s) of antimicrobial agent(s)
and/or antioxidant(s) incorporated in the preparation; and
(11) the statement “This preparation is sterile”.
Storage
Ophthalmic preparations should maintain their integrity throughout their shelf-
life when stored at the temperature indicated on the label. Special storage
recommendations or limitations are indicated in individual monographs.
Requirements for specific types of ophthalmic

preparations
Ophthalmic drops
Definition
Ophthalmic drops (eye drops) are sterile aqueous or oily solutions, suspen-
sions, or emulsions intended for instillation into the conjunctival sac.
Ophthalmic drops should be clear and practically free from particles
when examined under suitable conditions of visibility.
953
“Water for injections” should be used in the manufacture of aqueous

ophthalmic drops.
The preparation of aqueous ophthalmic drops requires careful consider-
ation of the need for isotonicity, a certain buffering capacity, the desired pH,
the addition of antimicrobial agents and/or antioxidants, the use of viscos-
ity-increasing agents, and the choice of appropriate packaging.
Ophthalmic drops are considered isotonic when the tonicity is equal to
that of a 0.9% solution of sodium chloride. The eye can usually tolerate
solutions equivalent to 0.5-1 .8% of sodium chloride.
Ideally, the pH of ophthalmic drops should be equivalent to that of tear
fluid,which is 7.4. However, the decision to add a buffering agent should
be based on stability considerations. The pH selected should be the
optimum for both stability of the active pharmaceutical ingredient and
physiological tolerance. If a buffer system is used, it must not cause pre-
cipitation or deterioration of the active ingredient. The influence on the
lachrymal flow should also be taken into account.
Visual inspection
Evidence of physical instability is demonstrated by the cloudiness of
aqueous solutions, due to the formation of a precipitate.
Containers
Ophthalmic drops are normally supplied in suitable multidose containers
that allow successive drops of the preparation to be administered. The con-
tainer should be fittedwith a tamper-evident device. The maximum volume
of the preparation in such a container should be no more than 10 ml, unless
otherwise specified and authorized. Multidose ophthalmic drop prepara-
tions may be used for up to 4 weeks after the container is initially opened.
Droppers supplied separately should also comply with 3.2 Test for sterility.
Ophthalmic drops may also be provided in suitable single-dose con-
tainers that will maintain the sterility of the contents and the applicator up
to the time of use.
It is recommended that single-dose containers for surgical use should not
include any antimicrobial agents.
Ophthalmic emulsions
Definition
Ophthalmic emulsions are generally dispersions of oily droplets in an
aqueous phase. There should be no evidence of breaking or coalescence.
954
Op'hthalmic susp'ensions
Definition
Ophthalmic suspensions contain solid particles dispersed in a liquid vehicle;
they must be homogeneous when shaken gently and remain sufficiently dis-
persed to enable the correct dose to be removed from the container. A sed-
iment may occur, but this should disperse readily when the container is
shaken, and the size of the dispersed particles should be controlled. The
active ingredient and any other suspended material must be reduced to a
particle size small enough to prevent irritation and damage to the cornea.
Visual inspection
Evidence of physical instability is demonstrated by the formation of agglom-
erates or precipitates in aqueous solutions (suspensions) that do not disperse
when the solution is shaken gently.
Ophthalmic ointments
Definition
Ophthalmic ointments are sterile, homogeneous, semi-solid preparations
intended for application to the conjunctiva or the eyelids.
They are usually prepared from non-aqueous bases, e.g. soft paraffin
(Vaseline), liquid paraffin, and wool fat. They may contain suitable addi-
tives, such as antimicrobial agents, antioxidants, and stabilizing agents.
Organoleptic inspection
Evidence of physical instability is demonstrated by:
— a noticeable change in consistency, such as excessive “bleeding” (sepa-

ration of excessive amounts of liquid) or formation of agglomerates or
grittiness;
— discoloration;
— emulsion breakdown;
— crystal growth;
— shrinking due to evaporation of water; or
— evidence of microbial growth.
Uniform consistency
Ophthalmic ointments should be of uniform consistency. When a sample is
rubbed on the back of the hand, no solid components should be noticed.
Containers
Ophthalmic ointments are normally supplied in small, sterilized, collapsible
tubes fitted with a tamper-evident applicator. The containers or the nozzles
955
of the tubes are shaped so that the ointment can be applied without cont-
aminating what remains in the tube. The content of such a container is
limited to not more than 5g of the preparation.

Suitable single-dose containers may also be used.
PARENTERAL PREPARATIONS
Definition
Parenteral preparations are sterile, pyrogen-free liquids (solutions, emulsions,
or suspensions) or solid dosage forms containing one or more active ingredi-
ents, packaged in either single-dose or multidose containers. They are intended
for administration by injection, infusion, or implantation into the body.
Preparations such as vaccines, human blood and products derived from
human blood, peritoneal dialysis solutions, and radioactive pharmaceuticals
require special formulation, methods of manufacture, or presentation appro-
priate to their particular use and may not comply with certain parts of this
monograph.
There are four main forms of parenteral preparations: injections, intravenous
infusions (large volume parenterals), powders for injections, and implants.
Certain injections and intravenous infusions may be presented in the form of
sterile concentrated solutions, which must be suitably diluted before use.
Parenteral preparations may contain excipients such as solvents, suspend-

ing agents, buffering agents, substances to make the preparation isotonic with
blood, stabilizers, or antimicrobial preservatives. The addition of excipients
should be kept to a minimum. When excipients are used, they should not
adversely affect the stability, bioavailability, safety, or efficacy of the active
ingredient(s), or cause toxicity or undue local irritation. There must be no

incompatibility between any of the components of the dosage form.
Water for injections used as the vehicle for aqueous injections. It should
is
be freshly distilled by the process described under “Aqua pro Injectione”,

be free from carbon dioxide, and comply with 3.4 Test for bacterial endotox-
ins. Sterilization at this stage may be omitted, provided that the solution or
preparation is immediately sterilized upon finalization. For non-aqueous injec-

tions, fixed oils of vegetable origin are used as vehicles.
Unless otherwise specified in the individual monograph, sodium chloride or
other suitable substance(s), may be added to an aqueous solution for injection
in order to render the preparation isotonic.
Manufacture
The manufacturing process should meet the requirements of Good Manufac-
turing Practice. The following information is intended to provide very broad
956
The quality of starting materials, the design and maintenance of the

equipment, and the method of manufacture must be such as to ensure the
stability of the active substance and the final product which is sterile and free
of pyrogens andparticulate matter. From the clinical viewpoint all parenteral
preparations must be pyrogen-free. For practical purposes, however, certain
categories of parenteral preparations may be exempted from the test for
bacterial endotoxins or the test for pyrogens as specified in the individual
monograph.
For the sterilization of parenteral preparations follow 5.8 Methods of ster-
ilization. Ideating inan autoclave is the method of choice for aqueous prepa-
rations and should therefore be used whenever possible.
When a parenteral preparation is liable to deterioration due to oxidation,
the operation of filling may be performed in an atmosphere of suitable inert
gas, such as nitrogen, whereby the air in the container is replaced by this gas.
controls during production of parenteral preparations should include monitor-
ing of environmental conditions (especially with respect to particulate and
microbial contamination), bacterial endotoxins, pH and clarity of solution,
freedom from particulate matter, and integrity of container (absence of leakage,
etc.). For dispersions controls should also include the particle size of the dis-
persed phase, and for powders for injections the uniformity of content and
mass, moisture content, and the ease of reconstitution of a solution or suspen-
sion. The presence of preservatives or other additives should be determined as
these can influence the choice of assay method.
Visual inspection
Inspect the solutions, reconstituted solutions, and intravenous infusions (except
dispersions). They should be clear and free from visible particulate matter.
Test for sterility

Parenteral preparations comply with 3.2 Test for sterility.
Test for bacterial endotoxins/pyrogens

All intravenous infusions and those injections and powders for injections where
the volume to be injected in a single dose is 15ml or more must comply with
3.4 Test for bacterial endotoxins or, where justified, with 3.5 Test for pyrogens.
In addition,where specified in the individual monographs for certain prepara-
tions where the active ingredients are of biological origin, injections and
powders for injections must comply with the appropriate test, irrespective of
the volume to be injected in a single dose.
For injections, the amount to be tested should relate to the volume of the
dose and should be specified in the individual monograph.
957
For powders for injections, the amount of powder to be tested and the
nature and volume of the liquid in which it is to be dissolved or suspended
should be specified in the individual monograph.
Containers
Parenteral preparations are usually supplied in glass ampoules, bottles or vials,
plastic bottles or bags, and prefilled syringes, which are coloured in the case of
light-sensitive substances.
Except where otherwise indicated in individual monographs, these con-
tainers should be made from material that is sufficiently transparent to
permit the visual inspection of the contents. They should not adversely
affect the quality of the preparation, allow diffusion of any kind into or
across the material of the container, or yield foreign substances into the
preparation.
Closures
Closures for parenteral preparation containers should be equipped with a firm
seal to prevent entry of microorganisms and other contaminants while per-
mitting the withdrawal of a part or the whole of the contents without removal
of the closure. They should not be made of components that react with the
contents, nor should they allow foreign substances to diffuse into the prepara-
tion. Plastic materials or elastomers of which the closure is composed should
be sufficiently firm and elastic to allow the passage of a needle with the least
possible shedding of particles. Closures for multidose containers should be
sufficiently elastic to allow the puncture to reseal when the needle is withdrawn
and protect the contents from airborne contamination. A tamper-evident con-
tainer is fitted with a device that reveals clearly whether it has ever been
opened.
Labelling

possible;
(3) theamount of the active ingredient(s) in a suitable dose volume and the
volume in the container; for powder for injections: the amount of the
active ingredient(s) in the container;

necessary;
958
For parenteral preparations that are solutions or dispersions, the concentration

of the active ingredient(s) should be given in terms of mass or biological activ-
ity per volume. For concentrated solutions, labels should state the composition
and the dilution to be carried out before use.
Requirements for specific types of parenteral preparations
Injections
Definition
Injections are sterile, pyrogen-free solutions or dispersions (emulsions or sus-
pensions) of one or more active ingredients in a suitable vehicle.
Whenever possible, an injection should be prepared using an aqueous
vehicle. If necessary, suitable non-aqueous solvents are indicated in the indi-
vidual monographs. Injections that are dispersions should remain sufficiently
stable so that, after shaking, a homogeneous dose can be withdrawn.
The use of single-dose injections is to be preferred and is essential when the
preparation is intended for administration by routes where, for medical reasons,
an antimicrobial preservative is not acceptable, e.g. intracisternal, intrathecal.
Single-dose preparations
Single-dose preparations should contain a sufficient quantity of the injec-
tion readily to permit the withdrawal and administration of the volume
specified on the label.
Multidose preparations
Multidose preparations should contain a suitable antimicrobial preservative
in appropriate concentrations, except in cases where the preparations them-
selves have adequate antimicrobial properties. The containers should be
equipped to ensure adequate protection of the contents after partial with-
drawal. In order to minimize the risk of contamination resulting from mul-
tiple penetrations of the closure, the contents of a multidose preparation
should normally not exceed 30 ml.
Intravenous infusions
Definition
Intravenous infusions are sterile, pyrogen-free aqueous solutions or emulsions
with water as continuous phase, usually prepared to be isotonic. They are
intended for administration in large volumes (usually 100ml or more), and
should not contain any antimicrobial preservatives.
959
On visual inspection, emulsions for intravenous injection should show no

evidence of phase separation. The particle size of the dispersed phase should
be controlled by the manufacturer.
Powders for injections
Definition
Powders for injections are solid substances (including freeze-dried materials),
distributed in their final containers and which, when shaken with the pre-
scribed volume of the appropriate sterile liquid, rapidly form either clear and
practically particle-free solutions or uniform suspensions. Powders for injec-
tions, after dissolution or suspension, comply with the requirements for injec-
tions or intravenous infusions, as appropriate.
Unifonnity of mass
Powders for injections (single-dose use) comply with the test for 5.2 Unifor-
mity of mass for single-dose preparations, unless otherwise specified in the
individual monograph.
A requirement for compliance with the test for 5.1 Uniformity of content
for single-dose preparations is specified in certain individual monographs
where the active ingredient than 40 mg. In such cases, compliance with
is less
the test for 5.2 Uniformity of mass for single-dose preparations may not be
required.
Imprlants
Definition
Implants are solid preparations containing one or more active ingredients. They
are of a size and shape suitable for parenteral implantation, and provide release
of the active ingredient(s) over an extended period of time. They are presented
in individual sterile containers.
vidual monographs.
SUPPOSITORIES
Definition
Suppositories are solid preparations which may contain one or more active
pharmaceutical ingredient(s) intended for rectal application. They are normally
used for local action or systemic absorption of the active ingredient(s). They
usually melt, soften, or dissolve at body temperature.
960
Suppositories are usually prepared from excipients or bases such as cocoa

butter, hard fat, glycerinated gelatin, hydrogenated vegetable oils and macro-
gols. They may also contain additives, such as adsorbents, surface-active
agents, viscosity-influencing agents, antioxidants, antimicrobials, and autho-
rized colouring agents.
Any excipients used for the preparation of suppositories must be proven
through product development studies not to affect adversely either the stabil-
ity of the finalproduct or the availability of the active ingredient(s) at the site
of action; incompatibility between any of the components of the dosage form
should be avoided.
Manufacture
facturing Practices, especially with regard to cross-contamination. The follow-
ing information is intended to provide very broad guidelines concerning the
main steps to be followed during production, indicating those that are the most
important.
designed to guarantee the effectiveness of each stage of production.
Where the active ingredient is suspended in the suppository base, appro-
priate limits should be set for the particle size.
Suppositories may be manufactured by moulding or compressing powdered
material into a suitable shape, or by encapsulating a semi-solid mass into soft
gelatin. Rectal capsules are in general similar to soft capsules, except that they
may have lubricating coatings.
Moulded suppositories are the most common type. They are usually
obtained by pouring the medicated mass, sufficiently liquefied by heating,
into suitable moulds. The suppositories solidify on cooling. In certain cases, it
is also possible to use the cold-moulding compression procedure in a suitable

press.
Packaging must be adequate to protect suppositories from light, excessive
temperature, moisture, and damage due to handling and transportation. It is
necessary to ensure that the suppositories can be released from the packaging
material easily and without damage.
Visual inspection
Suppositories are elongated, smooth and have a uniform texture and appear-
ance. They may also consist of several layers.
Evidence of physical and/or chemical instability is demonstrated by notice-
able changes in:
— surface texture or form; and

— colour and odour.
961
Disintegration
Suppositories should comply with 5.4 Disintegration test for suppositories,
unless intended for modified release. Unless otherwise stated in the individual
monograph, for each of the three suppositories, examine the state of the sample
after 30 minutes for fat-based suppositories and rectal capsules, and after 60
minutes for water-soluble based suppositories.
Uniformity of mass
See the general requirements under 5.2 Uniformity of mass for single-dose
preparations. Not more than two of the individual masses should deviate from
the average mass by more than 5%, and none by more than 10%.
See the general requirements under 5.1 Uniformity of content for single-dose
preparations. Preparations with an active ingredient content of less than 2 mg,
or less than 2% of the total mass, comply with the test, unless otherwise
described in the individual monograph and authorized. If the preparation has
more than one active ingredient, the requirement applies only to those active
ingredients that fall into the above category. If the test for uniformity of content
is prescribed for all active ingredients, the test for uniformity of mass is not
required.
Containers
Suppositories should be supplied in a well-closed container. The container
material should not adversely affect the quality of the preparation, nor should
it allow diffusion into or across the material of the container or yield foreign
substances into the preparation.
Labelling
established by Good Manufacturing Practices.

(3) the amount of the active ingredient(s) in each suppository and the
number of suppositories in the container;
necessary;
(7) directions for use, warnings and precautions that may be necessary;
962
placing the product on the market; and
(9) if applicable, the names and concentrations of the antimicrobial agents
and/or antioxidants incorporated in the preparation.
Storage
Suppositories should maintain their shape throughout their shelf-life when
stored at the temperature indicated on the label.
TABLETS
Definition
Tablets are solid dosage forms containing one or more active ingredients. They
are obtained by single or multiple compression (in certain cases they are
moulded) and may be uncoated or coated. They are usually intended for oral
administration, but preparations for alternative applications, such as implants,
solution-tablets for injections, irrigations, or for external use, vaginal tablets,
etc., are also available in this presentation. These preparations may require a
special formulation, method of manufacture, or form of presentation, appro-
priate to their particular use. For this reason they may not comply with certain
sections of this monograph.
The different categories of tablet that exist include soluble tablets, effer-
vescent tablets, tablets for use in the mouth, and modified-release tablets.
Unless otherwise specified in the individual monograph, tablets are normally
circular in shape, and their surfaces are flat or convex. Tablets may have lines
or break-marks, symbols, or other markings. They should be sufficiently hard
to withstand handling, including packaging, storage, and transportation,
without crumbling or breaking.
Tablets may contain excipients such as diluents, binders, disintegrating
agents, glidants, lubricants, substances capable of modifying the behaviour of
the dosage forms and the active ingredient(s) in the gastrointestinal tract,
colouring matter, and flavouring substances. When such excipients are used,
it is necessary to ensure that they do not adversely affect the stability,
dissolution rate, bioavailability, safety, or efficacy of the active ingredient(s);

there must be no incompatibility between any of the components of the dosage
form.
Manufacture
facturing Practice, especially with regard to cross-contamination. The following
information is intended to provide very broad guidelines concerning the main
steps to be followed during production, indicating those that are the most
important.
963


mixing is carried out in a manner that ensures homogeneity. Sometimes, the
physical characteristics of the mixture allow it to be directly compressed, but
it is usually necessary to granulate before compression, either by wet granula-
tion or precompression (slugging).
The granulate and powders normally need to be mixed with lubricants
and/or disintegrating agents. The use of excessive amounts of lubricants should
be avoided since these will deleteriously affect the tablets. The final tablet
mixture is volume trically fed into the die cavity to ensure that the tablets are
of a uniform mass when compressed. The tablets may be coated, either in
coating pans or by an air-suspension technique. A hydrophobic subcoat applied
to the core of sugar-coated tablets may reduce dissolution.
controls during tablet production should include the particle size of the active
ingredient(s), the homogeneity and moisture content of the mixture and/or
granulate, the size of granules, the flow of the final mixture, and the dimen-
sions (thickness, diameter), uniformity of mass, hardness and/or crushing force,
friability, disintegration, or dissolution rate (e.g. for modified-release tablets) of
the finished dosage form. Attention should be paid to the uniformity of mass
of tablet cores before coating.
Packaging must be adequate to protect the tablets from light, moisture, and
damage during packaging and transportation.
Visual inspection
Unpack and inspect at least 20 tablets. They should be undamaged, smooth,
and usually of uniform colour.
— presence of excessive powder and/or pieces of tablets at the bottom of

the container (from abraded, crushed, or broken tablets);
— cracks or capping, chipping in the tablet surfaces or coating, swelling,
mottling, discoloration, fusion between tablets;
— the appearance of crystals on the container walls or on the tablets.
Uniformity of mass
Tablets comply with the test for 5.2 Uniformity of mass for single-dose prepa-
rations, unless otherwise specified below or in the individual monograph.
964
A requirement for compliance with the test for 5.1 Uniformity of content for
single-dose preparations is specified in certain individual monographs for sugar-
coated or enteric-coated tablets, where the test for 5,2 Uniformity of mass for
single-dose preparations does not apply. In addition, a requirement is specified
in certain individual monographs where the active ingredient is 5% or less of
the total formulation. In such cases the test for 5.2 Uniformity of mass for
single-dose preparations is not required.
Dissolution test
Where a requirement for the “Dissolution test” is specified in the individual
monograph, the 5.3 Disintegration test for tablets and capsules is not required.
Labelling

(3) the amount of the active ingredient(s) in each tablet and the number of
tablets in the container;

necessary;
Storage
Tablets should be kept in well-closed containers and protected from light, mois-
ture, crushing, and mechanical shock. Any special storage conditions should be
stated on the label. Tablets should be able to withstand handling, including
packaging and transportation, without losing their integrity. Moisture-sensitive
forms, such as effervescent tablets, should be stored in tightly closed contain-
ers or moisture-proof packs and may require the use of separate packages con-
taining water-adsorbent agents, such as silica gel.
Additional special packaging, storage, and transportation recommendations
are specified in the individual monograph.
965
Requirements for specific types of tablets
Uncoated tablets
Definition
The majority of uncoated tablets are made in such a way that the release of
active ingredients is unmodified. A broken section, when examined under a
lens, shows either a relatively uniform texture (single-layer tablets) or a strati-
fied texture (multilayer tablets), but no signs of coating.
Disintegration test
Uncoated tablets, except effervescent tablets, tablets for use in the mouth, and
chewable tablets, comply with 5.3 Disintegration test for tablets and capsules.
Operate the apparatus for 15 minutes, unless otherwise specified in the indi-
vidual monograph, and examine the state of the tablets.
Soluble tablets (tablets for solutions)

Definition
Soluble tablets are uncoated tablets that dissolve in water to give a clear
solution.
Disintegration test
Soluble tablets comply with 5.3 Disintegration test for tablets and capsules. Use
water at room temperature, and operate the apparatus for 5 minutes, unless
otherwise specified in the individual monograph.
Effervescent tablets
Definition
Effervescent tablets are uncoated tablets generally containing acid substances
and carbonates or hydrogen carbonates that react rapidly in the presence of
water to release carbon dioxide. They are intended to be dissolved or dispersed
in water before administration.
Labelling
The label should state: “Not to be swallowed directly”.
Disintegration test
Effervescent tablets comply with 5.3 Disintegration test for tablets and cap-
sules. Place one tablet in a 250-ml beaker containing 200ml of water at room
temperature. Numerous bubbles of gas are evolved. When the evolution of gas
around the tablet or its fragments ceases, the tablet should have disintegrated,
being either dissolved or dispersed in the water so that no agglomerates remain.
Repeat the operation on five additional tablets. The tablets comply with the
966
test if each of the six tablets used in the test disintegrates within 5 minutes,
unless otherwise specified in the individual monograph.
Tablets for use in the mouth (sublingual, buccal) and chewable tablets
Definition
Tablets for use in the mouth and chewable tablets are usually uncoated. They
are formulated to effect a slow release and local action of the active ingredi-
ent(s) (forexample, compressed lozenges) or the release and absorption of the
active ingredient(s) under the tongue (sublingual tablets) or in other parts of the
mouth (buccal) for systemic action.
Coated tablets
Definition
Coated tablets are tablets covered with one or more layers of mixtures of sub-
stances such as natural or synthetic resins, polymers, gums, fillers, sugars, plasti-
cizers, polyols, waxes, colouring matters, flavouring substances, and sometimes
also active ingredients. A broken section, when examined under a lens, shows a
core which is surrounded by a continuous layer of a different texture.
The tablets may be coated for a variety of reasons such as protection of the
active ingredients from air, moisture, or light, masking of unpleasant tastes and
odours, or improvement of appearance. The substance used for coating is
usually applied as a solution or suspension.
Three main categories of coated tablet may be distinguished: sugar-coated,
film-coated,and certain modified-release tablets.
Sugar-coated tablets
Uniformity of mass
The test for 5.2 Uniformity of mass not apply
for single-dose preparations, does
to sugar-coated tablets (see in-process controls under “Manufacture”).
Disintegration test
Sugar-coated tablets comply with 5.3 Disintegration test for tablets and cap-
sules. Operate the apparatus for 60 minutes, unless otherwise specified in the
individual monograph, using water, and examine the state of the tablets. If any
of the tablets has not disintegrated, repeat the test on an additional six tablets,
using hydrochloric acid (0.1 mol/1) VS.
All six tablets must disintegrate.
Film-coated tablets
Definition
A film-coated tablet is covered with a thin layer of resins, polymers, and/or
plasticizers capable of forming a film.
967
Disintegration test
Film-coated tablets comply with 5.3 Disintegration test for tablets and capsules.
Operate the apparatus for 30 minutes, and examine the state of the tablets.
Modified-release tablets
Definition
Modified-release tablets are coated, uncoated, or matrix tablets containing
excipients or prepared by procedures which, separately or together, are
designed to modify the rate of release of the active ingredient(s) in the gas-
trointestinal tract.
Extended-retease tablets
Definition
Extended-release tablets are designed to slow the rate of release of the active
ingredient(s) in the gastrointestinal tract.
vidual monographs.
Delayed-release tablets (enteric-coated tablets)

Definition
Delayed-release tablets are intended to resist gastric fluid but disintegrate in
intestinal fluid. This is achieved by using coating substances such as cellacefate
(cellulose acetate phthalate) and anionic copolymers of methacrylic acid and its
esters. It is sometimes necessary to apply more than one layer.
Uniformity of mass
The test for 5.2 Uniformity of mass for single-dose preparations does not apply
to delayed-release tablets.
Disintegration test
Delayed-release tablets comply with 5.3 Disintegration test for tablets and cap-
sules, using hydrochloric acid (0.1 mol/1) VS as the immersion fluid. Operate the
apparatus for 2 hours, unless otherwise specified in the individual monograph
(but in any case for not less than 1 hour), and examine the state of the tablets.
No tablet should show from fragments of
signs of either disintegration (apart
coating) or cracks that would allow the contents to escape. Replace the acid by
phosphate buffer solution, pH 6.8, TS. Operate the apparatus for 60 minutes
and examine the state of the tablets.
968
TOPICAL SEMI-SOLID DOSAGE FORMS

Definition
Topical semi-solid dosage forms are normally presented in the form of creams,
gels, ointments, or pastes. They contain one or more active ingredients dis-
solved or uniformly dispersed in a suitable base and any suitable excipients
such as emulsifiers, viscosity-increasing agents, antimicrobial agents, antioxi-
dants, or stabilizing agents. Preparations susceptible to the growth of microor-
ganisms should contain a suitable antimicrobial agent in an appropriate
concentration unless the preparations themselves have adequate antimicrobial
properties. Assurance must be provided through product development studies
that such excipients do not adversely affect either the stability of the final
product or the availability of the active ingredient(s) at the site of action; there
must be no incompatibility between any of the components of the dosage form.
The choice of a base for semi-solid dosage forms depends on many factors:
the therapeutic effect desired, the nature of the active ingredient(s) to be incor-
porated, the availability of the active ingredient(s) at the site of action, the shelf-
life of the finished product, and the environmental conditions in which the
product is intended to be administered. In many cases, a compromise has to
be made in order to achieve the required stability. For example, drugs that
hydrolyse rapidly are more stable in hydrophobic bases than in water-
containing bases, even though they may be more effective in the latter.
The base should neither irritate nor sensitize the skin, nor should it delay
wound healing. It should be smooth, inert, odourless, physically and chemi-
cally stable, and compatible with both the skin and the active ingredient(s) to
be incorporated. It should normally be of such a consistency that it spreads and
softens easily when stress is applied.

It may be necessary for a topical semi-solid dosage form to be sterile, for ex-
ample, when it is intended for use on large open wounds or severely injured skin.
Manufacture
facturing Practice. The following information is intended to provide very broad
Throughout manufacturing, certain procedures should be validated and mon-
itored by carrying out appropriate in-process controls. They should be designed
to guarantee the effectiveness of each stage of production. Appropriate limits
should be set for the particle size of the active ingredient(s), which should be
controlled during production. Particular care should be paid to environmental
conditions, especially with respect to microbial and cross-contamination.*
'
Warning: Semi-solid dosage forms should not be diluted. If a dilution is nevertheless necessary,
same type of base should be used in order to obtain a homo-
this requires special attention; the
geneous mixture.
969
Packaging must be adequate to protect topical semi-solid dosage forms from

light, moisture, and damage due to handling and transportation. The use of
flexible tubes of suitable metal or plastic is preferred. Preparations for nasal,
should be supplied in containers adapted for appro-
aural, vaginal, or rectal use
priate delivery of the product to the site of application, or should be supplied
with a suitable applicator.
Organoleptic inspection
— a noticeable change in consistency, such as excessive “bleeding” (sepa-

ration of excessive amounts of Uquid) or formation of agglomerates and
grittiness;
— discoloration;
— emulsion breakdown;
— crystal growth;
— shrinking due to evaporation of water; or
— evidence of microbial growth.
A noticeable change in odour is also a sign of instability.
Sterility
Preparations required to be sterile should comply with 3.2 Test for sterility.
Uniform consistency
Topical semi-solid dosage forms should be of uniform consistency. When a
sample is rubbed on the back of the hand, no solid components should be
noticed.
Containers
The container material should not adversely affect the quality of the prepara-
any kind into or across the material of the container
tion or allow diffusion of
into the preparation. The container should be
fitted with a closure that mini-
mizes microbial contamination and is equipped with a device that reveals
whether the container has ever been opened.
Labelling

possible;
970
(3) the amount of the active ingredient(s) in a specified quantity of suitable

base or vehicle, and the quantity of preparation in the container;
necessary;
(7) directions for use, warnings, and precautions that may be necessary;
(10)
placing the product on the market;
(9) the name and quantity of antimicrobial agent incorporated in the prepa-
ration; and
if applicable, the statement that the preparation is “sterile”.
Storage
Topical semi-solid dosage forms should be kept in well-closed containers. The
preparation should maintain its pharmaceutical integrity throughout shelf-life
when stored at the temperature indicatedon the label; the temperature should
normally not exceed 25 °C. Special storage recommendations or limitations are
indicated in individual monographs.
Requirements for specific types of topical semi-solid

dosage forms
Creams
Definition
Creams arehomogeneous, semi-solid preparations consisting of opaque emul-
sion systems. Their consistency and rheological properties depend on the type
of emulsion, either water-in-oil (w/o) or oil-in-water (o/w),and on the nature
Creams are intended for application to the
of the solids in the internal phase.
skin or certain mucous membranes for protective, therapeutic, or prophylactic
purposes, especially where an occlusive effect is not necessary. The term
“cream” is most frequently used to describe soft, cosmetically acceptable types
of preparations.
Generally, o/w creams are prepared at an elevated temperature and then
cooled down to room temperature in order for the internal phase to solidify.
The semi-solid form of a w/o cream is attributable to the character of the exter-
nal phase.
Hydrophobic creams (w/o)

Hydrophobic creams are usually anhydrous and absorb only small amounts
of water. They contain w/o emulsifying agents such as wool fat, sorbitan
esters, and monoglycerides.
971
Hydrophilic creams (o/w)

Hydrophilic creams contain bases that are miscible with water. They also
contain o/w emulsifying agents such as sodium or triethanolamine soaps,
sulfated fatty alcohols, and polysorbates combined, if necessary, with w/o
emulsifying agents. These creams are essentially miscible with skin
secretions.
Gets
Definition
Gels are usually homogeneous, clear, semi-solid preparations consisting of a
liquidphase within a three-dimensional polymeric matrix with physical or
sometimes chemical cross-linkage by means of suitable gelling agents.
Gels are applied to the skin or certain mucous membranes for protective,
therapeutic, or prophylactic purposes.
Hydrophobic gels
Hydrophobic gel (oleogel) bases usually consist of liquid paraffin with poly-
ethylene or fatty oils gelled with colloidal silica or aluminium or zinc soaps.
Hydrophilic gels
Hydrophilic gel (hydrogel) bases usually consist of water, glycerol, or propy-
lene glycol gelled with suitable agents such as tragacanth, starch, cellulose
derivatives, carboxyvinyl polymers, and magnesium aluminium silicates.
Ointments'
Definition
Ointments are homogeneous, semi-solid preparations intended for external
application to the skin or mucous membranes. They are used as emollients or
for the application of active ingredients to the skin for protective, therapeutic,
or prophylactic purposes and where a degree of occlusion is desired.
Ointments are formulated using hydrophobic, hydrophilic, or water-emul-
sifying bases to provide preparations that are immiscible, miscible, or emulsi-
fiable with skin secretions. They can also be derived from hydrocarbon (fatty),
absorption, water-removable, or water-soluble bases.
Hydrophobic ointments
Hydrophobic (lipophilic) ointments are usually anhydrous and can absorb
only small amounts of water. Typical bases used for their formulation are
water-insoluble hydrocarbons such as hard, soft, and liquid paraffin, veg-
etable oil, animal fats, waxes, synthetic glycerides, and polyalkylsiloxanes.
’
Ophthalmic ointments are described in the separate monograph for "Ophthalmic Preparations”.
972
Water- emulsifying ointments

Water- emulsifying ointments can absorb large amounts of water. They typ-
ically consist of a hydrophobic fatty base in which a w/o agent, such as
wool fat, wool alcohols, sorbitan esters, monoglycerides, or fatty alcohols

can be incorporated to render them hydrophilic. They may also be w/o
emulsions that allow additional quantities of aqueous solutions to be incor-
porated. Such ointments are used especially when formulating aqueous
liquids or solutions.
Hydrophilic ointments
Hydrophilic ointment bases are miscible with water. The bases are usually
mixtures of liquid and solid polyethylene glycols (macrogols).
Pastes
Definition
Pastes are homogeneous, semi-solid preparations containing high concentra-
tions of insoluble powdered substances (usually not less than 20%) dispersed
in a suitable base. The pastes are usually less greasy, more absorptive, and stiffer
in consistency than ointments because of the large quantity of powdered ingre-
dients present. Some pastes consist of a single phase, such as hydrated pectin,
and others consist of a thick, rigid material that does not flow at body tem-
perature. The pastes should adhere well to the skin. In many cases they form
a protective film that controls the evaporation of water.
973
CopyrigfiJed i
Monographs
Dosage Forms:
Specific monograph
CopyrigfiJed i
Dosage Forms: Specific monographs

ACIDI ACETYLSALICYLICI COMPRESSI
ACETYLSALICYLIC ACID TABLETS
Category. Analgesic; antipyretic; nonsteroidal antiinflammatory; antimigraine
drug.
Storage. Acetylsalicylic acid tablets should be kept in a tighdy closed

container.
Additional information. Strength in the current WHO Model list of essen-

tial medicines: 100-500mg.
Usually no odour of acetic acid is perceptible on opening the container.
Requirements
Comply with the monograph for “Tablets”.
Acetylsalicylic acid tablets contain not less than 95 0 % and not more than
.
105 0 % of the amount of C 9 Hg04

. stated on the label.
Identity tests
A. Place a quantity of the powdered tablets equivalent to 10 mg of Acetylsali-
cylic acid on a suitable white test plate or on a watch-glass placed on a white
background, and add 1 drop of ferric chloride (25g/l) TS; no violet colour is
produced.
B. Place a quantity of the powdered tablets equivalent to 10 mg of Acetylsali-

cylic acid on a suitable white test plate or a watch-glass placed on a white
background, and add drop of potassium hydroxide/ethanol TSl. After 1
1
minute add 1 drop of ferric chloride (25g/l) TS; a violet colour is produced.
Salicylic acid. To a quantity of the powdered tablets equivalent to 0.2 g

of Acetylsalicylic acid add 4ml of ethanol (~750g/l) TS and shake. Dilute to
100 ml with cool water (not exceeding 10 °C) and filter immediately. Transfer
50ml of the filtrate to a comparison tube, add 1ml of freshly prepared ferric
ammonium sulfate TSl, mix, and allow to stand for 1 minute. Separately and
concurrently, place 3 ml of a freshly prepared solution of salicylic acid R con-
taining 0.1 mg per ml into a second comparison tube, add 2ml of ethanol
(-750 g/1) TS, 1ml of freshly prepared ferric ammonium sulfate TSl, and suffi-
cient water to produce 50 ml.
977
Any violet colour produced in the first tube is not more intense than that pro-
duced in the second tube (0.3%).
Assay. Weigh and powder 20 tablets. To a quantity of the powder equivalent

to about 0.5 g of Acetylsalicylic acid, accurately weighed, add 30 ml of carbon-
ate-free sodium hydroxide (0.5mol/l) VS, and boil gendy for 10 minutes. Back-
titrate the excess alkali with hydrochloric acid (0.5 mol/1) VS, using phenol
red/ethanol TS as indicator. Repeat the procedure without the powdered tablets

being examined and make any necessary corrections.
Each ml of carbonate-free sodium hydroxide (0.5 mol/1) VS is equivalent to

45.04mg of C 9 H 8 O 4 .
ALLOPURINOLI COMPRESSI
ALLOPURINOL TABLETS
Category. Drug used for the treatment of gout.

tial medicines: lOOmg.
Requirements
Allopurinol tablets contain not less than 90 0 % and not more than 110 0 %
. . of
the amount of C5H4N4O stated on the label.
Identity tests
A. Triturate a quantity of the powdered tablets equivalent to about 0.1 g of

Allopurinol with 10ml of sodium hydroxide (0.1 mol/1) VS. Filter, acidify the
with acetic acid (~60g/l) TS, and allow to stand for 10-15 minutes.
filtrate
Separate the precipitate, wash it with 3 ml of dehydrated ethanol R and

4ml of ether R.Allow to dry in air for 15 minutes, then dry at 105°C for 3
hours. Keep half of the residue for test C. Carry out the examination with
the residue as described under 1.7 Spectrophotometry in the infrared region.
from allopurinol RS or with the reference spectrum of allopurinol.
978
B. The absorption spectrum of the solution obtained in the “Assay”, when

250 nm.
C. To the residue obtained in test A add 5 ml of sodium hydroxide (50g/l) TS,

1.0 ml of alkaline potassiomercuric iodide TS, heat to boiling, and allow to
stand; a yellow precipitate is produced.
6 volumes of 2-butanol R, 2 volumes of ammonia (-260 g/1) TS, and 2 volumes
of ethylene glycol monomethyl ether R as the mobile phase. Apply separately
to the plate lOpml of each of the following 2 solutions. For solution (A) shake
a quantity of the powdered tablets equivalent to about 0.25 g of AUopurinol
with a mixture of 1.0 ml of diethylamine R and 9 ml of water, filter, and use the
For solution (B) use 0.05 mg of aminopyrazole-4-carboxamide hemisul-
filtrate.
fateRS per ml of ammonia (-260 g/1) TS. After removing the plate from the
chromatographic chamber, allow it to dry in a current of air, and examine the
chromatogram in ultraviolet light (254 nm).

to about 0.1 g of AUopurinol add 20 ml of sodium hydroxide (0.05 mol/1) VS and
shake for 20 minutes. Then add 80ml of hydrochloric acid (0.1 mol/1) VS and
shake for 10 minutes. Dilute to 250ml with hydrochloric acid (0.1 mol/1) VS,
filter, and dilute 10ml of the filtrate to 250ml with the same acid. Measure the
absorbance of a 1-cm layer at the maximum at about 250 nm against a solvent

cell containing hydrochloric acid (0.1 mol/1) VS.
Calculate the percentage content of C5H4N4O using the absorptivity value of

56.3 (Ai°(';„ = 563).
AMPHOTERICINI B PULVIS AD INJECTIONEM

AMPHOTERICIN B POWDER FOR INJECTIONS
Description. A yellow to orange powder; odourless or almost odourless.
Storage. Amphotericin B powder for injections should be protected from light

and stored at a temperature between 2 and 8°C.
979
Labelling. The label should state whether any buffering agents and preserva-
tives are added. Further, it should indicate that it is intended for intravenous or
intrathecal administration, and that it should be protected from light. Expiry
date.

tial medicines: 50 mg.
Requirements
The powder for injections and the reconstituted solution for injections comply
with the monograph for “Parenteral preparations”.
Definition. Amphotericin B powder for injections is a sterile powder of

amphotericin B mixed with sodium desoxycholate.
In water, it yields a colloidal dispersion.
Amphotericin B powder for injections may contain suitable buffers and pre-
servatives. The powder is sterilized by a suitable method (see 5.8 Methods
of sterilization).
Identity tests
A. Dissolve a quantity of the powder for injections equivalent to 25 mg of
Amphotericin B in 5 ml of dimethyl sulfoxide R, add sufficient methanol R
to produce 50ml, and dilute 2 ml to 200 ml with methanol R. The absorp-
tion spectrum of the resulting solution, when observed between 300 nm and
450 nm, exhibits three maxima at about 362 nm, 381 nm, and 405 nm. The
ratio of the absorbance of a 1-cm layer at 362 nm to that at 381 nm is about
0.6; the ratio of the absorbance at 381 nm to that at 405nm is about 0.9.
B. Dissolve a quantity of the powder for injections equivalent to 1 mg of

Amphotericin B ml of dimethyl sulfoxide R and introduce 5ml of phos-
in 2
phoric acid (-1440 g/1)TS to form a lower layer; a blue ring is immediately
formed at the interface of the two liquids. Mix the two liquids; a strong blue
colour is produced. Add 15 ml of water and mix; the colour of the solution
changes to pale yellow.
Loss on drying. Dry the powder for injections to constant mass at 60 °C under
reduced pressure (not exceeding 0.6 kPa or 5 mm of mercury); it loses not more
than 80mg/g.
pH value. pH of a suspension of the powder for injections containing 10 mg

of Amphotericin B per ml of carbon-dioxide-free water R, 7. 2-8.0.
980
Assay
Mix the contents of 10 containers and carry out the assay as described.
Triturate a quantity of thepowder for injections equivalent to about 0.06g of

Amphotericin B, accurately weighed, with dimethylformamide R and add, with
shaking, sufficient dimethylformamide R to produce 100 ml. Dilute 10 ml to
100 ml with dimethylformamide R and carry out the assay as described under
3.1 Microbiological assay of antibiotics, using Saccharomyces cerevisiae (NCTC
10716, or ATCC 9763) as the test organism, culture medium Cm3 with a final
pH of 6.1, sterile phosphate buffer, pH
TSl, an appropriate concentration
10.5,
of Amphotericin B (usually between 0.5 and lO.Opg/ml), and an incubation
temperature of 29-33 °C. The precision of the assay is such that the fiducial
limits of error of the estimated potency (P = 0.95) are not less than 95% and
not more than 105% of the estimated potency.
The upper fiducial limit of error is not less than 750pg/g, calculated with
bacterial endotoxins; contains not more than 1.0 lU of endotoxin RS per mg.

AMPICILLINI CAPSULAE
AMPICILLIN CAPSULES
Storage. Ampicillin capsules should be kept in a tightly closed container and

stored at a temperature not exceeding 25 °C.
Labelling. The label should state whether the active ingredient is in the anhy-
drous form or is the trihydrate, and the quantity should be indicated in terms
of the equivalent amount of ampicillin. Expiry date.

tial medicines: 250 mg, 500 mg.
Requirements
Comply with the monograph for "Capsules”.
981
Ampicillin capsules contain not less than 90 0 % and not more than 110 0 %
. .
of the amount of C 16H 19N 3 O 4 S stated on the label.
Identity tests

acetone R, 10 volumes of water, 10 volumes of toluene R, and 2.5 volumes
of glacial acetic acid R
mobile phase. Apply separately to the plate
as the
2 pi of each of the following two solutions. For solution (A) shake a quan-
tity of the contents of the capsules equivalent to 50 mg of Ampicillin with
10ml of a mixture of 4 volumes of acetone R and 1 volume of hydrochlo-

ric acid (0.1 mol/1) VS, filter, and use the clear filtrate. For solution (B) dis-
solve 25 mg of ampicillin RS in 5 ml of the same mixture of solvents. After

air, spray lighdy with triketohydrindene/ethanol TS, dry at 90 °C for 15
minutes, and examine the chromatogram in daylight.

B. Shake a quantity of the contents of the capsules equivalent to lOmg of Ampi-

cillin with 3 ml of water and filter. To the filtrate add 0.1 g of hydroxylamine
hydrochloride R and about 0.4 ml of sodium hydroxide (~80g/l) TS and allow

to stand for 5 minutes. Add 1.3ml of hydrochloric acid (~70g/l) TS and 0.5ml
of ferric chloride (25g/l) TS; a violet-red to violet-brown colour is produced.
C. Shake a quantity of the contents of the capsules equivalent to 0.5 g of Ampi-

cillinwith 5 ml of water for 5 minutes, filter, wash the residue with ethanol
(-750 g/1) TS, and dry it under reduced pressure (not exceeding 0.6kPa or
5mm of mercury) for 1 hour. Place 2 mg of the residue in a test-tube, add 1

drop of water followed by 2 ml of sulfuric acid (-1760 g/1) TS, and mix; the
solution is colourless. Immerse the test-tube for 1 minute in a water-bath;
the solution remains colourless. Place 2 mg in a second test-tube, add 1 drop
of water and 2 ml of formaldehyde/sulfuric acid TS, and mix; the solution
is colourless to slightly pink. Immerse the test-tube for 1 minute in a water-
bath; an orange-yellow colour is produced.
Loss on drying. Dry a quantity of the contents of the capsules equivalent to

0.1 g of the active ingredient at 60°C under reduced pressure (not exceeding
0.6kPa or 5 mm of mercury) for 3 hours. For capsules containing anhydrous
Ampicillin the loss is not more than 40mg/g, and for capsules containing
Ampicillin trihydrate the loss is not less than lOOmg/g and not more than
150mg/g of the initial quantity taken.
982
Assay. To a quantity of the mixed contents of 20 capsules equivalent to about

0.12 g of Ampicillin, accurately weighed, add 400 ml of water and shake for 30
minutes. Dilute to 500ml with water and filter. Transfer 10ml of the filtrate to
a 100-ml volumetric flask, add 10ml of buffer borate, pH 9.0, TS and 1ml of
acetic anhydride/dioxan TS, allow to stand for 5 minutes at room temperature,
and dilute to volume with water. Transfer two 2-ml aliquots of this solution
into separate stoppered tubes. To one tube add 10 ml of imidazole/mercuric
chloride TS, mix, stopper the tube, and place in a water-bath at 60 °C for exactly
25 minutes. Cool the tube rapidly to 20 °C {solution A). To the second tube add
10ml of water and mix {solution B).
Without delay, measure the absorbance of a 1-cm layer at the maximum at

about 325 nm, against a solvent cell containing a mixture of 2 ml of water and
10ml of imidazole/mercuric chloride TS for solution A and water for solution B.
From the difference between the absorbance of solution A and that of solution B,
calculate the amount of C 16 H 19N 3 O 4S in the substance being examined by com-
parison with ampicillin RS. In an adequately calibrated spectrophotometer the
AMPICILLINI NATRICI PULVIS

AD INJECTIONEM
AMPICILLIN SODIUM POWDER
FOR INJECTIONS
Storage. Ampicillin sodium powder for injections should be protected from

lightand stored at a temperature not exceeding 25 °C. The reconstituted solu-
tion should be used immediately after preparation.
Labelling. The label should state the dose as the equivalent amount of anhy-
drous ampicillin. Expiry date.

tial medicines: 500 mg in vials.
Requirements
983
Definition. Ampicillin sodium powder for injections is a sterile powder of

ampicilHn sodium.
The powder is sterilized by a suitable method (see 5,8 Methods of sterilization).
The container of Ampicillin sodium powder for injections contains not less than
90 0 % and not more than 110 0 % of the amount of CigH, 9N 304 S stated on
. .
the label.
Identity tests

spectrum obtained from ampicillin sodium RS or with the reference spectrum
of ampicillin sodium.

acetone R, 10 volumes of water, 10 volumes of toluene R, and 2.5 volumes
of glacial acetic acid R as the mobile phase. Apply separately to the plate
2 pi of each of the following twosolutions. For solution (A) shake a quan-
tity of the powder mg of Ampicillin sodium
for injections equivalent to 5
with 10 ml of a mixture of 4 volumes of acetone R and 1 volume of
hydrochloric acid (0,1 mol/1) VS, filter, and use the clear filtrate. For solution
(B) dissolve 25 mg of ampicillin sodium RS in 5 ml of the same mixture of
solvents. After removing the plate from the chromatographic chamber,
allow it to dry in air, spray lightly with triketohydrindene/ethanol TS, dry
at 90 “C for 15 minutes, and examine the chromatogram in daylight.

C. To a quantity of the powder for injections equivalent to 2 mg of Ampicillin

sodium in a test-tube add 1 drop of water followed by 2 ml of sulfuric acid
1 minute in a water-bath; the solution remains colourless. Place the same
quantity of powder in a second test-tube, add 1 drop of water and 2 ml of

formaldehyde/sulfuric acid TS, and mix; the solution is colourless. Immerse
the test-tube for 1 minute in a water-bath; a dark yellow colour is produced.
the powder for injections yields the characteristic reactions. If reaction B is
to be used, ignite a small quantity of the powder for injections and dissolve
the residue in acetic acid (-60 g/1) TS.
984
Specific optical rotation. Use a solution containing a quantity of the powder

for injections equivalent to 5mg of Ampicillin sodium per ml in acetate stan-
dard buffer TS and calculate with reference to the anhydrous substance;
[a]g>‘‘= = -H260 to-H290°.
Clarity of solution. A freshly prepared solution of the powder for injections

equivalent to 1 g of Ampicillin sodium in 10 ml of water is clear. A similar solu-
tion in 10 ml of hydrochloric acid (1 mol/1) VS is also clear.

Fischer method, Method A, using a quantity of the powder for injections equiv-
alent to 0.5 g of Ampicillin sodium; the water content is not more than 20 mg/g.
pH value. pH of a solution containing a quantity of the powder for injections

equivalent to 0.1 g of Ampicillin sodium per ml of carbon-dioxide-free water R,
8 0 - 10 0
. . .
Assay
Dissolve a quantity of thepowder for injections equivalent to about 0.12g of

Ampicillin sodium, accurately weighed, in sufficient water to produce 500 ml.
Transfer 10ml of this solution to a 100-ml volumetric flask, add 10ml of buffer
borate, pH
9.0, TS and 1ml of acetic anhydride/dioxan TS, allow to stand for
5 minutes at room temperature, and dilute to volume with water. Transfer two
2-ml aliquots of this solution into separate stoppered tubes. To one tube add
10ml of imidazole/mercuric chloride TS, mix, stopper the tube, and place in a
water-bath at 60 °C for exactly 25 minutes. Cool the tube rapidly to 20 °C {solu-
tion A). To the second tube add 10ml of water and mix {solution B).

about 325 nm against a solvent cell containing a mixture of 2 ml of water and
calculate as a percentage the amount of C 16H 19N 3 O 4 S in the powder for injec-
tions by comparison with ampicillin RS. In an adequately calibrated spec-
trophotometer the absorbance of the reference solution should be 0.29 + 0.02.
ampicillin.
985
ARTEMETHERI CAPSULAE
ARTEMETHER CAPSULES
Storage. Artemether capsules should be kept in a hermetically closed con-

tainer and stored in a cool place.
Additional information. Available strengths: 40 mg, 50 mg.
Requirements
Comply with the monograph for “Capsules”.
Artemether capsules contain not less than 90 0

. % and not more than 110 0 % .
of the amount of CigHjeOs stated on the label.
Identity tests
• Either tests A and B, or tests B, C, and D may be applied.
A. To a quantity of the contents of the capsules equivalent to 0.040 g of

Artemether add 40 ml of acetone R, shake to dissolve, and filter. Evaporate
the filtrate at low temperature and dry overnight over desiccant silica gel R.
Carry out the examination with the residue as described under 1.7 Spec-
concordant with the spectrum obtained from artemether RS or with the ref-
erence spectrum of artemether.
C. To a quantity of the contents of the capsules equivalent to 0.08 g of

Artemether add 40ml of dehydrated ethanol R, shake to dissolve, and filter.
Evaporate half of the filtrate to about 1 ml (keep the remaining filtrate for
test D), add O.lOg of potassium iodide R and heat; a yellow colour is
produced.

about 5 ml. Place a few drops of the mixture on a white porcelain dish and
add 1 drop of vanillin/sulfuric acid TSl; a pink colour is produced.
Related substances
986
g
other than the principal peak, is not greater than that obtained with solution
C (0.5%). Not more than one peak is greater than half the area of the princi-
pal peak obtained with solution C (0.25%). The sum of the areas of all the
peaks, other than the principal peak, is not greater than twice the area of the
principal peak obtained with solution C (1.0%). Disregard any peak with an
area less than 0.1 times the area of the principal peak in the chromatogram
obtained with solution C,

light petroleum R1 and 3 volumes of ethyl acetate R as the mobile phase.

Apply separately to the plate 10 pi of each of the following 5 solutions in
acetone R. For solution (A) shake a quantity of the contents of the capsules
equivalent to about 20 mg of Artemether with 2 ml of acetone R, filter, and
use the filtrate. Prepare similarly solution (B) with the equivalent of about
0.05 mg of Artemether per ml, solution (C) with the equivalent of about
0.025 mg of Artemether per ml, and solution (D) with the equivalent of
about O.lOmg of Artemether per ml. For solution (E) use O.lOmg of
artemether RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air, and spray with vanillin/sulfuric acid TSl.
Any spot obtained with solution A, other than the principal spot, isnot more
intense than that obtained with solution B (0.5%). Furthermore, not more than
one such spot is more intense than that obtained with solution C (0.25%).
Assay

groups (5pm). As the mobile phase, use a mixture of 62 volumes of ace-
tonitrile R and 38 volumes of water.
Prepare the following solutions in the mobile phase. For solution (A) mix
the contents of 20 capsules, shake a quantity equivalent to about 0.05
987
of Artemether, accurately weighed, with 2 ml of acetone R, and filter.
Evaporate the filtrate to dryness and dissolve the residue in 5 ml of the

mobile phase. For solution (B) use 10 mg of artemether RS per ml, and for
solution (C) dilute a suitable volume of solution A to obtain a concentration
equivalent to 0.05 mg of Artemether per ml.


from solutions A and B, and calculate the percentage content of C 16H 26 O 5 .
B. Mix the contents of 20 capsules and transfer a quantity equivalent to about

13mg of Artemether, accurately weighed, to a 100-ml volumetric flask and
dilute to volume with dehydrated ethanolR. Shake the flask for 15 minutes
and filter,discarding the first 10ml of the
filtrate. Accurately measure 5 ml
of the clear filtrate 50-ml volumetric flask and dilute to volume with
into a
hydrochloric acid/ethanol (1 mol/1) VS. Stopper the flask and place it in a
water-bath at 55 °C for 5 hours. Allow to cool to room temperature. For the
blank use 5 ml of dehydrated ethanol R diluted with sufficient hydrochloric
acid/ethanol (1 mol/1) VS to produce 50 ml.

against a solvent cell containing the blank. Calculate the percentage content
of C 16H 26O 5 in the capsules being examined by comparison with artemether
RS, similarly and concurrently examined.
Dissolution. Carry out the test as described under 5.5 Dissolution test for
solid oral dosage forms.
ARTEMETHERI COMPRESSI
ARTEMETHER TABLETS
Additional information. Available strengths: 40 mg, 50 mg.
Requirements
988
Artemether tablets contain not less than 90 0. % and not more than 1 10 0 % of
.
the amount of CKSH26O5 stated on the label.
Identity tests
• Either tests A and B, or tests B, C, and D may be applied.
A. To a quantity of the powdered 0.040g of Artemether

tablets equivalent to
add 40 ml of acetone R, shake to dissolve, and filter. Evaporate the filtrate
at low temperature and dry overnight over desiccant silica gel R. Carry out
the examination with the residue as described under 1.7 Spectrophotome-
try in the infrared region. The infrared absorption spectrum is concordant
with the spectrum obtained from artemether RS or with the reference spec-
trum of artemether,
C. To a quantity of the powdered tablets equivalent to 0.08 g of Artemether

add 40 ml of dehydrated ethanol R, shake to dissolve, and filter. Evaporate
half of the filtrate to about 1 ml (keep the remaining filtrate for test D), add
0.10 g of potassium iodide, and heat; a yellow colour is produced.

about 5 ml. Place a few drops of the mixture on a white porcelain dish and
add 1 drop of vanillin/sulfuric acid TSl; a pink colour is produced.
Related substances
Inject alternately 20|il each of solutions A and C.

989

light petroleum R1 and 3 volumes of ethyl acetate R as the mobile phase.

acetone R. For solution (A) shake a quantity of the powdered tablets equiv-
alent to about 20 mg of Artemether with 2 ml of acetone R, filter, and use
the filtrate. Prepare similarly solution (B) with the equivalent of about
0.05 mg of Artemether per ml, solution (C) with the equivalent of about
0.025 mg of Artemether per ml, and solution (D) with the equivalent of
about 0.10 mg of Artemether per ml. For solution (E) use 0.10 mg of
artemether RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air, and spray with vanillin/sulfuric acid TSl.

(0.25%).
Assay

Prepare the following solutions in the mobile phase. For solution (A) weigh
and powder 20 tablets, shake a quantity of the powder equivalent to about
0.05 g of Artemether, accurately weighed, with 2 ml of acetone R, and filter.
Evaporate the filtrate to dryness and dissolve the residue in 5 ml of the
mobile phase. For solution (B) use 10 mg of artemether RS per ml, and for
equivalent to 0.05 mg of Artemether per ml.


from solutions A and B, and calculate the percentage content of C]6H260s.
990
B. Weigh and powder 20 tablets. Transfer a quantity of the powder equivalent

to about 13mg of Artemether, accurately weighed, to a 100-ml volumetric
flask and dilute to volume with dehydrated ethanol R. Shake the flask for
15 minutes and filter, discarding the first 10ml of the filtrate. Accurately
measure 5 ml of the clear filtrate into a 50-ml volumetric flask and dilute to
volume with hydrochloric acid/ethanol (1 mol/1) VS. Stopper the flask and
place it in a water-bath at 55 °C for 5 hours. Allow to cool to room tem-
perature. For the blank use 5 ml of dehydrated ethanol R diluted with suf-
ficient hydrochloric acid/ethanol (1 mol/1) VS to produce 50 ml.

against a solvent cell containing the blank. Calculate the percentage content
of C 16H 26O 5 in the tablets being examined by comparison with artemether
ARTEMETHERI INJECTIO
ARTEMETHER INJECTION
Description. A clear, colourless or almost colourless, oily solution.
Storage. Artemether injection should be kept protected from light and stored
in a cool place.
Labelling. The oil used in the formulation should be indicated.
Additional information. Strength in the current WHO

Model list of essen-
tial medicines: 80 mg/ml in 1-ml ampoule; other available strengths: 40 mg/ml
(paediatric formulation), 60 mg/ml, 100 mg/ml (adult formulation).
Artemether injection is normally intended for intramuscular administration.
Requirements
for extractable volume for parenteral preparations, 3,4 Test for bacterial endo-
toxins, and 5.7 Visual inspection of particulate matter in injectable preparations.
991
Definition. Artemether injection is a sterile solution of artemether in a suit-

able oil for injection.
The solution is sterilized by a suitable method (see 5,8 Methods of sterilization).
Artemether injection contains not less than 95 0 . % and not more than 105 0 % .
of the amount of CigH 2605 stated on the label.
Identity tests
A. To a the injection equivalent to 0.050 g of Artemether add 25ml

volume of
of acetone R, mix, and filter. Evaporate the filtrate at low temperature and
dry overnight over desiccant silica gel R. Carry out the examination as
artemether RS or with the reference spectrum of artemether.
C. To a volume of Artemether injection equivalent to about 30 mg of

Artemether add 6 ml of dehydrated ethanol R. Place a few drops of the
Related substances
A. Note-. This test cannot be performed if arachis oil is present in the

formulation.

992

lightpetroleum R1 and 3 volumes of ethyl acetate R as the mobile phase.

acetone R. For solution (A) dilute a volume of the injection with acetone
R to obtain a concentration equivalent to 10 mg of Artemether per ml.
Prepare similarly solution (B) with the equivalent of about 0.05 mg of
Artemether per ml, solution (C) with the equivalent of about 0.025 mg of
Artemether per ml, and solution (D) with the equivalent of about O.lOmg
of Artemether per ml. For solution (E) use O.lOmg of artemether RS per ml.
in air, and spray with vanillin/sulfuric acid TSl. Examine the chromatogram
in daylight.
(0.25%).
Assay

Prepare the following solutions in the mobile phase. For solution (A) dilute
a volume of the injection to obtain a concentration equivalent to lOmg of
Artemether per ml, for solution (B) use 10 mg of artemether RS per ml, and
for solution (C) dilute a suitable volume of solution A to obtain a concen-
tration equivalent to 0.05 mg of Artemether per ml.


from solutions A and B, and calculate the percentage content of C16H26O5.
993
B. Dilute an accurately measured volume of the injection equivalent to about

0.08 g with sufficient ethanol R to produce 100 ml. Dilute 5 ml of this solu-
tion with the same solvent to 50ml and mix. Transfer a further 5ml of the
diluted solution to a 50-ml volumetric flask and dilute to volume with
hydrochloric acid/ethanol (1 mol/1) VS. Stopper the flask and place it in a
water-bath at 55 + 1 °C for 5 hours. Allow to cool to room temperature.

about 254 nm. {Note-. If arachis oil is used in the formulation of Artemether
injection subtract the value of 0.025 from the absorbance value determined;
the correction in absorbance value for other oils would have to be estab-
lished.) Calculate the percentage content of C 16H 26 O 5 in the formulation
being examined by comparison with artemether RS, similarly and concur-
rently examined.
ARTEMISININI CAPSULAE
ARTEMISININ CAPSULES
Storage. Artemisinin capsules should be kept in a cool place.
Additional information. Available strength: 250 mg.
Requirements
Artemisinin capsules contain not less than 90 0 . % and not more than 110 0 % .
of the amount of C 15 H 22 O 5 stated on the label.
Identity tests
A. To a quantity of the contents of the capsules equivalent to 0.040 g of

Artemisinin add 40ml of acetone R, shake to dissolve, and filter. Evaporate
the filtrate at low temperature and dry overnight over desiccant silica gel R.
Carry out the examination with the residue as described under 1.7 Spec-
concordant with the spectrum obtained from artemisinin RS or with the
reference spectrum of artemisinin.
994
C. To a quantity of the contents of the capsules equivalent to 10 mg of

Artemisinin add 20 ml of dehydrated ethanol R, shake to dissolve, filter, and
evaporate to dryness. To half of the residue (keep the remaining residue for
test D) add ml of dehydrated ethanol R, 1.0ml of potassium iodide
0.5
(80g/l) TS, 2.5 ml of sulfuric acid (-100 g/1) TS, and 4 drops of starch TS; a
violet colour is immediately produced.
D. Evaporate the remaining filtrate from test C to dryness on a water-bath.

Dissolve the residue in 0.5 ml of dehydrated ethanol R, add about 0.5ml
of hydroxylamine hydrochloride TS2 and 0.25ml of sodium hydroxide
(-80 g/1) TS. Heat the mixture in a water-bath to boiling, cool, add 2 drops
Related substances
cally bonded octadecylsilyl groups (3 pm). The mobile phases for gradient
Time Mobile phase A Mobile phase B Comment

17-30 60 => 100 40 => 0 Linear gradient
30-35 100 ^ 60 0 => 40 Return to initial conditions
Prepare the following solutions. For solution (A)mix the contents of 20 cap-
sules, shake a quantity equivalent to about 10 mg of Artemisinin, accurately
weighed, with 2 ml of acetone R, and filter. Evaporate the filtrate to dryness
and dissolve the residue in 1.0 ml of a mixture of 8 volumes of acetonitrile
R and 2 volumes of water.
For solution (B) use 50 pg of Artemisinin per ml in a mixture of 6 volumes

of acetonitrile R and 4 volumes of water.
995
acetonitrile
less than 2.0.

with solution B (0.5%). Not more than one peak is greater than half the area
of the principal peak obtained with solution B (0.25%). The sum of the areas
of all peaks, other than the principal peak, is not greater than twice the area
of the principal peak obtained with solution B (1.0%). Disregard any peak
with an area less than 0.1 times the area of the principal peak in the chro-
matogram obtained with solution B.

the plate 10 pi of each of the following 5 solutions in toluene R. For solu-
tion (A) take a quantity of the contents of the capsules equivalent to about
0.10 g of Artemisinin, add 10 ml of toluene R, shake vigorously, filter into a
10-ml volumetric flask, and dilute to volume with toluene R. Prepare simi-
larly solution (B)containing the equivalent of about 0.05 mg of Artemisinin
per ml, solution (C) the equivalent of about 0.025 mg of Artemisinin per ml,
and solution (D) with the equivalent of about O.lOmg of Artemisinin per
ml. For solution (E) use O.lOmg of artemisinin RS per ml. After removing
the plate from the chromatographic chamber, allow it to dry in air, spray
with anisaldehyde/sulfuric acid TS, and heat the plate to 105°C for 7
minutes. Examine the chromatogram in daylight.

(0.25%).
996
Assay
A. Determine by 1.14,4 High performance liquid chromatography, using a

Prepare the following solutions in the mobile phase. For solution (A) mix
the contents of 20 capsules, shake a quantity equivalent to about l.Omg of
Artemisinin, accurately weighed, with 2 ml of acetone R, and filter. Evapo-
rate the filtrate to dryness and dissolve the residue in 1,0ml. For solution (B)
use l.Omg of artemisinin RS per ml.
For the system suitability test prepare solution (C) containing l.Omg of
artemisinin RS per ml and l.Omg of artenimol RS per ml in a mixture of 8
acetonitrile

is
less than 2.0.

from solutions A and B, and calculate the percentage content of C]5H2205.
B. Mix the contents of 20 capsules and transfer a quantity equivalent to about

0.05 g of Artemisinin, accurately weighed, to a 100-ml volumetric flask and
dilute to volume with ethanol (-750 g/1) TS. Shake the flask, filter, and
discard the first 20ml of the filtrate. Dilute 10ml of the filtrate to 100ml
with the same solvent Accurately transfer 10 ml to a 50-ml volumetric flask,
dilute to volume with sodium hydroxide (0.05mol/l) VS, mix thoroughly,
and warm to 50 °C in a water-bath for 30 minutes. Cool to room
temperature.

stance being tested by comparison with artemisinin RS, similarly and
997
ARTEMISININI COMPRESSI
ARTEMISININ TABLETS
Storage. Artemisinin tablets should be kept in a cool place.
Requirements
Artemisinin tablets contain not less than 90 0. % and not more than 110 0 % .
of the amount of C 15 H 22 O 5 stated on the label.
Identity tests
A. To a quantity of the powdered tablets equivalent to 0.040 g of Artemisinin

with the spectrum obtained from artemisinin RS or with the reference
spectrum of artemisinin.
C. To a quantity of the powdered tablets equivalent to 10 mg of Artemisinin

add 20ml of dehydrated ethanol R, shake to dissolve, filter, and evaporate
to dryness. To half of the residue (keep the remaining residue for test D)
add 0.5 ml of dehydrated ethanol R, 1.0 ml of potassium iodide (80g/l) TS,
2.5 ml of sulfuric acid (-100 g/1) TS, and 4 drops of starch TS; a violet colour
is immediately produced.
D. Evaporate the remaining filtrate from test C to dryness on a water-bath.

998

Related substances
cally bonded octadecylsilyl groups (3 pm). The mobile phases for gradient
'lime Mobile phase A Mobile phase B Comment

17-30 60 => 100 40 => 0 Linear gradient
30-35 100 ^ 60 0 ^ 40 Return to initial conditions
Prepare the following solutions. For solution (A) weigh and powder 20
tablets, take a quantity of the powder equivalent to about lOmg of
Artemisinin, accurately weighed, add 2 ml of acetone R, shake, and filter.
Evaporate the filtrate and dissolve the residue in 1.0 ml of a

to dryness
mixture of 8 volumes of acetonitrile R and 2 volumes of water. For solution
(B) use 50 pg of Artemisinin per ml in a mixture of 6 volumes of acetonitrile
R and 4 volumes of water.
acetonitrile
less than 2.0.
999

from solutions A and B, and calculate the content of related substances as
a percentage. In the chromatogram obtained with solution A, the area of
with solution B (0.5%). Not more than one peak is greater than half the
area of the principal peak obtained with solution B (0.25%). The sum of the
areas of all peaks, other than the principal peak, is not greater than twice
the area of the principal peak obtained with solution B (1.0%). Disregard
the chromatogram obtained with solution B.

tion (A) take a quantity of the powdered tablets equivalent to about O.lOg
of Artemisinin, add 10 ml of toluene R, shake vigorously, filter into a 10-ml
volumetric flask, volume with toluene R. Prepare similarly solu-
and dilute to
tion (B) with the equivalent of about 0.05mg of Artemisinin per ml, solu-
tion (C) with the equivalent of about 0.025 mg of Artemisinin per ml, and
solution (D) with the equivalent of about 0.10 mg of Artemisinin per ml. For
solution (E) use 0.10 mg of artemisinin RS per ml. After removing the plate
from the chromatographic chamber, allow it to dry in air, spray with
anisaldehyde/sulfuric acid TS, and heat the plate to 105 °C for 7 minutes.

(0.25%).
Assay

1.0 mg of Artemisinin, accurately weighed, with 2 ml of acetone R, and filter.
Evaporate the filtrate to dryness, and dissolve the residue in 1.0ml. For solu-
tion (B) use 1.0 mg of artemisinin RS per ml.
1000
acetonitrile
less than 2.0.

B. Weigh and powder 20 tablets. Transfer a quantity of the powder equivalent

to about 0.05 g of Artemisinin, accurately weighed, to a 100-ml volumetric
flaskand dilute to volume with ethanol (~750g/l) TS. Shake the flask, filter,
and discard the first 20 ml of the filtrate. Dilute 10 ml of the filtrate to
100 ml with the same solvent. Accurately transfer 10 ml to a 50-ml volu-
metric flask, dilute to volume with sodium hydroxide (0.05 mol/1) VS, mix
thoroughly, and warm to 50 °C in a water-bath for 30 minutes. Cool to room
temperature.

produce 50ml. Calculate the percentage content of C,5H2205 in the sub-
stance being tested by comparison with artemisinin RS, similarly and
ARTEMOTILI INJECTIO
ARTEMOTIL INJECTION
Description. A clear, colourless to slightly yellowish, oily solution.
Storage. Artemotil injection should be kept protected from light.
1001
Labelling. The oil used in the formulation should be indicated.
Additional information. Available strengths: 50mg/ml (paediatric formula-

tion), 75mg/ml, 150mg/ml (adult formulation).
Artemotil injection is normally intended for intramuscular administration.
Requirements
Complies with the monograph for “Parenteral preparations”, and with 5.6 Test
for extractable volume for parenteral preparations, 3.4 Test for bacterial endo-
toxins, and 5.7 Visual inspection of particulate matter in injectable preparations.
Definition. Artemotil injection is a sterile solution of artemotil in an oil
suitable for injection.
The solution is sterilized by a suitable method (see 5.8 Methods of sterilization).
Artemotil injection contains not less than 95 0. % and not more than 105 0 % .
of the amount of C] 7 H 2s 05 stated on the label.
Identity tests
A. To a volume of the injection equivalent to O.OSOg of Artemotil add 25ml of

acetone R, mix, and filter. Evaporate the filtrate at low temperature and dry
overnight over desiccant silica gel R. Carry out the examination as described
tion spectrum is concordant with the spectrum obtained from artemotil RS
or with the reference spectrum of artemotil.
C. To a volume of the injection equivalent to about 30 mg of Artemotil add

6ml of dehydrated ethanol R. Place a few drops of the mixture on a white
porcelain dish and add 1 drop of vanillin/sulfuric acid TSl; a pink colour is
produced.
Related substances
matography, using the conditions given below under Assay.
1002
Inject alternately 20|il each of solutions A and C.


tion (A) dilute a volume of the injection with toluene R to obtain a con-
centration equivalent to 10 mg of Artemotil per ml. Prepare similarly
solution (B) with the equivalent of about 0.05 mg of Artemotil per ml, solu-
tion (C) with the equivalent of about 0.025 mg of Artemotil per ml, and solu-
tion (D) with the equivalent of 0.10 mg of Artemotil per ml. For solution (E)
use 0.10 mg of artemotil RS per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in air, and spray with vanillin/sulfu-
ric acid TSl. Examine the chromatogram in daylight.
(0.25%).
Assay. Determine by 1.14.4 High performance liquid chromatography, using

a stainless steel column (25cm x 4mm) packed with particles of silica gel, the
groups (5pm). As the mobile phase, use a mixture of 62 volumes of acetoni-
trile R and 38 volumes of water.
Prepare the following solutions in acetonitrile R. For solution (A) dilute

a volume of the injection to obtain a concentration equivalent to 10 mg of
Artemotil per ml; for solution (B) use 10 mg of artemotil RS per ml; and for
equivalent to 0.05 mg of Artemotil per ml.
1003
Inject alternately 20|il each of solutions A and B.
solutions A and B, and calculate the percentage content of C 17 H 28 O 5 .
ARTENIMOLI COMPRESSI
ARTENIMOL TABLETS
Storage. Artenimol tablets should be kept in a cool place and protected from
light.
Requirements
Artenimol tablets contain not less than 90 0 . % and not more than 110 0 % of
.
the amount of C 15 H 24 O 5 stated on the label.
Identity tests
A. To a quantity of the powdered tablets equivalent to 0.040 g of Artenimol

with the spectrum obtained from artenimol RS or with the reference spectrum
of artenimol.
C. To a quantity of the powdered tablets equivalent to 10 mg of Artenimol add

20ml of dehydrated ethanol R, shake to dissolve, filter, and evaporate to
dryness. To half of the residue (keep the remaining residue for test D) add
0.5ml of dehydrated ethanol R, 1.0 ml of potassium iodide (80g/l) TS,
2.5 ml of sulfuric acid (-100 g/1) TS, and 4 drops of starch TS; a violet colour
is produced.
1004
D. Evaporate the remaining filtrate from test C

on a water-bath.
to dryness
of hydroxylamine hydrochloride TS2 and 0.25ml of sodium hydroxide
a deep reddish-brown colour is immediately produced.
Related substances
stainless steel
which has been modified with chemi-
particles of silica gel, the surface of
cally bonded As the mobile phase for gradient
octadecylsilyl groups (3 pm).
elution, use a mixture of 6 volumes of acetonitrile R and 4 volumes of water
for the first 17 minutes; then run a gradient, which should reach 100% ace-
tonitrile within 13 minutes.
Prepare the following solutions. For solution (A) weigh and powder 20
tablets, shake a quantity of the powder equivalent to about 10 mg of Arten-
imol, accurately weighed, with 2 mland filter. Evaporate the
of acetone R,
filtrate to dryness and dissolve the residue in 1.0 ml of methanol R with son-
ication. For solution (B) dissolve 50 pg of Artenimol per ml in methanol R

with sonication.
For the system suitability test prepare solution (C) by dissolving 1.0 mg of
artemisinin RS per ml and 1.0 mg of artenimol RS per ml in methanol R with
sonication.
The not valid unless the relative retention of a-artenimol compared

test is
less than 2.0.
matograms from solutions A and B, and calculate the content of the related
substances as a percentage. In the chromatogram obtained with solution A,
the area of any peak, other than the twin peak, is not greater than that
1005
obtained with solution B (0.5%). Not more than one peak is greater than
half the area of the twin peak obtained with solution B (0.25%). The sum
of the areas of all the peaks, other than the twin peak,
is not greater than
twice the area of the twin peak obtained with solution B (1.0%). Disregard
any peak with an area less than 0.1 times the area of the twin peak in the
chromatogram obtained with solution B.

tion (A) shake a quantity of the powdered tablets equivalent to about 20 mg
of Artenimol, with 2 ml of acetone R, and filter. Use the filtrate. Prepare sim-
ilarly solution with the equivalent of about 0.05 mg of Artenimol per ml,
(B)
solution (C) with the equivalent of about 0.025 mg of Artenimol per ml,
and solution (D) with the equivalent of about 0.10 mg of Artenimol per
ml. For solution (E) use 0.10 mg of artenimol RS per ml. After removing the
plate from the chromatographic chamber, allow it to dry in air, and spray
with vanillin/sulfuric acid TSl. Examine the chromatogram in daylight.
Any spot obtained with solution A, other than the principal spot,is not more
(0.25%).
Assay

1.0 mg of Artenimol, accurately weighed, with 2 ml of acetone R, and filter.
tion (B) use 1.0 mg of artenimol RS per ml.
acetonitrile
1006

is
less than 2.0.
matograms from solutions A and B, and calculate the percentage content of
C15H24O5.
B. Weigh and powder 20 tablets. To a quantity of the powder equivalent to

about 0.05 g of Artenimol, accurately weighed, add sufficient ethanol (-750
g/1) TS to produce 100 ml, shake, and filter. Discard the initial 20 ml of the
filtrate and dilute 10 ml to 100 ml with the same solvent. Accurately trans-
fer 10ml to a 50-ml volumetric flask, dilute to volume with sodium hydrox-
ide (0.05 mol/1) VS, mix thoroughly, and warm to 50 °C in a water-bath for
30 minutes. Cool to room temperature.

stance being tested by comparison with artenimol RS, similarly and
ARTESUNATI COMPRESSI
ARTESUNATE TABLETS
Storage. Artesunate tablets should be kept in a cool place.

1007
Requirements
Artesunate tablets contain not less than 90 0 . % and not more than 1 10 0 % of
.
the amount of C19H28O8 stated on the label.
Identity tests
A. To a quantity of the powdered

tablets equivalent to 0.050 g of Artesunate
with the spectrum obtained from artesunate RS or with the reference spec-
trum of artesunate.

ethyl acetate R and 95 volumes of toluene R as the mobile phase. Apply
separately to the plate 2|il of the following 2 solutions in toluene R. For
solution (A) shake a quantity of the powdered tablets equivalent to about
0.10 mg of Artesunate in dehydrated ethanol R, filter, and evaporate. Dis-
solve the residue in 1.0ml of toluene R. For solution (B) use O.lOmg of arte-
sunate RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry in air, spray with anisaldehyde/methanol TS, and
heat the plate to 120°C for 5 minutes. Examine the chromatogram in ultra-

C. To a quantity of the powdered tablets equivalent to 0.1 g of Artesunate add

40ml of dehydrated ethanol R, shake to dissolve, and filter. To half of the
filtrate (keep the remaining filtrate for test D) add about 0.5ml of hydroxy-
lamine hydrochloride TS2 and 0.25 ml of sodium hydroxide (~80g/l) TS.
Heat the mixture in a water-bath to boiling, cool, add 2 drops of hydrochlo-
ric acid (~70g/l) TS and 2 drops of ferric chloride (50g/l) TS; a light redvio-
let colour is produced.
about 5 ml. Place a few drops of the mixture on a white porcelain dish, add
1 drop of vanillin/sulfuric acid TSl, a reddish-brown colour is produced.
1008
Related substances

area of the principal peak obtained with solution C (0.5%). The sum of the
areas of all the peaks, other than the principal peak, is not greater than twice
the area of the principal peak obtained with solution C (2.0%). Disregard
the chromatogram obtained with solution C.

light petroleum Rl, 36 volumes of ethyl acetate R and 1 volume of glacial

of the following 3 solutions in dichloromethane R. For solution (A) shake a
quantity of the powdered tablets equivalent to about lOmg of Artesunate
with 2 ml of dichloromethane R, filter, and use the filtrate. Prepare similarly
solution (B) with the equivalent of about 0.05 mg of Artesunate per ml, and
solution (C) with the equivalent of about 0.025 mg of Artesunate per ml.
in air, and spray with vanillin/sulfuric acid TSl. Examine the chromatogram
in daylight.

(0.5%).
Assay

stainless steel column (12.5 cm x 3.5mm) packed with particles of silica gel,
cylsilyl groups (5pm). As the mobile phase, use a mixture of equal volumes
of acetonitrile R and buffer pH 3.0 (dissolve 1.36g of potassium dihydrogen
1009
phosphate R in 1000ml of water and adjust the pH to 3.0 with phosphoric

acid (-1440 g/1) TS).
Prepare the following solutions in acetonitrile R. For solution (A) weigh

4.0 mg of Artesunate, accurately weighed, with 2 ml of acetone R, and filter.
tion (B) use 4.0mg of artesunate RS per ml, and for solution (C) dilute solu-
tion A to obtain a concentration equivalent to 0.04 mg of Artesunate per ml.
Operate with a flow rate of 0.6ml per minute. Maintain the column tem-
perature at 30 °C and use an ultraviolet spectrophotometer set at a wave-
length of about 216nm.


about 0.5 g of Artesunate, accurately weighed, add 50ml of neutralized
ethanol TS, shake thoroughly, filter, and discard about 10ml of the initial
filtrate. Titrate 25ml of the filtrate with sodium hydroxide (0.05 mol/1) VS,
using 2 drops of phenolphthalein/ethanol TS as indicator.

C19H28O8.
ATROPINI SULFATIS COMPRESSI

ATROPINE SULFATE TABLETS
Category. Antispasmodic drug.

Requirements
1010
Atropine sulfate tablets contain not less than 90 0. % and not more than
110 0 %
. of the amount of (Ci7H23N03)2,H2S04,H20 stated on the label.
Identity tests

chloroform R, 4 volumes of acetone R, and 1 volume of diethylamine R as

the mobile phase. Apply separately to the plate 5 pi of each of the follow-
ing two solutions. For solution (A) shake a quantity of the powdered tablets
equivalent to lOmg of Atropine sulfate with 2ml of ethanol (~750g/l) TS,
centrifuge, and use the supernatant liquid. For solution (B) dissolve 25 mg of
atropine sulfateRS in 5 ml of ethanol (-750 g/1) TS. After removing the plate
from the chromatographic chamber, heat it at 105 °C for 20 minutes, allow
to cool, and spray with potassium iodobismuthate TS2. Examine the chro-

B. Triturate a quantity of the powdered tablets equivalent to 1 mg of Atropine

sulfate with 1 drop of ammonia (-260 g/1) TS, add 2 ml of chloroform R, and
triturate again thoroughly. Filter the chloroform layer and evaporate. To the
residue add about 0.2 ml of fuming nitric acid R and evaporate to dryness
on yellow residue is obtained. To the cooled residue add
a water-bath; a
2 ml of acetone R and about
0.2 ml of potassium hydroxide/methanol TS;
a deep violet colour is produced.
C. A filtered solution of the powdered tablets in water yields the reactions

described under 2.1 General identification tests as characteristic of sulfates.
Assay. Weigh and powder 20 tablets. Transfer a quantity of the powder equiv-
alent to about 2.5mg of Atropine sulfate, accurately weighed, into a 50-ml
volumetric flask, add 30 ml of water, shake well, dilute to volume, and filter.
Discard the first few ml of filtrate and use the successive clear filtrate as the test
solution. For the reference solution use 25 mg of atropine sulfate RS, accurately
weighed and previously dried to constant mass at 120 °C, dissolve in sufficient
water to produce 25 ml, and mix well. Dilute 5 ml of this solution to 100 ml
with water (= 50 pg of anhydrous atropine sulfate per ml). Transfer 2 ml of each
of the test solution and the reference solution to two 60-ml separating funnels con-
taining 10ml of chloroform R. Add 2ml of bromocresol green TSl, shake for 2
minutes, and allow to stand until two layers are formed.
1011
Measure the absorbance of the chloroform layers of the test solution and the ref-
erence solution at the maximum at about 420 nm against a solvent cell contain-
ing chloroform R.
Calculate the amount in mg of (Ci7H23N03)2,H2S04,H20 in the sample being

examined using the following formula; \.Q27(M/'tO)(AJAf), in which M is the
mass in mg of atropine sulfate RS in the reference solution and and As are the
absorbances for the test solution and the reference solution, respectively.
Uniformity of content. Individually transfer 10 powdered tablets to 10 sep-

arate stoppered test-tubes, to each add 6ml of water, accurately measured,
shake thoroughly for 30 minutes, centrifuge, and use the clear solution as the
test solution. For the reference solution weigh accurately 25 mg of atropine sulfate
RS, previously dried to constant mass at 120 °C, dissolve in sufficient water to
produce 25ml, and mix well. Dilute 5ml of this solution to 100ml with water
(= 50 pg of anhydrous atropine sulfate per ml). Transfer 2 ml of each of the solu-
tions to he examined and the reference solution to two 60-ml separating funnels con-
taining 10ml of chloroform R. Add 2ml of bromocresol green TSl, shake for 2
minutes, and allow to stand until two layers are formed.
Measure the absorbance of the chloroform layers of the solutions to be examined

and the reference solution at the maximum at about 420 nm against a solvent cell
containing chloroform R. Calculate the content of (Ci7H23N03)2,H2S04,H20 in
mg, using the following formula: \.Q27(M/'iO)(AJAf), in which M
is the mass
in mg of atropine sulfate RS in the reference solution and A„ and are the

absorbances for the solutions examined and the reference solution, respectively.
The tablets comply with the test for 5.1 Uniformity of content for single-dose
preparations.
BENZYLPENICILLINI KALICI PULVIS

AD INJECTIONEM
BENZYLPENICILLIN POTASSIUM
POWDER FOR INJECTIONS
Storage. Unless otherwise recommended by the manufacturer, the reconsti-

tuted solution should be used within 24 hours when stored at a temperature
not exceeding 20 °C or within 7 days (14 days if a buffering agent is present)
when stored at a temperature between 2 and 8°C.
1012
Labelling. The designation on the container should state the nature of the
buffering agent. Expiry date.

tial medicines: 600
mg and 3g of Benzylpenicillin.
Requirements
with the monograph for “Parenteral preparations’’.
Definition. Benzylpenicillin potassium powder for injections is a sterile
powder of benzylpenicillin potassium.
Benzylpenicillin potassium powder for injections usually contains a suitable

buffering agent. The powder is sterilized by a suitable method (see 5.8 Methods
of sterilization).
The container of Benzylpenicillin potassium powder for injections contains not

less than 90 0 % and not more than 110 0 % of the amount of Ci 5 H] 7 KN 204 S
. .
stated on the label.
Identity tests

infrared region.
spectrum obtained from benzylpenicillin potassium RS or with the reference
spectrum of benzylpenicillin potassium.
B. Dissolve a quantity of the powder for injections equivalent to 0.1 g of Ben-

zylpenicillin potassium in sufficient phosphate buffer, pH 7.0, (0.067 mol/1)
TS to produce 100 ml. Dilute 10 ml with the same reagent to produce
100 ml {solution A). To 10 ml of solution A add 0.5ml of a solution prepared
by diluting 1 ml of penicillinase TS to 10 ml with water and allow to stand
at 30 °C for 10 minutes {solution B). Place 5 ml of each of solutions A and B in
two separate test-tubes and add to each tube 10 ml of acetate buffer, pH 4.6,
TS and 5 ml of iodine (0.0005 mol/1) VS. After mixing the contents of each
tube add 0.1ml of starch TS; the mixture obtained with solution A is blue
and that with solution B remains colourless.
C. To a quantity of the powder for injections equivalent to 2 mg of Ben-

zylpenicillin potassium in a test-tube add 1 drop of water followed by 2 ml
of sulfuric acid (~1760g/l) TS and mix; the solution is colourless. Immerse
the test-tube for 1 minute in a water-bath; the solution remains colourless.
1013
Place a further similar quantity of the powder for injections in a second test-
tube, add 1 drop of water and 2 ml of formaldehyde/sulfuric acid TS, and
mix; the solution is brownish yellow. Immerse the test-tube for 1 minute in
a water-bath; a reddish brown colour is produced.
D. Ignite a small quantity of the powder for injections, dissolve the residue in
water, and filter; on addition of 2 ml of sodium hydroxide (-80g/l) TS to the
filtrate it yields the reaction described under 2.1 General identification tests
as characteristic of potassium.
Clarity and colour of solution. A solution of the powder for injections

equivalent to 0.2 g of Benzylpenicillin potassium in 10ml of carbon-dioxide-
free water R is clear and colourless. (Keep this solution for the “pH value”.)
Loss on drying. Dry the powder for injections to constant mass at 105 °C; it
loses not more than lOmg/g.
pH value. pH of the solution prepared above for the test of clarity and colour,
5.5-7.S.
Assay
Dissolve a quantity of the powder equivalent to about 50 mg of Benzylpeni-

cillin potassium, accurately weighed, in sufficient water to produce 1000 ml.
Transfertwo 2-ml aliquots of this solution into separate stoppered tubes. To
one tube add 10ml of imidazole/mercuric chloride TS, mix, stopper the tube,
and place in a water-bath at 60 °C for exacdy 25 minutes. Cool the tube rapidly
to 20 °C {solution A). To the second tube add 10 ml of water and mix {solution B).

B, calculate the amount of C16H17KN2O4S in the powder for injections by
comparison with benzylpenicillin sodium RS similarly and concurrently
examined, taking into account that each mg of benzylpenicillin sodium RS
(Ci6Hi7N2Na04S) is equivalent to 1.045mg of benzylpenicillin potassium
(Ci 6 Hi 7KN204S). In an adequately calibrated spectrophotometer the absorbance
of the reference solution should be 0.62 + 0.03.
benzylpenicillin.
1014
CARBAMAZEPINI COMPRESSI
CARBAMAZEPINE TABLETS
Category. Antiepileptic agent.
Storage. Carbamazepine tablets should be kept in a tightly closed container.

Requirements
Carbamazepine tablets contain not less than 90 0 % and not more than
.
110 0 % of the amount of C] 5 Hi 2 N 20 stated on the label.

.
Identity tests
Transfer a quantity of the powdered tablets equivalent to about 0.5 g of Car-

bamazepine to a 50-ml beaker, add 10 ml of warm acetone R, and shake. Filter
while still warm, evaporate the filtrate to dryness on a water-bath, and dry at
80 °C. Dissolve in acetone R, allow to recrystallize, and use the crystals for the
following tests.
A. Carry out the examination with the crystals as described under 1.7 Spec-
concordant with the spectrum obtained from carbamazepine RS or with the
reference spectrum of carbamazepine.
C. To about 0.1 g of the crystals add about 2 ml of nitric acid (-1000 g/1) TS and
heat in a water-bath for 1 minute; an orange colour is produced.
D. Melting temperature of the crystals, about 189 °C.
Apply separately to the plate 10 pi of each of the following 3 solutions. For solu-
1015
tion (A) shake a quantity of the powdered tablets equivalent to about 0.20 g of
Carbamazepine with three 10-ml quantities of chloroform R and filter. Evapo-
rate the combined filtrates to dryness and dissolve the residue in 10 ml of chlo-
roform R. For solution (B) use 20 mg of carbamazepine RS per ml of chloroform
R. For solution (C) use O.Ohmg of iminodibenzyl R per ml of methanol R. After
for 15 minutes, spray with potassium dichromate TS3, and examine the chro-

to about O.Obg of Carbamazepine add 25ml of ethanol (-750 g/1) TS and boil
for a few minutes. Stir the hot mixture in a closed flask for 10 minutes and filter
through a sintered glass filter. Wash the flask with ethanol (-750 g/1) TS, filter,
and dilute the cooled filtrate with sufficient ethanol (~750g/l) TS to produce
100ml. Dilute 5ml to 250ml with the same solvent. Measure the absorbance
of a 1-cm layer at the maximum at about 285 nm against a solvent cell con-
taining ethanol (-750 g/1) TS.
Calculate the percentage content of Ci5H]2N20 using the absorptivity value of

49.0 (All = 490).
CHLOROQUINI PHOSPHATIS COMPRESSI

CHLOROQUINE PHOSPHATE TABLETS
Categoiy. Antiamoebic drug; antimalarial drug.

tial medicines: lOOmg, 150mg.
Requirements
Chloroquine phosphate tablets contain not less than 93 0

107 0 %. of the amount of CisH26ClN3,2H3P04 stated on the label.
1016
Identity tests
B. Shake a quantity of the powdered tablets equivalent to 5 mg of Chloroquine

phosphate with 1 ml of water and filter. To the filtrate add 1 ml of silver
nitrate (40g/l) TS; ayellow precipitate is produced. To a portion of the pre-
cipitate add a few drops of nitric acid (-130 g/1) TS; a clear solution is
obtained. To another portion of the precipitate add a few drops of ammonia
(-100 g/1) TS and shake; the yellow precipitate dissolves but a small amount
of white precipitate remains.
of the following four solutions. For solution (A) shake a quantity of the pow-
dered tablets equivalent to 0.5 g of Chloroquine phosphate with 50 ml of water
for 30 minutes, centrifuge, and use the supernatant liquid; if necessary, filter
through a glass-fibre paper. For solution (B) dilute 5 ml of solution A to 100 ml
with water, and for solution (C) dilute 25ml of solution B to 50ml with water.
For solution (D) dissolve 8 mg of chloroquine sulfate RS in 1 ml of water. After
and examine the chromatogram in ultraviolet light (254nm).
intense than that obtained with solution B, and not more than one such spot
is more intense than that obtained with solution C.
alent to about 0.5 g of Chloroquine phosphate, accurately weighed, to a sepa-
rating funnel, add 20 ml of sodium hydroxide (1 mol/1) VS, and extract with four
quantities, each of 25 ml, of chloroform R. Filter each extract through a glass-
fibre paper washed with chloroform R and kept moistened with the solvent.
Evaporate the combined chloroform extracts to about 1 0 ml, add 40 ml of glacial
acetic acid Rl, and titrate with perchloric acid (0.1 mol/1) VS as described under
2.6 Non-aqueous titration, Method A.

C. sH2<;C1N3,2H3P04.
1017
CHLOROQUINI SULFATIS COMPRESSI

CHLORO QUINE SULFATE TABLETS
Category. Antiamoebic drug; antimalarial drug.

tial medicines: lOOmg, 150mg.
Requirements
Comply with the monograph for "Tablets”.
Chloroquine sulfate tablets contain not less than 93 0 . % and not more than
107 0 %
. of the amount of C]8H26C1N3,H2S04 stated on the label.
Identity tests
B. Shake a quantity of the powdered tablets equivalent to 0.1 g of Chloroquine

sulfate with 10 ml of water and 1ml of hydrochloric acid (~70g/l) TS, and
filter. To the filtrate add 1ml of barium chloride (50g/l) TS; a white
of the following four solutions. For solution (A) shake a quantity of the pow-
dered tablets equivalent to 0.4 g of Chloroquine sulfate with 50 ml of water
for 30 minutes, centrifuge, and use the supernatant liquid; if necessary, filter
through a glass-fibre paper and use the clear filtrate. 5 ml
For solution (B) dilute
of solution A to 100 ml with water, and for solution (C) dilute 25 ml of solu-
tion B to 50 ml with water. For solution (D) dissolve 8 mg of chloroquine sulfate
RS in 1 ml of water. After removing the plate from the chromatographic
light (254 nm).
intense than that obtained with solution B, and not more than one such spot
is more intense than that obtained with solution C.
1018
alent to about 0.5g of Chloroquine sulfate, accurately weighed, to a separating
funnel, add 20 ml of sodium hydroxide (1 mol/1) VS, and extract with four quan-
tities, each of 25ml, of chloroform R. Filter each extract through a glass-fibre
paper previously washed with chloroform R and kept moistened with the
solvent. Evaporate the combined chloroform extracts to about 10 ml, add 40 ml
of glacial acetic acid Rl, and titrate with perchloric acid (0.1 mol/1) VS as
described under 2.6 Non-aqueous titration. Method A.

C,sH2,C1N3,H2S04.
CHLORPHENAMINI HYDROGENOMALEATIS
COMPRESSI
CHLORPHENAMINE HYDROGEN
MALEATE TABLETS
Other name. Chlorpheniramine hydrogen maleate tablets.
Category. Antiallergic drug.
Storage. Chlorphenamine hydrogen maleate tablets should be kept in a tightly

closed container.

Requirements
Chlorphenamine hydrogen maleate tablets contain not less than 90 0 % and .
not more than 110 0 % of the amount of C,sHi 9 ClN 2 ,C 4 H 404 stated on the
.
label.
Identity tests
1019
A. Triturate a quantity of the powdered tablets equivalent to 25 mg of Chlor-

phenamine hydrogen maleate with 20ml of hydrochloric acid (~70g/l) TS.
Separately dissolve 25 mg of Chlorphenamine hydrogen maleate RS in
20ml of hydrochloric acid (~70g/l) TS. To each solution add sufficient
sodium hydroxide (~80g/l) TS to render them alkaline to a pH of about 11,
then extract with two 50-ml portions of hexane R. Collect the extracts in
beakers and evaporate to dryness. Carry out the examination with the
residues as described under 1.7 Spectrophotometry in the infrared region.
The infrared absorption spectrum of the sample being examined is concor-
dant with the spectrum obtained from Chlorphenamine hydrogen maleate
RS.
B. See the test described below under “Related substances”. Under an ultravi-
olet light the two with solution A correspond in
principal spots obtained
position, appearance, and intensity with those obtained with solution B.
After spraying, the principal spot obtained with solution A corresponds to
that obtained with solution B.
C. To a quantity of the powdered tablets equivalent to 40 mg of Chlor-

phenamine hydrogen maleate add about 20 ml of water, warm the mixture,
and filter. To the filtrate add 10 ml of a saturated solution of trinitrophenol
R in water and warm on a water-bath for 5 minutes; a precipitate is pro-
duced. Filter, wash the precipitate with water, collect the precipitate,
and dry it at 105°C for 1 hour; melting behaviour, about 196°C with
decomposition.
chromatography, using silica gel R2 as the coating substance and heating the
coated plate at 105 °C for 30 minutes. Use as the mobile phase a mixture of 5
volumes of ethyl acetate R, 3 volumes of methanol R, and 2 volumes of acetic
acid (~60g/l) TS. Apply separately to the plate 2 pi of each of the following four
solutions. For solution (A) extract a quantity of the powdered tablets equiva-
lent to 5 mg Chlorphenamine hydrogen maleate with chloroform R, filter,
of
evaporate the dryness, and dissolve the residue in 1 ml of chloroform
filtrate to
R. For solution (B) dissolve 25 mg of chlorphenamine hydrogen maleate RS in

5 ml of chloroform R. For solution (C) extract a quantity of the powdered tablets
equivalent to 50 mg of Chlorphenamine hydrogen maleate with chloroform R,
filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of chlo-
roform R. For solution (D) dilute 0.2 ml of solution C to 100 ml with chloro-
form R. After removing the plate from the chromatographic chamber, allow it
to dry in air, and examine the chromatogram in ultraviolet light (254 nm) for
identification purposes, then spray it with potassium iodobismuthate TS2.
1020
Assay. Weigh and powder 20 tablets. Shake a quantity of the powder equiv-
alent to about 3 mg of Chlorphenamine hydrogen maleate, accurately weighed,
with 20 ml of sulfuric acid (0.05 mol/1) VS for 5 minutes. Add 20 ml of hexane
R, shake carefully, and filter the acid layer into a second separator. Extract the
hexane layer with two quantities, each of 10ml, of sulfuric acid (0.05mol/l) VS,
filtering each acid layer into the second separator, and wash the filter with sul-
furic acid (0.05 mol/1) VS. Add sodium hydroxide (1 mol/1) VS to the acid
extracts and washings to make the solution just alkaline to litmus paper R, add
2 ml in excess, and extract with two quantities, each of 50 ml, of hexane R.
Wash each hexane extract with the same 20 ml of water, and extract in suc-
cession with 20ml, 20ml, and 5 ml of sulfuric acid (0.25 mol/1) VS. Dilute the
combined acid extracts to 50 ml with sulfuric acid (0.25mol/l) VS and dilute
10 ml to 25 ml with the same acid.

about 265 nm. Calculate the content of Ci 5 Hi 9 ClN 2 ,C 4 H 404 using the absorp-
,
tivity value of 21.2 (Ai°cm = 212).

arate stoppered test-tubes, and shake with 20ml of sulfuric acid (0.05mol/l) VS
for 5 minutes. Add 20 ml of hexane R, shake carefully, and filter the acid layer
into a second separator. Extract the hexane layer with two quantities, each of
10ml, of sulfuric acid (0.05mol/l) VS, each acid layer into the second
filtering
separator, and wash the filter with Add sodium
sulfuric acid (0.05 mol/1) VS.
hydroxide (1 mol/1) VS and washings to make the solution
to the acid extracts
just alkaline to litmus paper R, add 2 ml in excess, and extract with two quan-
tities, each of 50 ml, of hexane R. Wash each hexane extract with the same
20ml of water, and extract in succession with 20ml, 20ml, and 5 ml of sulfuric
acid (0.25 mol/1) VS. Dilute the combined acid extracts to 50 ml with sulfuric
acid (0.25 mol/1) VS, dilute 10 ml to 25 ml with the same acid.

about 265 nm. Calculate the content of Ci 6 Hi 9 ClN 2 ,C 4 H 404 using the absorp-
,
tivity value of 21.2 (AT™ = 212).
preparations.
CLOXACILLINI NATRICI CAPSULAE

CLOXACILLIN SODIUM CAPSULES
Categoiy. Antibacterial drug.
Storage. Cloxacillin sodium capsules should be kept in a tightly closed container.
1021
Labelling. The designation on the container should state the quantity of

Cloxacillin sodium in terms of the equivalent amount of cloxacillin. Expiry date.

tial medicines: the equivalent of 500
mg of cloxacillin.
Requirements
Cloxacillin sodium capsules contain not less than 90 0 % and not more than
.
110 0 %
. of the amount of C] 9 HigClN 305 S stated on the label.
Identity tests


volumes of acetone R and 70 volumes of a solution containing 154g/l of
ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial
acetic acid R, as the mobile phase. Apply separately to the plate 1 pi of each
of the following three solutions. For solution (A) shake a quantity of the
contents of the capsules equivalent to 0.25 g of Cloxacillin sodium with
50ml of water, filter, and use the clear filtrate. For solution (B) dissolve
25 mg of cloxacillin sodium RS in 5 ml of water. For solution (C) dissolve
25 mg of each of cloxacillin sodium RS, dicloxacillin sodium RS, and flu-
sodium RS together in 5 ml of water. After removing the plate
cloxacillin
from the chromatographic chamber, allow it to dry in air, and expose it to
the vapour of iodine R until spots appear. Examine the chromatogram in
daylight.

if the chromatogram obtained with solution C shows three distinctly sepa-
rated spots.
C. Place a quantity of the contents of the capsules equivalent to 2 mg of

Cloxacillin sodium into a test-tube, and add 2 mg of disodium chro-
motropate R and 2 ml of sulfuric acid (~1760g/l) TS. Immerse the tube in a
suitable bath at 150 °C for 3-4 minutes; a red-violet colour is produced.
1022
=
D. Ignite a quantity of the contents of the capsules equivalent to 20 mg of

Cloxacillin sodium and dissolve the residue in acetic acid (~60g/l) TS. The
solution yields reaction B described under 2.1 General identification tests as
Specific optical rotation. Use a filtered solution containing a quantity of the

contents of the capsules equivalent to 10 mg of CloxaciUin sodium per ml, and
calculate with reference to the anhydrous substance; [a]™ +163 to + 172°.

Fischer method, Method A, using a quantity of the contents of the capsules
equivalent to 0.25 g of Cloxacillin sodium; the water content is not more than
50mg/g.
pH value. pH of a filtered solution containing a quantity of the contents

of the capsules equivalent to O.lOg of CloxaciUin sodium per ml of
carbon-dioxide-free water R, 5. 0-7.0.
Assay. Mix the contents of 20 capsules and transfer a quantity equivalent to

about 0.25 g of Cloxacillin sodium, accurately weighed, into a glass-stoppered
flask. Add 70 ml of water, shake the flask for 15 minutes, and dilute with suf-
ficient water to produce 500 ml. Filter and dilute 10 ml of the filtrate to 100 ml
with water. Transfer two 2.0-ml aliquots of this solution into separate stop-
pered tubes. To one tube add 10 ml of imidazole/mercuric chloride TS, stopper
the tube, and place in a water-bath at 60 °C for exactly 25 minutes. Cool the
tube rapidly to 20 °C {solution A). To the second tube add 10.0ml of water and
mix {solution B).

calculate theamount of C 19 H 18 CIN 3 O 5 S in the substance being examined by
comparison with cloxacillin sodium RS. In an adequately calibrated spec-
1023
CLOXACILLINI NATRICI PULVIS

AD INJECTIONEM
CLOXACILLIN SODIUM POWDER
FOR INJECTIONS
Storage. Cloxacillin sodium powder for injections should be protected from

light.
Labelling. The designation on the container should state the quantity of

Cloxacillin sodium in terms of the equivalent amount of cloxacillin. Expiry date.

tial medicines: the equivalent of 500
mg of cloxacillin in vials.
Cloxacillin sodium powder for injections is hygroscopic.
Requirements
Definition. Cloxacillin sodium powder for injections is a sterile powder of

cloxacillin sodium.
The powder is sterilized by a suitable method (see 5.8 Methods of sterilization).
The container of Cloxacillin sodium powder for injections contains not less
than 90 0 % and not more than 110 0 % of the amount of C] 9 Hi 7 ClN 3 Na 05 S
. .
Identity tests


1024
=
volumes of acetone R and 70 volumes of a solution containing 154g/l of

ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial
acetic acid R, as the mobile phase. Apply separately to the plate 1 pi of each
of the following three solutions. For solution (A) shake a quantity of the
powder for injections equivalent to 0.25 g of Cloxacillin sodium with 50ml
of water, filter, and use the clear filtrate. For solution (B) dissolve 25 of
mg
cloxacillin sodium RS in 5ml of water, and for solution (C) dissolve 25 mg
of each of cloxacillinsodium RS, dicloxacillin sodium RS, and flucloxacillin
sodium RS together in 5 ml of water. After removing the plate from the chro-
matographic chamber, allow it to dry in air and expose it to the vapour of
iodine R until spots appear. Examine the chromatogram in daylight.

if the chromatogram obtained with solution C shows three distinctly sepa-
rated spots.
C. Place a quantity of the powder for injections equivalent to 2 mg of

Cloxacillin sodium in a test-tube and add 2 mg of disodium chromotropate
R and 2 ml of sulfuric acid (~1760g/l) TS. Immerse the tube in a suitable
bath at 150 °C for 3-4 minutes; a red-violet colour is produced.
D. Ignite a quantity of the powder for injections equivalent to 20 mg of

Cloxacillin sodium and dissolve the residue in acetic acid (~60g/l) TS. The
solution yields reaction B described under 2.1 General identification tests as
Specific optical rotation. Use a solution containing a quantity of the powder

for injections equivalent to 10 mg of Cloxacillin sodium per ml, and calculate
with reference to the anhydrous substance; [a]o -t-163 to -l- 173°.

equivalent to 0.2 g of Cloxacillin sodium in 10 ml of carbon-dioxide-free water
R is clear and colourless. (Keep this solution for the “pH value”.)

Fischer method, Method A, using a quantity of the powder for injections equiv-
alent to 0.25 g of Cloxacillin sodium; the water content is not less than 35 mg/g
and not more than 45 mg/g.
5.0-7.0.
Assay
1025
Transfer a quantity of the powder for injections equivalent to about 0.1 g of

Cloxacillin, accurately weighed, to a 500-ml flask and dilute to volume with
water. Dilute 25 ml to 100 ml with water. Transfer two 2-ml aliquots of this
solution into separate stoppered tubes. To one tube add 10ml of
imidazole/mercuric chloride TS, mix, stopper the tube, and place in a water-
bath at 60 °C for exactly 25 minutes. Cool the tube rapidly to 20 °C {solution A).
To the second tube add 10 ml of water and mix {solution B).

amount of Ci9Hi7ClN3Na05S in the substance being examined by
calculate the
comparison with cloxacillin sodium RS. In an adequately calibrated spec-
bacterial endotoxins; contains not more than 0.40 lU of endotoxin RS per mg
of cloxacillin.
CODEINI PHOSPHATIS COMPRESSI

CODEINE PHOSPHATE TABLETS
Category. Opioid analgesic, antidiarrhoeal, antitussive.
Additional information. Strength in the current WHO

Model list of essen-
tial medicines: 30 mg. Codeine phosphate tablets are prepared either from the
hemihydrate or the sesquihydrate.
Requirements
Codeine phosphate tablets contain not less than 90 0 and not more than . %
110 0 % of the amount of Ci8H2iN03,H3P04 stated on the label.
.
Identity tests
A. To powdered tablets equivalent to about 0.1 g of Codeine

a quantity of the
phosphate add 15ml of water and 5 ml of sulfuric acid (~100g/l) TS, and
1026
allow to stand for 1 hour. Filter, if necessary, and wash any undissolved
residue with a few ml of water. Render the filtrate alkaline with ammonia
(~100g/l) TS and extract with several small portions of chloroform R. Evap-
orate the combined extracts to dryness on a water-bath and dry the residue
at 80 °C for 4 hours. Carry out the examination with the residue as described
tion spectrum is concordant with the spectrum obtained from the reference
spectrum of codeine phosphate.
Shake a quantity of the powdered tablets equivalent to about 0.06g of

codeine phosphate with four 10-ml portions of ethanol (~750g/l) TS and
filter. Evaporate the combined filtrate to dryness on a water-bath and use
the residue for the following tests.
B. Dissolve 10 mg of the residue in 5 ml of sulfuric acid (~1760g/l) TS, add 1
drop of ferric chloride (25g/l) TS and, if necessary, heat gently; a violet-blue

colour is produced. Add a few drops of nitric acid (~130g/l) TS; the colour
changes to dark red.
C. Dissolve 20mg of the residue in 1.0ml of water and add 1 drop of ferric
chloride (25g/l) TS; a precipitate is formed but no blue tinge is observed in
the solution (distinction from morphine).
D. Dissolve lOmg of the residue in 2.0ml of carbon-dioxide-free water R and

add a few drops of silver nitrate (40g/l) TS; a yellow precipitate is produced.
Divide the solution with the precipitate into 2 portions. To 1 portion add a
few drops of nitric acid (-130 g/1) TS; the precipitate dissolves to a clear solu-
tion.To the other portion add a few drops of ammonia (-100 g/1) TS and
shake well; again the precipitate dissolves to a clear solution.

to about 0.2 g of Codeine phosphate add sufficient water to produce a thin sus-
pension, then add 20 ml of a mixture of 1 part of sulfuric acid (-100 g/1) TS and
3 parts of water. Shake occasionally over a period of 2 hours, then allow to
stand for 12 hours, and filter. Add 5 ml of sodium hydroxide (-80 g/1) TS and
extract with successive quantities of 15ml, 10ml, 10ml, and 5ml of chloroform
R. Evaporate the combined extracts to dryness on a water-bath, dissolve the
residue in 20 ml of glacial acetic acid Rl, and titrate with perchloric acid
Each ml of perchloric acid (0.05mol/l) VS is equivalent to 19.87 mg of

C]8Fl2iN03,H3P04.
1027
COLCHICINI COMPRESSI
COLCHICINE TABLETS
Category. Drug used for the treatment of gout.

Requirements
Colchicine tablets contain not less than 90 0 % and not more than 110 0 %
. . of
the amount of C 22 H 25NO 5 stated on the label.
Identity tests
A. Triturate a quantity of the powdered tablets equivalent to about 20 mg of

Colchicine with 20 ml of water. Allow the solids to settle and filter the super-
natant liquid into a separatory funnel. Shake with 30ml of chloroform R.
Evaporate the chloroform layer to dryness using mild heat. Carry out the
examination with the residue as described under 1.7 Spectrophotometry in
the spectrum obtained from colchicine RS or with the reference spectrum of
colchicine.
B. The absorption spectrum of the solution obtained in the “Assay”, when

observed between 230 nm and 380 nm, exhibits two maxima at about
243 nm and 350 nm. The ratio of the absorbance at 243 nm to that at
C. Suspend a quantity of the powdered tablets in 1.5ml of ethanol (~750g/l)

TS and filter. Place a few drops of the filtrate on a porcelain dish and evap-
orate to dryness on a water-bath. Mix the residue with 3 drops of sulfuric
acid (~1760g/l) TS; a lemon yellow colour is produced. Add 1 drop of nitric
acid (~130g/l) TS; the colour changes to greenish blue, turning rapidly to
reddish, and finally becoming yellowish. Following this add about 0.5ml of
sodium hydroxide (-200 g/1) TS; the colour turns to red.
chromatography, using a suitable aluminium oxide R as the coating substance,
containing a substance that fluoresces at about 254 nm, and a mixture of
125 volumes of chloroform R, 100 volumes of acetone R, and 2 volumes of
1028
ammonia (-260 g/1) TS as the mobile phase. Apply separately to the plate 2 ml
of each of the following 2p solutions. For solution (A) shake a quantity of the
powdered tablets equivalent to about 5 mg of Colchicine with 5 ml of chloro-
form R, filter, and evaporate the filtrate to dryness in a current of air. Dissolve
the residue as completely as possible in about 0.1 ml of ethanol (-750 g/1) TS.
Allow and use the supernatant liquid. For solution (B) dilute 1 volume
to settle
of solution A to 20 volumes with ethanol (-750 g/1) TS. After removing the plate
from the chromatographic chamber, allow it to dry in air, and examine the chro-
Assay
The operations described below must be carried out in subdued light.
Weigh and powder 20 To a quantity of the powder equivalent to about

tablets.
0.5 mg of Colchicine add 10 ml of dehydrated ethanol R and shake for 30
minutes. Centrifuge, separate, and wash the residue with dehydrated ethanol
R. Combine the extract and washings and dilute to 50 ml with the same solvent.
against a solvent cell containing dehydrated ethanol R.
Calculate the percentage content of C 22 H 25 NO 5 using the absorptivity value of

42.5 (A!"^ = 425).
Place 1 and add 10 ml of dehydrated ethanol R. Crush

tablet in a centrifuge tube
the tablet to a fine powder, shake for 30 minutes, centrifuge, and wash the
residue with dehydrated ethanol R. Combine the extract and washings and
dilute to produce a solution of 0.01 mg/ml of dehydrated ethanol R. Measure
the absorbance of a 1-cm layer at the maximum at about 350nm against a
solvent cell containing dehydrated ethanol R.
Calculate the tablet content of C 22 H 25 NO 5 in mg using the absorptivity value

of 42.5 (AlcT = 425). The tablets comply with the test for 5.1 Uniformity of
content for single-dose preparations.
1029
DAPSONI COMPRESSI
DAPSONE TABLETS

The tablets may be coloured.
Requirements
Dapsone tablets contain not less than 93 0 % and not more than 107 0 %
. . of
the amount of C 12 H 12 N 2 O 2 S stated on the label.
Identity tests
A. To a quantity of the powdered tablets equivalent to 0.1 g of Dapsone add

50ml of methanol R, shake, and filter. Dilute 0.5 ml of the filtrate to 200ml
with methanol R. The absorption spectrum of the resulting solution, when
observed between 230 nm and 350 nm, exhibits maxima at about 260 nm
and 295 nm; the absorbances of a 1-cm layer at these maximum wavelengths
are about 0.36 and 0.6, respectively.
C. Shake a quantity of the powdered tablets equivalent to 0.05 g of Dapsone

with 5 ml of warm acetone R, filter, evaporate the filtrate, and dry at 105°C
for 30 minutes. Dissolve the residue in 2 ml of hydrochloric acid (~70g/l)
TS, cool in ice, and add 4ml of sodium nitrite (lOg/1) TS. Allow to stand for
2 minutes then pour the mixture into 2 ml of freshly prepared 2-naphthol
TSl containing 1 g of sodium acetate R; an orange-red precipitate is
produced.
8 volumes of toluene R and 4 volumes of acetone R as the mobile phase. Apply
separately to the plate 1 pi of each of the following two solutions. For solution
(A) shake a quantity of the powdered tablets equivalent to 10 mg of Dapsone
1030
with 10ml of methanol R, filter, and use the clear filtrate. For solution (B) dis-
solve 5 mg of dapsone RS in 5 ml of methanol R. Further apply 10 pi of the fol-
lowing three solutions. For solution (C) shake a quantity of the powdered
tablets equivalent to 0.1 g of Dapsone with 10ml of methanol R, filter, and
use the clear filtrate. For solution (D) dilute 1 ml of solution C to 100 ml with
methanol R, and for solution (E) dilute 1 ml of solution D to 5 ml with methanol
R. After removing the plate from the chromatographic chamber, allow it to dry
in air and spray first with sodium nitrite/hydrochloric acid TS and then, while
still damp, with Al-(l-naphthyl)ethylenediamine hydrochloride (lg/1) TS, and
examine the chromatogram in daylight.
intense than that obtained with solution D, and not more than two such spots
are more intense than that obtained with solution E.
Assay. Weigh and powder 20 tablets. Dissolve a quantity of the powder equiv-
alent to about 0.25g of Dapsone, accurately weighed, in a mixture of 15ml
of water and 15ml of hydrochloric acid (-70g/l) TS. Carry out the assay as
described under 2.7 Nitrite titration, titrating with sodium nitrite (0.1 mol/1) VS.

C12FI12N2O2S.
DEXAMETHASONI COMPRESSI
DEXAMETHASONE TABLETS

tial medicines: 500 pg, 4 mg.
Requirements
Dexamethasone tablets contain not less than 90 0 . % and not more than
110 0 % of the amount of C 22 H 29 FO 5
Identity tests
1031
To a quantity of the powdered tablets equivalent to about 20 mg of Dexa-

methasone add 50ml of chloroform R and shake for 30 minutes. Filter, evapo-
rate the filtrate to dryness, and dry the residue at 105 °C for 2 hours. Use the
residue for the following tests.
concordant with the spectrum obtained from dexamethasone RS or with
the reference siaectrum of dexamethasone.

using kieselguhr R1 as the coating substance and a mixture of 9 volumes of
acetone R and 1 volume of formamide R to impregnate the plate, dipping
it about 5 mm into the liquid. After the solvent has reached a height of at
least 16cm, remove the plate from the chromatographic chamber and allow
it to stand at room temperature until the solvent has completely evaporated.
Use the impregnated plate within 2 hours, carrying out the chromatogra-
phy in the same direction as the impregnation. Use chloroform R as the
mixture of 9 volumes of chloroform R and 1 volume of methanol R con-
taining (A) 2.5mg of the residue per ml, (B) 2.5mg of dexamethasone RS
per ml, and (C) a mixture of equal volumes of solutions A and B. Develop
the plate for a distance of 15 cm. After removing the plate from the chro-
matographic chamber, allow it to dry in air until the solvents have evapo-
rated, heat at 120°C for 15 minutes, and spray the hot plate with sulfuric
acid/ethanol TS. Heat at 120 °C for a further 10 minutes, allow to cool, and
examine the chromatogram in daylight and in ultraviolet light (365 nm).

ance, and intensity with that obtained with solution B. A single spot is
C. Transfer a solution containing about 0.2 mg of the residue in 2.0 ml of

ethanol (~750g/l) TS to a stoppered test-tube, add 10ml of phenylhy-
drazine/sulfuric acid TS, mix, heat in a water-bath at 60 °C for 20 minutes,
and cool immediately. The absorbance of a 1-cm layer at the maximum at
about 423 nm is not less than 0.40.
D. Transfer about 10 mg to a porcelain crucible, add 45 mg of magnesium oxide

R, and ignite until an almost white residue is obtained. Allow to cool, add
2.0 ml 0.05ml of phenolphthalein/ethanol TS, and 1.0 ml of
of water,
hydrochloric acid (~70g/l) TS. Filter, to the filtrate add a freshly prepared
mixture of 0.10ml of sodium alzarinsulfonate (lg/1) TS and 0.10ml of

zirconyl nitrate TS, mix, and allow to stand for 5 minutes. Repeat the test
without the substance being examined. A yellow colour is produced in the
solution of the substance being examined and the reagent blank turns red.
1032

to about 4.0 mg of Dexamethasone, add 15ml of water, and shake with four
quantities, each of 25 ml, of chloroform R. Filter the chloroform layers through
cotton wool, previously washed with chloroform R, and add sufficient chloro-
form R to produce 200ml. Transfer 10ml of the resulting solution to a glass-
stoppered 50-ml conical flask, carefully evaporate to dryness, and dissolve the
residue in 20ml of dehydrated ethanol R. Add 2.0ml of blue tetrazolium/
ethanol TS and displace the air in the flask with oxygen-free nitrogen R. Imme-
10ml of aldehyde-free ethanol (-750 g/1) TS in a similar manner.
Calculate the percentage content of C22H29FO5 by comparison with dexa-

methasone RS, similarly and concurrently examined.
To 1 tablet add 15 ml of water and shake until the tablet is completely disinte-
grated. Extract with four quantities of 1 ml of chloroform R, filter the chloro-
form layers through cotton wool, previously washed with chloroform R, and
add sufficient chloroform R to produce 50 ml. Transfer a volume of this solu-
tion, equivalent to about 200 |ig of Dexamethasone, to a glass-stoppered 50-ml
conical flask, carefully evaporate to dryness, and dissolve the residue in 20ml
of dehydrated ethanol R. Add 2.0 ml of blue tetrazolium/ethanol TS and dis-
place the air in the flask with oxygen-free nitrogen R. Immediately add 2.0ml
of tetramethylammonium hydroxide/ethanol TS and again displace the air with
oxygen-free nitrogen R. Stopper the flask, mix the contents by gende swirling,
and allow to stand for 1 hour in a water-bath at 30 °C. Cool rapidly, add suffi-
cient aldehyde-free ethanol (-750 g/1) TS to produce 25 ml, and mix. Measure
the absorbance of a 1-cm layer at the maximum at about 525 nm against a
solvent cell containing a solution prepared by treating 10 ml of aldehyde-free
ethanol (-750 g/1) TS in a similar manner.
Calculate the tablet content of C22H29FO5 in mg by comparison with dexam-

ethasone RS, similarly and concurrently examined. The tablets comply with the
test for 5.1 Uniformity of content for single-dose preparations.
1033
DIETHYLCARBAMAZINI
DIHYDROGENOCITRATIS COMPRESSI
DIETHYLCARBAMAZINE DIHYDROGEN
CITRATE TABLETS
Categoiy. Antifilarial drug.
Storage. Diethylcarbamazine dihydrogen citrate tablets should be kept in a

tightly closed container.

Requirements
Diethylcarbamazine dihydrogen citrate tablets contain not less than 93 0 . % and

not more than 107 0 %
of the amount of CioH 2 iN 30 ,C 6 Hs 07 stated on the
. label.
Identity tests
A. To a quantity of the powdered tablets equivalent to 0.15g of Diethylcarba-

mazine dihydrogen citrate add 15 ml of ethanol (-750 g/1) TS, shake for
5 minutes, filter, and evaporate the filtrate to dryness. To the residue add
10ml of sodium hydroxide (-80 g/1) TS and extract with three 10-ml quan-
tities of chloroform R. Dry the combined extracts over anhydrous sodium
sulfate R, filter, evaporate the filtrates, and carry out the examination as
diethylcarbamazine dihydrogen citrate RS similarly treated or with the ref-
erence spectrum of diethylcarbamazine.
B. To a quantity of thepowdered tablets equivalent to 0.2 g of Diethylcarba-

mazine dihydrogen add 10 ml of water, shake, and filter. Transfer the
citrate
filtrate to add 1 ml of sodium hydroxide (-400 g/1) TS,
a separatory funnel,
and extract with 20 ml, 15 ml, and 10 ml of chloroform R. Keep the aqueous
layer for test C. Evaporate the combined chloroform extracts on a water-
bath and, towards the end, by drying with the aid of a current of air. Dis-
solve the oily residue in 10 ml of ethyl acetate R, warming the mixture to
50 °C, and pour it into 2 ml of a solution containing 1 g of maleic acid R in
10ml of acetone R, warming again to 50°C. Cool, rub the sides of the tube
with a glass rod to induce crystallization, collect the white precipitate on a
1034
sintered-glass filter, wash twice with 1 ml of acetone R and once with 5 ml

of ethyl acetate R, and dry in a desiccator; melting point, 126-128 °C.
C. Filter the aqueous layer from test B, Add

drop of phenolphthalein/ethanol
1
TS and neutralize with sulfuric acid (~100g/l) TS. Then add 2 ml of mer-
curic sulfate TS, heat to boiling and add, drop by drop, potassium perman-
ganate (lOg/1) TS; the violet colour is discharged and a white precipitate is
produced.
iV-Methylpiperazine. Carry out the test as described under 1.14.1 Thin-layer

volumes of ethanol (-750 g/1) TS, 3 volumes of glacial acetic acid R, and 1 volume
of water as the mobile phase. Apply separately to the plate 5|il of each of the
following two solutions. For solution (A) shake a quantity of the powdered
tablets equivalent to 0.5g of Diethylcarbamazine dihydrogen citrate with 10ml
of methanol R, filter, and use the clear filtrate. For solution (B) dissolve 5 mg of
N-methylpiperazine R in 100ml of methanol R. After removing the plate from
the chromatographic chamber, allow it to dry in air, spray with a mixture of 3
volumes of platinic chloride (60 g/1) TS, 97 volumes of water, and 100 volumes
of potassium iodide (60 g/1) TS, and examine the chromatogram in daylight.
The spot obtained with solution B is more intense than any spot, correspond-
ing in position and appearance, obtained with solution A.
alent to about 0.75g of Diethylcarbamazine dihydrogen citrate, accurately
weighed, to a separatory funnel, add 10ml of water and 10ml of sodium
hydroxide (-200 g/1) TS, and shake to dissolve. Extract with four 25-ml quan-
tities of chloroform R, washing each extract with the same two 20-ml quanti-
ties of water, and with a third quantity if the second is alkaline to
phenolphthalein/ethanol TS. Extract the combined chloroform extracts with

25ml of sulfuric acid (0.05mol/l) VS and then with 15ml and 10ml of water.
Combine the acid and water extracts, warm to remove the chloroform, cool,
and back-titrate the excess of acid with sodium hydroxide (0.1 mol/1) VS using
bromocresol green/ethanol TS as indicator.
Each ml of sulfuric acid (0.05mol/l) VS is equivalent to 39.14mg of

CloFl2lhl30,C6El807.
1035
DILOXANIDI FUROATIS COMPRESSI

DILOXANIDE EURO ATE TABLETS

Requirements
Diloxanide furoate tablets contain not less than 90 0 . % and not more than
110 0 %
. of the amount of C] 4 HnCl 2 N 04 stated on the label.
Identity tests
To a quantity of the powdered tablets equivalent to about 0.2 g of Diloxanide
furoate add 20 ml of dichlorome thane R and shake. Filter, evaporate the filtrate
to dryness, and use the dried residue for the following tests.
concordant with the spectrum obtained from diloxanide furoate RS or with
the reference spectrum of diloxanide furoate.
B. Melting temperature of the residue, about 115°C.
96 volumes of dichloromethane R and 4 volumes of methanol R as the mobile
phase. Apply separately to the plate 5 pi of each of the following 2 solutions.
For solution (A) shake a quantity of the powdered tablets equivalent to about
0.5 g of Diloxanide furoate with 5 ml of chloroform R, centrifuge, and use the
supernatant liquid. For solution (B) dilute 1 volume of solution A to 20 volumes
of chloroform R, further dilute 1 volume of this solution to 20 volumes with
the same solvent. After removing the plate from the chromatographic chamber,
(254 nm).

to about 0.04g of Diloxanide furoate add 150ml of ethanol (-750 g/1) TS and
1036
shake for 30 minutes. Add sufficient ethanol (~750g/l) TS to produce 200 ml,
mix, and filter. Dilute 10 ml of the filtrate to 250 ml with the same solvent.
against a solvent cell containing ethanol (-750 g/1) TS.
Calculate the percentage content of C14H11CI2NO4 using the absorptivity value

of 70.5 (ASL = 705).
DOXYCYCLINI HYCLATIS COMPRESSI

DOXYCYCLINE HYCLATE TABLETS
Storage. Doxycycline hyclate tablets should be kept in a tightly closed

container.
Additional information. Strength in the current WHO Model list of

essential medicines: 100 mg of doxycycline.
Requirements
Doxycycline hyclate tablets contain not less than 90 0 . % and not more than
110 0 % of
. the amount of C22H24N2O8 stated on the label, if Assay method A
is applied.
Identity tests

using silica gel R3 as the coating substance. Adjust the pH of a solution of
0.1 g of disodium edetate R per ml to 9.0 with sodium hydroxide (-400 g/1)
TS, and spray this evenly onto the plate. Allow the plate to dry in a hori-
zontal position for not less than1 hour. Just before use, dry the plate in an
oven at 110°C for 1 hour. Use a mixture of 59 volumes of dichlorome thane

R,35 volumes of methanol R, and 6 volumes of water as the mobile phase.
Apply separately to the plate 1 pi of each of the following 3 solutions. For
solution (A) shake a quantity of the powdered tablets equivalent to about
5 mg of Doxycycline hyclate with 5 ml of methanol R, filter, dilute the fil-
trate to 10 ml with the same solvent, and use the resulting solution. For solu-
tion (B) dissolve 5 mg of doxycycline hyclate RS in methanol R and dilute
1037
to 10ml with the same solvent. For solution (C) dissolve 5mg of doxycy-
cline hyclate RS and 5 mg of tetracycline hydrochloride RS in methanol R,
and dilute to 10 ml with the same solvent. After removing the plate from
the chromatographic chamber, allow it to dry in a current of air, and

if the chromatogram obtained with solution C shows two clearly separated
spots.
B. To a quantity of the powdered tablets equivalent to about 5 mg of Doxy-

cycline hyclate add about 2ml of sulfuric acid (~1760g/l) TS; an intense
yellow colour is produced.
To a quantity of the powdered tablets equivalent to about 0.1 g of

Doxycycline hyclate add 10 ml of water, filter, and use the filtrate for the
following tests.
C. To 2.0 ml of the filtrate add 1 drop of ferric chloride (25g/l) TS; a dark red-
D. To 1.0 ml of the filtrate add 5 drops of silver nitrate (40g/l) TS; a white, curdy
precipitate is formed which dissolves in 1.0 ml of ammonia (~100g/l) TS.
Light-absorbing impurities. To a quantity of the powdered tablets equiva-

lent to about O.lOg of Doxycycline hyclate add 10ml of a mixture of 1 volume
of hydrochloric acid (1 mol/1) VS and 99 volumes of methanol R, shake, and
filter, discarding the first 2 ml of filtrate. Measure the absorbance of a 1-cm layer
at 490 nm; the absorbance does not exceed 0.2.
Assay
A. Weigh and powder 20 Carry out the test as described under 1.14.4
tablets.
High performance liquid chromatography, using a stainless steel column
(25 cm X 4.6mm) packed with particles of styrene-divinylbenzene copolymer
(8-10 |im). As the mobile phase, use a solution prepared as follows: trans-
fer 60.0 g of rerr-butanol R with the aid of 200 ml of water to a 1000-ml vol-
umetric flask. Add 400ml of buffer borate, pH 8.0, TS, 50ml of a solution
of 10mg of tetrabutylammonium hydrogen sulfate R per ml adjusted to pH
8.0with sodium hydroxide (~80g/l) TS, and 20 ml of sodium edetate (20g/l)
TS adjusted to pH 8.0 with sodium hydroxide (~80g/l) TS. Dilute to
1000 ml with water.
1038
Prepare the following solutions in hydrochloric acid (0.01 mol/1) VS: for solu-
tion (A) use a quantity of the powdered tablets sufficient to give 0.80 mg of
Doxycycline hyclate per ml; solution (B) 0.80 mg of doxycycline hyclate RS
per ml; solution (C) 0.80 mg of 6-epidoxycycline hydrochloride RS per ml;
solution (D) 0.80 mg RS per ml; for solution
of metacycline hydrochloride
(E) mix 4.0ml of solution B with 1.5ml of solution C and 1.0 ml of solution
D, and dilute to 25 ml with hydrochloric acid (0.01 mol/1) VS; and for solu-
tion (F) mix 2.0ml of solution C and 2.0ml of solution D and dilute to
100 ml with hydrochloric acid (0.01 mol/1) VS.
Operate with a flow rate of about 0.9ml per minute. As a detector use an
ultraviolet spectrophotometer set at a wavelength of about 254 nm.
Inject 20 pi of solution E. The test is not valid unless the resolution between
the firstpeak (metacycline) and the second peak (6-epidoxycycline) is not
lessthan 1.25, and the resolution between the second peak and the third
peak (doxycycline) is not less than 2.0. If necessary, adjust the Krt-butanol
R content in the mobile phase. The test is not valid unless the symmetry
factor for the third peak is not more than 1.25. If necessary adjust the inte-
grator parameters.

from solutions A and B, and calculate the percentage content of C22H24N2O8
in the tablets, taking into account the declared content of C22H24N2O8 in
doxycycline hyclate RS.

about 50 mg, accurately weighed, add 50ml of dimethylformamide R and
shake for 1 hour. Centrifuge, and carry out the assay with the supernatant
liquid as described under 3.1 Microbiological assay of antibiotics, using
Bacillus cereus (NCTC 10320 or ATCC 11778) as the test organism, culture
medium CmlO with a final pH of 6.6, potassium dihydrogen phosphate
(13.6g/l) TS as the buffer, an appropriate concentration of Doxycycline
(usually between 0.2 and 2.0 lU per ml), and an incubation temperature of
35-39 °C. The precision of the assay is such that the fiducial limits of error
of the estimated potency {P = 0.95) are not less than 95% and not more than
105%. The upper fiducial limit of error is not less than 97.0% and the lower
fiducial limit of error is not more than 110.0% of the content stated on the
label expressed in mg, with 870 lU being equivalent to 1 mg of doxycycline.
1039
EPHEDRINI SULFATIS INJECTIO

EPHEDRINE SULFATE INJECTION
Description. A clear, colourless solution.
Category. Anti-asthmatic drug.
Storage. Ephedrine sulfate injection should be protected from light.

tial medicines: 50 mg/ml.
Requirements
Definition. Ephedrine sulfate injection is a sterile solution of ephedrine sulfate

in water for injections.
The solution is sterilized by a suitable method (see 5.8 Methods of

sterilization).
Ephedrine sulfate injection contains not less than 95 0 . % and not more than
105 0 % of the amount of (CioHi 5 NO) 2 ,H 2 S 04
Identity tests
To a volume of the injection equivalent to 0.1 g of Ephedrine sulfate add 5ml

of ethanol (-750 g/1) TS and evaporate to dryness on a water-bath with the aid
of a stream of air. Use the residue for tests A and C.
concordant with the spectrum obtained from ephedrine sulfate RS similarly
treated or with the reference spectrum of ephedrine sulfate.
B. Measure the optical rotation of the injection; it is levorotatory.
C. Dissolve lOmg of the residue in 1ml of water and add 0.1ml of copper(ll)
sulfate (80 g/1) TS, followed by 2 ml of sodium hydroxide (-80 g/1) TS; a
violet colour is produced. To this solution add 2 ml of 1 -butanol R and shake;
a reddish violet colour is produced in the butanol layer.
1040
D. The injection yields reaction A described under 2.1 General identification

pH value. pH of the injection, 4. 5-7.0.
of 80 volumes of 2-propanol R, 15 volumes of ammonia (-260 g/1) TS, and
5 volumes of chloroform R as the mobile phase. Apply separately to the plate
10 pi of each of the following two solutions. For solution (A) dilute a volume
of the injection equivalent to 0.1 g of Ephedrine sulfate to 5 ml with methanol
R, and for solution (B) dilute 0.5ml of solution A to 100 ml with methanol R.
air, spray with a mixture of 0.2 g of trike tohydrindene hydrate R dissolved in
95ml of 1-butanol R and 5ml of acetic acid (~120g/l) TS, and heat to 110°C
for 5 minutes. Examine the chromatogram in daylight.
Any secondary spot obtained with solution A is not more intense than that
obtained with solution B. Disregard any spot of lighter colour than the
background.
Assay. Transfer an accurately measured volume of the injection equivalent to

about 0.25 g of Ephedrine sulfate to a separator, dilute, if necessary, with water
to a volume of about 10 ml, add 3g of sodium chloride R to saturate the solu-
tion, then add 5 ml of sodium hydroxide (1 mol/1) VS, and extract four times,
each with 25ml of chloroform R. Wash the combined chloroform extracts with
10 ml of a saturated solution of sodium chloride R, and filter through purified
cotton saturated with chloroform R. Shake the aqueous wash solution with
10ml of chloroform R and add it to the main chloroform extract. Add 0.25ml
of methyl red/ethanol TS and titrate with perchloric acid/dioxan (0.1 mol/1) VS,
as described under 2.6 Non-aqueous titration. Method A.
Each ml of perchloric acid/dioxan (0.1 mol/1) VS is equivalent to 21.43 mg of

(C.„Hi5NO)2,H,SO,.
ERGOMETRINI HYDROGENOMALEATIS
INJECTIO
INJECTION
Description. A colourless to faintly yellow solution.
Category. Oxytocic.
1041
Storage. Ergometrine hydrogen maleate injection should be kept in a single-

dose container, protected from light, and stored in a cool place.
Labelling. Expiry date.

tial medicines: 200pg/ml.
Requirements
Definition. Ergometrine hydrogen maleate injection is a sterile solution of

ergometrine hydrogen maleate in water for injections.
Ergometrine hydrogen maleate may contain a suitable stabilizer. The solution

is by “Heating in an autoclave” or by another
sterilized suitable method (see
5.8 Methods of sterilization).
Ergometrine hydrogen maleate injection contains not less than 90 0 . % and not
more than 110 0 % of . the amount of Ci 9 H 23N 302 ,C H 404
4 stated on the label.
Identity tests
B. To a volume of the injection equivalent to 0.1 mg of Ergometrine hydrogen

maleate, add 0.5 ml of water and 2 ml of 4-dimethylaminobenzaldehyde
TSl; a blue colour is slowly produced.
pH value. pH of the injection, 2. 7-3. 5.
Related substances. Carry out the test protected from light as described
under 1.14.1 Thin-layer chromatography, using a suspension of silica gel R1 in
sodium hydroxide (0.1 mol/1) VS as the coating substance and a mixture of 9
volumes of chloroform R and 1 volume of methanol R as the mobile phase.
Apply separately to the plate 5 pi of each of the following five solutions. For
solution (A) evaporate a volume of the injection equivalent to 1 mg of
Ergometrine hydrogen maleate to dryness at 20 °C under reduced pressure (not
exceeding O.dkPa or 5mm of mercury) and dissolve the residue in 0.25ml of
methanol R. Prepare solutions (B), (C), (D), and (E) in methanol R containing
0.1 mg/ml, 0.2mg/ml, 0.4mg/ml, and 4mg/ml, respectively, of ergometrine
hydrogen maleate RS. After removing the plate from the chromatographic
1042
light (365 nm) and in daylight after spraying with 4-dimethylaminobenzalde-

hyde TS2.
Assess the intensity of every spot, other than the principal spot, obtained with
solution A by reference to the spots obtained with solutions B, C, and D; the
total of intensities so assessed does not exceed 10% of the intensity of the prin-
cipal spot. In addition, no single spot, other than the principal spot, obtained
with solution A is more intense than that obtained with solution B.
Assay
The solutions must be protected from light throughout the assay.
Dilute an accurately measured volume of the

injection with sufficient water to
produce a concentration of about 0.04 mg of Ergometrine hydrogen maleate per
ml.To 3 ml add 6ml of 4-dimethylaminobenzaldehyde TSl, mix, cool to room
temperature, and allow to stand for 30 minutes.

against a solvent cell containing a reagent blank. Calculate the amount in mg
of C] 9 H 23N 302 ,C 4 H 404 in the sample being examined by comparison with
ergometrine hydrogen maleate RS.
ERGOMETRINI HYDROGENOMALEATIS
COMPRESSI
TABLETS
Category. Oxytocic.
Storage. Ergometrine hydrogen maleate tablets should be kept in a tightly

closed container.

tial medicines: 200 pg.
Requirements
1043
Ergometrine hydrogen maleate tablets contain not less than 90 0 . % and not
more than 110 0 % of. the amount of Ci 9 H 23N 302 ,C H 404
Identity tests
B. Extract a quantity of thepowdered tablets equivalent to 2 mg of Ergometrine

hydrogen maleate with 20 ml of water, filter, and wash the residue with suf-
ficient water to produce 20 ml; the solution has a blue fluorescence. To 2 ml
add 4 ml of 4-dimethylaminobenzaldehyde TSl; a blue colour is slowly
produced.
Related substances. Carry out the test protected from direct light as
described under 1.14.1 Thin-layer chromatography, using a suspension of silica
gel R1 in sodium hydroxide (0.1 mol/1) VS as the coating substance and a

mixture of 9 volumes of chloroform R and 1 volume of methanol R as the
mobile phase. Apply separately to the plate 5 pi of each of the following five
solutions. Eor solution (A) triturate a quantity of the powdered tablets equiva-
lent to 1 mg hydrogen maleate with 0.2 ml of domiphen
of Ergometrine
bromide add 2 ml of methanol R, centrifuge, and remove the super-
(lOg/1) TS,
natant liquid. Extract the residue with two further quantities, each of 1 ml, of
methanol R. Evaporate the combined extracts to dryness at 20 °C under reduced
pressure (O.fikPa or 5mm of mercury), and dissolve the residue in 0.25 ml of
methanol R; centrifuge if necessary. Solutions (B), (C), (D), and (E) are solutions
in methanol R containing 0.1 mg per ml, 0.2 mg per ml, 0.4mg per ml, and
4 mg per ml, respectively, of ergometrine hydrogen maleate RS. After remov-
Assess the intensity of each spot, other than the principal spot, obtained with
solution A by reference to the spots obtained with solutions B, C, and D; the
total of the intensities so assessed does not exceed 10% of the intensity of the
principal spot In addition, no single spot, other than the principal spot, obtained
with solution A is more intense than the spot obtained with solution B.
alent to about 2 mg of Ergometrine hydrogen maleate, accurately weighed, with
50ml of tartaric acid (lOg/1) TS for 30 minutes, centrifuge, and use the super-
natant liquid. Dilute a suitable volume to produce a solution containing
0.040mg per ml of ergometrine hydrogen maleate. To 3ml add 6ml of 4-
dimethylaminobenzaldehyde TSl, mix, cool to room temperature, and allow
to stand for 30 minutes.
1044
Measure the absorbance of a 1-cm layer at the maximumabout 545 nm

at
against a solvent cell containing a reagent blank composed of 6ml of 4-dimethy-
laminobenzaldehyde TSl and 3 ml of water. Calculate the amount in mg of
Ci 9 H 23 N 302 ,C 4 H 404 in the sample being examined by comparison with a solu-
tion containing 0.04 mg/ml of ergometrine hydrogen maleate RS.

arate stoppered test-tubes, add 10ml of tartaric acid (lOg/1) TS, shake for 30
minutes, and centrifuge. Dilute a suitable volume to produce a solution con-
taining 0.040mg per ml of ergometrine hydrogen maleate. To 3ml of a clear
solution add 6ml of 4 dimethylaminobenzaldehyde TSl, mix, cool to room
temperature, and allow to stand for 30 minutes.

against a solvent cell containing a reagent blank composed of 6ml of 4-dimethy-
laminobenzaldehyde TSl and 3 ml of water. Calculate the amount in mg of
C] 9 H 23 N 302 ,C 4 H 404 in the sample being examined by comparison with a solu-
tion containing 0.04 mg/ml of ergometrine hydrogen maleate RS.
preparations.
ERYTHROMYCINI ETHYLSUCCINATIS
COMPRESSI
ERYTHROMYCIN ETHYLSUCCINATE TABLETS
Storage. Erythromycin ethylsuccinate tablets should be kept in a tightly closed

container.

tial medicines: 250
mg of erythromycin.
Requirements
Identity tests
1045
To a quantity of the powdered tablets equivalent to about 0.25 g of Ery-

thromycin ethylsuccinate add 20 ml of chloroform R and shake. Filter, evapo-
and use the dried residue for tests A, C, and D.
rate the filtrate to dryness,
concordant with the spectrum obtained from erythromycin ethylsuccinate
RS or with the reference spectrum of erythromycin ethylsuccinate.

methanol R and 15 volumes of chloroform R as the mobile phase. Apply
separately to the plate 10 pi of each of the following 2 solutions. For solu-
tion (A) shake a quantity of the powdered tablets equivalent to about 30 mg
of Erythromycin ethylsuccinate with 10ml of methanol R by mechanical
means for 30 minutes. Centrifuge a portion of this mixture and use the clear
supernatant liquid. For solution (B) use 3 mg of erythromycin ethylsuccinate
RS per ml of methanol R. After removing the plate from the chromato-
graphic chamber, allow it to dry in a current of air, and spray with a mixture
of 90 volumes of dehydrated ethanol R, 5 volumes of anisaldehyde R, and
5 volumes of sulfuric acid (-1760 g/1) TS. Heat the plate at 100°C for 10
minutes and examine the chromatogram in daylight.

C. To 5 mg of the residue add about 2 ml of sulfuric acid (~1760g/l) TS and

shake gently; a reddish brown colour is produced.
D. Dissolve about 3 mg of the residue in 2.0ml of acetone R and add about

2 ml of hydrochloric acid (-420 g/1) TS; an orange colour is produced, which
changes to orange-red and finally to violet-red. Add 2.0 ml of chloroform R
and shake; the chloroform layer turns to blue.

to about 0.1 g (lOOOOOIU) of erythromycin, accurately weighed, add sufficient
methanol R to produce lOOml, shake, and allow the sediment to setde. Care-
fully transfer 40ml of the clear solution to a 100- ml volumetric flask, dilute to
volume with sterile phosphate buffer, pH 8.0, TSl or TS2, and allow to stand,
protected from light, for 5 hours at 20-25 °C. Carry out the assay as described
under 3.1 Microbiological assay of antibiotics, using Bacillus puniilus (NCTC
pH of 8. 0-8.1, sterile phosphate buffer, pH 8.0, TSl orTS2, an appropriate con-
centration of Erythromycin (usually between 5 and 15 lU per ml), and an incu-
bation temperature of 35-39 °C. The precision of the assay is such that the
fiducial limits of error of the estimated potency (P = 0.95) are not less than 95%
1046
and not more than 105%. The upper fiducial limit of error is not less than 95.0%
and the lower fiducial limit of error is not more than 110.0% of the content
stated on the label, expressed in mg, with lOOOIU being equivalent to 1 mg of
erythromycin.
ERYTHROMYCINI STEARATIS COMPRESSI

ERYTHROMYCIN STEARATE TABLETS
Storage. Erythromycin stearate tablets should be kept in a tightly closed

container.

tial medicines: 250 mg of erythromycin.
Requirements
Identity tests
A. To a quantity of the powdered tablets equivalent to about 0.2 g of Ery-

thromycin stearate add 20 ml of water and shake. Decant the supernatant
liquid and discard. Add 10 ml of methanol R to the residue, shake, filter, and
evaporate to dryness. Carry out the examination with the dried residue as
erythromycin stearate RS or with the reference spectrum of erythromycin
stearate.

methanol R and 15 volumes of chloroform R as the mobile phase. Apply
separately to the plate 20 pi of each of the following 2 solutions. For solu-
tion (A) shake a quantity of the powdered tablets equivalent to about
0.05 g of Erythromycin stearate with 10 ml of methanol R by mechanical
means for 30 minutes. Centrifuge a portion of this mixture and use the clear
1047
supernatant liquid. For solution (B) use 5 mg of erythromycin stearate RS

per ml of methanol R. After removing the platefrom the chromatographic
chamber, allow it to dry in a current of air, and spray with dichlorofluores-
cein TS. Heat the plate at 100 °C for 10 minutes and examine the chro-

Next, spray the plate with a mixture of 90 volumes of dehydrated ethanol

R, 5 volumes of anisaldehyde R, and 5 volumes of sulfuric acid (-1760 g/1)
TS. Heat the plate at 100 °C for 10 minutes and examine the chromatogram
in daylight.

C. To a quantity of the powdered tablets equivalent to about 10 mg of Ery-

thromycin stearate add 2.0 ml of acetone R and about 2 ml of hydrochloric
acid (-420 g/1)TS and shake; a pale orange colour is produced, which
changes to red or violet-red. Add 2.0ml of chloroform R and shake; the
chloroform layer acquires a violet colour.
D. Shake a quantity of the powdered tablets equivalent to about 0.1 g of

Erythromycin stearate with 10 ml of chloroform R, filter, and evaporate the
filtrate to dryness on a water-bath. Gently heat the residue with 10 ml of
water and 5 ml of hydrochloric acid (-70 g/1) TS until the solution boils; oily
globules rise to the surface. Cool, remove the fatty layer, and heat it with
3.0 ml of sodium hydroxide (0.1 mol/1) VS. Allow to cool; the solution sets
to a gel. Add 10ml of hot water, shake, heat the mixture for 2-3 minutes
and shake again; the solution froths. To 1.0ml of the resulting solution add
2.0 ml of calcium chloride (55 g/1) TS; a granular precipitate is produced,
which is insoluble in hydrochloric acid (-250 g/1) TS.

to about 0.1 g (lOOOOOIU) of erythromycin, accurately weighed, add sufficient
methanol R to produce 100ml, shake, and allow the sediment to setde. Care-
fully transfer 40ml of the clear solution to a 100- ml volumetric flask, dilute to
volume with sterile phosphate buffer, pH 8.0, TSl or TS2, and allow to stand,
protected from light, for 5 hours at 20-25 °C. Carry out the assay as described
under 3.1 Microbiological assay of antibiotics, using Bacillus puniilus (NCTC
pH of 8. 0-8.1, sterile phosphate pH 8.0, TSl orTS2, an appropriate con-
buffer,
centration of Erythromycin (usually between 5 and 15 lU per ml), and an incu-
bation temperature of 35-39 °C. The precision of the assay is such that the
fiducial limits of error of the estimated potency (P = 0.95) are not than 95%
less
1048
and not more than 105%. The upper fiducial limit of error is not less than 95.0%
and the lower fiducial limit of error is not more than 110.0% of the content
stated on the label, expressed in mg, with lOOOIU being equivalent to 1 mg of
erythromycin.
ETHAMBUTOLI HYDROCHLORIDI COMPRESSI

ETHAMBUTOL HYDROCHLORIDE TABLETS

tial medicines: 100-400 mg.
Requirements
Ethambutol hydrochloride tablets contain not less than 90 0 % and not more .
than 110 0 % of the amount of CJ0H24N2O2, 2HC1 stated on the label.

.
Identity tests
To powdered tablets equivalent to about 0.1 g of Ethambutol

a quantity of the
hydrochloride add 10ml of methanol R and shake. Filter the extract and evap-
orate to dryness. Use the residue for tests A and C.
concordant with the spectrum obtained from ethambutol hydrochloride RS
or with the reference spectrum of ethambutol hydrochloride.

using silica gel R1 as the coating substance and a mixture of 100 volumes
of methanol R and 1.5 volumes of ammonia (-260 g/1) TS as the mobile

phase. Apply separately to the plate 10 pi of each of the following 2 solu-
tions. For solution (A) shake a quantity of the powdered tablets equivalent
to about 10 mg of Ethambutol hydrochloride with 10 ml of water, filter, and
use the filtrate. For solution (B) use 1.0 mg of ethambutol hydrochloride RS
1049
itto dry in air, and expose it to the vapour of iodine R until spots appear.

C. The residue yields reaction A described under 2.1 General identification tests
as characteristic of chlorides.
Aminobutanol. Carry out the test as described under 1.14.1 Thin-layer chro-
volumes of ethyl acetate R, 35 volumes of glacial acetic acid R, 5 volumes of
hydrochloric acid (-420 g/1) TS, and 1 volume of water as the mobile phase.
Apply separately to the plate 2 pi of each of the following 2 solutions. For solu-
tion (A) shake a quantity of the powdered tablets equivalent to about 0.5 g of
Ethambutol hydrochloride with 10 ml of methanol R for 5 minutes, filter, and
use the filtrate. For solution (B) use 0.5 mg of aminobutanol R per ml of
methanol R. After removing the plate from the chromatographic chamber,
allow it to dry in air, heat at 105°C for 5 minutes, cool, spray with triketohy-
drindene/cadmium TS, and heat again at 90 °C for 5 minutes. Examine the
chromatogram in daylight.
Any spot corresponding to aminobutanol obtained with solution A is not more


to about 0.2 g of Ethambutol hydrochloride add 10 ml of sodium hydroxide
(-80 g/1) TS and extract with 5 portions, each of 20 ml, of chloroform R.
Evaporate the combined extracts to a volume of about 25 ml, filter, and add
100ml of glacial acetic acid Rl. Add 1-naphtholbenzein/acetic acid TS and
titration. Method A.

C 10 H 24N 2 O 2 2HC1.
,
1050
GLYCERYLIS TRINITRATIS COMPRESSI

GLYCERYL TRINITRATE TABLETS
Other name. Nitroglycerin tablets.
Storage. Glyceryl trinitrate tablets should be kept in a tightly closed container,

preferably made of glass, with a screw closure lined with aluminium or tin foil,
protected from light, and stored at a temperature not exceeding 20 °C. To
prevent loss of potency, the tablets should be kept in the original container.
Each container should hold not more than 100 tablets. After each use the con-
tainer should be closed immediately.
Labelling. The designation on the container should state that the tablets are
for sublingual use. Expiry date.

tial medicines: 500 pg.
CAUTION: Undiluted glyceryl trinitrate can explode by percussion or excessive heat.

Avoid keeping isolated small amounts.
Requirements
Definition. Glyceryl trinitrate tablets are tablets for sublingual use.
Glyceryl trinitrate tablets contain not less than 80 0 % and not more than
.
120 .0% of the amount of C 3H N 3 O 9

Identity tests
A. Shake a quantity of the powdered tablets equivalent to 0.50 mg of Glyceryl

trinitrate with 5 ml of dehydrated ethanol R, filter, and evaporate the filtrate
to dryness using a stream of air. To the residue add 3-4 drops of sodium
hydroxide (~80g/l) TS and 3 ml of ferrous sulfate (15g/l) TS and shake; a
greenish brown precipitate is produced.
B. Extract a quantity of the powdered tablets equivalent to 5 mg of Glyceryl

trinitrate with 3 ml of ethanol (-750 g/1) TS and To the filtrate add care-
filter.
fully 1 ml of diphenylamine/sulfuric acid TS in amanner to form a lower

layer; a dark blue colour is produced at the interface of the two layers.
1051
Test for the absence of decomposition. Shake a quantity of the powdered

tablets equivalent to 0.50 mg of Glyceryl trinitrate with 5 ml of water and filter.
To the filtrate add 0.1 g of potassium iodide R, 2 drops of sulfuric acid
(-100 g/1) TS, and 1ml of starch TS; the liquid remains colourless. Add 1ml of
sodium hydroxide (-80 g/1) TS and heat gently to boiling. Cool and add 3 ml of
sulfuric acid (-100 g/1) TS; a dark blue colour is immediately produced.

to about 1 mg of Glyceryl trinitrate, accurately weighed, add 4.5ml of glacial
acetic acid R and 0.5ml of water, shake for 1 hour, and centrifuge. For the ref-
erence solution, dissolve 133.5 mg of potassium nitrate R, previously dried at
105 °C, in sufficient water to produce 50 ml. Transfer 10 ml to a volumetric flask
and add sufficient glacial acetic acid R to produce 100 ml. To 1 ml of each of the
clear sample and reference solutions add 2 ml of phenoldisulfonic acid TS, mix, and
allow to stand for 15 minutes. Then add 8 ml of water, mix well, allow to cool,
and add slowly with swirling 10ml of ammonia (-260 g/1) TS. Cool and dilute
to 20 ml with water.
Measure the absorbances of both solutions at the maximum at about 405 nm

against a solvent cell containing a reagent blank. Calculate the amount in mg
of C3H5N3O9 sample being examined by comparison with the reference
in the
solution. Each ml of the potassium nitrate reference solution is equivalent to
0.2000 mg of C3H5N3O9.
Disintegration test. Complies with the test for 5.3 Disintegration test for
tablets and capsules. Time period: 2 minutes.
Uniformity of content. For the sample solution, place 10 powdered tablets in

10 separate centrifuge tubes containing a few glass beads, add 4.5 ml of glacial
acetic acid R and 0.5ml of water, shake for 1 hour, and centrifuge.
For the reference solution, dissolve 133.5 mg of potassium nitrate R, previously

dried at 105 °C, in sufficient water to produce 50 ml. Transfer 10 ml to a volu-
metric flask and add sufficient glacial acetic acid R to produce 100 ml.
For tablets containing 0.4 to 0.6 mg of Glyceryl trinitrate, use 1ml of the clear
sample solution and 1 ml volumes of the reference solution
of a mixture of equal
and glacial acetic acid R. To both add 2 ml of phenoldisulfonic acid
solutions
TS, mix, and allow to stand for 15 minutes. Then add 8ml of water, mix well,
allow to cool, and add slowly with swirling 10 ml of ammonia (-260 g/1) TS.
Cool and dilute to 20ml with water.
For tablets containing 0.2 to 0.5 mg of Glyceryl trinitrate, use 2 ml of the clear
sample solution and 2 ml of a mixture of 3 volumes of glacial acetic acid R and

1 volume of the reference solution. To both solutions add 2 ml of phenoldisulfonic
acid TS, mix, and allow to stand for 15 minutes. Then add 8 ml of water, mix
1052
well, allow to cool,and add slowly with swirling 10 ml of ammonia (-260 g/1)
TS. Cool and dilute to 20 ml with water.
For tablets containing less than O.Zmg of Glyceryl trinitrate, use 2 ml of the clear
sample solution and 2 ml of a mixture of 7 volumes of glacial acetic acid R and
1 volume of the reference solution. To both solutions add 2 ml of phenoldisulfonic
acid TS, mix, and allow to stand for 15 minutes. Then add 8 ml of water, mix
well, allow to cool, and add slowly with swirling 10 ml of ammonia (-260 g/1)
TS. Cool and dilute to 20ml with water.
Measure the absorbances in the sample and reference solutions of all three con-
centrations at the maximum at about 405 nm against a solvent cell containing
a reagent blank. Calculate the amount in mg of C3H5N3O9 in the sample being
examined by comparison with the reference solution. Each ml of the potassium
nitrate reference solution is equivalent to 0.2000 mg of C3H5N3O9.
preparations.
GRISEOFULVINI COMPRESSI
GRISEOFULVIN TABLETS

tial medicines: 125mg, 250mg.
Requirements
Griseofulvin tablets contain not less than 95 0 . % and not more than 105 0 % .
of the amount of Ci/Hi/ClOg stated on the label.
Identity tests
A. Shake a quantity of the powdered tablets equivalent to 0.125g of Criseo-

fulvin with 20 ml of chloroform R and 1 g of anhydrous sodium sulfate R,
and filter. Evaporate the filtrate to dryness and dry under reduced pressure
1053
(not exceeding 0.7 kPa) for 1 hour. Carry out the examination with the
from griseofulvin RS or with the reference spectrum of griseofulvin.

ethylmethylketone R and 1 volume of xylene R as the mobile phase. Apply
separately to the plate 10 pi of each of the following two solutions. For solu-
tion (A) shake a quantity of the powdered tablets equivalent to 5 mg of
Griseofulvin with 10ml of chloroform R, filter, and use the clear filtrate. For
solution (B) dissolve 5 mg of griseofulvin RS in 10 ml of chloroform R. After
air, and examine the chromatogram in ultraviolet light (254 nm).

C. To a quantity of the powdered tablets equivalent to 5 mg of Griseofulvin

add 1 mlof sulfuric acid (-1760 g/1) TS; a yellow-orange colour is produced.
Add 1 drop of potassium dichromate (100 g/1) TS; the colour of the solution
changes to wine-red.
Loss on drying. Dry a quantity of the powdered tablets equivalent to 0.1 g of

Griseofulvin at 60 °C under reduced pressure (not exceeding 0.6kPa or 5mm of
mercury) for 3 hours; it loses not more than 50mg/g.

to about 0.08 g of Griseofulvin, accurately weighed, add 150 ml of ethyl acetate
R and boil under a reflux condenser for 15 minutes. Gool, add sufficient dehy-
drated ethanol R to produce 200ml, shake, and centrifuge. Dilute 2.0ml of the
supernatant liquid to 100ml with dehydrated ethanol R.
Measure the absorbance of this solution in a 1-cm layer at the maximum of

about 291 nm and calculate the content of CuHuClOg using the absorptivity
value of 68.6 (Ailm = 686).
Dissolution. Garry out the test as described under 5.5 Dissolution test for
1054
IBUPROFENI COMPRESSI
IBUPROFEN TABLETS
Category. Non-steroidal anti-inflammatoiy drug.

Requirements
Ibuprofen tablets contain not less than 90 0 . % and not more than 110 0 % of .
the amount of CisHigOj stated on the label.
Identity tests
To a quantity of the powdered about 0.8 g of Ibuprofen

add 20 ml of acetone R, filter, and allow the filtrate to evaporate without
heating. To the residue add 10 ml of acetone R, allow to crystallize, separate
the crystals, dry in air, and use the dried crystals for the following tests.
A. Carry out the examination with the dried crystals as described under 1.7
isconcordant with the spectrum obtained from ibuprofen RS or with the
reference spectrum of ibuprofen.
B. Dissolve 25 mg of the dried crystals in sufficient sodium hydroxide

(0.1 mol/1) VS to produce 100ml. The absorption spectrum of the resulting
solution, when observed between 230 nm and 350 nm, exhibits maxima
at about 265 nm and 273 nm, minima at about 245 nm and 271 nm, and a
shoulder at about 259 nm.
C. Melting temperature of the dried crystals, about 76 °C.
15 volumes of hexane R, 5 volumes of ethyl acetate R, and 1 volume of glacial
acetic acid R as the mobile phase. Apply separately to the plate 5 pi of each of
the following 3 solutions. For solution (A) shake a quantity of the powdered
tablets equivalent to about 0.2 g of Ibuprofen with three 10- ml quantities of
chloroform R, filter, evaporate the combined filtrates to a volume of about
1 ml, and add sufficient chloroform R to produce 2 ml. For solution (B) dilute 1
1055
volume of solution A to 100 volumes with chloroform R. After removing the

plate from the chromatographic chamber, allow it to dry in air, and spray very
lightly with a solution of 10 mg of potassium permanganate R per ml of sulfu-
ric acid (-100 g/1) TS. Heat again at 120 °C for 20 minutes and examine the chro-

to about 0.5 g of Ibuprofen add 60 ml of chloroform R and shake for 15 minutes.
Filter through a fine glass microfibre paper (e.g. Whatman GF/F) under reduced
pressure. Wash the residue with 2 quantities, each of 20ml of chloroform R,

and evaporate the combined filtrates in a current of air until just dry. Dissolve
the residue in 100 ml of neutralized ethanol TS and titrate with sodium hydrox-
ide (0.1 mol/1) VS, determining the end-point potentiometrically.

C]3His02.
INDOMETACINI COMPRESSI
INDOMETACIN TABLETS
Category. Non-steroidal anti-inflammatory drug.
Requirements
Indometacin tablets contain not less than 90 0 . % and not more than 110 0 . %
of the amount of C 19 H 16CINO 4 stated on the label.
Identity tests
A. To a quantity of the powdered equivalent to about 0.1 g of

tablets
Indometacin add 5ml of chloroform R and
shake. Filter and evaporate the
filtrate to dryness. Dry °C under reduced pressure (not
the residue at 70
exceeding 0.6 kPa or 5 mm
of mercury). Carry out the examination with the
1056

from indometacin RS or with the reference spectrum of indometacin.
B. To a quantity of the powdered tablets equivalent to about 0.05 g of

Indometacin add 60ml of ethanol (-750 g/1) TS and shake. AUow to stand
for 10 minutes, shake again, and dilute with sufficient ethanol (-750 g/1) TS
to produce 100 ml. Filter, discard the first 10 ml of filtrate, then dilute 5 ml
of the filtrate to 100 ml with the same solvent. The absorption spectrum of
a maximum at about 318nm.
C. To powdered tablets equivalent to about 25mg add 10ml

a quantity of the
of water, 2 drops of sodium hydroxide (-200 g/1) TS, shake, and filter. To
the add 1.0 ml of sodium nitrite (10 g/1) TS, allow to stand for
filtrate
5 minutes, and carefully add about 0.5ml of hydrochloric acid (-250 g/1) TS;
slurry in sodium dihydrogen phosphate (45 g/1) TS. As the mobile phase, use a
mixture of 7 volumes of ether R and 3 volumes of light petroleum Rl. Apply
separately to the plate 5 pi of each of the following 2 solutions. For solution
(A) shake a quantity of the powdered tablets equivalent to about 0.1 g of
Indometacin with 5ml of chloroform R, filter, and use the filtrate. For solution
(B) dilute 1 volume of solution A to 20 volumes with chloroform R, further
dilute 1 volume of this solution to 10 volumes with the same solvent. After
and examine the chromatogram in ultraviolet light (254 nm).

of about 0.05g of Indometacin add 10 ml of water and allow to stand for 15
minutes, swirling occasionally. Add 75 ml of methanol R, shake well, add suf-
ficient methanol R to produce 100ml, and filter. To 5ml of the filtrate add a
mixture of equal volumes of methanol R and phosphate buffer pH 7.2, TS to
produce 100 ml. Measure the absorbance of a 1-cm layer at the maximum at
about 318 nm against a solvent cell containing the above solvent mixture.
Calculate the percentage content of C, 9 H] 6 C 1 N 04 using the absorptivity value

of 19.3 (Ai°i= 193).
1057
ISONIAZIDI COMPRESSI
ISONIAZID TABLETS

Requirements
Isoniazid tablets contain not less than 90 0 % and not more than 110 0 %
. . of
the amount of CgH/NsO stated on the label.
Identity tests
A. To a quantity of the powdered tablets equivalent to about 0.1 g of Isoniazid

add 10ml of ethanol (-750 g/1) TS and shake for 15 minutes. Centrifuge and
decant the supernatant liquid. Extract the remaining liquid with two further
10-ml quantities of ethanol (~750g/l) TS and evaporate the combined
extracts to dryness. Carry out the examination with the residue as described
tion spectrum is concordant with the spectrum obtained from isoniazid RS
or with the reference spectrum of isoniazid.
B. To a quantity of the powdered tablets equivalent to about 0.1 g of Isoniazid

add 2.0 ml of water, shake, and filter. Then add a mixture composed of
1.0 ml of silver nitrate (40 g/1) TS and 1.0 ml of ammonia (-100 g/1) TS;
bubbles of nitrogen evolve, the mixture turns from yellow to black and a
metallic silver mirror appears on the sides of the test-tube.
C. To a quantity of the powdered about 1 mg of Isoniazid

add 50ml of ethanol (~750g/l) TS, shake, and filter. To 5ml of the filtrate
add 0.1 g of sodium tetraborate R and 5ml of l-chloro-2,4 dinitroben-
zene/ethanol TS, evaporate to dryness on a water-bath, and continue
heating for a further 10 minutes. To the residue add 10ml of methanol R
and mix; a reddish violet colour is produced.
5 volumes of ethyl acetate R, 2 volumes of acetone R, 2 volumes of methanol
R, and 1 volume of water as the mobile phase. Apply separately to the plate
1058
10|J,1 of each of the 3 following solutions. For solution (A) shake a quantity
of the powderedtablets equivalent to about 0,1 g of Isoniazid with 10ml of
methanol filter, and use the filtrate. For solution (B) use lOmg of isoniazid
R,
RS per ml methanol R. For solution (C) dilute 1 volume of solution A to 100
of
volumes with methanol R. After removing the plate from the chromatographic
light (254 nm).
Assay. Weigh and powder 20 tablets. Dissolve a quantity of the powdered

tablets equivalent to about 0.4 g of Isoniazid as completely as possible in water,
filter, and wash the residue with sufficient water to produce 250 ml. Place
50ml of the resulting solution in a titration vessel, add 50 ml of water, 20ml of

hydrochloric acid (-250 g/1) TS, and 0.2 g of potassium bromide R, and titrate
with potassium bromate (0.0167 mol/1) VS as described under 2.7 Nitrite
titration.
Each ml of potassium bromate (0.0167 mol/1) VS is equivalent to 3.429mg of

C.H7N3O.
MEBENDAZOLI COMPRESSI
MEBENDAZOLE TABLETS

Requirements
Mebendazole tablets contain not less than 90 0 % and not more than
. 1 10 0 %
.
of the amount of C 16H 13N O 3

1059
Identity tests
B. Shake a quantity of the powdered tablets equivalent to 0.04 g of Mebenda-

zole with 2 ml of sodium hydroxide (-80g/l) TS and heat the yellowish
coloured suspension until dissolved; the solution is yellow. Add a few drops
of copper (II) sulfate (160g/l) TS; a greenish precipitate is produced. Add a
few drops of ammonia (~100g/I) TS; the colour of the precipitate turns to
greenish blue.
C. To a quantity of thepowdered tablets equivalent to 0.04 g of Mebendazole

add 2ml of sulfuric acid (~1760g/l) TS; a yellow solution is produced. Care-
fully dilutewith 3 ml of water; the yellow colour disappears. Filter and add
1ml of silver nitrate (40g/l) TS; a white precipitate is formed which does
not dissolve in an excess of ammonia (-lOOg/1) TS.
90 volumes of chloroform R, 5 volumes of methanol R, and 5 volumes of anhy-
drous formic acid R as the mobile phase. Apply separately to the plate 10 pi of
each of the following four solutions. For solution (A) shake a quantity of the
powdered tablets equivalent to 50 mg of Mebendazole with a mixture of 1ml
of anhydrous formic acid R and 9 ml of chloroform R, filter, and use the clear
filtrate. For solution (B) dilute 5ml of solution A to 10ml using the same mixture
of solvents, and for solution (C) dilute 0.5 ml of solution A
10ml using the to
same mixture of solvents. For solution (D) dissolve 12.5 mg of mebendazole RS
in 5ml of the same mixture of solvents. After removing the plate from the chro-
alent to about 0.1 g of Mebendazole, accurately weighed, to a 100-ml volu-
metric flask with 50 ml of anhydrous formic acid R. Heat in a water-bath at
50 °C for 15 minutes. Cool, add water to volume, mix, and filter through a sin-
tered-glass filter. Transfer 10ml of the filtrate to a 250-ml separator, add 50ml
of water and 50ml of chloroform R. Shake for about 2 minutes, allow the
phases to separate, and transfer the chloroform layer to a second 250-ml sep-
arator. Wash the aqueous layer with two portions, each of 10 ml, of chloroform
R, adding the washings to the second separator, and discard the aqueous layer.
Wash the combined chloroform extracts with a mixture of 4 ml of hydrochlo-
1060
ric VS and 50 ml of a previously diluted solution of 5 ml of anhy-

acid (0.1 mol/1)
drous formic acid R with 45 ml of water. Transfer the chloroform layer to a
100-ml volumetric flask. Extract the aqueous washings with two portions, each
of 10 ml, of chloroform R, add these chloroform extracts to the chloroform solu-
tion in the volumetric flask, dilute with 2-propanol R to volume, and mix.
Further dilute 5 ml of this solution to 100ml with 2-propanol R, and mix. For
the reference solution transfer 20 mg of mebendazole RS, accurately weighed, to
a 100-ml volumetric flask and add 90 ml of chloroform R, 7 ml of 2-propanol R,
and 2 ml of a mixture of 0.2 ml of anhydrous formic acid R and 1.8 ml of water.
Shake until the solid has dissolved, add 2-propanol R to volume, and mix.
Transfer 5 ml of this solution to a 200-ml volumetric flask, dilute with 2-
propanol R to volume, and mix. For the reagent blank mix 45ml of chloroform
R with 1 ml of a mixture of 0.1ml of anhydrous formic acid R and 0.9ml of
water using a 100-ml volumetric flask, dilute to volume with 2-propanol R, and
mix. Transfer 5ml of this solution to a second 100-ml volumetric flask, dilute
to volume with 2-propanol R, and mix.
Without delay measure the absorbance of the sample and the reference solutions
in a 1-cm layer at the maximum at about 247 nm against a solvent cell con-
taining the reagent blank. Calculate the amount in mg of CisHiaNsOa in the
sample being examined using the following formula: 20C(A„/Aj), in which C is
the concentration, in mg per ml, of mebendazole RS in the reference solution, and
A„ and A, are the absorbances for the sample and reference solutions, respectively.
MELARSOPROLI INJECTIO
MELARSOPROL INJECTION
Description. Melarsoprol injection is a clear, colourless to almost colourless
solution.
Category. Antitrypanosomal drug.
Storage. Melarsoprol injection should be protected from light.

tial medicines: 3.6%.
Melarsoprol (formula: C 12 H 15ASN 2 OS 2) has a relative molecular mass of 398.3

and the chemical name: 2-[4-(4,6-Diamino-l,3,5-triazin-2-ylamino)phenylj-
l,3,2-dithiarsolan-4-yl-methanol; CAS Reg. No. 494-79-1.
1061
Requirements
Definition. Melarsoprol injection is a sterile solution of melarsoprol in propy-

lene glycol, containing 5% of water.
The injection is normally prepared by heating equimolar amounts of melarsen

oxide and dimercaprol in propylene glycol and adding water. The solution is
sterilized by “Heating in an autoclave” or by any other suitablemethod (see
5.8 Methods of sterilization).
Melarsoprol injection contains not less than 3 4. % and not more than 3 8 % of
.
C12H15ASN6OS2.
Identity tests
A. To a volume of the injection equivalent to 35 mg of Melarsoprol, add 5 ml
of dilute hypophosphorous acid TS and heat on a water-bath; a yellow
B. To a volume of the injection as for test A add 4 ml of sodium hydrox-

similar
ide (-300 g/1) TS and heat to boiling; the evolved vapours turn red litmus
paper R to blue.
C. To a further volume of the injection as for test A add 2 ml of water, 1 ml of

ammonia (-260 g/1) TS, and 3 ml of silver nitrate (40 g/1) TS; a yellow pre-
cipitate is produced which is soluble in nitric acid (-1000 g/1) TS.
Relative density, dll = 1.050-1.056.
Inorganic arsenic. To a volume of the injection equivalent to 0.18g of Melar-

soprol add 4 ml of sodium hydroxide (1 mol/1) VS, shake, and add 3 ml of sul-
furic acid (-190 g/1) TS; a precipitate is produced. Shake, dilute to 50 ml with
water, and mix. Allow to stand for 2-3 minutes, filter, and dilute 10 ml of the
clear filtrate to 100 ml with water. Use 30 ml of this solution and proceed as
described under 2.2.5 Limit test for arsenic; the arsenic content is not more than
30mg/g.
Clarity and colour of solution. The injection is clear and not more intensely
1062

Fischer method, Method A, using 0.5g of the injection; the water content is not
less than 40mg/g and not more than 60mg/g.

about 0.18g of Melarsoprol to a long-necked, 200-ml flask. While cooling, add
a mixture of 12 ml of sulfuric acid (-1760 g/1) TS and 40 ml of hydrogen perox-
ide (-330 g/1) TS. Heat gently and carefully until white vapours appear in the
neck of the flask (heating time: 45-60 minutes). Cool slighdy, add 8g of potas-
sium sulfate R and 0.05 g of starch R, and heat to boiling until decolorization
occurs (heating time: 60-90 minutes). Cool, and add slowly 70 ml of water.
Transfer to a conical flask, rinse with 50ml of water, add 0.05ml of phenolph-
thale in/ ethanol TS, then add slowly while cooling 30-40 ml of sodium hydrox-
ide (-300 g/1) TS until a light pink colour is obtained. Add, drop by drop, sulfuric
acid (-190 g/1) TS until decolorized, then add 3g of sodium hydrogen carbon-
ate R and 50ml of water. Titrate with iodine (0.05mol/l) VS.
Each ml of iodine (0.05 mol/1) VS is equivalent to 19.92 mg of Ci 2 H, 5 AsNgOS 2 .
METRONIDAZOLI INJECTIO
METRONIDAZOLE INJECTION
Description. Metronidazole injection is a colourless solution.
Category. Antibacterial drug; antiamoebic drug.
Storage. Metronidazole should be kept in a single-dose container, protected

from light.

tial medicines: 5mg/ml.
Requirements
Definition. Metronidazole injection is a sterile solution of metronidazole in

water for injections.
The solution is sterilized by a suitable method (see 5.8 Methods of sterilization).
Metronidazole injection contains not less than 90 0 . % and not more than
110 0 %
. of the amount of CgHgNaOg stated on the label.
1063
Identity tests
A. Dilute a volume of the injection equivalent to 20 mg of Metronidazole
to 100 ml with a mixture of solvents composed of 1ml of sulfuric acid
(~1760g/l) TS in 350 ml of methanol R. Further dilute 1ml of this solution
to 10 ml using the same mixture of solvents. The absorption spectrum, when
20pg/ml solution of metronidazole RS in the same mixture of solvents.
B. To a volume of the injection equivalent to 5 mg of Metronidazole, add

0.05 g of 4-dimethylaminobenzaldehyde R dissolved in 2 ml of hydrochloric
acid (~70g/l) TS; a yellowish colour is produced. Add O.OSg of zinc R
powder; the colour changes to red-orange.
chromatography, using silica gel R4 as the coating substance and acetone R as
the mobile phase. Apply separately to the plate lOpl of each of the following
two solutions. For solution (A) evaporate a volume of the injection equivalent to
O.OSg of Metronidazole to dryness on a water-bath and dissolve the residue in
5 ml of a mixture of equal volumes of methanol R and chloroform R. For solution
(B) dissolve 20 mg of 2-methyT5-nitroimidazole R in 10 ml of the same mixture
of solvents. After removing the plate from the chromatographic chamber, allow
Assay. Transfer an accurately measured volume of the solution equivalent

to about lOmg of Metronidazole to a 100-ml volumetric flask. Add 80ml of
hydrochloric acid (0.1 mol/1) VS and shake. Dilute to volume with the same acid
and mix well. Filter through a dry filter-paper, discarding the first few ml of fil-
trate. Dilute accurately 5.0ml of the filtrate to 200ml with hydrochloric acid
(0.1 mol/1) VS and mix well.
Measure the absorbance of the resulting solution in a 1-cm layer at the

maximum at about 277 nm. Calculate the content of C6H9N3O3, using the
absorptivity value of 37.7 (Al°m = 377).
Bacterial endotoxins. Carry out the test as described under “3.4 Test for bac-
terialendotoxins”. Dilute the injection, if necessary, with water LAL to give a
solution containing 5 mg per ml (solution A). Solution A contains not more than
3.5lU of endotoxin per ml. Carry out the test using the maximum valid dilu-
tion of solution A calculated from the declared sensitivity of the lysate used in
the test.
1064
METRONIDAZOLI COMPRESSI
METRONIDAZOLE TABLETS
Category. Anti-amoebic drug; antibacterial drug.

tial medicines: 200-500 mg.
Requirements
Definition. Metronidazole tablets may be film-coated and not necessarily

circular in shape.
Metronidazole tablets contain not less than 95 0. % and not more than 105 0 % .
of the amount of CeHgNsOg stated on the label.
Identity tests
To a quantity of the powdered tablets equivalent to 60 mg of Metronidazole
add 20 ml of water, shake, and filter. Evaporate the filtrate to a smaller volume,
allow to crystallize, separate the crystals, dry at 105°C for 1 hour, and use the
dried material for tests A and B.
A. Dissolve 20 mg of the dried material in 100 ml of a mixture of solvents com-

posed of 1 ml of sulfuric acid (~1760g/l) TS in 350 ml of methanol R. Further
dilute 1ml of this solution to 10 ml using the same mixture of solvents. The
absorption spectrum, observed between 220 nm and 350 nm, is qual-
when
itatively similar to that of a 20pg/ml solution of metronidazole RS in the
same mixture of solvents.
B. To 25 mg of the dried material add 0.05 g of 4-dimethylaminobenzaldehyde

R dissolved in 2 ml of hydrochloric acid (~70g/l) TS; a yellowish colour is
produced. Add 0.05 g of zinc R powder; the colour changes to red-orange.
chromatography, using silica gel R4 as the coating substance and acetone R as
the mobile phase. Apply separately to the plate lOpl of each of the following
two solutions. For solution (A) shake a quantity of the powdered tablets equiv-
alent to 0.2 g of Metronidazole with 5 ml of a mixture of equal volumes of
methanol R
and chloroform R for 5 minutes, filter, and use the clear filtrate. For
solution (B) dissolve 20 mg of 2-methyl-5-nitroimidazole R in 10ml of the same
mixture of solvents. After removing the plate from the chromatographic chamber,
allow it to dry in air, and examine the chromatogram in ultraviolet light (254nm).
1065
Assay. Weigh and powder 20 tablets. Transfer a quantity of the powder, equiv-
alent to about 0.2 g of Metronidazole, accurately weighed, to a sintered-glass
filtering crucible, and extract with 6 quantities, each of 10 ml, of hot acetone R.
Cool, add to the combined extracts 50 ml of acetic anhydride R and 0.1ml of

brilliant green/acetic acid TS, and titrate with perchloric acid (0.1 mol/1) VS as
described under 2.6 Non-aqueous titration. Method A. Repeat the procedure
without the powdered tablets being examined and make any necessary
corrections.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 17.12 mg of C6H9N3O3.
MORPHINI SULFATIS COMPRESSI

MORPHINE SULFATE TABLETS
Category. Opioid analgesic.

tial medicines: lOmg.
Requirements
Morphine sulfate tablets contain not less than 90 0 % and not more than .
110 0 % of the amount of (Ci7Hi9N03)2,H2S04,5H20 stated on the label.

.
Identity tests
A. To a quantity of the powdered about 0.1 g of Morphine

sulfateadd 10ml of ethanol (~750g/l) TS and shake for 15 minutes. Cen-
trifugeand decant the supernatant liquid. Extract the remaining liquid with
two further 10-ml quantities of ethanol (-750 g/1) TS and evaporate the com-
bined extracts to dryness. Carry out the examination with the residue as
absorption spectrum is concordant with the reference spectrum of morphine
sulfate.
1066
B. To a quantity of the powdered tablets equivalent to about 20 mg of Mor-

phine sulfate add 5ml of water, shake, and filter. To the filtrate add 0.05ml
of ferric chloride (25g/l) TS; a blue colour is produced.
C. To a quantity of the powdered tablets equivalent to about 20 mg of Mor-

phine sulfate add 5 ml of sulfuric acid (0.05 mol/1) VS, shake, and filter. To
the filtrate add 0.5 ml of a saturated solution of potassium iodate R; an amber
colour is produced which reaches maximum intensity after 5 minutes. Add
0.5ml of ammonia (-260 g/1) TS; the colour darkens almost to black.
D. To a quantity of the powdered tablets equivalent to about 20 mg of

Morphine sulfate add 5 ml of water, shake, and filter. The filtrate yields the
reactions described under 2.1 General identification tests as characteristic of
sulfates.
Assay. Weigh and powder 20 tablets. To a quantity of the powdered tablets

equivalent to about 0.4 g of Morphine sulfate add 25 ml of water, 5 ml of sodium
hydroxide (1 mol/1) VS, and 1 g of ammonium sulfate R, swirl to dissolve. Add
20ml of ethanol (-750 g/1) TS and extract with successive quantities of
40ml, 20 ml, 20 ml, and 20ml of a mixture of 3 volumes of chloroform R and
1 volume of ethanol (-750 g/1) TS. Wash each extract with the same 5 ml of
water, filter, and evaporate the solvent. Dissolve the residue in 10ml of
hydrochloric acid (0.05 mol/1) VS, boil, cool, add 15 ml of water and a few drops
of methyl red/ethanol TS. Titrate the excess acid with sodium hydroxide
(0.05mol/l) VS.
Each ml of hydrochloric acid (0.05mol/l) VS is equivalent to 18.97mg of

(C,7H«N03)2,H2S04,5H20.
NICLOSAMIDI COMPRESSI
NICLOSAMIDE TABLETS
Labelling. The designation on the container should state that the tablets
should be chewed thoroughly before being swallowed.

Chewable Niclosamide tablets may contain sweetening and flavouring agents.
1067
Requirements
Niclosamide tablets contain not less than 95.0% and not more than 105.0%
of the amount of C13H8CI2N2O4 stated on the label.
Identity tests
Heat a quantity of the powdered tablets equivalent to 0.5 g of Niclosamide with
25ml of ethanol (~750g/l) TS, filter while hot, and evaporate to dryness on a
water-bath.
• The residue complies either with test A alone or with tests B, C, and D.
concordant with the spectrum obtained from niclosamide RS or with the
reference spectrum of niclosamide.
B. Using a small flame heat 0.05 g of the residue in a test-tube. Collect and dis-
solve the sublimate in 5 ml of water and add a few drops of ferric chloride
(25g/l) TS; a violet colour is produced.
C. To 0.05 g of the residue add 5 ml of hydrochloric acid (1 mol/1) VS and 0.1 g

of zinc R powder and heat in a water-bath for 10 minutes. Cool and filter.
To the filtrate add 0.5ml of sodium nitrite (lOg/1) TS and allow to stand for
10 minutes. Add 2 ml of ammonium sulfamate (25g/l) TS, shake, allow to
stand for 10 minutes, and add 2 ml of W-(l-naphthyl)ethylenediamine
hydrochloride (5g/l) TS; a deep red colour is produced.
D. Dissolve 0.1 g of the residue in 1ml of acetic anhydride R and boil for 10
minutes. Cool and add 10ml of water. Collect the precipitate on a filter,
wash with water, recrystallize from ethanol (-750 g/1) TS, and dry at
105 °C; melting temperature of the acetyl derivative, about 178 °C.
2-Chloro-4-nitroaniline. To a quantity of the powdered tablets equivalent

to 0.1 g of Niclosamide add 20ml of methanol R and boil for 2 minutes. Cool,
add sufficient hydrochloric acid (1 mol/1) VS to produce 50 ml, and filter. To
10ml of the filtrate add 1 ml of sodium nitrite (1 g/1) TS and allow to stand for
10 minutes. Add 1 ml of ammonium sulfamate (25 g/1) TS, shake, allow to stand
for 10 minutes, and add 1ml of N-(l-naphthyl)ethylenediamine hydrochloride
(5 g/1) TS; the colour produced is not more intense than that produced by a
similarly and simultaneously treated solution containing 10 pg of 2-chloro-4-

nitroaniline R.
1068
5-ChIorosalicylic acid. To a quantity of the powdered tablets equivalent to

0.5 g of Niclosamide add 10 ml of water and boil. Cool, filter, and add to the
filtrate a few drops of ferric chloride (25g/l) TS; no red or violet colour is
produced.

to about 0.3 g of Niclosamide, accurately weighed, add 60 ml of dimethylfor-
mamide R and titrate with tetrabutylammonium hydroxide (0.1 mol/1) VS,
aqueous titration. Method B.

32.71 mg of C 13H 8 CI2N 2 O 4.
NITROFURANTOINI COMPRESSI
NITROFURANTOIN TABLETS
Additional infonnation. Strength in the current, WHO Model list of essen-

Nitrofurantoin tablets may have an enteric sugar coating.
Requirements
Nitrofurantoin tablets contain not less than 90.0% and not more than 1 10.0%
of the amount of CsHeNÔs stated on the label.
Identity tests
A. To a quantity of the powdered tablets equivalent to 0.1 g of Nitrofurantoin

add 10ml of acetic acid (~300g/l) TS, boil for a few minutes, and filter while
hot. Cool to room temperature, collect the precipitate, and dry at 105 °C for
1 hour. Carry out the examination with the residue as described under 1.7

is concordant with the spectrum obtained from nitrofurantoin RS or with
the reference spectrum of nitrofurantoin.
1069
B. Carry out the test in subdued light.
The absorption spectrum of the final solution obtained in the “Assay”, when
266nm and 367 nm. The ratio of the absorbance at 367 nm to that at
266nm is between 1.36 and 1.42.
C. To a quantity of thepowdered tablets equivalent to 25 mg of Nitrofuran-

toin add a mixture of 25 ml of water and 1ml of ammonia (-260 g/1) TS,
shake, and filter; a yellow-orange colour is produced. Add 5 ml of silver
nitrate (40 g/1) TS; a yellow precipitate is produced (distinction from fura-
zolidone and nitrofural).
9 volumes of nitromethane R and 1 volume of methanol R as the mobile phase.
Apply separately to the plate 10 pi of each of the following two solutions. For
solution (A) shake a quantity of the powdered tablets equivalent to 0.1 g of
Nitrofurantoin with 10 ml of a mixture of1 volume of dimethylformamide R
and 9 volumes of acetone R, and use the clear filtrate. For solution (B)
filter,
dilute 1ml of solution A to 100ml with acetone R. After removing the plate
from the chromatographic chamber, allow it to dry in air, heat it at 105°C for
5 minutes, and examine the chromatogram in ultraviolet light (254 nm). Spray
the warm plate with phenylhydrazine/hydrochloric acid TS, heat it again at
105 °C for 10 minutes, and examine the chromatogram in daylight.
By either method of visualization any spot obtained with solution A, other than
the principal spot, is not more intense than that obtained with solution B.
Assay
Weigh and powder 20 tablets. Add a quantity of the powder equivalent to

about 0.12g of Nitrofurantoin, accurately weighed, to 50ml of dimethylfor-
mamide R, shake for 5 minutes, and add sufficient water to produce 1000 ml.
Mix and dilute 5 ml to 100 ml with an aqueous solution containing 1.8 g of
sodium acetate R and 0.14ml of glacial acetic acid R in 100ml, and filter.
Measure the absorbance of the filtrate in a 1-cm layer at the maximum at about
367 nm, using as the blank the mixture as prepared above of sodium acetate
and glacial acetic acid. Calculate the content of CsH(;N 405 using the absorp-,
tivity value of 76.5 = 765).
1070
NYSTATINI COMPRESSI
NYSTATIN TABLETS
Storage. Nystatin tablets should be kept in a tightly closed container.

tial medicines: 500 000 lU.
Nystatin tablets may be coated.
Requirements
Identity tests
A. To powdered tablets equivalent to 0.1 g of Nystatin add a
a quantity of the
mixture of 5 ml of glacial acetic acidR and 50 ml of methanol R, shake, add
sufficientmethanol R to produce 100 ml, and filter. Dilute 1 ml of the filtrate
to 100 ml with methanol R. The absorption spectrum of the resulting solu-
tion, when observed between 240 nm and 350 nm, exhibits 3 maxima at
about 291 nm, 305nm, and 319nm; the ratio of the absorbance of a 1-cm
layer at 291 nm to that at 305 nm is between 0.61 and 0.73, and the ratio of
the absorbance at 319nm to that at 305 nm is between 0.83 and 0.96.
B. To a quantity of the powdered tablets equivalent to 0.05 g of Nystatin add

2 ml of sulfuric acid (-1760 g/1) TS; a brown-violet colour is produced.
C. To a quantity of the powdered tablets equivalent to 0.05 g of Nystatin add

2 ml of ethanol (-750 g/1) TS, shake, and filter. To the filtrate add 1ml of
hydrochloric acid (-250 g/1) TS, and 2 drops of a solution composed of 1 ml
of ferric chloride (25 g/1) TS and 10 ml of water; the yellow colour of the
solution becomes more intense.

Nystatin at 60 °C under reduced pressure (not exceeding 0.6 kPa or 5 of mm
mercury) for 3 hours; it loses not more than 50mg/g.
Assay
1071
Weigh and powder 20 tablets. To a quantity of the powder equivalent to about

200 000 lU of Nystatin, accurately weighed, add 50 ml of dimethylformamide
R and shake for 1 hour. Centrifuge, and dilute 10ml of the clear, supernatant
liquid to 200ml with sterile phosphate buffer pH 6.0, TS3. Carry out the assay
as described under 3.1 Microbiological assay of antibiotics, using Petri dishes
or rectangular trays filled to a depth of 1-2 mm
with culture medium Cm3
having a final pH of 6.0-6.2 and inoculated with Saccharomyces cerevisiae (NCYC
87; ATCC 9763) as the test organism, adding an appropriate concentration of
Nystatin (usually between 25 and 300 lU per ml) and incubating at a tempera-
ture of 29-33 °C. The precision of the assay is such that the fiducial limits of
error of the estimated potency [P = 0.95) are not less than 95% and not more
than 105%.
The upper is not less than 97.0% and the lower fiducial
fiducial limit of error
limit of error is not more than 110.0% of the activity prescribed or stated on
the label and expressed in International Units.
Disintegration. Carry out 5.3 Disintegration test for tablets and capsules,
but using hydrochloric acid (0.1 mol/1) VS instead of water, and operate the
apparatus for 30 minutes. If any tablets have not disintegrated, wash them
rapidly by immersion in water and continue the test using phosphate standard
buffer, pH 6.8, TS; all tablets must have disintegrated within a further 30
minutes.
SALES PERORALES AD REHYDRATATIONEM

ORAL REHYDRATION SALTS
Definition. Oral Rehydration Salts (ORS) are dry mixtures of powders con-
taining per packet:
sodium chloride NaCl 2.6g

trisodium citrate dihydrate C 6H 5 Na 307 2 H 20
, 2.9g
potassium chloride KCl 1.5g
anhydrous glucose C 6H 12 O 6 13.5g
Before administration the contents of each packet should he dissolved in 1 litre of water.
Category. Used for the prevention and treatment of dehydration due to diar-
rhoea, including maintenance therapy.
1072
Storage. Oral Rehydration Salts should be kept in a sealed packet; if a free-

flowing powder is required, it should be kept in an airtight packet, preferably
made of aluminium laminate.
Labelling. The designation on the packet of Oral Rehydration Salts should

state; (1) the total net mass and the mass of the contents of each constituent,
both expressed in grams, (2) volume of water to reconstitute the
the required
solution, (3) directions for the preparation of the solution and its administra-
tion, and (4) a warning that any solution that remains unused 24 hours after
preparation is to be discarded.
Additional information. In the formulation of Oral Rehydration Salts

trisodium citrate dihydrate may be replaced by 2.5 g/1 of sodium hydrogen
carbonate, NaHCOj (sodium bicarbonate). However, as the stability of the
latter formulation under tropical conditions is very poor, it is recommended
only in Oral Rehydration Salts manufactured for immediate use, or where

sodium hydrogen carbonate is packaged in separate packets. These formula-
tions would also allow the use of 14.85 g/1 of glucose monohydrate,
CgH, 206 ,H 20 instead of anhydrous glucose.
,
The title of the two formulations could be distinguished by: “ORS-citrate” or

“OSR-hydrogen carbonate” (bicarbonate). The title Oral Rehydration Salts
(ORS) used without qualification implies that the product is the citrate formu-
lation as defined above.
Oral Rehydration Salts may contain suitable pharmaceutical aids, in minimal

quantities, to improve the flow characteristics and/or the flavour.
Requirements
These specifications apply only to ORS-citrate.
One to three single doses may represent a complete treatment; therefore, the contents of
each packet should comply with the following requirements.
less than 90 0 % and not more than 1 10 0 %

Oral Rehydration Salts contain not . .
of the equivalent amounts of sodium NaR potassium K*, chlorides Ch, citrate
on the label, and not less than
of the relevant constituents stated
90 0 % and not more than 110 0 % of the amount of anhydrous glucose
. .
CsH ,206 stated on the label.
Identity tests
A. Melts when heated; first becomes yellow then brown, swells up and burns,
evolving an odour of burnt sugar.
1073
Dissolve the entire contents of one packet in Z50ml of water to prepare the test
solution to be used in tests B, C, D, E, and F.
B. The test solution yields reaction A described under 2.1 General identifica-
tion tests as characteristic of sodium.
C. To 5 ml of the test solution add 4 drops of sodium cobaltinitrite (lOOg/1) TS;

a yellow-orange precipitate is produced (potassium).
D. A 5-ml aliquot of the test solution yields reaction A described under 2.1
General identification tests as characteristic of chlorides.
E. A 5-ml aliquot of the test solution after neutralization yields reaction A

described under 2.1 General identification tests as characteristic of citrates.
E Add a few drops of the test solution to 5 ml of hot potassio-cupric tartrate

TS; a copious red precipitate is produced (glucose).
Uniformity of mass. Weigh the contents of 20 packets selected at random

and determine the average mass. Not more than two of the individual masses
deviate from the average mass by more than 5% and none deviates by more
than 10%.
Loss on drying. Dry to constant mass at 50 °G; it loses not more than 20 mg/g.
pH value. pH of the solution reconstituted as directed on the label, 7. 0-8. 8.
Assays
Carry out all the assays on quantities taken from a single packet. If the
quantity of one packet is insufficient to carry out all the assays, take another
packet for the assay for citrates and for the assay for glucose from the same
batch.
Prepare the following solution (= solution A) for the assays for sodium, potas-
sium, and chlorides. Dissolve about 8g of ORS, accurately weighed, in suffi-
cient water to produce 500ml.
Sodium. Dilute 3 ml of solution A to 500 ml with water and determine by

flame photometry as described under 1.8 Atomic spectrometry; emission and
absorption at a wavelength of 589nm. For the preparation of the reference solu-
tions, use a stock standard solution prepared by dissolving sodium chloride R,
previously dried to constant mass at 130°C, in 1000ml of water to contain
508. 4mg of NaCl (0.2 mg of Na* per ml).
1074
Each g of sodium chloride and of trisodium citrate dihydrate is equivalent to

0.3934 g and 0.2345 g of Na“^, respectively.
Potassium. Dilute 3 ml of solution A to 500 ml with water and determine by

flame photometry as described under 1.8 Atomic spectrometry; emission and
absorption at a wavelength of 767 nm. For the preparation of the reference solu-
tions, use a stock standard solution prepared by dissolving potassium chloride
R, previously dried to constant mass at 130 °C, in 1000ml of water to contain
190. 6mg of KCl (0.1 mg of K* per ml).
Each g of potassium chloride is equivalent to 0.5245 g of K*.
Chlorides. Titrate 20ml of solution A with silver nitrate (0.1 mol/1) VS, using
potassium chromate (lOOg/1) TS as indicator.
Each ml of silver nitrate (0.1 mol/1) VS is equivalent to 3.545 mg of Ch.
Each g of sodium chloride and of potassium chloride is equivalent to 0.6066g

and 0.4756g of Ch, respectively.
Citrates. Disperse about 2.8 g of ORS, accurately weighed, in 80 ml of glacial

about 50 °C, cool, dilute to 100 ml with glacial acetic acid
acetic acid Rl, heat to
Rl, and allow to stand for 10 minutes. To 20ml of the supernatant liquid add
0.25ml of 1-naphtholbenzein/acetic acid TS and titrate with perchloric acid
(0.1 mol/1) VS as described under 2.6 Non-aqueous titration, Method A.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 6.303 mg of CeHsO/^".

Each g of sodium citrate is equivalent to 0.6430 g of C^HsOy^.
Glucose. Dissolve about 8.0 g of ORS, accurately weighed, in 40 ml of water,

add 0.2ml of ammonia (~100g/l) TS, and dilute to 50ml with water. Mix and
allow to stand for 30 minutes. Determine the “Optical rotation’’ and calculate
the quantity, in g, of anhydrous glucose C^HiyOg by multiplying the observed
rotation in degrees by 0.9477.
PARACETAMOLI COMPRESSI
PARACETAMOL TABLETS
Other name. Acetaminophen tablets.
Category. Non-opioid analgesic; antimigraine drug.
Storage. Paracetamol tablets should be kept in a tightly closed container.
1075

Requirements
Definition. Paracetamol tablets may not necessarily be circular in shape.
Paracetamol tablets contain not less than 95.0% and not more than 105.0%
of the amount of C 5H 9NO 2 stated on the label.
Identity tests
To a quantity of the powdered tablets equivalent to 1 g of Paracetamol add
40ml of acetone R, filter, evaporate the filtrate to dryness, and use the residue.
• The residue complies either with test A alone or with tests B and C. (Keep
the remaining residue for “4-Aminophenol”.)
concordant with the spectrum obtained from paracetamol R or with the
reference spectrum of paracetamol.
C. To 0.1 g of the residue add 2 ml of hydrochloric acid (-250 g/1) TS, and heat
to boiling for 1 minute. Following this add 10ml of water and 1 drop of
potassium dichromate (lOOg/1) TS, and shake; a violet colour slowly devel-
ops and does not become red.
4-Aminophenol. Dissolve 0.5 g of the residue in a mixture of equal volumes

of methanol R and water and dilute to 10 ml with this mixture of solvents. Add
0.2 ml of alkaline sodium nitroprusside TS, mix, and allow to stand for 30
minutes. Prepare similarly a reference solution containing 0.5 g of 4-aminophenol-
free paracetamol R and 0.5ml of a solution containing 0.05 mg/ml of 4-
aminophenol R in the same mixture of solvents. The colour of the test solution
is not more intense than that of the reference solution (0.05mg/g).
65 volumes of chloroform R, 25 volumes of acetone R, and 10 volumes of
1076
toluene R as the mobile phase, but allowing the solvent front to ascend 14 cm
above the line of application in an unsaturated chamber.
Powder not less than 10 tablets and prepare the following six solutions. For
solution (A) transfer a quantity of the powder equivalent to 200 mg of Parac-
etamol to a ground-glass-stoppered15-ml centrifuge tube, add 10ml of ethanol
(-750 g/1) TS, shake mechanically for 30 minutes, centrifuge at 1000 revolutions
per minute for 15 minutes or until a clear supernatant liquid is obtained, decant,
and apply 40 pi. For solution (B) dissolve 20 mg of paracetamol RS per ml in
ethanol (-750 g/1) TS and apply 40 pi. For solution (C) transfer a quantity of the
powder equivalent to Ig of Paracetamol to a ground-glass-stoppered 15-ml
centrifuge tube, add 5 ml of peroxide-free ether R, shake mechanically for 30
minutes, centrifuge at 1000 revolutions per minute for 15 minutes or until a
clear supernatant liquid is obtained, decant, and apply 200 pi. For solution (D)
dilute 1 ml of solution C with sufficient ethanol (-750 g/1) TS to produce 10ml
and apply 40 pi. For solution (E) transfer 0.5ml of solution B to a 10-ml volu-
metric flask, add 25 mg of 4'-chloroacetanilide R, dilute to volume with ethanol
(-750 g/1) TS, and apply 40 pi. For solution (F) dissolve 10 mg of 4'-chloroac-
etanilide R in sufficient ethanol (-750 g/1) TS to produce 20 ml, further dilute
1 ml to 10 ml with the same solvent, and apply 40 pi. After removing the plate

and examine the chromatogram in ultraviolet light (254nm).
The spot obtained with solution C, other than the principal spot, is not more
intense than the corresponding spot (same Revalue) obtained with solution F.
Any spot obtained with solution D, other than the principal spot, is not more
intense than that obtained with solution The test is valid only if the
F. chro-
matogram obtained with solution E shows two clearly separated spots.
Assay. Weigh and powder 20 tablets. Add a quantity of the powder equiva-
lent to about 0.15g of Paracetamol, accurately weighed, to 50 ml of sodium
hydroxide (0.1 mol/1) VS, dilute with 100 ml of water, shake for 15 minutes, and
add sufficient water to produce 200ml Mix, filter, and dilute 10ml of the fil-
trate to 100 ml with water. Add 10 ml of this solution to 10 ml of sodium
hydroxide (0.1 mol/1) VS and dilute to 100ml with water.
Measure the absorbance of the resulting solution in a 1-cm layer at the

maximum at about 257 nm and calculate the content of C 8H 9NO 2 using the
absorptivity value of 71.5 (Al°cm = 715).
1077
PENTAMIDINI ISETIONATIS PULVIS

AD INJECTIONEM
PENTAMIDINE ISETIONATE POWDER
FOR INJECTIONS
Category. Antileishmaniasis drug.
Labelling. The designation on the container should state that Pentamidine ise-
tionate injection should be used immediately after preparation. Expiry date.

Pentamidine isetionate is hygroscopic. The injection deteriorates on storage.
Requirements
Definition. Pentamidine isetionate powder for injections is a sterile powder

of pentamidine isetionate.
The powder is sterilized by a suitable method (see 5.8 Methods of sterilization).
The container of Pentamidine isetionate powder for injections contains not

less than 90 0 % and not more than 110 0 %
. . of the amount of
C]9H24N402,2C2H604S Stated on the label.
Identity tests
• Either test A alone or tests B and C may by applied.

spectrum obtained from pentamidine isetionate RS or with the reference spec-
trum of pentamidine isetionate.
B. The absorption spectrum of a solution containing a quantity of the powder

for injections equivalent to lOmg Pentamidine isetionate per ml of
hydrochloric acid (0.01 mol/1) VS, when observed between 230nm and
350 nm, exhibits a maximum at about 262 nm; the absorbance of a Tcm
layer at this wavelength is about 0.47.
1078
C. To a quantity of the powder for injections equivalent to 0.5 g of Pentami-

dine isetionate add5ml of water and heat to 80 °C to dissolve. Add 10ml
of sodium hydroxide (~50g/l) TS, cool in ice, and filter. To 2 ml of the fil-
trate add 0.2ml of nitric acid (~1000g/l) TS followed by 0.2 ml of ceric
ammonium nitrate TS; a red-orange colour is produced.

equivalent to 0.5 g of Pentamidine isetionate in 10 ml of carbon-dioxide-free
water R is clear and colourless. (Keep this solution for the “pH value”.)
4.5-6.5.
chromatography, using silica gel R6, activated at 105°C for 1 hour, as the
separately to the plate 10 pi of each of the following two solutions. For solu-
tion (A) dissolve a quantity of the powder for injections equivalent to 0.1 g of
Pentamidine isetionate in 2ml of methanol R. For solution (B) dilute 1ml of
solution A to 200 ml with methanol R. After removing the plate from the chro-
Assay
Use Method A as described under 2.10 Determination of nitrogen, with a quan-

tity of the powder for injections equivalent to about 0.4 g of Pentamidine ise-
tionate, accurately weighed, and 9 ml of nitrogen-free sulfuric acid (-1760 g/1)
TS.
Each ml of sulfuric acid (0.05mol/l) VS is equivalent to 14.82 mg of

Cl9H24hl402,2C2H604S.
1079
PETHIDINI HYDROCHLORIDI COMPRESSI

PETHIDINE HYDROCHLORIDE TABLETS
Category. Opioid analgesic.

Requirements
Pethidine hydrochloride tablets contain not less than 90 0 % and not more than
.
110 0 %
. of the amount of C] 5 H 2 iN 02 ,HCl stated on the label.
Identity tests
A. To a quantity of the powdered about 0.05 g of Pethi-

dine hydrochloride add 20ml of chloroform R, shake, and filter. Evaporate
the filtrate and dry the residue under reduced pressure (not
to dryness
exceeding O.hkPa or 5mm of mercury). Carry out the examination with the
The infrared absorption spectrum is concordant with the reference spectrum
of pethidine hydrochloride.
B. To a quantity of the powdered tablets equivalent to about 0.2 g of Pethidine

hydrochloride add 20 ml of water, shake, and filter. To 5 ml of the filtrate
(keep the remaining filtrate for tests C and D) add 5 ml of trinitrophenol/
ethanol TS and shake; a yellow, crystalline precipitate is produced. Filter,
wash with water, and dry the crystals at 105 °C for 2 hours; melting
temperature, about 190 °C.
C. Evaporate 1 ml of the filtrate from test B to dryness on a water-bath, dis-

solve the residue in 1 ml of formaldehyde/sulfuric acid TS, and heat gently;
the colour of the solution turns to pink, changing to violet-red and showing
a red fluorescence when held in front of a strong light.
D. Dilute 5 ml of the filtrate from test B with 5 ml of water; it yields the reac-
tions described under 2.1 General identification tests as characteristic of
chlorides.
1080
of 9 volumes of acetone R and 1 volume of 2-phenoxyethanol R to impregnate

the plate, dipping it about 5 mm into the liquid. After the solvent has reached
remove the plate from the chromatographic chamber,
a height of at least 16 cm,
and dry it air. Use the impregnated plate immediately, carrying
in a current of
out the chromatography in the same direction as the impregnation.
Shake together 100 volumes of light petroleum Rl, 8 volumes of 2-

phenoxyethanol R, and 1 volume of diethylamine R, allow to settle, and use
this solution as the mobile phase. Apply separately to the plate 5 pi of each of
the following 2 solutions. For solution (A) shake a quantity of the powdered
tablets equivalent to about 0.1 g of Pethidine hydrochloride with 5 ml of water,
filter, shake the with 0.5ml of sodium hydroxide (-200 g/1) TS and 2 ml
filtrate
of ether R, allow the layers to separate, and use the upper layer. For solution
(B) dilute 0.5ml of solution A to 50 ml with ether R. After removing the plate
from the chromatographic chamber, allow it to dry in air for 10 minutes, return
it to the chromatographic chamber, and repeat the development. Remove the
plate, allow it to dry in air for 10 minutes, and spray with dichlorofluorescein
TS. Allow to stand for 5 minutes and spray with water until the background
is white to pale yellow.
Examine the chromatogram in daylight. The chromatogram shows red to

orange spots.
Examine the chromatogram without delay in ultraviolet light (365 nm). The
chromatogram shows spots with intense yellow fluorescence.

equivalent to about 0.5 g of Pethidine hydrochloride add 40 ml of water, 2.0ml
of sodium hydroxide (-200 g/1) TS, and extract immediately with quantities of
25 ml, 10 ml, and 10 ml of chloroform R. Wash each extract with the same
15 ml of water and filter into a dry flask. To the combined extracts, which should
be clear and free from droplets of water, add 0.15ml of 1-naphtholbenzein/
acetic acid TS and titrate with perchloric acid (0.05 mol/1) VS as described under
2.6 Non-aqueous titration. Method A.
Each ml of perchloric acid (0.05mol/l) VS is equivalent to 14.19mg of

C,5H2,N02,HC1.
1081
PHENOBARBITALI COMPRESSI
PHENOBARBITAL TABLETS

tial medicines: 15-lOOmg.
Requirements
Phenobarbital tablets contain not less than 90 0 . % and not more than 1 10 0 %
.
of the amount of C 12 H 12N 2 O 3 stated on the label.
Identity tests
• Either tests A, C, and D or tests B, C, and D may be applied.
To a quantity of the powdered about 0.4 g of Phenobar-

bital add 10ml of dehydrated ethanol R, shake, and filter. Evaporate the filtrate
to dryness and dry the residue at 105 °C for 1 hour. Use the residue for the fol-
lowing tests.
concordant with the spectrum obtained from phenobarbital RS or with the
reference spectrum of phenobarbital. If the spectra obtained are not concor-
dant, heat the residue in a sealed tube at 105°C for 1 hour and prepare a
new spectrum of the residue.
B. Melting temperature of the residue, about 174 °C.
C. Dissolve 20 mg of the residue in 5 ml of methanol R, add 1 drop of cobalt(II)

chloride (30g/l) TS and 3-4 drops of ammonia (~100g/l) TS; a violet colour
is produced.
D. To 0.20 g of the residue add about 2 ml of sulfuric acid (-1760 g/1) TS, 20 mg
of sodium nitrate R, and allow to stand for 30 minutes; a yellow colour is
produced.

equivalent to about 0.2g of Phenobarbital add 40ml of methanol R and 15ml
of a freshly prepared solution of 30 mg of anhydrous sodium carbonate R
per ml. Titrate with silver nitrate (0.1 mol/1) VS, determining the end-point
potentiometrically.
1082
Each ml of silver nitrate (0.1 mol/1) VS is equivalent to 23.22 mg of C 12 H 12 N 2 O 3 .
Disintegration test. Complies with the 5.3 Disintegration test for tablets and
capsules. Time period; 30 minutes.
PHENOXYMETHYLPENICILLINI
KALICI COMPRESSI
PHENOXYMETHYLPENICILLIN
POTASSIUM TABLETS
Storage. Phenoxymethylpenicillin potassium tablets should be kept in a

tightly closed container.
Labelling. The designation on the container indicates the quantity of active

ingredient in terms of the equivalent amount of phenoxymethylpenicillin. It
also indicates whether the tablets are to be chewed before swallowing. Expiry
date.

Requirements
Phenoxymethylpenicillin potassium tablets contain not less than 90 0 . % and

not more than 110.0% of the amount of Ci 6 H]sN 205 S stated on the label.
Identity tests
A. Shake a quantity of the powdered tablets equivalent to 80 mg of Phe-
noxymethylpenicillin potassium with water, dilute to 250 ml with water,
and filter. The absorption spectrum of the filtrate, when observed between
230 nm and 350 nm, exhibits maxima at268nm and 274 nm and a minimum
at 272 nm.
B. Shake a quantity of the powdered tablets equivalent to Ig of Phe-

noxymethylpenicillin potassium with 5ml of water, filter, and add 3 drops
of hydrochloric acid (~70g/l) TS. Mix, cool in ice, add 1 drop of sodium
nitrite (lOOg/1) TS and a solution composed of 50mg of 2-naphthol R dis-
1083
solved in a mixture of 2 ml of sodium hydroxide (~80g/l) TS and 3 ml of

water; no scarlet-red precipitate is formed (absence of procaine penicillin).
C. Ignite a quantity of the powdered tablets equivalent to 0.5 g of Phe-

noxymethylpenicillin potassium, add 5 ml of hydrochloric acid (~70g/l) TS,
boil, cool, and filter; the filtrate made alkaline yields the reaction described
under 2.1 General identification tests as characteristic of potassium.

Phenoxymethylpenicillin potassium under reduced pressure (not exceeding
O.dkPa or 5 mm of mercury) at 60 °C for 3 hours; it loses not more than
15mg/g.
Phenoxyacetic acid. Carry out the test as described under 1.14.1 Thin-layer
90 volumes of diisopropyl ether R, 7 volumes of anhydrous formic acid R, and
1 volume of water mobile phase. Apply separately to the plate 10 pi of
as the
each of the following two solutions. For solution (A) shake a quantity of the
powdered tablets equivalent to 0.25 g of Phenoxymethylpenicillin potassium
with 25 ml of a mixture of equal volumes of methanol R and water, filter, and
use the clear filtrate. For solution (B) dissolve 10 mg of phenoxyacetic acid R in
the same mixture of removing the plate from the chromato-
solvents. After
graphic chamber, allow it warm air and spray with a
to dry in a current of
mixture of 0.15g of potassium permanganate R dissolved in 100ml of sulfuric
acid (0.5 mol/1) VS. Examine the chromatogram in daylight.
Any spot corresponding to phenoxyacetic acid obtained with solution A is not

alent to about 125 mg of Phenoxymethylpenicillin potassium, accurately
weighed, with 300 ml of water for 30 minutes, add sufficient water to produce
500 ml, dilute 25 ml to 100 ml with water, and filter. Transfer two 2-ml aliquots
of the filtrate into separate stoppered tubes. To one tube add 10ml of imida-
zole/mercuric chloride TS, mix, stopper the tube, and place it in a water-bath
at 60 °C for exactly 25 minutes. Cool the tube rapidly to 20 °C {solution A). To
the second tube add 10ml of water and mix {solution B).

10ml of imidazole/mercuric chloride TS A
and water for solution B.
for solution
calculate the amount of C] 6 H] 8 N 205 S in the sample being examined by com-
parison with phenoxymethylpenicillin potassium RS. In an adequately cali-
brated spectrophotometer the absorbance of the reference solution should be
0.63 + 0.03.
1084
Dissolution. For tablets other than those that are intended to be chewed
before swallowing, carry out the test as described under 5.5 Dissolution test
for solid oral dosage forms, using as the dissolution medium, 500ml of disso-
lution buffer, pH 6.8, TS and rotating the paddle at 75 revolutions per minute.
At 30 minutes withdraw a sample of about 5 ml of the medium directly through
an in-line filter. Measure the absorbance of the filtered sample, suitably diluted
if necessary, at the maximum at 268 nm. At the same time measure the
absorbance at the maximum at 278 nm of a suitable solution of phe-

noxymethylpenicillin potassium RS in dissolution buffer, pH 6.8, TS.
For each of the six tablets tested, calculate the total amount of phenoxyme-
thylpenicillin, Ci6HigN205S, in the medium from the absorbances obtained and
from the declared content of Ci6HiaN205S in phenoxymethylpenicillin potas-
sium RS. The average amount of phenoxymethylpenicillin, Ci6HiaN205S, in
solution is not less than 85% of the amount declared on the label. If the amount
obtained for one of the six tablets is less than 80%, repeat the test using a
further six tablets; the average amount for aU 12 tablets tested is not less than
85%.
PHENYTOINI NATRICI COMPRESSI

PHENYTOIN SODIUM TABLETS

tial medicines: 25 mg, 50 mg, 100 mg.
Requirements
Phenytoin sodium tablets contain not less than 90 0. % and not more than
110 0 %
. of the amount of C]5HnN2Na02 stated on the label.
Identity tests
To a quantity of the powdered tablets equivalent to about 0.1 g of Phenytoin

sodium add 20 ml of water, shake, and filter. Acidify the filtrate with hydrochlo-
ric acid (~70g/l) TS and extract with chloroform R. Wash the chloroform extract
with water, dry with anhydrous sodium sulfate R, and evaporate to dryness.
Use the residue for tests A and B.
1085
trophotometry in the infrared region. The infrared absorption spectrum
isconcordant with the spectrum obtained from phenytoin RS or with the
reference spectrum of phenytoin.

chloroform R and 1 volume of acetone R as the mobile phase. Apply sepa-
rately to the plate 10 pi of each of the following 2 solutions in chloroform
R. For solution (A) use 1 mg of the residue per ml. For solution (B) use 1 mg
of phenytoin RS per ml. After removing the plate from the chromatographic
chamber, allow it to dry, and examine the chromatogram in ultraviolet light
(254 nm).

C. To a quantity of thepowdered tablets equivalent to about 40 mg of Pheny-

toin sodium add 2.0ml of ammonia (~100g/l) TS and heat until boiling
begins. Add 1 drop of copper(ll) sulfate (160g/l) TS and shake; a blue-violet
solution with a blue-green precipitate is produced. Allow to stand for 3
minutes, filter, and wash with water; pink needles remain on the filter.
D. To a quantity of the powdered tablets equivalent to about 40 mg of Pheny-

toin sodium add 5 ml of water, shake, and filter. The filtrate yields reaction
B described under 2.1 General identification tests as characteristic of sodium.
Benzophenone. Carry out the test as described under 1.14.1 Thin-layer chro-
volumes of hexane R and 30 volumes of dioxan R as the mobile phase. Apply
separately to the plate 5 pi of each of the following 2 solutions. For solution (A)
shake a quantity of the powdered tablets equivalent to about 0.1 g of Phenytoin
sodium with 5 ml of methanol R, warm on a water-bath while shaking, filter,
and use the filtrate. For solution (B) use 0.10 mg of benzophenone R per ml of
methanol R. After removing the plate from the chromatographic chamber, allow
Any spot corresponding to benzophenone obtained with solution A is not more

Assay. Weigh and powder 20 tablets. Transfer to a separatory funnel a quan-

tity powdered tablets equivalent to about 0.3 g of Phenytoin sodium, add
of the
25ml and shake. Add 50ml of ether R, shake, and add 10 drops of
of water,
bromophenol blue/ethanol TS. Titrate with hydrochloric acid (0.1 mol/1) VS,
shaking vigorously, until the colour of the aqueous layer turns to bluish-grey.
Transfer the aqueous layer to a stoppered conical flask. Wash the ether layer
1086
with 5 ml of water and combine the washing with the aqueous layer in the
conical flask. Add 20 ml of ether R and continue the titration with hydrochlo-
ric acid (0.1 mol/1) VS, shaking vigorously, until the colour of the aqueous layer
turns to pale green.

Ci5HiiN2Na02.
PIPERAZINI ADIPATIS COMPRESSI

PIPERAZINE ADIPATE TABLETS
Category. Intestinal anthelminthic drug.

tial medicines: the equivalent of 500mg of piperazine hydrate.
600 mg of piperazine adipate is approximately equivalent to 500 mg of piper-

azine hexahydrate.
Requirements
Piperazine adipate tablets contain not less than 93 0 % and not more than
.
107 0 % . of the amount of C 4HioN 2 ,C 6 Hio04 stated on the label.
Identity tests
A. See the test described below under “Related substances”. Examine the chro-
matograms after spraying with the two triketohydrindene hydrate reagent
solutions. The principal spot obtained with solution B corresponds in posi-
tion, appearance, and intensity with that obtained with solution C.
B. Shake a quantity of the powdered tablets equivalent to 1 g of Piperazine

adipate with 20 ml of water for 5 minutes and filter. To 10 ml of the filtrate
(keep the remaining filtrate for test C) add 5 ml of hydrochloric acid

(-250 g/1) TS and allow to stand for 10 minutes; a crystalline precipitate is
formed. Filter,wash the precipitate 2-3 times with 3-ml portions of cold
water, and dry at 105 °C; melting temperature, about 152 °C (adipic acid).
1087
C. To the remaining filtrate obtained in test B add 0.5 g of sodium nitrite R.

Heat to boiling and cool if necessary to induce
in ice for 15 minutes, stirring
crystallization. Filter, wash with 10 ml of ice-water, and dry the precipitate
at 105 °C; melting temperature, about 158 °C (N,N'-dinitrosopiperazine).
chromatography, using silica gel R1 as the coating substance and a freshly pre-
pared mixture of 2 volumes of ammonia (-260 g/1) TS and 8 volumes of acetone
R as the mobile phase. Prepare 500 ml of the following mixture of solvents
for solutions B, C, D, E, and F: mix 2 volumes of ethanol (-750 g/1) TS with 3
volumes of ammonia (-260 g/1) TS. Apply separately to the plate 5 pi of each
of the following six solutions. For solution (A) shake a quantity of the pow-
dered tablets equivalent to Ig of Piperazine adipate with a mixture of 6ml of
ammonia (-260 g/1) TS diluted to 10ml with water, and use the clear fil-
filter,
trate. For solution (B) dilute 1ml of solution A to 10ml with the mixture of
solvents. For solution (C) dissolve 0.1 g of piperazine adipate RS in sufficient
mixture of solvents to produce 10ml. For solution (D) dissolve 25mg of ethyl-
enediamine R in sufficient mixture of solvents to produce 100 ml. For solution
(E)dissolve 25 mg of triethylenediamine R in sufficient mixture of solvents to
produce 100ml. For solution (F) dissolve 12.5 mg of triethylenediamine R in
5 ml of test solution A and dilute to 50ml with the mixture of solvents. After
removing the plate from the chromatographic chamber, allow to dry at 105°C
and spray successively with a 3 mg/ml solution of triketohydrindene hydrate R
dissolved in a mixture of 3 volumes of glacial acetic acid R and 100 volumes of
1-butanol R and then with a 1.5mg/ml solution of triketohydrindene hydrate
R in ethanol (-750 g/1) TS. Dry the plate at 105°C for 10 minutes and examine
intense than that obtained with solution D. Spray the plate with iodine
(0.05mol/l) VS, allow to stand for 10 minutes, and examine the chromatogram
in daylight.
Any spot corresponding to triethylenediamine obtained with solution A is not

more intense than that obtained with solution E. The test is valid only if the
chromatogram obtained with solution F shows two clearly separated spots.
Disregard any spot remaining on the line of application.

to about 0.2 g of Piperazine adipate, accurately weighed, add 10 ml of water
and shake for 1 hour. Filter and wash the residue with two portions, each of
10 ml, of water. To the combined extract and washings add 5 ml of sulfuric acid
(-1760 g/1) TS and 100 ml of trinitrophenol (7 g/1) TS, heat on a water- bath for
15 minutes, and allow to stand for 1 hour. Filter, wash the residue with
successive quantities of trinitrophenol (7 g/1) TS, using 10ml each time, and
1088
finally wash with dehydrated ethanol R. Dry the residue to constant mass at
lOS^C.
Each g of residue is equivalent to 426.8 mg of C4HioN2,C6Hio04.
PIPERAZINI CITRATIS COMPRESSI

PIPERAZINE CITRATE TABLETS
Category. Intestinal anthelminthic drug.
Storage. Piperazine citrate tablets should be kept in a tightly closed container.

Requirements
Piperazine citrate tablets contain not less than 93 0 % and not more than
.
107 0 %
. of the amount of (C4HioN2)3,2CsHa07 stated on the label.
Identity tests
A. See the test described below under “Related substances”. Examine the chro-
matograms after spraying with the two triketohydrindene hydrate reagent
solutions. The principal spot obtained with solution B corresponds in posi-
tion, appearance, and intensity with that obtained with solution C.
B. Mix a quantity of the powdered tablets equivalent to 0.2 g of Piperazine

citrate with 5 ml of hydrochloric acid (~70g/l) TS and filter. To the filtrate
add 0.5 g of sodium nitrite R and shake to dissolve. Cool in ice for 15
minutes, stir if necessary to induce crystallization, filter, wash with 10 ml of
ice-water, and dry the precipitate at 105°C; melting temperature, about
158 °C (N,N’-dinitrosopiperazine).
C. To a quantity of the powdered tablets equivalent to 0.5g of Piperazine

citrate add 10 ml of water, shake, and filter. The filtrate yields reaction A
described under 2.1 General identification tests as characteristic of citrates.
chromatography, using silica gel R1 as the coating substance and a freshly pre-
pared mixture of 2 volumes of ammonia (-260 g/1) TS and 8 volumes of acetone
1089
R as the mobile phase. Prepare 500ml of the following mixture of solvents for
solutions B, C, D, E, and F: mix 2 volumes of ethanol (~750g/l) TS with 3
volumes of ammonia (-260 g/1) TS. Apply separately to the plate 5 pi of each
of the following six solutions. For solution (A) shake a quantity of the pow-
dered tablets equivalent to Ig of Piperazine with a mixture of 6ml of
citrate
ammonia (-260 g/1) TS diluted to 10 ml with water,filter, and use the clear
filtrate. For solution (B) dilute 1 ml of solution A to 10 ml with the mixture of
solvents. For solution (C) dissolve 0.1 g of piperazine citrate RS in sufficient

mixture of solvents to produce 10 ml. For solution (D) dissolve 25 mg of ethyl-
enediamine R in sufficient mixture of solvents to produce 100 ml. For solution
(E)dissolve 25 mg of triethylene diamine R in sufficient mixture of solvents to
produce 100ml. For solution (F) dissolve 12.5mg of triethylenediamine R in
5 ml of test solutionA and dilute to 50ml with the mixture of solvents. After
removing the plate from the chromatographic chamber, allow to dry at 105°C
and spray successively with a 3 mg/ml solution of trike tohydrindene hydrate R
dissolved in a mixture of 3 volumes of glacial acetic acid R and 100 volumes of
1-butanol R and then with a 1.5 mg/ml solution of triketohydrindene hydrate
R in ethanol (~750g/l) TS. Dry the plate at 105°C for 10 minutes, and examine
intense than that obtained with solution D. Spray the plate with iodine
(0.05mol/l) VS, allow to stand for 10 minutes, and examine the chromatogram
in daylight.
Any spot corresponding to triethylenediamine obtained with solution A is not

more intense than that obtained with solution E. The test is valid only if the
chromatogram obtained with solution F shows two clearly separated spots.
Disregard any spot remaining on the line of application.

to about 0.2 g of Piperazine citrate, accurately weighed, add 10 ml of a mixture
of 1 part of hydrochloric acid (-70g/l) TS and 3 parts of water, and shake for
1 hour. Filter and wash the residue with two portions, each of 10ml, of water.
To the combined extract and washings add 100 ml of trinitrophenol (7 g/1) TS,
heat on a water-bath for 15 minutes, and allow to stand for 1 hour. Filter, wash
the residue with successive quantities of trinitrophenol (7 g/1) TS, using 10ml
each time, and finally wash with dehydrated ethanol R. Dry the residue to
constant mass at 105 °C.
Each g of residue is equivalent to 393.5 mg of (C4HioN2)3,2C6H807.
1090
PRAZIQUANTELI COMPRESSI
PRAZIQUANTEL TABLETS

tial medicines: 150mg, 600mg.
Requirements
Praziquantel tablets contain not less than 90 0 % and not more than 110 0 %
. .
of the amount of C 19 H 24N 2 O 2 stated on the label.
Identity tests
To a quantity of the powdered about 0.1 g of Praziquan-

tel add 10 ml of chloroform R, shake, and filter. Evaporate the filtrate to dryness
and dry the residue at 50 °C under reduced pressure (not exceeding 0.6kPa or
about 5mm of mercury). Use the residue for tests A and D.
concordant with the spectrum obtained from praziquantel RS or with the
reference spectrum of praziquantel.
C. The absorption spectrum of the solution obtained in the “Assay”, when

264 nm and 272 nm.
D. Melting temperature of the residue, about 138 °C.
Apply separately to the plate, in a current of nitrogen R, 10 pi of each of the
following 2 solutions. For solution (A) shake a quantity of the powdered tablets
equivalent to about 0.25 g of Praziquantel with 5ml of chloroform R, filter, and
1091
use the filtrate. For solution (B) use 0.05 g of praziquantel RS per ml of chloro-
form R. Then apply 2 pi of each of the following 2 solutions in chloroform R

containing (C) 0.5 mg of praziquantel RS per ml, and (D) 1.0 mg of praziquan-
tel RS per ml. Allow the mobile phase to ascend 7 cm. After removing the plate

place in a chamber with iodine vapours, and allow to stand for 20 minutes.
Examine the chromatogram immediately in daylight.
intense than that obtained with solution C, except one spot above the main
spot which is not more intense than that obtained with solution D.

to about 25 mg of Praziquantel add 50 ml of ethanol (-750 g/1) TS, shake, and
dilute to volume with the same solvent. Filter and discard the first 5 ml of the
filtrate. Measure the absorbance of a 1-cm layer at the maximum at about
264 nm against a solvent cell containing ethanol (-750 g/1) TS. Calculate the
percentage content of C19H24N2O2 by comparison with a solution containing
0.50 mg of praziquantel RS per ml of ethanol (-750 g/1) TS.
PREDNISOLONI COMPRESSI
PREDNISOLONE TABLETS

tial medicines: Img, 5 mg.
Requirements
Prednisolone tablets contain not less than 90 0 . % and not more than 110 0 . %
of the amount of C21H28O5 stated on the label.
Identity tests
To a quantity of the powdered tablets equivalent to about 0.05 g of Pred-

nisolone add 10ml of acetone R, shake, and filter. Evaporate the filtrate to
dryness and use the residue for the “Identity tests” and “Related substances”.
1092
concordant with the spectrum obtained from prednisolone RS or with the
reference spectrum of prednisolone.

using kieselguhr R1 as the coating substance and a mixture of 9 volumes of
acetone R and 1 volume of formamide R to impregnate the plate, dipping
it about 5 mm into the liquid. After the solvent has reached a height of at
least 16cm, remove the plate from the chromatographic chamber and allow
it to stand at room temperature until the solvent has completely evaporated.
Use the impregnated plate within 2 hours, carrying out the chromatogra-
phy in the same direction as the impregnation. Use chloroform R as the
mixture of 9 volumes of chloroform R and 1 volume of methanol R con-
taining (A) 2.5mg of the residue per ml, and (B) 2.5mg of prednisolone RS
per ml. Develop the plate for a distance of 15 cm. After removing the plate
have evaporated, heat at 120 °C for 15 minutes, and spray the hot plate with
sulfuric acid/ethanol TS. Heat at 120 °C for a further 10 minutes, allow to
cool, and examine the chromatogram in daylight and in ultraviolet light
(365 nm).

C. To 5 mg of the residue add 1.0ml of ethanol (-750 g/1) TS and shake. Then
add 1.0ml of potassio-cupric tartrate TS and heat to boiling; an orange pre-
cipitate is produced slowly.
R and 1 volume of methanol R containing (A) 15 mg of the residue per ml, and
(B) 0.30 mg of the residue per ml. After removing the plate from the chro-
and heat at 105 °C for 10 minutes. Cool, and examine the chromatogram in

equivalent to about 20 mg of Prednisolone, add 15 ml of water, shake with four
1093
quantities, each of 25 ml, of chloroform R, and filter the chloroform layer

through cotton wool previously washed with chloroform R. Add sufficient
chloroform R to the filtrate to produce 250 ml and dilute 25 ml to 100 ml with
the same solvent. Transfer 10ml of the resulting solution to a glass-stoppered,
50-ml conical flask, carefully evaporate to dryness, and dissolve the residue in
20ml of dehydrated ethanol R. Transfer 20ml of dehydrated ethanol R to a
similar flask to serve as the blank. To each of the flasks add 2.0ml of blue tetra-
zolium/ethanol TS and mix. Then add to each flask 2.0 ml of tetramethyl-
ammonium hydroxide/ethanol TS, mix, and allow to stand in the dark for
90 minutes. Measure the absorbance of a 1-cm layer at the maximum at about
525 nm against a solvent cell containing the blank.
Calculate the percentage content of C 21 H 28 O 5 by comparison with prednisolone

For tablets containing I mg of Prednisolone. Individually transfer 10 powdered
tablets to 10 separate 100-ml volumetric flasks, add 50 ml of ethanol (-750 g/1)
TS, shake, and dilute to volume with the same solvent.
For tablets containing 5 mg of Prednisolone. Individually transfer 10 powdered

tablets to 10 separate 100-ml volumetric flasks, add 50 ml of ethanol (-750 g/1)
TS, shake, and dilute to volume with the same solvent. Dilute 2.0ml to 10ml
with ethanol (-750 g/1) TS. Measure the absorbance of a 1-cm layer of the solu-
tions at the maximum at about 242 nm.
Calculate the tablet content of C 21 H 28 O 5 in mg by comparison with pred-

nisolone RS. The tablets comply with the test for 5.1 Uniformity of content for
single-dose preparations.
PREDNISOLONI ET NATRII
PHOSPHATIS INJECTIO
PREDNISOLONE SODIUM
PHOSPHATE INJECTION
Description. A colourless solution.
Storage. Prednisolone sodium phosphate injection should be protected from

lightand stored at a temperature not exceeding 15 °C.
1094
Labelling. The designation on the container should state the amount of active
ingredient as the equivalent quantity of prednisolone in a suitable dose volume.
It should also state whether any buffering agent is added.

tial medicines: 20 mg, 25 mg in vials.
Prednisolone sodium phosphate injection contains a suitable stabilizing agent,

and may contain a suitable buffering agent. The solution is sterilized by
“Filtration” or by another suitable method (see 5.8Methods of sterilization).
Requirements
Definition. Prednisolone sodium phosphate injection is a sterile solution of

prednisolone sodium phosphate in water for injections.
Prednisolone sodium phosphate injection contains not less than 90 0 % and not
.
more than 110 0 % . of the equivalent amount of prednisolone C21H28O5 stated

on the label.
Identity tests
using silica gel R4 as the coating substance and 6 volumes of 1 -butanol R,
2 volumes of acetic anhydride R, and 2 volumes of water as the mobile
phase, prepared immediately before use. Apply separately to the plate 5|il
of each of the following four solutions. For solution (A) dilute a volume of
the injection to obtain a concentration of the equivalent of 2 mg of pred-
nisolone per ml. For solution (B) dissolve 27 mg of prednisolone sodium
phosphate RS in 10 ml of water. For solution (C) prepare a mixture of equal

volumes of solutions A and B. For solution (D) mix 2 ml of solution B with
2 ml of a solution containing 29 mg of betamethasone sodium phosphate RS
in 10 ml of water. After removing the plate from the chromatographic
chamber, allow it to dry in air until the odour of solvent is no longer per-
ceptible, heat to 110°C for 10 minutes, and examine the chromatogram in

ance, and intensity with that obtained with solution B. The chromatogram
obtained with solution D shows two principal spots with almost the same
Revalues. Additional spots due to pharmaceutical aids may be observed in
the chromatograms obtained with solutions A and C.
1095
B. Evaporate a volume of the injection equivalent to 2 mg of prednisolone

to dryness on a water-bath. Dissolve the residue in 2 ml of sulfuric acid
(-1760 g/1) TS and allow to stand for 2 minutes; a red colour is produced.
Assay. Dilute an accurately measured volume of the injection equivalent to

about 20mg of prednisolone with sufficient water to produce 200ml. To 25ml
of this solution add 2.5 g of sodium chloride R and 1ml of hydrochloric acid
(0.1 mol/1) VS, mix, and shake with three quantities, each of 25ml, of chloro-
form R. Wash each chloroform extract with 1 ml of hydrochloric acid (0.1 mol/1)
VS, add the washings to the aqueous solution, and discard the chloroform
layers. Extract the aqueous solution with two quantities, each of 10ml, of trib-
utyl phosphate R and dilute the combined extracts to 25ml with methanol R.
Transfer 2 ml to a stoppered tube and add 10 ml of isoniazid TS, heat to 50 °C
for 3 hours, protecting the solution from light, and cool.
Measure the absorbance in a 1 -cm layer at the maximum at about 405 nm. Sim-
ilarly prepare a blank solution, omitting the injection to be examined. Repeat
the procedure using 25ml sodium phosphate RS
of a solution of prednisolone
containing the equivalent of O.lOmg/ml of prednisolone. Determine the latter
by diluting an aliquot with water, measuring the absorbance of the dilution at
the maximum at about 247 nm. Calculate the content of C 21 H 28 O 5 in the injec-
tion being examined by comparison with the absorbances obtained and the
exact strength of the solution of prednisolone sodium phosphate RS, using 419
as the h\°tra at the maximum at 247 nm.
PREDNISOLONI ET NATRII SUCCINATIS

PULVIS AD INJECTIONEM
PREDNISOLONE SODIUM SUCCINATE
Description. A white powder or friable lumps; odourless.
Labelling. The designation on the container should state the dose as the equiv-
alent amount of prednisolone. It should also state the nature of the buffering
agent. Expiry date.

tial medicines: 20 mg, 25 mg in vials.
1096
Requirements
Definition. Prednisolone sodium succinate powder for injections is a sterile

powder of prednisolone sodium succinate prepared from prednisolone succi-
nate with the aid of sodium hydroxide or sodium carbonate.
Prednisolone sodium succinate powder for injections usually contains a suit-

able buffering agent. The powder is sterilized by a suitable method (see 5.8
Methods of sterilization).
The container of Prednisolone sodium succinate powder for injections contains

not less than 90 0 % and not more than 110 0 % of the equivalent amount of
. .
prednisolone C21H28O5 stated on the label.
Identity tests
A. Dissolve a quantity of the powder for injections equivalent to 40 mg of pred-

nisolone in 20ml of water, add 3ml of hydrochloric acid (~70g/l) TS, and
extract with 25 ml of chloroform R. Filter the extract into a beaker, evapo-
rate to dryness on a water-bath, and dry the residue at 60 °C for 1 hour.
Carry out the examination as described under 1.7 Spectrophotometry in the
spectrum obtained from prednisolone succinate RS or with the reference spec-
trum of prednisolone succinate.

using silica gel R4 as the coating substance and 6 volumes of 1 -butanol R,
2 volumes of acetic anhydride R, and 2 volumes of water as the mobile
phase, prepared immediately before use. Apply separately to the plate 5 pi
of each of the following two solutions. For solution (A) dissolve a quantity
of the powder for injections to obtain a concentration equivalent to 2 mg of
prednisolone per ml. For solution (B) dissolve 28 mg of prednisolone succi-
nate RS in 10 ml of water. After
removing the plate from the chromato-
graphic chamber, allow it to dry in air until the odour of solvent is no longer
perceptible, heat to 110 °C for 10 minutes, and examine the chromatogram
in ultraviolet light (254 nm).

1097
C. To a quantity of the powder for injections equivalent to 2 mg of pred-

nisolone add 2ml of sulfuric acid (~1760g/l) TS and allow to stand for
2 minutes; a red colour is produced.
D. Ignite a quantity of the powder for injections equivalent to 20 mg of pred-

nisolone and dissolve the residue in acetic acid (~60g/l) TS. The solution
yields reaction B described under 2.1 General identification tests as charac-
teristic of sodium.
Clarity of solution. A solution containing a quantity of the powder for injec-

tions equivalent to 0.35g of prednisolone in 10ml of carbon-dioxide-free water
R is clear. (Keep this solution for the “pH value’’.)
Loss on drying. Dry the powder for injections at 60 °C under reduced pres-
sure (not exceeding 0.6kPa or 5mm of mercury) over phosphorus pentoxide R
for 3 hours; it loses not more than 20mg/g.
pH value. pH of the solution prepared above for the test of clarity, 6. 5-8.0.
Assay
Dissolve a portion of the powder for injections equivalent to about 0.05g of

prednisolone, accurately weighed, in 5 ml of water and dilute with sufficient
ethanol (~750g/l) TS to produce 200ml. Dilute 4ml to 100ml with ethanol
(-750 g/1) TS. Transfer 20ml of the resulting solution to a glass-stoppered
50-ml conical flask {solution A). Separately, dissolve 64 mg of prednisolone suc-
cinate RS, accurately weighed, in 100ml of ethanol (-750 g/1) TS, add 5ml of
water, and dilute with sufficient ethanol (-750 g/1) TS to produce 200 ml. Dilute
4ml to 100ml with ethanol (-750 g/1) TS. Transfer 20ml of the resulting solu-
tion to a glass-stoppered 50-ml conical flask {solution B). To each flask contain-
ing solutions A and B and a third one containing 20ml of ethanol (-750 g/1) TS
to serve as a blank, add 2 ml of blue tetrazolium/ethanol TS, mix, then add
2 ml of tetramethylammonium hydroxide/ethanol TS, mix, and allow to stand
in the dark for 90 minutes.
Without delay measure the absorbance of solutions A and B against the blank,
using a suitable spectrophotometer at a maximum wavelength of about 525 nm.
Calculate theamount of C2)H2805 in the substance being examined using the

formula 5C(0.7827)(A„/A,) where C is the concentration in mg per ml of pred-
nisolone succinate RS, 0.7827 is the ratio of the relative molecular mass of pred-
nisolone to that of prednisolone succinate, and A„ and A, are the absorbances
of solutions A and B, respectively.
1098
prednisolone.
PRIMAQUINI DIPHOSPHATIS COMPRESSI

PRIMAQUINE DIPHOSPHATE TABLETS
Labelling. The designation on the container indicates the quantity of active

ingredient in terms of the equivalent amount of primaquine.

tial medicines: 7.5mg and 15mg of primaquine base.
The tablets may be coated.
Requirements
Primaquine diphosphate tablets contain not less than 90 0

110 0 %
. of the amount of C] 5 H 2 iN 30 stated on the label.
Identity tests
• Either tests A and D or tests B, C, and D may he applied.
A. To powdered tablets equivalent to 60 mg of Primaquine

a quantity of the
add a mixture of 10ml of water and 2ml of sodium hydroxide (~80g/l) TS,
and shake with two 20-ml quantities of chloroform R. Wash the chloroform
extracts with water, dry with anhydrous sodium sulfate R, and evaporate
to dryness. Dissolve the residue in 2 ml of chloroform R and carry out the
examination as described under 1.7 Spectrophotometry in the infrared
region.The infrared absorption spectrum is concordant with the spectrum
obtained from primaquine diphosphate RS similarly treated or with the ref-
erence spectrum of primaquine.
B. Shake a quantity of the powdered tablets equivalent to 15mg of Primaquine

with 100ml of hydrochloric acid (0.01 mol/1) VS and filter. The absorption
spectrum of this solution, when observed between 310 nm and 450 nm,
exhibits two maxima at about 332 nm and 415nm. The absorbances of a
1-cm layer at these wavelengths are between 45 and 52, and 27 and 35,
1099
respectively. Dilute 5 ml of the solution to 50 ml with hydrochloric acid

(0.01 mol/1) VS. The absorption spectrum of this solution, when observed
between 215nm and 310nm, exhibits three maxima at about 225nm,
265 nm, and 282 nm. The absorbances of a 1-cm layer at these wavelengths
are between 495 and 515, 335 and 350, and 330 and 345, respectively.
C. Shake a quantity of the powdered tablets equivalent to 25 mg of Primaquine

with 10 ml of water and filter. To 2 ml of the filtrate add 3 ml of water and
1 ml of ceric ammonium sulfate/nitric acid TS; a deep violet colour is imme-

diately produced. (Keep the remainder of the filtrate for test D.)
D. To the filtrate remaining from test C add 3 ml of nitric acid (-130 g/1) TS; it

alent to about 0.15g of Primaquine, accurately weighed, to a separatory funnel,
add 20 ml of water and 5 ml of sodium hydroxide (-80 g/1) TS, and extract with
four 25-ml quantities of chloroform R. Evaporate the combined chloroform
extracts to a volume of about 10 ml, add 40 ml of glacial acetic acid Rl, and
titrate with perchloric acid (0.1 mol/1) VS, determining the end-point potentio-
metrically as described under 2.6 Non-aqueous titration. Method A.
Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 12.97 mg of C 15 H 21 N 3 O.
PROBENECIDI COMPRESSI
PROBENECID TABLETS

Requirements
Probenecid tablets contain not less than 95 0 % and not more than 105 0 % of
. .
the amount of C13H19NO4S stated on the label.
Identity tests
1100
Triturate a quantity of the powdered tablets equivalent to 0.5g of Probenecid

with ethanol (~750g/l) TS, filter, concentrate the filtrate to a small volume by
evaporation, cool, and filter. RecrystaUize the residue from ethanol (-457 g/1)
TS and use it for the following two tests:

infrared region.
spectrum obtained from probenecid RS or with the reference spectrum of
probenecid.
B. Melting temperature of the residue, about 199 °C.
C. The absorption spectrum of the solution prepared for the assay, when
225 nm and 248 nm; the absorbance of a 1-cm layer at the maximum of
248 nm is between 310 and 350.
15 volumes of Tpropanol R and 3 volumes of ammonia (~17g/l) TS as the
mobile phase. Apply separately to the plate 5 pi of each of the following two
solutions. For solution (A) shake a quantity of the powdered tablets equivalent
to 0.2 g of Probenecid with 10 ml of a mixture of 1 volume of ammonia
(~17g/l) TS and 9 volumes of ethanol (~750g/l) TS, centrifuge, and use the
supernatant liquid. For solution ml of solution A to 100 ml with the
(B) dilute 1
same mixture of solvents. After removing the plate from the chromatographic
light (254 nm).

to about 0.2 g of Probenecid, accurately weighed, add 200ml of ethanol
(-750 g/1) TS and 5 ml of hydrochloric acid (1 mol/1) VS. Heat on a water-bath
at 70 °C for 30 minutes, shaking the flask occasionally, cool, add sufficient
ethanol (-750 g/1) TS to produce 250ml, and filter. To 5 ml of the filtrate, add
5ml of hydrochloric acid (0.1 mol/1) VS, and dilute to 250ml with ethanol
(-750 g/1) TS.

about 248 nm and calculate the content of C] 3 Hi 9 N 04 S, using the absorptivity
value of 33.2 (A\°t = 332).
Disintegration test. Complies with the test for 5.3 Disintegration test for
tablets and capsules. Time period: 30 minutes.
1101
PROCAINI BENZYLPENICILLINI
PROCAINE BENZYLPENICILLIN
Storage. Procaine benzylpenicillin powder for injections should be protected

from light and stored at a temperature not exceeding 25 °C.
Labelling. The designation on the container should state the nature of the
buffering agent. Expiry date.

tial medicines: 1
g and 3g.
Requirements
The powder for injections and the reconstituted preparation for injections
comply with the monograph for “Parenteral preparations”.
Definition. Procaine benzylpenicillin powder for injections is a sterile powder

of procaine benzylpenicillin.
Procaine benzylpenicillin powder for injections usually contains a suitable

buffering agent. The powder is sterilized by a suitable method (see 5.8 Methods
of sterilization). The reconstituted preparation is a suspension.
The container of Procaine benzylpenicillin powder for injections contains not

less than 90 0 % and not more than 110 0 % of the amount of total penicillins
. .
calculated as Ci(sHigN204S,Ci3H2oN202 and not less than 36 0 % and not more .
than 44 0 % of C13H20N2O2 both stated on the label.

.
Identity tests
A. To a quantity of the powder for injections equivalent to 2 mg of Procaine
benzylpenicillin in a test-tube add 0.05ml of water followed by 2 ml of
sulfuric acid (~1760g/l) TS and mix; the solution almost colourless.
is
Immerse the test-tube for 1 minute in a water-bath; the solution remains

almost colourless. Place the same quantity of the powder for injections in a
TS, and mix; the solution is almost colourless and after a few minutes the
1102
colour changes to yellow-brown. Immerse the test-tube for 1 minute in a

water-bath; a reddish brown colour is produced.
B. Dissolve a quantity of the powder for injections equivalent to 10 mg of Pro-

1 0 ml of water and add 0.5 ml of neutral red/ ethanol
caine benzylpenicilKn in
TS. Add sodium hydroxide (0.01 mol/1) VS to give a permanent
sufficient
orange colour and then add 1 ml of penicillinase TS; the colour changes
rapidly to red.
C. A quantity of the powder for injections equivalent to 50 mg of Procaine ben-

zylpenicillin yields the reaction described for the identification of primary
aromatic amines under 2.1 General identification tests, producing a bright,

Fischer method. Method A, using a quantity of the powder for injections equiv-
alent to 0.5 g of Procaine benzylpenicillin; the water content is not less than
Assays
Mix the contents of 10 containers and carry out the assays as described.
Total penicillins. Suspend a quantity of the powder for injections equivalent

to about 0.045 g of Procaine benzylpenicillin, accurately weighed, in sufficient
water to produce 1000 ml. Transfer two 2-ml aliquots of this suspension into
separate stoppered tubes. To one tube add 10ml of imidazole/mercuric chlo-
ride TS, mix, stopper the tube, and place in a water-bath at 60 °C for exactly
25 minutes. Cool the tube rapidly to 20 °C {solution A). To the second tube add
10ml of water and mix {solution B).

calculate the amount of Ci6HiaN204S,Ci3H2oN202 in the substance being exam-
ined by comparison with 0.050 g of benzylpenicillin sodium RS, taking into
account that each mg of benzylpenicillin sodium RS (CisH]7N2Na04S) is equiv-
alent to 1.601 mg of C](;HiaN204S,Ci3H2oN202. In an adequately calibrated spec-
Procaine. Suspend a quantity of the powder for injections equivalent to about

0.5 g of Procaine benzylpenicillin, accurately weighed, in 10ml of water, add
5ml of sodium carbonate (75g/l) TS, and extract with four successive quan-
tities, each of 25 ml, of chloroform R, filter the chloroform extracts, and evap-
1103
orate to a small volume on a water-bath. Add 20 ml of hydrochloric acid

(0.1 mol/1) VS and distil off the remaining chloroform. Cool, add 0.25ml of
methyl red/ethanol TS, and back-titrate with sodium hydroxide (0.1 mol/1) VS.

C13H20N2O2.
benzylpenicillin.
PYRANTELI EMBONATIS COMPRESSI

PYRANTEL EMBONATE TABLETS

tial medicines: 250 mg of pyrantel.
Requirements
Pyrantel embonate tablets contain not less than 90 0 . % and not more than
110 0 %
. of the amount of CiiHi 4 N 2 S,C 23 Hi <;05 stated on the label.
Identity tests
To a quantity of the powdered tablets equivalent to about 0.05g of Pyrantel

embonate add a mixture of 10 ml of chloroform R, 10 ml of methanol R, and
about 1ml of ammonia (~260g/l) TS, shake, and filter. Evaporate the filtrate to
dryness on a water-bath, dissolve in a small volume of methanol R, and allow
to recrystallize. Separate the crystals, dry at 80 °C for 2 hours, and use the dried
crystals for the “Identity tests” and “Related substances”.
A. Carry out the examination with the dried crystals as described under 1.7
is concordant with the spectrum obtained from pyrantel embonate RS or
with the reference spectrum of pyrantel embonate.
1104
C. Dissolve 5mg of the dried crystals in 1.0ml of hydrochloric acid (~70g/l) TS

and add 1 .0 ml of formaldehyde/sulfuric acid TS; a violet-red colour is produced.
D. Dissolve about 2mg of the dried crystals in 2ml of sulfuric acid (~1760g/l)
TS; a yellow colour is produced which changes to orange and finally to red.
20 volumes of ethyl acetate R, 5 volumes of methanol R, and 1.5 volumes
of diethylamine R as the mobile phase. Apply separately to the plate 100ml of
each of 3 solutions in a mixture of 5 volumes of chloroform R, 5 volumes of
methanol R, and 0.5 volume of ammonia (-260 g/1) TS containing (A) 20 mg
of the dried crystals per ml, (B) 20 mg pyrantel embonate RS per ml, and (C)
0.20 mg of the dried crystals per ml. After removing the plate from the chro-
matographic chamber, allow it to dry in a current of air for 10 minutes, and
Assay
The operations described below must be carried out in subdued light and
without any prolonged interruptions, preferably using low-actinic glassware.
Weigh and powder 20 tablets. To a quantity of the powdered tablets equiva-

lent to about 0.1 g add 10ml of dioxan R and 10ml of ammonia (100 g/1) TS,
and shake for 10 minutes. Dilute to 100ml with perchloric acid (-140 g/1) TS,
filter, discard the first 10 ml of the filtrate, and transfer 5 ml of the subsequent
filtrate to a 50-ml volumetric flask. Dilute to volume with perchloric acid
(-140 g/1) TS and mix. Transfer 25ml to a 250-ml separatory funnel, add 100ml
of chloroform R, and shake well. Drain off the chloroform layer into a second
separatory funnel. Repeat the extraction of the aqueous phase with a second
100-ml portion of chloroform R, and combine the chloroform extracts into the
same separatory funnel. Add 40 ml of hydrochloric acid (0.05 mol/1) VS to the
combined chloroform extracts and shake well. Drain off the chloroform phase
into a third separatory funnel and extract with a further 40-ml portion of
hydrochloric acid (0.05 mol/1) VS, discarding the chloroform phase. Combine
the aqueous phases in a 100-ml volumetric flask, rinse the separatory funnel
volume with hydrochloric acid
draining into the volumetric flask, and dilute to
(0.05mol/l) VS. Measure the absorbance of a 1-cm layer at the maximum at
about 31 1 nm against a solvent cell containing hydrochloric acid (0.05 mol/1) VS.
1105
Calculate the percentage content of CnH]4N2S,C23Hi605 by comparison with

pyrantel embonate RS, similarly and concurrently examined.
PYRAZINAMIDI COMPRESSI
PYRAZINAMIDE TABLETS

Requirements
Pyrazinamide tablets contain not less than 93 0 . % and not more than 107 0 % .
of the amount of C5H5N3O stated on the label.
Identity tests
A. Shake a quantity of the powdered tablets equivalent to 0.25 g of Pyrazi-

namide with 20 ml of dehydrated ethanol R, filter, and evaporate the filtrate
to dryness. Dry the residue at 105 °C for 30 minutes and carry out the exam-
ination as described under 1.7 Spectrophotometry in the infrared region.
The is concordant with the
infrared absorption spectrum of the residue
spectrum obtained from pyrazinamide RS or with the reference spectrum of
pyrazinamide.
B. Shake a quantity of the powdered tablets equivalent to 0.050 g of Pyrazi-

namide with 50ml of water and filter. Dilute 1ml of the filtrate to 100ml
with water. The absorption spectrum of this solution, when observed
between 230 nm and 350 irm, exhibits two maxima at about 268 nm and
310nm. The ratio of the absorbance of a 1-cm layer at 268 nm to that at
310nm is between 11.6 and 12.0.
C. To a quantity of the powdered tablets equivalent to 0.06g of Pyrazinamide

add 5 ml of sodium hydroxide (-80 g/1) TS and heat on a water-bath; vapours
are evolved. Insert moistened pH-indicator paper R into the vapours; the
colour of the paper changes to an alkaline range, and an odour of ammonia
is perceptible.
1106
6 volumes of 1-butanol R, 2 volumes of glacial acetic acid R, and 2 volumes of
water as the mobile phase, but allowing the solvent front to ascend 10 cm above
the line of application. Apply separately to the plate 20 pi of each of the fol-
lowing two solutions. For solution (A) shake a quantity of the powdered tablets
equivalent to 0.1 g of Pyrazinamide with 50ml of a mixture of 9 volumes of
chloroform R and 1 volume of methanol R, and filter. Evaporate the filtrate to
dryness and dissolve the residue in sufficient mixture of solvents to produce
10ml. For solution (B) dilute 1 volume of solution A to 500 volumes with the
same mixture of solvents. After removing the plate from the chromatographic
chamber, allow it to dry in air, and examine the chromatogram immediately in
Assay. Weigh and powder 20 tablets. Add a quantity of the powder equiva-
lent to about 0.1 g of Pyrazinamide, accurately weighed, to 200 ml of water,
allow to stand for 10 minutes, swirling occasionally, mix in an ultrasonic bath
for 10 minutes, and dilute to 500 ml with water. Filter and discard the first
20ml of filtrate. Dilute 5 ml of the filtrate to 100 ml with water.

about 268 nm. Calculate the content of C5H5N3O, using the absorptivity value
of 65.0 (A;°™ = 650).
QUININI DIHYDRO CHLORIDI INJECTIO

QUININE DIHYDROCHLORIDE INJECTION
Description. Quinine dihydrochloride is an almost colourless to light yellow
solution.
Storage. Quinine dihydrochloride injection should be protected from light.
Labelling. The designation on the container should state that the solution
must be diluted to a strength not exceeding 30 mg per ml before
administration.
Additional information. Care must be taken to ensure slow intravenous

administration.
1107
Strength in the current WHO Model list of essential medicines: 300mg/ml.
Requirements
Definition. Quinine dihydrochloride injection is usually a concentrated, sterile

solution of quinine dihydrochloride in water for injections.
The solution is sterilized by “Heating in an autoclave” or any other suitable

method (see 5.8 Methods of sterilization).
Quinine dihydrochloride injection contains not less than 95 0 and not more . %
than 105 0 . %
of the amount of C2oH24N2Q2,2HCl stated on the label.
Identity tests
A. Dilute a volume of the injection to a concentration of 0.5 mg of Quinine
dihydrochloride per ml. To 10 ml of this solution add 0.05 ml of sulfuric acid
(~100g/l) TS; a strong blue fluorescence is produced. (Keep the solution for
test B.)
B. To the solution prepared for test A add 0.15ml of bromine TSl and 1 ml of
ammonia (~100g/l) TS; an emerald-green colour is produced.
C. The injection yields reaction A described under 2.1 General identification

mixture of 10 volumes of chloroform R, 8 volumes of acetone R, and 2.5 volumes

of diethylamine R as the mobile phase. Apply separately to the plate 5 pi of each
of the following four solutions. For solution (A) dilute a volume of the injection
with ethanol (-750 g/1) TS to obtain a concentration equivalent to 10 mg of
Quinine dihydrochloride per ml. For solution (B) dissolve 12.5mg of quinine R
in 50ml of ethanol (-750 g/1) TS. For solution (C) dissolve 12.5mg of cinchoni-
dine R in 50 ml of ethanol (-750 g/1) TS. For solution (D) mix 1 ml of solution B
with 1ml of solution C. After removing the plate from the chromatographic
chamber, allow it to dry in a current of air for 15 minutes and repeat the devel-
opment. Heat the plate at 105°C for 30 minutes, allow to cool, spray with potas-
sium iodoplatinate TS, and examine the chromatogram in daylight.
intense than that obtained with solution B or solution C. If any spot is obtained
1108
g
with solution A
immediately below the principal spot, it is disregarded. The
test is valid only if the chromatogram obtained with solution D shows two
distinctly separated spots.
Limit of dihydroquinine. Dilute an accurately measured volume of the injec-

tion equivalent to 0.2 g of Quinine dihydrochloride with water to 20ml. Add
0.5g of potassium bromide R, 15ml of hydrochloric acid (-70g/l) TS, and
Titrate the iodine liberated by excess potassium bromate in the solution with
has reached a light yellow colour. Repeat the procedure without the injection
solution being examined and make any necessary corrections.
Each ml of potassium bromate (0.0167 mol/1) VS is equivalent to 19.87 mg of

C2 oH 24N202,2HC1. Express the results of both the above determination and the
assay in percentages. The difference between the two is not more than 10%.

about 0.5 of Quinine dihydrochloride to a separator and add 20ml of water
and 5 ml of sodium hydroxide (-200 g/1) TS. Extract with successive quantities,
each of 10 ml, of chloroform R until complete extraction of the alkaloid is
effected. Wash each extract with the same two quantities, each of 5ml, of
water. Evaporate the combined chloroform layer, dissolve the residue in 50ml
of acetic anhydride R, add 10ml of glacial acetic acid R, and titrate with per-
Method A.

C 2 „H 24N 202 2 HC 1
,
.
STREPTOMYCINI SULFATIS
STREPTOMYCIN SULFATE
Description. A white or almost white powder; odourless or with a slight
odour.
Categoiy. Antituberculosis drug.
1109
Storage. Unless otherwise recommended by the manufacturer, the reconsti-

tuted suspension should be used within 4 days when stored at a temperature
between 2 and 8°C and protected from light.
Labelling. The designation on the container should state the dose in the
equivalent amount of streptomycin. It should also state the nature of the buffer-
ing agent, preservatives, and stabilizers. Expiry date.

tial medicines: 1 g of streptomycin base.
Requirements
Definition. Streptomycin sulfate powder for injections is a sterile powder of

streptomycin sulfate.
Streptomycin sulfate powder for injections may contain suitable buffers, preser-
and stabilizers. The powder is sterilized by a suitable method (see 5.8
vatives,
Methods of sterilization).
Identity tests
A. Carry out the test as described under 1.14.1 Thin-layer chromatography, but
using the coating prepared as follows: to 0.3 g of carbomer R add 240ml of
water, mix, and shake moderately for 1 hour; then add gradually while stir-
ring a sufficient volume of sodium hydroxide (~80g/l) TS to adjust to pH

7.0. To this mixture add 30 g of silica gel R3 and coat the plate with a layer
0.75mm thick. Heat the plate to 100 °C for 1 hour, cool, and use immedi-
ately. Use potassium dihydrogen phosphate (70g/l) TS as the mobile phase.
Apply separately to the plate 10|il of each of the following three solutions.
For solution (A) dissolve a quantity of the powder for injections equivalent
to 5 mg ml of water. For solution (B) dissolve
of Streptomycin sulfate in 5
lOmg of streptomycin sulfate 10ml of water. For solution (C) dissolve
RS in
1 mg of kanamycin monosulfate RS and 1 mg of framycetin sulfate RS in
1 ml Develop the plate for a distance of 12 cm. After remov-
of solution B.
ing the platefrom the chromatographic chamber, allow it to dry in a current
of warm and spray with a mixture of equal volumes of naphthalene-1, 3-
air,
diol/ethanol TS and sulfuric acid (-635 g/1) TS. Heat the plate to 150 °C for
5-10 minutes and examine the chromatogram in daylight.
1110

if the chromatogram obtained with solution C shows three clearly separated
spots,
B. Dissolve a quantity of the powder for injections equivalent to 0.1 g of Strep-

tomycin sulfate in 4 ml of water, add 1 ml of sodium hydroxide (-80g/l) TS,
and heat on a water-bath for 5 minutes. Cool and add 1.5 ml of hydrochlo-
ric acid (~70g/l) TS and 1 drop of ferric chloride (25g/l) TS; an intense violet
colour is produced.
C. Dissolve a quantity of the powder for injections equivalent to 0.1 g of Strep-

tomycin sulfate in 2 ml of water, add 1 ml of 1-naphthol TSl and 2 ml of a
mixture of equal volumes of sodium hypochlorite (~40g/l) TS and water;

a red colour is produced.
D. A solution containing a quantity of the powder for injections equivalent to

50 mg of Streptomycin sulfate yields reaction A described under 2.1 General
identification tests as characteristic of sulfates.

equivalent to 2.5 g of Streptomycin sulfate in 10 ml of carbon-dioxide-free water
R is clear and colourless. (Keep this solution for the “pH value”.)
Loss on drying. Dry the powder for injections at 60 °C under reduced pres-
sure (not exceeding 0.6kPa or 5 mm of mercury) for 3 hours; it loses not more
than 70mg/g.
5.0-8.0.
Assays
Mix the contents of 10 containers and carry out the assays as described.
Potency. Carry out the assay as described under 3.1 Microbiological assay of
antibiotics, using either (a) Bacillus subtilis (NCTC 8236, or ATCC 1 1774) as the
test organism, culture medium Cml with a final pH of 7. 9-8.0, sterile phos-
phate buffer, pH 8.0, TSl or TS2, an appropriate concentration of streptomycin
(usually between 5 and 20IU/ml), and an incubation temperature of 36-39 °C,
or (b) Bacillus subtilis (ATCC 6633) as the test organism, culture medium Cml
with a final pH of 8. 0-8.1, sterile phosphate buffer, pH 8.0, TSl or TS2, an
appropriate concentration of streptomycin (usually between 3 and 151U/ml)
and an incubation temperature of 35-37 °C. The precision of the assay is such
that the fiducial limits of error of the estimated potency (P = 0.95) are not less
than 95% and not more than 105% of the estimated potency.
nil
The upper fiducial limit of error is not less than 720 lU per mg calculated with
reference to the dried substance. Using the average contents and estimated
potency, calculate the total number of units of streptomycin in the container.
In places where the microbiological determination is not feasible perform the following
alternative assay.
Streptomycin sulfate. Dissolve a quantity of the powder for injections equiv-

alent to about 0.1 g of Streptomycin sulfate, accurately weighed, in sufficient
water to produce 100 ml. To 5 ml add 5 ml of sodium hydroxide (0.2 mol/1) VS
and heat in a water-bath for exactly 10 minutes. Cool in ice for exactly 5
minutes. Add 3ml of ferric ammonium sulfate TS2, as well as sufficient water
to produce 25ml, mix, and allow to stand for exactly 20 minutes after the addi-
tion of the last reagent.
Immediately measure the absorbance of a 1-cm layer at the maximum at about

525 nm, against a solvent cell containing a solution prepared in the same
manner but omitting the powder for injections being examined. Calculate the
content of (C2]H39N70j2)2,3H2S04 in the powder for injections, using the
absorptivity value of 1.18 = 11.8). Using the result and the average mass
of contents, calculate the total content of streptomycin in the container, using
the conversion 800 mg of streptomycin being equivalent to 1 g of Streptomycin
sulfate. The container of Streptomycin sulfate powder for injections contains
not less than 90.0% and not more than 115,0% of the amount of C21H39N7O12
streptomycin.

1112
Monographs
Radiopharmaceuticals
CopyrigfiJed i
Radiop’harniaceuticals
RADIOPHARMACEUTICALS
The handling and testing of radiopharmaceuticals (radioactive pharmaceuticals)
require specialized techniques in order that correct results may be obtained and
hazards to personnel minimized. All operations should be carried out or super-
vised by personnel who have received expert training in handling radioactive
materials.
Definitions
Nuclide
A species of atom characterized by its mass number, atomic number, and
nuclear energy state, provided that the mean life in that state is long enough
to be observable.
Radioactivity
The property of certain nuclides of emitting radiation by the spontaneous trans-
formation of their nuclei into those of other nuclides.
EXPLANATORY NOTE. The term “disintegration” is widely used as an

alternative to the term “transformation”. Transformation is preferred as it
includes, without semantic difficulties, those processes in which no parti-

cles are emitted from the nucleus.
Radionuclide
A nuclide that is radioactive.
Units of radioactivity
The activity of a quantity of radioactive material is expressed in terms of the
number of nuclear transformations taking place in unit time. The SI unit of
activity is the becquerel (Bq), a special name for the reciprocal second (s“'). The
expression of activity in terms of the becquerel therefore indicates the number
of disintegrations per second. One curie (Ci) equals 3.7 x 10“Bq.
The conversion factors between becquerel and curie and its submultiples
are given in “Units of measurement”.
Half-life period
The time in which the radioactivity decreases to one-half its original value.
EXPLANATORY NOTE. The rate of radioactive decay is constant and char-

acteristic for each individual radionuclide. The exponential decay curve is
described mathematically by the equation
N=
1115
where N is the number of atoms at elapsed time t, is the number of atoms

when = t 0, and 'k is the disintegration constant characteristic of each indi-
vidual radionuclide. The half-life period is related to the disintegration
constant by the equation
0.693
Radioactive decay corrections are calculated from the exponential equation,

or from decay tables, or are obtained from a decay curve plotted for the
particular radionuclide involved (see Fig. 1).
Radioactive concentration
The radioactive concentration of a solution refers to the radioactivity in a unit
volume of the solution. As with all statements involving radioactivity, it is nec-
essary to include a reference date of standardization. For radionuclides with
30 days, the time of standardization should be
a half-life period of less than
expressed to the nearest hour. For radionuclides with a half-life period of less
than one day, a more precise statement of the reference time is required.
Specific radioactivity (or specific activity)

The specific activity of a preparation of a radioactive material is the radioac-
tivity per unit mass of the element or of the compound concerned.
0 0.5 1 1.5 2 2.5 3 3.5

Half-lives
Figure 1. Master Decay Chart.
1116
Radiafkarmaceutkak
EXPLANATORY NOTE. It is usual to specify the radionuclide concerned

and also it is necessary to express the time thus: “ImCi of iodine-131 per
mg of D-iodohippuric acid at 12.00 hours UT on 1 January 1979", or “40MBq
of selenium-75 per mg of selenomethionine on 1 January 1979".
Specific radioactivity is often not determined directly but is calculated
from a knowledge of the radioactive concentration of the solution and of
the chemical concentration of the radioactive compound. Thus, if a solution
contains xmCi of per ml, and if the is entirely in the chemical form
of sodium o-iodohippurate of which the concentration is ymg per ml, then

at that time the specific activity is:
x/y mCi of iodine-131 per mg of o-iodohippuric acid.
Where necessary, the radiochemical purity of the preparation (see below)

must be taken into account.
The term employed in radiochemical work is “specific activity". As the
word “activity" has other connotations in a pharmacopoeia, the term should,
where necessary, be modified to “specific radioactivity" to avoid ambiguity.
Radionuclidic purity
The radionuclidic purity of a preparation is that percentage of the total radioac-
tivity that is present in the form of the stated radionuclide.
EXPLANATORY NOTE. Some radionuclides decay into nuclides that are

themselves radioactive: these are referred to as mother (or parent) and
daughter radionuclides, respectively. Such daughter radionuclides are often
excluded when calculating the radionuclidic purity; for example, iodine-131
will always contain its daughter xenon-131m, but this would not be con-
sidered an impurity because its presence is unavoidable.
In employing the definition, the radioactivity must be measured in
appropriate units: that is, in the number of nuclear transformations that
occur in unit time (in terms of curies or becquerels). If, for example, a prepa-
ration stated to be iodine-125 is known to contain 99mCi of iodine-125 and
1 mCi and no other radionuclide, then the preparation is said

of iodine-126,
to be of 99% It will be noted that the relative amounts
radionuclidic purity.
of iodine-125 and iodine-126, and hence the radionuclidic purity, will
change with time. An expression of radionuclidic purity must therefore
contain a statement of the time, such as: “Not more than 1% of the total
radioactivity is due to iodine-126 at the reference date stated on the label”.
In the case of radionuclides with a half-life period of less than 30 days the
reference hour should also be included.
It is clear that, in order to give a statement of the radionuclidic purity of
a preparation, the activities (and hence the identities) of every radionuclide
present must be known. There are no simple and certain means of identi-
fying and measuring all the radionuclidic impurities that might be present
in a preparation. An expression of radionuclidic purity must either depend
1117
upon the judgement of the person concerned, or it must be qualified by ref-

erence to the method employed, for example: “No radionuclidic impurities
were detected by gamma scintillation spectrometry using a sodium iodide
detector.”
Radiochemical purity
The radiochemical purity of a preparation is that percentage of the stated
radionuclide that is present in the stated chemical form. As radiochemical
purity may change with time, mainly because of radiation decomposition, the
time at which the radiochemical purity limit is applicable must be specified.
EXPLANATORY NOTE. If, for example, a preparation of cyanocobalamin

f^Co) is stated to be 99% radiochemically pure, then 99% of the cobalt-57
is present in the form of cyanocobalamin. Radiochemical impurities might
include such substances as cobaltous f^Co) ion or hydroxycobalamin f ^Co).
The possible presence of radionuclidic impurities is not taken into
account in the definition. If the radionuclidic impurity is not isotopic with
the stated radionuclide, then it cannot possibly be in the identical chemical
form. If the radionuclidic impurity is isotopic with the stated radionuclide,
it could be, and indeed is likely to be, in the same chemical form.
Radiochemical impurities may arise during the preparation of the mate-
rial or during storage, because of ordinary chemical decomposition or, what
is often more important, because of radiation decomposition (that is,

because of the physical and chemical effects of the radiation).
Production and handling of radiopharmaceuticals

The following paragraphs concern special considerations applying to the mono-
graphs on radiopharmaceuticals. The facilities for the production, use, and
storage of radiopharmaceuticals are generally subject to licensing by national
authorities. They often have to comply with 2 sets of regulations, those con-
cerned with pharmaceutical preparations and those concerned with radioactive
materials. Each producer or user must be thoroughly cognizant of the national
requirements pertaining to the articles concerned.
Carriers
The mass of radioactive material usually encountered in radioactive pharma-
ceuticals is often too small to be measured by ordinary chemical or physical
methods. Since such small amounts may not be subject to the usual methods
of separation and purification, a carrier, in the form of inactive material, either
isotopic with the radionuclide, or non-isotopic, but chemically similar to the
radionuclide, may be added during processing and dispensing to permit ready
handling. Thus sodium phosphate carrier is present in “Natrii Phosphatis (®^P)
Injectio” and rhenium is used as a carrier in certain colloidal preparations of
technetium-99m. The amount of carrier added must be sufficiently small for it
1118
.
Radiop’harniaceuticak
not to cause undesirable physiological effects. The mass of an element formed

in a nuclear reaction may be exceeded by that of the inactive isotope present
in the target material or in the reagents used in the separation procedures.
Radioactive preparations in which no carrier is intentionally added during
the manufacture or processing are often loosely referred to as carrier-free.
Detection and measurement

Radioactive transformations may involve the emission of charged particles, the
process of electron capture, or the process of isomeric transition. The charged
particles emitted from the nucleus may be alpha particles (helium nuclei of
mass number 4) or beta particles (electrons of negative or positive charge, |3-
or P+ respectively, the latter known as positrons). The emission of charged par-

ticles from the nucleus may be accompanied by gamma rays, which are of the
same physical nature as X-rays. Gamma rays are also emitted in the process of
isomeric transition (i.t.). X-rays, which may be accompanied by gamma rays,
are emitted in the process of electron capture (e.c.). Positrons are annihilated
on contact with matter. Each positron annihilated is accompanied by the emis-
sion of 2 gamma rays, each with an energy of 0.511 Me V.
The physical characteristics of radionuclides are summarized in Table 1
The methods employed for the detection and measurement of radioactiv-

ity are dependent upon the nature and energy of the radiation emitted. Radioac-
tivity may be detected and/or measured by a number of different instruments
based upon the action of radiation in causing the ionization of gases and solids,
or the fluorescence of certain solids and liquids, or by the effect of radiation on
a photographic emulsion.
In general, a counting assembly consists of a sensing unit and an electronic
scaling device. The sensing unit may be a Geiger-Mtiller tube, a proportional
counter, a scintillation detector in which a photomultiplier tube is employed in
conjunction with a scintillator, or a solid-state semiconductor.
Geiger-Muller counters and proportional counters are generally used for the
measurement of the beta emitters. Scintillation counters employing liquid or
solid phosphors may be used for the measurement of alpha, beta, and gamma
emitters. Solid-state devices may also be used for alpha, beta, and gamma mea-
surements. The electronic circuitry associated with a detector system usually
consists of a high-voltage supply, an amplifier, a pulse-height selector, and a
sealer, a ratemeter, or other readout device. When the electronic scaling device
or the sealer in a counting assembly is replaced by an electronic integrating
device, the resultant assembly is a ratemeter. Ratemeters are used for the
purpose of monitoring and surveying radioactivity and are somewhat less
precise as measuring instruments than the counters. Ionization chambers are
often used for measuring gamma-ray activities and, provided they are thin-
walled, for measuring X-rays.
Radiation from a radioactive source is emitted in all directions. Procedures
for the standardization and measurement of such sources by means of a count
of the emissions in all directions are known as 47t-counting; those based on
1119
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5.27
Physical
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Cobalt-60
Table
1120
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5.0% 2.0%
13.7
a. CO r<] ^ o o cN) Lo oo i\
o
G
LOK On^t-hvO'ÔO
cocNj ooo^'omcocNi
O CO VO N. CO
OCD cq^LO^Kcq^ O CO
O CM CO CO CN of™"'Xe)
-C VO K 0.506 0.523 0.621 0.630
RS OO CDOOOOoX o o o o o o o
e disintegrations
Vi 0 :2
—
*2
^
fs., 16.0%
“is O OO vq CM 3.5% 6.5% 3.0%

LO
X O K CO X
E
to
5jd 0 relate
ii u
y cu
S 8
iiR (percentages
si K O
M-
'=r VO VO
O O
CO
y y CM CO CO vq cq 0.84 1.01 1.07 1.09
(2 i w < o o o o
c>
o ^
a y
,>Ny p-
H -o
uL 0 h
2.29
=5
s
o
u
_c
Iodine-132
1122
<
Radiopharmaceuticals
0^
0.4% CO 80.7%
0.9%
X-rays)
X
K
(Au
vP \0 vO ^ ^ ^ ^
2.7%
98.7%
4.9% 5.2%
(N) T— o% d^
CO CO o CO CO ^
CNJ
q
19.2% -1.1%
-0.1%
-73%
'OI\C<jLO^cdcdc<jt\^'^’^ O CM O O LO O
0.067-0.080
KCOOCMLO^LOCMOSCO^CM
cmk-^^ldcooN-Oncocmo ^
o
»- CO CM LO CO ON CM CM
0.651 0.668 0.670 0.672
Khvqqq^qqq^qq-^
OOOOO^T-^^^^^CM o qo
3J
M- ON CO CO ON ON CO
CD O q q q q
CD O ^ ^ ^
0.077 0.192 0.268
0^
18.4% 10.8% K CM
-V.O
q K d^ 100%
2.6% 2.9%
CM
T-H
O
CM CO
LO CM q
ID
M- CM
CO CO N. \0
K ^
1.10 1.26 1.29 1.57 K CM -S q O O
q T-H
q
O CD
u I
e.c.
<6 cci.
CO ~o h
On "O
64.4
CM M-
.ercury-197
1123
transition:
internally nO
c^-
CO K O
^ C5^
emitted
photons
CO ^ LOCO ôôô^csiloCNjÔo^COo^
^ ^ O O O K
o ^
transitions
^
CO O CO
CN) \o -1
o
energy
Electromagnetic
photon K^' O On O hv
CO K O NO
On^ ^^t-hNOCSiOnÔCOCO'^
I\K ^•^CO'O'^CNICNI'^N.CNNO
O cNO o^^cO'^LoNqi\rvcqoN
MeV
2
’>
CN
O O O O oo ooooooooooo
transition
and
probabilities
CO ^ O?
energies
Particle
transition
NO cs
Lo NO CO 2^
energy ^ CO CSI -g
of
Type decay^
Half-life
period^
Nuclide
2 2
1124
^ ^
^ i\
Sp nP nP u
o% O
0.3% 3.0% \q On CN 0.1%
0\ CO O
X-rays)
K
(In
vP
On LO 0^ CO
1.1% 2.9%
^ O CO
CS| On ^ 21% 73%
0.024-0.028
^'OONLOO'^ '-'O ^'=d-0'=^-o

cmcoon^coo cNCOO-^ csi^co T-H
CO CO
CN LO
CO \o
^ ^
hv
0.066 0.097
T-Hr-HT-lCNJCNl'^'Ô _2pcNjcqo O O ^ 0.255
oooooo oo >0000 ooo T-H T-H
100% 100%
100%
1.709
'^’"’In
Daughter
p" e.c. e.c.
d d d
118.5
14.3
115
Phosphorus-32
SeIenium-75
Tin-113
1125
transitions
internally ^
CO cq
o 'd K
emitted
X X X
X X
<u
X
photons
X S? X X VO
transitions ^
° VO LO ^ o CO
(N
Lo
I O d
CO O O O CO o
energy
year,
Electromagnetic
photon
VO <N
^co'=foKco->-Hr\coO'^co
CSIVOO^^^COKO'^VOO
=
CO CO VO CO
ooOT-i^T-HT-i^T^CNicqcq a
MeV
oo oooo oo dddddddddddd day;
d
a
3
PS
hour;
transition
and
probabilities
o =
h
energies
minute;
transition
Particle
min
energy
X
second;
transition.
of =
s
Type decay^
isomeric
millisecond;
i.t.
Half-life
"O =
period^
VO
ms
capture;
microsecond;
electron
=
=
5 ==
o
Nuclide
w3 C l^s e.c.
'C
H X
1126
a count of the emissions in a solid angle of 2 ti steradians are known as 27t-

counting; and those based on a fraction of the emissions defined by the solid
angle subtended from the detector to the source are known as counting in a
fixed geometry. It is customary by
to assay the radioactivity of a preparation
comparison with a standardized preparation using identical geometry condi-
tions. The validity of such an assay is critically dependent upon the repro-
ducibility of the spatial relationships of the source to the detector and its
surroundings and upon the accuracy of the standardized preparation. In the
primary standardization of radionuclides coincidence techniques are employed
in preference to simple 4n-counting whenever the decay scheme of the radionu-
clide permits. One of the most commonly employed coincidence techniques is
471-beta/gamma coincidence counting, which is used for nuclides in which

some or all of the disintegrations are followed by prompt photon emission. An
additional adjacent detector, sensitive only to photons, is used to measure the
efficiency in the 47t-counter of those disintegrations with which the photons
are coincident. 47t-Gamma/gamma coincidence counting techniques are often
employed for the standardization of pure gamma emitters.
The construction and performance of instruments and accessory apparatus
vary. The preparation of samples must be modified to obtain satisfactory results
with a particular instrument. The operator must follow carefully the manufac-
turer’s instructions for optimum instrument performance and substantiate
results by careful examination of known samples. Proper instrument function-
ing and reliability must be monitored on a day-to-day basis through the use of
secondary reference preparations.
Radioactivity due to materials of construction, to cosmic rays, and to
spontaneous discharges in the atmosphere contributes what is known as the
background activity. All sample radioactivity measurements must be corrected by
subtracting background activity.
In the counting of samples at high activity levels, corrections must be made
also for loss of counts due to inability of the equipment to resolve pulses arriv-
ing in close succession. Such coincidence-loss corrections must be made prior
to the subtraction of background correction.
The corrected count rate, R, is given by the formula
where r is the observed count rate, and t is the resolving time,
A radioactivity count is a statistical value, i.e. it is a measure of nuclear

decay probabilities, and is not exactly constant over any given time interval.
The magnitude of the standard deviation is approximately equal to the square
root of the number of counts. In general, at least 10000 counts are necessary
to obtain a standard deviation of 1%.
1127
Absorption
Ionizing radiation is absorbed in the material surrounding the source of the
radiation. Such absorption occurs in air, in the sample itself (self-absorption),

in sample coverings, in the window of the detection device, and in any special
absorbers placed between the sample and the detector. Since alpha particles
have a short range of penetration in matter, beta particles have a somewhat
greater range, and gamma rays are deeply penetrating, identification of the type
and energy of radiation emitted from a particular radionuclide may be deter-
mined by the use of absorbers of varying thickness. In practice, this method is
little used, and then only in connection with beta emitters. However, variations
in counting rate due to variations in thickness and density of sample contain-
ers can be a major problem with beta emitters and with X-ray emitters, such
as iodine-125. Plastic tubes, in which variations of density and thickness are
minimal, are, therefore, often employed.
The absorption coefficient (p), which is the reciprocal of the "thickness”
expressed in mg/cm^, or the half-thickness (the thickness of absorber required
to reduce the radioactivity by a factor of two), is commonly determined to
characterize the beta radiation emitted by a radionuclide.
Method
The following procedure is used for the identification test in “Natrii Phosphatis
(^^P) Injectio” for the measurement of beta activity and for calculation of the
absorption coefficient of half-thickness:

Place the radioactive substance, suitably mounted under a suit-
for counting,
able counter. Make count rate determinations individually and successively,
using at least 6 different thicknesses of aluminium chosen from a range of
foil
10 to 200mg/cm^ and a single absorber with a thickness of at least 800mg/cm^.

The sample and absorbers should be as close as possible to the detector in order
to minimize scattering effects. Obtain the net beta count rate at the various
absorbers used by subtracting the count rate found with the thickest absorber
(800mg/cm^ or more). Plot the logarithm of the net beta count rate as a func-
tion of the total absorber thickness. The total absorber thickness is the thick-
ness of the aluminium plus the thickness of the counter window (as stated by
the manufacturer), plus the air-equivalent thickness (the distance, expressed in
cm, of the sample from the counter window multiplied by 1 ,205), all expressed
in mg/cm^. An approximately straight line results.
Choose two of the absorber thicknesses (q and tT) that are 20mg/cmôr
more apart and that fall on the plot, and calculate the absorption coefficient (p)
from the equation
where q is the thinner absorber, q is the thicker absorber, and A,j and A,^ rep-
resent the net beta count rate with q and q absorbers, respectively. Alterna-
tively the half-thickness may be read directly from the plot.
1128
Radiafkarniaceutkak
The choice of absorber thickness depends on the radionuclide. For radionu-

clides other than phosphorus-32, which have higher or lower beta energy,
greater or lesser absorber thicknesses are necessary.
For characterization of the radionuclide, the absorption coefficient or the
half- thickness should be within ±5%
of that found for a sample of the same
radionuclide of known purity when
determined in parallel.
The count rate at zero total may be determined by
absorber thickness
plotting a curve identical with the one described for determination of the
absorption coefficient and extrapolating the straight line plot to zero absorber
thickness, taking into consideration the thickness, expressed in mg/cm^, of
sample coverings, the air, and the counter window.
Radiation spectrometry
Crystal scintillation spectrometry
When the energy of beta or gamma radiation is dissipated within materials
known as scintillators, light is produced in an amount proportional to the
energy dissipated. This quantity of Light may be measured by suitable means,
and is proportional to the energy absorbed in the scintillator. The light emitted
under the impact of a gamma photon or a beta particle is converted into an
electric output pulse by a photomultiplier. Scanning of the output pulses with
a suitable pulse-height analyser results inan energy spectrum of the source.
The scintillators most commonly used for gamma spectrometry are single
crystals of thallium-activated sodium iodide. Gamma-ray scintillation spectra
show one or more sharp, characteristic photoelectric peaks, corresponding to
the energies of the gamma radiation of the source. They are thus useful for
identification purposes and also for the detection of gamma-emitting impuri-
ties in These peaks are accompanied by other peaks due to sec-
a preparation.
ondary effects of radiation on the scintillator and its surroundings, such as
backscatter, positron annihilation, coincidence summing, and fluorescent X-
rays. In addition, broad bands known as the Compton continua arise from the
scattering of the gamma photons in the scintillator and in surrounding materi-
als. Calibration of the instrument is achieved with the use of known samples
of radionuclides whose energy spectra have been characterized. The shape of

the spectrum produced will vary with the instrument used, owing to such
factors as differences in the shape and size of the crystal, in the shielding mate-
rials used, the distance between the source and the detector, and in the types
of discriminator employed in the pulse-height analysers. When using the spec-

trum for identification of radionuclides it is, therefore, necessary to compare
the spectrum with that of a known sample of the radionuclide obtained in the
same instrument under identical conditions.
Certain radionuclides, for example, iodine-125, emit characteristic X-rays of
well-defined energies that will produce photoelectric peaks in a suitable gamma
spectrometer. Beta radiation also interacts with the scintillators, but the spectra
are continuous and diffuse and generally of no use for identification of the
radionuclide or for the detection of beta-emitting impurities in a preparation.
1129
Semiconductor detector spectrometry

Gamma-ray and beta-particle spectra may be obtained using solid-state detec-
tors, The peaks obtained do not suffer to the same extent the broadening
shown in crystal scintillation spectrometry, and the resolution of gamma
photons of similar energies is very much improved. However, the efficiencies
of such detectors are much lower.
The energy required to create an electron-hole pair or to promote an elec-
tron from the valence band to the conduction band in a semiconductor is far
less than the energy required to produce a photon in a scintillation crystal. In
gamma-ray spectrometry a lithium-drifted germanium detector can provide an

energy resolution of 0.33% for the 1.33 MeV photon of cobalt-60, as compared
with 5.9% with a 7.6-cm x 7.6-cm thallium-activated sodium iodide crystal.
Licjuid scintillation counting
For weak beta emitters like '“'C and ^H, where self-absorption of the low-
energy beta particles is significant, the preferred counting method is by liquid
scintillation,which can occasionally be employed also for emitters of X-rays,

alpha particles, and gamma-rays. If the sample to be counted is dissolved in, or
mixed with, a solution of an appropriate scintillator material, the decay energy
from the sample is converted into light photons. These are sensed by a photo-
multiplier, which converts them into an electric pulse, whose intensity is pro-
portional to the energy of the initial radiation. Thus, simultaneous counting of
several radionuclides differing in the energy of emitted radiation can be effected
with suitable discriminators (pulse-height analysers), providing the energy sep-
aration is sufficient. Detection efficiencies approaching 95% for '^'C and 60%
for are reached because self-absorption is minimized.
The scintillator solute usually consists of a polycyclic aromatic compound,
such as /^-terphenyl or 2,5-diphenyloxazole (primary solute), together with
a secondary solute, such as l,4-di[2-(4-methyl-5-phenyloxazole)]benzene
(Dimethyl-POPOP), that shifts the wavelength of the light emitted to match
the highest sensitivity of the photomultiplier tube. Water-immiscible solvents,
such as toluene, or water-miscible solvents, such as dioxan, can be used. To
facilitate the counting of aqueous solutions, special solvents have been devel-
oped. Alternatively, samples may be counted as suspensions in scintillator gels.
As a means of attaining compatibility and miscibility with aqueous specimens
to be assayed, many additives, such as surfactants and solubilizing agents, are
also incorporated into the scintillator. For accurate determination of sample
radioactivity, care must be exercised to prepare a sample that is truly homoge-
neous. The presence of impurities and colour in the solution causes a decrease
in the number and energy of photons reaching the photomultiplier tube; such
a decrease is known as quenching. Accurate radioactivity measurement requires
correcting for count-rate loss due to quenching. Solutions containing organic
scintillators are prone to photoexcitation and samples may need to be prepared
in subdued light and kept in darkness before counting.
1130
Radiation shielding
Adequate shielding must be used to protect laboratory personnel from ioniz-
ing radiation, and measuring instruments must be suitably shielded from back-
ground radiation.
Alpha and beta radiations are readily shielded because of their limited range
of penetration, although the production oi Bremsstrahlung by the latter must be
taken into account (see below). The range of alpha and beta particles varies
inherently with their kinetic energy. The alpha particles are monoenergetic and
have a range of a few centimetres in air. The absorption of beta particles, owing
to their continuous energy spectrum and scattering, follows an approximately
exponential function. The range of beta particles in air varies from centimetres
to metres.
The secondary radiation produced by beta radiation upon absorption by
shielding materials is known as Bremsstrahlung and resembles soft X-rays in its
property of penetration. The higher the atomic number or density of the
absorbing material, the greater the intensity of the Bremsstrahlung produced.
Elements of low atomic number produce low-energy Bremsstrahlung, which is
readily absorbed; therefore, materials of low atomic number or of low density,
such as aluminium, glass, or transparent plastic, are used to shield sources of
beta radiation.
Gamma-ray radiation is deeply penetrating. Attenuation of gamma-ray radi-
ation in matter is exponential and is given in terms of half-value layers. The
half-value layer is the thickness of shielding material necessary to decrease the
intensity of radiation to half its initial value. A shield of 7 half-value layers is
of a thickness that will reduce the intensity of radiation to less than 1 % of its
unshielded intensity of activity. Gamma-ray radiation is commonly shielded

with lead.
Intensity of gamma-ray radiation is diminished according to the inverse
square of the intervening distance between the source and the point of refer-
ence. Radioactive materials of multimillicurie strength can be handled safely in
the laboratory by using proper shielding and/or by arranging for the maximum
practicable distance between the source and the operator by means of remote-
handling devices.
Determination of radionuclidic purity

For gamma emitters the most useful method of examination for radionuclidic
purity is gamma spectrometry. It is not, however, a completely certain method,
because:
{a) beta-emitting impurities are, in general, not detected;

{b) when sodium iodide detectors are employed, the photoelectric peaks due
to impurities may be obscured by those due to the major radionuclide,
or, in other words, the degree of resolution of the instrument is insuffi-
cient; this problem can be overcome through the use of high-resolution.
1131
solid-state, semiconductor detectors, such as a lithium-drifted germa-

nium (Ge:Li) detector;
(c) unless the instrument has been calibrated with a standard source of
known radionuclidic purity under identical conditions of geometry, it is
determine whether additional peaks are due to impurities

difficult to
or whether they result from such secondary effects as backscatter,

coincidence summation, or fluorescent X-rays.
The range of gamma spectrometry may be extended in two ways: first, by

observing changes in the spectrum of a preparation with time (this is especially
useful in detecting the presence of long-lived impurities in a preparation of

a short-lived radionuclide); secondly, by the use of chemical separations,
whereby the major radionuclide may be removed by chemical means and the
residue examined for impurities, or whereby specific impurities may be sepa-
rated chemically and then quantified. It is evident that chemical means will not
separate an impurity that is isotopic with the major radionuclide.
Requirements for radionuclidic purity

Requirements for radionuclidic purity are specified in two ways:
1. By expression of a minimum level of radionuclidic purity. Unless other-

wise stated in the individual monograph, the gamma-ray spectrum, as
determined by simple gamma spectrometry employing a sodium iodide
detector, should not be significantly different from that of a standardized
solution of the radionuclide before the expiry date is reached. As
discussed above, it is difficult to set more precise requirements for a
minimum level of radionuclidic purity.
2. By expression of maximum levels of specific radionuclidic impurities in
the individual monographs. In general, such impurities are those that are
known to be likely to arise during the production of the material - for
example, mercury-203 in a preparation of mercury-197.
It is evident that while the above requirements are necessary, they are not in
themselves sufficient to ensure that the radionuclidic purity of a preparation is
sufficient for human use. A duty must remain with the manufacturer to
examine his products in detail, and especially to examine preparations of short-
lived radionuclides for long-lived impurities after a suitable period of decay.
In this way, the manufacturer may satisfy himself that the manufacturing
processes employed are producing materials of appropriate purity. In particu-
lar, the radionuclidic composition of certain preparations is determined by the
chemical and isotopic composition of the target material, which is irradiated

with neutrons, and trial preparations are advisable when new batches of target
material are employed.
1132
Radiof’harniaceuticals
Determination of radiochemical purity

Radiochemical purity can be studied by a variety of techniques, but 1,14.2 Paper
chromatography and 1.14.1 Thin-layer chromatography are of particular impor-
tance. After completion of the separation, the distribution of radioactivity on
the chromatogram is determined. The weight of substance applied to the chro-
matogram is often extremely small (because of the great sensitivity of detec-
tion of the radioactivity) and particular care has to be taken in interpretation
with regard to the formation of artefacts. Instead of chromatography, elec-
trophoresis may be used for separation (see 1.15 Electrophoresis). As men-
tioned above, the addition of carriers (i.e. the corresponding non-radioactive
compounds) for both the radiopharmaceutical itself and the suspected impur-
ities issometimes helpful. There is, however, a danger that when an inactive
carrier of the radiopharmaceutical is added it may interact with the radio-
chemical impurity, leading to underestimation of these impurities. Another
useful technique involves monitoring the biological distribution of the injected
radiopharmaceutical in suitable test animals.
Determination of chemical purity

Chemical purity refers to the proportion of the preparation that is in the spec-
chemical form regardless of the presence of radioactivity; it
ified may be deter-
mined by normal methods of analysis.
The chemical purity of a preparation is often no guide to its radiochemical
purity. Preparations, especially those resulting from exchange reactions (for
example, a preparation of o-iodohippuric acid in which some of the iodine
atoms are replaced by atoms of iodine-131), may be of high chemical purity
but may contain impurities of high specific activity (that is, a tiny weight of an
impurity may be associated with a relatively large amount of the radionuclide).
In general, chemical impurities in preparations of radiopharmaceuticals are
objectionable only if they are toxic or if they modify the physiological processes
that are under study.
Tests for sterility and pyrogens

A number of monographs for radiopharmaceuticals contain the requirement
that the product be sterile and free of pyrogens. The half-life of radiopharma-
ceutical products is such that, as a rule only tests for pyrogens can be com-
pleted prior to release. Tests for sterility must, in general, be completed
retrospectively.
Sterility tests
The manufacturer should begin the sterility test as soon as possible and read
the results after release.
A particular responsibility falls upon the manufacturer of radiopharmaceu-
ticals to validate the sterilization process by all suitable measures, which may
1133
include careful and frequent calibration of sterilizers and the use of biological
and chemical indicators of the efficiency of the sterilization process.
Pyrogen tests
The manufacturer also bears a particular responsibility to ensure that all
substances used in the preparation of radiopharmaceuticals are handled in a

manner that ensures their freedom from pyrogens. Pyrogen tests are specified
in certain monographs where there are special dangers.
Addition of bacteriostatic agents

Injections of radiopharmaceuticals are commonly supplied in containers that
are sealed to permit the withdrawal of successive doses on different occasions.
The International Pharmacopoeia normally requires that such injections should
contain a suitable bacteriostatic agent in a suitable concentration.
Many common bacteriostatic agents (for example, benzyl alcohol) are grad-
ually destroyed by the effect of radiation in aqueous solutions. The rate of
destruction dependent upon a number of factors, including the nature of the
is
radionuclide and the radioactive concentration of the solution. It is, therefore,

not always possible to prescribe an effective bacteriostatic agent for an injec-
tion of a radiopharmaceutical and for certain preparations the addition of an
agent is undesirable; for this reason the inclusion of bacteriostatic agents is not
mandatory. The nature of the bacteriostatic agent, if present, must be stated on

the label; if no bacteriostatic agent is present, this must also be stated. Radio-
pharmaceuticals whose expiry periods are greater than one day and that do not
contain a bacteriostatic agent should preferably be supplied in single-dose
containers.
Other requirements
Radiopharmaceuticals administered parenterally should comply with the
relevant requirements for injections in the International Pharmacopoeia, except
that they are not subject to the requirements concerning volume of injection in
a single-dose container.
Expiry date
The special nature of a radiopharmaceutical requires that it be assigned an
expiry period (or an expiry date), beyond which its continued use is not rec-
ommended. The expiry period so designated begins with the date at which the
radioactivity is expressed on the label, and may be stated in terms of days,
weeks or months. For longer-lived radionuclides, the expiry period does not
exceed 6 months. The expiry period depends on the radiochemical stability and
the content of longer-lived radionuclidic impurity in the preparation under con-
sideration. At the end of the expiry period, the radioactivity will have decreased
1134
to the extent where insufficient radioactivity remains to serve the intended

purpose or where the dose of active ingredient must be increased so much that
undesirable physiological responses occur. In addition, chemical or radiation
decomposition may have reduced the radiochemical purity to an unacceptable
extent. Also the radionuclidic impurity content may be such that an unaccept-
able radiation dose would be delivered to the patient. The use of products
beyond their expiry periods is, therefore, inadvisable.
Labelling
In general, the label should include:
(1) the name of the preparation;

(2) a statement that the product is radioactive;
(3) the name and location of the manufacturer;

(4) the total radioactivity present at a stated date and hour (whenever the
half-life period is more than 30 days only the date need be stated);
(5) the expiry date or the expiry period;

(6) a number or other indication by which the history of the product may
be traced (for example, batch or lot number);
(7) in the case of solutions, the total volume of the solution;
(8) special storage requirements with respect to temperature and light
NOTE: In the case of a solution, instead of a statement of the total radioactiv-

ity, a statement of the radioactive concentration (for example, in mCi or MBq
per ml of the solution) may be given.
The shipment of radioactive substances is subject to special national and
international regulations as regards their packaging and outer labelling.
Storage
Radiopharmaceuticals should be kept in well-closed containers and stored in
an area assigned The storage conditions should be such that
for the purpose.
the maximum which persons may be exposed is reduced
radiation dose rate to
to an acceptable Care should be taken to comply with national regula-
level.
tions for protection against ionizing radiation. Glass containers may darken
under the effect of radiation.
1135
CopyrigfiJed i
Methods of Analysis
Methods of Analysis: Contents

1. Physical and Physicochemical Methods 1141
1.1 Measurement of mass 1141
1.2 Determination of melting temperature, melting
range, congealing point, boiling point, and boiling
range 1143
1.2.1 Melting temperature and melting range 1144
1.2.2 Congealing point 1147
1.2.3 Boiling point 1147
1.2.4 Boiling range 1148
1.3 Determination of mass density and relative
density 1150
1.4 Determination of optical rotation and specific
rotation 1151
1.5 Determination of refractive index 1154
1.6 Spectrophotometry in the visible and ultraviolet
regions 1154
1.7 Spectrophotometry in the infrared region 1160
1.8 Atomic spectrometry: emission and absorption 1164
1.9 Fluorescence spectrophotometry 1166
1.10 Turbidimetry and nephelometry 1169
1.11 Colour of liquids 1170
1.12 Powderfineness and sieves 1171
1.13 Determination of pH 1172
1.14 Chromatography 1175
1.14.1 Thin-layer chromatography 1176
1.14.2 Paper chromatography 1180
1.14.3 Column chromatography 1182
1.14.4 High performance liquid chromatography 1184
1.14.5 Gas chromatography 1193
1.15 Electrophoresis 1199
1.16 Phase solubility analysis 1202
2. Chemical methods 1206

2.1 General identification tests 1206
2.2 Limit tests 1211
2.2.1 Limit test for chlorides 1211
2.2.2 Limit test for sulfates 1211
2.2.3 Limit test for heavy metals 1212
2.2.4 Limit test for iron 1214
2.2.5 Limit test for arsenic 1216
1138
Methods of Analysis
2.3 Sulfated ash 1218

2.4 Oxygen flask method 1218
2.5 Complexometric titrations 1220
2.6 Non-aqueous titration 1223
2.7 Nitrite titration 1226
2.8 Determination of water by the Karl Fischer method 1227
2.9 Determination of methoxyl 1228
2.10 Determination of nitrogen 1229
3. Biological methods 1230

3.1 Microbiological assay of antibiotics 1230
3.2 Test for sterility 1236
3.2.1 Test for sterility of non-injectable
preparations 1240
3.2.2 Sterility testing of antibiotics 1241
3.3 Microbial purity of pharmaceutical preparations 1243
3.4 Test for bacterial endotoxins 1245
3.5 Test for pyrogens 1247
3.6 Test for histamine -like substances (vasodepressor
substances) 1249
3.7 Undue toxicity 1250
4. Methods for materials of plant origin 1251

4.1 Determination of ash and acid-insoluble ash 1251
4.2 Determination of iodine value 1251
4.3 Determination of peroxides in fixed oils 1252
4.4 Determination of saponification value 1252
4.5 Determination of unsaponifiable matter 1253
4.6 Determination of acid value 1254
4.7 Determination of hydroxyl value 1254
5. Pharmaceutical technical procedures 1255

Introduction 1255
5.1 Uniformity of content for single-dose
preparations 1255
5.2 Uniformity of mass for single-dose preparations 1256
5.3 Disintegration test for tablets and capsules 1258
5.4 Disintegration test for suppositories 1259
5.5 Dissolution test for solid oral dosage forms 1262
5.6 Test for extractable volume for parenteral
preparations 1273
1139
5.7 Visual inspection of particulate matter in

injectable preparations 1274
5.8 Methods of sterilization 1276
1140
Methods of Analysis
Methods of Analysis
1. Physical and physicochemical methods
1.1 Measurement of mass
Tests that involve the measurement of mass require the use of balances of
capacity and sensitivity corresponding to the degree of accuracy sought.
When weighing quantities of 50 mg or more that are to be “accurately
weighed”, an analytical balance of 100-200g capacity and 0.1 mg sensitivity is
required. When weighing quantities of less than 50 mg that are to be “accu-
rately weighed”, an analytical balance of 20 g capacity and 0.001 mg sensitiv-
ity, usually called an analytical microbalance, is required.
Balances of lower sensitivity are used for other tests in which a measure-
ment of mass is involved.
Apparatus
Analytical balances should possess adequate capacity and sensitivity. They may
be either of the equal-arm type, requiring the use of a set of calibrated weights,
or of any other suitable type (for example, analytical microbalances using mag-
netic measurement) provided that their performance is periodically checked by
means of a reference set of calibrated weights.
The analytical balance should be so constructed as to support its full capac-
ity without developing undue stress and its sensitivity should not be altered by
repeated weighings of the full-capacity load. It should preferably be equipped
with a damping device (for example, a magnetic or air damper) that causes the
beam to come quickly to rest (aperiodic balance).
The analytical balance may be constructed for manual placement of all
weights or, preferably, be equipped with a weight-loading device for the whole
or part of the balance range. In the latter case it should be equipped with loading
registers clearly indicating the load applied. Furthermore, the analytical balance
may be equipped with an optical scale projection system, usually encompass-
ing a part of the balance range (e.g. where the displacement of the projected
scale relative to the datum line gives a direct reading of weight), or a read-out
device of any other type.
The type of analytical balance having constant sensitivity over the whole
is the constant-load, single-pan balance. It has a set of weights
capacity range
suspended from a counterpoised beam; in the process of weighing, these are
removed from the beam by a manually operated mechanical device until equi-
librium is reached.
The analytical balance should be constructed in a proper housing with suit-
able openings to permit the placement of weighed material. The openings

should be constructed in such a way as to exclude air currents. Desiccants may
be placed inside the housing (e.g. silica gel, anhydrous calcium chloride) for the
maintenance of a relatively dry atmosphere.
1141
Sets of calibrated weights used with balances that require manual placement
of weights and sets of weights used to check the sensitivity of balances of another
type should be kept in a case made of suitable material and properly lined.
Placement of balance
The analytical balance should be placed upon a firm foundation that is as free
from mechanical vibration as possible, preferably on an antivibration table of
proper design. Alternatively, it may be placed on a concrete slab resting upon
piers that are either sunk into the ground or connected to the construction ele-
ments of the building; or it may be placed upon a stout table or shelf protected
by shock absorbers, such as cork mats or sheet rubber.
The balance should also be protected from humidity and acid fumes, prefer-
ably by placing it in a separate room of the laboratory. It should not be near a
window or radiator, in direct sunlight, or in a position where draughts may
come into contact with it.
The balance should be equipped with a levelling device and an indicator of
proper position. Proper adjustment of levelling should be frequently checked.
Checking of sensitivity
The sensitivity of the balance should be periodically checked by a qualified
expert.
Recommended procedure
Checking the stability of the equilibrium position
Before the balance is used, its equilibrium position without load should be
checked several times. After each test, the balance has to be arrested.
The equilibrium position of the balance under load should also be deter-
mined from time to time, for example, with one-tenth of the full load and with
the full load. The difference between equilibrium positions found in two suc-
cessive determinations made with equal loads should not exceed 0.1 mg for
analytical balances and 0.001 mg for analytical microbalances.
Operation of the balance

When the balance is not in use the balance beam and pan supports should be
raised. The doors of the housing should always be kept closed.
To release the balance, the beam and pans should be lowered very carefully.
Objects to be weighed must be allowed to attain the temperature of the
balance before weighing is The object to be weighed, as well as the
started.
weights, should always be placed on the pan as centrally as possible. During a
weighing or on any occasion when objects are being added to or removed from
the pans, both the beam arrests and the pan supports must be raised. Substances
must be weighed in suitable containers such as beakers, weighing bottles, or
crucibles. Liquids and volatile or hygroscopic solids must be weighed in tightly
1142
Methods of Analysis
closed vessels, such as stoppered weighing bottles. No chemicals or objects that

might injure the balance pans should be placed directly upon them.
When small quantities of a substance (for example, the sulfated ash) must
be weighed in a large vessel and a fairly long period elapses between the two
weighings, atmospheric pressure and temperature may alter sufficiently to
affect the buoyancy and thus cause an appreciable error. In two-pan balances,
this error may be eliminated by using another vessel of similar shape and
weight for taring.
The pans of the balance should be periodically lightly brushed with a camel-
hair or similar brush toremove any dust that may have collected.
The weights should be handled only by means of a pair of forceps, which
should possess tips covered with suitable material.
1.2 Determination of melting temperature, melting range,

congealing point, boiling point, and boiling range
The thermodynamically true melting point of a substance (the triple point) is a
physical constant that is indicative of the identity and purity of the material. It
is defined as the temperature at which the solid, liquid, and gaseous phases of
the substance are in equilibrium in an evacuated, closed system. Under normal
atmospheric pressure the solid and liquid phases of a substance are in equilib-
rium at a temperature that differs somewhat from the triple point but, since
pressure effects on the solid-liquid transition temperature are minimal, this dif-
ference does not, in general, exceed a few hundredths of a degree Celsius.
Methods for the determination of equilibrium melting points are laborious
and require complicated equipment. The usual practice is, therefore, to esti-
mate melting points by dynamic rather than equilibrium methods. The melting
points thus determined usually differ significantly from the corresponding triple
points. The magnitude of the deviation varies with the particular method
employed, with the criterion adopted as the “melting point”, and possibly with
the substance under examination. Melting points determined by the capillary
method of the International Pharmacopoeia are typically about one degree higher
than the thermodynamically true melting points.
Determination of melting points (called subsequently melting temperatures)
is used in pharmacopoeia! specifications primarily for identification of the sub-
stance concerned. The validity of the identification is greatly enhanced if the

so-called mixed-melting point procedure is applied. This involves an additional
determination to demonstrate that the substance being examined and a mixture
prepared of equal parts of this substance and an authentic specimen (reference
substance) of the substance melt at the same temperature. If the two substances
are not identical the mixture normally melts at a significantly lower tempera-
ture than the substance being examined, and the melting range is relatively
broad.
The presence of impurities in a substance results in a more or less pro-
nounced lowering of its melting point. Even more significant is that impurities
present in the substance may cause its melting range to be extended. In most
1143
cases where melting behaviour is used as a criterion of purity the International

Pharmacopoeia, therefore, prescribes determination of the melting range rather
than the melting point.
Similarly for liquids, determinations of boiling point and boiling range give
information that contributes to the identification and purity estimation of liquid
compounds. Practical considerations again dictate the use of methods that yield
apparent constants that may differ from the thermodynamically true values.
However, if the prescribed experimental conditions are closely adhered to, the
results obtained are of sufficient reproducibility.
1.2.1 Melting temp>erature and melting range

A. Determination of melting temperature and melting range of
pulverizable substances
The melting range of a substance is the range between the corrected tempera-
ture at which the substance begins to collapse or form droplets on the wall of
a capillary tube and the corrected temperature at which it is completely melted
as shown by the disappearance of the solid phase.
The statement in a monograph "melting range a-b°C” means that the
melting range determined by the method below must fall within these limits.
The melting temperature of a substance is the corrected temperature at which
it is completely melted as shown by the disappearance of the solid phase.
Apparatus
A suitable apparatus for the determination consists of a glass vessel with appro-
priate liquid, a controlled source of heat, a thermometer, a capillary tube, and
a magnifying glass.
The glass vessel should have a suitable construction, contain an appropri-
ate liquid and be fitted with a stirring device capable of rapid mixing of the
liquid (certain liquid silicones are suitable). The controlled source of heat should
be capable of raising the temperature of the liquid heating medium at the
required rate.
Standardized thermometers should cover the range -10 to -1-360 °C, the
length of one degree on the scale being not less than 0.8 mm. These ther-
mometers should preferably be of the mercury-in-glass, solid-stem type with
a cylindrical bulb and made of approved thermometric glass suitable for the
range covered; each thermometer should have a safety chamber.
Thermometers used for determination of melting temperatures may be cal-
ibrated for total or partial immersion. A total- immersion thermometer should read
correctly when immersed at least to the end of the liquid column in the
it is
medium, which is to be measured. A partialimmersion ther-

the temperature of
mometer should read correctly when it is immersed to a prescribed depth, and
when the emergent liquid column is under prescribed conditions. When total-
immersion thermometers are used partially immersed, an auxiliary thermome-
ter is required for the determination of the emergent-stem correction. These
1144
Methods of Analysis
two thermometers should be surrounded with a glass tube above the surface
of the heating material.
The capillary tube should be made of borosilicate glass, closed at one end,
and have the following dimensions: thickness of the wall, about 0.10-0. 15mm;
length, suitable for the apparatus used; internal diameter, 0.9-1. 1mm.
A suitable magnifying glass should be used for observation of the capillary tube.
Other apparatus or methods may be used provided they are capable of equal
accuracy and have been calibrated against the method of the International Phar-
macopoeia by means of the WHO Melting Point Reference Substances*.
Spread a small quantity of the finely powdered substance in a thin layer and
dry it in a vacuum desiccator over silica gel, desiccant, R, phosphorus pentox-
ide R, or other suitable desiccant for 24 hours, or at a temperature specified in
the monograph.
Transfer a quantity of the dried powder to a dry capillary tube and pack the
powder, for example, by tapping the tube on a hard surface, so as to form a
tightly packed column about 3 mm
in height. Introduce the capillary tube into
the heated bath at a temperature 5°C below the expected lower limit of the
melting range, the rise of temperature being regulated beforehand to 1 °C per
minute, unless either the temperature of the introduction of the capillary tube
into the bath or the rate of temperature rise are otherwise specified in the mono-
graph. The capillary tube should be fitted in the bath in such a way that its closed
end is at the level of the middle of the bulb of the standard thermometer.
When a
thermometer calibrated for partial immersion is used, care must be
taken that it is immersed exactly to its immersion mark when the readings are
taken.
Unless otherwise specified in the monograph, readings are taken of the tem-
perature at which is observed to collapse or form droplets on the
the substance
wall of the tube and of the temperature at which it is completely melted as
indicated by the disappearance of the solid phase.
To the temperature readings add the correction for deviation of the stan-
dard thermometer. When thermometers calibrated for total immersion are used
partially immersed, add to the readings of the standard thermometer also the
emergent-stem correction, which is obtained as follows:
Before starting the determination of the melting range, an auxiliary ther-

mometer is attached so that the bulb touches the standard thermometer at
a point midway between the graduation for the expected melting point and
the surface of the heating material. When the substance has melted the tem-
perature is read on the auxiliary thermometer. The correction to be added
^
A with melting points according to the International Pharmacopoeia in the tem-
set of substances
perature range +69 to +263 °C. The substances are available from the WHO Collaborating Centre
for Chemical Reference Substances, Apoteket AB, Produktion &: Laboratorier, Centrallaborato-
riet, ACL, Prismavagen 2, SE-141 75 Kungens Kurva, Sweden.
1145
to the temperature reading of the standard thermometer is calculated from

the following formula:
0.00015 N{T-t)
where T is the temperature reading of the standard thermometer;

t is the temperature reading of the auxiliary thermometer;
N isthe number of degrees of the scale of the standard thermometer
between the surface of the heating material and the level of the
mercury.
When needed, the emergent-stem correction for thermometers calibrated for

partialimmersion may be calculated from the same formula as above, but
replacing T by T^, which is the mean temperature of the emergent-stem of the
thermometer at the time of calibration.
Both the above-mentioned corrections for emergent-stem and any deviation
of the standard thermometer may conveniently be replaced by calibration of
the apparatus by means of the WHO
Melting Point Reference Substances.
B. Determination of melting point of fats, waxes, etc.

The melting point of fats, waxes, etc. is the corrected temperature at which
the column of substance in the capillary tube becomes transparent or moves
upwards, when tested by the method described below.
Apparatus
A similar apparatus to that described under A for the determination of melting
temperature and melting range of pulverizable substances should be used with
the following modifications:
— water should be used in the heating vessel;

— an accurately standardized thermometer should cover the range -10 to
-t-100°C;
—a capillary tube should have the same dimensions as described
glass
under A but be open at both ends; soft glass capillary tubes may be used.
Unless otherwise specified in the monograph, melt the substance at as low a
temperature as possible, and then suck the liquid up to a height of about
10mm in the capillary tube. Cool the charged tube at 10 °C or lower for 24
hours. If the monograph specifies that the melting temperature is to be deter-
mined without previous melting of the substance, charge the capillary tube by
pushing it into the unmelted substance so that a column about 10 mm long is
forced in. The determination may then be carried out immediately. Attach the
tube to the thermometer in the water-bath by means of a rubber band or oth-
erwise so that the lower end of the capillary tube is at the level of the middle
of the bulb of the thermometer, and the distance between the lower end of the
1146
Methods of Analysis
capillary tube and the water about 20 mm. Heat the bath with constant
level is
stirring, the heating being regulated so that the temperature rise, at a temper-
ature of 5 °C below the expected melting temperature, is about 1 °C per minute.
1.2.2 Congealing f'oint
The congealing point of a liquid or of a melted solid is the highest temperature

at which it solidifies. The congealing point of the liquid is the same as the
melting temperature of the solid, but since the liquid may be cooled to a
temperature below its congealing point without assuming the solid form, the
method described below is used to determine the congealing point of a liquid
or of a melted solid.
Apparatus
A suitable apparatus consists of a test-tube of about 2 cm internal diameter and
about 10 cm in length, suspended by means of a bored cork inside a larger tube,
about 3 cm in diameter and 12 cm in length, a vessel with water or suitable
freezing mixture, and an accurately standardized thermometer.
Unless otherwise specified in the monograph, place in the inner test-tube about
10ml of the liquid, or lOg of the melted solid, to be tested and cool together
the inner and the outer tubes in water or in a suitable freezing mixture to a
temperature about 5 °C below the expected congealing point of the liquid; with
the thermometer gently stir the liquid until it begins to solidify. At first there
is a gradual fall in temperature. Then, as the solid phase forms, the tempera-
ture remains constant for some time or rises before becoming constant. The
highest temperature observed is regarded as the congealing point. If the liquid
does not start to congeal within 2°C of the expected temperature, congelation
may be induced by adding a small crystal of the substance to the liquid or by
rubbing the inner walls of the test-tube with the thermometer.
1.2.3 Boiling f>oint
The boiling point of a liquid is the corrected temperature at which the liquid
boils under normal atmospheric pressure when determined by the method
described below.
Apparatus
A suitable apparatus for the determination consists of a vessel with appropri-
ate liquid, a source of heat, and a thermometer, as described under A for the
determination of melting temperature and melting range for pulverizable sub-
stances; also needed are a thin-walled test-tube of glass of external diameter
about 4mm and length suitable for the apparatus used and a thin-walled cap-
1147
illary tube of glass of internal diameter not exceeding 1mm, which should be
closed by fusing about 2 mm from one end.
Transfer 3-4 drops of the liquid to be tested (or the equivalent quantity of a
solid compound) to the test-tube. Place the capillary tube (fused end down) in
the test-tube and introduce the test-tube into the heating bath in such a way
that its lower end is at the level of the middle of the bulb of the thermometer.
Heat the bath rapidly with constant stirring to a temperature about 10 °C below
the expected boiling point, then regulate the heating so that the temperature
rise is1-2 °C per minute. During the heating bubbles begin to escape from the
lower end of the capillary tube, slowly at first but then more rapidly as the tem-
perature approaches the boiling point. Read the temperature at which bubbles
are released in an even rapid stream and then decrease the heating so that the
temperature of the bath falls 1-2 °C per minute. Read the temperature at which
the release of bubbles ceases. The boiling point is taken as the average of the
two temperatures, corrections for emergent-stem of the thermometer and for
deviation from normal atmospheric pressure being applied as necessary. Obtain
the emergent-stem correction as described under A for the determination of
melting temperature and melting range of pulverizable substances. If the deter-
mination is made at a barometric pressure that deviates from lOl.SkPa
(760mmHg), add to the temperatures the following correction:
k(p-pi)
where p is the standard barometric pressure;
Pi is the barometric pressure read on a mercury barometer, without

taking into account the temperature of the air; and
k is the boiling temperature increment, as indicated below.
For pressures read on a barometer calibrated in kPa, use the following data:
^=101.3
k = 0.3 (boiling temperature increment produced by a rise of pressure of 1 kPa),
unless otherwise specified in the monograph.
For pressures read on a barometer calibrated in mmHg, use the following data:
p = 760
k = 0.04 (boiling temperature increment produced by a rise of pressure of
1 mmHg), unless otherwise specified in the monograph.
1.2.4 Boiling range
The boiling range (distillation range) is the corrected range of temperature,

within which the whole or a specified portion of a liquid distils, under normal
atmospheric pressure, when determined by the method described below.
1148
Methods of Analysis
Apparatus
A suitable apparatus for the determination consists of a distillation flask, a con-
denser, a receiver, a heat sourcewith heat shields, and a thermometer.
The 50-60 ml capacity is made of heat-resistant glass.
distillation flask of
Flasks with the following dimensions are suitable: neck 10-12 cm long and
14-1 6 mm in internal diameter with a side-arm, 10-12 cm long and about 5 mm
in internal diameter, attached at about the midway point of the neck and
forming an angle of 70-75 ° with the lower portion of the neck.
The condenser is either a straight glass condenser, made of heat-resistant
glass, 55-60 cm in length with a water jacket about 40 cm in length, or a con-
denser of another design having equivalent condensing capacity. The lower end
of the condenser may be bent to provide a delivery tube, or a bent adapter may
be attached to it to serve as a delivery tube.
The receiver consists of a 25-50-ml measuring cylinder graduated in subdi-
visions of 0.5 ml.
The heat source consists of a small gas burner, preferably of a Bunsen type,
or an electric heater or mantle capable of adjustment comparable to that possi-
ble with the gas burner. If a gas burner is used, a suitable shield is placed around
the flask near its bottom. The shield is made of heat-resistant material in the form
of a square with sides of 14-1 6 cm and with a perforation in the centre. The
diameter of the latter should be such that when the flask is set into it, the portion
of the flask below the upper surface of the shield has a capacity of 3-4 ml.
The thermometer should preferably be calibrated for partial immersion of
100 mm as described under A for the determination of melting temperature
and melting range of pulverizable substances; otherwise a total-immersion ther-
mometer may be used with appropriate emergent-stem correction. When the
thermometer is placed in position, the stem should be located in the centre of
the neck of the flask, and the top of the bulb should be just below the bottom
of the outlet to the side-arm.
Place in the flask 25 ml any
of the liquid to be tested, taking care not to allow
of the liquid to enter the side-arm, and add 0. 3-0.5 g of glass-beads or other
suitable substance. Shield the burner and the flask from external air currents
and apply heat so that the vapour rises only slowly into the neck of the flask
and between 5-10 minutes elapse before the first drop of distillate falls from
the condenser. Continue the distillation at the rate of 2-3 ml per minute, col-
lecting the distillate in the receiver. Read the temperature when the first drop
of distillate falls from the condenser, and again when the last quantity of liquid
evaporates from the bottom of the flask or when the specified percentage has
distilled over.
The boiling ranges (distillation ranges) indicated in the monographs are

applied at a barometric pressure of 101.3kPa (760mmHg). If the determination
is made at some other barometric pressure, correct the observed temperature
readings for any difference in the barometric pressure by allowing 0.1 °C for
1149
each 0.36kPa (2.7mmHg) of the difference, adding if the pressure is lower, or

subtracting if the pressure is higher.
1.3 Determination of mass density and relative density

The mass density (p) of a substance is the mass of one unit volume of the sub-
stance. In terms of SI base units, mass density is expressed in kilograms per

cubic metre. However, in the International Pharmacopoeia the mass density is
expressed in kilograms per (which is equivalent to grams per millilitre) at

litre
a temperature of 20 and it is corrected for buoyancy (i.e. reduced to

°C (p 2 o)
vacuum conditions). For pharmacopoeial purposes the mass density of liquids
is not measured directly but calculated from their relative density.
The relative density dll is the ratio of the mass of the substance in air at
20 °C to that of an equal volume of water at the same temperature. The term
“relative density” is equivalent to the formerly used term “specific gravity
determined at 20 °C”.
The relative density df denotes the ratio of the mass of the substance in air
at 20 °C to that of an equal volume of water at 4°C. As the relative density of
water at 20 °C is 0.998234, these values are related by the following equation:
df = 0.998234 dfo
Determine the relative density (îjo) using a hydrostatic balance (only when the
precision indicated in the monograph is three decimal digits) or a pycnometer.
If mass density p 2 o (in kg/1 or g/ml) is referred to in the mono-
the value of
graph, carry out the measurement of relative density and from the value
obtained calculate the mass density according to the formula:
P2o = 0.99703 iiS-t 0.0012
Use of a hydrostatic balance

Use an instrument of suitable construction placed on a horizontal support. The
plummet (diver) should be suspended on a thin wire, made preferably of plat-
inum. To calibrate the instrument equilibrate the plummet in the air, then
immerse it in the cylinder filled with water and equilibrate again by placing
suitable riders (weights) at appropriate notches along the beam. The plummet
should swim freely in the liquid. Fill the cylinder with the test liquid and carry
out the measurement in a similar way. Take care that the length of the
immersed portion of the suspending wire is similar in all measurements. The
weight that has to be added to obtain the equilibrium in the test liquid (or to
be subtracted in the case of liquids of density lower than that of water) gives
direcdy the measure of its relative density.
1150
Methods of Analysis
Use of a pycnometer
Use a pycnometer of form of a capacity of not less than 5 ml. Weigh
suitable
accurately the empty, dry pycnometer, and fill it with the test liquid brought
previously to a temperature of about 20 °C. Hold the filled pycnometer at a
temperature of 20 + 1 °C for about 30 minutes, adjust the liquid to the mark

using, if necessary, a small strip of filter-paper to remove the excess and to wipe
the inlet from the inside, and weigh accurately. Calculate the weight of the
liquid in the pycnometer. Remove the liquid, clean and dry the pycnometer,
repeat the measurement with carbon-dioxide-free water R, also at 20 ± 1 °C,
and calculate the weight of water in the pycnometer. The ratio of the weights
of the test liquid and of water gives the relative density (djo).
1.4 Determination of optical rotation and specific rotation

Many substances possess the inherent property to rotate the plane of incident
polarized light; this property is called optical activity. The measurement of
optical activity is used for pharmacopoeial purposes mainly to establish the iden-
tity of the substance. It may also be employed to test the purity of the substance
(absence of optically non-active foreign substances) and as an assay procedure.
Optical rotation
The optical rotation is the angle through which the plane of polarization is
rotated when polarized light passes through a layer of a liquid. Substances are
described as dextrorotatory or levorotatory according to whether the plane of
polarization is rotated clockwise or counterclockwise, respectively, as deter-
mined by viewing towards the light source. Dextrorotation is designated (-I-)
and levorotation is designated (-).

In the International Pliamacop’oeia the optical rotation (a) is expressed in angular
degrees. In the SI, the angle of optical rotation is expressed in radians (rad).
The optical rotation is measured on wave-

a layer of suitable thickness at the
length specified in the monograph. If the sodium D line is
sodium specified, the
light ofwavelength 589.3 nm (a mean value for a doublet at 589.0 nm and
589. 6nm) should be used. The wavelength of the mercury green line at
546.1 nm is also frequently used. If the wavelength specified lies in the ultravi-
olet range, the use of a photoelectric polarimeter is necessary.
The measurement of optical rotation should be carried out at the tempera-
ture indicated in the monograph, usually at 20-25 °C. Some substances have
large temperature coefficients and for them special care should be taken to
adjust the temperature indicated.
Specific optical rotation (specific rotation)

The specific optical rotation of a liquid substance is the angle of rotation mea-
sured as specified in the monograph, calculated with reference to a layer
100 mm thick, and divided by the relative density (specific gravity) measured
at the temperature at which the rotation is measured.
1151
The specific optical rotation of a solid is the angle of rotation measured as

specified in the monograph, and calculated with reference to a layer 100mm
thick of a solution containing 1 g of the substance per ml.
,
Specific rotation =
lOOOOî
= —
10000a
—
Ic lap
where a is the observed rotation, / is the length of the observed layer in mm,
c is the number of g of substance contained in 100ml of solution, d is the
relative density and p is the number of g of substance contained in 100 g of
solution.
In the International Pharmacopoeia the specific optical rotation is expressed as
[a]i where t is and X the wavelength. For solid substances the
the temperature
solvent, if from water, and the concentration are further described.
different
The general directions concerning wavelength and temperature as given above
for optical rotation refer equally to the measurement of specific optical rota-
tion. In the SI specific optical rotation (optical rotatory power) is given in m^-
rad/kg and molar optical rotatory power (a„) in m^ rad/mol.
Apparatus
Optical rotation is measured with a polarimeter. The zero point of the
polarimeter is determined with the tube empty but closed for liquid substances
and with the specified solvent for solutions of solid substances.

filled
Generally, a polarimeter accurate to 0.05° of angular rotation, and capable

of being read with the same precision, suffices for pharmacopoeial purposes;
in some cases, a polarimeter accurate to 0.01 ° of angular rotation, and read with
comparable precision, may be required.
Polarimeters for visual measurement: commercial instruments are normally
constructed for use with sodium light or a mercury-vapour lamp. The manu-
facturer’s instructions relating to a suitable light source should be followed.
Photoelectric polarimeters: where it is directed in the individual monograph
to determine the optical rotation photoelectrically, use a photoelectric polarime-
ter capable of an accuracy of at least 0.01 °.
Measurement of optical rotation

The accuracy and precision of optical rotatory measurements will be increased
if they are carried out with due regard for the following general considerations.
Optical elements of the instrument must be and in exact
brilliantly clean
alignment. The match point should lie close to the normal zero mark. The light
source should be rigidly set and well aligned with respect to the optical bench.
It should be supplemented by a filtering system capable of transmitting light of
a sufficiently monochromatic nature. Precision polarimeters are generally
designed to accommodate interchangeable discs to isolate the from D line
sodium light or the 546.1 nm line from the mercury spectrum. With polarime-
ters not thus designed, cells containing suitably coloured liquids may be
employed as filters.
1152
Methods of Analysis
Observations should be accurate and reproducible to the extent that differ-

ences between replicates, or between observed and true values of rotation (the
latter value having been established by calibration of the polarimeter scale with
suitable standards), shall not exceed one-fourth of the range given in the indi-
vidual monograph for the rotation of the substance being tested.
Polarimeter tubes should be filled in such a way as to avoid creating or
leaving air bubbles, which interfere with the passage of the beam of light. Inter-
ference from bubbles is minimized by the use of tubes in which the bore is
expanded at one end. However, with tubes of uniform bore, such as semi-micro
or micro tubes, care is required for proper filling.
In closing tubes having removable end-plates fitted with gaskets and caps,
the latter should be tightened only enough to ensure a leak-proof seal between
the end-plate and the body of the tube. Excessive pressure on the end-plate
may set up strains that result in interference with the measurement. In deter-
mining the optical rotation of a substance of low rotatory power, it is desirable
to loosen the caps and tighten them again between successive readings in the
measurement of both the rotation and the zero point. In this way, differences
arising from end-plate strain will generally be revealed and appropriate adjust-
ments to eliminate the cause can be made.
The requirements for optical rotation and specific rotation apply to the
dried, anhydrous, or solvent-free material in all those monographs in which
standards for loss on drying, water, or solvent content are given. In calculating
the result, the loss on drying, water, or solvent content determined by the
method specified in the monograph should be taken into account.
If the substance is a solid, weigh a suitable portion and transfer it to a volu-
metric flask by means of water, or other solvent if specified in the monograph,
reserving a portion of the solvent for the blank determination. Add enough
solvent to bring the meniscus close to, but still below, the mark, and adjust the
temperature of the flask contents by suspending the flask in a constant-
temperature bath. Add solvent to the mark, and mix. Transfer the solution to
the polarimeter tube, preferably within 30 minutes from the time the substance
was dissolved, taking care to standardize the elapsed time in the case of sub-
stances known to undergo racemization or mutarotation. During the elapsed
time interval, maintain the solution at the required temperature.
If the substance is a liquid, adjust its temperature, if necessary, and transfer
it directly to the polarimeter tube.
When a polarimeter is used for visual measurement, make at least 6 readings
of the observed rotation at the required temperature. Take half the readings in a
clockwise and the other half in a counterclockwise direction. Substitute the
reserved solvent for the solution, and make an equal number of readings on it.
In the case of liquid substances, carry out blank determinations on the empty,
dry tube. The zero correction is the average of the blank readings, and is sub-
1153
tracted from the average observed rotation if the two figures are of the same sign,
or added if they are opposite in sign, to give the corrected observed rotation.
When a photoelectric polarimeter is used, a smaller number of readings is
required, depending on the type of instrument.
1.5 Determination of refractive index

The refractive index {n) of a substance is the ratio of the velocity of light in a
vacuum to its velocity in the substance. It varies with the wavelength of the
light used in its measurement and with the temperature. It is, therefore, nec-
essary to specify these conditions (kJJ. In practice it is usually convenient to
measure the refraction with respect to air and the substance, rather than with
respect to a vacuum and the substance, since, for pharmacopoeial purposes,
this has no significant influence on the observed values.
The refractive index may also be defined as the ratio of the sine of the angle
of incidence to the sine of the angle of refraction.
The measurement of is employed for pharmacopoeial
the refractive index
purposes mainly to establish the identity of liquid substances. It may also be
used to test the purity of such substances.
Refractive indices are usually stated in terms of sodium light of wavelength
589. 3nm (line D) at a temperature of 20± 0.5°C (hd°)-
The accuracyof the measurement should be related to the requirements of
the monograph. For pharmacopoeial purposes it is usually adequate to express
the refractive index to three decimal places.
Apparatus
Commercial refractometers are normally constructed for use with white light
but are calibrated to give the refractive index in terms of the sodium light of
wavelength 589.3 nm (line D).
The optical parts of the apparatus should be kept brilliantly clean. The
working surfaces of prisms should be free from scratches.
Subject to the directions given above, the manufacturer’s instructions relat-
ing to a suitable light source should be followed.
The instrument should be calibrated against a standard provided by the
manufacturer; the temperature control of the liquid being examined and the
cleanliness of the prism should be checked frequently by determining
the refractive index of distilled water, which is 1.3330 at 20 °C and 1.3325 at
25 °C.
1.6 Spectrophotometry in the visible and

ultraviolet regions
Absorption spectrophotometry measurement of the absorption, by sub-

is the
stances, of electromagnetic radiation of definite and narrow wavelength range,
essentially monochromatic.
1154
Methods of Analysis
The spectral range used in the measurements described below extends from
the short wavelengths of the ultraviolet through the visible region of the spec-
trum. For convenience of reference, this range may be regarded as consisting
of two regions, the ultraviolet (1 90-380 nm) and the visible (380-780 nm).
Spectrophotometry in the visible region (formerly the term colorimetry was
commonly measurement of absorption
used) is the of visible light, which is
usually not monochromatic but restricted by the use of pigmented or interfer-

ence filters.
The ultraviolet and visible spectra of a substance generally do not have a

high degree of specificity. Nevertheless, they are highly suitable for quantita-
tive assay, and for many substances they are useful as additional means of
identification.
General agreement has not yet been reached on the definition of terms
used in spectrophotometry. The terms in italics used in connexion with spec-
trophotometric tests in the International Pharmacop’oeia are defined as follows:
Absorbance (A) - The logarithm, to the base 10, of the reciprocal of the trans-
mittance (T). The term internal transmission density may be used as a
synonym of absorbance; descriptive terms formerly used included
optical density, absorbancy, and extinction.
Transmittance (T) - The ratio of the radiant flux transmitted by the test sub-
stance to that of the incident radiant flux. Terms formerly used include
transmittancy and transmission.
Absorptivity (a) - The quotient of the absorbance (A) divided by the product
of the concentration of the substance (c), expressed in g per litre, and the
absorption path length (b) expressed in cm (a =A/bc). Two terms closely
related to absorptivity are specific extinction and specific absorption
The term “specific extinction”
coefficient. as generally used in
pharmacopoeias, denotes the quotient of the absorbance {A) divided by
the product of the concentration of the substance (c), expressed in g per
100 ml, and the absorption path length (b) expressed in cm; therefore
£icm = 10^- The term “specific absorption coefficient”, tentatively pro-
posed by the Commission on Physicochemical Symbols, Terminology
and Units of the International Union of Pure and Applied Chemistry
(lUPAC), is defined as the quotient of absorbance (A) divided by the
product of concentration (c) and the absorption path length (/); when the
symbol asi is used for specific absorption coefficient, which in the SI
should be expressed in m^ per kg, asi = 100a. The term “absorptivity” is
not to be confused with absorbancy index or extinction coefficient.
Molar absorptivity (e) - The quotient of the absorbance (A) divided by the
product of the concentration of the substance (c), expressed in moles per
litre, and the absorption path length (b) in cm. It is also the product of
the absorptivity (a) and the molecular weight of the substance. The term
“molar absorption coefficient (linear)” recommended by the Commission
on Physicochemical Symbols, Terminology and Units of lUPAC is
defined as the quotient of the internal transmission density (absorbance)
1155
of the substance divided by the product of the concentration of the sub-

stance and the absorption path length, and according to the SI should be
expressed in per mole. The terms formerly used for molar absorp-
tivity include molar absorbancy index and molar extinction coefficient.
Absorption spectrum - The relationship of absorbance and wavelength or any
functions of these, frequently represented in a graphic form.
The use of absorption spectrophotometry in the visible and ultraviolet

regions for assay procedures is based on the fact that the absorptivity of a sub-
stance is usually a constant independent of the intensity of the incident radia-
tion, the internal cell length, and the concentration, with the result that
concentration may be determined photometrically.
Deviations from the above may be caused by either physical, chemical, or
instrumental variables. Deviations due to instrumental error might be caused
by by polychromatic radiation. Apparent failure
slit-width effects, stray light, or
may also result from a concentration change in solute molecules because of
association between solute molecules or between solute and solvent molecules,
or dissociation or ionization.
Apparatus
In essence all types of spectrophotometer are designed to permit substantially
monochromatic radiant energy to be passed through the test substance in a
suitable form and to allow measurement of the fraction of that energy that is
transmitted. The spectrophotometer comprises an energy source, a dispersing
device with slits for selecting the wavelength band, a cell or holder for the test
substance, a detector of radiant energy, associated amplifiers, and measuring
and recording devices. Some instruments are manually operated, while others
are equipped for automatic operation. Instruments are available for use in
the visible region of the spectrum, usually 380 nm to about 700 nm, and in
the visible and ultraviolet regions of the spectrum, usually 190 nm to about
700 nm.
Both double-beam and single-beam instruments are commercially available
and either is suitable. Depending on the type of apparatus used, the results may
be displayed on a scale, on a digital counter, or by a recorder or printer.
The apparatus should be maintained in proper working condition. The
housing of the optical system should minimize any possibility of errors due
to stray light; this is particularly relevant in the short-wave region of the
spectrum.
Cells usually used in the spectral range discussed are 1-cm absorption cells
with glass or silica windows. Other path lengths may also be used. The cells
used and the blank should be matched, and must have the
for the test solution
same spectral transmittance when containing only the solvent. If this is not the
case, an appropriate correction must be applied.
1156
Methods of Analysis
Spectrophotometer calibration
Spectrophotometers should be regularly checked for accuracy of calibrations.
Where a continuous source of radiant energy is used, both the wavelength and
photometric scales should be calibrated; where a spectral line source is used,
only the photometric scale need be checked.
A number of sources of radiant energy have spectral lines of suitable inten-
sity, adequately spaced throughout the spectral range selected. The exact values
for the position of characteristic lines in quartz-mercury arc are 253.7, 302.25,
313.16, 334.15, 365.48, 404.66 and 435.83 nm. The wavelength scale may also
be calibrated bymeans of suitable glass filters that have useful absorption bands
through the visible and ultraviolet regions. Standard glass containing didymium
(a mixture of praseodymium and neodymium) has been widely used. Glass
containing holmium is considered superior. The exact values for the position
of characteristic maxima in holmium glass filters are 241.5 + 1, 287.5 + 1, 360.9
+ 1and 536.2 + 3nm. Holmium glass filters are obtainable from some national
institutions and from commercial sources. The performance of an uncertified
filter should be checked against one that has been properly certified. The wave-
length scale may also be calibrated using holmium perchlorate TS. The exact
values for the position of characteristic maxima of this solution are as follows:
241.15nm, 278. 2nm, 361. 5nm and 536.3 nm. It should be noted that the posi-
tion of characteristic maxima of holmium perchlorate solutions and holmium
glass filters may differ slightly.
For the calibration of the photometric scale the tolerance generally permit-
ted is ±1% of the absorptivity. For checking this scale potassium dichromate
TS may be used. The exact values of absorbance and specific extinction for a
solution of potassium dichromate containing exactly 60.06mg in 1000 ml of sul-
furic acid (0.005 mol/1) VS at an absorption path length of 1.000 cm and the
permitted tolerances for A are given below:
Wavelength 235 nm 257 nm 313nm 350nm

(minimum) (maximum) (minimum) (maximum)
/I 0.748 0.865 0.292 0.640

Permitted tolerance 0.740-0.756 0.856-0.874 0.289-0.295 0.634-0.646
E\’L 124.54 144.02 48.62 106.56
A number of standard inorganic glass filters of known transmittance produced

for checking the photometric scale are also available from some national insti-
tutions and from commercial sources but may require periodic calibration.
Operation of spectrophotometers
Detailed instructions for operating spectrophotometers are supplied by the
manufacturers. To achieve significant and valid results, the operator of a spec-
trophotometer should be aware of its limitations and of potential sources of
error and variation. The instruction manual should be followed closely on such
matters as care, cleaning, and calibration of the instrument, and instructions for
1157
operation. Where double-beam recording instruments are used the cell con-
taining solvent only is placed in the reference beam.
The cleanliness of absorption cells should receive particular attention.
Usually, after treatment with an appropriate cleansing medium, the cells should
be rinsed with distilled water and then with a volatile organic solvent to
promote drying. Test solutions should not be left in the cells longer than nec-
essary for carrying out the measurement. When handling the cells, special care
should be taken never to touch the external surfaces through which the light
beam passes. When the solvent and the test solution are transferred to the cells,
care is to be taken that the liquids do not contaminate the outer surfaces.
Solvents for use in the ultraviolet region

Many solvents are suitable for tests and assays using spectrophotometry in the
ultraviolet region. Water, alcohols, chloroform, lower hydrocarbons, ethers, and
dilute solutions of ammonium hydroxide, sodium hydroxide, sulfuric acid and
hydrochloric acid can be used for this purpose. The solvents differ as to the
lower wavelength at which the decrease in transparency prevents their use. Pre-
cautions should be taken to use solvents free from contaminants absorbing in
the relevant spectral region. Specially purified solvents for spectrophotometric
determinations are available commercially from several sources but need only
be used when the spectral characteristics of the usual analytical grade of solvent
are inadequate for a particular purpose.
The absorbance of the solvent cell and its contents should not exceed 0.4
per cm of path length when measured with reference to air at the same wave-
length. The solvent in the solvent cell should be of the same batch as that used
to prepare the solution and must be free wavelength
from fluorescence at the
of measurement. Ethanol (-750 g/1), dehydrated ethanol, methanol, and cyclo-
hexane used as solvents should have an absorbance, measured in a 1-cm cell at
240 nm with reference to water, not exceeding 0.10.
Identification tests in the ultraviolet region

The monographs describing qualitative tests involving spectrophotometry in
the ultraviolet region specify the concentration of the solution and the path
length. In such tests it is more convenient to use a recording instrument. If the
conditions stated are not appropriate for a particular instrument, the thickness
of the solution should be varied and not the concentration.
Some identification tests involving spectrophotometry require the use of ref-
erence substances, generally an International Chemical Reference Substance
(ICRS). The reference substance is then to be prepared and simultaneously mea-
sured under conditions identical for all practical purposes to those used for the
test substance. Unless otherwise specified in the monograph, in making up the
solution of the reference substance, a solution of about the desired concentra-
tion (i.e. within 10% of the value) should be prepared. Identical conditions for
the measurement include the following: wavelength setting, slit-width adjust-
ment, cell placement, and correction and transmittance levels.
1158
Methods of Analysis
A useful approach for identification tests in the ultraviolet region is to quote

the ratio of absorbance values at two maxima. This procedure minimizes the
influence of instrumental variations on the test and obviates the need for a ref-
erence substance.
Quantitative determinations in the ultraviolet region

Spectrophotometric assays usually call for a comparison of the absorbance
produced by the solution of the test substance, prepared as specified in the
monograph, with the absorbance of a solution of a reference substance. In
such cases the spectrophotometric measurements are made first with the
solution prepared from the reference substance and secondly with the solution
prepared from the substance to be examined. The second measurement is
carried out as quickly as possible after the first, using the same experimental
conditions,
Spectrophotometric assays are usually carried out at a peak of spectral
absorption for the compound concerned. The monographs give the commonly
accepted wavelength for peak spectral absorption of the substance in question.
It is known that different spectrophotometers may show minor variations in
the apparent wavelength of this peak. Good practice demands that use be made
of the peak wavelength actually found in the individual instrument, rather than
monograph, provided the two do not differ
the specific wavelength given in the
by more than ±0.5nm in the 240-280 nm range, by more than ±1 nm in the
280-320 nm range, and by more than ± 2nm above 320 nm. If the difference is
greater, recalibration of the instrument may be indicated.
The solution of the reference substance, generally an ICRS, is to be prepared
and measured in the same manner as described for “Identification tests in the
ultraviolet region”. The calculations should be made on the basis of the exact
amount weighed and, if the reference substance used has not previously been
dried, on the dried or anhydrous basis. Specific instructions in individual mono-
graphs indicate the manner in which a reference substance is to be dried or
treated prior to use. These instructions are to be followed unless otherwise
specified in the individual test or assay, or in the labelling.
To ensure that the conditions used are appropriate the monograph may also
specify the value of absorbance of a 1-cm layer of the reference substance. In
this case the determination carried out against the reference substance is con-
sidered valid when the observed value of absorbance is within the range of
values specified in the monograph.
For quantitative work, a manually scanning instrument is frequently used.
When a recording instrument is used for that purpose special attention should
be paid to proper calibration of the absorbance scale at the wavelength used.
Quantitative determinations are usually carried out at wavelengths above
235 nm. If the measurements are to be made at a wavelength in the 190-
210nm range, special precautions should be observed, for example, purging the
cellcompartment with nitrogen, use of solvents of special spectrophotometric
quality, and using cells that are transparent in this region.
1159
When measuring the absorbance at an absorption maximum the spectral

slit-width must be small compared with the half width of the absorption band,
otherwise an erroneously low absorbance will be measured. Particular care is
needed for certain substances and the instrumental slit-width used should
always be such that further reduction does not result in an increased absorbance
reading. Problems may be encountered, owing to diffraction of the light beam,
at slit-widths below 0.01mm.
When the assays are carried out with routine frequency it is permissible to
omit the use of a reference substance and use instead a suitable standard curve
prepared with the respective ICRS. This may be done when, for the substance
tested, the absorbance is proportional to the concentration within the range of
about 75-125% of the final concentration used in the assay. Under these cir-
cumstances, the absorbance found in the assay may be interpolated on the stan-
dard curve, and the assay result calculated therefrom. Such standard curves
should be confirmed frequently, and always when a new apparatus or new lots
of reagents are put into use. In the event of uncertainty or dispute, direct com-
parison with an ICRS must be made.
Quantitative determinations in the visible region

Spectrophotometric assays in the visible region, as in the ultraviolet region,
usually call for simultaneous comparison of the absorbance produced by the
assay preparation with that produced by a standard preparation containing
approximately an equal quantity of a reference substance.
For spectrophotometric assays in the visible region the recommendations
given under “Quantitative determinations in the ultraviolet region”, including
those concerning the use of standard curves, should be followed with suitable
modifications, where necessary. In this region, observed wavelengths should
not differ by more than 5nm from that specified in the monograph.
1 .7 Spectrophotometry in the infrared region

The infrared region of the electromagnetic spectrum used in pharmaceutical
analysis covers the range 4000-250 cm'' (2.5-40 pm).'
Spectrophotometric measurements in the infrared region are used mainly as
an identification test. The infrared spectrum is unique for any given chemical
compound with the exception of optical isomers, which have identical spectra
in solution. Polymorphism and other factors, such as variations in crystal size
and orientation, the grinding procedure, and the possible formation of hydrates
may, however, be responsible for a difference in the infrared spectrum of a
given compound in the solid state. The infrared spectrum is usually not greatly
affected by the presence of small quantities of impurities (up to several percent)
in the tested substance. For identification purposes the spectrum may be com-
’
The values given in brackets are wavelengths; the preceding values are wave-numbers, which
are the reciprocals of the wavelengths.
1160
Methods of Analysis
pared with that of a reference substance, concomitandy prepared or with a

standard reference spectrum.
The terms absorption spectrum, absorbance, transmittance, absorptivity, and
specific extinction are described in 1.6 Spectrophotometry in the visible and
ultraviolet regions.
Apparatus
Spectrophotometers for the infrared region are basically similar to those used
for the visible and ultraviolet regions of the spectrum, but may differ as to the
energy sources, optical materials, and detection devices. Furthermore, in some
instruments the monochromator may be located between the test substance
and the detector.
Spectrophotometers suitable for use for identification tests should operate
in the range4000-670 cm“’ (2.5-15 pm). They should be checked frequently to
ensure that they meet the standards of performance laid down by the manu-
facturer of the instrument, including the reliability of the wavelength scales,
which should be checked by use of a polystyrene film.
For the use of the attenuated total reflectance technique, the instru-
ment should be equipped with a suitable attachment, which may be a single-
The attachment consists of a reflecting
reflection or a multi-reflection one.
element and a suitable mounting permitting its alignment in the spectropho-
tometer for maximum transmission.
Use of solvents
The solvent used in infrared spectrophotometry must not affect the material,
usually sodium chloride, of which the cell is made.
No solvent in appreciable thickness is completely transparent throughout
the infrared spectrum. Carbon tetrachloride R is practically transparent (up
to 1mm in thickness) from 4000 to 1700 cm“* (2.5 to 6pm). Chloroform R,
dichlorome thane R, and dibromomethane R are other useful solvents. Carbon
disulfide IR (up to 1mm in thickness) is 250 cm“'
suitable as a solvent to
(40 pm), except in the 2400-2000 cm“' (4. 2-5. 0 pm) and the 1 800-1300 cm“'
(5. 5-7.5 pm) regions, where it has strong absorption. Its weak absorption in the
875-845 cm”' (11. 4-11. 8pm) region should also be noted. Other solvents have
narrow regions of transparency.
relatively
Preparation of the substance to be tested

To determinethe infrared absorption spectrum of a substance, the latter has to
be suitably prepared. Liquid substances may be tested directly or in a suitable
solution. For solid substances, the usual methods of preparation include dis-
persing the finely ground solid specimen in mineral oil, or incorporating it in a
transparent disc or pellet obtained by mixing it intimately with previously dried
potassium halide and pressing the mixture in a die, or preparing a solution in
a suitable solvent. Preparation of the substance for the attenuated total
reflectance technique is described separately.
1161
The following procedures may be used for the preparation of the substance:
Method I. Use a capillary film of the liquid held between two sodium
chloride plates or a filled cell of suitable thickness.
Method 2. with the minimum
Triturate a small quantity of the substance
amount of a smooth,
suitable mineral oil or other suitable liquid to give a
creamy 2-5 mg of the substance being tested is often sufficient to
paste;
prepare a satisfactory mull, which should be semi-transparent to light.
Compress a portion of the mull between two flat sodium chloride or
other suitable plates.
Method 3. Triturate the solid substance with dry, finely powdered potassium
halide (potassium bromide IR, potassium chloride IR); the proportion of
substance to the halide should be about 200 for example, 1.5 mg
1 to
in 300 mg of the halide - in the case of prism instruments, and about 1
to 300 - for example, 1.0 mg in 300 mg of the halide - in the case of
grating instruments. The amount taken should be such that the weight
of substance per area of the disc is about 5-15 pg per mm^, varying with
the molecular weight and to some degree with the type of apparatus
used. Insert a portion of the mixture in a special die and subject it under
vacuum to a high pressure. Commercial dies are available and the man-
ufacturer’s instructions should be followed. Mount the resultant disc in
a suitable holder. Several factors, for example, inadequate or excessive
grinding, moisture or other impurities in the halide carrier, may give rise
to unsatisfactory discs. Unless its preparation presents particular diffi-
culties, a disc should be rejected if visual inspection shows lack of uni-

formity or if the transmission at about 2000 cm“' (5 pm) in the absence
of a specific absorption band is less than 75% without compensation.
Method 4. Prepare a solution of the liquid or solid substance in a suitable
solvent and choose a concentration and cell thickness to give a satisfac-
tory spectrum over a sufficiently wide wavelength range.
Identification by reference substance

Prepare the substance under examination and the reference substance by the
same method, and record the spectrum of each from about 4000 to 670cm“'
(2.5pm to 15pm) on an infrared spectrophotometer. The concentration of the
substance should be such that the strongest peak attributable to the substance
reaches to between 5% and 25% transmittance.
If the positions and relative intensities of the absorbance maxima in the
spectrum of the substance under examination are not concordant with those
of the spectrum of the reference substance in the case of the curves obtained
by methods 2 or 3, this may be due to differences in crystalline form. When
such difficulties are suspected, the substance should, where possible, be exam-
ined in solution. If examination in solution is not practicable, attempts should
be made to obtain, by recrystallization, the reference substance and the sub-
stance under examination in the same crystalline form.
1162
Methods of Analysis
If the spectrum of mineral oil used in Method 2 interferes with regions of

interest, an additional dispersion of the substance in a medium such as a suit-
able fluorinated hydrocarbon oil or hexachlorobutadiene may be prepared and
the spectrum recorded in the regions where the mineral oil shows strong
absorption.
Identification by reference spectrum

Prepare the substance being examined exactly as described in the note accom-
panying the International Reference Spectrum and record the spectrum from
about 4000 to 670 cm“* (2.5 to 15 pm) using an instrument that is being checked
frequently to ensure that it meets the standards of performance laid down by
the manufacturer. To allow for a possible difference in wavelength calibration
between the instrument on which the International Reference Spectrum was
obtained and that on which the spectrum of the substance is to be recorded,
reference absorbance maxima of a polystyrene spectrum are superimposed on
the International Reference Spectrum at about 2851 cm“' (3.51pm), 1601 cm“'
(6.25pm) and 1028cm“' (9.73pm). Similar reference maxima should be super-
imposed on the spectrum of the substance. Taking these polystyrene maxima
is considered to be positive if the principal
into account, the identification
absorbance maxima in the spectrum of the substance being tested are concor-
dant with the corresponding maxima in the relevant International Reference
Spectrum. When comparing the two spectra, care should be taken to allow for
the possibility of differences in resolving power between the instrument on
which the International Reference Spectrum was prepared and the instrument
being used to examine the substance. An International Reference Spectrum of
polystyrene that was recorded on the same instrument as the collection of Inter-
national Reference Spectra should be used for assessing these differences. It
should be noted that the greatest variation due to differences in resolving power
is likely to occur in the region between 4000 and 2000 cm“* (2.5 and 5 pm).
Attenuated total reflectance technique

To determine the infrared absorption spectrum of a substance by the attenu-
ated total reflectance technique the solid substance has usually to be finely pul-
verized. The powder may then be packed directly against the prism of
either
the attachment or adhesive tape may be used to facilitate the contact. The pow-
dered substance is spread on the adhesive side of an adhesive tape to form an
almost translucent layer and the tape is pressed on the reflecting element, on
the side with powder in contact. Next, either the backing plate is attached or
moderate pressure is applied in a suitable clamp for 1-2 minutes. Finally, the
reflecting element is placed in the holder. The tape used in the procedure should
preferably contain a natural rubber adhesive. In the case of some plastic mate-
rials it may be placed directly on the reflecting element.
Proper alignment of the attachment in the apparatus should be carefully
controlled.
1163
1.8 Atomic spectrometry: emission and absorption

Atomic emission spectrometry, flame photometry, and atomic absorption spec-
trometry are analytical techniques that measure the concentration of chemical
elements in a sample. When elements are transformed into atomic vapour at
high temperatures, emission or absorption of light may occur and this can be
accurately measured at a unique resonant wavelength, which is characteristic
of the emission/absorption lines of the elements concerned.
Using this basic process, the concentrations of most elements may be esti-
mated by measuring the amount of radiation either emitted by the sample
(emission spectrometry) or absorbed by the sample following production by a
primary radiation source (absorption spectrometry).
In emission spectrometry/flame photometry, a small proportion of the
atoms undergo excitation with a subsequent and characteristic emission of radi-
ation, at wavelengths in the ultraviolet or visible regions, which is proportional
to the number of excited atoms.
Thus under controlled conditions, it is possible with either technique to
relate the measured light intensity (emission or absorption) to the concentra-
tion of individual elements in the sample.
The major advantage of emission spectrometry is the relatively low cost and
simplicity of instrumentation, since it requires no additional source of primary
radiation. The fraction of excited atoms produced by most elements at normal

flame temperatures is small and hence, in practice, emission spectrometry is
limited to a few elements such as sodium, potassium, lithium, and calcium.

Sensitivity may be increased, however, by using higher flame temperatures or
other more effective means of excitation.
In comparison, absorption spectrometry offers greater sensitivity, since
absorption is proportional to the concentration of ground state atoms,which
are produced numbers by most elements at normal operating flame
in sufficient
temperatures. The disadvantage of this technique is the high initial cost of
instrumentation, because of the requirement for a primary source of radiation
(an individual hollow cathode lamp, one for each element to be determined).
Both techniques are comparative in nature and require the concomitant
preparation and use of standard reference solutions of all elements to be
determined.
Apparatus
The emission spectrometer consists essentially of a burner - an atomic gener-
ator of the element to be determined (flame, furnace, plasma, arc, etc.), a suit-
able filter monochromator, and a detector. The absorption spectrometer also
or
includes a source of radiation (a hollow cathode tube) where the cathode con-
sists of the same element as that to be determined.
Use of solvents
For flame spectrometry, the ideal solvent is one that produces neutral atoms
and interferes the least with the emission or absorption processes. Differences
1164
Methods of Analysis
in surface tension or viscosity between the and reference solutions may

test
cause difficulties in the aspiration or atomization rates and significant changes
in the signals generated. The solvent of choice for the preparation of test and
reference solutions should, therefore, be water, although organic solvents, such
as flammable solvents, either alone or mixed with water, may also be used if
precautions are taken to ensure that they do not interfere with the stability of
the flame. When mineral acids are necessary for the dissolution of the element,
care should be taken to avoid interference from the acidic anion. A dilute solu-
tion of hydrochloric acid is the preferred solvent for this purpose.
Calibration
To calibrate the instrument, introduce water or the blank solution into the
atomic vapour generator (flame) and adjust the reading of the instrument, either
to zero for emission spectrometers or to indicate maximum transmission for
absorption spectrometers. For emission measurements, introduce the most con-
centrated standard solution into the flame and adjust the sensitivity to full-scale
deflection. Refer to the manufacturer’s instructions for instruments with an
absorbance scale.
Follow the manufacturer’s instructions for the operation of the spectrometer,
and use the wavelength indicated in the individual monograph. Use Method 1,
unless otherwise specified in the monograph.
Method 1: External standard method

Prepare the solution of the substance to be tested as specified in the mono-
graph. Prepare concurrently, adding any reagents in the same concentration as
for the solution above, not fewer than three reference solutions of the element
to be determined that cover the expected concentration range of the solution
under test. Similarly, prepare a blank solution.
After calibration, introduce each reference solution into the instrument three
times, recording the steady reading obtained. If the generator is a flame, wash
the apparatus after each introduction with water or the blank solution to check
that the reading returns to its initial setting. If the generator is a furnace, fire it
after each introduction. Prepare a curve by plotting the mean of each group of
three readings against the concentration.
Introduce the solution to be tested into the instrument three times, and
record the readings. Determine the concentration of the element using the
mean of the readings and interpolating from the curve.
Method 2: Standard addition method

Prepare in at least three similar volumetric flasks a series of solutions contain-
ing equal quantities of the substance to be tested as specified in the monograph
and increasing volumes of the reference solution containing known concentra-
1165
tions of the element to be determined. The concentrations chosen should be

expected to give responses in the linear part of the curve. One of the solutions
of the substance to be tested should contain no added reference solution.
After calibration, introduce each solution into the instrument three times,
recording the steady reading obtained. If the generator is a flame, wash the
apparatus after each introduction with water or the blank solution to check that
the reading returns to its initial setting. If the generator is a furnace, fire it after
each introduction.
By using a least-squares fit, calculate the linear equation of the graph and
derive from it the concentration of the element determined in the test solution.
Alternatively, plot the mean of the readings on a graph against the added quan-
tity of the element and determine the concentration of the element in the test
solution by extrapolating the straight line joining the points on the graph to an
extended concentration axis.
1.9 Fluorescence spectrophotometry

Fluorescence spectrophotometry is the measurement of the fluorescence, i.e.
photoluminescence, emitted by a substance while it is being exposed to ultra-

violet, visible, or other electromagnetic radiation. In general, the light emitted
by fluorescent solutions is of maximum intensity at a wavelength longer than
that of the absorption band causing excitation, usually by some 20 or 30nm.
The intensity of the light emitted by a fluorescent solution is, in certain
circumstances, a simple function of the concentration of the solute and can,
therefore, be used for analysis. It is difficult, however, to measure absolute
fluorescence intensity, and measurements are usually made by reference to dilu-
tions of a properly selected reference substance. The general scheme in fluo-
rescence spectroscopy is, therefore, to excite with radiation at the wavelength
of maximum absorption, and to measure or compare the intensity of the fluo-
rescent light with that of a reference solution. The fluorescent light should be
carefully freed from scattered incident light.
Terms
Fluorescence intensity is an empirical expression of fluorescence activity, com-
monly given in terms of arbitrary units proportional to detector response.
The fluorescence emission spectrum is the relationship between the intensity of
the emitted radiation and the wavelength and is frequently represented in a
graphic form.
The fluorescence excitation spectrum is between the maximum
the relationship
intensity of radiation emitted by an activated substance and the wavelength of
the incident radiation and is frequently represented in a graphic form.
Apparatus
Measurement of fluorescence intensity can be made with a simple filter fluo-
rimeter (sometimes the term fluorometer is used). Such an instrument consists
1166
Methods of Analysis
of a radiation source, a primary sample chamber, a secondary filter, and

filter, a
a fluorescence detection system. In most such fluorimeters, the detector is
placed on an axis at 90 ° from that of the incident beam. This right-angle geom-
etry permits the incident radiation to pass through the test solution without
contaminating the output signal received by the fluorescence detector.
However, the detector unavoidably receives some of the incident radiation as
a result of the inherent scattering properties of the solutions themselves, or if
dust or other solids are present. used to eliminate this residual scatter.
Filters are
The primary filter wavelength radiation capable of causing exci-

selects short
tation of the test substance, while the secondary filter is normally a sharp cut-
off filter that allows the longer wavelength fluorescence to be transmitted but
blocks the scattered excitated radiation.
Most fluorimeters use photomultiplier tubes as detectors; many types are
available, each having special characteristics with respect to spectral region of
maximum sensitivity, gain, and electrical noise. After amplification of the pho-
tocurrent its value is read visually on a measuring device or recorded.
A fluorescence spectrophotometer differs from a filter fluorimeter in that
filters are replaced by monochromators, of either the prism or the grating type.
For analytical purposes, the fluorescence spectrophotometer is superior to the
filter fluorimeter in wavelength selectivity, flexibility, and convenience.
Many radiation sources are used in fluorimeters and fluorescence spec-
trophotometers. Mercury lamps are relatively stable and emit energy mainly
at discrete wavelengths. Tungsten lamps provide an energy continuum in the
The high pressure xenon arc lamp is often used in fluorescence
visible region.
spectrophotometers because it has an energy continuum extending from the
ultraviolet into the infrared.
In fluorescence spectrophotometers the monochromators are equipped with
slits. A narrow slit provides high resolution and spectral purity, while a large
slit sacrifices these for high intensity. Choice of slit-width is determined by the
wavelength separation between incident and emitted radiation, as well as by

the degree of sensitivity needed.
The used in fluorescence measurements may be rectangular cells similar
cells
to those used in absorption spectrophotometers, except that they are polished

on all 4 vertical sides and on the bottom, or
cells in the shape of round tubes
with flat polished bottoms may be used. A convenient size is 2-3 ml, but some
instruments can be fitted with small cells holding 0. 1-0.3 ml, or with a capil-
lary holder requiring even less solution.
Standardization
Fluorimeters and fluorescence spectrophotometers should be standardized
daily with a stable fluorophore to assure proper reproducibility of response.
The changes are due to instrumental variables such as differences in lamp inten-
sity and photomultiplier sensitivity. The fluorophore may be a pure specimen
of the fluorescent substance under test or another readily purified fluorescent
substance with absorption and fluorescence bands similar to those of the test
substance. Quinine in dilute sulfuric acid is often a convenient fluorophore for
1167
blue fluorescence, sodium fluorescein for green fluorescence, and rhodamine for
red fluorescence.
Calibration of the wavelength scale

The wavelength scale of the fluorescence spectrophotometer should be
periodically calibrated.
Preparation of solution
The solvent used for the measurement should be properly selected. The solvent
itself, its purity and its pH may markedly affect the intensity and spectral dis-
tribution of fluorescence.
Test solutions prepared for fluorescence spectrophotometry are usually 10
times to 100 times less concentrated than those used in absorption spec-
trophotometry. In analytical applications, it is necessary that the fluorescence
intensity be linearly related to the concentration in the range used for mea-
surements; but if a solution is too concentrated, a significant part of the inci-
dent light absorbed by the substance near the cell surface, thus resulting in
is
a reduction of the light reaching the centre. That is, the substance itself acts as
an “inner filter”. Fortunately, fluorescence spectrophotometry is inherently a
very sensitive technique, and concentrations of 10“^-10“^ mol/1 are frequently
used.
Owing to the usually very narrow range within which the fluorescence
is proportional to the concentration of the fluorescent substance, the ratio
{c-d)/{a-b), where a is the fluorescence intensity read for the reference sub-
stance, b the reading for the corresponding blank, c the fluorescence intensity
read for the test substance, and d the reading for the corresponding blank,
should be not less than 0.40 and not more than 2.50. It is then necessary to
make a working curve of fluorescence intensity corrected for a solvent blank
versus concentration.
Measurement technique
Fluorescence measurements are sensitive to the presence of solid particles in
the test solution. Such impurities may reduce the intensity of the exciting beam
or give misleadingly high readings because of multiple reflections in the cell. It
is, therefore, wise to eliminate solid particles by centrifugation; filtration may

also be used, but some filter-papers may contain fluorescent impurities.
Oxygen dissolved in the solvent has a strong quenching effect. The inten-
sity of fluorescence is, therefore, increased by use of a degassing procedure,
such as bubbling nitrogen or another inert gas through the test solution.
Temperature regulation is often important in fluorescence spectrophotom-
etry. For some substances, fluorescence efficiency may be reduced by as much
as 1-2% per degree of temperature rise. In such cases, if maximum precision
is desired, temperature-controlled sample cells are essential. For routine analy-
sis, it may be sufficient to make measurements rapidly enough so that the test
solution does not heat up appreciably from exposure to the intense light source.
1168
Methods of Analysis
Many fluorescent compounds are light-sensitive. Exposed in a fluorescence

spectrophotometer, they may be photodegraded into more or less fluorescent
products. Such effects may be detected by observing the detector response in
relation to time, and may be reduced by attenuating the light source with filters
or screens.
1.10 Turbidimetry and nephelometty

Turbidimetry is the measurement of the degree of attenuation of a radiant beam
incident on particles suspended in a medium, the measurement being made in
the directly transmitted beam. It may be measured with a standard photoelec-
tric filter photometer or spectrophotometer with illumination at an appropri-

ate wavelength.
Nephelometty is the measurement of the light scattered by suspended par-
ticles, the measurement usually being made perpendicularly to the incident
beam.
Turbidimetry or nephelometty may be useful for the measurement of pre-
cipitates formed by the interaction of very dilute solutions of reagents, or other
particulate matter, such as suspensions of bacterial cells. In order that consistent
results may be achieved, all variables must be carefully controlled. Problems due
to birefringence may be encountered, particularly with bacterial cells. Where

proper control is possible, extremely dilute suspensions may be measured.
Terms
Transmittance (T) - The ratio of the radiant flux transmittedby the test substance
to that of the incident radiant flux. Terms formerly used include transmittancy
and transmission.
Turbidance (S) - A measure of the light-scattering effect of suspended
particles.
Turbidity (t) - In light-scattering measurements, the turbidity is the measure
of the decrease in incident beam intensity per unit length of a given suspension.
Apparatus
Turbidity may be measured with a standard photoelectric filter photometer or
spectrophotometer, preferably with illumination in the red-orange region of the
spectrum (for example, by using a blue filter).
Nephelometric measurements require an instrument with a photocell placed
so as to receive scattered rather than transmitted light; as this geometry applies
also to fluorimeters in general, the latter can be used as nephelometers by
proper selection of filters.
Instrumental measurement
For instrumental measurement it is advisable to ensure that settling of the parti-
cles being measured will be negligible. This is usually accomplished by including
a protective colloid in the liquid suspending medium. It is important that results
1169
be interpreted by comparison of readings with those representing known con-

centrations of suspended matter, produced under exactly the same conditions.
Visual comparison
Carry out turbidity comparison in tubes that are matched as closely as possi-
ble in internal diameter and in all other respects. Flat-bottomed comparison
tubes of transparent glass of about 70ml capacity and about 23mm internal
diameter are suitable. For turbidity comparison the tubes should be viewed hor-
izontally, against a dark background, with the aid of a light source directed from
the sides of the tubes.
1.11 Colour of liquids

The test for colour of liquids is carried out by comparing the test solution pre-
pared as specified in the monograph with a standard colour solution indicated
in the monograph. The composition of the standard colour solution is selected
depending on the hue and intensity of the colour of the test solution corre-
sponding to the limits permitted in the specifications.
Unless otherwise specified in the monograph, carry out the comparison in flat-
bottomed tubes of transparent glass that are matched as closely as possible in

internal diameter and in all other respects (tubes of about 16mm internal diam-
eter are suitable). Use 10 ml of the test solution and 10 ml of the standard colour
solution; the depth of liquid should be about 50 mm. The colour of the test
solution is not more intense than the standard colour when viewed down the
vertical axis of the tubes in diffused light against a white background.
Stock colour standard solutions

Yellow stock standard TS
To 9.5ml of cobalt colour TS, add 1.9 ml of copper colour TS, 10.7 ml of dichro-
mate colour TS, 4.0 ml of iron colour TS, dilute to 100.0 ml with sulfuric acid
(~10g/l) TS, and mix.
Red stock standard TS

To 40.5 ml of cobalt colour TS, add 6. 1 ml of copper colour TS, 6.3 ml of dichro-
mate colour TS, 12.0ml of iron colour TS, dilute to 100.0ml with sulfuric acid
Green stock standard TS

To 3.5 ml of cobalt colour TS, add 20.1 ml of copper colour TS, 10.4 ml of dichro-
mate colour TS, 4.0 ml of iron colour TS, dilute to 100.0 ml with sulfuric acid
1170
Methods of Analysis
Brown stock standard TS

To 35.0mlof cobalt colour TS, add 17.0 ml of copper colour TS, 8.0 ml of dichro-
mate colour TS, dilute to 100.0 ml with iron colour TS, and mix.
Standard colour solutions

The standard colour solution is prepared by suitably diluting the stock stan-
dard solutions (yellow, red, green, and brown stock standard TS) with sulfuric
acid (~10g/l) TS. The designation of the standard colour solution is composed
of two letters indicating the stock standard solution (Yw for yellow, Rd for red,
Gn for green, and Bn for brown) and of a number describing the dilution, as
given below:
Dilution number Stock standard Sulfuric acid

for standard solution (~10g/l) TS
colour solutions (ml) (ml)
0 0.78 99.22
1 1.56 98.44
2 3.12 96.88
3 6.25 93.75
4 12.50 87.50
5 25.00 75.00
6 50.00 50.00
7 100.00 -
Standard colour solution numbers 4-7 may be stored in sealed glass contain-
ers, protected from sunlight but the more dilute standard colour solutions
should be prepared as required.
1.12 Powder fineness and sieves

A. Powders
The degree of coarseness or fineness of a powder is differentiated by the
nominal aperture size of the mesh of the sieve through which the powder is
able to pass, expressed in |im.

The following terms are used in the description of powders:
Coarse powder (ZOOO/355)- A powder of which all the particles pass through
a No. 2000 sieve, and not more than 40% through a No. 355 sieve.
Moderately coarse powder (7'IO/250). A powder of which all the particles pass
through a No. 710 sieve, and not more than 40% through a No. 250
sieve.
Moderately fine powder (3$ Ml 80). A powder of which all the particles pass
through a No. 355 sieve, and not more than 40% through a No. 180
sieve.
1171
Fine powder (ISO). A powder of which all the particles pass through a No.
180 sieve.
Very powder ('IZ5).
fine A powder of which all the particles pass through a
No. 125 sieve.
When the fineness of a powder is described by means of a number, it is
intended that all the particles of the powder should pass through the sieve dis-
tinguished by that number.
B. Sieves
The wire sieves used in sifting powdered drugs are distinguished by numbers,
which indicate the nominal aperture size expressed in pm.
The sieves are made of wires of uniform circular cross-section, in accordance
with the specifications given in Table 2.
The nominal size of aperture of wire mesh sieves has been selected princi-
pally from among those recommended in the ISO standard 565-1972.
1.13 Determination of pH
A value characteristic of an aqueous solution is its pH value, which represents
conventionally its acidity or alkalinity.
The pH of a solution is the negative logarithm of the hydrogen ion activity,
which may be measured potentiometrically. Formerly, pH was regarded as the
negative logarithm of the hydrogen ion concentration. As it is known that not
all hydrogen ions are necessarily equally active, this concentration may be dif-
ferent from the hydrogen ion activity. However, if the activity coefficient is
Table 2. Wire mesh sieves
Number of sieve Nominal size Nominal diameter Approximate

(^m) of aperture of wire screening area
(mm) (mm) (%)
2000 2.00 0.90 48

710 0.710 0.450 37
500 0.500 0.315 38
355 0.355 0.224 38
250 0.250 0.160 37
212 0.212 0.140 36
180 0.180 0.125 35
150 0.150 0.100 36
125 0.125 0.090 34
90 0.090 0.063 35
75 0.075 0.050 36
45 0.045 0.032 34
1172
Methods of Analysis
close to 1, as is true in dilute solutions, the values of hydrogen ion activity and
hydrogen ion concentration become nearly identical.
The determination of the pH by measurement of the
value is carried out
potential difference between electrodes immersed in standard and test solu-
tions. The standard solutions used are assigned a definite pH value by
convention.
In the measurement of pH, glass electrode finds wide applicability as it
shows an immediate response to rapid changes of hydrogen ion concentrations
even in poorly buffered solutions. Since the mechanism of this electrode
involves no electron exchange it is the only electrode sensitive to hydrogen ions
that is not disturbed by oxidizing or reducing agents.
The pH values of solutions or suspensions that are only partially aqueous
and that can be considered only as “apparent pH values” can also be measured
by using the proper electrode and by suitably standardizing the pH-meter.
As pH values are dependent on temperature, the measurements are carried
out at selected constant temperatures.
Solutions used in determinations of pH are prepared with carbon-dioxide-
free water R.
pH Scale
The difference in pH between two solutions, X and S, at the same temperature
may be defined operationally as follows:
The electromotive force £„ of the cell
PtlH 2 solution
l X 13.5 mol/1 KCll reference electrode
and the electromotive force, £„ of the cell
PtlH 2 solution S
l 13.5 mol/1 KCll reference electrode
are measured, both cells being at the same temperature throughout and the ref-
erence electrodes and bridge solutions being identical in the two cells.
The pH of solution X, denoted by pH (X), is then related to the pH of solu-

tion S, denoted by pH (S), by the equation:
Ex Es
pH(X) = pH(S) +
2.3026 RTjF
where R denotes the gas constant, T the thermodynamic temperature (in K),
and F the Faraday constant. Thus defined, the quantity pH is a dimensionless
number.
Numerical values of the factor 2.3026 RT/F at several temperatures are given
below:
1173
Temperature 2.3026 RT/F

(in °C) (mV)
10 56.18
15 57.17
20 58.17
25 59.16
30 60.15
Potentiometric determination ofp>H
For the practical determination of pH, a potentiometric method is usually

employed. When glass electrodes are used they should be stored in a suitable
liquid, usually water.
It is often found more convenient to measure pH by means of a glass elec-
trode instead of the hydrogen electrode. In some solutions, especially those
containing oxidizing agents, the glass electrode can be used when the hydro-
gen electrode cannot. The accuracy and reproducibility of ±0.005 usually
obtainable with the hydrogen electrode are, however, rarely obtainable with
the glass electrode, and never outside the pH range 2-10.
When the glass electrode is used, the best accuracy is obtained by assum-
ing that over a short range of pH there is a linear relation between pH and mea-
sured electromotive force, but that the proportionality factor relating them is
not necessarily exactly 2.3026 RF/T. This method of using the glass electrode
necessitates calibration by means of two solutions of known pH, near to, and
preferably bracketing, the pH to be measured. There are at present various suit-
able solutions, the pH values of which are reliably known to an accuracy of

±0.005 (see Table 3).
Calibration of apparatus
The apparatus is calibrated with standard buffer solutions to check the linear-
ity of the response of the electrode at different pH values and to detect a faulty
Table 3. Standard buffer solutions and their pH values
Standard buffer solutions pH values at various temperatures
20 °C 25 °C 30 °C 35 °C 40 °C
Potassium tetraoxalate standard TS 1.675 1.679 1.683 1.688 1.694

Potassium hydrogen tartrate standard TS - 3.557 3.552 3.549 3.547
Potassium hydrogen phthalate standard TS 4.002 4.008 4.015 4.024 4.035
Phosphate standard buffer, pH 6.8, TS 6.881 6.865 6.853 6.844 6.838
Phosphate standard buffer, pH 7.4, TS 7.429 7.413 7.400 7.389 7.380
Sodium tetraborate standard TS 9.225 9.180 9.139 9.102 9.068
Sodium carbonate standard TS 10.062 10.012 9.966 9.925 9.889
1174
Methods of Analysis
glass electrode. The standardization of the apparatus with only a single solu-
tion may be completely erroneous and therefore at least two standard buffer
solutions should be used for calibration. The presence of a faulty electrode will
be detected by failure to obtain a reasonably correct value (+0.04 unit) for the
pH of the second standard solution when the apparatus has been standardized
in terms of the first standard. A cracked electrode will yield pH values that are
essentially the same for both solutions. If the difference between the known
and the observed pH values for the second solution exceeds +0.04, another glass
electrode should be substituted. If the difference persists, fresh standard solu-
tions should be prepared.
After the apparatus has been calibrated, thoroughly wash the electrodes and
the cup. Fill the cup with a portion of the solution to be tested and obtain a
preliminary value for the pH. In general, this value will drift and is regarded as
an approximation. Subsequent readings taken on additional portions of the
same solution will yield successively more constant pH values. In the case of
solutions that are well buffered, 3 portions may be sufficient to yield pH values
that are reproducible to +0.04 unit and that show drifts of less than +0.04 unit
in 1 or 2 minutes. In the case of very dilute or unbuffered solutions, as many
as 6 portions of the test solution may be required, and the pH values may con-
tinue to drift and be reproducible to only +0.05 unit.
If a precision greater than 0.1 pH unit is desired, the temperature of the stan-
dard solutions, the glass and calomel electrodes, and the test solutions must be
within 2°C of one another, and the electrodes, standard solutions, test solu-
tions, and wash water must be kept at the temperature of measurement for at
least 2 hours prior to making the measurement in order to reduce to a negligi-
ble value the effects of thermal or electrical hysteresis of the electrodes.
Standard buffer solutions

Standard buffer solutions are used in the determination of pH values. They are
prepared with carbon-dioxide-free water R. They should be stored in botdes
of chemically resistant glass or in botdes made of polyethylene.
Unless otherwise specified, standard buffer solutions should not be used
later than 3 months after preparation. If growth of microorganisms starts in the
solutions they should immediately be discarded and the bottles thoroughly
cleaned and sterilized before refilling.
1.14 Chromatography
Chromatographic processes involve, in general, the distribution of a solute
between two phases, one of which is the mobile phase and the other the sta-
tionary phase. The stationary phase may act by adsorption, by partition (where,
for example, a liquid that is immiscible with the mobile phase may be coated
1175
on the by ion exchange, or by gel permeation. In

surface of a solid support),
practice, the chromatographic process in many pharmaceutical applications
may be a complex synthesis of several physical phenomena; certainly many
chromatographic procedures that are referred to as partition methods may be
significantly influenced by adsorption effects.
Chromatography is thus simply a method of separation and of itself does
not permit either identification or quantitative measurement. It is the combi-
nation of chromatography with appropriate methods of detection and mea-
surement that results in the loosely but conveniendy termed “chromatographic
method of analysis”.
For convenience the types of chromatography that are useful in pharma-
ceutical analysis may be considered as falling into three broad groups. In the
planar methods, chromatography is effected by allowing the mobile phase to
flow over and through a layer of the adsorbent (paper and thin-layer chro-
matography). In column methods, the adsorbent is packed into a column,
which may be of the traditional open type or may be closed and designed to
withstand considerable pressures so that the mobile phase may be pumped
through the column at high speed (high-pressure liquid chromatography -
sometimes known as high-performance or high-speed liquid chromatography).
Gas chromatography is a special case of column chromatography in that the
mobile phase is a gas rather than a liquid and the solutes must be volatile
or rendered so by elevated temperatures and/or by conversion to volatile
derivatives.
No
one method of chromatography is adequate for all purposes, since each
has advantages and disadvantages. Most of the planar methods are simple
its
and and require inexpensive equipment (although some complex

effective
accessories are available); these methods are valuable for identification and
screening purposes but are less suitable for precise quantitative applications.
Older column methods may be used without great expense but are often time-
consuming and tedious to carry out. High-pressure liquid and gas chromato-
graphic methods need specialized apparatus but may afford rapid and effective
separations that are suitable for precise quantitative measurement of compo-
nents. Such methods are particularly valuable for the determination of small
amounts of impurities. Limitations are currently imposed on the value of high-
pressure liquid chromatography by the lack of universally applicable methods
of detection and on that of gas-liquid chromatography by the non-volatility or
thermal instability of many compounds.
1.14-1 Thin-layer chromatography

In thin-layer chromatography the adsorbent is a thin, uniform layer (usually
about 0.24 mm
thick) of a dry, finely powdered material applied to a suitable
support, such as a glass plate or an aluminium or plastic foil. The mobile phase
is allowed to move across the surface of the plate (usually by capillary action)
and the chromatographic process may depend upon adsorption, partition, or a

combination of both, depending on the adsorbent, its treatment, and the nature
1176
Methods of Analysis
of the solvents used. During the chromatographic procedure the plate is con-
tained in a chromatographic chamber (usually made of glass to permit obser-
vation of the movement of the mobile phase up the plate), which is usually
saturated with the solvent vapour. Solid supports frequently used are silica gel,
kieselguhr, alumina, and may be added suitable substances,

cellulose; to these
for example, calcium sulfate to promote adhesion to the support. The prepared
layer may be impregnated with buffering materials to afford acidic, neutral, or
basic layers, or with other material, such as silver nitrate, designed to modify
its properties. In certain cases the layer may consist of an ion-exchange mate-
rial. This wide range of possible layers, used in conjunction with different
solvent systems allows an almost infinite variation of separating power that

makes thin-layer chromatography such a useful technique in pharmaceutical
analysis.
As an adjunct to identification, thin-layer chromatography may be used
by comparing the behaviour of the material to be identified with that of a
standard substance, usually an authentic specimen of the substance being
examined. If the two substances move identical distances during the
chromatographic process and if the two substances, when mixed together and
then subjected to chromatography, move as a single substance, it may be pre-
sumed that the two substances are identical. This presumption may be
strengthened by repeating the procedure using a different system of chro-
matography; in general, if two substances behave identically in as many as
three fundamentally different systems the presumption of identity becomes
very strong.
For identification purposes it convenient to define the relative distance that
the unknown material moves in relation to the distance moved either by
the solvent front or by a standard reference material. On a developed chro-
matogram, the ratio of the distance travelled on the adsorbent by a given com-
pound to that travelled by the leading edge oft he solvent (or mobile phase),
both measured from the point of application of the test substance, is referred
to as the Rf value of the substance in the given chromatographic system. The
ratio of the distances moved by the compound and a stated reference substance
is referred to as the Revalue. In practice values may vary considerably accord-
ing to the exact experimental conditions so that the Revalue determined against
the reference substance subjected to chromatography on the same surface gives
a more reliable numerical value. Even more reliable, however, is comparison
with an authentic specimen as described above and this is the procedure usually
used for pharmacopoeial purposes.
To determine the position of a colourless substance on a developed chro-
matogram it is usually necessary to treat the chromatogram with a reagent that
will either char the separated substances or convert them to coloured or fluo-
rescent derivatives. A convenient alternative that is frequently applicable is to
carry out the chromatography on a surface impregnated with a substance that
fluoresces strongly when examined under short-wave ultraviolet light. Areas
of the plate occupied by substances absorbing at the same wavelengthshow
as dark spots on the fluorescent background. In special cases, other means of
1177
recognition may be used, for example, the detection of radioactivity where

labelled compounds are being separated or a microbiological response where
antibiotics are concerned.
The most valuable use of thin-layer chromatography in pharmaceutical
work is to provide a means of assessing low levels of impurities in medicinal
substances. For this purpose the substance is applied to the chromatographic
surface and, after chromatography, any secondary spots to be seen in the chro-
matogram after appropriate visualization are compared for size and intensity
with those of low loadings of expected impurities that have simultaneously
been subjected to chromatography on the same plate. Such a procedure
requires that the expected impurities be available and in certain monographs
the use of authentic specimens of impurities is called for. Frequently, such
impurities are not available and in such cases it is often possible to compare
secondary spots arising from trace impurities with the spot obtained by car-
rying out chromatography on the same plate using an appropriately low
loading of the substance being examined. This expedient is not always pos-
sible since impurities and the substance being examined may respond in dif-
ferent ways to the method of revelation used, but it often provides an
acceptable criterion by which the level of impurity in the substance may be
judged. A third procedure that is sometimes advocated is to apply such an
amount of the substance being examined that, after chromatography, no
secondary spots will appear if the sample is acceptably pure. This is the least
satisfactory of the three methods since ability to see a secondary spot is a
subjective matter and because the intensity of spots on a chromatogram may
vary considerably from one occasion to another depending on the exact
conditions of chromatography.
For quantitative procedures the spot may be removed from the plate, the
substance eluted with a suitable solvent and then determined by a sufficiently
sensitive method, such as a spectrophotometric measurement, either directly
or after a chemical reaction. In certain cases, quantitation can also be achieved
by measurement of the spot intensity with the aid of a scanning densitometer
and subsequent comparison of the intensity with the intensities obtained from
standard amounts of the same substance similarly treated.
Separations effected by thin-layer chromatography may sometimes be
improved by multiple development (when the chromatogram is allowed to dry
and then subjected again to the same system of chromatography), by continu-
ous development (when the mobile phase is allowed to evaporate continuously
from the upper end of the adsorbent surface), or by two-dimensional chro-
matography (when the chromatographic plate is allowed to dry, turned at right
angles, and then subjected to further chromatography, frequently in a different
solvent system from that used for the initial chromatography). Caution should
be exercised in interpreting the results of chromatograms where such interme-
diate drying processes are used, however, since decomposition, such as oxida-
tion, of the substance being chromatographed may occur on the plate. The
process of two-dimensional chromatography is especially valuable in judging
whether any chemical change is taking place during the development process.
1178
Methods of Analysis
If a mixture of substances is developed first in one direction and then at right

angles with the same solvent the separated substances will lie in a diagonal line
across the plate if no artefacts are being produced.
In thin-layer chromatography the adsorbent is usually spread in a thin even

layer on a support plate. This may be undertaken in the analytical laboratory
but it is also possible and convenient to obtain commercially prepared chro-
matographic surfaces that are attached to glass or to plastic or metal foil. Unfor-
tunately, so sensitive may the chromatographic process be to minor changes in
conditions that these various commercially available materials are not always
interchangeable one with another or with an apparently similar laboratory-
coated plate. A factory-coated silica gel plate prepared by a given manufacturer
may give different separation characteristics from a laboratory-coated plate pre-
pared using the same manufacturer’s coating substance, and instances exist
where a perfectly satisfactory method of separation devised using laboratory-
prepared plates fails when using precoated plates and vice versa. Great caution
must, therefore, be exercised when changing from one type of chromatographic
plate to another and the suitability of a given type of plate should always be
assessed before relianceis placed upon it.
The chromatographic chamber in which chromatography takes place should

be protected from light if it is suspected that the materials to be examined may
be unstable in light. In chromatographic chamber should always
any case, the
be in a position where the direct rays of the sun cannot fall on it since the rays
may be refracted to different degrees owing to imperfections in the glass walls
of the chamber. This may give rise to areas of elevated temperature on the chro-
matographic plate and result in erratic flow of the mobile phase.
The method given below assumes the use of a laboratory-prepared chromato-
graphic plate but a precoated plate, activated if necessary, may be used pro-
vided that it has been shown to be suitable for the particular application.
The equipment consists of:
• a device for spreading on plates a uniform layer of coating substance of

the desired thickness;
• plates 200 mm long and wide enough to accommodate the required
number of solutions to be examined and the reference solutions;
• a chromatographic chamber of transparent material, usually glass, with
a tightly fitting lid, of a size suitable for the plates used.
Prepare a slurry of the coating substance and, using the spreading device, coat
the carefully cleaned plates with a layer about 0.25mm thick, unless otherwise
specified in the monograph. Allow the coated plates to dry in air and heat to
activate, unless otherwise specified in the monograph, at 110°C for 30 minutes,
then allow to cool. If the plates are not to be used immediately, store them in
a desiccator containing silica gel, desiccant, R. Remove a narrow strip (2-5 mm)
of the coating substance from the vertical sides of the plate.
1179
Unless otherwise specified in the monograph, work under saturated

chamber conditions. To achieve such conditions, line the chromatographic
chamber with filter-paper and pour into the chamber a sufficient quantity of
the mobile phase to saturate the filter-paper and form a layer about 5 mm deep.
Close the chamber and allow to stand for at least 1 hour at room temperature.
Method
All operations during which the plate is exposed to the air should preferably
be carried out at a relative humidity of 50-60%. Apply the volume of the solu-
tion as specified in the monograph as a compact spot, preferably not more than
4 mm in diameter. Application may be made using a micropipette, a syringe,
or other suitable means The spot should be placed about
. 1 .5 cm from the lower
edge and not less than 2 cm from the vertical sides of the plate. Where more
than one chromatogram is run on the same plate, the spots should be placed
not less than 1.5 cm apart and form a line parallel with the lower edge of the
plate. When the solvent has evaporated, place the plate in the chromatographic
chamber, ensuring that the plate is as nearly vertical as possible and that the
starting points are above the level of the mobile phase. Close the chamber and
maintain it at a constant temperature. Allow the mobile phase to ascend,
usually 10-15 cm, remove the plate, mark the position of the solvent front and
dry as specified in the monograph.
1.14.2 Pafaer chromatography

In paper chromatography the stationary phase is a sheet of paper of suitable
texture and thickness, which may sometimes be impregnated with a liquid
phase that is immiscible with the mobile phase.
Chromatographic separations on paper are usually considerably slower than

on thin-layer plates and the method is, in general, not so versatile as thin-layer
chromatography since the degree of variation of the stationary phase is much
more restricted. Neither is it possible to use the variety of corrosive detection
reagents that is commonly employed when the adsorbent is an inorganic mate-
rial coated on a glass plate. Nevertheless, paper chromatography is still a useful
technique and certain very effective separations that were originally devised
using paper have never been successfully transferred to the thin-layer plate. For
semi-quantitative and quantitative work it is considerably easier and more
effective to cut out a required area of paper and to elute a separated compo-
nent than it is to remove completely the powder layer for elution, as is neces-
sary with thin-layer chromatography.
The concepts of Rf and R, values referred to in the discussion on thin-layer
chromatography apply equally well to paper chromatography. Because of the
nature of the adsorbent it is possible to carry out paper chromatography in
either a descending or an ascending mode.
1180
Methods of Analysis
Descending paper chromatography
The apparatus consists of a glass chamber of suitable dimensions to accom-
modate the chromatographic paper used, ground at the top to take a closely
fitting glass lid. The lid has a central hole about 1.5 cm in diameter closed by a
heavy glass rod or a stopper. In the upper part of the chamber is suspended a
solvent trough with a device, usually a glass rod, for holding the chromato-
graphic paper. On either side of the trough, parallel to and slightly above its
upper edges, are two glass guide rods to support the paper in such a manner
that no part of it is in contact with the walls of the chamber. The chromato-
graphic paper consists of suitable filter-paper, cut into strips of sufficient length,
and of any convenient width between 2.5 cm and the length of the trough; the
paper is cut so that the mobile phase runs in the direction of the grain of the
paper.
Method Place in the bottom of the chromatographic chamber a layer 2-3 cm

deep of the stationary phase specified in the monograph. Close the chamber,
and allow to stand for 24 hours at constant temperature. All operations during
which the paper is exposed to the air should preferably be carried out at a
relative humidity of about 50%. Maintain the chamber under these conditions
throughout the subsequent procedure. Draw a fine pencil line horizontally
across the paper at such a distance from one end that when this end is secured
in the solvent trough and the remainder of the paper is hanging freely over the
guide rod the line is a few centimetres below the guide rod and parallel to it.
Using a micropipette, syringe, or other suitable means, apply to a spot on the
pencil line the volume of the solution specified in the monograph. If the total
volume to be applied would produce a spot more than 10 mm
in diameter,
apply the solution in portions, allowing each to dry before the next application.
When more than one chromatogram is to be run on the same strip of paper,
space the solutions along the pencil line at points not less than 3 cm apart. Insert
the paper in the chamber, close the lid, and allow to stand for 90 minutes.
Introduce into the solvent trough, through the hole in the lid, a sufficient
quantity of the mobile phase specified in the monograph, close the chamber
and allow development to proceed for the prescribed distance or time. Remove
the paper from the chromatographic chamber and allow to dry in air. The paper
should be protected from bright light during the development and drying
processes.
Ascending paper chromatography

The apparatus consists of a glass chamber of suitable dimensions to accom-
modate the chromatographic paper used, ground at the top to take a closely
fitting glass lid. In the top of the chamber is a device that suspends the chro-
matographic paper and is capable of being lowered without opening the

chamber. In the bottom of the chamber is a dish to contain the mobile phase
into which the paper may be lowered. The chromatographic paper consists of
1181
suitable filter-paper, cut into strips of sufficient length and not less than 2.5cm
wide; the paper is cut so that the mobile phase runs in the direction of the grain
of the paper.
Method Place in the dish a layer 2-3 cm deep of the mobile phase specified in
the monograph. If specified in the monograph, pour the stationary phase
between the walls of the chamber and the dish. Close the chromatographic
chamber and allow to stand for 24 hours at constant temperature. Maintain the
chamber at this temperature throughout the subsequent procedure. AU
operations during which the paper is exposed to the air should preferably be
carried out at a relative humidity of about 50%. Draw a fine pencil fine
horizontally across the paper 3 cm from one end. Using a micropipette, syringe,
or other suitable means, apply to a spot on the pencil line the volume of the
solution specified in the monograph. If the total volume to be applied would
produce a spot more than 10 mm
in diameter apply the solution in portions,
allowing each to dry before the next application. When more than one
chromatogram is to be run on the same strip of paper, space the solutions along
the pencil line at points not less than 3 cm apart. Insert the paper into the
chamber, close the lid, and allow to stand for 90 minutes. Lower the paper into
the mobile phase specified in the monograph, and allow development to
proceed for the prescribed distance or time. Remove the paper from the
chamber and allow to dry in air. The paper should be protected from bright
light during the development and drying processes.
1.14-3 Column chromatograp>hy

In adsorption column chromatography the solid support (e.g. activated alumina,
powdered cellulose, silicic acid, or kieselguhr) is packed as a dry solid or as a
slurry into a tube (glass, plastic, or other suitable material) having a restricted
orifice (usually protected by a sintered glass disc) for efflux of the mobile phase.
A solution of the material to be subjected to chromatography is added to the
top of the column and allowed to flow into the adsorbent; the solvent that con-
stitutes the mobile phase is then introduced in the top of the column and
allowed to flow downwards either by gravity or by application of positive pres-
sure; during this procedure care should be taken to ensure that the top of the
column does not become dry. The effluent solution (known as the eluate) is
monitored in either a continuous fashion (for example, with a flow-through
ultraviolet absorption cell) or a stepwise fashion (for example, by collection of
fractions at intervals determined by time or by volume or weight of eluate and
subsequent examination of each fraction for the separated component). The
need to examine individually many fractions in order to obtain a completely
quantitative assessment of a substance has resulted in a decline in the use of
classical column chromatographic procedures in recent years; where they do
continue to be used, there is a natural tendency to favour methods of detec-
tion and determination that are readily adaptable to automated processes.
1182
Methods of Analysis
In p’artition column chromatograp’hy, a liquid stationary phase, which should be

substantially immiscible with the mobile phase, is adsorbed to the surface of
the solid adsorbent. Chromatography is carried out as described for adsorption
column chromatography, the mobile phase being saturated with the stationary
phase before it is used for elution. Usually the solid adsorbent in partition chro-
matography is polar and the adsorbed stationary phase is also polar with
respect to the mobile phase. The most widely used adsorbent in this connex-
ion is a siliceous earth having an appropriate particle size to permit ready flow
of the mobile phase. In certain cases reverse-fahase partition chromatography is
useful; in this case the polar adsorbent is rendered non-polar by silanizing or

other means, such as treatment with paraffins, and the adsorbed stationary
phase is less polar than the mobile phase.
In these partition systems the degree of partition of a compound is governed
by its between the two liquid phases and, in the case
distribution coefficient
of compounds by the pH of the more polar of the two phases.
that dissociate,
Selective elution of components of a mixture can often be achieved by succes-
sive changes in the mobile phase or by changing the pH of the stationary phase
by using a mobile phase consisting of a solution of an appropriate acid or base
in an organic solvent.
Ion-exchange chromatography may be considered a case of chromatography
where the solid phase contains an ion-exchange material, usually called ion-
exchange resin.
Ion-exchange is defined as the reversible interchange between the ion
present in the solution and the counter-ion of the resinous polymer, modified
cellulose, or bonded silica gel support; it may be exemplified for the HVNa^
exchange of a strongly acidic cation-exchange resin as;
RSO3H -t Na* ^ RSOsNa -t H*

and for a C170H“ strongly basic anion-exchange resin as:
R'NfCHsfÔH-tCr ^ R'N(CH3)3Cl-tOH-
The selection of strong or weak depend on

resins, of either type, will largely
the pH at which the exchange is and on the types of cation
to be carried out
or anion that are to be exchanged. However, the strongly acidic and basic
exchange resins will serve in most analytical applications. Their specific capac-
itymay vary from 2 to 5 millimoles per gram (dry basis). In practice, a
large(200-300%) excess of resin is used over the calculated stoichiometric
requirement.
The laws governing the exchange reaction are complex, being in part
described by mass action, ionic charge, and activity relationships. The selec-
used to indicate the preference of the ion-exchange resin for
tivity coefficient is
the uptake of 2 (or more) ions from solution. Generally speaking, the resin will
take up divalent (or higher) ions in preference to monovalent ions, and in the
case of a choice between ions of the same valence, the resin will take up the
heavier ion preferentially.
1183
Treatment of the ion-exchange resin and preparation of the column. Usually the
ion-exchange resin is immersed in water and allowed to swell for 24 hours; it
is then packed into a suitable column and, in the case of an anion-exchange

resin, converted to the basic form by passing sodium hydroxide (~80g/l) TS
through the column at a rate of about 3 ml per minute until the effluent is free
of chloride, followed by carbon-dioxide-free water, R to remove alkalinity. In
the case of a cation-exchange resin, conversion to the acidic form is achieved
by passing hydrochloric acid (~70g/l) TS through the column, followed by
carbon-dioxide-free water R until the washings are neutral.
The prepared column is used in a similar manner to that described for adsorp-
tion column chromatography except that there is usually no need to monitor the
effluent;according to the type of resin chosen and the type of material being
determined the volume of effluent detailed in the particular application is col-
lectedand titrated with acid or base as appropriate, using a suitable indicator.
After the determination has been completed, the ion-exchange column may
be regenerated by washing either with sodium hydroxide (~80g/l) TS, for an
anion-exchange column, or hydrochloric acid (~70g/l) TS, for a cation-exchange
column, followed by water until a neutral reaction is obtained.
1.14-4 High frerformance liquid chromatografahy

Introduction
High-performance liquid chromatography (HPLC) is a separation technique
that can be used for the analysis of organic molecules and ions. HPLC is based
on mechanisms of adsorption, partition, ion exchange or size exclusion,
depending on the type of stationary phase used. HPLC involves a solid sta-
tionary phase, normally packed inside a stainless-steel column, and a liquid
mobile phase. Separation of the components of a solution results from the dif-
ference in the relative distribution ratios of the solutes between the two phases.
HPLC can be used to assess the purity and/or determine the content of many
pharmaceutical substances. It can also be used to determine enantiomeric com-
position, using suitably modified mobile phases or chiral stationary phases.

Individual separation mechanisms of adsorption, partition, ion exchange and
size exclusion rarely occur in isolation, since several principles act to a certain
degree simultaneously.
Apparatus
The apparatus consists of a pumping system, an injector, a chromatographic
column, stationary and mobile phases, connecting tubing and fittings, a detec-
tor, and a data collection device (computer, integrator or recorder).
Pumping system
HPLC pumping systems are required to deliver metered amounts of mobile
phase at a constant flow rate. Pumping systems that deliver solvent from one
or more reservoirs are available. Pressure fluctuations should be minimized, e.g.
1184
Methods of Analysis
by passing the pressurized solvent through a pulse-dampening device. Tubing

and connections should be capable of withstanding the pressures developed by
the pumping system. Many HPLC pumps are fitted with a facility for “bleed-
ing” the system of entrapped air bubbles.
Modern computer- or microprocessor-controlled pumping systems are
capable of accurately delivering a mobile phase of either constant (isocratic
elution) or varying (gradient elution) composition, according to a defined pro-
gramme. In the case of gradient elution, solvent mixing can be achieved on
either the low or high-pressure side of the pump(s). Depending on the flow rate
and composition of the mobile phase, operating pressures of up to 42000 kPa
(6000 psi) can be generated during routine analysis.
Injector
The sample solution is usually introduced into the flowing mobile phase at or
near the head of the column using an injection system based on an injection
valve design which can operate at high pressure. Such an injection system has
a fixed-loop or a variable volume device which can be operated manually or
by an auto-sampler. Partial filling of loops may lead to poorer injection volume
precision.
Chromatograp’hic column
Columns are usually made of polished stainless steel, are between 50 and
300mm long, and have an internal diameter of between 2 and 5 mm. They are
commonly filled with a stationary phase with a particle size of 5-10 pm.
Columns with internal diameters of less than 2 mm are often referred to as
microbore columns. Ideally, the temperature of the mobile phase and the
column should be kept constant during an analysis. Most separations are per-
formed at ambient temperature but columns may be heated using, for instance,
a water-bath, a heating block or a column oven in order to achieve better
efficiency. Normally, columns should not be heated above 60 °C because of
the potential for stationary phase degradation or changes occurring to the
composition of the mobile phase.
Stationary phases
Separation of pharmaceuticals is usually achieved by partition of compounds
in the test solution between the mobile and the stationary phases. HPLC
systems consisting of polar stationary phases and nonpolar mobile phases are
described as normal-phase chromatography; those with nonpolar stationary
phases and polar mobile phases are called reversed-phase chromatography.
There are many types of stationary phases used in HPLC including:
— unmodified silica, alumina, or porous graphite, used in normal-phase

chromatography, where separation is based on differences in adsorption;
— a variety of chemically modified supports prepared from polymers, silica,
or porous graphite, used in reverse-phase HPLC, where separation is
1185
based principally on partition of the molecules between the mobile phase

and the stationary phase;
— resins or polymers with acid or basic groups, used in ion-exchange chro-
matography, where separation is based on competition between the ions
to be separated and those in the mobile phase;
— porous silica or polymers, used in size-exclusion chromatography, where
separation is based on the relative molecular mass of the molecules.
Most separations are based on partition mechanisms using chemically modi-

fied silica as the stationary phase and polar solvents as the mobile phase
(reverse-phase HPLC). The surface of the support, e.g. the silanol groups of
silica, is reacted with various silane reagents to produce covalently bonded silyl
derivatives covering a varying number of active sites on the surface of the
support. The nature of the bonded phase is an important parameter for deter-
mining the separation properties of the chromatographic system.
Commonly used bonded phases are shown below.
Common bonded phases

octyl Si-(CH2)7-CH3 Cs
octadecyl Si-(CH2)i7-CH3 Cl8
phenyl Si-(CH2)3-C,H5 C«H5
cyanopropyl Si-(CH2)3-CN CN
aminopropyl Si-(CH2)3-NH2 NHj
diol Si-(CH2)3-OCH(OH)-CH2-OH
For the separation of enantiomers, special chemically modified stationary

phases (chiral chromatography) are available, e.g. cyclodextrins, albumins, etc.
As a guide, silica-based reverse-phase columns are generally considered to
be stable in mobile phases with an apparent pH in the range 2. 0-8.0, but the
column manufacturer’s instructions should be consulted before using the
column. Columns containing particles of polymeric materials such as styrene
divinylbenzene copolymer are stable over a wider pH range.
Analysis using normal-phase HPLC with unmodified silica, porous graphite
or polar chemically modified silica (e.g. cyanopropyl or diol) as the stationary
phase and a nonpolar mobile phase employed in certain cases.
is still
For analytical separations the particle size of the most commonly used sta-
tionary phases varies between 3 pm and 10 pm. The particles may be spherical
or irregular, of different porosities and specific surface area. In the case of
reversed-phase, the extent of bonding of the stationary phase is expressed as
the carbon-loading. Furthermore, stationary phases may be “end-capped”, i.e.
the number of residual silanol groups is reduced by methylation. These para-

meters contribute to the chromatographic behaviour of a particular stationary
phase. Tailing of peaks, particularly for basic substances, can occur when
residual silanol groups are present.
1186
Methods of Analysis
Mobile phases
The choice of mobile phases is based on the desired retention behaviour and
the physicochemical properties of the analyte.
For normal-phase HPLC using unmodified stationary phases, lipophilic sol-
vents should be employed. The presence of water in the mobile phase must be
avoided as this will reduce the efficiency of the stationary phase. In reverse-
phase HPLC aqueous mobile phases, with and without organic modifiers, are
used.
The mobile phase should be filtered through suitable membrane-type filters
with a porosity of 0.45 pm to remove mechanical particles. Multicomponent
mobile phases should be prepared by measuring the required volumes (unless
masses are specified) of the individual components, followed by manual or
mechanical mixing. Alternatively, the solvents may be delivered by the indi-
vidual pumps or proportioning valves of the liquid chromatograph and mixed
according to the desired proportion. Solvents are normally degassed by sparg-
ing with helium or by sonification before pumping to avoid the creation of gas
bubbles in the detector cell.
If an ultraviolet detector is employed, the solvents used for the preparation

of the mobile phase should be free of stabilizers and transparent at the
wavelength of detection. Adjustment of the pH, if necessary, should be made
using the aqueous component of the mobile phase and not the mixture. Buffers
of high molarity should be avoided in the preparation of mobile phases. If
buffers are used, the system should be rinsed with an adequate mixture of
water and the organic modifier of the mobile phase to prevent crystallization
of salts.
Mobile phases may contain other components, e.g. a counter-ion for ion-
pair chromatography or a chiral selector for chiral chromatography using an
achiral stationary phase.
Connecting tubing and fittings
The column may never be achieved because

potential efficiency of an analytical
of the design limitations of injectors and detectors. The connections between
injector/column, column/detector, and/or detector/detector may compromise
the overall efficiency of the system and any fittings should be of the “zero dead
volume” (ZDV) type. It is recommended that minimum lengths of capillary
tubing with a maximum internal diameter of 0.25mm be used for these fittings
to minimize band spreading.
Detectors
Ultraviolet/visible (UV/vis) absorption spectrometers are the most commonly
used detectors for pharmaceutical analysis. In specific cases, fluorescence
spectrophotometers, differential refractometers, electrochemical detectors,
light-scattering detectors, mass spectrometers, or other special detectors may
be used. Where an analytepossesses a chromophoric group that absorbs UV/vis
radiation, the UV/vis detector is the most appropriate because of its sensitiv-
1187
ity and stability. Such a detector is not suitable for detecting analytes with very
weak chromophores.
A variant on the UV/vis type of detector, which is becoming increasingly
popular because of its ability to furnish detailed spectral information, is the
diode array spectrophotometer. This type of detector acquires absorbance data
over a certain UV/vis range and can provide chromatograms at multiple, selec-
table wavelengths, together with spectra for the eluted peaks. In addition, the
detector and accompanying computer programmes can be used to assess the
spectral homogeneity of peaks, which may provide information on the chro-
matographic purity of the peaks. This can be especially useful in method devel-
opment and validation.
Enhanced sensitivity may be achieved in certain cases by using pre-column
or post-column derivatization techniques. (These techniques are to be avoided
for the control of impurities.)
Data collection devices
Signals from the detector may be collected on chart recorders or electronic inte-
grators that vary in complexity and in their ability to process, store, and
reprocess chromatographic data. The data storage capacity of these devices is
usually limited.
Modern data stations are computer based and have a large storage capacity
to collect, process, and store data for possible subsequent reprocessing.
Analytical reports can often be customized to the needs of the analyst.
Integration of peak areas and the setting of threshold levels are not normally
problematic in an assay since the peak of the substance to be analysed should
be free of interference. However, in a test for impurities, the selection of the
peak area integrator parameters becomes very important, particularly when
baseline separations are not always attainable. If baseline separations cannot
be obtained, valley-to-valley integration should be employed.
HPLC allows limits to be set for individual impurities and for the sum of
impurities, but there is a level at which peaks should not be integrated. This
“disregard level” is set in relation to the area of the peak in the chromatogram
of the prescribed reference solution and is usually equivalent to 0.05% of the
substance being examined.
System suitability
test represents an integral part of the method and is
The system suitability
used to ensure the adequate performance of the chosen chromatographic
system.
Efficiency, capacity factor, resolution factor, and symmetry factor are the
parameters that are normally used in assessing the column performance; these
terms are defined below. Factors that can affect chromatographic behaviour
include mobile phase composition, temperature, ionic strength and apparent
pH, flow rate, and column length, and stationary phase characteristics such as
porosity, particle size and type, specific surface area, and, in the case of
1188
Methods of Analysis
reversed-phase supports, the type of chemical modification, carbon loading,

and end-capping.
Efficiency (N)
The efficiency of a chromatographic column is defined in terms of the number
of theoretical plates (A/) and can be calculated using the following formula:
t^
N = 5.54-V
Wh^
where = retention time or the baseline distance between the point of injec-
tion and the perpendicular dropped from the maximum of the
peak of interest.
Wh = the width of the peak of interest determined at half peak height,
measured in the same units as in..
The number of theoretical plates can be expressed per metre (N'):
where / = length of column in metres.
Capacity factor (mass distribution ratio, DJ

The capacity factor or mass distribution ratio is defined as follows:
amount of solute in stationary phase

™ amount of solute in mobile phase
This factor determines the retention of a solute and can be calculated from the
chromatogram using the following formula:
~ ^m)
j-j _ (^R
where Jr = retention time of the solute

iM = retention time of an unretained component
A low Dm value indicates that the peak elutes close to the solvent front, which
may compromise selectivity. A minimum Dm value of 1 is recommended for
the peak of interest.
The retention time of the test substance can be varied, if necessary, by
changing the relative proportion or composition of solvents in the mobile
phase. Generally, an increase in the proportion of a more polar solvent will lead
to a shorter retention time on a normal-phase column and a longer retention
time on a reversed-phase column.
1189
Resolution factor (RJ

The resolution between two peaks of similar height in a chromatogram can be
calculated using the following formula:
l-18(t;^2 ~ tg^])
J,
_
(Wm+Wi,2)
where tRi and tp^ = retention times or baseline distances between the point of
injection and the perpendicular dropped from the
maximum of each of the two peaks.
Whj and Wb2 = the respective peak widths determined at half peak height,
measured in the same units as and tp2 -
The value of Rs for a baseline separation between peaks of similar height should
be at least 1.5.
Relative retention
The relative retention (t) is calculated as an estimate using the following
formula:
^Rl
“ t(v1
where Jr2 = retention time of the peak of interest

tRi = retention time of the reference peak
tM = retention time of an unretained component
The unadjusted relative retention (tc) is calculate from the expression:
tc = te/ tRl
Unless otherwise indicated, values for relative retention stated in the mono-
graphs correspond to unadjusted relative retention.
Symmetry factor (A,)

The symmetry factor for a peak can be calculated using the following formula:
where = peak width at 5% of peak height, measured from the baseline.

d = baseline distance between the perpendicular dropped from the
peak maximum and the leading edge of the peak at 5% of the
peak height, measured in the same units as W^.
Values of which are greater than 2 may lead to incorrect integration, result-
ing in erroneous quantitation. The main factors that influence peak symmetry
depend upon retention, solvent effects, incompatibility of the solute with the
mobile phase, or development of an excessive void at the inlet of the column.
1190
Methods of Analysis
In reversed-phase chromatography, adsorption phenomena due to the presence

of residual silanol groups in the stationary phase may lead to tailing (poor peak
symmetry).
Repeatability
Unless otherwise stated in the “Assay” of the individual monograph, the rela-
tive standard deviation of peak areas or peak heights for a series of injections
of reference solutions bracketing groups of test solutions should not exceed
2 0 %.
.
In a “Related substances" test the relative standard deviation of peak areas

or peak heights for three replicate injections of the reference solution should
not exceed 5.0%, unless otherwise stated in the individual monograph.
In a series of injections the relative standard deviation of the retention time
of the principal peak should not exceed 1.0%.
Adjustment of chromatographic conditions

The extent to which the various parameters of a chromatographic test may be
adjusted to satisfy the system suitability criteria without fundamentally mod-
ifying the methods are described below for information. The chromatographic
conditions described have been validated during the elaboration of the mono-
graph. The system suitability tests are included to ensure the separation
required for satisfactory performance of the test or assay. Nonetheless, since
the stationary phases are described in a general way and there is such a variety
available commercially, with differences in chromatographic behaviour, some
adjustments of the chromatographic conditions may be necessary to achieve
the prescribed system suitability requirements and to achieve the indicated
retention time. The retention time must be within +10.0 per cent of that indi-
cated in the test and/or assay. With reverse-phase liquid chromatographic
methods, in particular, adjustment of the various parameters will not always
result in satisfactory chromatography. In that case, it may be necessary to
replace the column with another of the same general type (e.g. octadecylsilyl
silica gel) that exhibits the desired chromatographic behaviour.
For critical parameters the adjustments are defined clearly in the monograph
to ensure the system suitability.
Multiple adjustments which may have a cumulative effect on the perfor-
mance of the system are to be avoided.
Composition of the mobile phase The amount of the minor solvent component
may be adjusted by +30% relative or +2% absolute, whichever is the
larger. [Examples: For a minor component that is at 10% of the mobile
phase, a 30% relative adjustment allows a range of 7-13%, whereas a
2% absolute adjustment allows a range of 8-12%; in this case, the rela-
tive value is the larger. For a minor component that is at 5% of the mobile
phase, a 30% relative adjustment allows a range of 3. 5-6.5%, whereas
a 2% absolute adjustment allows a range of 3-7%; in this case, the
1191
absolute value is the larger.] No other component is altered by more than

10% absolute,
pH of the acjueous component of the pH, unless otherwise stated
mobile phase +0.2
in the monograph, or +1.0 pH when neutral substances are to be examined.
Concentration of salts In the buffer component of a mobile phase: +10%.
Detector wavelength No adjustment permitted.
Stationary phase
column length:+70%,
±25%,
column internal diameter:
particle size:maximal reduction of 50%, no increase permitted.
Flow rate ±50%. When the retention time of the principal peak is indicated
in a monograph, the flow rate has to be adjusted, if the column internal
diameter has been changed. No decrease in flow rate is permitted,
however, if the monograph uses apparent number of theoretical plates
in the qualification section.
Temperature ±10%, to a maximum of 60 °C.
Injection volume may be decreased, provided detection and repeatability of
the peak(s) to be determined are satisfactory.
Gradient elution The configuration of the equipment employed may signifi-
candy alter the resolution, retention time and relative retentions

described in the method. Should this occur, it may be due to excessive
dead volume (dwell volume) which is the volume between the point at
which the two eluants meet and the top of the column.
To equilibrate the column, allow the mobile phase to flow through the chro-
matographic system until the baseline is stable at the flow rate specified in the
individual monograph (about 30 minutes). Prepare the prescribed test and ref-
erence solutions as directed. Inject the prescribed reference solution and, if nec-
essary, adjust the detector and/or recorder response to produce an adequate
peak size. For chart recorders and integrators this should be at least 50% of the
full-scale deflection of the principal peak in the chromatograms obtained with
the reference solution. Ensure that the criteria for system suitability are met.
The reference solution should be injected at the start, at regular intervals
during, and at the end of a series of assays (e.g. every 2-4 samples). Both the
reference and the test solutions should be injected in duplicate.
component composition of a complex mixture, a “nor-
In determining the
malization” procedure, based on the calculation of individual peak areas as a
percentage of the total area of all the peaks, may be used where the relative
response factors of the individual components are similar. The response factor
is relative, being the response of the equal mass of one substance relative to
that of another according to the conditions described in the test. For example,
when an HPLC test with UV/vis detection is used for the control of impurities,
the wavelength of detection should be such that the substance and its impuri-
ties have similar responses. If an impurity has a significantly different response
1192
Methods of Analysis
(more than ±20%) from that of the substance being examined, the preferred
manner of limiting this impurity is to use a reference substance of the impu-
rity. If a reference substance is not available, the response factors of the poten-
tial impurities relative to those of the substance being examined are determined
during method development. Subsequently, the derived correction factors (i.e.

the reciprocals of the response factors) are applied, if necessary, as described in
the individual monograph.
If gradient elution is specified in the monograph, an instrument equipped
with a special pumping system capable of delivering a mobile phase of con-

tinuously varied composition is needed. The mobile phase composition
changes from the initial composition within a fixed period of time, as specified
in the monograph. Where the mobile phase composition is varied at a linear
gradient elution, perform a blank run to identify any interfering peaks by inject-
ing the solvent specified for preparing the test solutions. Allow sufficient time
for equilibration when the mobile phase is reset to the initial composition for
the next injection.
1.14-5 Gas chromatograp>hy

Gas chromatography may be regarded as a form of column chromatography in
which the mobile phase is a gas (referred to as the carrier gas) rather than a
liquid solvent. The stationary phase may be an active adsorbent such as
alumina, silica gel, or carbon (gas-solid chromatography) or it may be a liquid
that is coated as a thin film on a finely divided inert solid support such as
diatomaceous earth, firebrick, glass beads, or other suitable material (gas-liquid

chromatography); if the chromatographic column is of very small diameter the
stationary phase may be coated on its inner wall to provide the so-called “open
tube” or “capillary” column. Certain materials are available that do not require
coating with a liquid phase, for example, polyaromatic porous beads, and these
are of great value for specific applications.
The substance is introduced in a vaporized state into the carrier gas stream
at the head of the column and it undergoes distribution between the gas and
liquid or solid stationary phase in a similar manner to that in other forms of
chromatography. The partition coefficient (K), which is defined as
quantity of solute in the stationary phase

^
quantity of solute in the mobile phase
will depend upon the nature of the solute, the nature and amount of the sta-
tionary phase, the temperature, and the carrier gas flow rate. It will be clear
then that the value of K will depend upon the particular column and precise
operating conditions used and, since these would be impossible to reproduce
exactly from one laboratory to another and from one make of instrument to
another, it is necessary to treat the operating conditions set down for pharma-
copoeia! purposes with a degree of flexibility that is not accorded to other,
nonchromatographic, test procedures.
1193
The essential features of the apparatus for effecting gas-liquid chromatog-

raphy (much more widely used for pharmaceutical applications than adsorp-
tion chromatography on a solid) are a source of carrier gas (usually contained
in compressed form in a cylinder fitted with a pressure-reducing valve), a
flowmeter through which the gas passes, and an injection port that may be
heated to a suitable temperature to volatilize but not decompose the substance
and through which the test solution is introduced into the flow of carrier gas,
preferably directly into the column packing. The constant flow of carrier gas is
then maintained as the chromatographic process takes place, the components
of the test solution being separated according to the value of K for each under
the particular conditions being used.
As the components emerge from the column they enter a suitable differen-
tial-type detector,which usually depends on changes of ionization in a flame
oron changes of thermal conductivity. Many other types of detectors exist and
some of these are useful for specific pharmaceutical applications, for example,
the electron capture detector that is of special value in the sensitive detection
of halogenated compounds. Electrical signals from the detector are passed to
an amplifier connected to a suitable recording device, such as a strip-chart
recorder, where signals may be plotted against time. A powerful though very
expensive means of detection is to use a mass spectrometer coupled to the gas
chromatograph. This is very sensitive and provides information that enables
unambiguous identification of substances issuing from the column.
The column is made of glass or metal (in the latter case care has to be taken
since certain organic substances undergo metal-catalysed degradation reactions
at elevated temperatures) and is contained in a temperature-controlled oven
capable of being maintained at temperatures ranging from just above ambient
temperature to about 300 °C according to the particular application. Ovens may
also be controlled in such a way that a steady rise of temperature may be main-
tained over a given period of time; such “temperature programming” is fre-
quently of value when complex mixtures of compounds having widely
different volatilization characteristics are being examined. Columns of differ-
ent lengths are commonly employed, varying from as little as 0.5 m to almost
3 m for packed and from 10 to 100 m for capillary columns; for many pharma-
ceutical applications a column of about 1.5 m is generally used and it may be
from 2 to 5mm in internal diameter.

The packing contained within columns for gas-liquid chromatography has
a profound effect on the quality and effectiveness of separations. The solid
support, which may vary in particle size from about 75|im to 250 pm (60-
200 mesh; although in any given column it should be closely defined within a
narrow range), must be as inert as possible, particularly when polar drugs are
to be chromatographed on supports coated with low concentrations of a liquid
of low polarity. Active sites on the solid support may result in peak tailing of
the solute, or even in its decomposition or rearrangement. Reactivity of the
support may be minimized by treatment with a silanizing reagent before it is
coated with the stationary phase. Injection residues might render the inlet part
of the column ineffective.
1194
Methods of Analysis
Liquid phases commonly used include macrogols (polyethylene glycols) and

esters, high molecular weight amides, silicone gums and fluids, and hydrocar-
bons. The silicone gums are substituted polysiloxanes and are a particularly
valuable series of stationary phases. The highest temperature at which the sta-
tionary phase is designed to be used should be carefully noted and respected
since “column bleeding” may occur and vitiate results if excessive temperatures
are used. Prior to use a new column should be conditioned by maintaining it
for several hours with a stream of carrier gas passing through it at a tempera-
ture somewhat higher than the temperature at which the column is subse-
quently to be used but certainly not above the highest recommended
temperature.
As carrier gas it is necessary to choose one that is inert and for flame-
ionization detection (the most commonly used method in pharmaceutical
analysis), nitrogen is very satisfactory. Though helium is also suitable and is
actually to be preferred for work with a thermal conductivity detector because
of its high thermal conductivity, it is expensive and not readily available in aU
parts of the world, whereas nitrogen is.
For quantitative applications of gas-liquid chromatography in the pharma-
copoeia an internal standard is usually used since the comparison of one chro-
matogram with another resulting from a second injection on to the column
could be subject to error. The addition of a suitable internal standard to the test
solution and to a standard solution eliminates this error since the ratio of area
or height (see below) of the peak due to the substance to be determined and
of that due to the internal standard is compared from one chromatogram to
another. In other applications it may
be more convenient to use a process of
normalization (particularly where an impurity is being assessed). In this case,
the area of the peak due to the sought-for impurity is expressed as a percent-
age of the total area of all peaks derived from the substance being examined
and its attendant impurities. Since the peak due to the principal component will
usually be at least two orders of magnitude greater than that due to a minor
impurity, it is necessary for such determinations to use a reliable automatic inte-
grator and a wide-range amplifier that will respond in a linear fashion to both
the major and the minor components. Peak areas may also be determined by
planimeter, graphically, or by comparing the weights of paper cut from under-
neath the various peaks. In certain circumstances, it may be valid to use
measurements of peak height rather than peak area, although the latter is
considered to be more accurate for quantitative determinations. When the mea-
surement of peak width is required, it is defined as the distance along the base
line that is between the two points of intersection of lines drawn tangentially
to the sides of the peak.
When the internal standard method is used the detailed procedure given
below is applicable. It will be noted that reference is made to 3 solutions. The
first of these (solution 1) contains an internal standard and an appropriate quan-
tity of the substance to be determined (in the case of an impurity determina-
tion this might be the impurity itself, if available, or a suitably low loading of
the substance in which impurities are to be sought). Chromatogram A thus
1195
obtained permits the relationship of response to the internal standard and to

the substance being determined to be established. The second solution (solu-
tion 2) consists only of the substance being examined; this enables the analyst
to confirm that there is no impurity present that will elute with the same reten-
tion time as the internal standard or to make allowance for the quantity present
if there is a coincident peak. The third solution (solution 3) consists of the sub-
stance being examined and the same
internal standard (the latter present at the
concentration as in solution Data derived from chromatograms A and C,
1).
corrected if made on chromatogram B, enable the

necessary for observations
quantities of minor constituents present in the substance being examined to be
determined.
When a normalization procedure is used, a single solution may suffice
since the total area of all minor peaks is to be expressed as a proportion of
the total peak area. If a specific minor peak is to be evaluated, a second solu-
tion containing the sought-after material will be necessary so that the appro-
priate peak on the chromatogram of the substance being examined may be
identified.
The length of the chromatographic column, the stationary phase, the solid
support, the temperature, the carrier gas, the detector, and any other relevant
details that are required for the determination are specified in the monograph.
Where a non-volatile material is to be injected on to the column, a suitable
interchangeable pre-column may be used.
In certain monographs a minimum column efficiency may be required. This
is defined by the expression 16 tl/Ly^, where
tg is the distance (in mm) along the baseline between the point of injection
and a perpendicular dropped from the maximum of the peak produced
by the internal standard specified in the monograph;
L is the length (in m) of the column;
is the peak width (in mm) of the peak produced by the internal standard.
When aqueous solutions are examined and a flame ionization detector is used,
the results are invalid if the water is eluted at the same time as any of the
required components.
Method
Using solution 1, determine experimentally suitable sensitivity settings of the
instrument and the volumes of solutions to be injected to produce an adequate
response. Adjustment of the concentration of the internal standard should be
made, if necessary, so that the recorder response produced by the internal stan-
dard is approximately the same as the response of the substance being deter-
mined. Prepare a differential chromatogram by injecting the selected volume of
solution 1 through the sample injection port on the chromatographic column.
1196
Methods of Analysis
maintained at a suitable temperature, and eluting with the carrier gas. Repeat
the determination twice more. In the same manner, prepare graphs using the
same volumes o£ solutions 2 and 3. Measure the peak areas or, where the sym-
metry factor lies between 0.95 and 1.05, the peak heights produced by the sub-
stance or substances being determined and the internal standard. If an impurity
peak has the same retention time as the internal standard, allowance is made
in assessing the responses of these constituents for the contribution of each.
From the values obtained calculate the proportion of the substance or
substances being determined.
The symmetry factor of a peak is calculated from the expression yJ2A,
where:
y, is the width of the peak at one-twentieth of the peak height;

A is the distance between the perpendicular dropped from the peak
maximum and the leading edge of the peak at one-twentieth of the peak
height.
The results of the determination are usually not valid unless the resolution
between measured peaks on the chromatogram is greater than 1.0. The reso-
lution is calculated from the expression 2{tgi. - tgf)/{Y„ T;.), where: -(-
tg„ and are the distances along the baseline between the point of injection
t[,[.
and perpendiculars dropped from the maxima of two adjacent peaks;

Y„ and Yi. are the respective peak widths.
Static Head-space Gas Chromatography

Static head-space gas chromatography is a technique particularly suitable for
separating and determining volatile compounds present in solid or liquid
samples. The method is based on the analysis of the vapour phase in equilib-
rium with the solid or liquid phase.
Static head-space injection systems include a thermostatically controlled
sample heating chamber in which closed vials containing solid or liquid samples
are placed for a fixed period of time to allow the volatile components of the
sample to reach equilibrium between the non-gaseous phase and the vapour
phase. After equilibrium has been established, a predetermined amount of the
head-space of the vial is flushed into the gas chromatograph.
Apparatus
The apparatus consists of a gas chromatograph provided with a device for intro-
ducing the sample that may be connected to a module that automatically con-
trols the pressure and the temperature. If necessary, a device for eliminating
solvents can be added.
The sample to be analysed is introduced into a container fitted with a suit-
able stopper and a valve-system which permits the passage of the carrier gas.
The container is placed in a thermostatically controlled chamber at a tempera-
ture set according to the substance to be examined.
1197
The sample is held at this temperature long enough to allow equilibrium to

be established between the solid or liquid phase and the vapour phase.
The carrier gas is introduced into the container and, after the prescribed
time, a suitable valve is opened so that the gas expands towards the chro-
matographic column taking the volatilized compounds with it.
Instead of using a chromatograph specifically equipped for the introduction
of samples, it is also possible to use airtight syringes and a conventional chro-
matograph. Equilibration is then carried out in a separate chamber and the
vapour phase is carried onto the column, taking the precautions necessary to
avoid any changes in the equilibrium.
Using the reference preparations, determine suitable instrument settings to
produce an adequate response.
Method 1: Direct calibration

Separately introduce into identical containers the preparation to be examined
and each of the reference preparations, as prescribed in the monograph, avoid-
ing contact between the sampling device and the samples.
Close the containers hermetically and place in the thermostatically con-
trolled chamber set to the temperature and pressure prescribed in the mono-
graph; after equilibration, carry out the chromatography under the prescribed
conditions.
Method 2: Standard additions

Add to a set of identical suitable containers equal volumes of the preparation
to be examined. Add to all but one of the containers suitable quantities of a
reference preparation containing a known concentration of the substance to be
determined so as to produce a series of preparations containing steadily increas-
ing concentrations of the substance.
Close the containers hermetically and place in the thermostatically con-
trolled chamber set to the temperature and pressure prescribed in the mono-
graph; after equilibration, carry out the chromatography under the prescribed
conditions.
Calculate the linear equation of the graph using a least-squares fit, and derive
from it the concentration of the substance to be determined in the preparation
to be examined.
Alternatively, plot on a graph the mean of readings against the added quan-
tity of the substance to be determined. Extrapolate the line joining the points
on the graph until it meets the concentration axis. The distance between this
point and the intersection of the axes represents the concentration of the sub-
stance to be determined in the preparation to be examined.
1198
Methods of Analysis
1.15 Electrophoresis
Electrophoresis is a physical method of analysis permitting the separation of
compounds that are capable of acquiring en electrical charge in a conducting
electrolyte. In this medium the ionized particles move more or less rapidly
under the influence of an electrical field.
The electrophoretic mobility is the rate of migration of the substance mea-
sured in cm/s under the influence of a potential gradient of IV/cm, and is
expressed in cm^-V“'-s”'.
The measurement of electrophoretic mobility is where
significant only
experimental conditions have been precisely defined. This mobility depends on
the characteristics of the substance, its nature, size, form, and electrical charge.
It also depends on the composition of the conducting liquid, its nature, con-
centration, pH, the presence of additional solvents and viscosity. The direction
of migration depends on the sign of the electrical charge of the particle as it
moves towards the electrode of opposite sign.
According to the methods used, the electrophoretic mobility is either mea-
sured directly or compared with that of a reference substance.
Moving boundary (free- flow) electrophoresis
This technique, used exclusively for the determination of the mobility, is par-
ticularly suitable for substances of high molecular weight with poor diffusion
properties.
The boundaries are usually measured both before and after the application
of an electrical field by a physical method, such as refractometry or conduc-
tometry. The concentration of the substance in the conducting liquid, the char-
acteristics of the latter and the details of the procedure, including quantitative
evaluation of the fractions, are specified in the monographs.
Zone electrophoresis (electrophoresis using a supporting

medium)
This method uses only small sample sizes. The nature of the supporting
medium (for example, paper, cellulose acetate, starch-gel, agar-gel, poly-
methacrylamide, mixed gel) introduces additional factors influencing the mobil-
ity. The rate of migration depends on the mobility of the particles and also on
the electroendosmotic current (in the case of carriers with polar properties), on
the currents due to evaporation (caused by heat generated through the Joule
effect), and on the gradient of the electrical field.
In practice, the mobility of the electrophoretic zones and their signs are
ignored; the zones are located by experience or by comparison with those given
by a reference substance treated in the same way.
After separation of the constituents the position of colourless substances
may be determined by treating the electropherogram with a reagent that will
convert them to coloured or fluorescent derivatives. For quantitative purposes.
1199
the spot (zone) may be carefully separated, the substance eluted with a suit-
able solvent and then determined by a sufficiently sensitive method, such as

spectrophotometric measurement, either directly or after a chemical reaction.
In another quantitative procedure after conversion to a coloured derivative, the
zone intensity can be measured with the aid of a scanning densitometer.
An apparatus for electrophoresis on a supporting medium is composed of:
• A source of direct current, preferably of stabilized voltage.

• A chamber for electrophoresis, generally in the form of a parallelepiped,
made from glass or rigid plastic material with an airtight lid ensuring the
maintenance of an atmosphere of saturated humidity. Two insulated
electrical leads are sealed through the walls of the chamber, one at either
end, each lead having an internal connector to which are attached elec-
trodes of platinum wire. The chamber should be fitted with suitable
safety devices to ensure that the electrical supply is disconnected when
the lid is removed. Two double troughs provided with a central length-
wise partition are inserted in the chamber, one at each end. Alternatively,
the troughs may be integral parts of the chamber. One platinum elec-
trode is laid along the bottom of each outer trough compartment. The
electrodes are connected through external insulated cables to an electri-
cal power source having an output of not less than 450V D.C. at
150 mA. The power source should be provided with a means of
indicating and controlling the voltage and of indicating the current
consumption. Additional circuitry may be incorporated to stabilize the
voltage.
• A holder device. When paper or cellulose acetate is used, the carrier strips
impregnated with the conducting liquid are stretched by an appropriate
arrangement and the ends immersed into the electrode troughs. In gel
electrophoresis, an adherent on an even layer of gel is placed on glass
and the electrical connexions are attached at each end.
• A device to locate and measure the spots.
Recommended procedures
Paper electrophoresis
A chamber about 50 cm long, 38 cm wide and 4.5 cm deep, with troughs about
37 cm long externally, 5cm wide and 2 cm in depth internally, is suitable.
The electrophoresis paper consists of suitable filter-paper (Whatman 3 MM
or similar grade is suitable) that has been washed chromatographically with a
suitable solvent if so specified in the monograph. The paper is cut into strips
of appropriate size and a baseline is drawn across the paper about 13 cm from
one end.
Fill the troughs of the apparatus with the conducting liquid specified in the
monograph. Place strips of electrophoresis paper (about 30 cm by 5 cm) in the
troughs so as to form bridges between the outer and inner compartments and
ensure that the electrodes are fully immersed in the conducting liquid in the
outer compartments.
1200
Methods of Analysis
Apply separately to points along the base line of the electrophoresis paper,
at least 1 cm from the edge of the paper and not less than 2.5 cm apart, the
volumes of solutions prepared as specified in the monograph.
Allow the spots to dry and then place the end of the paper nearest the base-
line in the inner compartment of the trough connected to the anode and the
other end in the inner compartment of the trough connected to the cathode.
Wet the paper with the conducting liquid, using a brush, starting from the ends
of the paper and working towards the baseline. Do not wet the strip that
includes the applied substance. Close the lid, allow the liquid to diffuse across
the baseline, if necessary cover the apparatus so as to exclude light, connect
the cables to the power supply and switch on the current. Adjust the voltage
to about 20 V per cm of paper between the troughs and allow electrophoresis
to proceed for the time indicated or until the marker substances have moved
the specified distances. Switch off the current, remove the paper, dry in a
current of air protected from light if necessary, and examine the resulting elec-
tropherogram under the conditions prescribed in the monograph. When the use
of marker substances is specified, the test is valid only if the marker substances
move to the specified distances from the baseline. If the intensity of any sub-
sidiary spots derived from the tested substance is less than that of the spot
obtained from the reference solution, the substance conforms to the require-
ments. When specified in the monograph, spray the paper uniformly on both
sides with the reagent, carry out any further prescribed treatment to complete
the reaction, and apply the same criteria to the resulting spots.
Electrophoresis on cellulose acetate strips

It is preferable to use a smaller chamber than for paper electrophoresis; one
measuring about 25 cm by 24 cm with troughs of 10 cm by 23 cm is suitable.

Use cellulose polyacetate strips of suitable quality, measuring 2.5 cm by
17 cm, which are immersed in the conducting liquid for approximately 1 hour
before use.
Apply the solutions, prepared as specified in the monograph and in the
volume indicated, 8 cm from one end of the strip, then carry out electrophore-
sis as described under paper electrophoresis. Colour the bands, wash them and
render them transparent by the methods specified in the monographs, which

also give the method of evaluation to be used.
Gel electrophoresis
The inert carrier consists of a 1-2 mm thick layer of an agar or starch gel of
suitable consistency and shaped as an elongated rectangle.
The conducting liquid is either incorporated into the gel layer or sprinkled
on it, until it is well moistened, after it has already been formed. The solution
of the substance is placed on the surface of the gel layer or inside the holes
bored for that purpose in the layer. The
connected at both its nar-
gel layer is
rower ends with two troughs containing the conducting liquid, the connection
being made by wicks composed of a double layer of absorbent lint moistened
1201
with the conducting liquid. The gel layer on its support and the connections
are then placed in a suitable chamber.
The electrophoretic process is effected by application of direct electric
current. To remove heat generated by the Joule effect of the current, water or
other suitable cooling liquid should be circulated in the course of the process
through the supporting plate.
When the process has been completed, the resulting spots or areas of migra-
tion are located by method (for example, as an inhibition zone after
a suitable
suitable incubation,if the tested substance is an antibiotic and an appropriate
testorganism has been incorporated into the gel layer, or by a chemical

method), as specified in the individual monographs.
1.16 Phase solubility analysis

Phase solubility analysis is a technique for quantitatively determining the purity
of a substance through the application of precise solubility measurements. At
a given temperature, a definite amount of a pure substance is soluble in a def-
inite quantity of solvent. The resulting solution is saturated in respect of the
particular substance, but the solution remains unsaturated in respect of other
substances even though such substances may be closely related in chemical
structure and physical properties to the particular substance being tested. Con-
stancy of solubility indicates that a material is pure or free from foreign sub-
stances except in the unique case where the percentage composition of the
material being tested is in direct ratio to the solubilities of the respective com-
ponents. Conversely, variability of solubility indicates the presence of an impu-
rity or impurities.
The standard solubility method consists of several distinct steps: ia) prepa-
ration of a series of separate systems composed of increasing quantities of
material and measured, constant amounts of solvent; {b) establishment of equi-
librium for each system at identical constant temperature and pressure; (c)
separation of the solid phase from the solutions; {d) determination of the
concentration of the material dissolved in the various solutions; (e) graphical
representation of the ratio of the weight of the dissolved materials per weight
of solvent against the ratio of the total weight of material per weight of solvent,
extrapolation and calculation. Alternatively, statistical procedures may be used
to evaluate the purity of the test substance from the data obtained.
Solvents
The following criteria are used in the selection of a proper solvent for phase
solubility analysis:
(1) The solvent should be of such volatility that it can be evaporated
under vacuum, but it should not be so volatile that difficulty is experi-

enced in transferring and weighing the solvent and its solutions.
Normally, solvents with boiling points between 60 °C and 150 °C are
suitable.
1202
Methods of Analysis
(2) The solvent should not adversely affect the substance. Solvents that
cause decomposition or react with the substance cannot be used. Sol-
vents that solvate or form salts should be avoided if possible.
(3) The solvent should be of known purity and composition. Mixed solvents
are permissible. Trace impurities may affect solubility greatly.
(4) For the method described below the test substance should be soluble to
the extent of not less than 4mg/g and not more than 50mg/g in the
solvent chosen. A solubility of 10-20mg/g is optimal.
Apparatus
Constant-temperature hath. For these tests use a constant-temperature bath
capable of maintaining a preselected temperature within ±0.1 °C. Normally
temperatures of 25 °C or 30 °C are selected. The bath should be equipped with
a horizontal shaft capable of rotating at approximately 25 revolutions per
minute, equipped with clamps to hold ampoules. Alternatively the bath may
contain a suitable vibrator, capable of vibrating at 100-120 vibrations per
second, equipped with a shaft with suitable clamps to hold the ampoules, or
other suitable device to achieve equilibrium in the ampoules.
Ampoules. Use 15-ml ampoules similar to that shown in Fig. 2. Other con-
tainers may be used provided that they are leakproof and are suitable in aU
other respects.
Solubility flasks. Use solubility flasks suitable for freeze-drying. A suitable
flask (with stopper) is shown in Fig. 2.
Balance.Use a suitable balance and technique to ensure that all weighings
are within +10 pg.
The method described below is generally applicable. However, in certain cases
other conditions (volume of solvent, etc.) than those specified here might be
preferable.
System composition
Weigh accurately not less than 7 marked, scrupulously cleaned, ampoules and
into each of them weigh, accurately, increasingly larger amounts of the test sub-
stance. The weight of the test substance is selected so that the first ampoule
contains slightly less material than will go into solution in 5 ml of the selected
solvent, and the second ampoule and subsequent ampoules slightly more than
the indicated solubility. Pipette 5.0ml of the solvent into each of the ampoules,
cool in a dry-ice/acetone mixture, and seal, using a double-jet air-gas burner
and taking care to save all the fragments of glass. Allow the ampoules and their
contents to come to room temperature, and weigh the individual sealed
ampoules plus any corresponding system com-
glass fragments. Calculate the
position, in mg of substance per g of solvent, for each ampoule by the formula
1000 (W2 - Wi)/{W3 - Wj), in which Wi is the weight of the empty ampoule.
1203
3rnin
X s
19 mm
Figure 2. Ampoule (left) and solubility flask (right) used in phase solubility analysis.
W 2 isthe weight of the ampoule plus test substance, and 3 W is the weight of
the ampoule plus test substance, solvent and separated glass.
Equilibration
The time required for equilibration varies with the substance, the method of
mixing (vibration or rotation) and the temperature. Normally equilibrium is
obtained more rapidly by the vibration method (1-7 days) than by the rota-
tional method (7-14 days).
In order to demonstrate that a state of equilibrium has been attained, the
following procedure frequently applicable: Bring about a supersaturated solu-
is
tion in one of the ampoules - the next to the last in the series - by warming it
to a temperature about 10°C above that of the constant-temperature bath,
taking care that not all the solid in the ampoule is dissolved. Thereafter, treat
this ampoule in exactly the same manner as the other ampoules. If the solu-
bility value obtained from it plots out in line with the other values, it indicates
that all the ampoules have attained equilibrium. However, failure of the solu-
bility value from the “supersaturated” ampoule to fall in line with the other sol-
ubility values does not necessarily prove that the other ampoules have not
1204
Methods of Analysis
attained equilibrium, since can occasionally be due to the tendency of certain

it
materials to remain in supersaturated solution. To attain equilibrium in such

cases, run a series of phase solubility analyses applying different equilibration
times so as to make certain that constant values for the slope of the solubility
curve have been achieved.
Solution composition
After equilibration, place the ampoules vertically in a rack in the constant-tem-
perature bath, with their necks above the water level, and allow the contents
to settle. Taking full precautions to minimize solvent evaporation, open the
ampoules, and remove a volume of 2.0 ml from each by means of a pipette
equipped with a small pledget of cotton-wool or with other suitable means of
serving as a filter. Remove the cotton-wool, transfer the aliquot of clear solu-
tion from each ampoule to a marked, tared solubility flask, and weigh each
flask plus its solution to obtain the weight of the solution. Cool the flasks in a
dry-ice/acetone bath, and then evaporate the solvent in a vacuum. Gradually
increase the temperature from 70 to 100°C, and dry the residue to constant
weight. Calculate the solution composition, in mg of substance per g of solvent,
by the formula 1000 ~ Ps), in which T is the weight of the solubil-
ity flask, p2 is the weight of the flask plus solution, and F3 is the weight of the
flask plus residue.
Calculation
Prepare a graphical representation of the results for each portion of the test
substance taken by plotting the ratio of the weight of the dissolved materials
per weight of solvent {Y axis or solution composition) against the ratio of the
total weight of material per weight of solvent {X axis or system composition).
As shown in Fig. 3, the points for those containers that represent a true solu-
tion should approach a straight line (AB) with a slope of 1, passing through
the origin; the points corresponding to saturated solutions should approach
another straight line (BC), the slope, S, of which represents the fraction of total
impurities present in the test substance. Failure of points to approach a straight
line usually indicates that equilibrium has not been achieved although this may
from ideal behaviour.
also be due to formation of a solid solution or departure
Calculate the percentage purity of the test substance by the formula 100 -
lOOS. The slope may be calculated by the equation S = (Yj - Yi)/(X2 - Xf) in
which Y2 and Yj represent solution compositions, and X2 and Xi represent the
respective system compositions, at convenient points on the second straight
line (BC).
The point B in the diagram represents the system where the main compo-
nent or, in rare cases, the least soluble of the components of the test substance
has reached the limit of its solubility. The solubility of this component is
obtained by extending the solubility line (BC) through the Y axis. The point of
interception on Y axis gives the
the solubility, in mg/g, which should be con-
stant for a given compound.
1205
Figure 3. Typical phase solubility diagram.
At point C
in the diagram the next component of the test substance has
reached the limit ofits solubility. The intercept obtained by extending the sol-
ubility line(CD) through the Y axis represents the combined solubilities of the
two components that have first reached their solubility limits. The solubility
of the second component can thus be obtained by subtraction.
Between points D and E in the diagram the solution is saturated in respect
of all components in the test substance, and its composition remains constant.
Under appropriate conditions the number of impurities present in the test
substance is shown by the number of inflexions of the solubility curve above
the first saturation point (B) and the solubilities of the respective components
can be obtained in a way similar to that described above.
2. Chemical methods
2.1 General identification tests
Acetylated substances
Place a quantity of the substance as specified in the monograph in a test-tube
(of maximum 18 mm diameter) and treat it with 3 drops of phosphoric acid
(-1440 g/1) TS. Close the tube with a stopper through which passes a smaller
test-tube filled with water and on the outside of which hangs a drop of lan-
thanum nitrate (30 g/1) TS.Heat the apparatus in a boiling water-bath for 5
minutes. Transfer the drop of lanthanum nitrate to a white porcelain spot plate
and mix with a drop of iodine (0.02 mol/1) VS. Place at the edge of the mixture
1206
Methods of Analysis
a drop of ammonia (~100g/l) TS. A blue colour slowly appears at the interface
of the two liquids and persists for a short time.
Amines, primary aromatic

Dissolve a quantity of the substance as specified in the monograph in 2 ml of
hydrochloric acid (~70g/l) TS with the aid of heat, if necessary. Cool in ice,
treat it with 4 ml of sodium nitrite (lOg/1) TS and pour the mixture into 2 ml
of 2-naphthol TSl containing 1 g of sodium acetate R. A heavy precipitate,
coloured as specified in the monograph, is produced.
Ammonia and volatile aliphatic amines

Dissolve a quantity of the substance as specified in the monograph, place the
g of magnesium oxide R; warm, if specified in
solution in a test-tube and add 1
the monograph. Alkaline vapours evolve gradually and turn manganese/ silver
paper R black, the reagent paper being placed in the upper part of the test-tube.
Ammonium
Carry out the test in an apparatus consisting of stoppered test-tubes A and B
connected by a bent glass tube to permit a stream of air to pass consecutively
through test-tubes A and B.
Place the solution as specified in the monograph and 0.2 g of magnesium
oxide R into test-tube A, and 1ml of hydrochloric acid (0.1 mol/1) VS contain-
ing 1 drop of methyl red/ethanol TS in test-tube B. Bubble air through the appa-
ratus. Evolved ammonia turns the colour of the solution in test-tube B to yellow.
On the addition of 1ml of sodium cobaltinitrite (lOOg/1) TS to this solution a
yellowish brown precipitate is formed.
Bismuth
A. Prepare the solution in hydrochloric acid (-250 g/1) TS as specified in the
monograph and dilute 10 times with water. A white precipitate is
formed, which turns dark brown on the addition of sodium sulfide TS.
B. Treat the solution in nitric acid (-1000 g/1) TS as specified in the mono-
graph with potassium iodide (80 g/1) TS. A black precipitate is formed,
which is soluble in an excess of the reagent to give a yellowish brown
or orange solution. Dilute this solution with several volumes of water

and heat; an orange or copper-coloured precipitate is obtained. The black
precipitate that is first formed on the addition of potassium iodide
(80 g/1) TS also becomes orange or copper-coloured when heated with
water.
Bromides
A. Prepare a solution as specified in the monograph, acidify with nitric acid
(-130 g/1) TS and add silver nitrate (40 g/1) TS. A yellowish curdy precipi-
1207
tate is produced, which is partially soluble in ammonia (-260 g/1) TS, but
almost insoluble in ammonia (-100 g/1) TS and in nitric acid (-1000 g/1) TS.
B. (For testing bromides or hydrobromides of insoluble or sparingly soluble
bases.) Prepare the solution as specified in the monograph, add ammonia
(-100 g/1) TS, filter, acidify the filtrate with nitric acid (-130 g/1) TS, and
proceed with test A.
C. Prepare the solution as specified in the monograph, acidify with sulfuric
acid (~100g/l) TS, and mix with chlorine TS. A brown solution results;
after shakingwith chloroform R it becomes colourless, whereas the chlo-
roform layer turns reddish.
Calcium
A. Prepare the solution as specified in the monograph and add to it ammo-
nium oxalate (25 g/1) TS. A white precipitate is formed, which is soluble
in hydrochloric acid (-250 g/1) TS but is practically insoluble in acetic acid
(-300 g/1) TS.
B. Treat 1 drop of a solution as specified in the monograph with 4 drops of
glyoxal bis(2-hydroxyanil) TS, and 1 drop of sodium hydroxide (-80 g/1)
TS. A reddish brown precipitate is formed, which dissolves in chloro-
form R to give a red solution.
Chlorides
(-130 g/1) TS and add silver nitrate (40g/l) TS. A white curdy precipitate
is produced, which is soluble in ammonia (-100 g/1) TS but is practically
insoluble in nitric acid (-1000 g/1) TS.
B. (For testing chlorides or hydrochlorides of insoluble or sparingly soluble
bases.) Prepare the solution as specified in the monograph, add ammonia
(-100 g/1) TS, filter and acidify the filtrate with nitric acid (-130 g/1) TS
and proceed with test A.
C. Mix the quantity of the substance as specified in the monograph with
an equal quantity of manganese dioxide R, moisten with sulfuric acid
(-1760g/l) TS and heat gently. The evolved chlorine is recognizable by
its greenish colour and produces a blue coloration of moistened
starch/iodide paper R. Carry out the reaction preferably under a hood.
Citrates
A. Treat at ambient temperature a neutral solution as specified in the mono-
graph with calcium chloride (55 g/1) TS. No precipitate is formed, but
on boiling, a white solid is produced, which is soluble in acetic acid
(-300 g/1) TS.
B. Boil a solution with mercuric sulfate TS as specified in the monograph
and filter if necessary. After the addition of a few drops of potassium
permanganate (10 g/1) TS to the filtrate, the colour is discharged and a
1208
Methods of Analysis
Ferrous salts
A. Prepare a solution as specified in the monograph and add potassium fer-
ricyanide (lOg/1) TS. A dark-blue precipitate is formed, which is practi-
cally insoluble in hydrochloric acid (~70g/l) TS.

B. Prepare a solution as specified in the monograph, acidify with sulfuric
acid (~100g/l) TS, and treat with o-phenanthroline (1 g/1) TS. An intense
red colour is produced, which is discharged by the addition of ceric
sulfate (35 g/1) TS.
Iodides
(-130 g/1) TS and add silver nitrate (40 g/1) TS. A yellow curdy precipi-
tate is formed, which is practically insoluble in ammonia (-100 g/1) TS
and in nitric acid (-lOOOg/1) TS.
B. (For testing iodides of insoluble or sparingly soluble bases.) Prepare the
solution as specified in the monograph, add ammonia (-100 g/1) TS, filter
and acidify the filtrate with nitric acid (-130 g/1) TS and proceed with
test A.
C. Prepare a solution as specified in the monograph, acidify with sulfuric
acid (-lOOg/1) TS and add potassium nitrite (100 g/1) TS. A brown solu-
tion results; after shaking with chloroform R, it becomes colourless,
whereas the chloroform layer turns violet.
Nitrates
A. Prepare a solution as specified in the monograph and treat it with ferrous
sulfate (15 g/1) TS. No brown colour appears unless sulfuric acid
(~1760g/l) TS is cautiously added to form a lower layer. A brown colour
isthen produced at the interface of the two liquids.
B. Add 2mg of the finely ground test substance to a mixture of 0.1ml of
nitrobenzene R and 0.2 ml of sulfuric acid (-1760 g/1) TS. Allow to stand
at room temperature for 5 minutes, cool in ice, and add slowly while
mixing 5ml of water and 3ml of sodium hydroxide (-400 g/1) TS. Add
5 ml of acetone R, shake and allow to separate. An intense violet colour
is produced in the upper phase.
Orthophosphates
A. Add drop by drop a quantity of nitric acid (-130 g/1) TS to 5 ml of ammo-
nium molybdate (95 g/1) TS until any precipitate that may appear dis-
solves. Divide this solution into 2 portions, add to one portion the test
solution acidified with nitric acid (~130g/l) TS as specified in the mono-
graph, and boil both portions. yellow precipitateA is formed with the
test solution while the other shows no more than a slight opalescence.
B. Prepare a neutral solution as specified in the monograph and add silver
nitrate (40 g/1) TS. A yellow precipitate is produced, which does not
1209
darken upon heating the solution to boiling. The precipitate is soluble in

ammonia (-100 g/1) TS and in nitric acid (-130 g/1) TS.
Potassium
Prepare an alkaline solution as specified in the monograph and treat it with
sodium tetraphenylborate (30g/l) TS. A white precipitate is produced.
Salicylates
Treat a neutral solution as specified in the monograph with ferric chloride
(25 g/1) TS. An intense reddish violet colour appears, which remains on the addi-
tion of a small amount of acetic acid (-300 g/1) TS but disappears on the addi-
tion of hydrochloric acid (-70 g/1) TS, with separation of a white crystalline
precipitate.
Sodium
A. Moisten a quantity of the substance with hydrochloric acid (-250 g/1) TS.
An intense yellow colour is produced when the solution is introduced
into a nonluminous flame.

NOTE: Perform test B if for technical reasons test A cannot be carried out.
B. Acidify a solution as specified in the monograph with acetic acid

(-60 g/1) TS, filter, if necessary, and treat it with uranyl/zinc acetate TS.
A yellow crystalline precipitate is produced.
Sulfates
A. Prepare a solution as specified in the monograph and add barium chlo-
ride (50 g/1) TS. A white precipitate is formed, which is practically insol-
uble in hydrochloric acid (-250 g/1) TS.
B. To monograph, add lead acetate (80 g/1) TS.
a solution as specified in the
A white precipitate is formed, which is soluble in ammonium acetate
(80 g/1)TS and in sodium hydroxide (-80 g/1) TS, but practically insolu-
ble in hot water.
Tartrates
A. Acidify a solution as specified in the monograph with acetic acid
(-300 g/1) TS and add 1 drop of ferrous sulfate (15 g/1) TS, a few drops of
hydrogen peroxide (-60 g/1) TS, and enough sodium hydroxide (-80 g/1)
TS to make the solution alkaline. A purple or violet colour is produced.
B. Mix a few ml of sulfuric acid (~1760g/l) TS with a few drops of resor-
cinol (20 g/1) TS and a few drops of potassium bromide (100 g/1) TS and
add 2 or 3 drops of a solution as specified in the monograph. Warm the
liquid in a water-bath for 5 to 10 minutes. An intense blue colour is pro-
duced. Cool the liquid and pour it into water. The solution becomes red.
1210
Methods of Analysis
2.2 Limit tests

Z.2. 1 Limit test for chlorides
The limit test for chlorides is provided to demonstrate that the content of chlo-
rides does not exceed the limit given in the individual monograph in terms of
micrograms of chloride ions per gram of the substance being tested. The stan-
dard solution against which the comparison of opalescence is made contains
250 (tg of Ch.
Carry out the test in matched flat-bottomed comparison tubes of transparent
glass of about 70 ml capacity and about 23 mm
internal diameter bearing a
45-ml and a 50-ml mark. Nessler cylinders complying with the above dimen-
sions are suitable. The expression "matched tubes” means tubes that are
matched as closely as possible in internal diameter and in all other respects.
Prepare a solution as specified in the monograph, transfer to a comparison
tube, dilute to 50 ml with water and add 1 ml of silver nitrate (40g/l) TS. Stir
immediately with a glass rod, and set aside for 5 minutes, protected from direct
sunlight. The opalescence produced is not greater than the similarly prepared
standard opalescence when viewed down the vertical axis of the tube in dif-
fused light against a black background.
Standard opalescence
Measure 5.0ml of hydrochloric acid CITS and 10 ml of nitric acid (-130 g/1) TS
into a comparison tube. Dilute to 50 ml with water, and add 1 ml of silver nitrate
(40 g/1) TS. Stir immediately with a glass rod and set aside for 5 minutes, pro-
tected from direct sunlight.
2.2.2 Limit test for sulfates

The limit test for sulfates is provided to demonstrate that the content of sul-
fates does not exceed the limit given in the individual monograph in terms of
micrograms of sulfates per gram of the substance being tested.
The solution against which the comparison of turbidity is made contains
480 pg of SOT more than the standard barium sulfate suspension.
glass of about 70ml capacity and about 23 mm
internal diameter bearing a 45-
ml and 50-ml mark. Nessler cylinders complying with the above dimensions
a
are suitable. The expression “matched tubes” means tubes that are matched as
closely as possible in internal diameter and in all other respects.
Prepare a solution as specified in the monograph, transfer to a comparison
tube, dilute to 45 ml with water and add 5 ml of barium sulfate suspension TS.
1211
Stirimmediately with a glass rod, and set aside for 10 minutes. The turbidity
produced is not greater than the similarly prepared standard turbidity when
viewed down the vertical axis of the tube in diffused light against a black
background.
Standard turbidity
Measure 1.00 ml of sulfuric acid (0.005 mol/1) VS and 3 ml of hydrochloric acid
(~70g/l) TS into a comparison tube. Dilute to 45ml with water, and add 5ml
of barium sulfate suspension TS. Stir immediately with a glass rod and set aside
for 10 minutes.
2 2.3 Limit
. test for heavy metals
The limit test for heavy metals is provided to demonstrate that the content of
metallic impurities that are coloured by hydrogen sulfide does not exceed the
heavy metals limit given in the individual monograph in terms of micrograms
of lead per gram of the test substance.
The test consists of two consecutive operations: preparation of the test solu-
tion,and the colour development by reaction with hydrogen sulfide, followed
by comparison of the colour obtained with that produced with standard lead
solution.
The preparation of the test solution is carried out, as specified in the mono-
graph, according to procedures 1 to 4 described below. A blank is prepared in
a similar manner.
The reaction with hydrogen sulfide is carried out by mixing the test solu-
tion with freshly prepared hydrogen sulfide TS. The comparison of the colour
thus obtained is carried out either by directly comparing the coloration of the
liquid in suitable comparison tubes (Method A) or by comparing the intensity

of coloration of spots obtained by filtering the liquid using an appropriate appa-
ratus (Method B).
Method A is generally applicable only when the amount of heavy metals in
the weight of test substance used exceeds 5 |tg; for amounts of 2-5 |tg of heavy
metals Method B should be used.
The standard lead solution used in the test; dilute lead PbTS contains 10 pg
of lead in 1 ml. When 0.1 ml of this solution is employed to prepare the stan-
dard for comparison with a solution of g of the substance being tested, the
1
standard solution thus prepared contains 1 pg of Pb and represents the equiva-

lent of 1 pg of lead per g of the substance tested.
Apparatus
For determination of heavy metals by Method A carry out the test in matched
flat-bottomed comparison tubes of transparent glass of about 70 ml capacity
and about 23mm internal diameter bearing a 40-ml and a 50-ml mark. Nessler
cylinders complying with the above dimensions are suitable. The expression
“matched tubes” means tubes that are matched as closely as possible in inter-
1212
Methods of Analysis
nal diameter and in all other respects. For mixing the solution use a stirring rod
preferably having a loop at the lower end.
For determination of heavy metals by Method B use a 50-ml syringe made
of suitable material (usually plastic) with a detachable plunger and a male Luer
conical joint of 9 mm internal diameter at the lower end (Millipore syringe XX
1 1 050 05 is suitable) to which an adaptor for filtration is attached.
The adaptor is made of suitable material (a filtration adapter Millipore SXOO

013 00 in polypropylene is suitable) and has a female joint for connecting it
with the syringe. It is devised so as to be separable into two parts to permit
the exchange of filters, the lower part containing a support for membrane filters
13mm in diameter. A suitable prefilter (Millipore prefilter AP 2001 300 is suit-
able) and a membrane filter made of mixed cellulose esters, 13mm in diame-
ter, with a pore opening of 3pm (a Millipore filter SSWP 013 00 is suitable) are
used for the filtration.
Preparation of test solution
Procedure i. Weigh the quantity of substance specified in the monograph, dis-
solve it in 25 ml of water, adjust the pH of the solution to 3-4 with acetic acid
(~60g/l) PbTS, orwith ammonia (~100g/l) PbTS, as necessary, then dilute to
40 ml with water and mix.
Procedure Z. Weigh the quantity of substance specified in the monograph,
dissolve it in about 30ml of solvent specified (ethanol (-750 g/1) TS, methanol
R, acetone R, or dioxan R may be used), add 0.5 ml of acetic acid (-300 g/1) TS,
and dilute to 40 ml with the solvent.
Procedure 3. Place the quantity of the substance specified in the monograph
in a suitable crucible, preferably made of silica, and carefully ignite at a low
temperature until the contents are thoroughly charred. The crucible may be
loosely covered with a lid during the charring. Add to the contents of the cru-
cible 2 ml of nitric acid (-1000 g/1) TS and 5 drops of sulfuric acid (-1760 g/1 TS),
and cautiously heat white fumes are evolved, and then ignite, preferably
until
in a muffle furnace, at 500 °C until all the carbon is burned off. Cool, add 2 ml
of hydrochloric acid (-250 g/1) TS, and slowly evaporate in a water- bath to
dryness. Moisten the residue with 1 drop of hydrochloric acid (-250 g/1) TS,
add 10 ml of hot water, and digest for 2 minutes. Add, drop by drop, ammonia
(-100 g/1) PbTS, until the pH of the solution is between 8 and 8.5, then add,
drop by drop, acetic acid (-60 g/1) PbTS, to adjust the pH to between 3 and 4.
Filter if necessary, wash the crucible and the filter with about 10ml of water,
dilute with water to 40 ml, and mix.

Procedure 4- Place the quantity of substance specified in the monograph in a
suitable crucible, preferably made of silica,mix it well with about 0.5 g of mag-
nesium oxide R and incinerate until a homogeneous white mass is obtained. If
after 15 minutes of incineration the residue is still coloured, let the crucible cool,
mix the contents well with a glass rod and resume heating. Next, dissolve the
residue in hydrochloric acid (-70 g/1) TS, add, drop by drop, a solution of
1213
ammonia (~100g/l) PbTS, until the pH of the solution is between 8 and 8.5,
then add, also drop by drop, acetic acid (~60g/l) PbTS, to adjust the pH to 3-4,
dilute with water to 40 ml, and mix.
filter,
Colour development and measurement

Method A
To 40ml of the liquid contained in the comparison tube add 10 ml of freshly
prepared hydrogen sulfide TS, mix and allow to stand for 5 minutes.
In another comparison tube place a volume of solution of dilute lead PbTS,
containing the lead equivalent of the heavy metals limit specified in the mono-
graph, dilute with water, adjust the pH with ammonia (-100 g/1) PbTS and
acetic acid (-60 g/1) PbTS to 3-4; dilute with water or the solvent used to
40ml, mix, add 10ml of freshly prepared hydrogen sulfide TS, mix and allow
to stand for 5 minutes.
Compare the colours by viewing down the vertical axis of the tube in dif-
fused light against a white background, or by another suitable method. The
colour of the test solution is not darker than that of the lead standard.
Method B
Take the filtration syringe, arrange the prefilter and the membrane filter as indi-
cated in the diagram for prefiltration, remove the plunger from the syringe, place
the test solution inside the syringe, replace the plunger, and filter the test solu-
tion slowly by exerting a regular pressure on the plunger. Collect the filtrate in
a beaker or a test-tube. Open the adapter and check whether the membrane
filter is from impurities. If not, replace it and repeat the operation in the
free
same manner. Then rearrange the prefilter and membrane filter as indicated in
Fig. 4. Adjust the pH of the filtrate with ammonia (-100 g/1) PbTS and acetic acid
(-60 g/1) PbTS to 3-4, add 10 ml of freshly prepared hydrogen sulfide TS, aU the
reagents previously filtered through a membrane filter, mix, allow to stand for
5 minutes, take out the plunger, place the solution inside the syringe, and filter
it through the membrane filter by exerting slowly a regular and moderate pres-
sure on the plunger. Open the adaptor and take out the membrane filter.
Take a volume of solution of dilute lead PbTS containing the lead equiva-
lent to the heavy metals limit specified in the monograph, dilute with water,
adjust the pH with ammonia (-100 g/1) PbTS and acetic acid (-60g/l) PbTS to
3-4, dilute with water to 40 ml, mix, and proceed as described above.
Compare the intensity of the coloration of spots obtained on the membrane
filters. The colour obtained from the test solution is not more intense than that
from the lead standard.
2 2.4 Limit
. test for iron
The limit test for iron is provided to demonstrate that the content of iron does
not exceed the limit given in the individual monograph in terms of micrograms
of iron per gram of the test substance.
1214
Methods of Analysis
A — Luer cone
B — Joint
C — Prefilter
D — Membrane filter
WflO 790S0
E — Support
Figure 4. Limit test for heavy metals: Method B
The standard solution against which the comparison of colour is made con-
tains 40 pg of iron.
glass ofabout 70ml capacity and about 23mm internal diameter bearing a 45-
ml and a 50-ml mark. Nessler cylinders complying with the above dimensions
are suitable. The expression “matched tubes” means tubes that are matched as
closely as possible in internal diameter and in all other respects.
Prepare the substance as specified in the monograph, or dissolve directly an
indicated quantity in 40ml of water, and transfer to a comparison tube. Add
2 ml of acid (180 g/1) FeTS and 2 drops of mercaptoacetic acid R; mix, make
citric
alkaline with ammonia (-100 g/1) FeTS, dilute to 50 ml with water, and allow
to stand for 5 minutes. The colour produced is not more intense than the sim-
ilarly prepared standard colour when viewed down the vertical axis of the tube
in diffused light against a white background.
1215
Standard colour
Measure 2 ml of iron standard FeTS and 40 ml of water into a comparison tube.
Add 2 ml of citric acid (180g/l) FeTS and 2 drops of mercaptoacetic acid R; mix,
make alkaline with ammonia (~100g/l) FeTS, dilute to 50 ml with water, and
allow to stand for 5 minutes.
2 2.5 Limit
. test for arsenic
The limit test for arsenic is provided to demonstrate that the content of arsenic
does not exceed the limit given in the individual monograph in terms of micro-
grams of arsenic per gram of the test substance.
To carry out the limit test for arsenic a solution is prepared from the test
substance by a procedure specified in the monograph. This procedure assures
that the solution in every case contains the whole of the arsenic (if any) present
in the substance.
The standard stain against which the comparison is made contains 10 pg
of As.
The procedure described may also be used to determine the amount of
arsenic in the substance by matching the depth of colour of the stain with a
series of standard stains. A stain equivalent to the 1 ml standard stain produced
by operating on 10 g of a substance indicates that the amount of arsenic is
1 hg/g-
In the statements of arsenic limits, the permitted amount of arsenic is
expressed as As.
Apparatus
A suitable type of apparatus is described below, though other acceptable con-
structions are available.
A wide-mouthed bottle of about 120 ml capacity, is fitted with a rubber bung
through which passes a glass tube. The latter, made from ordinary glass tubing,
has a total length of200mm and an internal diameter of exactly 6.5mm (exter-
nal diameter about 8 mm), is drawn out at one end to a diameter of about
1 mm, and has a hole not less than 2 mm
in diameter blown in the side of the
tube, near the constricted part. The tube is passed through the bung fitting the
bottle so that, when inserted in the bottle containing 70 ml of liquid, the con-
stricted end of the tube is above the surface of the liquid and the hole in the
side is below the bottom of the bung. The upper end of the tube is cut off
square, and is either slightly rounded off or ground smooth.
Two rubber bungs (about 25mm x 25mm), each with a hole bored centrally
and true and exactly 6.5 mm
in diameter, are fitted with a rubber band or spring
clip for holding them tightly together. Alternatively, the two bungs may be
replaced by any suitable construction satisfying the conditions of the test, as
described below.
1216
Methods of Analysis
Pack tiie glass tube lightly with cotton-wool, previously moistened with lead
acetate (80g/l) TS and dried, so that theupper surface of the cotton-wool is not
less than 25 mm below the top of the tube.
Insert the upper end of the tube into the narrow end of one of the pair of
rubber bungs, either (1) to a depth of about 10 mm
in the case of the tube with
the rounded-off end or (2) so that the ground end of the tube is flush with the
larger end of the bung. Place a piece of mercuric bromide paper AsR flat on the
top of the bung, and place the other bung over it. Secure the assembly by means
of a rubber band or spring clip, in such a manner that the borings of the two
bungs (or the boring of the upper bung and the glass tube) meet to form a true
tube 6.5 mm in diameter interrupted by a diaphragm of mercuric bromide paper
AsR.
Instead of this method of attaching the mercuric bromide paper AsR, any
other method may be used provided (1) that the whole of the evolved gas
passes through the paper, (2) that the portion of the paper in contact with the
gas is a circle 6.5 mm
in diameter, and (3) that the paper is protected from sun-
light during the test.
Place the solution, prepared as specified in themonograph, in the wide-
mouthed bottle, add 1 g of potassium iodide AsR and lOg of granulated zinc
AsR, and place the prepared glass tube assembly quickly into position. Allow
the reaction to proceed for 40 minutes. Compare any yellow stain that is pro-
duced on the mercuric bromide paper AsR, with a standard stain, produced in
a similar manner with a known quantity of dilute arsenic AsTS. Make the com-
parison in daylight and immediately after simultaneous preparation of the test
and standard stains; the stains fade on keeping.
The most suitable temperature for carrying out the test is generally
about 40 °C but, as the rate of evolution of the gas varies somewhat with dif-
ferent batches of granulated zinc AsR, the temperature may be adjusted to

obtain a regular, but not too violent, evolution of gas. The reaction may be
accelerated by placing the apparatus on a warm surface, care being taken to
ensure that the mercuric bromide paper AsR remains quite dry throughout the
test.
Between successive tests, the tube must be washed with hydrochloric acid
(-250 g/1) AsTS, rinsed with water, and dried.
Standard stain
Prepare a solution by adding 10 ml of stannated hydrochloric acid (-250 g/1)
AsTS and 1 ml of dilute arsenic AsTS, to 50 ml of water. The resulting solution,
when treated as described in the general test, yields a stain on the mercuric
bromide paper AsR, referred to as the standard stain.
1217
2.3 Sulfated ash

Accurately weigh about Ig of the substance, or the quantity specified in the
monograph, into a suitable dish (usually platinum) and moisten with sulfuric
acid (~1760g/l) TS. Heat gently to remove the excess of acid and ignite at about
800 °C until all the black particles have disappeared; again moisten with sulfu-
ric acid (-1760 g/1) TS and reignite. Add a small amount of ammonium car-
bonate R and ignite to constant weight.
2.4 Oxygen flask method

The oxygen flask method for the determination of halogens and sulfur in
organic compounds consists of a combustion procedure followed by appropri-
ate titrimetric determination. Combustion of the organic material in oxygen
yields water-soluble inorganic products, which are determined as directed for
the individual element.
Apparatus
The combustion is carried out in a suitable conical flask into the stopper of
which is fused one end of a piece of platinum wire. A flask of 500ml is used,
unless otherwise specified in the monograph. Towards the other end of the
wire, a piece of platinum gauze is attached to provide a means of holding the
substance clear of the absorbing liquid during combustion.
CAUTION: The analyst should wear safety glasses and use a suitable safety
shield between himself and the apparatus. The flask must be scrupulously clean
and freefrom aU traces of organic solvents.
Wrap the test substance in a piece of halide-free filter-paper about 5 cm long
and 3 cm wide, secure the package in the platinum gauze, and insert one end
of a narrow strip of filter-paper into it. Moisten the neck of the flask with water,
place the specified absorbing liquid in the flask, fill the flask with oxygen, light
the free end of the narrow strip of filter-paper and immediately insert the
stopper. Hold the stopper firmly in place. When vigorous burning has begun,
tilt the flask to prevent incompletely burned material falling into the liquid.
Immediately after combustion is completed, shake the flask intermittently for

10 minutes, place a little water around the rim of the flask, carefully withdraw
the stopper, and rinse the stopper, platinum wire, platinum gauze, and sides of
the flask with water. Complete the analysis of this solution as specified in the
monograph.
Pulverizable substances should be finely ground and thoroughly mixed
before the specified quantity is weighed.
For liquids, use capsules of suitable material (e.g. methylcellulose). Place the
specified quantity on about 15 mg of ashless filter-paper flock contained in one
1218
Methods of Analysis
part of a capsule of suitable size, close the capsule, inserting one end of a narrow
strip of filter-paper between the two parts, and secure the capsule in the plat-
inum gauze.
Determination of bromine and chlorine

Using the oxygen flask method described above, burn the quantity of the sub-
stance specified in the monograph. The absorbing liquid consists of 17 ml of
hydrogen peroxide (~60g/l) TS and 3 ml of water. When the process is com-
plete, rinse the stopper, platinum wire, platinum gauze, and sides of the flask
with 40 ml of water.
Add 5 drops of bromophenol blue/ethanol TS and then, by drops, sodium
hydroxide (0.1 mol/1) VS until the colour changes from yellow to blue. Then
add 1 ml of nitric acid (3g/l) TS and 5 drops of diphenylcarbazone/ethanol TS
as indicator, and titrate with mercuric nitrate (0.01 mol/1) VS until the solution
turns light violet.
Each ml of mercuric nitrate (0.01 mol/1) VS is equivalent to 1.598mg of Br
or 0.709 mg of Cl.
Determination of fluorine
Using the oxygen flask method described, burn the quantity of the substance
specified in the monograph. The absorbing liquid consists of 15 ml of water.
When the process is complete, rinse the stopper, platinum wire, platinum
gauze, and sides of the flask with 40 ml of water.
Add 0.6ml of sodium alizarinsulfonate (1 g/1) TS and then, by drops, sodium
hydroxide (0.1 mol/1) VS until the colour changes from pink to yellow. Add
5 ml of acetate buffer, pH 3.0, TS and titrate with thorium nitrate (0.005 mol/1)
VS until the yellow colour changes to pinkish yellow.
Each ml of thorium nitrate (0.005 mol/1) VS is equivalent to 0.380 mg of E
If a difficulty arises in observing the colour change of the indicator, a pre-
liminary test with a solution containing known quantities of inorganic fluoride
should be performed.
Determination of iodine
specified in the monograph. The absorbing liquid consists of 10 ml of sodium
hydroxide (0.2 mol/1) VS. When the process is complete, rinse the stopper, plat-
inum wire, platinum gauze, and sides of the flask with 25 ml of potassium
acetate TS which 15 drops of bromine TSl are added. Then rinse with
to
40 ml of water and add, by drops, formic acid (-1080 g/1) TS until discoloration,
20ml of sulfuric acid (0.05 mol/1) VS, 0.5 g of potassium iodide R, and allow
to stand for 5 minutes. Titrate the liberated iodine with sodium thiosulfate
TS as indicator towards the end of the titration.
(0.05 mol/1) VS, adding starch
Each ml of sodium thiosulfate (0.05mol/l) VS is equivalent to 1.06mg of I.
1219
Determination of sulfur
specified in the monograph. The absorbing liquid consists of 12.5ml of hydro-
gen peroxide (~60g/l) TS. When the process is complete, rinse the stopper, plat-
inum wire, platinum gauze, and sides of the flask with 40 ml of water. Boil the
solution for 10 minutes, cool, add 2 ml of acetic acid (-300 g/1) TS, and 20 ml of
ethanol (-750 g/1) TS. Titrate with barium nitrate (0.01 mol/1) VS using 2 drops
of thorin (2 g/1) TS and 2 drops of methylthioninium chloride (0.2 g/1) TS as indi-
cator until the yellow colour changes to pink.
Each ml of barium nitrate (0.01 mol/1) VS is equivalent to 0.321 mg of S.
2.5 Complexometiic titrations
Complexing agents of value as titrants are aminopolycarboxylic acids that

possess the characteristic group
,CHjCOOH
'CHjCOOH
Such compounds are capable of forming chelate complexes with many

cations in which is bound in a ring structure. The ring results from
the cation
the formation of a salt-like bond between the cation and the carboxyl groups
together with a coordinate bond through the lone pair of electrons of the nitro-
gen atom. If the ring is five-membered, the chelate thus formed is likely to have
high stability, so the most useful chelating titrants are those that favour the
formation of such rings. This is the case with edetic acid (ethylene diaminete-
traacetic acid, EDTA); the commorJy used disodium salt is known as disodium
edetate. With most metals carrying more than unit positive charge, edetic acid
forms highly water-soluble 1:1 complexes of such a structure that at least 3 five-
membered chelate rings are formed, thus conferring high stability on the
complex. In some cases, coordinate bonds other than those resulting from dona-
tion of the nitrogen lone pair of electrons may be formed with the carbonyl
oxygens of the remaining carboxylic acid groups. Thus the complexes formed
by calcium and by trivalent aluminium may be represented:
CH- - CO - 0 - CO - CH-
/ \ / \
\ CHj-CO-0~/ ^
“O-CO-CHj /
CHj— CHj
and
1220
Methods of Analysis
CH, -CO-Ov P-CO-CH,
The stability ofsuch complexes is markedly dependent on pH. Most diva-

lent metals form complexes that are stable in alkaline solution but alkaline earth
chelates decompose below about pH 8, whereas many divalent metal com-
plexes (zinc and lead, for example) are also stable in quite acid solution. Triva-
lent metal complexes, by virtue of the additional stability conferred by an
increased number of chelate rings, are often stable even in strongly acid solu-
however, some of these metals may be precipitated
tions. In alkaline solutions,
as hydroxides in the presence of edetic acid, not because of instability of the
complex but because of the more powerful effect of the very low solubility
product of the metal hydroxide.
Stability constants of the edetic acid chelates of some metals, as recorded
by Schwarzenbach for 0.1 mol/1 solutions at 20°C are as follows:
Na 1.7
Li 2.8
Mg 8.7
Ca 10.6
Fe"+ 14.3
A1 15.5'
Zn 16.1
Pb 17.6
Hg^" 20.4
Fe=*+ 25.1
“
The aluminium chelate is slow to form so that this metal is usually determined by back-titration.
In order to determine the equivalence point in titration of metal ions with edetic
acid, it is necessary to use a suitable indicator that will react to the presence of
free metal ions in solution. The indicator originally used by Schwarzenbach for
titration of calcium ions was murexide (ammonium purpurate) but this is now
rarely used. Perhaps the most widely used of indicators has been Mordant Black
11 (also known under several trade names). This has a blue colour in ammo-
niacal solution but yields red complexes with many metal ions in such solu-
tions; the metal complexes so formed are generally weaker than the
corresponding edetic acid complexes, so that titration with edetates will readily
remove the metal from the indicator complex and a colour change to full blue
signifies total titration of the metal present in solution. Mordant Black 11 is fre-
1221
quently used as a mixture with methyl orange, which serves to provide a

screened (more readily detectable) end-point.
Many other substances have been proposed and used as indicators for com-
plexometric titrations, but the present discussion must be limited to a consid-
eration of those that are of potential value in pharmaceutical applications.
Calcon and calconcarboxylic acid give a very sharp colour change from wine-
red to full blue when a calcium solution is titrated with sodium edetate in the
pH range 12-14. If magnesium is present it is precipitated as hydroxide at this
pH and, providing the alkali is added before the indicator, does not interfere.
Neither of them, however, is very stable in alkaline solution and they should
preferably be added at a late stage in the titration.
Another widely used indicator is xylenol orange; this is a conventional acid-
base indicator into which iminodiacetic acid groups have been introduced, thus
permitting the substance to act as a metal-complexing indicator. The indicator
gives a clear colour change from pink-violet to yellow at the end-point in titra-
tions of aluminium, bismuth, lead, mercury, and zinc, and may be used from
pH 2-6 according to the metal being titrated.
Aluminium
Dissolve the quantity of substance, accurately weighed, as specified in the
monograph, in 2 ml of hydrochloric acid (1 mol/1) VS and 50 ml of water, unless
other conditions of solution are given in the monograph. Add 50 ml of disodium
edetate (0.05 mol/1) VS and neutralize to methyl red/ethanol TS with sodium
hydroxide (1 mol/1) VS. Heat the solution to boiling and maintain in a boiling
water-bath for at least 10 minutes, cool, add about 50 mg of xylenol orange
indicator mixture R and 5g of methenamine R and titrate the excess edetate
with lead nitrate (0.05 mol/1) VS until the yellow solution turns pink-violet. Each
ml of disodium edetate (0.05 mol/1) VS is equivalent to 1.349 mg of Al.
Bismuth
monograph, in the minimum quantity of nitric acid (-130 g/1) TS, add 50 ml of
water and adjust the pH to between 1 and 2 by dropwise addition of either
nitric acid (-130 g/1) TS or ammonia (-100 g/1) TS. Add about 50mg of xylenol
orange indicator mixture Rand slowly titrate with disodium edetate (0.05 mol/1)
VS until the solution turns from pink-violet to a full yellow. Each ml of dis-
odium edetate (0.05 mol/1) VS is equivalent to 10.45 mg of Bi.
Calcium
monograph, in a few millilitres of water, acidified with a minimum quantity of
hydrochloric acid (-70 g/1) TS if necessary, and then dilute to about 100ml with
water. Titrate with disodium edetate (0.05 mol/1) VS to within about 2 ml of the
1222
Methods of Analysis
expected equivalence point, add 4ml of sodium hydroxide (~300g/l) TS and

0.1 g of calcon indicator mixture R or of calcon carboxylic acid indicator mixture
R and continue the titration until the solution turns from pink to a full blue.
Each ml of disodium edetate (0.05 mol/1) VS is equivalent to 2.004 mg of Ca.
Lead
monograph, in 5-1 0 ml of water, acidified with a minimum quantity of acetic
acid (-300 g/1) TS if necessary, and then dilute to about 50 ml with water. Add
about 50 mg of xylenol orange indicator mixture R and sufficient methenamine
R (about 5g) to turn the solution red and titrate with disodium edetate
(0.05 mol/1) VS until the solution turns from deep violet to full yellow. Each ml
of disodium edetate (0.05mol/l) VS is equivalent to 10.35mg of Pb.
Magnesium
monograph, in 5-1 0 ml of water, acidified with a minimum quantity of
TS if necessary, and then dilute to about 50 ml with
hydrochloric acid (-70 g/1)
water. Add 10ml of ammonium chloride buffer, pH 10.0, TS and lOOmg of
Mordant Black 11 indicator mixture R and titrate with disodium edetate
(0.05mol/l) VS until the solution turns from violet to green. Each ml of dis-
odium edetate (0.05mol/l) VS is equivalent to 1.215mg of Mg.
Zinc
monograph, in 5-1 0 ml of water, acidified with a minimum quantity of acetic
acid (-300 g/1) TS if necessary, and then dilute to about 50 ml with water. Add
about 50 mg of xylenol orange indicator mixture R and sufficient methenamine

R (about 5g) to turn the solution red and titrate with disodium edetate
(0.05 mol/1) VS until the solution turns from pink-violet to full yellow. Each ml
of disodium edetate (0.05 mol/1) VS is equivalent to 3.268 mg of Zn.
2.6 Non-aqueous titration
Acids and bases have long been defined as substances that, when dissolved in
water, furnish hydrogen and hydroxyl ions, respectively. This definition, intro-
duced by Arrhenius, fails to recognize the fact that properties characteristic of
acids or bases may also be developed in other solvents. A more generalized
definition is that of Bronsted, who defined an acid as a proton donor, and a
base as a proton acceptor. Even broader is the definition of Lewis, who defined
an acid as any material that an electron pair, a base as any mater-
will accept
ial and neutralization as the formation of a
that will donate an electron pair,
coordination bond between an acid and a base.
The apparent strength of an acid or base is determined by the extent of its
reaction with a solvent. In aqueous solution all strong acids appear equally
1223
strong because they react with the solvent to undergo almost complete con-
version to hydronium ion (HaO^) and the acid anion. In a weakly protophilic
solvent such as acetic acid, the extent of formation of the acetonium ion
(CH3C00H2‘^) due to the addition of a proton provides a more sensitive dif-
ferentiation of the strength of acids and shows that the order of decreasing
strength for acids is and nitric.
perchloric, hydrobromic, sulfuric, hydrochloric,
Acetic acid reacts incompletely with water to form hydronium ion and is,
therefore, a weak acid. In contrast, it dissolves in a base such as ethylenedi-
amine, and reacts so completely with the solvent that it behaves as a strong
acid.
This so-called levelling effect is observed also for bases. In sulfuric acid
almost all bases appear to be of the same strength. As the acid properties of
the solvent decrease in the series sulfuric acid, acetic acid, phenol, water, pyri-
dine and butylamine, bases dissolved in them become
weaker progressively
and the differences between bases are accentuated. In order of decreasing
strength, strong bases of value for non-aqueous titrations are potassium
methoxide, sodium methoxide, lithium methoxide, and tetrabutylammonium
hydroxide.
Many water-insoluble compounds acquire enhanced acidic or basic proper-
ties when dissolved in organic solvents. Thus the choice of the appropriate
solvent permits the determination of a variety of such materials by non-
aqueous depending upon which part of a compound is phys-
titration. Further,
iologically active, it is often possible to titrate that part by proper selection of
solvent and titrant. Pure compounds can be titrated directly, but it is often nec-
essary to isolate the active ingredient in pharmaceutical preparations from inter-
fering excipients and carriers.
The types of compounds that may be titrated as acids include acid halides,
acid anhydrides, carboxylic acids, amino acids, enols such as barbiturates and
xanthines, imides, phenols, pyrroles, and sulfonamides. The types of com-
pounds that may be titrated as bases include amines, nitrogen-containing het-
erocyclic compounds, quarternary ammonium compounds, alkali salts of
organic acids, alkali salts of inorganic acids, and some salts of amines. Many
salts of halogen acids may be titrated in acetic acid or acetic anhydride after
the addition of mercuric acetate, which removes halide ion as the unionized
mercuric halide complex. In the case of hydrochlorides of weak bases not
containing acetylatable groupings it is also possible to titrate in acetic anhy-
dride without the addition of mercuric acetate and using an indicator such as
malachite green or crystal violet. Titrations carried out in the presence of an
excess of acetic anhydride must be applied cautiously, however, since any reac-
tion of the anhydride with the substance being titrated may give rise to low
results.
In the titration of a basic compound, a volumetric solution of perchloric acid

is usually used, although perchloric acid in dioxan may be
in glacial acetic acid
an acidic compound, a volumetric solu-
useful in special cases. In the titration of
tion of lithiummethoxide in a methanol-toluene solvent is often used. For
many applications it is convenient to use a solution of tetrabutylammonium
1224
Methods of Analysis
hydroxide in toluene; sodium methoxide, formerly in wide use, may often give
rise to troublesome gelatinous precipitates.
Because of interference by carbon dioxide, solvents for acidic compounds

must be protected from excessive exposure to the atmosphere by a suitable
cover or by an inert atmosphere during the titration. A blank determination
should be carried out and the volume generally should not exceed 0.01 ml of a
0.1 mol/1 titrant for each ml of solvent.
The end-point may be determined visually by colour change, or potentio-
metrically. If the calomel reference electrode is used, it is advantageous to
replace the aqueous potassium chloride solution in the salt bridge with lithium
perchlorate/acetic acid TS for titrations in acidic solvents, or potassium chlo-
ride in methanol for titrations in basic solvents. It should be recognized that
certain indicators in common use (crystal violet, for example) undergo a series
of colour changes and, in establishing a non-aqueous titration method for a par-
ticular use, care should be taken to ensure that the colour change specified as
the end-point of the titration corresponds to the maximum value of d£/dV
(where E is the electromotive force and V the volume of titrant) in a potentio-
metric titration of the substance under consideration.
When using titrants prepared with solvents that may have a relatively high
coefficient of expansion, for example, glacial acetic acid, toluene, etc., care
should be taken to compensate for differences in temperature that may exist
between the time the titrant is used and that at which it was standardized.
A (for bases and their salts)
Method
Prepare a solution as specified in the monograph or dissolve the substance being
examined in a suitable volume of glacial acetic acid Rl, previously neutralized
to crystal violet/acetic acid TS, warming and cooling if necessary. Alternatively
the titration blank for the solvent and indicator may be established in a separate
determination. When the substance is a salt of a hydrohalic acid, add 10 ml of
mercuric acetate/acetic acid TS. When the end-point is determined visually by
colour change, add 2-3 drops of crystal violet/acetic acid TS, and titrate with
perchloric acid of the specified concentration (mol/1) to the appropriate colour
change of the indicator. When a different indicator is specified in the monograph,
this indicator should also be used for the neutralization of the glacial acetic acid
Rl, and mercuric acetate/acetic acid TS, and the standardization of the titrant.
When the equivalence point is determined potentiometrically, the indicator
is omitted and neutralization of the solution and standardization of the titrant
are also carried out potentiometrically. A glass electrode and a saturated calomel
cell (containing potassium chloride (350 g/1) TS) as reference electrode, are used.
The junction between the calomel electrode and the titration liquid should have
a reasonablylow electrical resistance and there should be a minimum of trans-
from one side to the other. Serious instability may result unless
fer of liquid
the connections between the potentiometer and the electrode system are in
accordance with the manufacturer's instructions.
1225
When the temperature at which the titration is carried out differs from
{t-T)
the temperature (?i) at which the titrant was standardized, multiply the volume
of the titrant required by [1 + 0.001(?i - tTj\ and calculate the result of the assay
from the corrected volume.
Method B (for acids)

The titrant, where the end-point is determined visu-
solvent and (in the case
ally) monograph.
the indicator to be used for each substance are specified in the
Protect the solution and titrantfrom carbon dioxide of the atmosphere
throughout the determination. This may conveniently be done by replacing the
air above the titration liquid with nitrogen.
Dissolve the substance being examined in a suitable volume of the solvent

previously neutralized to the indicator, warming and cooling if necessary, or
prepare a solution as specified in the monograph. Titrate to the appropriate
colour change of the indicator. Carry out a blank determination and make any
necessary corrections. The titrant is standardized using the same solvent and
indicator as specified for the substance.
When the equivalence point is established potentiometrically, the indicator
is omitted and neutralization of the solution and standardization of the titrant
are also carried out potentiometrically.
A and a saturated calomel reference electrode in which the
glass electrode
aqueous potassium chloride (350 g/1) TS has been replaced by a saturated solu-
tion of potassium chloride R in methanol R are used. The junction between the
calomel electrode and the titration liquid should have a reasonably low elec-
trical resistance and there should be a minimum of transfer of the liquid from
one side to the other. Serious instability may result unless the connections
between the potentiometer and the electrode system are made in accordance
with the manufacturer’s instruction.
2.7 Nitrite titration

Nitrite titration is a titration method used particularly for the assay of primary
aromatic amines.
The apparatus usually used in an electrometric procedure for nitrite titra-
tion is composed of an open titration vessel containing two platinum electrodes
connected to a suitable circuit. The electrodes should have a potential differ-
ence of 50-100 mV. The circuit should include a device for measuring current
with a sensitivity of 0.1 to InA, usually with an indicating needle. The titra-
tion vessel should be provided with a suitable mechanical or magnetic stirring
device, or a stream of nitrogen passing through the solution may be used to
mix this solution. Electrodes made of platinum wire 0.5 mm in diameter and
about 20 mm long are suitable. Before each use, the electrodes should be
cleaned by immersing them for a few seconds in boiling nitric acid (~1000g/l)
TS, to which about 1 mg/ml of ferric chloride R has previously been added, and
then thoroughly rinsing them with water.
1226
Methods of Analysis
Place 20 ml of hydrochloric acid (-250 g/1) TS and 50 ml of water in the titra-
tion vessel, add the quantity of the test substance and, if indicated, a catalyst,
as specified in the monograph and stir to dissolve; cool to about 15 °C and
titrate slowly with sodium nitrite (0.1 mol/1) VS, placing the burette tip below
the surface of the solution. During the addition of the titrant, stir the solution
continuously and gently, without pulling a vortex of air under the surface, and
maintain the temperature of the solution at about 15 °C.
When the titration is within 1 ml of the estimated end-point, add the titrant
in 0.1 ml portions, allowing not less than 1 minute to elapse before adding sub-
sequent portions. Initially, the needle of the measuring device deflects at every
addition of reagent and then returns to its original position. No deflection is
observed when the end-point of the determination is reached.
2.8 Determination of water by the Karl Fischer method

The titrimetric determination of water by the Karl Fischer method depends on
the reaction that takes place quantitatively between water and a reagent con-
sisting of sulfur dioxide and iodine in anhydrous pyridine and usually methanol.
The reaction is carried out in a suitable solvent such as methanol or acetic acid.
The reagents and solutions used in the determination of water by this
method are sensitive to water and precautions must be taken throughout to
prevent exposure to atmospheric moisture.
The titration vessel is fitted with two platinum electrodes, a gas inlet tube
if needed, a stopper, which accommodates the burette tip, and a vent tube pro-
tected by The substance to be titrated is introduced through an inlet

a desiccant.
tube or side-arm, which can be closed by an airtight stopper. The Karl Fischer
reagent TS is protected from light and stored in a bottle into which is fitted an
automatic burette. The reagent is pumped into the burette by means of a hand
bellows, the access of moisture being prevented by a suitable arrangement of
desiccant tubes. Stirring is accomplished magnetically or by means of a stream
of suitably dried nitrogen passed through the solution during the titration.
The end-point is obtained by using an electrical circuit composed of a
microammeter, platinum electrodes, and a 1.5-V or 2-V battery connected
across a variable resistance of about 2000 n. The resistance is adjusted so that
an initial current passes through the platinum electrodes in series with a
microammeter. After each addition of reagent, the pointer of the microamme-
ter is deflected but quickly returns to its original position. At the end of the
reaction a deflection is obtained that persists for 10-15 seconds. Alternatively,
the end-point can also be determined by a voltametric method. A potential dif-
ference of 30-50 mV is applied to the platinum electrodes to serve as a constant
polarizing current and the solution is titrated with the reagent. The potential
difference is monitored by means of a microvoltmeter. The end-point is reached
when the voltmeter indicates a stable decrease of voltage. In the voltametric
method the end-point may also be obtained graphically by plotting the voltage
1227
versus the volume of the reagent, and establishing the beginning of the drop in
potential.
Direct titration (Method A)
Add about 20ml of dehydrated methanol R, unless otherwise specified in the
monograph, to the titration vessel and titrate to the end-point with Karl Fischer
reagent TS. Quickly transfer the specified quantity of substance, accurately
weighed, to the titration vessel. Stir for 1 minute and titrate again to the end-
point with Karl Fischer reagent TS.
Backtitration (Method B)
Add about 10ml of dehydrated methanol R, unless otherwise specified in the
monograph, to the titration vessel and titrate to the end-point with Karl Fischer
reagent TS. Quickly transfer the specified quantity of substance, accurately
weighed, to the titration vessel, followed by an accurately measured amount
of Karl Fischer reagent TS, sufficient to give an excess of about 1 ml.Allow to
stand protected from light for 1 minute or the time specified in the monograph,
stirring from time to time.
Titrate the excess of Karl Fischer reagent TS to the end-point with dehy-
drated methanol R, to which has been added an accurately known amount of
water, usually equivalent to about 2.5mg/ml.
2.9 Determination of methoxyl

The contents of methoxy-groups in an organic substance are assayed by react-
with concentrated hydriodic acid; methyl iodide produced in
ing the substance
the reaction is absorbed into a suitable absorbing liquid, and its
distilled off,
amount determined titrimetricaUy.
Apparatus
The apparatus consists of a 25-ml round-bottomed boiling flask into which is
sealed a capillary side-arm, 1 mm in diameter, to provide an inlet for a stream

of carbon dioxide. The flask is also fitted with an air condenser, approximately
25 cm in height and about 9 mm
in internal diameter. A suitable scrubber device
containing about 2 ml of water is placed over the condenser. Add 5 ml of anti-
mony sodium tartrate (50g/l) TS to the scrubber. The outlet from the scrubber
terminates in a tube that dips below the surface of the absorbing liquid in the
first of two receivers connected in series. For greater convenience in use and
cleaning, separate parts of the apparatus are connected by means of ground
glass conical or ball joints.
1228
Methods of Analysis
Place a quantity of the substance being tested, accurately weighed, as specified
in the monograph, in the boiling flask with a boiling rod. Add 2.5 ml of melted
phenol R and 5ml of hydriodic acid (~970g/l) TS, and connect the flask to the
condenser. Add potassium acetate TS to each of the two receivers, about 6ml
to the first one and about 4 ml to the second, and to each receiver 6 drops of
bromine R. Pass a slow uniform stream of carbon dioxide R through the cap-
illary side-arm of the boiling flask, and heat the liquid gently by means of a
mantled microburner or another suitable device, at such a rate that the vapours
of the boiling liquid rise halfway up the condenser. For most substances 30
minutes are sufficient to complete the reaction and sweep out the apparatus.
Wash the contents of both receivers into a 250-ml conical flask containing 5 ml
of sodium acetate (150 g/1) TS.
Adjust the volume of the liquid to approximately 125 ml, and add 6 drops
of formic acid (~1080g/l) TS. Rotate the flask until the colour due to the
bromine is discharged, then add 12 drops of formic acid (-1080 g/1) TS, stopper
the flask and mix the contents thoroughly so as to remove any free bromine
including the vapour above the liquid, and allow the solution to stand for 1-2
minutes; add g of potassium iodide R and 5 ml of sulfuric acid (-100 g/1) TS,
1
and titrate the liberated iodine with sodium thiosulfate (0.1 mol/1) VS using
starch TS as indicator. Repeat the operation without the substance being tested,
and deduct the volume of sodium thiosulfate (0.1 mol/1) VS used from the
volume required in the determination of methoxyl.
methoxyl (CH3O).
2.10 Determination of nitrogen

Procedure for macrodetermination (Method A)
Place the amount of test substance specified in the monograph in a 200-ml long-
necked flask, add 1 g of a mixture composed of 10 parts of potassium sulfate R
or anhydrous sodium sulfate R and 1 part of copper(ll) sulfate R, followed by
nitrogen-free sulfuric acid (-1760 g/1) TS, using the quantities of the substance
and of monograph. Heat the mixture over a small
sulfuric acid specified in the
flame until a clear green solution obtained and boil gently for a further 30
is
minutes unless otherwise specified in the monograph; precautions should be

taken to prevent the upper part of the flask from becoming overheated. Cool,
dilute to 75-85 ml with water, using suitable precautions, and add a piece of
granulated zinc R and a solution of 15g of sodium hydroxide R and 2g of
sodium thiosulfate R in 25 ml of water. The quantity of sodium hydroxide
should be increased, if necessary, to ensure that, before distillation, the mixture
is strongly alkaline. Immediately connect the flask to a distillation apparatus,
mix the contents, distil the liberated ammonia into 16ml of boric acid (50 g/1)
TS, and titrate with sulfuric acid (0.05 mol/1) VS using methyl red/ethanol TS
1229
as indicator. Repeat the operation without the substance being tested; the dif-
ference between the titrations represents the ammonia liberated by the sub-
stance being tested. Each ml of sulfuric acid (0.05 mol/1) VS is equivalent to
1.401mg of N.
Procedure for microdetermination (Method B)

Place the amount of test substance specified in the monograph in a suitable
digestion tube, add 3 drops of copper(ll) sulfate (190g/l) TS and 1 ml of nitro-
gen-free sulfuric acid (-1760 g/1) TS and boil gently for 10 minutes; cool; add
1 g of anhydrous sodium sulfate R and 10 mg of selenium R, boil gentiy for 1
hour and cool. Transfer the contents of the digestion tube to (or attach it to)
an ammonia microdistillation apparatus, add 6ml of sodium hydroxide
(-400 g/1) TS and pass steam through the flask; distil for 7 minutes, collecting
the distillate in a mixture of 5 ml of boric acid (50
g/1) TS, 5 ml of water, and 1
drop of methyl red/methylthioninium chloride TS and titrate with hydrochlo-
ric acid (0.015mol/l) VS. Repeat the operation without the substance being
between the titrations represents the ammonia liberated

tested; the difference
by the substance being tested. Each ml of hydrochloric acid (0.015 mol/1) VS is
equivalent to 0.210mg of N.
3. Biological methods
3.1 Microbiological assay of antibiotics
The potency (activity) of an antibiotic product is expressed as the ratio of the

dose that inhibits the growth of a suitable susceptible microorganism to the
dose of an International Biological Standard, an International Biological Refer-
ence Preparation, or an International Chemical Reference Substance of that
antibiotic that produces similar inhibition. Properly validated secondary refer-
ence materials may also be used in the assay. To carry out the assay a com-
parison is made between the inhibition of the growth of microorganisms
produced by known concentrations of the reference material and that produced

by measured dilutions of the test substance. This response can be measured by
the diffusion method, as described below, or in a liquid medium by the tur-
bidimetric method.
An International Unit is the specific activity contained in such an amount
(weight) of the relevant International Biological Standard or International Bio-
logical Reference Preparation as the WHO Expert Committee on Biological
Standardization may from time to indicate as the quantity exactly equivalent
to the unit accepted for international use. In some cases, when owing to the
properties of the material, difficulties are experienced in weighing with ade-
quate accuracy small amounts of the relevant International Biological Standard
or International Biological Reference Preparation, International Units are defined
on the basis of the total contents of the material in an ampoule or a vial. A
defined number of International Units is then assigned to the total contents of
1230
Methods of Analysis
an ampoule or a vial; this material has to be carefully removed with an appro-

priate solvent and the final volume of the solution has to be accurately adjusted.
International Chemical Reference Substances do not have defined units of
biological activity. The potency of those products for which biological assays
are required are in such cases expressed in terms of an equivalent weight of the
pure substance.
Use Petri dishes or rectangular trays filled to a depth of 3-4 mm, unless other-
wise indicated in the monograph, with a culture medium that has previously
been inoculated with a suitable inoculum of a susceptible test organism pre-
pared as described below. The nutrient agar may be composed of two separate
layers of which only the upper one may be inoculated. The concentration of
the inoculum should be so selected that the sharpest zones of inhibition and
suitable dose response at different concentrations of the standard are obtained.
When using the inoculum prepared as described below, an inoculated medium
containing 1 ml of inoculum per 100ml of the culture medium is usually suit-
able. When the inoculum consists of a suspension of vegetative organisms, the
temperature of the molten agar medium must not exceed 48-50 °C at the time
of inoculation. The dishes or trays should be specially selected with flat
bottoms. During the filling they should be placed on a flat, horizontal surface
so as to ensure that the layer of the medium will be of a uniform thickness.
With some test organisms, the procedure may be improved if the inoculated
plates are allowed to dry for 30 minutes at room temperature before use, or
refrigerated at 4°C for several hours.
For the application of the test solution, small sterile cylinders of uniform
size,approximately 10 mm
high and having an internal diameter of approxi-
mately 5mm, made of suitable material such as glass, porcelain, or stainless
steel, are placed on the surface of the inoculatedmedium. Instead of cylinders,
holes 8-1 0 mm in diameter may be bored in the medium with a previously ster-
ilized borer. Other methods of application of the test solution may also be used.
The arrangement on the plate should be such that overlapping of zones is
avoided.
Solutions of the reference material of known concentration and corre-
sponding dilutions of the test substance, presumed to be of approximately the
same concentration, are prepared in a sterile buffer of a suitable pH value. To
assess the validity of the assay at least 3 different doses of the reference mate-
rial should be used together with an equal number of doses of the test sub-
stance having the same presumed activity as the solutions of the reference
material. The dose levels used should be in geometric progression, for example,
by preparing a series of dilutions in the ratio 2:1. Once the relationship
between the logarithm of concentration of the antibiotic and the diameter of
the zone of inhibition has been shown to be approximately rectilinear for the
system used, routine assays may be carried out using only 2 concentrations of
the reference material and 2 dilutions of the test substance. Where a mono-
1231
graph gives directions for the initial preparation of a solution of the substance,
this solution is then diluted as necessary with the appropriate sterile buffer.
The solutions of the reference material and the test substance are preferably
arranged in the form of a Latin square when rectangular trays are employed.
When Petri dishes are used, the solutions are arranged on each dish so that the
solutions of the reference material and those of the test substance alternate
around the dish and are placed in such a manner that the highest concentra-
tions of the reference material and of the test substance are not adjacent. The
solutions are placed in the cylinders or holes by means of a pipette that deliv-
ers a uniform volume of liquid. When the holes are used the delivered volume
should be sufficient to fill them almost completely.
The plates are incubated at a suitable temperature, the selected temperature
being controlled at +0.5 °C, for approximately 16 hours, and the diameters or
areas of the zones of inhibition produced by the varied concentrations of the
standard and of the test substance are measured accurately, preferably to the
nearest 0.1mm of the actual zone size, by using a suitable measuring device.
From the results, the potency of the tested substance is calculated. Suitable pub-
lications on the statistics of bioassays are listed below.
Conditions for the assay of individual antibiotics and suitable test organ-
isms are given in the monographs. The choice of an appropriate strain of test
organism may be critical for the assay. For easy reference, examples of suitable
test organisms for a number of antibiotics are shown in Table 4. The designa-
tions of the test strains are as follows:
NCTC - National Collection of Type Cultures, Central Public Health Lab-

Avenue, London NW9 5HT, England
oratory, Colindale
NCYC - National Collection of Yeast Cultures, AFRC Food Research Insti-
tute, Colney Lane, Norwich NR4 7UA, England
ATCC American Type Culture Collection, 10801 University Boulevard,
-
Manassas, Virginia 20110-2209, USA
Other suitable strains of test organisms can be used. Additional information

regarding sources of suitable strains may be obtained from Quality Assurance and
Safety: Biologicals, World Health Organization, 1211 Geneva 27, Switzerland.
Precision of the assay

In order to determine whether or not a substance satisfies the requirements for
potency specified in the monograph, the assay should, if necessary, be repeated
until the required precision has been attained. This precision is such that the
fiducial limits (P = 0.95) of the mean estimated potency, expressed as a per-
centage of the mean estimated potency, should be within the required range
given in the individual monographs.
Calculation of results
The following publications contain suitable methods that may be used to carry
out the statistical evaluation of the microbiological assay of antibiotics:
1232
Methods of Analysis
1. Bliss Cl. Statistics ofhioassay. New York, Academic Press, 1952.

2. Bliss Cl. Statistics in biology, vol. 1. New York, McGraw Hill, 1967.
3. Bliss Cl. Statistics in biology, vol. II. New York, McGraw Hill, 1970.
4. Finney DJ. Statistical methods in biological assays. London, Griffin,
1964.
5. Hewitt W. Microbiological assay. New York, Academic Press, 1977.
6. Philippe J. Les metliodes statisticjues en pharmack et en cliintie. Paris, Masson,
1967.
The methods of carrying out the statistical evaluation of the microbiological

assay of antibiotics are also described in many national and regional
pharmacopoeias.
Culture media
The formulae for the culture media (Cm) referred to in Table 4 are described
in Reagents, test solutions, and volumetric solutions. In each instance the final
pH is adjusted to that stated in the table.
Preparation of inoculum
The test organism is grown for 7
Bacillus cereus; Bacillus pumilus; Bacillus subtilis.
days at a temperature of 37-39 °C on the surface of culture medium Cml (pH
€.5-6.6 after sterilization) to which has been added 1 pg of manganese sulfate
R per ml. Using sterile water, the growth, which consists mainly of spores, is
washed off, heated for 30 minutes at 70 °C, and suitably diluted - for example,
to give between 10^ and 10® spores per ml. The spore suspension may be stored
for long periods at a temperature not exceeding 4°C.
Bordetella broncliiseptica. The test organism is grown overnight on culture
medium Cm2 (pH 6. 5-6.6 after sterilization) at a temperature of 35-37 °C. A
suspension is prepared by washing off the growth and diluting with sterile
water or saline TS such that a 1-cm layer

to a suitable opacity, for example,
transmits 50% of the incident light when examined
650 nm. The suspension at
may be stored for up to 2 weeks at a temperature not exceeding 4°C.
A freshly prepared inoculum may be replaced by a suitable suspension of
the inoculum in a suitable vehicle, such as sterile peptone (1 g/1) TS2 that has
been stored frozen at-70°C and subsequently thawed.
Micrococcus luteus. The test organism isgrown overnight on culture medium
Cml (pH 6.5-6. 6 after sterilization) at a temperature of 35-37 °C. A suspension
is prepared by washing off the growth and diluting with saline TS to a suitable
opacity, for example, such that a 1-cm layer of a 1 in 50 dilution transmits 80%
of the incident light when examined at 650 nm. The suspension may be stored
for up to 2 weeks at a temperature not exceeding 4°C.
been stored frozen at -70 °C and subsequently thawed.
1233
Table 4- Test organisms and conditions of assay of individual antibiotics
Antibiotic Test organism Culture Phosphate Concentration Incubation

medium; buffer, (weight or temperature
final pH sterile, International (in °C)
pH-^, TS Units per ml)^
Bacitracin Micrococcus luteus Cml; 7.0 IM lU 35-37

NCTC 7743; 7.0-7.1
ATCC 10240
Micrococcus luteus Cml; 6.0 IM lU 30-31
NCTC 7743; 6.5-6.6
ATCC 10240
Cefalexin Staphylococcus aureus Cml; 6.0 10^0 (ig 32-35
NCTC 6571; 6.5-6.6
ATCC 9144
Staphylococcus aureus Cml; 6.0 10-40 gg 32-35
ATCC 6538-P 6.5-6.6
Cefalotin Staphylococcus aureus Cml; 6.0 0.5-2 lU 32-35
NCTC 6571; 65-6.6
ATCC 9144
Staphylococcus aureus Cml; 6.0 0.5-2 lU 32-35
ATCC 6538-P 6.5-6.6
Chlortetracycline Bacillus pumilus Cml; 4.5 2-20 lU 37-39
NCTC 8241; 6.5-6.6
ATCC 14884
Bacillus cereus Cml; 4.5 0.05-0.2 lU 30-33
ATCC 11778 5.9-6.0
Cloxacillin Bacillus suhtilis Cml; 7.0 5-20 ng 37-39
NCTC 8236; 6.5-6.6
ATCC 11774
Staphylococcus aureus Cml; 6.0 2-8 ng 32-35
ATCC 6538-P 6.5-6.6
Dicloxacillin Staphylococcus aureus
NCTC 6571; Cml; 6.0 2.5-10 ng 37-39
ATCC 9144 6.6
ATCC 6538-P 65-6.6
Erythromycin Bacillus pumilus
NCTC 8241; Cml; 8.0 5-25 lU 37-39
ATCC 14884 8.0-8.1
Micrococcus luteus Cml; 8.0 0.5-1 .5 lU 35-37
ATCC 9341 8.0-8.1
Neomycin Bacillus pumilus Cml; 8.0 2-14 lU 37-39
NCTC 8241; 8.0-8.1
ATCC 14884
Staphylococcus aureus Cml; 8.0 2-20 lU 35-37
ATCC 29737 7.8-8.0
Staphylococcus Cml; 8.0 0.5-2 lU 35-37
epidermidis
ATCC 12228 8.0-8.1
Novobiocin Bacillus suhtilis Cml; 6.0 1-5 lU 30-33
NCTC 10315; 6.5-6.6
1234
Methods of Analysis
Table 4- Continued
Antibiotic Test organism Culture Phosphate Concentration Incubation

medium; buffer, (weight or temperature
final pH sterile, International (in °C)
pH", TS Units per ml)*’
Micrococcus lutcus Cml; 6.0 10-50 lU 30-35

ATCC 9341 65-6.6
Nystatin Saccharomyces Cm3; 25-300 lU 35-37
cerevisiae
NCYC 87; 6.0-6.2

ATCC 9763
Oxacillin Bacillus suhtilis Cml; 7.0 2.5-10 fig 37-39
NCTC 8236; 6.5-6.6
ATCC 11774
ATCC 6538-P 6.5-6.6
Oxytetracycline Bacillus pumilus Cml; 4.5 2-20 lU 37-39
NCTC 8241; 6.5-6.6
ATCC 14884
Bacillus cereus Cml; 4.5 0.5-2 lU 30-33
ATCC 11778 5.9-6.0
Polymyxin B Bordetella Cm2; 6.0, TS3 20-100 lU 35-37
bronchiseptica
NCTC 8344; 7.2-7.3

ATCC 4617
Bordetella Cm2; 7.2 50-200 lU 35-37
bronchiseptica
NCTC 8344; 12-15
ATCC 4617
Escherichia coli Cml; 7.2 5-100 lU 35-37
ATCC 10536 65-6.6
Streptomycin Bacillus subtilis Cml; 8.0 5-20 lU 37-39
NCTC 8236; 7.9-8.0
ATCC 11774
Bacillus subtilis Cml; 8.0 3-15 lU 35-37
ATCC 6633 8.0-8.1
Tetracycline Bacillus pumilus Cml; 4.5 2-20 lU 37-39
NCTC 8241; 6.5-6.6
ATCC 14884
Bacillus cereus Cml; 4.5 0.5-2 lU 30-33
ATCC 11778 5.9-6.0
Phosphate buffers, sterile, of suitable pH. Buffers designated as TS, TSl, or TS2 may be used.
Range within which suitable concentrations may be found.
‘
The preparation of the solution of the reference material and of the corresponding dilution of the
test substance is done as described in the monograph with the aid of dimethylformamide R and
phosphate buffer, sterile, pH 6.0 TS3.
1235
Saccharoniyces cerevisiae. The test organism is grown overnight on culture

medium Cm3 (pH 6.0-6.2 after sterilization) at a temperature of 35-37 °C. A
suspension is prepared by washing off the growth with saline TS and diluting
to a suitable opacity, for example, such that a 1-cm layer transmits 50% of the
incident light when examined at 650 nm. The suspension may be stored for up
to 2 weeks at a temperature not exceeding 4°C.
Staphylococcus aureus. The test organism is grown overnight on culture
medium Cml (pH 6. 5-6.6 after sterilization) at a temperature of 35-37 °C. A
suspension is prepared by washing off the growth with saline TS and diluting
to a suitable opacity, for example, such that a 1-cm layer transmits 50% of the
incident light when examined at 650 nm.
the inoculum in a suitable vehicle, such as sterile peptone (5 g/1) TS that has
3.2 Test for sterility

This test for sterility is applicable only to parenteral preparations. For oph-
thalmic and other non-injectable preparations, surgical dressings, catgut and
other surgical sutures, appropriate alternative test conditions must be chosen.
Sterility testing should be carried out under strict conditions specifically
designed to avoid microbial contamination of the material being tested, for example
by using a laminar flow technique. Precautions should also be taken to ensure that
any microorganisms expected to be revealed by the test remain unmodified. Ade-
quate performance of the testing technique should be ensured periodically.
A batch (i.e. one sterilized load, or a quantity of containers filled under
aseptic conditions in one working sessionat one work station) is defined as a
collection of sealed containers whose contents and physical aspect are homo-
geneous in every respect. The risk of microbial contamination is thus assumed
to be equal for each container in the batch.
Therefore, once a sample has been tested and is found to be free of conta-
mination, the same degree of purity should apply to the whole batch. Assur-
ance that this is the case depends strongly on compliance with Good
Manufacturing Practices (GMP)' throughout the entire manufacturing process.
’
WHO good manufacturing practices: main principles for pharmaceutical products. Quality assur-
ance of pharmaceuticals. A compendium of guidelines and related materials, Volume 2, Updated
edition. Geneva, World Health Organization, 2004, However, many national authorities have
incorporated good manufacturing practices of their own into their legislation, and these should
be followed when available.
1236
Methods of Analysis
Sampling
Number of containers Minimum number of samples

in the batch to be tested
not more than 100 10% or 4 containers (whichever is greater)

between 100 and 500 10 containers
more than 500 2% or 20 containers (whichever is less)
Culture media
Culture media that have proven to be the most suitable for use in sterility tests
are: fluidsodium mercaftoacetate and soya-bean casein digest media. Fluid sodium mer-
captoacetate (sodium thioglycolate) medium (culture medium Cm4) is primarily
intended for the culture of anaerobic bacteria but will also detect aerobic bac-
teria. Soya-bean casein digest medium (culture medium Cm5) is primarily intended
for the culture of aerobic bacteria but is also suitable for fungi. Other media
may be used provided that they have been shown to sustain the growth of a
wide range of microorganisms and that they comply with the test for “effec-
tiveness of themedium” in the presence of the preparation to be tested.
The growth-promoting quality of each new batch of media should be tested
as follows: take separate representative samples of the medium and add inocula
of reference strains of appropriate microorganisms (e.g. Staphylococcus aureus
ATCC 6538 P (NCIMB 8625, CIP 53.156), Bacillus subtilis ATCC 6633, Clostrid-
ium sporogenes ATCC 19404, Candida albicans ATCC 2091), each containing
approximately 100 viable microorganisms. The media thus inoculated with the
reference strains should then be incubated according to the conditions speci-
fied in the recommended procedure. The test media are satisfactory if clear evi-
dence of growth appears in all inoculated media within 7 days.
Antibacterial effects of the sample

Before testing, possible antibacterial action of the substance or material under
examination should be determined and the growth-promoting properties of the
media in the presence and in the absence of the test sample should be com-
pared. This comparison should be carried out under test conditions using ref-
erence strains, as described above in the section “Culture media”. If the growth
of the reference strains is delayed or weakened in the presence of the product,
the antibacterial action of the product should be suppressed by means of fil-
tration, dilution, or neutralization. The effectiveness of this process should be
confirmed by repeating the test.
Thetest for sterility may be carried out by membrane filtration or by direct
inoculation. The technique of membrane filtration is to be preferred whenever
the nature of the product permits, i.e. for aqueous preparations able
to be fil-
tered, especially in large volumes, for alcoholic or oily preparations, and for
1237
preparations miscible with or soluble in aqueous or oily solvents that have not
shown any antimicrobial effect when tested. Whichever test is used, the media
should be observed at regular intervals throughout the specified incubation
period.
Membrane filtration
Use membrane filters of assured quality, preferably certified by the manufac-
turer, with a nominal pore size not greater than 0.45 pm. For example, use cel-
lulose nitrate filters for aqueous, oily, and weakly alcoholic liquids and acetate
filters for strongly alcoholic liquids. Use filter discs with a diameter of about
50mm in a previously sterilized filter assembly. If filters of a different

diameter are used, adjust the volumes of the dilutions and the washings
accordingly.
Before the test, filter a small quantity of a suitable sterile diluent, such as a
1 g/litre neutral solution of meat or casein peptone (culture media Cml or Cm5),
through the prepared filtration apparatus. Transfer the contents of the con-
tainer(s) to be examined to the apparatus. If possible, transfer the entire con-
tents of the containers or the minimum quantity laid down for the test by direct
inoculation. If necessary, dilute the product to be tested with the chosen sterile
diluent. Carry out the filtration without delay.

If the product to be tested has known antimicrobial properties, wash the
membrane by filtering through it not less than three successive quantities of

the chosen sterile diluent, each of approximately 100 ml, with the addition of
a suitable neutralizing substance. Oily liquids may be diluted before filtration
with a suitable sterile solvent, such as isopropyl myristate, shown to have
minimal antimicrobial effects.
Preferably, transfer one membrane to each of the media used or, where this
is not possible, transfer the one membrane cut aseptically into two equal parts
to the different media. Incubate the culture media with the membranes for not
lessthan 7 days at 30-35 °C if it is intended mainly to detect bacteria and at
20-25 °C if it is specifically intended to detect fungi. If the product or the treat-
ment to which it has been subjected is suspect, a longer incubation time than
7 days may be necessary.
Direct inoculation
Transfer directly a suitable quantity (see the following tables) of the product
under examination, taken from a sealed container, to culture media intended
for the detection of aerobic and anaerobic bacteria as well as media for the
detection of fungi.
1238
Methods of Analysis
Liquids
Quantity in the container Quantity of sample needed
less ml
than 1 entire contents of container
between 1 ml and 4 ml half contents of container
between 4 ml and 20 ml 2 ml
20ml or more (including large- 10% of contents
volume parenterals)
Solids
Quantity in the container Quantity of sample needed
less than 50 mg entire contents of container

between 50 mg and 200 mg 50% of contents of container
200mg or more lOOmg
There should be a sufficient quantity of culture medium to ensure that its nutri-
tive properties are unaffected by the addition of the product under examina-
tion. In order to ensure homogeneous
distribution and to eliminate antibacterial
activity, unless otherwise indicated in the monograph, transfer the product
under examination in such a way that liquids are diluted approximately 10-fold
and solids approximately 100-fold.
In the case of an oily liquid, an emulsifying agent may be added to the
culture medium, such as 0.5-1% m/v of polysorbate 80 or 0.1% m/v of if-tert-
octylphenoxy)polyoxyethanol. Other emulsifying agents shown not to have
any antimicrobial action under test conditions may also be used in appropriate
concentrations.
Incubate the inoculated media for not less than 14 days. Maintain the tem-
perature at 30-35 °C for media mainly intended to detect bacteria and at 20-
25 °C for media specifically intended to detect fungi. Gently shake at regular
intervals if the medium contains an oily liquid. If medium is used to detect
one
both aerobic and anaerobic bacteria, keep the shaking to a minimum in order
to maintain anaerobic conditions.
Interpretation of results
Ifno microbial growth appears in any of the culture media by the end of the
incubation period, the product complies with the test for sterility.
If microbial growth appears in the initial test cultures and/or in subcultures,

the organisms should be isolated and identified. A second test should then be
made to verify the results.
• If there is evidence - from the material used, the kind of organisms

detected, the monitoring or the control tests - that the test was carried out
1239
under inadequately aseptic conditions, the test is invalid and should be

repeated with the same corresponding number of items and quantities.
• If the microbial growth not due to lack of aseptic conditions, the test
is
should be repeated on twice the number of containers used for the original
test. If no microbial growth is detected, the product examined complies
with the test for sterility. If microbial growth is detected in the second test,
the product examined does not comply with the test for sterility.
3.Z.1 Test for sterility of non-injectable fareprarations
The methods are described under 3.2 Test for sterility.
Certain dosage forms such as ophthalmic drops, ophthalmic ointments,

topical semi-solid dosage forms to be applied to damaged skin, etc., require
specific sampling plans and preparation of samples.
For the membrane filtration method and for direct inoculation of the
medium, determine the number of containers according to the sampling
method proposed. The quantity taken should be not less than the minimum
and not more than the maximum quantity given below. The quantity used for
each medium, which should be taken from the mixed sample, is also specified
in the table.
Sampling plan
Type of preparation Total pooled for each Quantity to be used

medium
Membrane filtration method

Aqueous solution 10-1 00 ml 5-10 ml
Preparations soluble in water, 1-lOg equivalent to 0.5-1 g
isopropyl myristate, or
other solvents
Direct inoculation method
Liquid preparations 10-100 ml 5-10 ml
Soluble preparations 1-lOg equivalent to 0.5-1 g
Insoluble preparations to be 1-lOg equivalent to 0.5-1 g
suspended or emulsified,
e.g. creams and ointments
If the membrane filtration method is used for ointments and creams, the prepa-
ration being tested may require additional heating up to 40 °C (or exception-
ally up to 45 °C with continuous stirring). The filtration process should be
carried out as quickly as possible. If heat is applied and/or a solvent combina-
tion is used in the sample preparation, the method should first be validated to
ensure that the specific expected bioburden is not affected by these conditions.
1240
Methods of Analysis
3.Z.2 Sterility testing of antibiotics

The test is designed to reveal the presence of contamination with live microor-
ganisms in antibiotics intended for parenteral administration or for other sterile
applications.
Test conditions
The test should be carried out under aseptic conditions in an area as free from
contamination as it is possible to achieve by the use of disinfecting agents, ger-
micidal lamps, and air filters. Germicidal lamps and disinfecting aerosols should
not be used during actual testing operations. The test manipulations should be
carried out in a filtered air environment or under a laminar flow hood, with
operators dressed in sterilized, static-free clothing, including head and foot
wear. The air pressure in the testing room should be greater than that of the
exterior area. The performance of the laminar flowhood should be monitored
by particulate count, settle plates, or slit-sampling devices, and the performance
of the filters and germicidal lamps checked routinely.
Membrane filtration apparatus

A suitable unit consists of a closed reservoir and a receptacle, separated by a
properly supported membrane of appropriate porosity. Membranes generally
have a nominal porosity of 0.45 pm, a diameter of
suitable for sterility testing
approximately 47 mm, and a flow rate of 55-75 ml of water per minute at a
pressure of 90kPa (700mmHg). The entire unit should preferably be assembled
and sterilized with the membrane in place, prior to use. If each entire mem-
brane is to be cultured, at least 2 filter units should be set up.
All air entering the filtering unit should be passed through an air-filter
capable of removing microorganisms.
Sampling
Take the sample in such a manner as to be representative of the material to be
tested. The amount taken should be sufficient to perform the tests and any
repeat tests thatmay be required. The sampling should be carried out in such
a way as to maintain intact the sterility of the material.
Culture media
The culture media used for sterility tests for bacteria and fungi should be
capable of supporting the growth of a wide variety of microorganisms, with
both aerobic and anaerobic growth characteristics, including the types found
in the environment of the manufacturing operations. More than one culture
medium will generally be needed to fulfil these criteria. The media that usually
give satisfactory results are fluid mercaptoacetate (thioglycolate) medium
(culture medium Cm4) and soybean-casein digest medium (culture medium
Cm5). Any other media that are used, however, should have at least demon-
strably equivalent growth-supporting properties.
1241
For testing the growth-supporting properties of each culture medium, strains

of microorganisms should be used with exacting nutritive and aerobic-anaerobic
requirements, in an inoculum containing only a small number of organisms (less
than 100). The media should be incubated at the temperatures at which they will
be used in the sterility test and growth should be evident after 24 hours.
Each lot of dehydrated medium obtained from a specialized manufacturer or
each lot of the medium prepared entirely in the laboratory should be tested for
its growth-supporting properties, since not every lotmay support the growth of
microorganisms to the desired extent. Differences may be caused by the occa-
sional presence of unsatisfactory components in a particular lot, or by the destruc-
tion of certain components by overheating or oversterilization of the medium.
Membrane filtration test procedure
Aseptically transfer a suitable amount of the solid test material (0.3-6g depend-
ing on the size of the container) into a sterile flask containing about 200 ml of
peptone (1 g/1) TSl, stopper the flask and swirl to effect rapid dissolution. If the
test material dissolves slowly or if the resulting solution will not filter rapidly,
the volume of the solvent may be increased to not more than 400ml. Immedi-
ately after the test material has dissolved, aseptically filter the solution, with the
aid of reduced pressure, through the membrane filter previously moistened with
sterile water or with peptone TSl. To speed up the process of filtration,
(lg/1)
the solution may be filtered using two filtering units simultaneously. To remove
the residual antibiotic from the membrane, wash it with sufficient peptone
(1 g/1) TSl to which, if so indicated in the monograph (in the case of penicillin
and cephalosporin antibiotics), sufficient penicillinase TS has been added.
Upon completion of the filtration, divide the membrane aseptically into two
approximately equal parts. Transfer one part of the membrane into a culture
vessel (a test-tube is suitable) containing 50-1 00 ml of culture medium Cm4
(fluid mercaptoacetate (thioglycolate) medium) and the other part of the mem-
brane into another culture vessel containing 50-100ml of culture medium Cm5
(soybean-casein digest medium).
A control test should be carried out at appropriate intervals to demonstrate
that residual antibiotic activity is being reduced by the above described
washing procedure to below the level that allows growth of an inoculum con-
taining 50-100 microorganisms susceptible to the antibiotic tested.
Direct test procedure

Depending on the size of the container take from 1-10 portions, each 0.3 g
of the test material, and aseptically transfer them into individual sterile vessels
(test-tubes are suitable) containing 50-1 00 ml of culture medium Cm6 (fluid
mercaptoacetate (thioglycolate) medium with penicillinase). Transfer portions
of similar size to another set of individual sterile vessels containing 50-1 00 ml
of culture medium Cm7 (soybean-casein digest medium with penicillinase).
1242
Methods of Analysis
Check the ability of the penicillinase contained in the medium to inactivate

all the penicillin in the test material by adding
one vessel containing culture
to
medium Cm6 the amount of material taken from one
container under test. Next
add 1.0ml of a dilution containing 50-100 microorganisms of a suitable strain
{Stap’kylococcus aureus, ATCC 6538-P is suitable) in culture medium Cm4. Typical
microbial growth must be observable after 24 hours of incubation at 30-32 °C.
Incubation
Incubate for 7 days the vessels containing fluid mercaptoacetate (thioglycolate)
media (culture media Cm4 and Cm6) at 30-32 °C and the vessels containing
soybean-casein digest media (media Cm5 and Cm7) at 22-25 °C. In the direct
test procedure, gently agitate the vessels at least once a day or until complete
dissolution occurs.
If other culture media are used, the incubation temperature and the period
of incubation may have to be appropriately modified.
Examine inoculated culture vessels at regular intervals and on the last day
of incubation for evidence of microbial growth. If such growth is observed it
should be confirmed by microscopic examination. It is desirable that the incu-

bation apparatus be equipped with a continuous temperature-recording device.
Interpretation of test results

Ifno evidence of growth is found in any of the culture vessels, except in the
positive growth control, the material meets the requirements of the test. If,
however, evidence of growth is found, a repeat test may be performed. If no
evidence of growth is then found in any of the culture vessels, except in the
positive growth control, the material meets the requirement of the test. The
material fails to pass the test if growth occurs in the repeat test.
The distinction between failure of the product to pass the test and a possible
invalidity of the test procedure requires the competent judgement of an expert.
3.3 Microbial purity of pharmaceutical preparations

Introduction
The following text is provided to give information and guidance, and is not regarded as
an analytical recjuirement.
Microbial contamination of a product may lead not only to spoilage of the
product, with the associated physical and chemical changes, but also to risk of
infection for the user. Therefore, oral and topical pharmaceutical products (cap-
sules, tablets, suspensions, creams, patches, etc.), which are not required to be
sterile, should be subject to controls for microbial contamination.
Quality assurance and manufacturing controls should be such that organ-
isms capable of proliferation and contamination of the product are within
acceptable limits. The microbial limits and batch testing regimes set for the
various categories of products should reflect the types of contamination most
1243
likely to be introduced during manufacture. The intended use of the product

also needs to be considered.
Microbial limits cannot be formulated to cover every possibility of conta-
mination that may occur. In assessing the results of microbiological testing, the
number and types of organisms present should be considered in the context of
the proposed use of the product.
Thus, in the manufacture, packaging, storage, and distribution of pharma-
ceuticals, suitable measures must be taken to ensure their microbial quality.
Limits for medicinal plant materials and methods for detection and estima-
tion of microorganisms are not included in this text. For such preparations
reference should be made to Quality control methods for medicinal plant materials
(WHO, 1998’): “Determination of microorganisms”.
General requirements
Category Dosage forms Recommended criteria

and materials
1 Preparations for Sterility (see individual monographs)

injection
Preparations for
ophthalmic use
2 Preparations for Total viable aerobic count not more than 10^
topical use and fungi per 1 ml

bacteria 1 g or
Preparations for the Not more than 10^
respiratory tract enterobacteria and certain other Gram-negative

(except those bacteria per 1 g or 1 ml
required to be Absence of Pseudomonas aeruginosa in 1 g or 1 ml
sterile) Absence of Stap^hylococcus aureus in 1 g or 1 ml
3 Preparations for oral Total viable aerobic count not more than 10^
administration aerobic bacteria per 1 ml
g or 1
Preparations for Not more than 10^ fungi per g or 1 ml

1
rectal 1 g or 1 ml
Absence of Escherichia coli in
4 administration Total viable aerobic count not more than lO'*
Preparations for raw aerobic bacteria per 1 g or 1 ml
oral Not more than 10^ enterobacteria and certain
administration other
containing Gram-negative bacteria per 1 g or 1 ml
materials of Not more than 10^ fungi per 1 g or 1 ml
natural origin Absence of Salmonella in 10 g orlOml
Absence of Escherichia coli in 1 g or 1 ml
Absence of Staphylococcus aureus in 1 g or 1 ml
aerobic bacteria per Ig or 1ml
^
This text is currently under revision.
1244
5
Methods of Analysis
3.4 Test for bacterial endotoxins

Introduction
The method for the detection of Gram-negative bacterial endotoxins is based
on the gelation of a lysate of amoebocytes (limulus amoebocyte lysate, LAL)
from the horseshoe crab, Limulus polyphemus or Limulus tackypkus. The addition
of a solution containing endotoxins to a solution of the lysate produces tur-
bidity, precipitation, or gelation of the mixture.
The rate of reaction depends on the concentration of endotoxin, the pH,
and the temperature. The reaction requires the presence of certain divalent
cations, an enzyme system, and protein capable of clotting, which are provided
by the lysate.
The bacterial endotoxin test (BET) is carried out in a manner that avoids
microbial contamination.
Before carrying out the test on the preparation to be examined, it is neces-
sary to verify:
— that the equipment used does not adsorb endotoxins;

— the sensitivity of the lysate;
— the absence of interfering factors.
All equipment used must be free of endotoxins.
• Unless otherwise prescribed, the solutions and dilutions used in the test
are prepared using water BET.
Pre-test. Adjust the solution to be examined to pH 6.5-7. using hydrochloric

acid (0.1 mol/1) BET, sodium hydroxide (0.1 mol/1) BET, or a suitable buffer, if
necessary. To each of the requisite number of chosen receptacles (for example,

slides or tubes) add a volume of the lysate and maintain the temperature at
37 ± 1 °C. Then add to each receptacle an equal volume of the solution to be
examined, mixing immediately and gently with the lysate. Incubate the reac-
tion mixture without vibration, avoiding loss of water by evaporation, for a set
period of time that has been determined under experimental conditions (usually
20-60 minutes), and check the results.
A positive result is indicated by the formation of a firm gel and no disinte-
gration when the receptacle is gently inverted. If no such gel is formed the result
is negative.
Validation. A cross-validation will need to be performed if the lysate used is
different to the ones described in the “Introduction”.

of the lysate. Prepare not fewer than four replicate series of two-
Sensitivity
fold dilutions of endotoxin RS to give concentrations of 2X, lA,, 0.5A, and 0.25A,
where A is the stated sensitivity of the lysate used. The final dilution in each
series must at the minimum give a negative result. Examine the dilutions and
a negative control solution consisting of water BET. Calculate the average of
the logarithms of the lowest concentration of endotoxin in each series of dilu-
1245
tions giving a positive result. The antilogarithm of this average gives the esti-
mated lysate sensitivity. If the latter does not differ by more than a factor of 2
from the stated sensitivity, the sensitivity is confirmed and is used for all tests
performed using the same lysate.

Interfering factors. Prepare as described under “Sensitivity of the lysate”, but
use untreated specimens of the preparation to be examined in which no endo-
toxins are detectable to prepare the dilutions of endotoxin RS. Use these spec-
imens at the maximum valid dilution (MVD) calculated from the expression:
endotoxin limit concentration

maximum , ,
valid dilution
, ,
= —— ; ;
sensitivity of the lysate
both values being expressed in International Units (lU) of endotoxin per

millilitre.
When the endotoxin limit concentration is specified in individual mono-

graphs in terms of lU of endotoxin per mg or per lU of product, multiply the
endotoxin limit by the concentration of the product in the solution tested (in
mg or lU of the product per ml of solution) to obtain the endotoxin limit con-
centration in lU of endotoxin per ml of solution tested. Where relevant, the
multiplication applies to a reconstituted solution of the product as stated on
the label.
The preparation to be examined may need to be treated if it contains inter-
fering factors and acts as an inhibitor or an activator as determined under exper-
imental conditions. Suitable treatments are dilution, filtration, neutralization,
dialysis, or addition of substances that displace adsorbed endotoxins. The sen-

sitivity of the lysate in the presence of the preparation to be examined should
not differ by more than a factor of 2 from the sensitivity of the lysate alone.
More sensitive lysates permit a greater dilution of the preparation to be exam-
ined and may contribute to the elimination of interference.
Interfering factors passing through a filter with a nominal separation limit
corresponding to a relative molecular mass of 10000 to 20000 may be sepa-
rated adequately by Asymmetric membrane filters of cellulose
ultrafiltration.
triacetate may be used. The presence of components causing false positive

results must be determined. The material containing the endotoxins that is
retained on the filter is rinsed with water BET or a suitable buffer, and the endo-
toxins are recovered in the water BET or the buffer. The test volume and the
final volume used for the recovery of the endotoxins are determined for each
preparation to be examined.
To establish if interfering factors have been eliminated without removing
endotoxins, repeat the test using the preparation to be examined, add endo-
toxin RS, and submit it to the chosen treatment.
Preparation to he examined. Prepare in duplicate as described under “Pre-test”,
using the maximum valid dilution of the preparation to be examined, this
having been treated if necessary for the elimination of interfering factors. At
the same time examine a negative control consisting of water BET and two
positive controls, both of which contain endotoxin RS at a concentration cor-
responding to twice the stated sensitivity of the lysate and one of which con-
1246
Methods of Analysis
tains the preparation to be examined at the same concentration as in the test

(if necessary treated for the elimination of interfering factors after the addition
of the endotoxin standard).
The test is valid if the negative and both positive controls give the appro-
priate result. The endotoxin limit concentrations of the preparations are given
in the individual monograph. The product conforms if it complies with the
endotoxin limit concentration. However, compliance with this requirement can
only be demonstrated by showing that the endotoxin concentration of the
product less than the endotoxin limit concentration.
is
The preparationto be examined does not comply with the test if a positive
result found for both test mixtures. If a positive result is found for one test
is
mixture and a negative result for the other, repeat the test.
The preparation to be examined complies with the test if a negative result
is found for both test mixtures.
3.5 Test for pyrogens

The pyrogen test is designed to limit the risk of a febrile reaction following par-
enteral administration of drugs. It is intended to be used for liquid products
that can be tolerated by the test rabbit in a dose of 10 ml per kg, injected intra-
venously, generally within a period of not more than 4 minutes. For products
that require preliminary preparation or are subject to special conditions of
administration, additional directions given in the monograph should be
followed.
Test animal
Use healthy, adult rabbits, preferably of the same variety. House the animals
individually in an area of uniform temperature (+2°C), possibly with uniform
humidity, and free from disturbances likely to excite them. The animals are
given ad libitum water and food, commonly used for laboratory animals. One
to 3 days before using an animal that has not previously been used for a
pyrogen test, condition it by conducting a training exercise as described under
the recommended procedure, omitting the injection.
Do not use animals for pyrogen tests more frequently than once every 48
hours. After a pyrogen test in the course of which a rabbit's temperature has
risen by 0.5 °C or more, or after a rabbit has been given a test substance that
was adjudged pyrogenic, at least 2 weeks must be allowed to elapse before the
animal is used again.
Temperature recording
Use an accurate thermometer graduated in 0.1 °C that has been tested to deter-
mine the time necessary to reach the maximum reading, or any other temper-
ature-recording device of equal sensitivity. Insert the temperature-sensing
device into the rectum of the test animal to a depth of about 6cm. If the tem-
perature-sensing device is to remain inserted throughout the sensing period.
1247
restrain the rabbit with a neck stock that allows it to assume a

lightly-fitting
natural resting posture. When a thermometer is used, allow sufficient time for
it to reach a maximum temperature, as previously determined, before taking
the reading.
Perform the test in the area where the animals are housed or under similar envi-
ronmental conditions. For 2 hours before the test and during the test, withhold
all food from the animals being used. Access to water may be allowed. The
animals should be placed under the conditions of the test at least 1 hour before
the injection.
minutes before the injection of the test material, deter-
Prior to the test, 40
mine the temperature of each animal by taking 2 measurements at an interval
of 30 minutes. The mean of the 2 temperatures serves as the “control temper-
ature” of the animal. The control temperature recorded for each rabbit consti-
tutes the temperature from which any subsequent rise following the injection
of the material is calculated.
In any one test, use only those animals the control temperatures of which
do not deviate by more than 1.0 °C from each other. Those animals for which
the 2 temperatures used to determine the control temperature have deviated
by more than ±0.2 °C from the mean should not be used in the test, nor should
any animal with a control temperature below 38.0 °C or above 39.8 °C.
Render the syringes, needles, and glassware free from pyrogens by heating
at 250 °C for not less than 30 minutes or by any other suitable method. Warm
the solution to be tested to approximately 38 °C.
Inject into a marginal vein of the ear of each of 3 rabbits 10ml of the solu-
tion per kg of body weight or the amount specified in the monograph. The
injection should last not longer than 4 minutes, unless otherwise specified in
the monograph.
When the injection has been completed, record the temperature of the
animal during a period of 3 hours, taking the measurements continuously or
every 30 minutes. The maximum temperature recorded for each rabbit is con-
sidered to be its response; if the temperature readings taken after the injection
are all below the control temperature, the response is treated as a zero tem-
perature rise.
If no rabbit shows an individual rise in temperature of 0.6°C or more above

its respective control temperature, and if the sum of the 3 temperature rises
does not exceed 1.4 °C, the tested material meets the requirements for the
absence of pyrogens. If 1 or 2 rabbits show a temperature rise of 0.6°C or more,
or if the sum of the temperature rises exceeds 1.4 °C, continue the test using 5
other rabbits. If not more than 3 of the 8 rabbits show individual rises in tem-
perature of 0.6 °C or more, and if the sum of the 8 temperature rises does not
exceed 3.7 °C, the tested material meets the requirements for the absence of
pyrogens.
1248
Methods of Analysis
3.6 Test for histaunine-like substances

(vasodepressor substances)
The test for vasodepressor substances is carried out in cats by comparing the
depression of arterial pressure caused by the test solution with that obtained
after administration of a solution of histamine.
Use healthy, adult cats, either males or non-pregnant females.
Determine the weight of the animal and place under general anaesthesia by
injection of chloralose R or a suitable barbiturate that allows the maintenance
of uniform blood pressure. Protect the animal from loss of body heat and main-
tain it so that the rectal temperature remains within physiological limits. Intro-
duce a tube into the trachea. Surgically expose the common carotid artery and
by blunt dissection separate it completely from all surrounding structures,
including the vagus nerve. Insert a cannula filled with heparinized saline TS
into the artery and connect it to a mercury manometer or another suitable
device arranged for making a continuous record of blood pressure. Surgically
expose the jugular or the femoral vein and insert into it another cannula filled
with heparinized saline TS through which can be injected solutions of hista-
mine and of the test substance.
Determine the sensitivity of the animal to histamine in the following way;
Start the recording kymograph or a similar recording device and inspect the
tracings for amplitude of excursion and relative stability of blood pressure.
Inject into the jugular or femoral vein histamine TS, in doses of 0.05 pg (dose
A), 0.1 pg (dose B) - repeated at least 3 times - and 0.15pg (dose C) of hista-
mine base per kg of animal weight. Administer the second and subsequent
injections not less than 1 minute after the blood pressure has returned to the
level recorded immediately before the previous injection. Repeat this series of
injections until, disregarding the first series of readings, a relatively uniform
decrease in blood pressure is obtained after doses B of histamine. The animal
should be used for the test only if the decrease after doses B is not less than
2.7 kPa (20 mm
Hg) and, moreover, if dose A causes smaller responses than
doses B whereas dose C gives greater responses than doses B.
Prepare the test solution as described in the monograph. During the course
of the test, take care to maintain a uniform rate of injection for both the test
solution and the standard solution. If the jugular vein is used, care should also
be taken that the injection of test solution and histamine standard are given in
equal volumes to avoid volume effects on blood pressure. When a common
cannula is used for both the standard and test solutions, each injection of the
standard and test solution should be immediately followed by an injection of
approximately 2.0ml of saline TS to flush any residues from the tubing.
Inject a dose B of the standard solution followed by an injection of the spec-
ified amount of the test solution and then another dose B of the standard solu-
tion. The second and third injections are given not less than 1 minute after the
1249
blood pressure has returned to the level recorded immediately before the pre-
ceding injection. If the response to the test solution is greater than that given
previously by dose A, repeat the series of injections twice and conclude the test
by giving dose C of standard solution. If the response to dose C is not greater
than that to dose B, the test is invalid.
The animal may be used in the test as long as it remains reasonably stable
and responsive to histamine and provided that {a) an injection of test substance
did not cause a greater depressor response than that caused by dose C and {b)
the response to dose C of the standard solution given after the administration
of the test substance does not become lower than the mean response to doses
of B previously injected.
The substance passes the test if the response or the mean of the responses
after the injection of the amount specified in the monograph is smaller than the
mean of the corresponding responses to dose B of the standard solution (0.1 pg
of histamine base per kg of animal weight), and no one single dose of the test
solution causes a greater depressor response than dose C of the standard solu-
tion (0.15 pg of histamine base per kg of animal weight).
3.7 Undue toxicity
The test is used to determine the absence of undue toxicity of antibiotics

intended for parenteral administration.
Use healthy mice of a have not previously been used for any
single strain that
test. Select 5 mice, eachweighing between 18 g and 22 g. Prepare the solution
of the test substance as specified in themonograph. Inject a test dose of 0.5ml
intravenously into a tail vein at a uniform rate, the injection occupying 5
seconds. Keep the mice under observation for 48 hours after the injection. The
product meets the requirements for freedom from undue toxicity if no animal
dies within 48 hours.
If 1 or 2 mice die within the observation period, repeat the procedure once,
using respectively 5 or 15 mice, healthy and not previously used for any test,
each weighing between 19.5g and 20.5 g. The product under test meets the
requirements for freedom from undue toxicity if no animal dies in the repeat
test within the observation period (48 hours).
1250
Methods of Analysis
4. Methods for materials of plant origin

4.1 Determination of ash and acid- insoluble ash
Determination of ash
Place about 3 g of the ground material, accurately weighed, or the quantity spec-
ified in the monograph, in a suitable tared dish (for example, of silica or plat-
inum), previously ignited, cooled and weighed. Incinerate the material by

gradually increasing the heat, not exceeding 450 °C, until free from carbon; cool,
and weigh. If a carbon-free ash cannot be obtained in this way, exhaust the
charred mass with hot water, collect the residue on an ashless filter-paper, incin-
erate the residue and filter-paper, add the filtrate, evaporate to dryness, and
ignite at a temperature not exceeding 450 °C. Calculate the content in mg of
ash per g of air-dried material.
Determination of acid-insoluble ash

Boil the ash for 5 minutes with 25ml of hydrochloric acid (~70g/l) TS; collect
the insoluble matter in a sintered crucible, or on an ashless filter-paper, wash
with hot water, and about 500 °C to constant weight. Calculate the
ignite at
content in mg of acid-insoluble ash per g of air-dried material.
4.2 Determination of iodine value

The iodine value of a substance is the weight of halogens expressed as iodine
absorbed by 100 parts by weight of the substance. The quantity of substance
used in the determination should be such that at least 70% of the iodine added,
as provided in the recommended procedure, is not absorbed. Unless otherwise
specified in the monograph, the quantity of the substance indicated in the fol-
lowing table should be used for the determination, depending on the expected
iodine value:
Iodine value Quantity of substance

in g
less than 20 1.0

20-60 0.5-0.25
60-100 0.25-0.15
more than 100 0.15-0.10
Place a quantity of the test substance, accurately weighed, as specified in the
monograph, in a dry 300-ml to 500-ml stoppered flask, add 15ml of carbon
tetrachloride R and dissolve. Add 25 ml of iodine bromide TS, insert the stopper.
1251
previously moistened with potassium iodide (80g/l) TS, shake the flask gently,
and keep in the dark for 30 minutes, unless otherwise specified in the mono-
graph. Add 20ml of potassium iodide (80g/l) TS and 150ml of water, and,
whilst shaking the contents of the flask, titrate with sodium thiosulfate
VS, adding starch TS as indicator towards the end of the titration.
(0.1 mol/1)
Note the number of ml required {a). At the same time carry out the operation
in exacdy the same manner, but without the substance being tested, and note
the number of ml of sodium thiosulfate (0.1 mol/1) VS required {b). Calculate
the iodine value from the following formula:
, ,
Iodine value
,
=
(i--^!)x
;
— r
——
0.01269x100
- ;
weight(ing) of substance
4.3 Determination of peroxides in fixed oils
Dissolve the quantity of test substance as specified in the monograph, usually
about 3g, accurately weighed, in 15 ml of chloroform R and 30 ml of glacial
acetic acid R in a 250-ml glass-stoppered flask. Add 1 ml of a freshly prepared
solution of 1.3g of potassium iodide R in 1 ml of water, stopper the flask, mix
by gentle swirling and set aside in the dark for 3 minutes. Add 100ml of water,
shake, and titrate with sodium thiosulfate (0.01 mol/1) VS, using starch TS as
indicator. Repeat the operation without the substance being tested and calcu-
late the difference between the titrations, the limit value being specified in the
monograph.
4.4 Determination of saponification value

The saponification value is the number of mg of potassium hydroxide required
to neutralize the fatty acids resulting from the complete hydrolysis of 1 g of the
substance.
In the procedure described, a 50-ml burette should preferably be used for
titration, as in the blank titration the volume of hydrochloric acid (0.5 mol/1) VS
used is exactly 35.5 ml when the concentration of ethanolic potassium hydrox-
ide is exactly 40g/l.
Place about 2g of the test substance, accurately weighed, or the quantity speci-
fied in themonograph, in a flask with a capacity of about 200 ml, add 25 ml of
potassium hydroxide/ethanol, TSl attach a reflux condenser, and heat in a
boiling water-bath for 30 minutes, or the time specified in the monograph, fre-
quently rotating the contents of the flask; immediately add 1 ml of phenolph-
thale in/ ethanol TS and titrate the excess of alkali with hydrochloric acid
(0.5 mol/1) VS. Note the number of ml of hydrochloric acid (0.5 mol/1) VS required
to titrate the sample (a). Repeat the operation without the substance being tested
1252
Methods of Analysis
and note the number of ml of hydrochloric acid (0.5 mol/1) VS required for neu-
tralization (b). Calculate the saponification value from the following formula:
^
Saponification value =
-it)
———
X 0.02805 X 1000
r -
weight(mg) of substance
;
4.5 Determination of unsaponifiable matter

The term “unsaponifiable matter” refers to those substances present in oils or
fats that are not saponified by alkali hydroxides and are extractable into ether.
Place a quantity of the test substance, accurately weighed, as specified in the
monograph, in a flask provided with a reflux condenser and boil in a water-
bath for 1 hour with 25 ml of potassium hydroxide/ethanol (0.5 mol/1) VS, with
frequent swirling of contents. Wash the contents of the flask into a separator
by means of 50ml of water and, while the liquid is still slightly warm, extract
by shaking vigorously with 3 successive quantities, each of 50ml, of ether R,
washing out the flask with the first quantity of ether R. (CAUTION: Ether
should be free of peroxides.) Take care to release frequently and carefully the
pressure that may build up inside the separator. Combine the ethereal solutions
in another separator containing 20 ml of water. (If the ethereal solutions contain
solid suspended matter, filter them into the separator through a fat-free filter-
paper and wash the filter-paper with ether R.) Gently rotate the separator for
a few minutes without violent shaking, allow and run
the liquids to separate,
off the aqueous layer. Wash the ethereal solution by shaking vigorously with
2 successive quantities, each of 20ml, of water; then treat 3 times with 20ml
of potassium hydroxide (0.5 mol/1) VS (NOTE: aqueous reagent), shaking vig-
orously on each occasion and washing with 20ml of water after each treat-
ment. Finally wash with
successive quantities, each of 20 ml, of water until the
aqueous layer no longer alkaline to phenolphthalein/ethanol TS. Transfer the
is
ethereal extract to a weighed flask, washing out the separator with ether R;
distil off the ether with the necessary precautions and add 3 ml of acetone R.
By the aid of a gentle current of air remove the solvent completely from the
flask, which is preferably held obliquely and rotated, almost entirely immersed,
in a water-bath at about 60 °C. Dry to constant weight at a temperature not

above 80 °C and dissolve the contents of the flask in 10 ml of ethanol (-750 g/1)
TS, previously neutralized to phenolphthalein/ethanol TS. Titrate with car-
bonate-free sodium hydroxide (0.1 mol/1) VS, using phenolphthalein/ethanol TS
as indicator. If the amount of carbonate-free sodium hydroxide (0.1 mol/1)
required does not exceed 0.2 ml, the amount weighed is to be taken as the
unsaponifiable matter. Calculate the unsaponifiable matter as a percentage of
the oil or fat. If the amount of carbonate-free sodium hydroxide (0.1 mol/1) VS
required exceeds 0.2 ml, the amount weighed cannot be taken as the unsaponifi-
able matter and the test must be repeated.
1253
4.6 Determination of acid value

The acid value is the number of mg of potassium hydroxide required to neu-
tralize the free acid in 1 g of the substance.
Accurately weigh about lOg of the substance, or the quantity specified in the
monograph, into a 250-ml flask, and add 50 ml of a mixture of equal volumes
of ethanol (-750 g/1) TS and ether R, which has been neutralized with potas-
sium hydroxide (0.1 mol/1) VS after the addition of 1ml of phenolphthalein/
ethanol TS. Heat, if necessary, until the substance has completely dissolved,
cool; titrate with potassium hydroxide (0.1 mol/1) VS, constantly shaking the
contents of the flask until a pink colour, which persists for 15 seconds, is
obtained. Note the number of ml required {a). Calculate the acid value from
the following formula:
a X 0.00561 X 1000
Acid value =
weight(ing) of substance
4.7 Determination of hydroxyl value

The hydroxyl value of a substance is the amount, in milligrams, of potassium
hydroxide required to neutralize any acid when combined by acylation in 1 g
of the substance under examination.
A
Method
To the quantity of the substance being examined (as specified in the individual
monograph) add 12g of stearic anhydride R and 10 ml of xylene R and heat
gently under a reflux condenser for 30 minutes. Allow to cool, add a mixture
of 40 ml of pyridine R and 4 ml of water, and heat again under a reflux con-
denser for 30 minutes. Titrate the hot solution with carbonate-free sodium
hydroxide (1 mol/1) VS, using phenolphthalein/ethanol TS as indicator. Repeat
the procedure, omitting the substance under examination.
The hydroxyl value is calculated from the expression 56.10 v/m, where v is
the difference, in ml, between the two titrations and ni is the quantity, in g, of
the substance taken.
Method B
Unless otherwise indicated in the individual monograph, weigh accurately the
quantity of the substance to be examined shown in the table under 4.7 Deter-
mination of hydroxyl value, place it in a 150-ml acetylation flask fitted with an
air condenser and add the corresponding volume of pyridine/acetic anhydride
TS.
1254
Methods of Analysis
Presumed hydroxyl value Quantity of substance Volume of

(g) pyridine/acetic
anhydride TS
(ml)
10-100 2.0 5,0

100-150 1.5 5.0
150-200 1.0 5.0
200-250 0.75 5.0
250-300 0.60 or 1.20 5.0 or 10.0
300-350 1.0 10.0
350-700 0.75 15.0
700-950 0.5 15,0
Heat the flask for 1 hour in a water-bath, maintaining the water

level of the
2-3 cm above the Remove the flask and con-
level of the liquid in the flask.
denser, allow to cool, and add 5 ml of water through the top of the condenser.
If a cloudiness appears, add sufficient pyridine R to produce a clear liquid,
noting the volume added. Shake the flask, place it in a water-bath for 10
minutes, remove, and allow to cool. Rinse the condenser and the walls of the
flask with 5ml of neutralized ethanol TS. Titrate with potassium hydroxide/
ethanol (0.5 mol/1) VS, using 0.2ml of phenolphthalein/ethanol TS as indicator.
Repeat the procedure, omitting the substance under examination.
Calculate the hydroxyl value from the expression (a + 28.05) v/m, where v
is the difference, in ml, between the two titrations, a is the acid value deter-
mined for the substance, and m is the quantity, in g, of the substance taken.
5. Pharmaceutical technical procedures

Introduction
In addition to general methods of analysis that are invoked within the general
monographs for different types of dosage forms, for example, dissolution
testing, this section contains other general texts of relevance during the devel-
opment and manufacture of pharmaceutical dosage forms and to which refer-
ence is made within the monographs of the International Pharmacop’oeia.
5.1 Uniformity of content for single-dose preparations
This test should be applied only where the declared quantity of active ingre-
dient in tablets, capsules, or suppositories is 5% or less of the total formulation
or, in the case of sugar-coated and enteric-coated tablets, where the test for 5.2
Uniformity of mass for single-dose preparations does not apply, or for certain
powders for injections when specified in individual monographs.
1255
Individually, determine the amount of active ingredient in each of 1 0 units using
the analytical method specified in the individual monograph for 5.1 Uniformity
of content for single-dose preparations or, where no specific method is stated,
refer to the procedure for the “Assay” in the appropriate monograph.
Requirements for tablets and powders for injections

Each within -1-15% of the average amount of the
single unit should contain
active ingredient. one individual unit deviates by more than ±15%
However, if
but is within ±25% amount of the active ingredient, examine a

of the average
further 20 units drawn from the same original sample as the first 10 units. The
preparation under test complies only if the amount of active ingredient found
in no more than one out of 30 units deviates by more than ±15% of the average
amount. None should deviate by more than ±25% of the average amount.
Requirements for capsules, oral powders and suppositories

Each single unit should contain within ±15% of the average amount of active
ingredient. However, if up to three individual units deviate by more than ± 15%
but are within ± 25% of the average amount of the active ingredient, examine
a further 20 units drawn from the same original sample as the first 10 units. The
preparation under test complies only if the amount of active ingredient found in
no more than three out of 30 units deviates by more than ± 15% of the average
amount. None should deviate by more than ± 25% of the average amount.
5.2 Uniformity of mass for single-dose preparations

Tablets
Uncoated tablets and film-coated tablets formulated to contain 5% or more of
the active ingredient should comply with the following test.
Weigh 20 tablets and calculate the average mass. When weighed singly, the
deviation of individual masses from the average mass should not exceed the
limits given below.
Average mass of tablet Deviation Number of tablets

%
less than 80 mg ±10.0 minimum 18
±20.0 maximum 2
80 mg to 250 mg ±7.5 minimum 18
±15.0 maximum 2
more than 250 mg ±5.0 minimum 18
±10.0 maximum 2
1256
Methods of Analysis
If film-coated tablets fail this test it may be because of variability in the thick-
ness (mass) of the coatings. In such a case, a test for 5.1 Uniformity of content
for single-dose preparations should be applied; if the tablets meet the require-
ment of this test, they can be considered acceptable.
Capsules
Weigh 20 and calculate the average mass. The mass
intact capsules individually,
of each capsule should be within ±10% of the average mass. If all the capsules
do not within these limits, weigh the 20 capsules again, taking care to pre-
fall
serve the identity of each capsule, and remove the contents as completely as
possible. For soft gelatin capsules, wash the shell with ether or some other suit-
able solvent and allow it to stand until the odour of the solvent is no longer
perceptible. Other means, such as a jet of compressed air, may be used to
remove the contents.
Weigh the emptied shells individually and calculate for each capsule the net
mass of its contents by subtracting the mass of the shell from the gross mass.
Determine the average net content from the sum of the individual net masses.
Then determine the difference between each individual net content and the
average net content. Deviation of individual net mass from the average net
mass should not exceed the limits given below.
Net mass of capsule contents Deviation Number of capsules

%
less than 300 mg ±10.0 minimum 18
+20.0 maximum 2
300 mg and over ±7.5 minimum 18
±15.0 maximum 2
Oral fôwders
Weigh 20 units and calculate the average mass. The deviation of individual
mass from the average mass should not exceed the limits given above under
“Capsules.”
Powders for injections
The test applies to powders for injections where the content is more than
40 mg. The recommended procedure is the same as described for capsules. The
deviation of individual net mass from the average net mass should not exceed
the limits given below.
1257
Deviation Number of containers

%
±10 minimum 18
+20 maximum 2
For preparations with a content of less than 40mg the test for 5.1 Uniformity
of content for single-dose preparations applies instead.
Supprositories
Weigh 20 suppositories and calculate the average mass. When the supposito-
ries are weighed singly, the deviation of individual mass from the average mass
should not exceed the limits given below.
Deviation Number of suppositories

%
±5 minimum 18
+10 maximum 2
5.3 Disintegration test for tablets and capsules

This test determines whether tablets and capsules disintegrate within a pre-
scribed time when placed in an immersion fluid under prescribed experimen-
tal conditions. Disintegration is defined as the state in which no residue of the
tablet or capsule, except fragments of undissolved coating or capsule shell,
remains on the screen of the test apparatus or, if any other residue remains, it
consists of a soft mass having no palpably firm, unmoistened core.
Disintegration apparatus
The apparatus consists of a circular basket-rack assembly, a suitable vessel for
the immersion fluid (such as a 1 -litre beaker), a thermostatic arrangement for
maintaining the fluid at the required temperature (normally 37 + 2°C), and a
device for raising and lowering the basket-rack in the immersion fluid at a con-
stant frequency of 28-32 cycles/min through a distance of 50-60 mm.
The basket-rack assembly consists of six open-ended cylindrical glass tubes
and a rack for holding them in a vertical position. The tubes are 75-80 long,mm
and have an inside diameter of about 21.5 mm
and a wall about 2 thick.mm
The tubes are held vertically by two superimposed plates, circular in shape and
made of transparent plastic material, each about 90 mm
in diameter and 6 mm
thick, perforated by six holes of a diameter that allows the tubes to be inserted.
The holes are equidistant from the centre of the plate and equally spaced one
from another. A piece of woven gauze, made of stainless steel wire about
0.635mm in diameter, with a mesh aperture of 2.0 mm
is attached to the under-
1258
Methods of Analysis
side of the lower plate. The upper plastic plate is covered with a stainless steel
plate, about 1 mm thick, of a diameter similar to that of the plastic plates. The
steel plate is perforated by six holes about 22mm in diameter, positioned to
coincide with those of the upper plastic plate. It is placed over the tubes and
consolidates the whole structure. The plates are held rigidly 75-80 mm apart
by vertical stainless steel rods at the periphery. A metal rod is fixed to the centre
of the upper plate. This enables the assembly to be attached to a suitable
mechanical device so that it may be lowered and raised.
The volume of the fluid in the immersion vessel should be such that, at the
highest point of the upward stroke, the wire mesh that forms the bottom of
mm below the surface of the fluid. At the lowest
the basket remains at least 25
point of the downward stroke, should descend to not less than 25 mm from
it
the bottom of the vessel. The time required for the upward stroke should be
equal to the time required for the downward stroke, and the change in stroke
direction should be a smooth transition rather than an abrupt reversal of
motion.
Where a disc is prescribed in the monograph, the following configuration
and dimensions apply: a cylindrical disc 20.7 + 0.15mm in diameter and 9.5 +
0.15 mm thick, made of transparent plastic with a relative density of 1.18 to
1.20. Each disc is pierced by five holes 2 mm in diameter, one in the centre and
the other four spaced equally on a circle of radius 6mm from the centre of the
disc. On the lateral surface of the disc, four equally spaced grooves are cut so
that on the upper surface of the disc they are 9.5mm wide and 2.55mm deep
and, at the lower surface, 1.6mm square.
Different designs of basket-rack assembly may be used, provided that
the specifications for the glass tubes and the stainless steel wire gauze are
maintained.
Recommended procedure (except for effervescent tablets)

Unless otherwise specified in the individual monograph, use water as the
immersion fluid at a temperature of 37 ± 2°C. Place one tablet or capsule in
each of the six tubes and, if prescribed, add a disc to each tube. Operate the
apparatus for the specified period of time, withdraw the assembly, and examine
the state of the tablets or capsules. All six tablets or capsules should disinte-
grate to pass the test.
5.4 Disintegration test for suppositories

The disintegration test determines whether suppositories soften or disintegrate
within a prescribed time when placed in an immersion fluid using the experi-
mental conditions described below.
Disintegration is considered to be achieved when:
— dissolution is complete;
— the components of the suppositories have separated, e.g. melted fatty
substances have collected on the surface of the liquid, insoluble powders
1259
have fallen to the bottom, and soluble components have dissolved or are
distributed in one or more of the ways described in Methods 1 and 2;
— there is softening of the test sample, usually accompanied
by an appre-
change of shape without complete separation of the components.
ciable
The softening process is such that a solid core no longer exists when
pressure is applied with a glass rod; or
— rupture of the gelatin shell or rectal capsule occurs resulting in release of
the contents.
Method i (for water-soluble, hydrodispersible and fat-based suppositories):

This test measures the time elapsed for a suppository placed in water to
disintegrate.
Apparatus
The apparatus (Fig. 1) consists of a 60-mm long cylinder of glass or transpar-
ent plastic and a metal device consisting of two perforated stainless steel discs,
held about 30 mm apart. each have 39 holes, 4mm in diameter,
These discs
which are evenly spaced in a concentric pattern. The diameter of the discs is
marginally inferior to that of the interior of the cylinder. Once inserted into the
cylinder, the metal device is attached to the rim of the cylinder by means of
three spring clips. The test is carried out using three such apparatuses, each
containing a single test sample. Each apparatus is placed in a beaker with a
minimum litres filled with water unless otherwise prescribed. The

capacity of 4
beaker is with a slow stirrer and a support that holds the apparatus ver-
fitted
tically 90 mm
below the surface of the water so that it can be inverted without
emerging from the water.
Unless otherwise described in the individual monograph, use water maintained
at a temperature of 36-37°C as the immersion fluid. The test requires three
suppositories and the procedure is applied to each of the suppositories.
Place the sample on the lower and then insert it
disc of the metal device
into the cylinder. Place the apparatus into the beaker and invert it every 10
minutes without removing it from the liquid. Repeat the operation with the
remaining two suppositories. Record the time required for the disintegration of
the suppositories.
Unless otherwise stated in the individual monograph, for each of the three
suppositories, examine the state of the sample after 30 minutes for fat-based
suppositories and rectal capsules, and after 60 minutes for water-soluble
suppositories.
Method Z (alternative for fat-based suppositories):
This test measures the time elapsed for a suppository placed in water to soften
to the extent that it no longer offers resistance when a defined weight is applied.
1260
Methods of Analysis
Spring clip
Figure 1. Apparatus for water-soluble, hydrodispersible, and fat-based suppositories

A. Horizontal view
B. Vertical view
Measurements in mm.
British Pharmacopoeia, 1999, Crown copyright material. Reproduced with the
permission of the Controller of Her Majesty's Stationery Office.
1261
Apparatus
The apparatus (Fig. 2) consists of a flat-bottomed glass tube about 140 mm long
with an internal diameter of 15.5mm and a two-part rod. The tube is closed
with a removable plastic cover that has an opening 5.2 mm in diameter. The
rod has two parts; both made of plastic, or the lower part made of plastic and
the upper of metal. The rod is 5mm in diameter and widens at the lower end
to a diameter of 12 mm. To the bottom of the lower end is fixed a metal needle
2 mm long and 1 mm in diameter. The upper part of the rod has an adjustable
sliding ring and a weighted disc is attached to the top. The two parts are held
tightly together for the manual version or separated for the automatic version.
The weight of the entire rod should be 30g ± 0.1 g.
Unless otherwise described in the individual monograph, use water maintained
at a temperature of 36-37°C as the immersion fluid. The test requires three
suppositories and the procedure is applied to each of the suppositories.
Place the glass tube containing 10ml of water and equili-
in the water-bath
brate at 36.5 + 0.5 °C. Fix the glass tubes vertically and immerse to a depth of
at least 7 cm below the surface but without touching the bottom of the water-
bath. Introduce a suppository, tip first, into the tube followed by the rod with
the free gliding plastic cover into the glass tube until the metal needle touches
the end of the suppository. Put the cover on the tube. Note the time which
flat
elapses until the rod sinks down to the bottom of the glass tube and the mark
ring reaches the upper level of the plastic cover.
Each of the three suppositories should melt within 30 minutes, unless oth-
erwise stated in the individual monograph.
5.5 Dissolution test for solid oral dosage forms

This test determines the amount of active ingredient(s) released from a solid
oral dosage form, such as a tablet or a capsule, using a known volume of dis-
solution medium within a predetermined length of time. This test method may
not be applicable to certain oral dosage forms.
Apparatus
All parts of the apparatus, including any metal that may come into contact with
the sample to be tested or the dissolution medium, should be made from a
chemically inert material and should not adsorb, react or interfere with the
preparation or the dissolution medium.
The dissolution assembly should be constructed in such a way that any
vibration is reduced to a minimum.
Use an apparatus that allows full visibility of all operations.
The apparatus “Paddle” (Fig. 3) consists of a cylindrical vessel of suitable
glass or other suitable transparent material with a hemispherical bottom and a
nominal capacity of 1000ml. The vessel is covered to prevent evaporation of
1262
Methods of Analysis
Figure 2. Alternative apparatus for fat-based suppositories

Measurements in mm.
the medium with a cover that has a central hole to accommodate the shaft of
the stirrer and other holes for the thermometer and for devices for withdrawal
of liquid. The with a blade at the lower end.
stirrer consists of a vertical shaft
The blade is constructed around the shaft so that it is flush with the bottom of
the shaft. When placed inside the vessel, the shaft’s axis is within 2 mm
of the
axis of the vessel and the bottom of the blade is 25 ± 2 mm
from the inner
bottom of the vessel. The upper part of the shaft is connected to a motor pro-
1263
Figure 3. Paddle
Measurements in mm.
British Pharniacop'oeia, 1999, Crown copyright material. Reproduced with the
permission of the Controller of Her Majesty’s Stationery Office.
vided with a speed regulator so that smooth rotation of the stirrer can be main-
tained without any significant wobble. The apparatus is placed in a water-bath
that maintains the dissolution medium in the vessel at 37 ± 0.5 °C.
The apparatus “Basket" (Fig. 4) consists of the same apparatus as described
for “Paddle”, except that the paddle stirrer is replaced by a basket stirrer. The
basket consists of two parts. The top part, with a vent, is attached to the shaft.
It is fitted with three spring clips, or other suitable attachments, that allow
1264
Methods of Analysis
WHO 0073
Figure 4. Basket
Measurements in mm.
British Pharmacoproda, 1999, Crown copyright material. Reproduced with the
permission of the Controller of Her Majesty’s Stationery Office.
removal of the lower part so that the preparation being examined can be placed
in the basket. These three spring clips firmly hold the lower part of the basket
concentric with the axis of the vessel during rotation. The lower detachable
part of the basket is made of welded-seam cloth, with a wire thickness of
0.254mm diameter and with 0.381 mm
square openings, formed into a cylinder
with a narrow rim of sheet metal around the top and the bottom. If the basket
is to be used with acidic media, it may be plated with a 2.5-pm layer of gold.
When placed inside the vessel, the distance between the inner bottom of the
vessel and the basket is 25 + 2 mm.
1265
Test conditions
The following specifications are given in the individual monographs:
— the apparatus to be used;

— the composition and volume of the dissolution medium;
— the rotation speed of the paddle or basket;
— the preparation of the sample and reference solutions;
— the time, the method, and the amount for sampling of the test solution
or the conditions for continuous monitoring;
— the method of analysis; and
— the limits of the quantity or quantities of active ingredient(s) required to
dissolve within a prescribed time.
Dissolution media
If a buffer is added to the dissolution medium, adjust its pH to within +0.05
units of the prescribed value. Prior to testing, if necessary, remove any dissolved
gases that could cause the formation of bubbles.
• Dissolution buffer pH 1.3, TS

Dissolve 2g of sodium chloride R in 800ml of deionized water, adjust the pH
to 1.3 with hydrochloric acid (~70g/l) TS, and dilute to 1000ml with water.

Dissolve 2g of sodium chloride R in 800ml of deionized water, adjust the pH
to 2.5 with hydrochloric acid (~70g/l) TS, and dilute to 1000ml with water.
Dissolve 7.507 g of glycine R and 5.844 g of sodium chloride R in 800 ml of
deionized water, adjust the pH to 3.5 with hydrochloric acid (~70g/l) TS,
and dilute to 1000 ml with water.

Dissolve 6.8 g of potassium dihydrogen phosphate R in 900 ml of deionized
water, adjust the pH to 4.5 either with hydrochloric acid (~70g/l) TS or
sodium hydroxide (~80g/l) TS, and dilute to 1000 ml with water.

Dissolve 5.53 g of disodium hydrogen phosphate R in 800ml of deionized
water, adjust the pH to 6.8 with sodium hydroxide (~80g/l) TS, and dilute

Dissolve 9.075g of potassium dihydrogen phosphate R in deionized water to
produce 1000ml (solution A). Dissolve 11.87g of disodium hydrogen phos-
phate R in sufficient water to produce 1000ml (solution B). Mix 300ml of
solution A with 700ml of solution B.
1266
Methods of Analysis
• Gastric fluid, simulated, TS

Dissolve 2.0 g of sodium chloride Rand 3.2 g of pepsin R in 7.0 ml of hydrochlo-
ric acid (-420 g/1) TS and sufficient water to produce 1000 ml. This test solu-
tion has a pH of about 1.2.
• Intestinal fluid, simulated, TS

Dissolve 6.8 g of potassium dihydrogen phosphate R in 250ml of water, mix,
and add 190 ml of sodium hydroxide (0.2 mol/1) VS and 400 ml of water. Add
10.0 g of pancreatin R, mix, and adjust the resulting solution with sodium
hydroxide (0.2 mol/1) VS to a pH of 7.5 ± 0.1. Dilute with sufficient water
to produce 1000ml.
Acceptance criteria
The requirements are met if the quantities of active ingredient(s) dissolved from
the dosage forms tested conform to the following table, unless otherwise spec-
ified in the individual monograph.
Stage Samples tested Acceptance criteria
s, 6 Each unit is not less than Q + 5%

S2 6 Average of 12 units (S^ + S 2 ) is equal to or greater than
Q, and no unit is less than Q-15%
S3 12 Average of 24 units (S^ + $2 + S 3) is equal to or greater
than Q; not more than 2 units are less than Q-15%;
no unit is less than Q-25%
Continue testing through the three stages unless the results conform at either
S] or S 2 . The quantity, Q, is the released labelled content of active ingredient
as a percentage as specified in the individual monograph; both the 5% and 15%
values in the acceptance table are percentages of the labelled content so that
these values and Q are in the same terms.
(See also guidance under Details of the procedure below.)
Ensure that the equipment has been calibrated within the past 6-12 months.
Place the volume of dissolution medium, as stipulated in the individual mono-
graph, in the vessel; assemble the apparatus and place it in the water-bath;
allow the temperature of the dissolution medium to reach 37 + 0.5 °C and

remove the thermometer.
When apparatus “Paddle” is used, allow either one tablet or one capsule of
the preparation to be tested to sink to the bottom of the vessel before starting
the rotation of the blade, taking care that no air bubbles are present on the surface
of the dosage form. In order to stop the dosage form from floating, anchor it to
the bottom of the vessel using a suitable device such as a wire or glass helix.
1267
When apparatus “Basket” is used, place either one tablet or one capsule of
the preparation to be tested in a dry basket at the beginning of each test. Lower
the basket into position before rotation.
Immediately start rotation of the blade or basket at the rate specified in the
individual monograph.
Withdraw a sample from a zone midway between the surface of the dis-
solution medium and the top of the rotating blade or basket, not less than
10mm below the surface’ and at least 10mm from the vessel wall, at the time
or time intervals specified (see under Details of the procedure, below).
Either replace the volume of dissolution medium with a volume equal to
that of the liquid removed, or compensate for the loss of liquid by calculation,
except where continuous measurement is used.
For filtration of the removed liquid, use an inert filter with a suitable pore size.
Use a filter that does not cause significant adsorption of the active ingredient from
the solution, and does not contain substances extractable by the dissolution
medium that would interfere with the specified method of analysis. Use cen-
trifugation as an alternative with conditions depending on the sample being tested.
Unless otherwise indicated, proceed in parallel with five additional tablets
or capsules.
Determine the quantity of active ingredient dissolved in the specified time
limit indicated in the individual monograph. The result should be expressed as
a percentage of the content stated on the label.
Details of the procedure are given below for those not familiar with the
method.
Details of the procedure

The following text is provided to give information and guidance, and is not regarded as
an analytical reeûirenient.
1. Verification and validation of equip>ment (six sets)
— Check the straightness of the shaft visually and with a ruler.

— Examine the paddle or basket for cracks in coating, applicable. if
— Check the paddle or basket for specified dimensions, particularly for any
deviation in the evenness of the blade and its distance from the axis.
— Mount the paddle or basket and check central position. its
— Check the apparatus to ensure the desired rotation speed can be main-
tained within the +4% throughout the
limits test.
— Check the level of vibration of the whole apparatus, preferably measured
with a vibration meter, and eliminate any sources of vibration to keep
the readings of displacement below 0.3 mm.
'
Recommended, 4.5 cm.
1268
Methods of Analysis
— Inspect the paddle or basket, and any portion of the apparatus which
will be in contact with the test solutions, for cleanliness; in particular
check the paddle/shaft joint for crevices or streaks.
— Inspect the vessels for cleanliness and for any abnormalities in dimen-
sion or shape, especially of the hemispherical bottom and internal radius.
Place in the dissolution test bath.
— Insert the paddle or basket into the unit and adjust it to the specified dis-
tance from the bottom of the vessel (25 + 2 mm).
— Adjust each vessel with a centring gauge. Mark the vessels to permit their
easy replacement without losing the correct centred position.
— Calibrate the system with suitable calibrators,’ according to a scheduled
periodic system.
2. Preparation of the dissolution medium

— Select the dissolution medium section (see 6).
— Check the pH of the dissolution medium to two decimal places, using a
suitably calibrated pH-meter.
— Dissolved gases can cause bubbles to form which may change the results
of the test. In such cases, dissolved gases should be removed prior to
testing using a suitable method (e.g. filtration under vacuum or in an
ultrasonic water-bath).
— Preheat the medium to 37 °C or slightly above, measuring the tempera-
ture with precalibrated precision thermometers.
3. Getting ready for the test
3.1 Apparatus
— Adjust the speed to that prescribed in the monograph and ensure that
it can be maintained within the limits +4%.
— Ensure there is no significant wobble on any rotating shaft.

— Recheck the centring of the vessels with a gauge and correct the if tilt
adjustments were altered.

— Set frame
vertical using markers or
limits, in order to insert the
collars,
sample to be tested without causing changes between each test.
— Prepare the samples ready for dropping into the
test vessels.
3.2 Water-bath or other suitable heating system

— Transfer the dissolution medium, accurately measured to +2%, to the
vessel,check the temperature, and check the volumetric procedure used
for subsequent determination as well as any weighing equipment
employed.
^
For example, calibrator tablets such as those available from United States Pharmacopeial Con-
vention Inc.
1269
— Use a transparent water-bath that allows for visual monitoring of the

process of disintegration/desegregation, and the possible presence of air.
— Adjust the bath temperature to maintain a temperature of 37 + 0.5 °C

in the vessels.
— Ensure that the in the bath
fluid above the top is of the medium level
in the vessels.
— Ensure that the position of the water-bath horizontal. is
3.3 Sampling procedures

• Documentation
— Determine the intervals of sampling as given in the sampling procedure,
and decide if a staggered or a simultaneous start for the six tests is
convenient.
— Determine whether the time interval allowed between sampling is suf-
(especially for manual sampling).
ficient
— Record information concerning:
all
(a) the reference material used and how it is prepared;

(b) the use of preevaluated correcting factors in the calculations for any
interference from excipients, if known;
(c) the preparation of samples for analysis: this must be checked to
ensure that it does not interfere with the analysis, e.g. solvent/pH
effects;
(d) the pH of the solution to be tested and whether it is the same as
that of the test medium used;
(e) the diluent for the assay if it is not the same as the dissolution
medium; and
(f) the possible effects, if known, from other solvents.
• Inspection
— In manual sampling, check that the syringe or other sampling devices
are clean.
— Observe that no interference occurs with the sampling probes.
— Ensure that the sample withdrawn from a zone midway between the
is
surface of the dissolution medium and the top of the rotating blade or
basket, not less than 10 mm from the vessel wall, and that each subse-
quent sample is taken in the same way.
3.4 Selecting and checking analytical procedures

— Use the method specified in the corresponding monograph.
— The correct should be used to avoid adsorption or interference,
filters
unless separation performed by centrifugation.

is
— Suitable tubing should be used in automated equipment to avoid

adsorption.
— a spectrophotometric method used, preferable to operate in the
If is it is
linear range of absorption (extinction) values at the specified wavelength.
1270
Methods of Analysis
4. Carrying out the test
— Examine the system to ensure that no bubbles are present. air

— For simultaneous sampling, add the sample to each of thetest vessels:
immediately the paddle/basket and stopwatch.
start
— For staggered sampling (paddle method), may be necessary to add the
it
test sample when the paddles are rotating. Drop the test sample as close
as possible to the centre of the vessel and start the stopwatch immedi-
ately. In order to stop the test sample from floating, anchor it to the
bottom of the vessel using a suitable device such as a wire or glass helix.
— Check the temperature of the water-bath and that of the dissolution
medium at the beginning and at the end of the test and note on the check
list (see also section 5).
— Proceed with the sampling.
5. At the end of the test
— Check the temperature of themedium in the vessels and record manu-

ally or automatically any deviations from the tolerance of ±0.5 °C.
— Record the speed of rotation.
— Ensure that all data are recorded or printed before discarding the samples
(possible problems may only be solved by duplication).
— Note any unusual appearance, such as a silvery hue which indicates the
release of dissolved gas.
— Note the condition of any undissolved part of the sample, such as test
position, form,
its (This valuable information for problem
etc. is solving.)
— Check the volume of the contents of one or two vessels to ascertain if
evaporation took place. This could affect the analytical results.
6. Suitable dissolution media

The preparation of suitable dissolution media is described above.
7 . Checklist
The following checklist is offered as an example for the purpose of monitoring the per-
formance of the system. It does not constitute a part of the recjuirements.
1271
Proposed checklist for performance of the dissolution test

(one sheet per test)
Name of product
International Nonproprietary Name (INN)
Proprietary Name
Date
Verified Further details

Spectrophotometer (values and
comments)
Cleanliness of cells
Description
Apparatus
Device(s)
- single/multi-spindle (3/6 vessels)
Cleanliness of paddle or basket
Cleanliness of vessel(s)
Dissolution medium (name and composition)
- pH of buffer
- de-aerated water
Filling of vessel and its position
Level of the dissolution medium

Position of paddle or basket (2.5 cm from
the bottom of the vessel)
Insertion of tablets
- time interval
- position
Initial speed
- after 15 minutes
- after 30 minutes
Initial temperature of dissolution medium
- after 15 minutes
- after 30 minutes
Sampling
Cleanliness of withdrawal device (e.g. needle)
Initial position of device 4.5cm from

(e.g, the
surface of the dissolution medium)
Position of device throughout the test
1272
Methods of Analysis
Results
Name of product
Date
Percentage of active ingredient dissolved/time
Test sample Time 1 Time 2 Time 3 Time 4 Time 5 Time 6

1
Mean value
Standard deviation
Relative standard
deviation (%)
5.6 Test for extractable volume for

parenteral preparations
The determination of extractable volume applies to the following preparations
for parenteral use: injections and parenteral infusions.
Injections
The volume of the injection in a single-dose container is usually sufficient to

permit withdrawal of the nominal dose. The single-dose container does not
hold a quantity relative to the declared volume that would present a risk should
the whole contents be administered.
Compliance with the requirement for extractable volume is assured by
making the filling volume greater than the nominal volume to a degree deter-
mined by the characteristics of the product.
Suspensions and emulsions must be shaken prior to withdrawal of the con-
tents and before proceeding with the determination of the density.
Oily or viscous preparations may need to be heated and thoroughly shaken
immediately before withdrawal of the contents.
Test for containers with a nominal volume of less than 5 ml

Take five containers. Choose a syringe with a capacity not greater than twice
the volume to be measured, fitted with a suitable needle.
1273
Withdraw as much of the contents as possible from one of the five con-
tainers to be used in the test, expel any bubbles and transfer this quantity,
without emptying the needle, into a dry tared container. Weigh the whole and
determine the mass of the contents. Repeat the procedure with the four
remaining containers.
Determine the density of the preparation at the temperature at which the
test is carried out. From the mass of the contents of each container, calculate
the corresponding volume by dividing by the density.
The preparation complies with the test for extractable volume if the volume
measured for each of the five containers is not less than its nominal value.
Test for containers with a nominal volume of 5 ml or more

Take five containers. Choose a syringe with a capacity not greater than twice
the volume to be measured, fitted with a suitable needle.
Withdraw as much of the contents as possible from one of the five con-
tainers to be used in the test, expel any bubbles and transfer this quantity,
without emptying the needle, into a dry measuring cylinder with a capacity
such that the volume to be measured is not less than 40% of the nominal
volume of the cylinder. Measure the volume transferred. Repeat the procedure
with the four remaining containers.
The preparation complies with the test for extractable volume if the volume
measured for each of the five containers is not less than its nominal value.
Parenteral infusions
Take one container. Transfer its contents into a dry measuring cylinder with a
capacity such that the volume to be measured is not less than 40% of the
nominal volume of the cylinder. Measure the volume transferred.
The volume measured is not less than the nominal value stated on the
container.
5.7 \^sual inspection of particulate matter in injectable

preparations
Particulate contamination of injections and parenteral infusions consists of
extraneous, mobile, undissolved particles unintentionally present in the solu-
tions. Disregard any gas bubbles.
The types of preparation for which compliance with this test is required are
stated in the individual monograph.
This test provides a simple method for the detection of visible particles. It
isperformed in accordance with the provisions of Good Manufacturing Prac-

tices.The test is not intended for use by a manufacturer for batch release pur-
poses. To ensure that a product will meet pharmacopoeial specifications with
respect to visible particulate matter, if and when tested, manufacturers should
1274
Methods of Analysis
Figure 5. Apparatus for visible particles

carry out a 100% inspection and rejection of unsatisfactory items prior to

release or use other appropriate means.
Subvisible particles and the nature of the particles are not identified by this
method.
Apparatus'
The apparatus (Fig. 5) consists of a viewing station comprising:
— a matt black panel of appropriate size held in a vertical position;

— a non-glare white panel of appropriate size held in a vertical position
next to the black panel;
— an adjustable lampholder fitted with a shaded, white-light source and
with a light diffuser (a viewing illuminator containing two 13-W fluo-
rescent tubes, each 525mm in length is suitable). The intensity of
illumination at the viewing point is maintained between 2000 lux and
3750 lux for clear glass ampoules. Fligher values are preferable for
coloured glass and plastic containers.
^
This method was developed by WHO in collaboration with Group 12 of the European Pharma-
copoeia Commission.
1275
Gently swirl or invert each individual container, making sure that no air bubbles
are introduced, and observe for about 5 seconds in front of the white panel.
Repeat the procedure in front of the black panel.
Record the presence of any particles. Repeat the procedure for a further 19
containers.
The preparation fails the test if one or more particles are found in more than
one container.
When the test is applied to reconstituted solutions from powder for injec-
tions, the test fails if particles are found in more than two containers.
5.8 Methods of sterilization
Sterilization is necessary for the complete destruction or removal of all

microorganisms (including spore-forming and non-spore-forming bacteria,
viruses, fungi, and protozoa) that could contaminate pharmaceuticals or other
materials and thereby constitute a health hazard. Since the achievement of the
absolute state of sterility cannot be demonstrated, the sterility of a pharma-
ceutical preparation can be defined only in terms of probability. The efficacy of
any sterilization process will depend on the nature of the product, the extent
and type of any contamination, and the conditions under which the final
product has been prepared. The requirements for Good Manufacturing Practice
should be observed throughout all stages of manufacture and sterilization.
Glassical sterilization techniques using saturated steam under pressure or
hot air are the most reliable and should be used whenever possible. Other ster-
ilization methods include filtration, ionizing radiation (gamma and electron-
beam radiation), and gas (ethylene oxide, formaldehyde).
For products that cannot be sterilized in the final containers, aseptic pro-
cessing is necessary. Materials and products that have been sterilized by one of
the above processes are transferred to presterilized containers and sealed, both
operations being carried out under controlled aseptic conditions.
Whatever method of sterilization is chosen, the procedure must be validated
for each type of product or material, both with respect to the assurance of steril-
ity and to ensure that no adverse change has taken place within the product.
Failure to follow precisely a defined, validated process could result in a non-
sterile or deteriorated product. A typical validation programme for steam or
dry-heat sterilization requires the correlation of temperature measurements,
made with sensory devices to demonstrate heat penetration and heat distribu-
tion, with the destruction of biological indicators, i.e. preparations of specific
microorganisms known to have high resistance to the particular sterilization
process. Biological indicators are also used to validate other sterilization
methods (see specific methods), and sometimes for routine control of individ-
ual cycles. Periodic revalidation is recommended.
1276
Methods of Analysis
Heating in an autoclave (steam sterilization)

Exposure of microorganisms to saturated steam under pressure in an autoclave
achieves their destruction by the irreversible denaturation of enzymes and
structural The temperature at which denaturation occurs varies
proteins.
inversely with the amount of water present. Sterilization in saturated steam
thus requires precise control of time, temperature, and pressure. As displace-
ment of the air by steam is unlikely to be readily achieved, the air should be
evacuated from the autoclave before admission of steam. This method should
be used whenever possible for aqueous preparations and for surgical dressings
and medical devices.
The recommendations for sterilization in an autoclave are 15 minutes at
121-124 °C (200 kPa).' The temperature should be used to control and monitor
the process; the pressure is mainly used to obtain the required steam temper-
ature. Alternative conditions, with different combinations of time and temper-
ature, are given below.
Temperature Approximate Minimum sterilization time

(°C) corresponding pressure (min)
(kPa)
126-129 250 (~2.5atm) 10

134-138 300 (~'3.0atm) 5
Minimum sterilization time should be measured from the moment when all
the materials to be sterilized have reached the required temperature through-

out. Monitoring the physical conditions within the autoclave during steriliza-
tion is essential. To provide the required information, temperature-monitoring
probes should be inserted into representative containers, with additional probes
placed in the load at the potentially coolest parts of the loaded chamber (as
established in the course of the validation programme). The conditions should
be within +2 °C and +10kPa (+0.1 atm) of the required values. Each cycle should
be recorded on a time-temperature chart or by other suitable means.
Aqueous solutions in glass containers usually reach thermal equilibrium
within 10 minutes for volumes up to 100 ml and 20 minutes for volumes up to
1000 ml.
Porous loads, such as surgical dressings and related products, should be
processed in an apparatus that ensures steam penetration. Most dressings are
adequately sterilized by maintaining them at a temperature of 134-138 °C for
5 minutes.
In certain cases, glass, porcelain, or metal articles are sterilized at 121-
124 °C for 20 minutes.
Fats and oils may be sterilized at 121 °C for 2 hours but, whenever possi-
ble, should be sterilized by dry heat.
'
1 atm = 101 325 Pa
1277
In certain cases (e.g. thermolabile substances), sterilization may be carried

out at temperatures below 121 °C, provided that the chosen combination of
time and temperature has been validated. Lower temperatures offer a different
level of sterilization; if this is evaluated in combination with the known micro-
bial burden of the material before sterilization, the lower temperatures may be
satisfactory. Specific conditions of temperature and time for certain prepara-
tions are stated in individual monographs.
The bioindicator strain proposed for validation of this sterilization process
is: spores oi Bacillus stearothermophilus (e.g. ATCC 7953 or CIP 52.81) for which
the D-value (i.e. 90% reduction of the microbial population) is 1.5-2 minutes
at 121 °C, using about 10® spores per indicator.
Dry-heat sterilization
In dry-heat processes, the primary lethal process is considered to be oxidation
of cell constituents. Dry-heat sterilization requires a higher temperature than

moist heat and a longer exposure time. The method is, therefore, more conve-
nient for heat-stable, non-aqueous materials that cannot be sterilized by steam
because of its deleterious effects or failure to penetrate. Such materials include
glassware, powders, oils, and some oil-based injectables.
Preparations to be sterilized by dry heat are filled in units that are either
sealed or temporarily closed for sterilization. The entire content of each con-
tainer maintained in the oven for the time and at the temperature given in
is
the table below. Other conditions may be necessary for different preparations
to ensure the effective elimination of all undesirable microorganisms.
Temperature Minimum sterilization time

(°C) (min)
160 180
170 60
180 30
Specific conditions of temperature and time for certain preparations are stated
in individual monographs.
The oven should normally be equipped with a forced air system to ensure
even distribution of heat throughout all the materials processed. This should
be controlled by monitoring the temperature. Containers that have been tem-
porarily closed during the sterilization procedure are sealed after sterilization
using aseptic techniques to prevent microbial recontamination.
The bioindicator strain proposed for validation of the sterilization process
is: spores ol Bacillus subtilis (e.g. var. niger KTCC 9372 or CIP 77.18) for which
the D-value is 5-10 minutes at 160°C using about 10® spores per indicator.
1278
Methods of Analysis
Filtration
Sterilization by filtrationis employed mainly for thermolabile solutions. These
may be sterilized by passage through sterile bacteria-retaining filters, e.g. mem-
brane filters (cellulose derivatives, etc.), plastic, porous ceramic, or suitable sin-
tered glass filters, or combinations of these. Asbestos-containing filters should
not be used.
Appropriate measures should be taken to avoid loss of solute by adsorption
onto the filter and to prevent the release of contaminants from the filter. Suit-
able filters will prevent the passage of microorganisms, but the filtration must
be followed by an aseptic transfer of the sterilized solution to the final con-
tainers which are then immediately sealed with great care to exclude any
recontamination.
Usually, membranes of not greater than 0.22 pm nominal pore size should
be used. The effectiveness of the filtration method must be validated if larger
pore sizes are employed.
To confirm the integrity of filters, both before and after filtration, a bubble
point or similar test should be used, in accordance with the filter manufacturer’s
instructions. This employs a prescribed pressure to force air bubbles
test
through the intact membrane previously wetted with the product, with water,
or with a hydrocarbon liquid.
All filters, tubes, and equipment used “downstream” must be sterile. Filters
capable of withstanding heat may be sterilized in the assembly before use by
autoclaving at 121 °C for 15-45 minutes depending on the size of the filter
assembly. The effectiveness of this sterilization should be validated. For filtra-
tion of a liquid in which microbial growth is possible, the same filter should
not be used for procedures lasting longer than one working day.
Exposure to ionizing radiation

Sterilization of certain active ingredients, drug products, and medical devices
in their final container or package may be achieved by exposure to ionizing
radiation in the form of gamma radiation from a suitable radioisotopic
source such as “Co (cobalt 60) or of electrons energized by a suitable electron
accelerator. Laws and regulations for protection against radiation must be
respected.
Gamma radiation and electron beams are used to effect ionization of the
molecules in organisms. Mutations are thus formed in the DNA and these reac-
tions alter replication. These processes are very dangerous and only well-
trained and experienced staff should decide upon the desirability of their use
and should ensure monitoring of the processes. Specially designed and purpose-
built installations and equipment must be used.
It is usual to select an absorbed radiation level of 25kGy’ (2.5Mrad)^,
although other levels may be employed provided that they have been validated.
’
kilogray
^
megarad
1279
Radiation doses should be monitored with specific dosimeters during the

entire process. Dosimeters should be calibrated against a standard source on
receipt from the supplier and at appropriate intervals thereafter. The radiation
system should be reviewed and validated whenever the source material is
changed and, in any case, at least once a year.
The bioindicator strains proposed for validation of this sterilization process
are: spores of Bacillus pumilus (e.g. ATCC 27142 or CIP 77.25) with 25kGy
(2.5Mrad) for which the D-value is about 3kGy (0.3Mrad) using 10^-10° spores
per indicator; for higher doses, spores of Bacillus cereus (e.g. SSI C 1/1) or
Bacillus sphaericus (e.g. SSI CiA) are used.
Gas sterilization
The active agent of the gas sterilization process can be ethylene oxide or
another highly volatile substance. The highly flammable and potentially explo-
sive nature of such agents is a disadvantage unless they are mixed with suit-
able inert gases to reduce their highly toxic properties and the possibility of
toxic residues remaining in treated materials. The whole process is difficult to
control and should only be considered if no other sterilization procedure can
be used. It must only be under the supervision of highly skilled staff.

carried out
The sterilizing efficiency of ethylene oxide depends on the concentration of
the gas, the humidity, the time of exposure, the temperature, and the nature of
the load. In particular,it is necessary to ensure that the nature of the packag-
ing such that the gas exchange can take place. It is also important to main-
is
tain sufficient humidity during sterilization. Records of gas concentration and

of temperature and humidity should be made for each cycle. Appropriate
sterilization conditions must be determined experimentally for each type of
load.
After sterilization, time should be allowed for the elimination of residual
sterilizing agents and other volatile residues, which should be confirmed by
specific tests.
Because of the difficulty of controlling the process, efficiency must be mon-
itored each time using the proposed bioindicator strains: spores of Bacillus sub-
tilis (e.g. var. niger ATCC 9372 or CIP 77.18) or of Bacillus stearothermophilus, (e.g.
ATCC 7953 or CIP 52.81). The same quantity of spores should be used as for
“Heating in an autoclave” and “Dry-heat sterilization”.
1280
Reagents, test solutions and
volumetric solutions
CopyrigfiJed i
Reagents, test solutions and volumetric solutions
Reagents, test solutions and

volumetric solutions
The reagents, test solutions and volumetric solutions mentioned in the Interna-
tional Pharmacopoeia, 4th edition, are described below. The reagents are denoted
by the abbreviation R, the test solutions by the abbreviation TS, and the vol-
umetric solutions, or solutions that are similarly standardized, by the abbrevi-
ation VS. Similarly named reagents differing in composition, purity, etc., are
distinguished by placing a numeral after the appropriate abbreviation. The des-
ignations AsR, AsTS, CITS, FeTS and PbTS refer to reagents of suitable purity
for use in the limit tests for arsenic, chlorides, iron, and heavy metals, respec-
tively. The designation IR refers to reagents of suitable purity for use in spec-
trophotometry in the infrared region. The designation Cm denotes culture
media for microbiological tests. The designation RS denotes International
Chemical Reference Substances.*
The concentrations are expressed in conformity with the Systeme interna-
tional d'Unites (SI) and they refer to the anhydrous substance. The reference to
SRIP indicates Specifications for reagents mentioned in the International Pharmacopoeia

(World Health Organization, Geneva, 1963). Designations used in SRIP butnow
discontinued are given in square brackets. The designation d denotes the rela-
tive density dfo, i.e., measured in air at 20 °C in relation to water at 20 °C.
Unless otherwise specified, all solutions indicated in the tests and assays of
The International Pharmacopoeia are prepared with water R.
Acacia (5g/I) TS. A solution in water containing about 5g of acacia R per

litre.
Acacia R. The dried gummy exudate from the stems and branches of Acacia
Senegal (L.) Willd. and of other species of Acacia of African origin.
Description. Rounded or ovoid tears of varying diameters from about 1 cm to 3
cm; yellowish white or pale amber; odourless.
Solubility. Very slowly soluble in twice its weight of water, leaving only a
very small residue of vegetable particles; practically insoluble in ethanol

TS and ether R.
(-750 g/1)
Ash. Not more than 50mg/g.
Acid-insoluble ash. Not more than 5.0mg/g.
Insoluble matter. Mix 5g of powdered or finely ground material with 100 ml of
water and 10ml of hydrochloric acid (~70g/l) TS and boil gendy for 15
minutes, stirring frequently. Filter while hot through a sintered glass cru-
^
International Chemical Reference Substances are available from the WHO Collaborating Centre for Chem-
ical Reference Substances, AgoteketAB, Produktion Ai Laboratorier, Centrallaboratoriet, ACL, Prismavd-
gen Z, S-141 75 Kungens Kurva, Sweden. (Fax: +4b 8 740 60 40; emaihwho. apl@apoteket.se).
1283
cible, wash the residue with hot water, and dry to constant weight at
105 °C; not more than 5mg/g.
Tannin. Dissolve 1 g in 10ml of water and add 0.1ml of ferric chloride (25g/l)
TS; no bluish black colour or blackish precipitate is produced.
Acetaldehyde R. Ethanal; C2H4O.

Description. A clear, colourless, flammable liquid.
Miscibility. Miscible with water and ethanol (-750 g/1) TS.
Refractive index, n™ = 1 .332
Relative density, dô = 0.788
Boiling point. About 21 °C.
p-Acetamidobenzalazine RS. International Chemical Reference Substance.
Acetate buffer, pH TS. A buffer mixture of pH 3.0.

3.0,
Procedure. Dissolve 12 g of sodium acetate R in water, add 6ml of glacial acetic
acid R and dilute with sufficient water to produce 100 ml.
Acetate buffer, pH 4.5, TS

Procedure. Dissolve 10. 9g of sodium acetate R in 100ml of water, add 8ml of
glacial acetic acid R, mix, and dilute to 1000 ml with water.

Procedure. Dissolve 5.4 g of sodium acetate R in 50 ml of water, adjust to pH 4.6
with glacial acetic acid R, and dilute to 100 ml with water.

Procedure. Dissolve 8.4 g of sodium acetate R in 100 ml of water, add 3.35 ml of

Procedure. Dissolve 13. 6g of sodium acetate R in 100ml of water, add 6ml of

Procedure. Dissolve 54.4 g of sodium acetate R in 50 ml of water, heating to
35 °C, if necessary. After cooling, slowly add 10ml of glacial acetic acid
R. Shake and dilute to 1000 ml with water.

Procedure. Dissolve lOOg of ammonium acetate R in 300 ml of water. Add
4.1ml of glacial acetic acid R, adjust the pH to 6.0 using either ammonia
(-100 g/1) TS or acetic acid (-300 g/1) TS, and dilute to 500ml with water.
1284
Acetate standard buffer TS

Procedure. To 10 ml of acetic acid (~60g/l) TS add 10 ml of sodium hydroxide
(1 mol/1) VS and dilute with sufficient carbon-dioxide-free water R to
produce 1000ml.
Acetazolamide RS. International Chemical Reference Substance.
Acetic acid (~90g/l) TS. Acetic acid (-300 g/1) TS, diluted with water to
contain about 90 g of C2H4O2 per litre (approximately 1.5 mol/1).
Acetic acid (~120g/l) TS. Acetic acid (-300 g/1) TS, diluted with water to
contain 120g of C2H4O2 per litre (approximately 2mol/l); d ~ 1.016.
Acetic acid (~300g/l) TS. A solution of glacial acetic acid R containing about
300 g/1 of C2H4O2 (approximately 5mol/l); d ~ 1.037.
Acetic acid (~60g/l) PbTS. Acetic acid (-60 g/1) TS that complies with the
following test: Evaporate 20ml of acetic acid (-60 g/1) TS almost to dryness
on a water-bath, add 25 ml of water and carry out the test for heavy metals.
The heavy metals limit is 3 pg/ml.
Acetic acid (-60 g/1) TS. Acetic acid (-300 g/1) TS, diluted to contain about
60 g/1 of C2H4O2 (approximately 1 mol/1); d - 1.008.
Acetic acid (0.07mol/I) VS. A solution prepared by diluting 4.2 ml of glacial

acetic acid R to 1000 ml with water.
Acetic acid (5.0 g/1) TS. Acetic acid (-300 g/1) TS, diluted with water to
contain about 5.0 g of C2H4O2 per litre; d - 1.0007.
Acetic acid, glacial, R. C2H4O2 (SRIP, 1963, p. 25); d- 1.048.
Acetic acid, glacial, Rl. Glacial acetic acid R, that complies with the fol-
lowing tests:
Substances reducing dichromate. To 10ml add 1.0ml of potassium dichromate
(0.0167mol/l) VS and cautiously add 10ml of sulfuric acid (-1760g/l) TS.
Cool the solution to room temperature and allow to stand for 30 minutes.
While swirling the solution, dilute slowly and cautiously with 50 ml of
water, cool, and add 1.5 ml of freshly prepared potassium iodide (80 g/1) TS.
Titrate the liberated iodine with sodium thiosulfate (0.1 mol/1) VS, adding
3 ml of starch TS as the endpoint is approached. Perform a blank titration
and make any necessary corrections. Not less than 0.60ml of sodium thio-
sulfate (0.1 mol/1) VS is consumed.
Substances reducing permanganate. Add 40ml to 10ml of water. Cool to 15 °C,
add 0.30 ml of potassium permanganate (0.02 mol/1) VS, and allow to stand
at 15 °C for 10 minutes; the pink colour is not entirely discharged.
1285
Acetic anhydride R. C4H6O3 (SKIP, 1963, p. 26).
Acetic anhydride/dioxan TS
Procedure. To 50 ml of dioxan R add 1 ml of acetic anhydride R (approximately
0.2 mol/1).
Acetone R. CsHgO (SRIP, 1963, p. 27).
Acetonitrile (400 g/1) TS

Procedure. Mix 1 volume of acetonitrile R with 1 volume of water. The result-
ing solution contains about 400 g/1 of C2H3N.
Acetonitrile R. Methyl cyanide, C2H3N.

Description. A clear, colourless liquid.
Miscibility. Freely miscible with water.
Acetyl chloride R. C2H3CIO. Contains not less than 98.0% of C2H3CIO in

both assay A and assay B (see below).
Description. A clear, colourless or very slightly yellow liquid.
Phosphorus compounds. Carefully treat 1 ml with 1 ml of water, add 1 ml of nitric
acid (-1000 g/1) TS, boil, cool, dilute with 20ml of water, add 10 ml of
ammonium molybdate/nitric acid TS and allow to stand at about 40 °C for
2 hours; no yellow precipitate is produced.
Assay. (A) Dissolve about Ig, accurately weighed, in 50 ml of carbonate-free
sodium hydroxide (1 mol/1) VS, and titrate with sulfuric acid (0.5mol/l) VS,
using phenolphthalein/ethanol TS as indicator. Each ml of carbonate-free

sodium hydroxide (1 mol/1) VS is equivalent to 7.850mg of C2H3CIO. (B)
Dilute the neutralized liquid from A to 250ml with water, mix, and titrate
50ml with silver nitrate (0.1 mol/1) VS, using potassium chromate (lOOg/1)
TS as indicator. Each ml of silver nitrate (0.1 mol/1) VS is equivalent to
7.850g of C2H3CIO.
Agar R. (SRIP, 1963, p. 27).
Albendazole RS. International Chemical Reference Substance.
Alcuronium chloride RS. International Chemical Reference Substance.
Allopurinol RS. International Chemical Reference Substance.
Alum R. Aluminium potassium sulfate dodecahydrate; KA1(S04)2, I2H2O

(SRIP, 1963, p. 29).
Aluminium chloride R. AlCl3,6H20 (SRIP, 1963, p. 30).
1286
Aluminium chloride TS
Procedure. Dissolve 65.0 g of aluminium chloride R in sufficient water to
produce 100 ml, add 0.5 g of charcoal R, stir for 10 minutes and filter. While
stirring, add to the filtrate sufficient sodium hydroxide (0.5mol/l) VS to
adjust the pH to 1.5.
Aluminium hydroxide R. Hydrated Al(OH)s.

Description. A white, odourless powder.
Solubility. Practically insoluble in water and in ethanol (~750g/l) TS.
Aluminium oxide R. AljOg.

A suitable grade for use in thin-layer chromatography.
Aluminium R. A1 (SRIP, 1963, p. 29); wire, granules, or sheets.
Aluminium
3- standard Al/ml) TS
(10|j.g
Procedure. Dissolve 17.6mg of alum R in 5ml of sulfuric acid (0.05mol/l) VS
Note. For the preparation of this test solution commercially available alu-
minium nitrate standard solution 1000 pg AlWml or aluminium nitrate non-
ahydrate can also be used.
4-
Amidotrizoic acid RS. International Chemical Reference Substance.
Amiloride hydrochloride RS. International Chemical Reference Substance.
Amino-2,4,6-triiodobenzoic acid RS. International Chemical Reference

Substance.
2-Amino-5-nitrothiazole R. C 3 H 3 N 3 O 2 S.
Description. Greenish yellow to orange -ye How, fluffy powder.
Solubility. Very slightly soluble in water; soluble in dilute mineral acids; slightly
Melting temperature. About 198 °C with decomposition.
Amino-6-chloro-l,3-benzenedisulfonamide R. C 6 H 8 CIN 3 O 4 S 2 .
Description. A white, odourless powder.

Solubility. Soluble in ammonia (-100 g/1) TS; practically insoluble in water.
Identification. The absorption spectrum of a 5pg/ml solution in methanol R
exhibits maxima at about 223 nm, 265 nm, and 312 nm. The absorptivity at
265 nm is about 64.0 = 640).
(Ej°(l„
Sulfated ash. Ignite 2g; not more than l.Omg/g.
4-Aminoantipyrine R. C11H13N3O. 4-Amino-2,3-dimethyl-l-phenyl-3-pyra-

zolin-5-one; ampyrone; aminopyrazolone.
Description. Pale yellow crystals or powder.
1287
Solubility. Sparingly soluble in water; freely soluble in ethanol (-750 g/1) TS;
very slightly soluble in ether R.
Melting temperature. About 108 °C.
4-Aminoantipyrine TSl
Procedure. Dissolve 0.125g of 4-aminoantipyrine R in 25ml of methanol R con-
taining 0.25ml of hydrochloric acid (-420 g/1) TS.
4-Aminoantipyrine TS2
Procedure. Dissolve about 0.1 g of 4-aminoantipyrine R in 30ml of water and
add a mixture of 10ml of sodium carbonate (200 g/1) TS and 2 ml of sodium
hydroxide (1 mol/1) VS; dilute with sufficient water to produce 100ml.
Note: 4-Aminoantipyrine TS2 must be freshly prepared.
4-Aminobenzoic acid R. C7H7NO2. Contains not less than 98.5% of

C7H7NO2.
White or slighdy yellow
Description. crystals or a crystalline powder; odourless.
Solubility. Soluble in 170 parts of water, in 9 parts of boiling water, in 8 parts
of ethanol (-750 g/1) TS, and in 50 parts of ether R.
Loss on drying. Dry at 105°C for 2 hours; it loses not more than 2.0mg/g.
Assay. Transfer to a beaker about 0.3 g, accurately weighed and previously
dried at 105 °C for 2 hours, add 5 ml of hydrochloric acid (-420 g/1) TS,
50ml and stir until dissolved. Cool to about 15°C, add about 25g
of water
of crushed ice and slowly titrate with sodium nitrite (0.1 mol/1) VS until a
glass rod dipped into the titrated solution produces an immediate blue ring
when touched to starch/iodide paper R. When the titration is complete, the
end-point is reproducible after the mixture has been allowed to stand for 1
minute. Each ml of sodium nitrite (0.1 mol/1) VS is equivalent to 13.71 mg
of C7H7NO2.
Storage. Store in a tightly closed container, protected from light.
2-AminobutanoI R. C^HnNO.
Description. A colourless or light yellow, clear liquid.
Miscibility. Miscible with water and methanol R.
Mass density. p 2o
= 0.944 - 0.950 kg/1.
Refractive index. nS* = 1.450 - 1.455.
Dissolve 0.05g in 4ml of ethanol (-750 g/1) TS, add 0.5ml of a
Identification.
2.5 mg/ml solution of triketohydrindene hydrate R and warm on a water-

bath; a violet colour is produced.
7 - [(2- Aminoethyl)amino]-l -cyclopropyl -6 -fluoro- 1,4 -dihydro -4-

oxoquinoline-3-carboxylic acid RS (ciprofloxacin ethylenediamine
compound). International Chemical Reference Substance.
1288
4-Aminophenol R. C^jH/NO.
3-Aminopyrazole-4-carboxamide hemisulfate RS. International Chemical

Reference Substance.
Amitriptyline hydrochloride RS. International Chemical Reference

Substance.
Ammonia (~100g/l) FeTS. Ammonia (~100g/l) TS that complies with the

following test: Evaporate 5ml of ammonia (~100g/l) TS nearly to dryness
on a water-bath, add 40 ml of water, 2 ml of citric and 2
acid (180g/l) FeTS,
drops of mercaptoacetic acid R; mix, make alkaline with ammonia (~100g/l)
FeTS, and dilute to 50 ml with water; no pink colour is produced.
Ammonia (~100g/l) PbTS. Ammonia (~100g/l) TS that complies with the

following test: Evaporate 5 ml of ammonia (~100g/l) TS to dryness on a
water-bath, add to the residue 1ml of hydrochloric acid (~70g/l) TS, and
evaporate to dryness. Dissolve the residue in 2 ml of acetic acid (~60g/l)
PbTS, dilute with water to 25ml and carry out the test for heavy metals.
Prepare the blank in a similar way. The heavy metals limit is 2 pg/ml.
Ammonia (~100g/l) TS. Ammonia (-260 g/1) TS, diluted to contain about
100 g/1 of NH3 (approximately 6mol/l); d ~ 0.956.
Ammonia (~17g/l) TS. Ammonia (~100g/l) TS, diluted to contain about

17 g of NH3 per litre (approximately 1 mol/1); d~ 0.992.
Ammonia (~260g/l) TS. [ammonia, strong, R]. (SRIP, 1963, p. 31); d~ 0.894.
Ammonia (-35 g/1) TS. Ammonia (-100 g/1) TS, diluted to contain about
35g of NH3 per litre (approximately 2 mol/1); d - 0.985.
Ammonia (~50g/l) TS. Ammonia (-260 g/1) TS, diluted with water to contain
about 50 g of NH3 per litre (approximately 3 mol/1); d - 0.977.
Ammonia buffer TS
Procedure. Dissolve 67. 5g of ammonium chloride R in 570ml of ammonia
(-260 g/1) TS and dilute with water to 1000 ml.
Ammonia buffer TS2

Procedure. Dissolve 67. 5g of ammonium chloride R in 650ml of ammonia
(-260 g/1) TS and dilute with water to produce 1000 ml.
1289
Ammonium acetate (lOOg/1) TS. A solution of ammonium acetate R con-

taining lOOg of CH2 7 NO 2 per litre.
Ammonium acetate (2g/l) TS. A solution of ammonium acetate R contain-

ing about 2 g of C H 7NO 2
2 per litre.
Note: Ammonium acetate (2g/l) TS must be freshly prepared.
Ammonium acetate (40g/l) TS. A solution of ammonium acetate R con-

taining about 38. 5g of C 2H 7 NO 2 per litre (approximately 0.5mol/l).
Note: Ammonium acetate (40g/l) TS must be freshly prepared.

taining about 50 g of C 2 H 7NO 2 per litre.

taining about 77g/l of C H 2 7 NO 2 (approximately 1 mol/1).
Ammonium acetate TS
Procedure. Dissolve 150g of ammonium acetate R in water, add 3ml of glacial
acetic acid R, and dilute with sufficient water to produce 1000 ml.
Note-. Ammonium acetate TS must be used within 1 week of preparation.
Ammonium acetate buffer, pH 4.62, TS

Procedure. Adjust the pH of ammonium acetate (100 g/1) TS to 4.62 using acetic
acid (~60g/l) TS.
Ammonium acetate R. CH 2 7 NO 2 (SRIP, 1963, p. 32).
Ammonium carbonate R. (NH 4 ) 2 C 03 (SRIP, 1963, p. 33).
Ammonium chloride (10p,g/ml NH4) TS

Procedure. Dissolve 0.296g, accurately weighed, of ammonium chloride R in
sufficient water to produce 1000 ml. Dilute 10ml of this solution to 100 ml.
Shelf-life. Use the solution within 2 weeks of its preparation.
Ammonium chloride (lOOg/1) TS. A solution of ammonium chloride R con-

taining about lOOg of NH4CI per litre.
Ammonium chloride (20 g/1) TS. A solution of ammonium chloride R con-

taining about 20 g of NH 4 CI per litre.
Ammonium chloride bufifer, pH 10.0, TS. A buffer mixture of pH 10.0.
Procedure. Dissolve 7.0g of ammonium chloride R in 57ml of ammonia
(-260 g/1) TS and dilute with sufficient water to produce 100 ml.
1290
Ammonium chloride buffer, pH 10.5, TS. A buffer mixtxire of pH 10.5.

Procedure. Dissolve 6.95g of ammonium chloride R in 75ml of ammonia
(-260 g/1) TS and dilute to 100 ml with water.
Ammonium chloride R. NH4CI (SKIP, 1963, p. 33).
Ammonium chloride TS (Nessler’s reagent)

Procedure. Dissolve 3.15g of ammonium chloride R in a sufficient quantity of
ammonia-free water R to produce 1000 ml.
Ammonium chloride, dilute, TS

Procedure. To 10 ml of ammonium chloride TS add a sufficient quantity of
ammonia-free water R to produce 1000 ml.
Ammonium dihydrogen phosphate. (NH 4)H 2 P 04
Ammonium mercurithiocyanate TS
Procedure. Dissolve 30 g of ammonium thiocyanate R and 27 g of mercuric chlo-
ride R in sufficient water to produce 1000 ml.
Ammonium molybdate (45 g/1) TS. A solution of ammonium molybdate R

containing about 47 g of (NH4)sMo7024 per litre.
Ammonium molybdate (95 g/1) TS. A solution of ammonium molybdate R

containing about 95 g/1 of (NH4)eMo7024.
Ammonium molybdate R. (NH4)6M07O24,4H2O (SRIP, 1963, p. 34).
Ammonium molybdate/nitric acid TS

Procedure. Dissolve 50 g of ammoniumR in 500 ml of nitric acid
sulfate
(-1000 g/1) TS using a 2000-ml conical flask or beaker. Dissolve separately
in a beaker 150 g of ammonium molybdate R in 400 ml of boiling water.
After cooling, pour this solution slowly, while stirring, into the acid solu-
tion and dilute with water to 1 000 ml. Allow to stand for 2-3 days, and filter.
Storage. Store in well-closed, brown glass bottles, and keep in a cool place.
Ammonium molybdate/sulfuric acid TS

Procedure. Dissolve 0.5 g of ammonium molybdate R in sufficient sulfuric acid
(-1760 g/1) TS to produce 10 ml.
Ammonium molybdate/vanadate TS
Procedure. Shake 4g of finely powdered ammonium molybdate R and 0.1 g of
finely powdered ammonium vanadate R with 70 ml of water. Add 20 ml of
nitric acid (-lOOOg/1) TS and dilute to 100ml with water.
1291
Ammonium nitrate (50g/I) TS. A solution of ammonium nitrate R contain-

ing about 50 g of NH4NO3 per litre.
Ammonium nitrate R. NH4NO3 (SKIP, 1963, p. 35).
Ammonium nitrate TS
Procedure. Dissolve 1.6g of ammonium nitrate R in 30 ml of water, add 3.0ml
of ammonia (-260 g/1) TS, and dilute with sufficient water to produce
100 ml.
Ammonium oxalate (25 g/1) TS. A solution of ammonium oxalate R con-

taining about 27 g/1 of C2HgN204.
Ammonium oxalate (50g/l) TS. A solution of ammonium oxalate R con-

taining about 50g of C2HgN204 per litre.
Ammonium oxalate R. C2HsN204,H20 (SKIP, 1963, p. 36).
Ammonium persulfate R. (NH4)2S20g (SRIP, 1963, p. 37).
Ammonium persulfate/phosphate buffer TS

Procedure. Dissolve lOg of ammonium persulfate R in sufficient phosphate
buffer, pH 7.4, TS to produce 100 ml.
Ammonium pyrrolidinedithio carbamate (lOg/1) TS

Immediately before use, wash lOg of ammonium pyrrolidinedithio-
Procedure.
carbamate R three times, each with 25 ml of isobutyl methyl ketone R, filter,
and dry the substance. Then dissolve l.Og in sufficient water to produce
100 ml.
Ammonium pyrrolidinedithiocarbamate R. Ammonium 1-pyrrolidinecar-

bodithioate; C5Hj2N2S2. Reagent grade quality.
Ammonium reineckate (lOg/1) TS. A solution of ammonium reineckate R

containing about lOg of NH4[Cr(NH3)2(SCN)4] per litre.
Ammonium reineckate R. NH4[Cr(NH3)2(SCN)4],H20 (SRIP, 1963, p. 39).
Ammonium sulfamate (25 g/1) TS. A solution of ammonium sulfamate R

containing about 25 g of NH4OSO2NH2 per litre.
Ammonium sulfamate (5 g/1) TS. A solution of ammonium sulfamate R con-

taining about 5g of NH4OSO2NH2 per litre.
Ammonium sulfamate (50g/l) TS. A solution of ammonium sulfamate R

containing 50 g of NH4OSO2NH2 per litre.
1292
Ammonium sulfamate R. NH OSO NH

4 2 2 (SKIP, 1963, p. 39).
Ammonium sulfate R. (NH 4 )2 S 04 (SKIP, 1963, p. 40).
Ammonium sulfide TS
Procedure. Prepare a saturated solution of hydrogen sulfide R in ammonia
(-100 g/1) TS. To 25ml of this solution add 50ml of ammonia (-lOOg/1) TS.
Ammonium thiocyanate (0.01 mol/1) VS. Ammonium thiocyanate R, dis-

solved in water to contain 0.7612 g of NH SCN 4 in 1000 ml.
Method of standardization. Ascertain the exact concentration of the solution fol-
lowing the method described under ammonium thiocyanate (0.1 mol/1) VS.

solved in water to contain 3.806g of NH4SCN in 1000 ml.
lowing the method described for ammonium thiocyanate (0.1 mol/1) VS.

solved in water to contain 7.612 g of NH 4 SCN in 1000 ml.
Method of standardization. Ascertain the exact concentration of the 0.1 mol/1
solution in the following manner: Place 30.0ml of silver nitrate (0.1 mol/1)
VS in a glass-stoppered flask. Dilute with 50 ml of water, add 2 ml of nitric
acid (~1000g/l) TS and then titrate with the ammonium thiocyanate solu-
tion to the first appearance of a red-brown colour, using 2 ml of ferric
ammonium sulfate (45 g/1) TS as indicator.
Ammonium thiocyanate (lOg/1) TS. A solution of ammonium thiocyanate

R containing 10 g of NH SCN4 per litre.
Ammonium thiocyanate (75 g/1) TS. A solution of ammonium thiocyanate

R containing about 75g/l of NH SCN 4 (approximately 1 mol/1).
Ammonium thiocyanate R. NH4SCN (SRIP, 1963, p. 40).
Ammonium thiocyanate/cobalt(II) nitrate TS

Procedure. Dissolve 20 g of ammonium thiocyanate R and 5 g of cobalt(ll) nitrate
R in 100ml of water. Add sufficient sodium chloride R to saturate the
solution.
Ammonium vanadate R. NH4VO3.

Descrifrtion. A white to slightly yellowish, crystalline powder.
Solubility. Slightly soluble in water; soluble in ammonia (-100 g/1) TS.
Amodiaquine hydrochloride RS. International Chemical Reference

Substance.
1293
Amoxicillin trihydrate RS. International Chemical Reference Substance.
Amphotericin B RS. International Chemical Reference Substance (containing

the declared content of tetraenes).
Ampicillin RS. International Chemical Reference Substance.
Ampicillin sodium RS. International Chemical Reference Substance.
Ampicillin trihydrate RS. International Chemical Reference Substance.
Amyl alcohol R. C 5 H 12 O (SRIP, 1963, p. 42).
Anhydrotetracycline hydrochloride RS. International Chemical Reference

Substance.
Aniline (25g/l) TS. A solution of aniline R containing about 25 g of CgHjN

per litre.
Aniline R. C^H/N (SRIP, 1963, p. 43).
Anisaldehyde R. 4-Methoxybenzaldehyde; CgHgOj.

Description. A colourless to pale yellow, oily liquid.
Solubility. Very slightly soluble in water, miscible with ethanol (-750 g/1) TS and
ether R.
Mass density.p 2 o = about 1.125 kg/1.
Anisaldehyde TS
Procedure. Mix in the following order 0.5ml of anisaldehyde R, 10ml of glacial
acetic acid R, 85ml of methanol R, and 5ml of sulfuric acid (-1760 g/1) TS.
Anisaldehyde/methanol TS
Procedure. Slowly add 10 ml of glacial acetic acid R and 5 ml of sulfuric acid
(~1760g/l) TS to 55ml of methanol R, and cool to room temperature. Sep-
arately add 0.5ml of anisaldehyde R to 30ml of methanol R. Mix the two
solutions thoroughly.
Storage. Keep anisaldehyde/methanol TS protected from light.
Note: Anisaldehyde/methanol TS should be freshly prepared.
Anisaldehyde/sulfuric acid TS
Procedure. Add 5 ml of anisaldehyde R to 10ml of sulfuric acid (-1760 g/1) TS.
Anthrone R. CmHiqO.
Solubility. Practically insoluble in water; slightly soluble in ethanol (-750 g/1) TS
and in sulfuric acid (-100 g/1) TS.
1294
Solubility in carbon tetrachloride R. Add 0.5g to 10ml of carbon tetrachloride R;

a clear, non-fluorescent solution is produced.
Anthrone TS
Procedure. Dissolve 35mg of anthrone R in 100ml of sulfuric acid (-1760 g/1)
TS.
Anthrone TS2
Procedure. Dissolve 200 mg of anthrone R in 100ml of sulfuric acid (-1760 g/1)
TS.
Antimony sodium tartrate (50g/l) TS. A solution of antimony sodium tar-
trate R containing about 50 g/1 of C 4 H 4 Na 07 Sb.
Antimony sodium tartrate R. C H Na 07 Sb.

4 4
Description. Hygroscopic, transparent or whitish scales or powder.

(-710 g/1) TS.
Antimony trichloride R. SbClj. Contains not less than 97.0% of SbCla.

Description. Colourless crystals.
Solubility. Very soluble in dehydrated ethanol R and in chloroform R (may form
a slightly turbid solution).
Chloroform- insoluble substances. Dissolve 5.0 g in 25 ml of chloroform R, filter
through a tared filtering crucible, wash the crucible with several portions
of chloroform R, and dry at 105°C; it leaves a residue of not more than
l.Omg.
Assay. Dissolve 0.5 g, accurately weighed, in 30 ml of water containing 4.0 g of
potassium sodium tartrate R, add 2g of sodium hydrogen carbonate R, and
titrate with iodine (0.1 mol/1) VS. Each ml of iodine (0.1 mol/1) VS is equiv-
alent to 11.41 mg of SbClg.
Note: In moist air fumes may be evolved.
Antimony trichloride TS
Procedure. Dissolve22 g of antimony trichloride R in 100 ml of ethanol-free
chloroform R, add 2.5ml of acetyl chloride R, and allow to stand for 30
minutes.
Aprotinin R. A polypeptide consisting of a chain of 58 amino acids. A com-

mercially available reagent of suitable grade.
Arachis oil R. Use arachis oil as described in the monograph for “Arachis oil”.
1295
Argon R. Ar. Contains not less than 99.995% of At.

Carbon monoxide. When used as described in the test for carbon monoxide in
medicinal gases, after passage of 10 litres of argon at a flow rate of 4 litres
per hour, not more than 0.05ml of sodium thiosulfate (0.002 mol/1) VS is
required for the titration (0.6pl/l).
Arsenic trioxide R. AS2O3 (SKIP, 1963, p. 44).
Arsenic trioxide Rl. Arsenic trioxide R that has been prepared according to
either of the following methods:
1. Recrystallize arsenic trioxide R from a boiling mixture of 20 parts of

hydrochloric acid (-420 g/1) TS and 5 parts of water. After cooling, collect
the crystals and recrystallize them from boiling water until the mother
liquor has a pH greater than 4.0. Dry the crystals to constant weight over
silica gel, desiccant, R.
2. Sublime arsenic trioxide R in an appropriate apparatus.
pH Value. Heat to boiling for a few minutes 1 .0 g in 20 ml of water, filter and

cool; the filtrate has a pH greater than 4.0.
Chlorides. Dissolve 10 mg water to produce 10 ml. Acidify with 1
in sufficient
drop of nitric acid (-1000 g/1) TS and add 2 drops of silver nitrate (0.1 mol/1)
VS; the solution remains clear and colourless for not less than 2 minutes.
Sulfides. To a solution of 5.0 g in a mixture of 10ml of sodium hydroxide
(-80 g/1) TS and 15 ml of water add 2 drops of lead acetate (80 g/1) TS; the
solution remains colourless.
Loss on drying.Dry to constant weight over silica gel, desiccant, R; it loses not
more than O.lmg/g.
Arsenic, dilute, AsTS. One millilitre contains lOpg of arsenic.

Procedure. Dilute 1 ml of strong arsenic AsTS with sufficient water to produce
100 ml.
Note: Dilute arsenic AsTS must be freshly prepared.
Arsenic, strong, AsTS

Procedure. Dissolve 0.132 g of arsenic trioxide R in 6 ml of sodium hydroxide
(-80 g/1) TS,by gentle heating. Dilute the cooled solution with 20 ml of
water, and add 50 ml of hydrochloric acid (-250 g/1) TS and sufficient water
to produce 100 ml.
Artemether RS. International Chemical Reference Substance.
Artemisinin RS. International Chemical Reference Substance.
ArtemotU RS. International Chemical Reference Substance.
1296
Artenimol RS. International Chemical Reference Substance.
Artesunate RS. International Chemical Reference Substance.
Atenolol RS. International Chemical Reference Substance.
Atenolol for column validation RS. International Chemical Reference

Substance.
Atropine sulfate RS. International Chemical Reference Substance.
Azathioprine RS. International Chemical Reference Substance.
Azo violet R. 4-(4-Nitrophenylazo)resorcinol; Magneson I: C12H9N3O4.

Description. A red powder.
Azo violet TS
azo violet
Procedure. Dissolve 0.2 g of R in a mixture of 1 volume of toluene R
and 2 volumes of cyclohexane R.
Bacitracin zinc RS. International Chemical Reference Substance.
Barium chloride (0.5 mol/1) VS. Barium chloride R, dissolved in water to

contain 104. 2g of BaCh in 1000ml.
Method of standardization. Ascertain the exact concentration of the 0.5mol/l
solution in the following manner: Place 10.0ml of sulfuric acid (0.5 mol/1)
VS in a flask, dilute with 40ml of water, add 2 drops of thorin (2g/l) TS and
titrate slowly with the barium chloride solution to a reddish colour.
Barium chloride (50g/l) TS. A solution of barium chloride R containing

about 52g/l of BaCl2 (approximately 0.25 mol/1).
Barium chloride R. BaCl2,2H20 (SRIP, 1963, p. 45).
Barium hydroxide (0.15 mol/1) VS. Barium hydroxide R dissolved in carbon-

dioxide-free water R to contain 25.7 g of Ba(OH)2 in 1000 ml.
Barium hydroxide (15g/l)TS. A solution of barium hydroxide R in carbon-

dioxide-free water R containing about 15 g of Ba(OH)2 per litre.
Note: Barium hydroxide (15g/l) TS must be freshly prepared.
Barium hydroxide R. Ba(0H)2,8H20 (SRIP, 1963, p. 46).
Barium nitrate (0.01 mol/I) VS. Barium nitrate R, dissolved in water to

contain 2.614g of Ba(N03)2 in 1000ml.
1297

solution in the following manner: Place 10.0ml of sulfuric acid (0.01 mol/1)
VS in a flask, dilute with 40 ml of water, add 2 drops of thorin (2g/l) TS and
2 drops of methylthioninium chloride (0.2 g/1) TS, and titrate slowly with
the barium nitrate solution from yellow to a pink colour.
Barium nitrate R. Ba(N03)2 (SKIP, 1963, p. 47).
Barium oxide R. BaO.

Description.White to yellowish-white lumps or powder. Absorbs moisture and
carbon dioxide on exposure to air.
Storage. Store in tightly closed containers.
Barium sulfate suspension TS

Procedure. Mix 15 ml of barium chloride (0.5 mol/1) VS with 55 ml of water
and 20ml of sulfate-free ethanol (-750 g/1) TS, add 5 ml of potassium sulfate
(174mg/l) TS, and dilute with sufficient water to produce 100ml.
Note. Barium sulfate suspension TS must be freshly prepared.
Beclometasone dipropionate RS. International Chemical Reference

Substance.
Beef extract R. A residue from beef broth obtained by extracting fresh, sound,
lean beef by cooking with water and evaporating the resulting broth at a
low temperature, usually under reduced pressure until a thick pasty residue
is obtained.
Benzalkonium chloride TS. A mixture of alkylbenzyldimethylammonium

chlorides. It contains in 1 litre not less than 470g and not more than 530g
of alkylbenzyldimethylammonium chlorides, calculated as C22H40CIN.
Description. A clear, colourless to pale yellow, syrupy liquid; odour, aromatic.
Miscibility. Miscible with water and ethanol (~750g/l) TS.
Assay. Dissolve 4g, accurately weighed, in sufficient water to produce 100 ml.
Transfer 25 ml to a separator, add 25 ml of chloroform R, 10 ml of sodium
hydroxide VS, and 10ml of freshly prepared potassium iodide
(0.1 mol/1)
(50 g/1) TS. Shake well, allow to separate, and run off the chloroform layer.
Shake the aqueous solution with 3 further quantities of chloroform R, each
of 10ml, and discard the chloroform solutions. Add 40ml of hydrochloric
acid (-420 g/1) TS, cool, and titrate with potassium iodate (0.05 mol/1) VS
until the solution becomes pale brown in colour. Add 2 ml of chloroform R
and continue the titration until the chloroform becomes colourless. Titrate
a mixture of 20ml of water, 6ml of potassium iodide (80g/l) TS, and 40ml
of hydrochloric acid (-420 g/1) TS with potassium iodate (0.05 mol/1) VS in
a similar manner; the difference between the titrations represents the
amount of potassium iodate (0.05 mol/1) VS required. Each ml of potassium
iodate (0.05mol/l) VS is equivalent to 35.40mg of C22H40CIN. Determine the
1298
mass density using a pycnometer as described in 1.3 Determination of mass

density and relative density and calculate in g/1 the proportion of C22H40CIN.
Note: A solution in water foams strongly when shaken.
Benzalkonium chloride TSl

ml of benzalkonium
Procedure. Dilute 2 chloride TS with sufficient water to
produce 100 ml.
Benzene R. (SKIP, 1963, p. 48).
Benznidazole RS. International Chemical Reference Substance.
Benzoic acid R. C/HgOj. Contains not less than 99.8% of C7H6O2.

Description. Colourless, light, feathery crystals or a white, microcrystalline
powder; odour, characteristic, faint.
Solubility. Slightly soluble in water; freely soluble in ethanol (-750 g/1) TS, and
ether R.
Methanol-insoluble substances. Dissolve 20 g in 200 methanol R and digest
ml of
under complete reflux for 30 minutes. Filter through a tared filtering cru-
cible, wash thoroughly with methanol R, and dry at 105 °C; it leaves a
residue of not more than 1.0 mg.
Assay. Dissolve about 0.5g, accurately weighed, in 15ml of ethanol (~750g/l)
TS, previously neutralized to phenol red/ethanol TS, add 20 ml of water and
titrate with sodium hydroxide (0.1 mol/1) VS, using phenol red/ethanol TS
as indicator. Repeat the operation without the substance being examined

and make any necessary correction. Each ml of sodium hydroxide (0.1 mol/1)
VS is equivalent to 12.21 mg of C7H6O2.
Benzophenone R. Diphenyl ketone; C,3H,oO.

A commercially available reagent of suitable grade.
Melting point. About 49°C.
Benzoyl chloride R. C7H5CIO (SRIP, 1963, p. 50).
Benzoyl peroxide, hydrous R. C14H10O4, x H2O.

Description.A white, amorphous or granular powder.
Note: For safety reasons it should be kept moistened with about 23% w/w
water.
Benzyl alcohol R. C7H8O.

Description. A colourless liquid; almost odourless.
Miscibility. Miscible with 25 parts of water; miscible with ethanol (-750 g/1) TS,
and ether R.
Boiling temperature. About 204 °C.
Mass density. p2o = about 1.05 kg/1.
1299
Benzyl benzoate R. C 14 H 12 O 2 .
Benzyl benzoate as described in the monograph for “Benzyl benzoate".

Description. A clear, colourless, oily liquid.
Benzylpenicillin potassium RS. International Chemical Reference

Substance.
Benzylpenicillin sodium R. C,6Hi7N2Na04S. Contains not less than 96.0%

and not more than 102.0% of C]eH]7N2Na04S, calculated with reference to
Description. A white or almost white, crystalline powder; odourless or with a
Solubility. Soluble in about 0.5 part of water; practically insoluble in ether R.
Benzylpenicillin sodium RS. International Chemical Reference Substance.
Benzylpenicillin sodium TS
Procedure. Dissolve 0.03 g of benzylpenicillin sodium R in sufficient phosphate
buffer, pH 7.0, TS, to produce 10ml. This solution contains not lessthan 3
mg/ml of benzylpenicillin sodium R.
Bephenium hydroxynaphthoate RS. International Chemical Reference

Substance.
Betamethasone RS. International Chemical Reference Substance.
Betamethasone sodium phosphate RS. International Chemical Reference

Substance.
Betamethasone valerate RS. International Chemical Reference Substance.
Biperiden hydrochloride RS. International Chemical Reference Substance.
Biperiden RS. International Chemical Reference Substance.
4,4'-Bis(dimethyIamino)benzophenone R. Tetramethyldiaminobenzophe-
none; C17H20N2O.
Other name. Michler’s ketone.
9,9'-Bisanthracene-10,10'(9H,9'H)-dione RS. International Chemical Ref-

erence Substance.
Bismuth oxynitrate R. Approximately 4BiN03(0H)2,Bi0(0H) (SRIP, 1963,

p. 50).
1300
Bismuth subnitrate R. Bismuth hydroxide nitrate oxide; Bi50(0H)9(N03)4,

Bismuth subnitrate is a basic salt, the composition of which varies with the
conditions under which it is prepared. It contains not less than 71.5% and
not more than 74.5% of Bi, calculated with reference to the dried substance.
Descrifetion. A white powder.
Solubility. Practically insoluble in water and ethanol (~750g/l) TS; soluble in
hydrochloric acid (-250 g/1) TS and nitric acid (-1000 g/1) TS.
Blue tetrazolium R. C40H32CI2NSO2. 3, 3'-Dianisole-bis-[4,4'-(3, 5-diphenyl)
tetrazolium chloride].
Description. Lemon-yellow crystals.
Solubility. Slightly soluble in water; freely soluble in ethanol (-750 g/1) TS, and
methanol R; practically insoluble in acetone R and ether R.
Molar absorptivity. Its molar absorptivity in methanol R at 252 nm, is not less
than 60000.
Suitability test. Prepare the following standard solution: Dissolve in ethanol
(-750 g/1) TS a suitable quantity of hydrocortisone R, previously dried at
105 °C for 3 hours and accurately weighed, and prepare by a step-by-step
dilution a solution containing about 30|ig/ml. Transfer 10-, 15- and 20-ml
quantities of the standard solution to separate glass-stoppered 50-ml conical
flasks. Add 10ml and 5ml, respectively, of ethanol (~750g/l) TS to the flasks
containing the 10- and 15-ml quantities of the standard solution and swirl
to mix. To each of the flasks, and to a fourth flask, representing the blank,
containing 20ml of ethanol (-750 g/1) TS, add 2.0 ml of a solution prepared
by dissolving 0.05g of the blue tetrazolium R in 10ml of ethanol (-750g/l)
TS, mix, and then add 2.0ml of tetramethylammonium hydroxide/ethanol
TS. Mix, allow the flasks to stand in the dark for 90 minutes, and determine
the absorbances at 525 nm against the blank. Plot the absorbances on the
abscissa and the amount of hydrocortisone on the ordinate scale of arith-
metic coordinate paper, and draw the curve of best fit; the absorbance of
each solution is proportional to the concentration and the absorbance of the
solution containing 200 pg of hydrocortisone is not less than 0.50.
Blue tetrazolium/ethanol TS
Procedure. Dissolve 0.5 g of blue tetrazolium R in sufficient aldehyde-free
ethanol (-750 g/1) TS, warming slightly if necessary, to produce 100 ml.
Blue tetrazolium/sodium hydroxide TS

Procedure. Immediately before use, mix 1 volume of a 2 mg/ml solution of blue
tetrazolium R in water with 3 volumes of a 0.12g/ml solution of sodium
hydroxide R in methanol TS.
Borate buffer, pH 8.0, TS

Procedure. Dissolve 0.25 g of boric acid R and 0.30 g of potassium chloride R in
50ml of carbon-dioxide-free water R, add 3.97ml of carbonate-free sodium
1301
hydroxide (0.2 mol/1) VS, and dilute with sufficient carbon-dioxide-free

water R to produce 200 ml,

Procedure. Dissolve 1.24g of boric acid R in about 100 ml of water, add 8.3ml
of sodium hydroxide (1 mol/1) VS, and add sufficient water to produce
200 ml.

Procedure. Dissolve 0.25 g of boric acid R and 0.30 g of potassium chloride R in
50 ml of carbon-dioxide-free water R, add 36.85 ml of carbonate-free sodium
hydroxide (0.2 mol/1) VS, and dilute with sufficient carbon-dioxide-free
water R to produce 200 ml,
Boric acid (50g/l) TS. A solution of boric acid R containing about 50g/l of
H3BO3.
Boric acid R. H3BO3. Contains not less than 99.0% of H3BO3.

Description. White crystals or scales of a somewhat pearly lustre or a white,
crystalline powder.
Solubility.Soluble in 20 parts of water, in 3 parts of boiling water, and in 16
parts of ethanol (~750g/l) TS.
Water-insoluble substances. 1.0 g dissolves in 30 parts of water; the solution is

Ethanol- insoluble substances. l.Og dissolves in 10ml of boiling ethanol (~750g/l)
TS; the solution is not more than faintly turbid.
Assay. Dissolve about 1 g, accurately weighed, in 30ml of water; add 50ml of
and titrate
glycerol R, previously neutralized to phenolphthalein/ethanol TS,
with carbonate-free sodium hydroxide (1 mol/1) VS, using phenolph-
thalein/ethanol TS as indicator. Each ml of carbonate-free sodium hydrox-
ide (1 mol/1) VS is equivalent to 61. 83 mg of H3BO3.
Brilliantgreen R. [4-[^-(Diethylamino)-a-phenylbenzylidene]-2,5-cyclohexa-
dien-l-ylidene]diethylammonium hydrogen sulfate; C27H34N2O4S; C.l.
42040; Malachite green G; C.L Basic green 1.
Description. Small, glistening, golden crystals.
Brilliant green/acetic acid TS

Procedure. Dissolve 0.5 g of brilliant green R in sufficient glacial acetic acid R1
to produce 100 ml.
Bromine AsTS
Procedure. Dissolve 30 g of potassium bromide R in 40 ml of water, add 30 g of
bromine R and dilute with sufficient water to produce 100 ml. The solution
complies with the following test: Evaporate 10 ml nearly to dryness on a
water-bath, add 50ml of water, 10ml of hydrochloric acid (-250 g/1) AsTS,
1302
and sufficient stannous chloride AsTS to reduce the remaining bromine, and
apply the general test for arsenic. The colour of the stain produced is not
more intense than that produced from a 1-ml standard stain, showing that
the amount of arsenic does not exceed 1 pg/ml.
Bromine R. Br2 (SKIP, 1963, p. 51).
Bromine TSl. A saturated solution of bromine R.
Bromocresol green R. C 2 iH] 4Br405 S (SKIP, 1963, p. 52).
Bromocresol green TSl

Procedure. Dissolve 0.05g of bromocresol green R and 1.021 g of potassium
hydrogen phthalate R in 6ml of sodium hydroxide (0.2 mol/1) VS, and dilute
to 100 ml with water. Filter if necessary.
Bromocresol green/ethanol TS
Procedure. Warm 0.1 g of bromocresol green R with 2.9 ml of sodium hydrox-
ide (0.05mol/l) VS and 5ml of ethanol (~710g/l) TS; after solution has been
effected, add a sufficient quantity of ethanol (~150g/l) TS to produce
250 ml.
Bromocresol purple R. C 2 iHigBr205 S (SRIP, 1963, p. 52).
Bromocresol purple/ethanol TS
Procedure. Dissolve 0.05g of bromocresol purple R in 100ml of ethanol
(-750 g/1) TS, and filter if necessary.
Bromophenol blue (1 g/1) TS. A solution of bromophenol blue R containing

about l.Og of C, 9 HioBr 405 S per litre.
Bromophenol blue R. Ci 9 H]oBr 405 S (SRIP, 1963, p. 52).
Bromophenol blue TS
Procedure. Dissolve 0.05 g of bromophenol blue R with gentle heating in
3.73 ml of sodium hydroxide (0.02 mol/1) VS and dilute to 100 ml with water.
Bromophenol blue/ethanol TS
Procedure. Warm 0.1 g of bromophenol blue R with 3.2 ml of sodium hydrox-
ide (0.05mol/l) VS and 5ml of ethanol (~710g/l) TS; after solution has been
250 ml.
Bromothymol blue R. C27H2aBr205S (SRIP, 1963, p. 53).
1303
Bromothymol blue/dimethylformamide TS
Procedure. Dissolve 1.0 g of bromothymol blue R in sufficient dimethylfor-
mamide R to produce 100 ml,
Bromothymol blue/ethanol TS
Procedure. Warm 0.1 g of bromothymol blue R with 3.2ml of sodium hydrox-
ide (0.05mol/l) VS and 5ml of ethanol (-710g/l) TS; after solution has been
250 ml.
Brown stock standard TS

Procedure. To 35.0ml of cobalt colour TS, add 17.0ml of copper colour TS,
8.0ml of dichromate colour TS, dilute to 100.0ml with iron colour TS,
and mix.
Bupivacaine hydrochloride RS. International Chemical Reference

Substance.
2 -Butanol R. C 4 H 10 O (SRIP, 1963, p. 53).
1 -Butanol R. [«-Butanol R]. C 4 H 10 O (SRIP, 1963, p. 54).

tert. -Butanol R. 2-Methylpropan-2-ol; (CH 3) 3 COH.
Description. A colourless liquid or solid.

Miscibility. Miscible with water, ethanol (-750 g/1) TS, and ether R.
Boiling range. Not less than 95% distils between 81 and 83 °C,
Melting range. 24-26 °C,
Mass density. p 2 o = 0.778-0.782 kg/1.
Residue on evaporation. Evaporate on a water-bath and dry to constant weight
at 105 °C; it leaves a residue of not more than 0.05mg/ml.
Butyl acetate R. C6H12O2.

Description. A clear, colourless, flammable liquid; odour, characteristic.
Miscibility. Slighdy miscible with water; miscible with ethanol (-750 g/1) TS.
Mass density. p 2 o = about 0.88 kg/1.
tert-Butyl methyl ether R. 1,1-Dimethylethyl methyl ether; CH

5 12 O,
Description.A clear, colourless liquid; inflammable.
Refractive index, nf? = 1.3756.
Relative density, df = 0.740-0.742.
1 -Butylamine R. 1-Aminobutane; C 4HnN.

Description. A colourless to pale yellow inflammable liquid.
Boiling range. Not less than 95% distils between 76 and 78 °C.
1304

Fischer method, using about 5ml of the liquid; not more than lOmg/ml.
Acid impurities. To 50ml add 5 drops of azo violet TS, and titrate quickly with
sodium methoxide (0.1 mol/1) VS to a deep blue endpoint, taking precau-
tions to prevent absorption of atmospheric carbon dioxide, e.g. by use of
an atmosphere of nitrogen; not more than 1.0 ml of sodium methoxide
(0.1 mol/1) VS is required for neutralization.
Butylated hydroxyanisole R. Use C],Hi(i02.

Butylated hydroxyanisole as described in the monograph in “Butylated hydrox-
yanisole'’.
Butylated hydroxytoluene R. 2,6-Di-iert-butyl-4-methylphenol, C15H24O.

Description. Colourless crystals, or a white, crystalline powder.
Solubility. Practically insoluble in water; freely soluble in ethanol (-750 g/1) TS;
very soluble in ether R.
Melting temperature. About 70°C.
Cadmium acetate R. (CH3C02)2Cd,2H20. Contains not less than 98.0% of

(CH3C02)2Cd,2H20.
Assay. Dissolve Ig, accurately weighed, in 50ml of water, add 25ml of
ammonia (-260 g/1) TS, and titrate with disodium edetate (0.1 mol/1) VS,
using methylthymol blue mixture R as indicator, until the blue solution
becomes Each ml of disodium edetate
colourless or grey. (0.1 mol/1) VS is
equivalent to 26.65mg of (CH3C02)2Cd,2H20.
Caesium chloride R. CsCl.

Caffeine RS. International Chemical Reference Substance.
Calcium acetate (0.25 mol/1) VS. Calcium acetate R, dissolved in water to

contain 44.04 g of Ca(C2H302)2,H20 in 1000ml.
Calcium acetate R. Ca(C2H302)2,H20 (SRIP, 1963, p. 56).
Calcium carbonate Rl. CaCOj (SRIP, 1963, p. 56).
Calcium carbonate R2. CaC03. Calcium carbonate Rl of suitable quality to

serve as a primary standard for the standardization of disodium edetate.
Calcium chloride (3.7g/l) TS. A solution of anhydrous calcum chloride R con-

taining about 9g of CaCl2 per litre.
1305
Calcium chloride (55 g/1) TS. A solution of hydrated calcium chloride R con-
taining about 55g/l of CaCl2 (approximately 0.5mol/l).
Calcium chloride, anhydrous, R. [calcium chloride R], CaCh (SRIP, 1963,

p. 58).
Calcium chloride, hydrated, R. CaCl2,6H20 (SRIP, 1963, p. 58).
Calcium fluoride R. Cap 2 .
Description. A white powder.

Solubility. Practically insoluble in water; slightly soluble in dilute acids.
Calcium folinate RS. International Chemical Reference Substance.
Calcium hydroxide R. Ca(OH)2 (SRIP, 1963, p. 59).
Calcium hydroxide TS
Procedure. Prepare a saturated solution of calcium hydroxide R.
Note: Calcium hydroxide TS must be freshly prepared.
Calcium standard (lOpg/ml Ca) TS

Procedure. Dissolve 2.50 g of dried calcium carbonate R2 in 15 ml of acetic acid
(-300 g/1) TS and dilute with water to 1000ml (solution A). Dilute 10.0ml
of this solution with water to produce 1000ml.
Calcium standard (lOOpg/ml Ca), ethanolic, TS

Procedure. Dilute 100.0 ml of solution A, described under calcium standard
(lOpg/ml Ca) TS, with sufficient ethanol (-750 g/1) TS to produce 1000ml.
Calcium sulfate R. CaS04,2H20 (SRIP, 1963, p. 62).
Calcium sulfate TS
Procedure. Shake 5g of calcium sulfate hemihydrate R for 1 hour with 100 ml
of water and filter.
Calcium sulfate, hemihydrate R. Plaster of Paris, CaS04, | H2O.

Description. A white powder which, when mixed with half its weight of water,
rapidly solidifies to a hard and porous mass.
Calcon carboxylic acid indicator mixture R

Procedure. Mix 0.1 g of calcon carboxylic acid R with 10 g of anhydrous sodium
sulfate R.
Calcon carboxylic acid R. 2-Hydroxy-l-(2-hydroxy-4-sulfo-l-naphthyl-azo)-

3-naphthoic acid; C2iHi4N207S,3H20.
Description. A dark-brown powder with a violet tint.
1306
Solubility. Practically insoluble in water; slightly soluble in methanol R and in

ethanol (-750 g/1) TS; freely soluble in solutions of alkali hydroxides.
Calcon indicator mixture R

Mix 0.1 g of calcon R with lOg
Procedure. of anhydrous sodium sulfate R.
Calcon R. Monosodium salt of 2-hydroxy-l-[(2-hydroxy-l-naphthyl)-

azo]naphthalene-4-sulfonic acid; C.l. Mordant Black 17, C.l. No. 15705, Eri-
ochrome Blue Black R, Solochrome Dark Blue; C 2 oH] 3 N Na 05 S.
2
Captopril disulfide RS. International Chemical Reference Substance.
Captopril RS. International Chemical Reference Substance.
Carbamazepine RS. International Chemical Reference Substance.
Carbidopa RS. International Chemical Reference Substance.
Carbomer R. Carbomer suitable for thin-layer chromatography. A high rela-

tive molecular mass cross-linked polymer of acrylic acid; it contains a large
proportion (56-68%) of carboxylic acid (-COOH) groups after drying at
80 °C for 1 hour.
pH value. The pH about 3.
of a 10 g/1 suspension is
Viscosity. Whilst stirring continuously, prepare a suspension containing 2.5 g in
500 ml of water. Maintain at 25 + 0.2 °C for 30 minutes, then add 0.2 ml of

phenolphthalein/ethanol TS, 1 ml of bromothymol blue/ethanol TS and,
whilst stirring, neutralize using a mixture of equal volumes of sodium
hydroxide (-400 g/1) TS and water until a uniform blue colour is obtained
(check the pH which must be 7. 3-7. 8). The dynamic viscosity of the neu-
tralized preparation is 30-40 Pas (300-400 poise).
Carbon dioxide detector tube. A cylindrical, sealed glass tube containing

adsorbent filters and suitable supports for hydrazine and crystal violet indi-
cators. The minimum value indicated is lOOpl/1 or less,with a relative stan-
dard deviation of at most +15%. Tubes can be verified with a calibration
gas containing the appropriate impurity, if a negative result is obtained.
Carbon dioxide R. COj.

Solubility. Soluble in about 1.3 parts by volume of water.
Carbon disulfide IR. Carbon disulfide R that complies with the following
test: The infrared absorption spectrum of a 1.0-mm layer, as described in
method 4 under 1.7 Spectrophotometry in the infrared region, and exam-

ined over the range 4000-670cm~' shows an absorbance of less than 0.1 in
1307
the regions 4000-3030 cm 2635-2440 cm 2000-1755 cm \ and 1265-

935 cm“', and an absorbance of less than 0.17 in the region 800-715 cm“‘.
Carbon disulfide R. CSj (SKIP, 1963, p. 62).
Carbon monoxide detector tube. A cylindrical, sealed glass tube containing

adsorbent filters and suitable supports for di-iodine pentoxide, selenium
dioxide and fuming sulfuric acid indicators. The minimum value indicated
is 5pl/l or less, with a relative standard deviation of at most +15%. Tubes
can be verified with a calibration gas containing the appropriate impurity,
if a negative result is obtained.
Carbon monoxide R. CO.

A commercially available gas of suitable grade.
Carbon tetrachloride R. CCI 4 (SKIP, 1963, p. 63).
Carboxymethylcellulose R. A suitable grade for column chromatography.
Cellulose Rl. Microcrystalline cellulose suitable for thin-layer chromatography.

Description. A fine, white homogeneous powder.
Particle size. Less than 30 p.m.
Note: A
suspension of about 25 g of cellulose Rl in 90 ml of water is used in
the preparation of the coating for thin-layer chromatographic plates.
Cellulose R2. Cellulose suitable for thin-layer chromatography.

Particle size. Less than 30 pm.
Note: A suspension of about 15 of cellulose R2 in 100 ml of water is used in the
preparation of the coating for thin-layer chromatographic plates.
Cellulose R3. Cellulose suitable for thin-layer chromatography.

Composition. Cellulose (particle size less than 30 pm) containing a fluorescent
indicator having an optimal intensity at 254 nm.
Note: A
suspension of about 25 g of cellulose R3 in 100ml of water is used in
the preparation of the coating for thin-layer chromatographic plates.
Cephaeline hydrochloride R. C28H3aN204,2HCl,7H20.

Specific optical rotation. Use a 20mg/ml solution; [a]n = +25°.
Cephalin TS
Procedure. Place a quantity between 0.5 and l.Og of acetone-dried ox brain R
into a centrifuge tube, add 20 ml of acetone R, and allow to stand for 2 hours.
Centrifuge for 2 minutes and decant the supernatant liquid. Dry the residue
1308
under reduced pressure, add to it 20 ml of chloroform R, and allow to stand

for 2 hours, shaking frequently. Separate the solid materialby filtration or
centrifugationand evaporate the chloroform under reduced pressure.
Suspend the residue in 5-1 0ml of sodium chloride (9g/l) TS. Solvents used
to prepare cephalin TS should contain a suitable antioxidant, for example,
a solution of 0.02 g/1 of butylated hydroxyanisole R.
Storage. Store in a freezer or keep in a freeze-dried state.
Note: The reagent must be used within 3 months.
Ceric ammonium VS
nitrate (0.01 mol/1)
Procedure. Dissolve 5.482 g of ceric ammonium nitrate R in sufficient nitric acid
(1 mol/1) VS to produce 1000 ml, and filter.

solution in the following manner: Measure accurately 2.0ml of freshly stan-
dardized ferrous ammonium sulfate (0.1 mol/1) VS into a flask and dilute
with water to about 100ml. Add 1 drop of nitrophenanthroline TS, and
titrate with the ceric ammonium nitrate solution to a colourless end-point.
From the volume of ferrous ammonium sulfate (0.1 mol/1) VS taken and
the volume of ceric ammonium nitrate solution consumed, calculate the
molarity.
Ceric ammonium nitrate R. Ce(N 03 ) 4,2 NH N 03

4 .
Description. Small orange-red monoclinic crystals.

Solubility.Very soluble in water.
Insoluble matter. To 5g, accurately weighed, add 10 ml of sulfuric acid
(-1760 g/1) TS, stir, and cautiously add 90 ml of water to dissolve. Heat to
boiling and digest in a covered beaker on a water-bath for 1 hour. Filter
through a tared filtering crucible, wash thoroughly, and dry at 105 °C. The
weight of the residue does not exceed 2.5 mg.
Assay. Dissolve 2.5g, accurately weighed and previously dried at 85 °C for 24
hours, in 10ml of sulfuric acid (-190 g/1) TS and add 40ml of water. Add a
few drops of o-phenanthroline TS and titrate with ferrous sulfate (0.1 mol/1)
VS. Each ml of ferrous sulfate (0.1 mol/1) VS is equivalent to 54.8 mg of
Ce(N03)4,2NH4N03.
Ceric ammonium sulfate (0.1 mol/I)VS

Procedure. Dissolve 65.0 g of ceric ammonium sulfate R in a mixture of 500ml
of water and 30 ml of sulfuric acid (-1760 g/1) TS. Allow to cool and dilute
solution in the following manner: Accurately weigh about 0.2 g of arsenic
trioxide R1 and dissolve by gently heating in 15 ml of sodium hydroxide
(0.2 mol/1) VS. Add to the clear solution 50 ml of sulfuric acid (-100 g/1) TS,
0.15ml of a 2.5mg/ml solution of osmium tetroxide R in sulfuric acid
(-100 g/1) TS, and 0.1ml of o-phenanthroline TS. Titrate the solution with
1309
the ceric ammonium sulfate solution until the red colour disappears. Titrate
slowly as the end-point is approached.
Ceric ammonium sulfate R. Ammonium cerium (IV) sulfate dihydrate,

Ce(S04)2,2(NH4)2S04,2H20. Contains not less than 95.0% of
Ce(S04)2,2(NH4)2S04,2H20.
Description. Yellow crystals or an orange -yellow, crystalline powder.
Solubility. Slowly soluble in water; insoluble in ethanol (-750 g/1) TS.
Assay. Dissolve about 1 g, accurately weighed, in 50 ml of sulfuric acid
(-100 g/1) TS, add 0.1 ml of alOmg/ml solution of osmium tetroxide R, and
titrate with sodium arsenite (0.05 mol/1) VS, using o-phenanthroline TS
as indicator. Each ml of sodium arsenite (0.05 mol/1) VS is equivalent to
63.26mg of Ce(S04)2,2(NH4)2S04,2H20.
Ceric ammonium sulfate/nitric acid TS

Procedure. Dissolve 5g of ceric ammonium sulfate R in sufficient nitric acid
(-130 g/1) TS to produce 100ml.
Ceric amonium nitrate TS

Procedure. Dissolve 6.25 g of ceric ammonium nitrate R in 10 ml of nitric acid
(15g/l)TS.
Shelf-life. Use within 3 days of preparation.
Ceric sulfate (O.lmoI/1) VS

Procedure. Dissolve ceric sulfate R, equivalent to 33.23 g of Ce(S04), in a mixture
of 28ml of sulfuric acid (~1760g/l) TS and 500ml of water, dilute to
1000 ml and mix. Allow the solution to stand for 48 hours and filter through
a sintered glass filter.

solution in the following manner: Place about 25ml, accurately measured,
in a glass-stoppered flask, dilute with 80ml of water, add 10ml of phos-
phoric acid (-105 g/1) TS and and allow the solu-
2.5 g of potassium iodide R,
tion to stand for 15 minutes. Add 1g of sodium carbonate R and titrate with
sodium thiosulfate (0.1 mol/1) VS, using starch TS as indicator.
Ceric sulfate (35 g/1) TS. A solution of ceric sulfate R containing about 33 g/1
of Ce(S04)2.
Ceric sulfate R. Usually Ce(S04)2,4H20 (SRIP, 1963, p. 63).
Charcoal R. (SRIP, 1963, p. 64).
Chloralose R. CgHnClsO^.
Description. A colourless, crystalline powder.
1310
Specific optical rotation. Use a 50 mg/ml solution in ethanol (-750 g/1) TS; [a]™
= +19°.
Chloramphenicol disodium disuccinate RS. International Chemical Refer-

ence Substance.
Chloramphenicol palmitate RS. International Chemical Reference

Substance.
Chloramphenicol sodium succinate RS. International Chemical Reference

Substance.
Chloramphenicol RS. International Chemical Reference Substance.
Chloraniline R. 4-Chloroaniline, CeHgClN.

Description. White or faintly coloured crystals.
Chloride standard (5 |ug/l) TS

Procedure. Weigh accurately 82.4 mg of sodium chloride R and dissolve in suf-
ficient water to produce 100ml. Dilute 1.0ml of this solution with water to
100 ml.
1-
Chlorine R. Clj (SRIP, 1963, p. 65).
Chlorine TS. A saturated solution of chlorine R in water.

Note: Chlorine TS must be freshly prepared.
2-
7 -Chloro-1 - cyclopropyl- 1 ,4-dihydro -4 -0X0 -6 -(pip erazin-1 -yl)

quinoline-3-carboxylic acid RS (ciprofloxacin by-compound A). Interna-
tional Chemical Reference Substance.
l-Chloro-2,4 dinitrobenzene R. C 6 H 3 CIN 2 O 4 .

Meltingpoint. About 144°C.
Chloro-2,4 dinitrobenzene/ethanol TS
Procedure. Weigh 5 g of TChloro-2,4 dinitrobenzene R and dissolve in sufficient
ethanol (-750 g/1) TS to produce 100ml.
5-Chloro-2-methylaminobenzophenone RS. International Chemical Ref-

erence Substance.
(4-Chloro-3-sulfamoyl)benzoic acid RS. International Chemical Refer-

ence Substance.
1311
2 -ChIoro- 4 -nitroaniline R. C6H5CIN2O2.

Description. A yellow to brown, crystalline powder.
Solubility. Slightly soluble in water; soluble in ethanol (-750 g/1) TS.
Melting range. 106-108°C,
4 -Chloroacetanilide R. CeHaClNO.
Description. Colourless, needle-shaped crystals or a white to pale yellow, crys-
talline powder.
Solubility. Practically insoluble in water; soluble in ethanol (~750g/l) TS and

ether R.
Chloroform R. CHCI3 (SKIP, 1963, p. 66).
Chloroform, ethanol-free, R
Procedure. Shake 20 ml of chloroform R gently but thoroughly with 20 ml of
water for 3 minutes, draw of the chloroform layer, and wash twice more
with 20-ml quantities of water. Finally filter the chloroform through a dry
filter-paper, shake it well with 5 g of powdered anhydrous sodium sulfate R
for 5 minutes, allow the mixture to stand for 2 hours, and decant or filter
the clear chloroform.
Chloroquine sulfate RS. International Chemical Reference Substance.
Chlorphenamine hydrogen maleate RS. International Chemical Reference

Substance.
Chlorpromazine hydrochloride RS. International Chemical Reference

Substance.
Chlortalidone RS. International Chemical Reference Substance.
Chlortetracycline hydrochloride RS. International Chemical Reference

Substance.
Chromic acid TS
Procedure. Dissolve 84 g of chromium trioxide R in 700 ml of water and add
slowly while stirring 400 ml of sulfuric acid (-1760 g/1) TS.
Chromium trioxide R. CrOj (SRIP, 1963, p. 68).
Ciclosporin RS. International Chemical Reference Substance.
Ciclosporin U RS. International Chemical Reference Substance.
1312
Cimetidine RS. International Chemical Reference Substance.
Cinchonidine R. C 19 H 22 N 2 O.
Solubility. Soluble in ethanol (-750 g/1) TS.
= -105 to -110°.
Cinchonine R. C]9H22N20 (SRIP, 1963, p. 69).
Ciprofloxacin hydrochloride RS. International Chemical Reference

Substance.
Ciprofloxacin RS. International Chemical Reference Substance.
Cisplatin RS. International Chemical Reference Substance.
Citrate buffer, pH 4.0, TS

Procedure. Dissolve 10. 5g of citric acidR in about 100ml of water, add 100ml
of sodium hydroxide (1 mol/1) VS, and dilute to 500 ml with water. Dilute
100 ml of hydrochloric acid (0.1 mol/1) VS with the solution prepared above
to produce 250 ml.
Citrate buffer, pH 5.4, TS

Procedure. Dissolve 2.101 g of citric acid R in water, add 20ml of sodium
hydroxide (1 mol/1) VS, and dilute with sufficient water to produce 100ml.
Mix 76.5ml of this solution with 23.5ml of sodium hydroxide (0.1 mol/1)
VS.
Citric acid (180g/l) FeTS. A solution of citric acid FeR containing about
183 g/1 of C^HsO;.
Citric acid (20g/I) TS. A solution of citric acid R containing about 20 g of

CeHaO/ per litre.
Citric acid FeR. Citric acid R that complies with the following test: Dissolve
0.5 g of citric acid R in 40 ml of water, add 2 drops of mercaptoacetic acid
R, mix, make alkaline with ammonia (-100 g/1) FeTS and dilute to 50 ml with
water; no pink colour is produced.
Citric acid PbR. Citric acid R free of lead.
Citric acid R. C,Hs 07 ,H 20 (SRIP, 1963, p. 69).
1313
Citric acid, copper-free, R. Citric acid R, that complies with the following
additional test: Dissolve 0.50 g in 20ml of water, make alkaline with
ammonia (~100g/l) TS, dilute to 50 ml with water, and add 1 ml of sodium

diethyldithiocarbamate (0,8g/l) TS; no yellow colour is produced.
Clindamycin hydrochloride RS. International Chemical Reference

Substance.
Clindamycin phosphate RS. International Chemical Reference Substance.
Clofazimine RS. International Chemical Reference Substance.
Clomifene citrate RS. International Chemical Reference Substance.
Clomifene citrate Z-isomer RS. See Zuclomifene RS.
Cloxacillin sodium RS. International Chemical Reference Substance.
Cobalt colour TS. A solution containing 60.0g/l of CoCl2 6 H 20 ,

.
Procedure. Prepare a solution containing 6.000 g of CoCl2 6H 20 in 100 ml by dilut-

,
ing the strong cobalt colour TS with sulfuric acid (~10g/l) TS, as necessary.
Cobalt colour, strong, TS

Procedure. Dissolve 8.0 g of cobaltous chloride R in 120 ml of sulfuric acid
(~10g/l) TS, filter the solution, if necessary, and determine the concentra-
tion of CoCl2 6 H 20
,
.
Assay. Dilute 5.0 ml withsufficient water to produce 100 ml. Transfer 10.0ml
of this solution to a glass-stoppered flask, add 10ml of water, 0.5ml of
hydrogen peroxide (-60g/l) TS and 10ml of sodium hydroxide (-80g/l) TS.
Add a few boiling chips to the flask, and boil the contents of the flask until
the excess of hydrogen peroxide is completely decomposed (approximately
10 minutes). Cool the flask, add 20 ml of water, 1 g of potassium iodide R,
and 25ml of hydrochloric acid (2 mol/1) VS. Close the flask with the stopper
and allow to stand until the precipitate dissolves. Titrate the liberated iodine
with sodium thiosulfate (0.01 mol/1) VS using starch TS as indicator. Each
ml of sodium thiosulfate (0.01 mol/1) VS is equivalent to 2.380mg of
CoCl2,6H20.
Cobalt(II) chloride (30g/I) TS. A solution of cobalt(II) chloride R containing

about 30 g of C 0 CI 2 per litre.
Cobalt(II) chloride (5g/l) TS. A solution of cobalt(II) chloride R containing

about 5g of C 0 CI 2 per litre.
Cobalt(II) chloride R. Cobaltous chloride; CoCl2 6H 20

,
(SRIP, 1963, p. 70).
1314
Cobalt(II) chloride TS
Procedure. Dissolve 6.5 g of cobalt(II) chloride R in a sufficient quantity of a
mixture of 2.5ml of hydrochloric acid (-250 g/1) TS and 97.5 ml of water to
produce 100 ml.
Cobalt(II) nitrate (lOg/I) TS

Procedure. Dissolve about 1.6g of cobalt(ll) nitrate R in sufficient water to
produce 100 ml.
Cobalt(II) nitrate (lOOg/1) TS. A solution of cobalt(ll) nitrate R containing

about lOOg of Co(N 03)2 per litre.
Cobalt(II) nitrate R. Co(N 03) 2 6 H 20

,
.
Description. Small red crystals.

Solubility. Very soluble in water.
Cobaltous chloride R. CoCl2,6H20 (SRIP, 1963, p. 70).
Cobaltous chloride TS
Procedure. Dissolve 6.5 g of cobaltous chloride R in a sufficient quantity of a
mixture of 2.5ml of hydrochloric acid (-250 g/1) TS and 97.5 ml of water to
produce 100 ml.
Codeine R. CisH 2 iN 03 ,H 20 .

ether R.
= -142 to - 146°.
Colbatous thiocyanate TS
Procedure. Dissolve 6.8 g of cobaltous chloride R and 4.3 g of ammonium thio-
cyanate R in sufficient water to produce 100 ml.
Colchicine RS. International Chemical Reference Substance.
Colecalciferol RS. International Chemical Reference Substance.
Congo red paper R. (SRIP, 1963, p. 72).
Copper colour TS. A solution containing 60.0 g/1 of CuS04,5H20.

Procedure. Prepare a solution containing 6.000g of CuS04,5H20 in 100ml by
diluting the strong copper colour TS with sulfuric acid (-10 g/1) TS, as
necessary.
1315
Copper colour, strong, TS

Procedure. Dissolve 8.0 g of copper(II) sulfate R in 120 ml of sulfuric acid
(~10g/l) TS, filter the solution, if necessary, and determine the concentra-
tion of CuS 04,5H20.
Assay. Dilute 5.0 ml withsufficient water to produce 100 ml. Transfer 10.0ml
of this solution to a glass-stoppered flask, add 20 ml of water, Ig of potas-
sium iodide R, and 5 ml of glacial acetic acid R. After 10 minutes, titrate the
liberated iodine with sodium thiosulfate (0.01 mol/1) VS, using starch TS as
indicator. Each ml of sodium thiosulfate (0.01 mol/1) VS is equivalent to
2.497mg of CuS04,5H20.
Copper edetate TS
Procedure. To 2 ml of a 20 mg/ml solution of copper(ll) acetate R, add 2 ml of
disodium edetate (0.1 mol/1) VS, and dilute to 50ml with water.
Copper standard (lOpg/ml Cu) TS

Procedure. Dissolve 0.393 g of copper(Il) sulfate R in sufficient water to produce
100ml and dilute 10.0ml of this solution to produce 1000ml.
Copper standard TSl

Procedure. Dissolve 1.965 g of copper(ll) sulfate R, accurately weighed, in suffi-
cient hydrochloric acid (0.1 mol/1) VS to produce 1000 ml.
Copper standard TS2

Procedure. Transfer3.0ml of copper standard TSl to a 1000-ml flask and dilute
with hydrochloric acid (0.1 mol/1) VS to produce 1000ml. This solution con-
tains 1.5pg of Cu per ml.
Copper tetramine hydroxide TS

Procedure. Dissolve 34.5 g of copper(Il) sulfate Rin 100 ml of water. Stir and add,
drop by drop, ammonia (-260 g/1) TS until the precipitate formed has com-
pletely dissolved. Keep the temperature below 20 °C and add slowly, while
stirring, 30ml of sodium hydroxide (-400 g/1) TS. Filter the precipitate
through a sintered glass filter (porosity 16-40 pm), and wash with water
until the filtrate is clear. Add 200ml of ammonia (-260 g/1) TS to the pre-
cipitate, stir, and filter.
Copper(II) acetate (45 g/1) TS. A solution of copper(II) acetate R containing

about 50 g of C4H6 Cu 04,H20 per litre.
Copper(II) acetate R. C4HgCu04,H20. Contains not less than 98.0% of

C4HsCu04,H20.
Description. Blue-green crystals or powder; odour, resembling that of acetic acid.
Assay. Dissolve 0.8 g, accurately weighed, in 50 ml of water, add 2 ml of acetic
acid (-300 g/1) TS and 3g of potassium iodide R, and titrate the liberated
1316
iodine with sodium thiosulfate (0.1 mol/1) VS, using starch TS as indicator,
until only a faint blue colour remains; add 2g of potassium thiocyanate
R and continue the titration until the blue colour disappears. Each ml
of sodium thiosulfate (0.1 mol/1) VS is equivalent to 19.97 mg of
C4H6Cu04,H20.
Copper(II) chloride R. CuCl2,2H20.

Description. Bluish green, deliquescent crystals.
Solubility. Freely soluble in water; soluble in ethanol (-750 g/1) TS; slightly
soluble in ether R.
Copper(II) chloride/ammonia TS
Procedure. Dissolve 22.5 g of copper(II) chloride R in 200 ml of water and add
100 ml of ammonia (-260 g/1) TS.
Copper(II) sulfate (lg/1) TS. A solution of copper(II) sulfate R containing

1 g of CUSO4 per litre.
Copper(II) sulfate (160g/l) TS. A solution of copper(II) sulfate R containing

about 160 g/1 of CUSO4.
Copper(II) sulfate (80g/l) TS. A solution of copper(II) sulfate R containing

about 80g of CUSO4 per litre (approximately 0.5mol/l).
Copper(II) sulfate R. CuS04,5H20 (SRIP, 1963, p. 72).
Copper(II) sulfate/ammonia TS
Procedure. Dissolve 50 g of copper(II) sulfate R in 1000 ml of ammonia (-35 g/1)
TS.
Copper(II) sulfate/pyridine TS
Procedure. Dissolve 4g of copper(II) sulfate R in 90 ml of water and add 30 ml
of pyridine R.
Note: Copper(II) sulfate/pyridine TS must be freshly prepared.
o-Cresol R. 2-Methylphenol; C/HsO.

Description. Colourless to pale brownish-yellow crystals or liquid; odour,
resembling that of phenol.
Miscibility. Miscible with ethanol (-750 g/1) TS, and ether R; miscible with about
50 parts of water.
Mass density. P20 = about 1.05 kg/1.
Refractive index, n™ = 1.540 - 1.550.

Freezing temperature. Not below 30.5 °C.
at 105 °C; it leaves a residue of not more than l.Omg/ml.
1317
Storage. Store in a tightly closed container, protected from light and oxygen.
Note: On exposure to light and air o-cresol R darkens in colour,
Cresol red R. CziHibOjS.

Description. A red-brown powder.
Solubility. Slightly soluble in water; soluble in ethanol (~750g/l) TS and in dilute
solutions of alkali hydroxides.
Cresol red/ethanol TS
Procedure. Warm 0.05 g of cresol red R with 2.65ml of sodium hydroxide
(0.05mol/l) VS and 5ml of ethanol (~710g/l) TS; after solution has been
effected, add sufficient ethanol (-150 g/1) TS to produce 250ml.
Crystal violet R. C25H30CIN3 (SRIP, 1963, p. 73).
Crystal violet/acetic acid TS. A solution of crystal violet R dissolved in

glacial acetic acid R1 containing about 5 g/1.
Culture medium Cml

Procedure. Dissolve 6.0 g of dried peptone R, 4.0 g of pancreatic digest of casein
R, 3.0 g of water-soluble yeast extract R, 1.5g of beef extract R, l.Og of glucose
hydrate R, and 10-20g of agar R in sufficient water to produce 1000 ml.
Note: The quantity of agar R used should permit the culture medium to be of
adequate firmness to support cylinders or to permit holes to be cut without
tearing the gel layer.
Culture medium Cm2

Procedure. Dissolve 17.0 g of pancreatic digest of casein R, 3.0 g of papaic digest
of soybean meal R, 5.0g of sodium chloride R, 2.5g of dipotassium hydro-
gen phosphate R, 2.5 g of glucose hydrate R, and 10-20g of agar R in about
500 ml of water. Heat the solution, add lO.Og of polysorbate 80 R and dilute
immediately with a sufficient amount of water to produce 1000 ml.
Culture medium Cm3

Procedure. Dissolve 9.4g of dried peptone R, 4.7 g of water-soluble yeast extract
R, 2.4g of beef extract R, 10. Og of sodium chloride R, 10. Og of glucose
hydrate R, and 15-25g of agar R in sufficient water to produce 1000ml.
1318
Culture medium Cm4. Fluid mercaptoacetate (thioglycolate) medium.

Procedure. Thoroughly grind in a mortar in the following order: 0.5 g of L-
cystine R, 2.5 g of sodium chloride R, 5.5 g of glucose hydrate R, 0.75 g of
agar R, 5.0 g of water-soluble yeast extract R and 15.0 g of pancreatic digest
of casein R. Add a small volume of hot water, transfer to a suitable con-
tainer, and add sufficient water to produce 1000 ml. Complete the solution
by heating in a boiling water-bath, taking care to ensure complete solution

of the L-cystine R. Add 0.3 ml of mercaptoacetic acid R or 0.5 g of sodium
mercaptoacetate R (the latter is preferred as a more stable compound) and
sufficient sodium hydroxide (1 mol/1) VS so that the pH of the final and ster-
ilized medium will be 7. 0-7. 2. Reheat the solution, but do not boil, filter, if
necessary, through cotton wool and add 1.0 ml of resazurin sodium (1 g/1)
TS. Distribute the solution into suitable vessels and sterilize by autoclaving
for 18-20 minutes at 121 °C and cool promptly to 25°C.
Storage. At 20-30 °C, avoiding excessive light.
Note: If the uppermost portion of the medium has changed to a pink colour
and this exceeds one-fifth of the depth of the medium, it is unsuitable for
use, but may be restored once by heating in steam.
Culture medium Cm5. Soybean-casein digest medium.

Procedure. Dissolve in water 17.0 g of pancreatic digest of case in R, 3.0 g of
papaic digest of soybean meal R, 5.0 g of sodium chloride R, 2.5 g of dipotas-
sium hydrogen phosphate R, and 2.5 g of glucose hydrate R. Warm the solu-
tion slightly, then cool it to room temperature and add sufficient water to
produce 1000 ml. Adjust the pH of the solution with sodium hydroxide
(1 mol/1) VS, if necessary, so that the pH of the final and sterilized medium
will be 7. 1-7.5. Filter, if necessary to clarify, distribute the solution into suit-
able vessels and sterilize in an autoclave for 18-20 minutes at 121 °C.
Culture medium Cm6. Fluid mercaptoacetate (thioglycolate) medium with

penicillinase.
Procedure. Use culture medium Cm4 with sufficient sterile penicillinase TS
added to inactivate the penicillin activity of the test material. Add penicilli-
nase TS to individual vessels containing sterile culture medium Cm4 using
an aseptic technique. Prior to use, or at the time of the test, incubate a rep-
resentative number of the vessels with culture medium Cm6 at 30-32 °C for
24-48 hours and examine for sterility.
Culture medium Cm7. Soybean-casein digest medium with penicillinase.

Procedure.Use culture medium Cm5 to which 5.0 ml of polysorbate 80 R has
been added before sterilization and with sufficient sterile penicillinase TS
added to inactivate the penicillin activity of the test material. Add penicilli-
nase TS to individual vessels containing sterile culture medium Cm5, using
an aseptic technique.
1319
Culture medium Cm8

Procedure. Dissolve lOg of dried peptone R, lOg of beef extract R, lOg of glyc-
erol R, 3.0g of sodium chloride R, and 17 g of agar R in sufficient water to
produce 1000ml. Adjust the pH with sodium hydroxide (0.05 mol/1) VS to
6.9-7. 1, and sterilize in an autoclave at 121 °C for 18-20 minutes.
Culture medium Cm9

Procedure. Dissolve lOg of dried peptone R, lOg of beef extract R, lOg of
glycerol R, and 3.0 g of sodium chloride R in sufficient water to produce
1000ml. Adjust the pH with sodium hydroxide (0.05mol/l) VS to 6. 9-7.1,
and sterilize in an autoclave at 121 °C for 18-20 minutes.
Culture medium CmlO

Procedure. Dissolve 1 g of water-soluble yeast extract R, 5g of ammonium
nitrate R, 5g of sodium dihydrogen phosphate R, 5g of anhydrous glucose
R, and 14g of agar R in sufficient water to produce 1000 ml.
Culture medium Cmll

Procedure.Dissolve lOg of dried peptone R, 6g of beef extract R, and 15g of
agar sufficient water to produce 1000 ml. Adjust the pH of the solution
R in
with sodium hydroxide (1 mol/1) VS, if necessary, so that the pH of the final
and sterilized medium will be 7.8. Filter, if necessary to clarify, distribute
the solution into suitable vessels and sterilize in an autoclave at 121 °C for
18-20 minutes.
Cyanide/oxalate/thiosulfate TS
Procedure. To 2.0ml of ammonia (~100g/l) TS, add in the following order;
1.5ml of ammonium oxalate (50g/l) TS, 15 ml of potassium cyanide (50g/l)
TS, 45ml of sodium acetate (60g/l) TS, 120ml of sodium thiosulfate
(320 g/1) TS, 75ml of sodium acetate (60g/l) TS, and 35 ml of hydrochloric
acid (1 mol/1) VS.
Note: Cyanide/oxalate/thiosulfate TS must be prepared immediately before use.
Cyanoethylmethyl silicone gum R. A suitable grade to be used in gas-liquid

chromatography.
Cyanogen bromide TS
Caution. Very toxic; avoid inhalation of vapours.
Procedure.Add drop by drop, while cooling, potassium cyanide (100 g/1) TS to
bromine TSl until the colour disappears.
Note: Cyanogen bromide TS must be prepared immediately before use.
Cyclohexane R. C(;Hi 2 (SRIP, 1963, p. 74).
Cyclohexane Rl. Cyclohexane R showing a fluorescence that is not more

intense than that of a solution of 2pg/ml of quinine R in sulfuric acid
1320
(0.05mol/l) VS when measured at 460 nm using a 1-cm layer and an excita-

tion beam at 365 nm.
1 -Cyclopropyl-l,4-dihydro-4-oxo-7-(piperazin-l-yl)quinoline-3-
carboxylic acid RS (ciprofloxacin desfluoro-compound). International
Chemical Reference Substance.
L-Cystine R. C 5H, 2 N 204 S 2 (SKIP, 1963, p. 75).
Cytarabine RS. International Chemical Reference Substance.
Dacarbazine RS. International Chemical Reference Substance.
Dacarbazine related compound A RS. International Chemical Reference

Substance.
Dacarbazine related compound B RS. International Chemical Reference

Substance.
Dactinomycin RS. International Chemical Reference Substance.
Dantron R. 1,8-dihydroxyanthraquinone; C 14H 8 O 4 .

Description.An orange, microcrystalline powder.
Solubility. Practically insoluble in water; slightly soluble in ethanol (-750 g/1) TS
and ether R.
Melting point. About 193 °C.
Dapsone RS. International Chemical Reference Substance.
Dexamethasone acetate RS. International Chemical Reference Substance.
Dexamethasone RS. International Chemical Reference Substance.
Dexamethasone sodium phosphate RS. International Chemical Reference

Substance.
Dextromethorphan hydrobromide RS. International Chemical Reference

Substance.
l,4-Di[2-(4-methyl-5-phenyloxazole)]benzene R.
3,3'-Diaminobenzidine tetrahydrochloride R. C]2Hi4N4,4HCl,2H20.

Description.An almost white or slightly pink powder.
Dimethyl-POPOP C 26 H 20N 2 O 2 . Suitable for scintillation counting.
1321
3,3'-Diaminobenzidine tetrahydrochloride (5g/I) TS. A solution of 3,3'-

diaminobenzidine tetrahydrochloride R containing 5g of CnHi^N^jdHCl
per litre.
Diammonium hydrogen phosphate (lOOg/I) TS. A solution of diammo-

nium hydrogen phosphate R containing about lOOg of (NH 4 HP 04 per litre. )2
Diammonium hydrogen phosphate R [ammonium phosphate R].

(NH4)2HP04(SR1P, 1963, p.38).
Diatomaceous support R
Description. White granules of silica consisting chiefly of the skeletons of
diatoms. The material is flux-calcined to sequester coloured metallic oxides
and is supplied commercially in various forms, such
in a colourless form, as;
acid-washed, silanized, and alkali-washed.
Diazepam RS. International Chemical Reference Substance.
Diazobenzenesulfonic acid TS
Procedure. To 0.9 g of sulfanilic acid R add 10 ml of hydrochloric acid (-250 g/1)
TS and sufficient water to produce 100ml. To 3 ml of this solution add 5 ml
of sodium nitrite (3 g/1) TS, cool in ice for 5 minutes, add a further 20ml of
sodium nitrite (3 g/1) TS, and again cool in ice; dilute with water to 100 ml,
keeping the solution cold.
Note: Diazobenzenesulfonic acid TS must be freshly prepared and should not
be used until at least 15 minutes after its preparation.
Diazomethane TS
Caution. Diazomethane is explosive in the gaseous state and its explosive
decomposition is easily initiated by rough surfaces. Do not use apparatus
with ground-glass joints or boiling stones of any kind. Diazomethane is
highly toxic and all operations should be conducted under a well-ventilated
hood.
Procedure. Prepare a solution of 0.4 g of potassium hydroxide R in 10 ml of
ethanol (~750g/l) TS and add it to a solution of 2.14g of iV-methyl-N-
nitrosotoluene-4-sulfonamide R in 30 ml of ether R while cooling in ice. If
a precipitate forms, add just sufficient ethanol (-750 g/1) TS to dissolve it.
After 5 minutes, gently distil the ethereal diazomethane solution from a

water-bath. Diazomethane TS contains about lOg of CH N
2 2 per litre.
Alternative procedures. Other procedures for the evolution of diazomethane,

using other starting materials, may also be employed, provided the result-
ing solution has the required concentration of CH N 2 2 .
Diazoxide RS. International Chemical Reference Substance.
1322
Dibromomethane R. Methylene bromide, CHjBrj.

Description. Colourless to yellowish liquid.
Miscibility. Miscible with ethanol (-750 g/1) TS, ether R, and acetone R.
Dibutyl ether R. Di-«-butyl ether, CsHisO.

Caution. Dibutyl ether R tends to form explosive peroxides, especially when
anhydrous.
Description. A colourless liquid.
Miscibility. Practically immiscible with water; miscible with ethanol (~750g/l)
TS and ether R.
Boiling range. 140-143°C.
Mass density, pjo = 0.769 kg/1.
Refractive index, no = 1.344.
Dibutyl phthalate R. Di-K-butyl phthalate, C 16H 22 O 4 .
Description. A clear, colourless or faintly coloured liquid.

Miscibility. Very slightly miscible with water; miscible with ethanol (-750 g/1)
TS and ether R.
Mass density. P 20 = 1.043 - 1.048 kg/1.
Sulfated ash. Not more than 0.2 mg/ml.
Dichloroethane R. 1,2-Dichloroethane, C 2 H 4 CI 2 (SRIP, 1963, p. 76).
Dichlorofluorescein R. C20H10CI2O5.
Description. A light orange-coloured, crystalline powder.
Solubility. Sparingly soluble in water; soluble in ethanol (-750 g/1) TS.
Dichlorofluorescein TS
Procedure. Dissolve 0.2g of dichlorofluorescein R in 100ml of methanol R.
Dichlororaethane R. Methylene chloride, CH 2 CI 2 .
Description. A clear colourless, mobile liquid.

Miscibility. with ethanol (-750 g/1) TS and ether R.
Freely miscible
Boiling range. Not 95% distils between 39 and 41 °C.
less than
Residue on evaporation. Leaves, after evaporationon a water-bath and drying at
105 °C, not more than 0.5mg/ml.
2 6 -Dichloroquinone chlorimide R.
,
C 6H 2 CI 3 NO (SRIP, 1963, p. 77).
2 6 -Dichloroquinone chlorimide/ethanol TS
,
Procedure. Dissolve 0.5 g of 2,6-dichloroquinone chlorimide R in sufficient

ethanol (-750 g/1) TS to produce 100 ml.
1323
Dichromate colour TS. A solution containing 4.904 g/1 of K2 Cr207 .
Procedure. Prepare a solution containing 490.35mg of K2 Ct 207 in 100ml by

diluting the strong dichromate colour TS with sulfuric acid (~10g/l) TS, as
necessary.
Dichromate colour, strong, TS

Procedure. Dissolve 6.0 g of potassium dichromate R in 120 ml of sulfuric acid
(-10 g/1) TS, filter the solution, if necessary, and determine the concentra-
tion of K2 Cr207 .
Assay. Dilute 5.0ml with sufficient water to produce 50ml. Transfer 10.0ml of
this solution to a glass-stoppered flask, add 10 ml of water, 2g of potassium
bicarbonate R, and 20 ml of sulfuric acid (-100 g/1) TS. Loosely close the flask
with its stopper. When the gas evolution has ceased, add 1 g of potassium
iodide R, keep the flask for 5 minutes in a dark place and titrate the liber-
ated iodine with sodium thiosulfate (0.1 mol/1) VS, using starch TS as indi-
cator. Each ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to 4.904mg
of K2 Cr207 .
Dicloxacillin sodium RS. International Chemical Reference Substance.
Dicoumarol RS. International Chemical Reference Substance.
Didanosine RS. International Chemical Reference Substance.
Didanosine for system suitability RS. International Chemical Reference

Substance.
Diethoxytetrahydrofuran R. CsHiÔa. Mixture of the cis and trans isomers.

Description. Colourless or slightly yellow, clear liquid.
Miscibility. Practically immiscible with water; miscible with ethanol (-750 g/1)
TS and ether R.
Mass density. P 20 = about 0.975kg/l.
Refractive index. n§^= 1.418.
Diethoxytetrahydrofuran/acetic acid TS
Procedure. Mix 1 ml of diethoxytetrahydrofuran R with sufficient glacial acetic
acid R to produce 100 ml.
Diethyl phthalate R. C12H14O4.

Mass density. P 20 = about 1.1 17 kg/1.
Refractive index. nf° = 1.500 - 1.505.
Diethylamine R. C 4 H„N. Contains not less than 99.5% of C 4 H„N.

Mass p 2 o = 0.702 - 0.704 kg/1.

density.
Refractive index, n^ = 1 .384 - 1 .386.
1324
Assay. Add about 3g, accurately weighed, to 50 ml of sulfuric acid (0.5 mol/1)
VS and titrate the excess acid with sodium hydroxide (1 mol/1) VS, using
methyl red/ethanol TS as indicator. Each ml of sulfuric acid (0.5mol/l) VS is
equivalent to 73.14mg of C^HuN.
Diethylaminoethylcellulose R. A suitable grade for column chromatography.
Diethylcarbamazine dihydrogen citrate RS. International Chemical Refer-

ence Substance.
Diethylene glycol R. C4H10O3.

Description. A colourless to faintly yellow liquid having a mild odour.
Miscibility. Freely miscible with water, ethanol (~750g/l) TS, ether R and
acetone R.
Boiling range. Between 240 and 250 °C.
Mass density (p2o)- 1.117
- 1.120kg/l.
Acidity. flask, add phenolphthalein/ethanol TS
Transfer 60 g to a 250-ml conical
and with potassium hydroxide/ethanol (0.02 mol/1) VS, to a pink
titrate
Not more than 2.5ml

colour that remains stable for at least 15 seconds.
should be consumed.
Diethylene glycol succinate R. A suitable grade to be used in gas-liquid

chromatography.
Diethylphenylenediamine sulfate TS
Procedure. To 250ml of water add about 2ml of sulfuric acid (-1760 g/1) TS and
50ml of disodium edetate (0.01 mol/1) VS. Dissolve 1.1 g of N,N-diethy\-p-
phenylenediamine sulfate R into this solution and dilute with sufficient
water to produce 1000 ml.
N,N-Diethyl-p-phenylenediamine sulfate R. Ai,Ai-Diethyl-l,4-phenylene-

diamine sulfate; CioHi6N2,H2S04.
Description. A white or slightly coloured powder.
Melting point. About 185°C, with decomposition.
Storage. N,W-Diethyl-/?-phenylenediamine sulfate R should be kept protected
from light.
Diethyltoluamide RS. International Chemical Reference Substance.
Digitonin R. C55H90O29 (SRIP, 1963, p. 78).
Digitonin TS
Procedure. Dissolve O.lOg of digitonin R in sufficient ethanol (~710g/l) TS to
produce 10 ml.
Note: Digitonin TS must be freshly prepared.
1325
Digitoxin RS. International Chemical Reference Substance.
Digoxin RS. International Chemical Reference Substance.
Diisopropyl ether R. Isopropyl ether; CeHnO.

Description. A colourless liquid; odour, characteristic.
Note. Diisopropyl ether is highly flammable.
Diloxanide furoate RS. International Chemical Reference Substance.
Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxyIate
RS. International Chemical Reference Substance.
Dimethyl 2,6-dimethyl-4-(2-nitrosophenyI)pyridine-3,5-dicarboxylate
RS. International Chemical Reference Substance.
Dimethyl sulfoxide R. C 2 H 6 OS.

Description. A colourless liquid; odourless or with a slight, but unpleasant odour.
Mass density. P20 = 1-10 kg/1.
Dimethylacetamide R. C4H9NO.
Boiling temperature. About 16°C.
Mass density. p2o = 0.94 kg/1.
Dimethylamine R. C 2 H 7N.
Description. A low-boiling liquid, 7°C; odour, characteristic.
Solubility. Soluble in water, ethanol (-750 g/1) TS, and ether R.
Dimethylamine/ethanol TS. A solution of dimethylamine R in ethanol

(-750 g/1) TS containing about 330 g/1 of C2H7N.
Assay. Dilute 2 ml to 10ml with ethanol (-750 g/1) TS. Transfer 2 ml to a flask
containing 50.0 ml of sulfuric acid (0.05 mol/1) VS and mix. Titrate the excess
acid with sodium hydroxide (0.1 mol/1) VS, using methyl red/ethanol TS as
indicator. Each ml of sulfuric acid (0.05 mol/1) VS is equivalent to 4.508 mg
of C2H7N.
4-DimethyIaminobenzaldehyde R [dimethylaminobenzaldehyde R].

C9H11NO (SRIP, 1963, p. 78).
4-Dim ethylaminobenzaldehyde TSl

Procedure. Dissolve 0.125g of 4-dimethylaminobenzaldehyde R in a cooled
mixture of 65 ml of sulfuric acid (-1760g/l) TS and 35 ml of water and add
0.2 ml of ferric chloride (25 g/1) TS.
Note: 4-Dimethylaminobenzaldehyde TSl must be freshly prepared.
1326
4-DimethylaminobenzaIdehyde TS2
Procedure. Dissolve 0.80 g of 4-dimethylaminobenzaldehyde R in a cooled
mixture of 80g of ethanol (-750 g/1) TS and 20g of sulfuric acid (~1760g/l)
TS.
4-DimethyIaminobenzaldehyde TS3
Procedure. Dissolve 0.5 g of 4-dimethylaminobenzaldehyde R in 50 ml of
ethanol (~750g/l) TS, add 1 ml of hydrochloric acid (~420g/l) TS, and dilute
with sufficient ethanol (-750 g/1) TS to produce 100 ml.
4-DimethylaminobenzaIdehyde TS4
Procedure. Dissolve 2g of 4-dimethylaminobenzaldehyde R in a mixture of 5 ml
of hydrochloric acid (-420 g/1) TS and 95ml of glacial acetic acid R.
without heating 2g of 4-dimethylaminobenzaldehyde R in
Procedure. Dissolve
a mixture of 45 ml of water and 55 ml of hydrochloric acid (-420 g/1) TS.
Procedure. Dissolve 0.2 g of 4-dimethylaminobenzaldehyde R in 20 ml of
ethanol (-750 g/1) TS and add 0.5 ml of hydrochloric acid (-420 g/1) TS. Shake
the solution with charcoal R and filter. The colour of this test solution is less
intense than that of iodine (0.0001 mol/1) VS.

Note: 4-Dimethylaminobenzaldehyde TS6 must be freshly prepared.
4-DimethyIaminocinnamaIdebyde R. CnHisNO.
Description. Orange crystals, or a crystalline powder; odour, characteristic.
Solubility. Practically insoluble in water; freely soluble in hydrochloric acid
(-70 g/1) TS; sparingly soluble in ethanol (-750 g/1) TS and ether R.
4-Dim etbylaminocinnamaldebyde TSl

Procedure. Dissolve 2g of 4-dimethylaminocinnamaldehyde R in a mixture of
100ml of hydrochloric acid (5mol/l) VS and 100ml of ethanol (-750 g/1) TS.
Storage. Store the solution at a temperature of about 0°C.
4-DimethyIaminocinnamaIdehyde TS2
Procedure. Dilute 20 ml of 4-dimethylaminocinnamaldehyde TSl with sufficient
ethanol (-750 g/1) TS to produce 100ml.
Note: 4-Dimethylaminocinnamaldehyde TS2 must be freshly prepared.
N,N-Dimethylaniline R. CgHuN.
Description. A colourless liquid darkening on storage.
Miscibility. Practically immiscible with water, miscible with ethanol (-750 g/1)
TS, and ether R.
Mass density. p 2 o = 0.96kg/l.
1327
Dimethylfomiamide R. C3H7NO.
Description. A clear and colourless liquid, having a characteristic odour.
Boiling range. Not less than 25% distils at between 152 and 156°C.
Mass density{p 2 o)- 0.945-0.947 kg/1.
Acidity and alkalinity. Dissolve Ig in 10 ml of water, add 2 drops of phenolph-
thale in/ ethanol TS; not more than 0.2 ml of carbonate-free sodium hydrox-
ide (0.01 mol/1) VS is required to produce a red colour. Add 0.3ml of
hydrochloric acid (0.01 mol/1) VS and 5 drops of methyl, red/ethanol TS; an

orange colour is produced.
N,N-Dimethyloctylamine R. Octyldimethylamine; C10H23N.

Dinitrobenzene R. C6H4N2O4 (SKIP, 1963, p. 79).
Dinitrobenzene/ethanol TS
Procedure. Dissolve 1 g of dinitrobenzene R in sufficient ethanol (-750 g/1) TS to
produce 100 ml.
2 4 -Dinitrochlorobenzene R. C6H4CIN2O4
,
(SKIP, 1963, p. 80).
Dinitrogen oxide R. N2O.

Dinonyl phthalate R. C26H42O4.

Description. A colourless to pale yellow, viscous liquid.
Mass density. P20 = 0.97 - 0.98 kg/1.
Fischer method, using about 2 ml of the liquid; not more than 1.0 mg/ml.
Acidity. Shake 5.0 g with 25ml of water for 1 minute. Allow to stand, filter the
aqueous layer and add to it 5 drops of phenolphthalein/ethanolTS; not more

than 0.3ml of carbonate-free sodium hydroxide (0.1 mol/1) VS is required
for the neutralization (0.5mg/g expressed as phthalic acid).
Dioxan R. 1,4-Dioxane, C4H8O2.

Caution. It is dangerous to determine the boiling range or the residue on evap-
oration before complying with the test for peroxides.
Miscibility. Miscible with water, ethanol (-750 g/1) TS and ether R.

Boiling range. Not less than 95% distils at between 101 and 105°C.
Melting temperature. Solidifies when cooled in ice and does not completely
remelt at temperatures below 10°C.
1328
. .
Residue on evap’oration. Evaporate on and dry to constant weight

a water-bath
at 105 °C; it leaves a residue of not more than 0.1 mg/ml.
Mass densityip 2 o)- About 1.031 kg/1.
Water. Determined by the Karl Fischer method, not more than 5.0 mg/ml.
Peroxide. Add 5ml 1 g of potassium iodide R dissolved in 10ml
to a mixture of
of water, 5ml of hydrochloric acid (~70g/l) TS, and 2 ml of starch TS, and
mix; not more than a faint blue or brown colour is produced.
Diphenoxylate hydrochloride RS. International Chemical Reference

Substance.
Diphenyl ether R. Phenyl ether, C^HioO.

Miscibility. Immiscible with water; freely miscible with glacial acetic acid R and
with most organic solvents.
M king
e ra nge 2 6-2 8 ° C
Diphenylamine R. C, 2 Hi]N (SRIP, 1963, p. 81).
Diphenylamine/sulfuric acid TS
Procedure. Dissolve l.Og of diphenylamine R in 100ml of sulfuric acid
(~1760g/l) TS.
Storage. Diphenylamine/sulfuric acid TS must be colourless and should be kept
Diphenylbenzidine R. C24H20N2.
Description. A white, or faintly grey-coloured, crystalline powder.
Solubility. Insoluble in water; slightly soluble in ethanol (-750 g/1) TS and
acetone R.
Nitrates. Dissolve 8 mg in a cooled mixture of nitrogen-free sulfuric acid
(~1760g/l) TS and 5ml of water; the solution is colourless or not more than
very pale blue.
1 , 5 -DiphenyIcarbazide R. Ci3FFi4N40.
Description. A white, crystalline powder, gradually turning pink on exposure to
air.
Diphenylcarbazide TS
Procedure. Dissolve 0.2 g of 1,5-diphenylcarbazide R in a mixture of 10ml of
glacial acetic acid R and 90 ml of ethanol (-710 g/1) TS.
Sensitivity test to chromate. Dilute 0.5ml of potassium dichromate (0.0167mol/l)
VS with water to 1000 ml. Dilute 5 ml of this solution to 50 ml with water.
1329
.
add 0.2 ml of hydrochloric acid (2 mol/1) VS and 0.5ml of diphenylcarbazide

TS; a reddish violet colour is produced.
Diphenylcarbazone R. C 13 H 12 N 4 O (SKIP, 1963, p. 81),
Diphenylcarbazone/ethanol TS. A solution of diphenylcarbazone R dis-

solved in ethanol (-750g/l) TS containing about 1 g/1 of C] 3 Hi 2 N 40 .
1,2-Diphenyletbylammonium 3-mercapto-2-metbylpropanoate RS.

International Chemical Reference Substance.
2,5-DiphenyIoxazole R. PPO, CisHnNO. Suitable for scintillation counting.
Dipotassium hydrogen phosphate R. K 2 HPO 4 (SRIP, 1963, p. 81).
Disodium chromotropate (lOg/1) TS. A solution of disodium chro-

motropate R containing about 9.5 g of CioH 6 Na 203 S 2 per litre.
Disodium chromotropate R [chromotropic acid sodium salt R].
6-iold6Na20 sS2,2H20
Description. A yellow to light-brown powder.
Solubility. Freely soluble in water; insoluble in ethanol (-750 g/1) TS.
Identification. To 0.5ml of a 2mg/ml solution add 10ml of water and 1 drop of
ferric chloride (25 g/1) TS; a green colour is produced.
Sensitivity. Dissolve 5mg in 10ml of a mixture of 9ml of sulfuric acid
(-1760 g/1) TS and 4ml of water. Separately dilute 0.5ml of formaldehyde
TS with water to make 1000ml. Transfer to each of two separate test-tubes
5ml of the disodium chromotropate solution, to one add 0.2ml of the
formaldehyde solution, and heat both tubes in a water-bath for 30 minutes;
a violet colour is produced in the tube containing the formaldehyde
solution.
Disodium chromotropate TS
Procedure. Dissolve 5 mg of disodium chromotropate R in 10 ml of a mixture of
9 ml of sulfuric acid (~1760g/l) TS and 4 ml of water.
Disodium edetate (0.01 mol/1) VS. Disodium edetate R, dissolved in water

to contain 3.342 g of C]oHi 4 N 2 Na 20 s in 1000 ml.
lowing an appropriate method, e.g. as described under disodium edetate
(0.05 mol/1) VS.
Disodium edetate (0.05 mol/1) VS. Disodium edetate R, dissolved in water

to contain 16.71 g of C]oHi 4 N 2 Na 20 a in 1000 ml.
Method of standardization. Ascertain the exact concentration by an appropriate
method. The following method is suitable: Transfer about 200mg of calcium
1330
carbonate R2, accurately weighed, to a 400-ml beaker, add 10ml of water,

and swirl to form a slurry. Cover the beaker with a watch glass, and intro-
duce 2 ml of hydrochloric acid (~70g/l) TS from a pipette inserted between
the lip of the beaker and the edge of the watch glass. Swirl the contents of
the beaker to dissolve the calcium carbonate. Wash down the sides of the
and the watch glass with water, and
beaker, the outer surface of the pipette,
dilutewith water to about 100ml. While stirring the solution, preferably
with a magnetic stirrer, add about 30 ml of the disodium edetate solution
from a 50-ml burette. Add 10ml of sodium hydroxide (~80g/l) TS and
0.3 g of calcon indicator mixture R or of calcon carboxylic acid indicator
mixture R and continue the titration with the disodium edetate solution to
a blue end-point. Each 5.005 mg of calcium carbonate is equivalent to 1ml
of disodium edetate (0.05 mol/1) VS.
Disodium edetate (0.1 mol/1) VS. Disodium edetate R, dissolved in water to

contain 33.42 g of CioHi4N2Na20a in 1000ml.
lowing an appropriate method, e.g. as described under disodium edetate
(0.05 mol/1) VS.
Disodium edetate (lOg/1) TS. A solution of disodium edetate R containing

about lOg of CioH^NaNaaQa per litre.
Disodium edetate (20g/l) TS. A solution of disodium edetate R containing

about 20 g of CioHi4N2Na208 per litre.
Disodium edetate (50g/l) TS. A solution of disodium edetate R containing

about 50 g of CioH,4N2Na203 per litre.
Disodium edetate R. C]oH]4N2Na20s,2H20 (SRIP, 1963, p. 82).
Disodium edetate RS. International Chemical Reference Substance.
Disodium hydrogen phosphate (lOOg/1) TS. A solution of disodium

hydrogen phosphate R containing about 100 g of Na2HP04 per litre.
Disodium hydrogen phosphate (28.4g/I) TS. A solution of anhydrous dis-

odium hydrogen phosphate R containing 28.4 g of Na2HP04 per litre.
Disodium hydrogen phosphate (40g/l) TS. A solution of disodium hydro-

gen phosphate R containing about 40 g of Na2HP04 per litre.
Disodium hydrogen phosphate R [sodium phosphate R]. Na2HP04,12H20

(SRIP, 1963, p. 192).
1331
Disodium hydrogen phosphate, anhydrous, R [sodium phosphate, anhy-

drous, R], Na2HP04 (SKIP, 1963, p. 193).
Dissolution media. See under 5.5 Dissolution test for solid oral dosage forms.
5,5'-Dithiobis(2-nitrobenzoic acid) R. 3-Carboxy-4-nitrophenyl disulfide;

C14H8H2O8S2.
5,5'-Dithiobis-2-nitrobenzoic acid/methanol TS
Procedure. Dissolve 0.198g of 5,5'-Dithiobis(2-nitrobenzoic acid) R in sufficient
methanol R to produce 500 ml.
Storage. Keep under refrigeration, and warm to room temperature before use.
Dithizone R. C13H12N4S (SRIP, 1963, p. 83).
Dithizone standard TS
Procedure. Dissolve 10 mg of dithizone R in 1000 ml of chloroform R.
Storage. Store the solution in a glass-stoppered, lead-free bottle, protected from
light, and kept at a temperature not exceeding 4°C.
Dithizone TS
Procedure. Dissolve O.lOg of dithizone R in sufficient ethanol (~750g/l) TS to
produce 100 ml.
Dithranol RS. International Chemical Reference Substance.
Domiphen bromide (lOg/1) TS. A solution of domiphen bromide R con-

taining about lOg of C22H4oBrNO per litre.
Domiphen bromide R. Dodecyldimethyl(2-phenoxyethyl)ammonium

bromide; C22H4oBrNO.
Description. Colourless or faintly yellow, crystalline flakes.
Solubility. Freely soluble in water and ethanol (-750 g/1) TS; soluble in acetone
R.
Dopamine hydrochloride RS.
Doxorubicin hydrochloride RS. International Chemical Reference

Substance.
Doxycycline hyclate RS. International Chemical Reference Substance.
Dragendorff reagent TS
Procedure. Shake vigorously to dissolve 0.85g of bismuth subnitrate R in 10ml
of glacial acetic acid R and 40ml of water {solution A). Dissolve 8g of potas-
1332
sium iodide R in 20ml Immediately before use, mix

of water {solution B).
together equal volumes of solutions A and B and glacial acetic acid R.

Storage. Solutions A and B must be protected from light.
Dragendorff reagent, modified, TS

Add 20 ml of acetic acid (~60g/l) TS to 4 ml of a mixture of equal
Procedure.
volumes of solutions A and B of Dragendorff reagent TS.
Note: Prepare this solution immediately before use.
Econazole nitrate RS. International Chemical Reference Substance.
Emetine hydrochloride RS. International Chemical Reference Substance.
Endotoxin RS. Second WHO International Standard for Endotoxin as estab-

lished in 1996, containing lOOOOIU per ampoule, approximately 1 pg of
freeze-dried endotoxin Escherichia coli, with 1 mg of PEG and 10 mg of
from
by the National Institute for Biological Standards and
lactose (distributed
Control (NIBSC), PO Box 1193, Blanche Lane, South Mimms, Potters Bar,
Herts EN6 3QH, England), or another suitable preparation, the activity of
which has been determined in relation to the WHO International Standard
using the gelation test.
Eosin Y (5g/l) TS. A solution of eosin Y R containing about 5g of

C oH 6Br Na 20 s
2 4 per litre.
Eosin YR. Sodium tetrabromofluorescein; C 2 oHgBr 4 N 205 .
Description. Red to brownish lumps or a powder.

Solubility. Freely soluble in water; sparingly soluble in ethanol (-750 g/1) TS.
Ephedrine sulfate RS. International Chemical Reference Substance.
4-Epianhydrotetracycline hydrochloride RS. International Chemical Ref-

erence Substance.
6-EpidoxycycIine hydrochloride RS. International Chemical Reference

Substance.
Epinephrine hydrogen tartrate R. Epinephrine hydrogen tartrate R as

described in the monograph for “Epinephrine hydrogen tartrate”, which
complies with the following test for the absence of levarterenol:
Levarterenol. Carry out descending 1.14.2 Paper chromatography. Mix 4
volumes of 1 -butanol R, 1 volume of and 5 volumes of
glacial acetic acid R,
water, shake and allow the two Use the lower layer as
layers to separate.
the stationary phase and the upper layer as the mobile phase. Apply to the
paper 20 pi of a solution containing 50 mg/ml of epinephrine hydrogen tar-
trate R, develop for 5 hours, dry the paper, and spray with a freshly pre-
1333
=
pared 4.4 mg/ml solution of potassium ferricyanide R dissolved in buffer

borate, pH 8.0, TS or another buffer having the same pH may be used; only
1 spot appears, which is pink.
4-Epitetracycline hydrochloride RS. International Chemical Reference

Substance.
Ergocalciferol RS. International Chemical Reference Substance.
Ergometrine hydrogen maleate RS. International Chemical Reference

Substance.
Ergosterol R. Provitamin D 2 ergosta-5,7,22-trien-3-ol; C 2 SH 44 O. Contains not

;
less than 95.0% of C 28 H 44 O.

Description. White or almost white needles or a crystalline powder.
Specific optical rotation. Use a 20mg/ml solution in chloroform R; [a]™ -133 °.
Ergotamine tartrate RS. International Chemical Reference Substance.
Erythromycin ethylsuccinate RS. International Chemical Reference

Substance.
Erythromycin lactobionate RS. International Chemical Reference Substance.
Erythromycin RS. International Chemical Reference Substance.
Erythromycin stearate RS. International Chemical Reference Substance.
Estrone RS. International Chemical Reference Substance.
Ethambutol hydrochloride RS. International Chemical Reference Substance.
Ethanol (~150g/l) TS. A solution of about 210ml of ethanol (~750g/l) TS

diluted with water to 1000 ml.
Ethanol (-375 g/1) TS. A solution of about 525 ml of ethanol (-750 g/1) TS
Ethanol (-457 g/1) TS

Procedure. Dilute 519ml of ethanol (-750g/l) TS with sufficient water to
produce 1000 ml.

Procedure. Dilute 623ml of ethanol (~750g/l) TS with sufficient water to
produce 1000 ml.
1334
Ethanol (~600g/l) TS. A solution of about 735 ml of ethanol (-750 g/1) TS

diluted with water to 1000 ml,



[ethanol (95 per cent) R] (SRIP, 1963, p. 84).
Ethanol (~750g/l), aldehyde-free TS

[ethanol, aldehyde-free, (95 per cent.) R]. (SRIP, 1963, p. 84).
Ethanol (~750g/l), sulfate-free, TS. Ethanol (-750 g/1) TS that complies with
the following test: Evaporate 25ml of ethanol (-750 g/1) TS to a volume of
about 2 ml, add a mixture of 3 ml of hydrochloric acid (-70 g/1) TS and
42ml of water, and 5 ml of barium sulfate suspension TS. Proceed as
described in 2.2.2 Limit test for sulfates. Sulfate-free ethanol (-750g/l) TS
contains not more than 20 pg/ml.
Ethanol, dehydrated, R. C2H5OH (SRIP, 1963, p. 85).
Ethanol, neutralized, TS
Procedure. To a suitable quantity of ethanol (-750g/l) TS add 0,5ml of phe-
nolphthalein/ethanol TS and just sufficient carbonate-free sodium hydrox-
ide (0.02 mol/1) VS or (0.1 mol/1)VS to produce a faint pink colour.
Note: Prepare neutralized ethanol TS just prior to use.
Ether R. C4H10O (SRIP, 1963, p. 85).
Ether, peroxide-free, R
Procedure. To 1000 ml of ether R add 20 ml of a solution of 30 g of ferrous sulfate
R in 55 ml of water and shake the mixture with 3 ml of sulfuric acid
(-1760 g/1) TS, Continue shaking until a small sample no longer produces a
blue colour when shaken with an equal volume of a 20 g/1 solution of potas-
sium iodide R and 0.1 ml of starch TS.
Ethinylestradiol RS. International Chemical Reference Substance.
Ethionamide RS. International Chemical Reference Substance.
Ethosuximide RS. International Chemical Reference Substance.
Ethyl acetate R. C4H8O2 (SRIP, 1963, p. 86).
1335
Ethyl iodide R. CH
2 5I (SKIP, 1963, p. 87).
Ethylene glycol monoethyl ether R. C4H10O2.

Miscibility. Miscible with water, ethanol (-750 g/1) TS, ether R, and acetone R.
Boiling range. Not less than 95% distils at between 133 and 135 °C.
Mass density{p 2 o)- About 0.93 kg/1.
Ethylene glycol monomethyl ether R. 2-Methoxyethanol; CsHgOj.

Mass density. p 2 o = about 0.96kg/l.
Ethylene oxide R. C2H4O.

Ethylene oxide TS
Procedure. Weigh 1.0 g of cold ethylene oxide stock solution R (equivalent to
2.5mg of ethylene oxide) into a cold flask containing 40 g of cold macrogol
200 TS. Mix and determine the exact mass, and dilute to a calculated mass
to obtain a solution containing 50 pg of ethylene oxide per 1.0 g of solution.
Weigh lO.Og into a flask and dilute with sufficient water to produce 50 ml
(lOpg/ml of ethylene oxide). Dilute 10ml of this solution to 50ml with
water ( 2 pg/ml of ethylene oxide).
Note-. Ethylene oxide TS should be prepared immediately before use.
Ethylene oxide stock solution R

Note-. All operations should be carried out in a fume-hood. The operator must protect
both hands and face by wearing polyethylene protective gloves and an appropriate
face mask.
Procedure. Into a dry, clean test-tube, cooled in a mixture of 1 part of sodium
chloride R and 3 parts of crushed ice, introduce a slow current of ethylene
oxide R gas, allowing condensation onto the inner wall of the test-tube.
Using a glass syringe, previously cooled to -10 °C, inject about 300 pi (cor-
responding to about 0.25 g) of liquid ethylene oxide R into 50ml of macro-
gol 200 TS. Determine the absorbed quantity of ethylene oxide by weighing
before and after absorption. Dilute to 100ml with macrogol 200 TS. Mix
well before use.
Assay. To 10 ml of a 500 g/1 suspension of magnesium chloride R in dehydrated
ethanol R add 20ml of hydrochloric acid/ethanol (0.1 mol/1) VS, stopper the
flask, shake to obtain a saturated solution, and allow to stand overnight to
equilibrate. Weigh 5g of the prepared ethylene oxide stock solution R (con-
and allow to stand for 30 minutes. Titrate
taining about 2.5 g/1) into the flask
with potassium hydroxide/ethanol (0.1 mol/1) VS, determining the end-point
potentiometricaUy. Carry out a blank titration, replacing the substance to be
1336
.
examined with the same quantity of macrogol 200 TS. Calculate the content
of ethylene oxide in mg/g.
Storage. Keep in a tightly closed container in a refrigerator at 4°C.
Ethylenediamine R. C 2 H 8 N 2 .
Description. A colourless to pale yellow, clear liquid; odour, ammonia -like.

Miscibility. Miscible with water and ethanol (-750 g/1) TS; slightly miscible with
ether R.
Storage. Store in a tightly closed container, protected from air and acidic vapours.
Ethylmethylketone R. C 4 HgO.
Description. A clear, colourless, mobile liquid; odour, characteristic.
B oiling range. 7 9-8 0 °C
1-EthyIquinaldinium iodide R. l-Ethyl-2-methylquinolinium iodide;

C,2H„1N.
Description. A yellow-green solid.
Solubility. Sparingly soluble in water.
1-EthyIquinaldinium iodide (15g/I) TS. A solution containing about 15g of

1-ethylquinaldinium iodide R per litre.
Etoposide RS. International Chemical Reference Substance.
Ferric ammonium sulfate (0.1 mol/I) VS. Ferric ammonium sulfate R,

dissolved in a mixture of sulfuric acid (-1760 g/1) TS and water to contain
48.22g of FeNH4(S04)2,12H20 in 1000ml.
Procedure. Dissolve 50 g of ferric ammonium sulfate R in a mixture of 300 ml of
water and 6ml of sulfuric acid (~1760g/l) TS. Dilute with sufficient water
to produce 1000ml.
solution in the following manner: Transfer 25ml to a glass-stoppered flask
and add 3 ml of hydrochloric acid (-420 g/1) TS and 2g of potassium iodide
R. Allow the solution to stand for 10 minutes and titrate the liberated iodine
with sodium thiosulfate (0.1 mol/1) VS, using starch TS as indicator. Perform
a blank determination and make any necessary corrections.
Storage. Store this solution in a tightly closed container, protected from light.
Ferric ammonium sulfate (45 g/1) TS. A solution of ferric ammonium sulfate
R containing about 45 g/1 of FeNH4(S04)2.
1337
Ferric ammonium sulfate R. FeNH 4 (S 04 ) 2 12 H 20

,
(SKIP, 1963, p. 88).
Ferric ammonium sulfate TSl

Procedure. Dissolve 0.2 g of ferric ammonium sulfate R in 50 ml of water, add
6ml of nitric acid (-1000 g/1) TS and sufficient water to produce 100ml.
Ferric ammonium sulfate TS2

Procedure. Dissolve 8.3 g of ferric ammonium sulfate R in sufficient sulfuric acid
(0.25 mol/1) VS to produce 1000 ml.
Ferric chloride (25 g/1) TS. A solution of ferric chloride R containing about
27 g/1 of FeCla.
Ferric chloride (50g/l) TS. A solution of ferric chloride R containing about

50g of FeCls per litre.
Ferric chloride (65 g/1) TS. A solution of ferric chloride R containing about
65g of FeCh per litre.
Ferric chloride R. FeCl3,6H20 (SRIP, 1963, p. 88).
Ferric chloride/ferricyanide/arsenite TS
Procedure. Prepare 3 separate solutions:
(1) Dissolve 2.7 g of ferric chloride R in 100 ml of hydrochloric acid (-70 g/1) TS.
(2) Dissolve 3.5 g of potassium ferricyanide R in 100ml of water. This solution
should be freshly prepared.
(3) Dissolve 3.8 g of arsenic trioxide R in 25ml of hot sodium hydroxide
(-80 g/1) TS. AUow to cool, add 50 ml of sulfuric acid (-100 g/1) TS, and dilute
Immediately before use mix 5 volumes of solution (1), 5 volumes of solution
(2), and 1 volume of solution (3).
Ferric chloride/potassium ferricyanide TS

Procedure. Dissolve 2g of ferric chloride R and O.lOg of potassium ferricyanide
R in sufficient water to produce 20 ml.
Note. Ferric chloride/potassium ferricyanide TS must be freshly prepared.
Ferricyanide standard (50pg/ml) TS

Procedure. Prepare a solution of potassium ferricyanide R in water to contain
7.8g of KsFefCN);; per 100ml. Dilute 1.0ml of this solution with sufficient
Note. Ferricyanide standard (50pg/ml) TS must be freshly prepared.
1338
Ferrocyanide standard (100|ig/ml) TS

Procedure. R in water to contain
Prepare a solution of potassium ferrocyanide
2.0g of K4pe(CN)6,3H20 per 100ml. Dilute 1.0ml of this solution with suf-
ficient water produce 100.0 ml.
to
Note. Ferrocyanide standard (lOOpg/ml) TS must be freshly prepared.
Ferroin TS
Procedure. Dissolve O.lSg of o-phenanthroline R in 10 ml of a solution of ferrous
sulfate R, prepared by dissolving 0.70 g of clear crystals of ferrous sulfate R
in 100 ml of water.
Note: The ferrous sulfate solution must be prepared immediately before dis-
solving the o-phenanthroline.
Storage. Ferroin TS should be stored in well-closed containers.
Ferrous ammonium sulfate (O.lmoI/1) VS

Procedure. Dissolve 40 g of ferrous ammonium sulfate R in 100 ml of sulfuric
acid (~190g/l) TS, and dilute to 1000 ml with carbon-dioxide-free water R.
solution in the following manner: To 25 ml add 10 ml of sulfuric acid
TS and 1 ml of phosphoric acid (-440 g/1) TS and titrate with potas-
(~100g/l)
sium permanganate (0.02 mol/1) VS. Each ml of potassium permanganate
(0.02 mol/1) VS is equivalent to 39.21 mg of (NH4)2Fe(S04)2.
Ferrous ammonium sulfate (Ig/I) TS. A solution of ferrous ammonium

sulfate R containing about 1 g/1 of Fe(NH4)2(S04)2.
Ferrous ammonium sulfate R. Fe(NH4)2(S04)2,6H20 (SRIP, 1963, p. 89).
Ferrous sulfate (O.lmol/I) VS

Procedure. Dissolve 2.8 g of ferrous sulfate R in 90 ml of freshly boiled and
cooled water, and add a sufficient quantity of sulfuric acid (~1760g/l) TS to
produce 100 ml.
solution in the following manner: To 40.0 ml of the ferrous sulfate solution
add 5ml of phosphoric acid (-1,440 g/1) TS and titrate immediately with
potassium permanganate (0.02 mol/1) VS.
Note: Standardize immediately before use.
Ferrous sulfate (15 g/1) TS. A solution of ferrous sulfate R in freshly boiled
and cooled water containing about 15g/l of FeS04 (approximately 0.1 mol/1).
Note: Ferrous sulfate (15g/l) TS must be freshly prepared.
Ferrous sulfate (7 g/1) TS. A solution of ferrous sulfate R in freshly boiled

and cooled water containing about 7 g of FeS04 per litre.
Note: Ferrous sulfate (7 g/1) TS must be freshly prepared.
1339
Ferrous sulfate R. FeSO^/HjO (SKIP, 1963, p. 90).
Ferrous sulfate/hydrochloric acid TS

Procedure. Dissolve 0,45 g of ferrous sulfate R in 50 ml of hydrochloric acid
(0.1 mol/1) VS and dilute with sufficient carbon-dioxide-free water to R
produce 100 ml.
Note-. Ferrous sulfate/hydrochloric acid TS should be prepared immediately
before use.
Firebrick, pink, R. A suitable grade for use in gas chromatography with an

average particle size of about 180-250 pm.
FlucIoxacUIin sodium RS. International Chemical Reference Substance.
Flucytosine RS. International Chemical Reference Substance.
Fludrocortisone acetate RS. International Chemical Reference Substance.
Fluoroquinolonic acid RS. International Chemical Reference Substance.
Fluorouracil RS. International Chemical Reference Substance.
Fluphenazine decanoate RS. International Chemical Reference Substance.
Fluphenazine enantate RS. International Chemical Reference Substance,
Fluphenazine hydrochloride RS. International Chemical Reference

Substance.
Folic acid RS. International Chemical Reference Substance.
Formaldehyde TS [formaldehyde R], (SRIP, 1963, p. 91).
Formaldehyde/sulfuric acid TS
Procedure. To 10ml of sulfuric acid (-1760 g/1) TS add 0.2ml of formaldehyde
TS.
Shelf-life. Use within 1 month after preparation.
Formamide R. CH3NO (SRIP, 1963, p. 92).
Formic acid (~1080g/l) TS

[formic acid R], CH O
2 2 (SRIP, 1963, p. 92) d~ 1.2.
Formic acid, anhydrous, R. CH O

2 2, d~ 1.22. Contains not less than 98.0%
of CH O 2 2.
Description. A colourless liquid; odour, pungent.
1340

Chlorides. Dilute 1 ml to 15 ml with water and proceed as described under 2.2.1
Limit test for chlorides. Anhydrous formic acid R contains not more than
0.50mg/g.
Sulfates. Dilute 0.5 ml to 15 ml with water and proceed as described under 2.2.2
Limit test for sulfates. Anhydrous formic acid R contains not more than
1.5mg/g.
at 105 °C; leaves not more than 0.5mg/g of residue.
Assay. To about 10 ml of water, quickly add about 1 ml
a tared flask containing
of the test liquid, and weigh. Dilute with 50 ml of water, and titrate with
carbonate-free sodium hydroxide (1 mol/1) VS, using phenolphthalein/
ethanol TS as indicator. Each ml of carbonate-free sodium hydroxide
(1 mol/1) VS is equivalent to 46.03 mg of CH O
2 2.
3-Formylrifamycin SV RS. International Chemical Reference Substance.
Framycetin sulfate RS. International Chemical Reference Substance.
Fuchsin TS
Procedure. Pour carefully 40 ml of sulfuric acid (~1760g/l) TS into 60 ml of water.
Allow to cool and add 100ml of a 1 g/1 solution of basic fuchsin R. Dilute
with water to 200 ml and allow to stand. An orange-yellow colour devel-
ops. Immediately before use dilute the solution with an equal volume of
glacial acetic acid R.
Fuchsin, basic, R [magenta, basic R]. A mixture of rosaniline hydrochloride,

(H 2 NCsH 4) 2 C:C 5H 3 (CH 3 ):NH 2 '^CL, and pararosaniline hydrochloride,
(H2NC,H4)2C:C,H4:NH2"CT.
Description. Crystals or crystalline fragments, with a glossy, greenish-bronze
lustre.
Solubility. Soluble in water, ethanol (~750g/l) TS, and amyl alcohol R.

O.lOg/g.
Sulfated ash. Ignite 1 g with 0.5 ml of sulfuric acid (-1760 g/1) TS: not more than
3-Omg/g.
Fuchsin, decolorized, TS
Procedure. Dissolve 1 g of basic fuchsin R in 600 ml of water and cool in an ice-
bath; add 20 g of sodium sulfite R dissolved in 100 ml of water; cool in an
ice-bath and add slowly, with constant stirring, 10 ml of hydrochloric acid
(-250 g/1) TS; dilute with water to 1000 ml. If the resulting solution is turbid,
it should be filtered and, if brown in colour, it should be shaken with suffi-
cient charcoal R (0.2-0.3g) to render it colourless, and then filtered imme-

diately. Occasionally, it is necessary to add 2-3 ml of hydrochloric acid
(-250 g/1) TS, followed by shaking, to remove a little residual pink colour.
1341
The solution resulting from any of the foregoing modifications should be

allowed to stand overnight before use. Decolorized fuchsin TS should be
Fuchsin/sulfurous add TS
Procedure. Dissolve O.lOg of fuchsin, basic, R in 50ml of water with gentle
heating.To the cooled solution add 20 ml of sodium metabisulfite (50g/l)
TS and 1 ml of hydrochloric acid (-420 g/1) TS. Dilute to 100ml with water,
mix, and allow to stand in the dark for 2 hours. Fuchsin/sulfurous acid TS
should be colourless and should not be used for a period longer than 24
hours.
Furosemide RS. International Chemical Reference Substance.
Gallamine triethiodide RS. International Chemical Reference Substance.
Gelatin R. Gelatin of suitable purity.
Gelatin TS. A solution of gelatin R dissolved in phosphate buffer, pH 7.0, TS

containing about 10 g/1.
Gentamidn sulfate RS. International Chemical Reference Substance.
Glibenclamide RS. International Chemical Reference Substance.
Glucose hydrate R. Monohydrate of a-D-glucopyranose, C6Hi206,H20. Con-

tains not less than 99.0% and not more than 101.5% of C^HoOg, calculated
Description. Colourless crystals or a white crystalline or granular powder;
odourless.
Solubility. 1 part of water and in about 60 parts of ethanol
Soluble in about
(-750 g/1) TS; more soluble in boiling water and in boiling ethanol (-750 g/1)
TS.
Acidity. Dissolve 5g in 50 ml of carbon-dioxide-free water R. It requires for
neutralization not more than 0.5 ml of carbonate-free sodium hydroxide
(0.02 mol/1) VS, phenolphthalein/ethanol TS being used as indicator.
Specific optical rotation. Dissolve 100 mg, previously dried to constant weight, in
1 ml of water, and add a few drops of ammonia (-100 g/1) TS; [a]o = -1-52
to-h53°.
Soluble starch or sulfites. Dissolveg in 10 ml of water and add
1 1 drop of iodine
TS; the liquid is coloured yellow.
Loss on drying.Dry to constant weight at 105 °C; it loses not less than 80mg/g
and not more than lOOmg/g.
Assay. Dissolve about 0.1 g, accuratelyweighed, in 50ml of water, add 30ml
of iodine (0.1 mol/1) VS, 10 ml of sodium carbonate (50 g/1) TS, and allow to
1342
stand for 20 minutes. Add TS and titrate

15 ml of hydrochloric acid (~70g/l)
the excess of iodine withsodium thiosulfate (0.1 mol/1) VS, using starch TS
as indicator.Perform a blank determination and make any necessary cor-
rections. Each ml of iodine (0.1 mol/1) VS is equivalent to 9.008mg of
C6H12O5.
Glucose, anhydrous, R. C,^,206- Use anhydrous glucose as described in the

monograph for “Glucose”.
Glycerol R. Propane-1, 2, 3-triol with small amounts of water, C3H8O3. Con-

tains not less than 970g/kg of C3H8O3.
Description. A clear, almost colourless, syrupy and hygroscopic liquid; odour-
less.
Miscibility. Miscible with water and ethanol (-750 g/1) TS; practically immisci-
ble with ether R.
Mass density(p 2 o). Not less than 1.256 kg/1.
Refractive indexing). Not less than 1.469.
Acrolein and other 1 ml of ammonia (-100 g/1)
reducing substances. Mix 1 ml with
TS and heat in a water-bath at 60 °C for 5 minutes; the liquid is not coloured
yellow. Remove from the water-bath and add 3 drops of silver nitrate
(40 g/1) TS; the liquid does not become coloured within 5 minutes.
Sulfated ash. Not more than 0.5 mg/ml.
Glycine R. Aminoacetic acid; C2H5NO2.

Solubility. Very soluble in water; slightly soluble in ethanol (-750 g/1) TS.
Assay. Determine the nitrogen by the Kjeldahl method in the test substance
previously dried at 105°C for 2 hours; between 18.4 and 18.8% of N is
found, corresponding to not less than 98.6% and not more than the equiv-
alent of 100.8% of C2H5NO2.
Insoluble matter. 10 g shows not more than 1.0 mg of insoluble matter (0.1 mg/g).
Chlorides. Not more than 0.1 mg of Cl/g.
Sulfates. Not more than 0.05 mg of SO^g.
Heavy Not more than 0.02 mg/g.
metals.
Iron. Not more than 0.01 mg of Fe/g, 3 ml of hydrochloric acid (-420 g/1) TS
being used to facilitate solution.
Glyoxal bis(2-hydroxyanil) R. 2,2'-(Ethanediylidenedinitrilo)diphenol,

C14H12N2O2.
Description. White crystals.
Solubility. Soluble in hot ethanol (-750 g/1) TS.
Melting temperature. 203-205 °C.
Glyoxal bis(2-hydroxyanil) TS. A solution of glyoxal bis(2-hydroxyanil) R

dissolved in ethanol (-750 g/1) TS containing about lOg/1 of C14H12N2O2.
1343
Green stock standard TS

Procedure. To 3.5 ml of cobalt colour TS, add 20.1ml of copper colour TS,
10.4ml of dichromate colour TS, and 4.0ml of iron colour TS; dilute to
100.0ml with sulfuric acid (~10g/l) TS, and mix.
Griseofulvin RS. International Chemical Reference Substance.
Haloperidol RS. International Chemical Reference Substance.
Helium R. He. Contains not less than 999.95 ml/1 of He.
Heparin RS. World Health Organization International Reference Material.

Heparin, porcine, mucosal. 5th International Standard 1998. (Ampoules
containing 2031 lU (distributed by the National Institute for Biological
Standards and Control (NIBSC), PO Box 1193, Blanche Lane, South Mimms,
Potters Bar, Herts EN6 3QH, England.))
Heparinized saline TS. A sterile solution of saline TS containing 50 Interna-

tional Units of heparin in 1 ml.
Heptane R. C 7 H,s (SRIP, 1963, p. 94).
Hexamethyldisilazane R. C^HigNSL.
Description. A clear, colourless liquid, having a characteristic odour.
Hexane R. «-Hexane, C^Hm.

Description. A colourless, mobile, highly inflammable liquid.
Boiling range. Distils completely over a range of 1 °C between 67.5 and 69.5 °C.
Mass density. P 20 = 0.658-0.659 kg/1
Hexylamine R. Hexaneamine; C^HjsN.

Boiling point. 127-131 °C.
Refractive index. nS* = about 1.418.
Histamine dihydrochloride R. C5H9N3,2HC1. Contains not less than 98.0%,

and not more than 101.0% of C 5 H 9 N 3 2 HC 1 calculated with reference to
, ,

Description. Colourless crystals or a white crystalline powder; odourless.
Solubility. Freely soluble in water and in methanol R; soluble in ethanol
(-750 g/1) TS.
1344
Loss on drying. Not more than 5.0mg/g.

of chloroformR and 25ml of ethanol (-750 g/1) TS. Titrate with carbonate-
free sodium hydroxide (0.2 mol/1) VS, using 0.5ml of thymolphthalein/
(0.2mol/l) VS is equivalent to 9.21 mg of C5H9N3,2HC1.
Histamine phosphate R. C5H9N3,2H3P04. Contains not less than 98.0%, and

not more than 101.0% of C,5H9N3,2H.3P04, calculated with reference to the
Description. Colourless, long, prismatic crystals; odourless. Stable in air.
Solubility. Soluble in about 5 parts of water; slightly soluble in ethanol

(-750 g/1) TS.
Water. Determined by the Karl Fischer method, using about l.Og, the water
content is 50-60 mg/g.
of chloroform R and 25 ml of ethanol (-750 g/1) TS. Titrate with carbonate-
free sodium hydroxide (0.2 mol/1) VS, using 0.5ml of thymolphthalein/
(0.2mol/l) VS is equivalent to 15.36mg of C5H9N3,2H3P04.
Histamine TS. A solution containing 1.0 mg/1 of histamine base.

Procedure. Prepare histamine TS by diluting strong histamine TS with a suffi-
cient quantity of saline TS.
Note: Histamine TS must be freshly prepared.
Histamine, strong, TS. A solution containing 1.00 g/1 of histamine base.

Procedure. Dissolve 138.1 mg, accurately weighed, of histamine phosphate R or
82.8 mg, accurately weighed, of histamine dihydrochloride R in sufficient
water to produce 50.0 ml.
Storage. Strong histamine TS should be stored at a temperature not exceeding
4-1 0°C, in dark glass botties with ground -glass stoppers, protected from light.
Shelf-life. Do not use longer than 30 days.
Holmium oxide R. H02O3. Contains not less than 99.9% of H02O3, the impu-
rities consisting of Et203and Dy203.
Description. A tan-coloured powder.
Solubility. Insoluble in water.
Holmium perchlorate TS
Procedure. Dissolve 40 g of holmium oxide R in sufficient perchloric acid
(-140 g/1) TS to produce 1000ml.
Hydrazine hydrate R. N2H4,H20. Contains not less than 98.0% of N2H4,H20.

1345
Miscibility. Miscible with water.

Residue on evaporation. Evaporate to dryness on a water-bath; it leaves a residue
of notmore than 5.0mg/g.
Assay. Dilute Ig to 200ml with water. Neutralize 20ml of this solution with
hydrochloric acid (-420 g/1) TS and add 10ml in excess. Add 5 ml of potas-
sium cyanide (100 g/1) TS, titrate with potassium iodate (0.05 mol/1) VS until
the brown colour which first forms becomes pale, add starch TS, and con-
tinue the titration until the blue colour disappears. Each ml of potassium
iodate (0.05 mol/1) VS is equivalent to 2.503 mg of N2H4,H20.
Hydrazine sulfate R. (NH 4 2 ,H 2 S 04 ) .
Description. Colourless crystals or a white, crystalline powder.

Solubility. Soluble in about 40 parts of water; practically insoluble in ethanol
(-750 g/1) TS.
Arsenic. Use a solution of lOg in35ml of boiling water and proceed as described
under 2.2.5 Limit test for arsenic; not more than 1 pg/g.
Hydriodic acid (~970g/l) TS [hydriodic acid R]. HI (SKIP, 1963, p. 95).
Hydrochloric acid (~330g/l) TS. A solution of hydrochloric acid (-420 g/1)

TS in water, containing approximately 330 g of HCl per litre; d~ 1.15 (about
9 mol/1).
Hydrochloric acid (~420g/l) TS [hydrochloric acid, saturated, R]. (SRIP,

1963, p. 96); 1.18.
Hydrochloric acid (-250 g/1) AsTS. Hydrochloric acid (-250 g/1) TS that
complies with the following tests A and B:
A. Dilute 10 ml with sufficient water to produce 50ml, add 5 ml of ammonium

thiocyanate (75 g/1) TS and stir immediately; no colour is produced.
B. To 50ml add 0.2ml of bromine AsTS, evaporate in a water-bath until
reduced to 16ml, adding more bromine AsTS if necessary to ensure that an
excess, as indicated by the colour, is present throughout the evaporation.
Add 50 ml of water and 5 drops of stannous chloride AsTS and apply the
general test for arsenic. The colour of the stain produced is not more intense
than that produced from a 0.2-ml standard stain, showing that the amount
of arsenic does not exceed 0.05pg/ml.
Hydrochloric acid (~250g/l) FeTS. Hydrochloric acid (-250g/l) TS that

complies with the following additional test: Evaporate 5 ml nearly to
dryness on a water- bath, add 40 ml of water, 2 ml of citric acid (180 g/1) FeTS,
and 2 drops of mercaptoacetic acid R; mix, make alkaline with ammonia
(-100 g/1) FeTS, and dilute to 50 ml with water; no pink colour is produced.
1346
Hydrochloric acid (~250g/l), stannated, AsTS

Procedure. Dilute 1 ml of stannous chloride AsTS with sufficient hydrochloric
acid (-250 g/1) AsTS to produce 100 ml,
Hydrochloric acid (~70g/l) TS

260 ml of hydrochloric acid (-250 g/1) TS with
Procedure. Dilute sufficient water
to produce 1000ml (approximately 2mol/l); d - 1.035.
Hydrochloric acid (0.0001 mol/1) VS. Hydrochloric acid (-250 g/1) TS,
diluted with water to contain 3.647 mg of HCl in 1000 ml.
lowing the method described under hydrochloric acid (1 mol/1) VS.
Hydrochloric acid (0.001 mol/I) VS. Hydrochloric acid (-250 g/1) TS, diluted
with water to contain 36.47 mg of HCl in 1000ml.
Hydrochloric acid (0.005 mol/1) VS. Hydrochloric acid (-250 g/1) TS, diluted
with water to contain 0.1824g of HCl in 1000ml.
Hydrochloric acid (0.01 mol/1) VS. Hydrochloric acid (-250g/l) TS, diluted
with water to contain 0,3647g of HCl in 1000 ml,
with water to contain 0.5470 g of HCl in 1000 ml.
with water to contain 0.7293 g of HCl in 1000 ml,
Hydrochloric acid (0.05mol/I) VS. Hydrochloric acid (-250g/l) TS, diluted

with water to contain 1.824g of HCl in 1000 ml.
Hydrochloric acid (0.1 mol/1) BET. Prepare from hydrochloric acid (-420 g/1)
TS and water BET. It is suitable if, after adjustment to pH 6.5-7. 5, it gives
1347
a negative result under the conditions prescribed in the 3.4 Test for bacterial
endotoxins.
Hydrochloric acid (O.lmol/I) VS. Hydrochloric acid (-250 g/1) TS, diluted
with water to contain 3.647 g of HCl in 1000 ml.
Hydrochloric acid (0.2mol/I) VS. Hydrochloric acid (-250 g/1) TS, diluted
with water to contain 7.293g of HCl in 1000ml.
Hydrochloric acid (0.5mol/I) VS. Hydrochloric acid (-250 g/1) TS, diluted
with water to contain 18.23g of HCl in 1000 ml.
Hydrochloric acid (1 moI/1) VS. Hydrochloric acid (-250 g/1) TS, diluted with
water to contain 36.47 g of HCl in 1000 ml.
Method of standardization. Ascertain the exact concentration of the 1 mol/1 solu-
tion in the following manner: Dissolve about 1.5g, accurately weighed, of
anhydrous sodium carbonate R, previously dried at 270 °C for 1 hour, in 50
ml of water and titrate with the hydrochloric acid solution, using methyl
orange/ethanol TS as indicator. Each 52.99 mg of anhydrous sodium car-
bonate is equivalent to 1ml of hydrochloric acid (1 mol/1) VS.
Hydrochloric acid (2 mol/1) VS. Hydrochloric acid (-250 g/1) TS, diluted with
water to contain 72.93 g of HCl in 1000ml.
Hydrochloric acid (5 mol/1) VS. Hydrochloric acid (-250 g/1) TS, diluted with
water to contain 182.35g of HCl in 1000ml.
Hydrochloric acid (-250 g/1) TS. A solution of hydrochloric acid (-420 g/1)
TS in water, containing approximately 250g/l of HCl; d - 1.12.
Hydrochloric acid CITS. One millilitre contains 50 pg of Cl.

Procedure. Dilute 14.3ml of hydrochloric acid (0.1 mol/1) VS with sufficient
1348
Hydrochloric acid, brominated, AsTS

Procedure. To 100 ml of hydrochloric acid (-250 g/1) AsTS add 1 ml of bromine
AsTS.
Hydrochloric acid/ethanol (1 mol/1) VS. Hydrochloric acid (-250 g/1) TS,

diluted with dehydrated ethanol R to contain 36.47g of HCl in 1000ml of
dehydrated ethanol R.
Hydrochloric acid/ethanol (0.1 mol/1) VS. Hydrochloric acid (-250 g/1) TS,
diluted with dehydrated ethanol R to contain 3.647g of HCl in 1000ml of
dehydrated ethanol R.
Hydrochloric acid/methanol (0.01 mol/1) VS. Hydrochloric acid (-250 g/1)

TS, diluted with methanol R to contain 0.3647g of HCl in 1000ml of
methanol R.
Hydrochlorothiazide RS. International Chemical Reference Substance.
Hydrocortisone acetate RS. International Chemical Reference Substance.
Hydrocortisone R. C 21 H 30 O 5 Use Hydrocortisone

. as described in the mono-
graph for “Hydrocortisone”.
Hydrocortisone RS. International Chemical Reference Substance.
Hydrocortisone sodium succinate RS. International Chemical Reference

Substance.
Hydrogen peroxide (~330g/I) TS [hydrogen peroxide (30 per cent.) R].

(SRIP, 1963, p. 97).
Hydrogen peroxide (~60g/l) TS. A solution in water containing about 60g

of H2 O 2 per litre.
Hydrogen sulfide R. H2 S (SRIP, 1963, p. 98).
Hydrogen sulfide TS. A saturated solution of hydrogen sulfide R in cold

water.
Note: Hydrogen sulfide TS must be freshly prepared.
1349
Hydroquinone R. C 6 H 4 (OH) 2 .
Description. Colourless or almost colourless crystals or a crystalline powder.

Solubility. Soluble in water, ethanol (~750g/l) TS, and ether R.
Note. Hydroquinone R darkens on exposure to air and light.
1 -Hydroxy-9 -an throne RS. International Chemical Reference Substance.
(-)-3-(4-Hydroxy-3-methoxyphenyl)-2-hydrazino-2-methylalanine RS.
(-)-3-(4-Hydroxy-3-methoxyphenyl)-2-methylalanine RS. International

Chemical Reference Substance.
Hydroxyethylcellulose R. Contains not less than 20% of C 2 H 5 O 2 calculated ,

Description. A white or yellowish, flaky, heterogeneous mass; odourless.
Solubility.Practically insoluble in ethanol (-750 g/1) TS; after soaking for several
hours in water, freely soluble in water.
Colour of solution. Transfer 2g to a 200-ml glass-stoppered, conical flask, add
200 ml of carbon-dioxide-free water R, shake, and allow to stand for 30
minutes. Repeat this operation until the substance has dissolved and filter
through sintered glass. Observe 5 ml of the filtrate; it is colourless (keep the
filtrate for the acidity or alkalinity test).
Loss on drying. To l.Og add 25ml of water, stir, and allow to stand. Repeat this
operation until dissolved. Evaporate on a water-bath and dry to constant
weight at 110°C; it loses not more than O.lOg/g. (Keep the dried substance
for the assay.)
Acidity or alkalinity. To 10 ml of the filtrate obtained from the test for colour of
solution, add 2 drops of bromothymol blue/ethanol TS; a yellow colour is
produced. Add 0.5ml of potassium hydroxide (0.01 mol/1) VS; a green or
blue solution is produced.
Assay. Place into a boiling flask as described under 2.9 Determination of
methoxyl, 0.5ml of acetic anhydride R, 0.05-0.10g of phenol R, 0.20g of
red phosphorus R, and 5.0 ml of hydriodic acid (-970 g/1) TS; connect the
flask to the condenser, pass a slow, uniform stream of carbon dioxide R
through the solution, and heat for 60 minutes. Cool for 10 minutes and add
0.035 g, accurately weighed, of the dried substance obtained in the test for
loss on drying. Proceed with this mixture as described under 2.9 Determi-
nation of methoxyl. For the calculation, take an average of 3 determinations.
Each ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to l.OlSmg of
C2H5O2.
Hydroxyethylcellulose TS
Procedure. Place 50ml of water in a 100ml beaker and add 2.0 g of hydrox-
yethylcellulose R. After 15 hours, stir the solution for 1 minute and cen-
1350
trifuge for 15 minutes. Using a pipette, separate 20ml of the supernatant

liquid.
Note: Hydroxyethylcellulose TS must be freshly prepared.
Hydroxylamine hydrochloride (200 g/1) TS. A solution of hydroxylamine

hydrochloride R containing about 200 g of NH20H,HC1 per litre.
Hydroxylamine hydrochloride (70 g/1) TS

Procedure. Dissolve 69.5 g of hydroxylamine hydrochloride R in sufficient water
to produce 1000ml (1 mol/1).
Hydroxylamine hydrochloride R. NH20H,HC1 (SRIP, 1963, p. 99).
Hydroxylamine hydrochloride TS
Procedure. Dissolve1 g of hydroxylamine hydrochloride R in 50 ml of water and
add 50ml of ethanol (~750g/l) TS and 1ml of bromophenol blue/ethanol

TS; then add sodium hydroxide (0.1 mol/1) VS until the solution becomes
green.
Hydroxylamine hydrochloride TS2

Procedure. Dissolve 3.5g of hydroxylamine hydrochloride R in 95ml of ethanol
(-535 g/1) TS, add 0.5ml of bromophenol blue (1 g/1) TS and sufficient potas-
sium hydroxide/ethanol (0.5 mol/1) TS until a greenish tint is developed.
Dilute the solution to 100 ml with ethanol (-535 g/1) TS.
8-Hydroxyquinoline R. 8-Quinolinol; C9H7NO.

Description. A white to yellowish white, crystalline powder.
Solubility. Practically insoluble in water and ether R; freely soluble in ethanol
(-750 g/1) TS, and acetone R.
8-Hydroxyquinoline/chloroform TS
Procedure. Dissolve 1 g of 8-hydroxyquinoline R in sufficient chloroform R to
produce 100 ml.
Hypophosphorous acid R. Phosphinic acid; H3PO2 (SRIP, 1963, p. 100).
Hypophosphorous acid, dilute, TS. A solution of hypophosphorous acid R

containing about lOOg of H3PO2 per 1000ml.
Hypoxanthine R. l,7-dihydro-6H-purin-6-one; C5H4N4O.

Solubility. Very slightly soluble in water, sparingly soluble in boiling water,
soluble in dilute acids and in dilute alkali hydroxide solutions.

Melting point. Decomposes without melting at about 150 °C.
1351
Thin-Layer Chromatography. Examine as prescribed in the monograph on

Mercaptopurine; the chromatogram shows only one principal spot.
Ibuprofen RS. International Chemical Reference Substance.
Idoxuridine RS. International Chemical Reference Substance.
Imidazole R. Glyoxaline, C3H4N2. Contains not less than 99.0% of C3H4N2.


Assay. Dissolve 0.3 g in 50ml of water and titrate with sulfuric acid (0.05 mol/1)
VS, using bromocresol green/ethanol TS as indicator. Each ml of sulfuric acid
(0.05 mol/1) VS is equivalent to 6.808 mg of C3H4N2.
Imidazole, reciystallized, R
Procedure. Dissolve25 g of imidazole R in 100 ml of hot toluene R and cool in
an ice-bath, while stirring. Filter off the crystals with suction using filter-
paper Whatman No. 54 or No. 541. Repeat the crystallization and filtration,
sucking as dry as possible. Slurry wash the resulting crystals with about
50ml of ether R and filter. Repeat this process and then wash the crystals
on the filter with ether R and suck as dry as possible. Transfer to a shallow
dish and dry at room temperature under reduced pressure (not exceeding
0.6kPa or about 5 mm of mercury) over silica gel, desiccant, R.
Storage. Store in a tightly closed container.
Imidazole/mercuric chloride TS
Procedure. Dissolve 8.25 g of recrystallized imidazole R in 60 ml of water and
add 10 ml of hydrochloric acid (5 mol/1) VS. Under continuous stirring add,
drop by drop, 10ml of mercuric chloride (2.7g/l) TS. If a cloudy solution
results, discard and prepare a further solution by adding the mercuric chlo-
ride solution more slowly. Adjust the pH to 6.80 ± 0.05 with hydrochloric
acid (5 mol/1) VS (about 4 ml is required) and add sufficient water to produce
100 ml.
Iminodibenzyl R. 10,ll-Dihydro-5H'-dibenz[f',/]azepine; C14H13N.

Imipramine hydrochloride RS. International Chemical Reference Substance.
Indinavir RS. International Chemical Reference Substance.
Indometacin RS. International Chemical Reference Substance.
1352
Iodide standard (20 ng I/ml) TS

Procedure. Dissolve 26.0 mg of potassium iodide R in sufficient water to produce
100 ml. Dilute 10 ml of this solution to 100 ml with water.
Iodine (0.0001 moI/1) VS. Iodine R and potassium iodide R, dissolved in

water to contain 25.38 mg of R and 36mg of KI in 1000ml.
lowing the method described under iodine (0.1 mol/1) VS.
Iodine (0.0005 mol/1) VS. Iodine R and potassium iodide R, dissolved in

water to contain 0.127g of h and 0.18g of KI in 1000ml.
lowing the method described under “Iodine (0.1 mol/1) VS”.
Iodine (0.005 mol/1) VS. Iodine R and potassium iodide R, dissolved in water
to contain 1.269g of Rand 1.80g of KI in 1000ml.
to contain 2.538 g of h and 3.6g of KI in 1000 ml.
to contain 5.076g of R and 7.2 g of KI in 1000 ml.
to contain 12.69g of h and 18. Og of KI in 1000ml.
Iodine (0.1 mol/I) VS. Iodine R and potassium iodide R, dissolved in water
to contain 25.38g of R and 36.0g of KI in 1000ml.
solution by titrating 25.0ml with sodium thiosulfate (0.1 mol/1) VS, using
starch TS as indicator.
Iodine bromide R. IBr.
Description. Blue-black or brownish-black crystals.

Solubility. Freely soluble in water, ethanol (-750 g/1) TS, ether R, and glacial
acetic acid R.
Storage. Store in a cool place, in a tightly closed container, protected from light.
1353
Iodine bromide TS
Procedure. Dissolve 20 g of iodine bromide R in sufficient glacial acetic acid R
to produce 1000ml.
Storage. Store in a tightly closed container, protected from light.
Iodine pentoxide R. Iodic anhydride; I2O5.

Iodine R. h (SRIP, 1963, p. 101).
Iodine TS
Procedure. Dissolve 2.6g of iodine R and 3g of potassium iodide R in sufficient
water to produce 100ml (approximately 0.1 mol/1).
lodine/chloroform TS
Procedure. Dissolve 5.0 g of iodine R in sufficient chloroform R to produce
100 ml.
lodine/ethanol TS
Procedure. Dissolve lOg of iodine R in sufficient ethanol (-750 g/1) TS to produce
1000 ml.
lohexol RS. International Chemical Reference Substance.
lopanoic acid RS. International Chemical Reference Substance.
lotroxic acid RS. International Chemical Reference Substance.
Iron colour TS. A solution containing 45.0 mg/ml of FeCl3,6H20.

Procedure. Prepare a solution containing 4.500g of FeCl3,6H20 in 100ml, dilut-
ing the strong iron colour TS with sulfuric acid (-10 g/1) TS, as necessary.
Iron colour, strong, TS

Procedure. Dissolve 6.6g of ferric chloride R in 120 ml of sulfuric acid (-10 g/1)
TS, filter the solution, if necessary, and determine the concentration of
FeCl3,6H20.
Assay. Dilute 5.0ml with water to produce 25.0ml. Transfer 10.0ml
sufficient
of this solution to a flask, and add 60 ml of water. Adjust the pH to 2-3 with
hydrochloric acid (1 VS and ammonia (-100 g/1) TS, using congo red
mol/1)
paper R. Heat the solution to approximately 45 °C, and titrate with disodium
edetate (0.05mol/l) VS, using 2ml of sulfosalicylic acid (175g/l) TS as indi-
cator, until the solution changes from a lilac tint to straw-yellow. Each ml
of disodium edetate (0.05 mol/1) VS is equivalent to 13.52 mg of FeCl3,6H20.
1354
Iron salicylate TS
Procedure. Dissolve 0.5 g of ferricammonium sulfate R in 250 ml of water con-
taining 10ml of sulfuric acid (~100g/l) TS and dilute with sufficient water
to produce 500 ml. To 100 ml of this solution add 50 ml of sodium salicylate
(11.5g/l) TS, 20 ml of acetic acid (~60g/l) TS and 80 ml of sodium acetate
(150 g/1) TS, and dilute with water to 500 ml.
Storage. Store in a well-closed container, protected from light.
Note. Iron salicylate must be freshly prepared.
Iron standard FeTS

Procedure. Dissolve 0.173g of ferric ammonium sulfate R in 100 ml of water,
add 5ml of hydrochloric acid (~70g/l) TS and sufficient water to produce
1000 ml. Each ml of this solution contains 20 pg of iron.
Iron, reduced, R. Fe (SRIP, 1963, p. 102).
Isobutyl methyl ketone R. 4-Methyl-2-pentanone; C^HoO.

Description. A clear, colourless liquid; odour, characteristic.
Boiling point. About 115°C.
Isoniazid R. Isoniazid as described in the monograph for “Isoniazid”.
Isoniazid RS. International Chemical Reference Substance.
Isoniazid TS
Procedure. Dissolve 0.1 g of isoniazid R in 150ml of methanol R, add 0.12ml of
hydrochloric acid (-420 g/1) TS, and dilute with methanol R to 200 ml.
Isopropylamine R. C3H9N.
Description. A colourless, volatile liquid with an ammoniacal odour.
Mass density. P 20 = about 0.69 kg/1.
Kanamycin monosulfate RS. International Chemical Reference Substance.
Kaolin, light, R. Kaolin as described in the monograph for “Kaolin”.
Kaolin suspension TS
Immediately before use mix equal volumes of cephalin TS and a sus-
Procedure.
pension containing 4g of kaolin R in 1000 ml of sodium chloride (9 g/1) TS.
Karl Fischer reagent TS. A freshly prepared solution contains up to

5.0 mg/ml of water. The solution should not be used if the water equivalent
falls below 2.5 mg of water per ml of the reagent.
1355
Procedure. Dissolve 63 g of iodine R in 100 ml of anhydrous pyridine R, cool in

ice, and pass sulfur dioxide R into the solution until a gain in weight of
32 g has occurred, taking care to avoid absorption of atmospheric moisture.
Add sufficient dehydrated methanol R to produce 500 ml and allow to stand
for 24 hours. Karl Fischer reagent TS may also be prepared by mixing com-
mercially available solutions of sulfur dioxide in pyridine and of iodine in
methanol, which are stable when properly stored, for example, protected
from light. The resulting solution should conform to the requirements stated
below.
Method of standardization. Ascertain the exact content of water in the following
manner: Add about 20 ml of dehydrated methanol R, to the titration vessel
and titrate to the endpoint with Karl Fischer reagent TS without recording
the volume required. Introduce in an appropriate form a suitable amount of
water, accurately weighed, and titrate again to the endpoint with Karl
Fischer reagent TS, recording the volume. Water might be introduced, for
example, as a solution in dry methanol, or under the form of a hydrated
compound. Calculate the water equivalent of the reagent in mg of water
per ml. Karl Fischer reagent TS deteriorates continuously and should be stan-
dardized immediately before use, or daily, as required.
Note: Ethylene glycol monoethyl ether R may be used in the preparation of the
reagent instead of dehydrated methanol R.
Ketamine hydrochloride RS. International Chemical Reference Substance.
Ketoconazole RS. International Chemical Reference Substance.
Kieselguhr R1 . Kieselguhr G.
A greyish-white powder, of an average particle size between 10
Description.
and 40pm, containing per kg about 150g of calcium sulfate, hemihydrate.
Kieselguhr R2. Kieselguhr GF254.

Description. A greyish-white powder, of an average particle size between 10
and 40 pm, containing per kg about 150 g of calcium sulfate, hemihydrate
and an adequate amount (usually about 15g/kg) of a fluorescent indicator
having a maximum absorption at 254 nm.
Kieselguhr R3
and 200 pm.
Kieselguhr R4
and 150 pm.
1356
Kieselguhr R5. Kieselguhr H.

A fine, greyish-white powder; the grey colour becomes more pro-
Description.
nounced on triturating the powder with water. The average particle size is
between 10 and 40 pm.
Lactobionic acid R. 4-O-p-D-Galactopyranosyl-D-gluconic acid; C12H22O12.

Lanthanum nitrate (30g/l) TS

Procedure. Dissolve 4.3 g of lanthanum nitrate R in 1 ml of nitric acid (~130g/l)
TS and sufficient water to produce 100 ml.
Lanthanum nitrate R. La(N03)3,6H20. Contains not less than 97.0% of

La(N03)3,6H20.
Description. Colourless crystals; deliquescent.
Assay. Dissolve about 0.75 g, accurately weighed, in 25 ml of water, add 3 ml
of nitric acid (-130 g/1) TS, 3g of methenamine R, and about20mg ofxylenol
orange indicator mixture R, and titrate with disodium edetate (0.05 mol/1)
VS becomes pure yellow in colour. If fading of the colour

until the solution
more methenamine
of the indicator occurs towards the end of the titration,
R should be added. Each ml of disodium edetate (0.05 mol/1) VS is equiva-
lent to 21.65mg of La(N03)3,6H20.
Lead acetate (80g/l) TS. A solution of lead acetate R in freshly boiled water
containing about 80 g/1 of C4Hg04Pb (approximately 0.25 mol/1).
Lead acetate paper R

Procedure. Dip white filter-paper into a mixture of 10 volumes of lead acetate
(80 g/1) TS and 1 volume of acetic acid (-60 g/1) TS. Allow to dry and cut the
paper into strips measuring 15mm x 40mm.
Storage. Lead acetate paper R should be kept in a well-closed container.
Lead acetate R. C4H604Pb,3H20 (SRIP, 1963, p. 105).
Lead nitrate (0.05 mol/1) VS. Lead nitrate R, dissolved in water to contain
16.56g of Pb(N03)2 in 1000ml.
solution by diluting 25.0 ml with 200 ml of water, add 10 ml of ammonia
buffer TS and about 20 mg of Mordant Black 11 indicator mixture R, and
titrate with disodium edetate (0.05 mol/1) VS.
Lead nitrate (0.1 mol/1) VS. Lead nitrate R, dissolved in water to contain
33.12g of Pb(N03)2 in 1000ml.
lowing the method described under “Lead nitrate (0.05 mol/1) VS”.
1357
=
Lead nitrate (lOOg/1) TS. A solution of lead nitrate R containing lOOg of

Pb(N03)2 per litre.
Lead nitrate paper R

Procedure. Dip strips of suitable filter-paper in a solution of 10 g of lead nitrate
R in 100ml of water, and allow to dry.
Lead nitrate R. Pb(N03)2 (SRIP, 1963, p. 107).
Lead subacetate TS. Contains not less than 16.7% m/m and not more than
17.4% m/m of Pb in a form corresponding approximately to the formula
CgHi40ioPb3.
Procedure. Dissolve 40. Og of lead acetate R in 90ml of carbon-dioxide-free
water R. Adjust the pH to 7.5 with sodium hydroxide (-400 g/1) TS. Cen-
trifuge and use the clear supernatant solution.
Storage. Lead subacetate TS should be stored in a well-closed container.
Lead(IV) oxide R. PbOg (SRIP, 1963, p. 105).
Lead, dilute, PbTS. One millilitre containslOpg of lead.

Procedure. Dilute 10 ml of strong lead PbTS with sufficient water to produce
100 ml.
Note: Dilute lead PbTS must be freshly prepared.
Lead, strong, PbTS. One millilitre contains lOOpg of lead.

Procedure. Dissolve 0.1598g of lead nitrate R in 5ml of nitric acid (-lOOOg/l) TS
and sufficient water to produce 1000 ml.
Levamisole hydrochloride RS. International Chemical Reference Substance.
Levarterenol hydrogen tartrate R. CsHi]N03,C4H(i0s,H20. Contains not

more than 99% of CgHiiN03,C4Hg06, calculated with reference to the
Solubility. Soluble in water; slightly soluble in ethanol (-750 g/1) TS; practically
Use a 50 mg/ml solution; [a]™
Specific optical rotation. -10 to -13°.
Fischer method. Method A, using about 0.5 g of the substance; not less than
Assay. Dissolve about 0.4 g, accurately weighed, in glacial acetic acid R, and
titrate with perchloric acid (0.1 mol/1) VS as described under 2.6 Non-
aqueous titration, Method A. Each ml of perchloric acid (0.1 ml/1) VS is

equivalent to 31.93mg of CgHnN03,C4Hg0g.
Levodopa RS. International Chemical Reference Substance.
1358
Levonorgestrel RS. International Chemical Reference Substance.
Levothyroxine sodium RS. International Chemical Reference Substance.
Lidocaine RS. International Chemical Reference Substance.
Limulus amoebocyte lysate. Reconstitute the lysate as stated on the label.
For each batch, confirm the stated sensitivity as prescribed under “Sensitiv-
ity of the lysate” in 3.4 The sensitivity of the
Test for bacterial endotoxins.
lysate is defined as the lowest concentration of endotoxin which yields a firm
gel under test conditions and is expressed in endotoxin units per millilitre.
Lincomycin hydrochloride RS. International Chemical Reference Substance.
Lindane RS. International Chemical Reference Substance.
Liothyronine RS. International Chemical Reference Substance.
Lithium carbonate R. Li 2 C 03 .
Descrifation. A white, granular powder; odourless.

Solubility. Sparingly soluble in water; very slighdy soluble in ethanol (-750 g/1) TS.
Lithium carbonate/trinitrophenol TS
Procedure. Dissolve 0.25g of lithium carbonate R and 0.5g of trinitrophenol R
in sufficient water to produce 100 ml.
Lithium chloride (lOg/1) TS. A solution of lithium chloride R containing

about lOg of LiCl per litre.
Lithium chloride R. LiCl.

Description. White, deliquescent crystals or granules.
Solubility. Freely soluble in water; soluble in acetone R, ethanol (-750 g/1) TS,
and ether R.
Lithium methoxide (0.1 mol/1) VS

Procedure. Dissolve 0.694 g of lithium R in 150 ml of methanol R and add suffi-
cient toluene R
to produce 1000 ml.
solution in the following manner: Dissolve about 0.15 g, accurately weighed,
of benzoic acid R in 25 ml of dimethylformamide R and titrate with the
lithium methoxide solution to a red end-point, using quinaldine
red/methanol TS as indicator, as described under 2.6 Non-aqueous titration.
Method B. Each 12.21 mg of benzoic acid is equivalent to 1ml of lithium
methoxide (0.1 mol/1) VS. Lithium methoxide solutions must be standard-
ized immediately before use.
1359
Lithium perchlorate R. LiClO^.

Description. Small crystals.
Solubility. Freely soluble in water; sparingly soluble in ethanol (-750 g/1) TS,
acetone R, ether R, and ethyl acetate R.
Lithium perchlorate/acetic acid TS

Procedure. Dissolve 10.64g of lithium perchlorate R in sufficient glacial acetic
acid R1 to produce 1000 ml.
Lithium R. Li.
Description. A soft metal whose freshly cut surface is silvery-grey, tarnishing

rapidly in air.
Solubility. Reacts violently with water, yielding hydrogen and a solution of

lithium hydroxide; soluble in methanol R, yielding hydrogen and a solution
of lithium methoxide; practically insoluble in ether R.
Litmus paper R. (SRIP, 1963, p. 109).
Litmus R. (SRIP, 1963, p. 108).
Litmus TS
Procedure. Boil lOg of litmus R with 40ml of ethanol (-710 g/1) TS for 1 hour
and pour away the clear liquid; repeat this operation twice with 30ml of
ethanol (-710g/l) TS. Digest the washed litmus with 100ml of water and
filter.
Loperamide hydrochloride RS. International Chemical Reference

Substance.
Macrogol lOOOR
Description. A white, waxy mass.
Viscosity. At 100 °C, about 17.3mm^s“*.
Macrogol 200 R
Description. A clear, colourless or almost colourless viscous liquid.
Solubility. Very soluble in acetone R and in ethanol (-750 g/1) TS; practically
insoluble in ether R and in fatty oils.
Macrogol 200 TS
Procedure. Pour 500 ml of macrogol 200 R into a 1000-ml, round-bottom flask.
Evaporate any volatile components using a rotation evaporator. Heat to
60 °C and apply a vacuum with a pressure of 1.5-2.5kPa for 6 hours.
Macrogol 20M R. Polyethylene glycol 20000. A suitable grade to be used in

gas-liquid chromatography.
1360
Macrogol 400 R. Polyethylene glycol 400. Macrogol 400 R is a polymer of

ethylene oxide and water, represented by the formula H(OCH 2 CH 2)„OH, in
which the average value of n lies between 8.2 and 9.1.
Description. Clear colourless (or practically colourless) viscous liquid having a
slight characteristic odour; slighdy hygroscopic.
Average molecular vaeight. Transfer to a pressure flask 2.1 g of macrogol 400 R,
accurately weighed, and 25.0 ml of phthalic anhydride/pyridine TS. Insert
the stopper in the flask, wrap the flask securely with cloth, and immerse it
in a water-bath maintained at 96-100 °C, to the same depth as the mixture

in the flask, for Remove the flask, retaining the cloth wrapping, and
1 hour.
allow to cool in air to room temperature. To the contents of the flask add
50ml of carbonate-free sodium hydroxide (0.5 mol/1) VS and 5 drops of phe-
nolphthalein/pyridine TS. Titrate with carbonate-free sodium hydroxide
(0.5 mol/1) VS to a pink end-point that remains for not less than 15 seconds.
Perform a blank determination in a similar manner. Calculate the average
molecular weight by multiplying by 4000 the weight, in g, of the test sub-
stance and dividing the result by the difference between the volume, in ml,
of carbonate-free sodium hydroxide (0.5 mol/1) VS consumed for the test
substance and the blank determination. The average molecular weight is
between 380 and 420.
Mass density (p 2 o)- 1.1 10-1. 140 kg/1.
Congealing point. Between 4 and 8°C, the congealing point being the average
of 4 consecutive temperature readings, the highest and lowest of which
differby not more than 0.4 °C.
pH Value. Between 4.5 and 7.5, in a 50g/l solution.
Acidity or alkalinity. Dissolve 5.0 g in 50 ml of water. Add a few drops of phenol
red/ethanol TS. If the solution turns yellow, titrate with sodium hydroxide
(0.01 mol/1) VS; if the solution turns red, titrate with hydrochloric acid
(0.01 mol/1) VS. Not more than 2.0 ml of titrant should be required in either
case.
Heavy metals. Mix 4g, accurately weighed, with 1 ml of hydrochloric acid (~70
g/1) TS and dilute with water to 25 ml. The limit is 50[xg/g.
Limit of monoethylene and dietliylene glycols. Dissolve 50g in 75ml of diphenyl

ether R in a 250-ml distillation flask. Slowly distil at a pressure of 100-
250 Pa (l-2mmHg) into a receiver that is graduated to 100 ml in 1-ml sub-
divisions, until 25ml of distillate have been collected. Add 25.0 ml of water
to the distillate, shake the receiving flask vigorously, and allow the layers to
separate. Cool the container in an ice-bath to solidify and facilitate the
removal of the layer of diphenyl ether R. Filter the water layer through filter-
paper into a glass-stoppered, 50-ml graduated cylinder. To the filtrate add
an equal volume of freshly distilled acetonitrile R, and shake the cylinder
until solution is complete. Pipette 10ml of the solution into 15ml of ceric
ammonium nitrate TS, mix, and within 2-5 minutes determine the
absorbance of the resulting solution at about 525 nm. Use a blank consist-
ing of 15 ml of ceric ammonium nitrate TS and 10 ml of acetonitrile (400 g/1)
1361
TS. Prepare a standard solution by mixing 10 ml of acetronile (400 g/1) TS,

to which 30 mg of diethylene glycol R have been added, and 15ml of ceric
ammonium nitrate TS and determine the absorbance within 2-5 minutes at
about 525 nm, using the same blank as above. The absorbance of the test
solution should not exceed that of the standard solution.
Macrogol p-isooctylphenyl ether R. C34HS2O11, /^’-ftrt-octylphenoxy poly-

ethoxy-ethanol. Use a suitable grade for the 3.2.2 Sterility testing of
antibiotics.
Magnesium (0.1 mg/ml Mg) TS

Procedure. Dissolve 1.014g of magnesium sulfate R in water, add 5ml of sulfu-
ric acid (-100 g/1) TS and dilute with water to 1000 ml.
Magnesium acetate R. C 4H 6Mg 04 4 H 20

,
.
Description. Colourless crystals. Deliquescent.

Magnesium chloride (O.lmol/I) VS

Procedure. Dissolve 20.5 g of magnesium chloride R in sufficient water to
produce 1000ml.
solution, carrying out the complexometric titration of magnesium under 2.5
Complexometric titrations using 25 ml of magnesium chloride solution.
Each ml of disodium edetate (0.1 mol/1) VS is equivalent to 20.33 mg of
MgCl2,6H20.
Magnesium chloride R. MgCl2,6H20 (SRIP, 1963, p. 110).
Magnesium oxide R. MgO.

Description. A white, very fine powder.
Solubility. Very slightly soluble in water; insoluble in ethanol (-750 g/1) TS.
Magnesium standard (lOpg/ml Mg) TS

Procedure. Dilute 10 ml of magnesium (0.1 mg/ml) TS with sufficient water to
produce 100 ml.
Magnesium sulfate (50g/l) TS. A solution of magnesium sulfate R contain-

ing about 50 g of MgS 04 per litre.
Magnesium sulfate R. MgS 04 7 H 20,

(SRIP, 1963, p. 111).
Magnesium sulfa te/sulfuric acid TS

Procedure. Dissolve 25 g of magnesium sulfate R in sufficient sulfuric acid
1362
Maleic acid R. C4H4O4.

Manganese dioxide R. MnOj (SKIP, 1963, p. 112).
Manganese sulfate (15g/l) TS. Manganese sulfate R, dissolved in water to

contain 15.0g/l of MnS 04 .
Manganese sulfate R. MnS04,H20.

Description. Pale-red, slightly efflorescent crystals.
Solubility. Soluble in about 1 part of water and 0.6 part of boiling water; prac-
tically insoluble in ethanol (-750 g/1) TS.
Manganese/sUver paper R
Procedure. To a mixture of equal volumes of silver nitrate (0.1 mol/1) VS and
manganese sulfate (15g/l)add drop by drop sodium hydroxide
TS,
(0.1 mol/1) VS is produced, and filter. Soak strips
until a persistent precipitate
of filter-paper (Whatman No. 1 is suitable) for 15 minutes in the solution,
dry them at ambient temperature, protected from light and acidic or alka-
line vapours. The manganese/silver paper R should be colourless.
Test for sensitivity. Place in a cylinder of about 40 ml capacity (height about
80mm, internal diameter about 30 mm) 1.0 ml of ammonium chloride
(lOpg/ml NH4) TS. Add 9ml of water and 1 g of magnesium oxide R. Imme-
diately stopper the flask using a polyethylene cap below which a man-
ganese/silver paper R is placed. Swirl the solution carefully so that
magnesium particles do not come into contact with the reagent paper. Keep
the cylinder at 50-60 °C for 1 hour. A true grey colour is produced on the
reagent paper.
Mebendazole RS. International Chemical Reference Substance.
Medroxyprogesterone acetate RS. International Chemical Reference

Substance.
Mefloquine hydrochloride RS. International Chemical Reference Substance.
Meglumine (lOOg/1) TS. A solution of meglumine R containing about lOOg

of C H 17NO 5
7 per litre.
Note: Meglumine (100 g/1) TS must be freshly prepared.
Meglumine R. C7H17NO5. Meglumine as described in the monograph for

“Meglumine”.
1363
Menadione R. 2-Methyl-l, 4-naphthoquinone, CiiHs02.

Description. Bright yellow crystals.
Mercaptoacetic add R
(thioglycolic acid R). C2H4O2S (SKIP, 1963, p. 206).
Mercuric acetate R. C4H6Hg04 (SKIP, 1963, p. 112).
Mercuric acetate/acetic acid TS

Procedure. Dissolve 50 g of mercuric acetate R in sufficient glacial acetic acid Rl,
that has been neutralized, if necessary, to crystal violet/acetic acid TS with
perchloric acid (0.1 mol/1) VS, to produce 1000ml.
Mercuric bromide AsTS

Procedure. Dissolve 5g of mercuric bromide R in sufficient ethanol (~750g/l) TS
to produce 100 ml.
Mercuric bromide paper AsR

Procedure. Use smooth, white filter-paper weighing 65-120g/m^. The thickness
of the paper in mm should be approximately equal numerically to the
weight expressed as above, divided by 400. Soak pieces of filter-paper, not
less than 25 mm in width, in mercuric bromide AsTS, decant the superflu-
ous liquid, suspend the paper over a non-metalKc thread, and allow it to dry,
Storage. Store the mercuric bromide paper AsR in stoppered bottles in the dark.
Note: Paper that has been exposed to sunlight or to vapours of ammonia must
not be used as it produces only a pale stain or no stain at all.
Mercuric bromide R. HgBt2 (SRIP, 1963, p. 113).
Mercuric chloride (2.7g/l) TS. A solution of mercuric chloride R containing

about 2.7 g of HgCl2 per litre.
Mercuric chloride (65g/l) TS. A solution of mercuric chloride R containing

about 65 g of HgCh per litre (approximately 0.25 mol/1).
Mercuric chloride R. HgCh (SRIP, 1963, p. 113).
Mercuric chloride/ethanol TS
Procedure. Dissolve 2g of mercuric chloride R in sufficient ethanol (-375 g/1) TS
to produce 100 ml.
Mercuric iodide R. Mercury diiodide; Hgh.

Description. A heavy, crystalline, scarlet-red powder; odourless.
1364
Solubility. Slightly soluble in water; sparingly soluble in ethanol (-750 g/1) TS,
acetone R, and ether R; soluble in solutions containing an excess of potas-
sium iodide R.
Storage. Mercuric iodide R should be stored protected from light.
Mercuric nitrate (O.Olmol/I) VS

Procedure. Dissolve about 3.5 g, accurately weighed, of mercuric nitrate R in a
mixture of 5 ml of nitric acid (-1000 g/1) TS and 500 ml of water, and dilute
with water to 1000 ml.
solution in the following manner: Transfer 20.0ml to a conical flask, add
2ml of nitric acid (-lOOOg/1) TS and 2ml of ferric ammonium sulfate (45 g/1)
TS. Cool to below 20 °C, and titrate with ammonium thiocyanate
(0.01 mol/1) VS to the first appearance of a permanent brownish colour.
Mercuric nitrate (0.02mol/I) VS

Procedure. Weigh accurately about 6.85 g of mercuric nitrate R, dissolve in a
mixture of 10ml of nitric acid (-130g/l) TS and 500ml of water, and dilute
with water to 1000 ml.
solution following the method described under mercuric nitrate (0.01 mol/1)
VS.
Mercuric nitrate R. Hg(N 03) 2 ,H 20 .
Caution. Mercuric nitrate R is poisonous.

Description. A white or slightly yellow, deliquescent, crystalline powder.
Solubility. Soluble in water in the presence of a small quantity of nitric acid
(-1000 g/1) TS.
Mercuric nitrate TS
Procedure. Dissolve 40 g of yellow mercuric oxide R in a mixture of 32 ml of
nitric acid (-lOOOg/1) TS and 15ml of water.
Storage. Keep in a container protected from light.
Mercuric oxide, yellow, R. HgO (SRIP, 1963, p. 114).
Mercuric sulfate TS
Procedure. Mix 5g of yellow mercuric oxide R with 40 ml of water and, while
stirring, add 20ml of sulfuric acid (~1760g/l) TS, then add 40ml of water
and stir until completely dissolved.
Mercury R. Hg (SRIP, 1963, p. 115).
Mercury/nitric acid TS
Procedure. Dissolve 3 ml of mercury R in 27 ml of cold fuming nitric acid R and
dilute the solution with an equal volume of water.
1365
Storage. The solution should be stored, protected from light, and for not more
than 2 months,
Metacycline hydrochloride RS. International Chemical Reference

Substance.
Methanesulfonic acid R
Molecular formula. CH4O3S
Description. Colourless and corrosive liquid, strong irritant.
Solubility. Miscible with water.

Density (d). -1.48.
Methanol R. CH3OH (SKIP, 1963, p. 117).
Methanol, dehydrated, R. Methanol R that complies with the following

requirement: Water, not more than l.Omg/g.
Methenamine R. Hexamethylenetetramine, CgH]2N4. Contains not less than

99.0% of CsH, 2N4.
Description. Colourless crystals or a crystalline powder; odourless.
Acidityand alkalinity. Dissolve 2.5g in 25ml of water. To 10ml add 3 drops of
phenolphthalein/ethanol TS; a pink colour is produced, which changes
to red after the addition of 1 drop of carbonate -free sodium hydroxide
(0.1 mol/1) VS. To a further 10ml aliquot add 3 drops of bromothymol
blue/ethanol TS; a blue colour is produced, which changes to green-blue
after the addition of 3 drops of hydrochloric acid (0.1 mol/1) VS.
of sulfuric acid (0.5 mol/1) VS, and boil until theodour of formaldehyde is
no longer with sodium hydroxide
perceptible. Titrate the excess of acid
(1 mol/1) VS, using methyl red/ethanol TS as indicator. Each ml of sulfuric
acid (0.5 mol/1) VS is equivalent to 35.05 mg of C5H12N4.
DL -Methionine RS. International Chemical Reference Substance.
Methotrexate RS. International Chemical Reference Substance.
Methyl green R. [a-[p-Dimethylamino)phenyl]-a-[4-(dimethyliminio)-2,5-

cyclohexadien-l-ylidene]-/:^-tolyl]trimethylammonium dichloride; Basic
blue 20; C.l. No. 42585; C26H33CI2N3.
Description. A green powder.
Solubility. Soluble in water; soluble in sulfuric acid (-1760 g/1) TS giving a yellow
solution and turning green on dilution.
1366
Methyl green/iodomercurate paper R

Procedure. Dip strips of suitable filter-paper in a solution of 4 g of methyl green
R in 100 ml of water and allow to dry in air. Then immerse the strips for 1
hour in a mixture composed of 14 g of potassium iodide R and 20 g of mer-
curic iodide R in 1 00 ml of water. Wash the strips with water until the wash-
ings are practically colourless and allow to dry in air.
Storage. Methyl green/iodomercurate paper R should be stored protected from
light.
Methyl orange ethanol TS

Procedure. Dissolve 0.04 g of methyl orange R in sufficient ethanol (-150 g/1) TS
to produce 100 ml.
Methyl orange R. Sodium salt of 4'-dimethylaminoazobenzene-4-sulfonic

acid, CMH^NsNaOsS (SRIP, 1963, p. 118).
Methyl orange/acetone TS. A saturated solution of methyl orange R in

acetone R.
N-Methyl-N-nitrosotoluene-4-sulfonamide R. CgH]oN 203 S.

Descrifation. A yellow, crystalline powder.
Solubility. Insoluble in water; soluble in ethanol (-750 g/1) TS and ether R.
Melting tenifperature. About 60°C.
N-Methylpiperazine R. CgHigNg.
Mass density. p2o =0.902 kg/1.
Refractive index, ng* = 1 .466.
Methyl red R. 4'-Dimethylaminoazobenzene-2-carboxylic acid, C15H15N3O2

(SRIP, 1963, p. 118).
Methyl red/ethanol TS
Procedure. Dissolve 25 mg of methyl red R in a mixture of 0.95 ml of sodium
hydroxide (0.05mol/l) VS and 5ml of ethanol (-750 g/1) TS, warm the solu-
tion slightly and after cooling dilute with sufficient ethanol (-375 g/1) TS to
produce 250 ml.
Methyl red/methylthioninium chloride TS

Procedure. Mix 20 ml of a 0.5 mg/ml solution of methyl red R in ethanol
(-150 g/1) TS with 0.4ml of a 20mg/ml solution of methylthioninium chlo-
ride R in water.
Methyl silicone gum R. A suitable grade to be used in gas-liquid chro-

matography.
1367
2-M ethyl-5 -nitroimidazole R. C H5N3O

4 2.
Methyl violet 2B R. Cl 42535; C.I. basic violet.

Melting point. About 137 °C, with decomposition.
Methylamine hydrochloride (20g/l) TS. A solution of methylamine

hydrochloride R containing about 20 g of CH5N,HC1 per litre.
Methylamine hydrochloride R. CHsNjHCl.

Description. Deliquescent tetragonal tablets.
Solubility. Soluble in water and dehydrated ethanol R; practically insoluble in
acetone R, ether R, and ethyl acetate R.
Methyldopa RS. International Chemical Reference Substance.
Methylisobutylketone R. Isopropylacetone, CeHi20. (SRIP, 1963, p. 119).
Methyltestosterone RS. International Chemical Reference Substance.
Methylthioninium chloride (0.2 g/1) TS

Procedure. Dissolve 23 mg of methylthioninium chloride R in sufficient water
to produce 100 ml.
Methylthioninium chloride (lg/1) TS. A solution of methylthioninium

chloride R containing about 1 g of CigHisClNsS per litre.
Methylthioninium chloride R [methylene blue]. C,6HisClN3S,3H20 (SRIP,

1963, p. 119).
Methylthymol blue mixture R

Procedure. Mix 1 part of methylthymol blue R with 100 parts of potassium
nitrate R.
Methylthymol blue R. Tetrasodium [3ff-2,l-benzoxathiol-3-ylidenebis[(6-

hydroxy-5-isopropyl-2-methyl-w-phenylene)methylenenitrilo]]tetraacetic
acid S,S-dioxide; C 37 H 44N 2Na 40 i 3 S.
Description. A brownish- black powder.

Solubility. Freely soluble in water; very slightly soluble in ethanol (-750 g/1) TS.
Metronidazole benzoate RS. International Chemical Reference Substance.
Metoclopramide hydrochloride RS. International Chemical Reference

Substance.
1368
Metronidazole RS. International Chemical Reference Substance.
Miconazole nitrate RS. International Chemical Reference Substance.
Molybdenum trioxide R. M0O3 (SRIP, 1963, p. 120).
Monoethanolamine (O.lmoI/1) VS. A solution of monoethanolamine R in

water to contain 6.108g of C2H7NO in 1000ml.
Monoethanolamine R. C2H7NO.
Description. A clear, colourless to faintly yellow, viscous liquid; odour,
ammoniacal.
Miscibility. Miscible with water, methanol R, and acetone R.
Mass density. p2o = 1.01 kg/1.
Mordant Black 1 1 indicator mixture R

Procedure. Mix 1 g of Mordant Black HR with lOOg of sodium chloride R.
Mordant Black HR
[eriochrome black R]. C.I. Mordant Black 11, C.I. No. 14645, Eriochrome Black
T, Solochrome Black; sodium salt of 2-(2-hydroxy-6-nitro-4-sulfo-l-naph-
thylazo)-l-naphthol, C2oHi2N3Na07S (SRIP, 1963, p. 84).
Morpholine R. Tetrahydro-l,4-oxazine; C4H9NO (SRIP, 1963, p. 121).
N-(1-Naphthyl)ethylenediamine hydrochloride (lg/1) TS. A solution of

A/-(l-naphthyl)ethyIenediamine hydrochloride R containing about Ig of
C]2 Hi 4N2,2HC1 per litre.
N-(1-Naphthyl)ethylenediamine hydrochloride (5g/l) TS. A solution of

W-(l-naphthyl)ethylenediamine hydrochloride R containing about 5g of
Ci 2 HhN 2,2HC1 per litre.
N-(1-Naphthyl)ethylenediamine hydrochloride R. Ci 2 HmN 2,2HC1 (SRIP,

1963, p. 124).
N-(1-Naphthyl)ethylenediamine hydrochIoride/1 -propanol TS

Procedure.To 7 ml of iV-(l-naphthyl)ethylenediamine hydrochloride (lg/1) TS
add 3 ml of 1 -propanol R.
N-(l -Naphthyl)ethylenediamine/ ethanol TS

Procedure. Dissolve 5g of N-(1-Naphthyl)ethylenediamine hydrochloride R
in a mixture of equal volumes of ethanol (-750 g/1) TS and water to produce
1000 ml.
1369
N-(1-Naphthyl)ethylenediamine hydrochloride/propylene glycol TS

Procedure. Dissolve 0.1 g of N-(l-naphthyl)ethylenediamine hydrochloride R in
30 ml of water and dilute to 100 ml with propylene glycol R.
Note. N-(1-Naphthyl)ethylenediamine hydrochloride/propylene glycol TS
must be freshly prepared.
Naloxone hydrochloride RS. International Chemical Reference Substance.
Naphthalene-1, 3-diol R. 1,3-Naphthalenediol; CjoHsOj.

Solubility. Freely soluble in water, ethanol (-750 g/1) TS, and ether R.
Naphthalene-1, 3-diol/ethanol TS
Procedure. Dissolve 0.2g of naphthalene- 1, 3-diol R in sufficient ethanol
1-
2-Naphthol R [P-naphthol R]. CjoHaO (SRIP, 1963, p. 122).
1-Naphthol R. C]oHsO.
2-
Description. Colourless crystals or a white, crystalline powder; odour,
characteristic.
Solubility. Soluble in 5 parts of ethanol (-750 g/1) TS (may form a slightly opales-
cent, colourless or almost colourless solution).
Naphthol TSl
Procedure. Dissolve O.lOg of 1-naphthol R in 3ml of sodium hydroxide
(-150 g/1) TS and dilute with sufficient water to produce 100 ml.
Note: 1-Naphthol TSl must be prepared immediately before use.
Naphthol TSl
Procedure. Dissolve 5g of 2-naphthol R, freshly recrystallized, in 40ml of
sodium hydroxide (-80 g/1) TS and add sufficient water to produce 100ml.
Note: 2-Naphthol TSl must be freshly prepared.
1-Naphtholbenzein R. C 27 H 20 O 3 .
Description. A reddish-brown powder.

Solubility. Practically insoluble in water; soluble in ethanol (-750 g/1) TS,
benzene R, ether R, and glacial acetic acid R.
1-Naphtholbenzein/acetic acid TS
Tnaphtholbenzein R
Procedure. Dissolve 0.2 g of in sufficient glacial acetic acid
R to produce 100ml.
1370
1-NaphthoI/ethanol TS
Procedure. Dissolve 0.05g of 1-naphthol R in 60ml of ethanol (-750 g/1) TS and
add sufficient water to produce 100 ml.
Neamine hydrochloride RS. International Chemical Reference Substance.
Nelfinavir mesilate RS. International Chemical Reference Substance.
Neostigmine metilsulfate RS. International Chemical Reference Substance.
Neutral red R. C.I. 50040; C.I. Basic Red; C 15 H 17 CIN 4 (SRIP, 1963, p. 124).
Neutral red/ethanol TS
Procedure. Dissolve 0.1 g of neutral red R in sufficient ethanol (~375g/l) TS to
produce 100 ml.
Nevirapine, anhydrous RS. International Chemical Reference Substance.
Nevirapine impurity B RS. International Chemical Reference Substance.
Niclosamide RS. International Chemical Reference Substance.
Nicotinamide RS. International Chemical Reference Substance.
Nicotinic acid RS. International Chemical Reference Substance.
Nifedipine RS. International Chemical Reference Substance.
Nifurtimox RS. International Chemical Reference Substance.
Niridazole RS. International Chemical Reference Substance.
Niridazole-chlorethylcarhoxamide RS. International Chemical Reference

Substance.
Nitric acid (~1000g/l) TS [nitric acid (70 per cent.) R]. (SRIP, 1963, p. 125); d
~ 1.41.
Nitric acid (~130g/I) TS

Procedure. Dilute 130ml of nitric acid (~1000g/l) TS with sufficient water to
produce 1000ml (approximately 2mol/l); d ~ 1.07.
Nitric acid (O.OSmoI/l) VS. Nitric acid (-1000 g/1) TS, diluted with water to
contain 3.151 g of HNO3 in 1000ml.
Method of standardization. Ascertain the exact concentration of the solution by
following the method described under nitric acid (1 mol/1) VS.
1371
Nitric acid (lmoI/1) VS. Nitric acid (~1000g/l) TS, diluted with water to
contain 63.10g of HNO 3 in 1000ml.
tion in the following manner: Dissolve 2g of anhydrous sodium carbonate
R in 50ml of water and titrate with the nitric acid solution using 0.1 ml of
methyl orange/ethanol TS as indicator until the solution just becomes
reddish yellow. Boil for 2 minutes; the solution reverts to yellow. Cool and
continue the titration until the reddish yellow colour is restored. Each ml of
nitric acid (1 mol/1) VS is equivalent to 0.0530 g of Na 2 C 03 .
Nitric acid (15g/l) TS. Nitric acid (~1000g/l) TS, diluted with water to
contain 15.0g/l of HNO3.
Nitric acid (3g/I) TS. Nitric acid (~1000g/l) TS, diluted with water to contain
3.0 g/1 of HNO3.
Nitric acid, fuming, R. HNO3 (SKIP, 1963, p. 126); d~ 1.5.
4-Nitroaniline R [y-nitroaniline R]. CgHeNjOj (SRIP, 1963, p. 127).
4-NitroaniIine TSl
Procedure. Dissolve 5g of 4-nitroaniline R in sufficient hydrochloric acid
(1 mol/1) VS to produce 1000 ml.
4-Nitroaniline TS2
Procedure. Dissolve 0.4 g of 4-nitroaniline R in 60 ml of hydrochloric acid
(1 mol/1) VS, cool to 15 °C, and add sufficient sodium nitrite (100 g/1) TS until
1 drop of the mixture turns starch/iodine paper R blue.
Note: 4-Nitroaniline TS2 must be freshly prepared.
Nitrobenzene R. CsRsNOj (SRIP, 1963, p. 128).
4-Nitrobenzoyl chloride R [/^-nitrobenzoyl chloride R]. C7H4CINO3 (SRIP,

1963, p. 128).
Nitrofurantoin RS. International Chemical Reference Substance.
Nitrogen monoxide R. NO.

Nitric oxide, washed with water.
Nitrogen monoxide and nitrogen dioxide detector tube. A cylindrical,

sealed glass tube containing adsorbent filters and suitable supports for an
oxidizing layer Cr(VI) salt and the diphenyl-benzidine indicator. The
minimum value indicated is 5 pl/1 or less, with a relative standard deviation
1372
of at most ±15%. Tubes can be verified with a calibration gas containing

the appropriate impurity, if a negative result is obtained.
Nitrogen R. N2 (SKIP, 1963, p. 129).
Nitrogen for chromatography R.

Contains not less than 99.95% v/v of N2.
Nitrogen, oxygen-free, R. Nitrogen R which has been freed from oxygen by

passing it through alkaline pyrogallol TS.
Nitromethane R. CH3NO2.
Description. A colourless, oily liquid.
Miscibility. Miscible with water, ethanol (~750g/l) TS, ether R, and di-methyl-
formamide R.
Refractive index, no = about 1.380.
Nitrophenanthroline R. 5-Nitro-l,10-phenanthroline; C12H7N3O2.

Nitrophenanthroline TS
Procedure. Dissolve 0.15g of nitrophenanthroline R in 15ml of freshly prepared
ferrous sulfate (7 g/1) TS.
l-Nitroso-2-naphthol-3, 6-disodium disulfonate (2g/I) TS. A solution of

l-nitroso-2-naphthol-3, 6-disodium disulfonate R containing about 2g of
C]oH5NNa208S2 per litre.
l-Nitroso-2-naphthol-3, 6-disodium disulfonate R [l-nitroso-2-naphthol-

3, 6-disodium sulfonate R]. CioH5NNa20gS2 (SRIP, 1963, p. 129).
Nonoxinol 9 RS. International Chemical Reference Substance.
Norethisterone acetate RS. International Chemical Reference Substance.
Norethisterone enantate RS. International Chemical Reference Substance.
Norethisterone RS. International Chemical Reference Substance.
Noscapine RS. International Chemical Reference Substance.
Nystatin RS. International Chemical Reference Substance.
1373
Octanoic acid R. Caprylic acid, CsHi602.

Boiling temperature. About 237 °C,
Mass density. p 2 o = about 0,92 kg/1,
Oil detector tube. A cylindrical, sealed glass tube containing adsorbent filters
and suitable supports for the sulfuric acid indicator. The minimum value
indicated mg/m^, with a relative standard deviation of at most +30%.
is 0.1
Tubes can be verified with a calibration gas containing the appropriate impu-
rity, if a negative result is obtained.
Olive oU R. A commercially available reagent of suitable grade.
Opalescence standard TSl

Procedure. Dilute 15ml of Opalescence stock standard TS with sufficient water
to produce 1000ml.
Shelf-life. Use within 24 hours after preparation.
Opalescence standard TS2

Procedure. Dilute 5.0 ml of Opalescence standard TSl with sufficient water to
produce 100 ml. Mix well and shake before use.
Note: Opalescence standard TS2 must be freshly prepared.
Opalescence standard TS3

Procedure. Dilute 10 ml of opalescence standard TSl with sufficient water to
produce 100 ml. Mix well and shake before use.
Note: Opalescence standard TS3 must be freshly prepared.
Opalescence stock standard TS

Procedure. Dissolve l.Og of hydrazine sulfate R in sufficient water to produce
100ml and allow to stand for 4-6 hours. To 25.0ml of this solution add a
solution of 2.5 g of methenamine R dissolved in 25.0ml of water, mix well,
and allow to stand for 24 hours.
Storage. Store in a glass container free from surface defects.
Shelf-life. Use within 2 months after preparation.
Oracet blue B R. Solvent blue 19; a mixture of l-methylamino-4-

anilinoanthraquinone (C 21 H 16N 2 O 2 ) and l-amino-4-anilinoanthraquinine
(C20H14N2O2).
Oracet blue B/acetic acid TS

Procedure. Dissolve 0.5g of oracet blue B R in sufficient glacial acetic acid R1 to
produce 100 ml.
1374
Osmium tetroxide R. OSO 4 .
Caution. The fumes are corrosive to the eyes, the mucous membranes, and the
skin.
Description. Yellow, needle-shaped crystals or a yellow, crystalline mass; hygro-
scopic; light sensitive; odour, pungent.
Solubility. Soluble in water, ethanol (-750 g/1) TS, and ether R.
Ox brain, acetone-dried, R
Procedure. Cut into small pieces a fresh ox brain previously freed from vascu-
lar and connective tissue. Place in acetone R for preliminary dehydration.
Complete the dehydration by pounding in a mortar 30 g of the material with
successive quantities, each of 75ml of acetone R, until a dry powder is
obtained after filtration. Dry at 37 °C for 2 hours or until the odour of

acetone is no longer perceptible.
Oxalic acid (0.05 g/I) TS. A solution of oxalic acid R containing 0.05 g of
C2H204 in 1000 ml
Procedure. Dissolve 0.07 g of oxalic acid R in sufficient water to produce
1000 ml.
Oxalic acid R. C2H204,2H20 (SRIP, 1963, p. 131).
Oxalic acid/sulfuric acid TS

Procedure. Dissolve 5g of oxalic acid R in a sufficient quantity of a cooled
mixture of equal volumes of sulfuric acid (~1760g/l) TS and water to
produce 100 ml.
Oxamniquine RS. International Chemical Reference Substance.
Oxytetracycline dihydrate RS. International Chemical Reference Substance.
Oxytetracycline hydrochloride RS. International Chemical Reference

Substance.
Pancreatic digest of casein R. (SRIP, 1963, p. 132).
Papaic digest of soybean meal R. (SRIP, 1963, p. 134).
Papaverine hydrochloride RS. International Chemical Reference Substance.
Paracetamol R. Paracetamol as described in the monograph for

“Paracetamol”.
Paracetamol, 4-aminophenol-free, R. Paracetamol as described in the

monograph for “Paracetamol”, or paracetamol recrystallized from water
until it complies with the following test:
1375
Dissolve 5g of the dried material in a mixture of equal volumes of methanol R

and water and dilute to 100ml with this solvent mixture. Add 1.0 ml of alka-
line sodium nitroprusside TS, mix, and allow to stand for 30 minutes; no
blue or green colour is produced.
Paraffin, liquid, R. (SKIP, 1963, p. 135).
Paraformaldehyde R. (CH20)„.
Description. A white, crystalline powder; odour, characteristic of formaldehyde.
Solubility. Slowly soluble in cold water, freely soluble in hot water, with evo-
lution of formaldehyde; practically insoluble in ethanol (-750 g/1) TS and
ether R.
Solubility in ammonia. Dissolve 1 g in 10ml of ammonia (-100 g/1) TS; a practi-
cally clear and colourless solution is produced.
Acidity or alkalinity. Shake Ig with 20ml of water for 1 minute and filter; the
nitrate is neutral to litmus paper R.
Paraldehyde R. 2,4,6-Trimethyl-s-trioxane; CgH] 203 .
Description. Liquid; odour, characteristic, aromatic.

Mass density. p 2 o = 0.994 kg/1.
Paromomycin sulfate RS. International Chemical Reference Substance.
Penicillinase R. An enzyme, usually obtained from culture filtrates of a strain

of Bacillus cereus,which has the specific property of inactivating penicillin
by bond linking the nitrogen of the thiazolidine to the adjacent
splitting the
carbonyl carbon and thus releasing a carboxyl group. It is precipitated from
solutions in water by acetone R, ethanol (-750 g/1) TS, and dioxan R, but
inactivated by several hours’ contact with these solvents; it is rapidly inac-
tivated by ethyl acetate R. In place of penicillinase R a sterile filtrate obtained
by fermentation of a penicillinase-producing organism in a suitable medium,
described below under “Preparation of penicillinase", may also be used
directly.
Description. Small, brown, easily pulverizable pieces or granules.
Solubility. Freely soluble in water, forming a slighdy opalescent solution.
lOg of pancreatic digest of casein R, 2.7 g
Preparation of penicillinase. Dissolve
of potassium dihydrogen phosphate R, and 5.9 g of sodium citrate R in
200ml of water, adjust the alkalinity to pH 7.2 with sodium hydroxide
(-200 g/1) TS, and dilute to 1000 ml with water. Dissolve 0.4 g of magnesium
sulfate R in 5 ml of water and add 1 ml of ferrous ammonium sulfate (1 g/1)
TS and sufficient water to produce 10 ml. Sterilize both solutions by heating
in an autoclave, cool, mix, distribute in shallow layers in conical flasks and
inoculate with a suitable strain {Bacillus cereus, NCTC 9946, is suitable).
Allow the flasks to stand at 18-37 °C until growth is apparent and then
1376
maintain at 35-37 °C for 16 hours, shaking constantly to ensure maximum

aeration. Centrifuge and sterilize the supernatant liquid by filtration through
a suitable membrane filter.
Penicillinase TS. A sterile aqueous solution of penicillinase R. To test the

activity of penicillinase TS, carry out the “Penicillinase assay”. The time
required for iodine decolorization is not more than 36 seconds.
Penicillinase assay. Carry out the assay in test-tubes of borosilicate glass, 15cm
long and about 20mm in internal diameter, immersed in a water-bath of
30 ± 1 °C. All reagents should have a temperature of 30 °C before use.
To the test-tube add the reagents in the following order: 1.6 ml of gelatin TS.
0.4ml of penicillinase TS to be tested, 1 drop of starch TS, and 1 ml of ben-
zylpenicillin sodium TS, blowing out the last reagent from a 1-ml pipette.
Start the stop-watch and after 15 seconds add 2.0ml of iodine (0.01 mol/1)
VS, recording the time of decolorization of iodine from the time of the addi-
tion of benzylpenicillin sodium TS. The activity of penicillinase TS is cal-
culated from the results of the assay. The time of decolorization of strictly
36 seconds corresponds to a penicillinase activity equivalent to a rate of
decomposition (at 30°C at pH 7.0) of 220mg of benzylpenicillin sodium R
per hour per ml of penicillinase TS.
Storage. Store between 0 and 2°C and use within 2-3 days. When dried from
the frozen state and kept in sealed ampoules, penicillinase TS may be stored
for several months.
Pentamidine isetionate RS. International Chemical Reference Substance.
n-Pentane R. C5H12.
Description. A colourless, volatile liquid.
Boiling point.About 36 °C.
d™ = about 1.359.
Relative density,
Transmittance. Not less than 20% at 200 nm, 50% at 210nm, 85% at 220nm,
93% at 230nm, and 98% at 240nm, determined using water in the refer-
ence cell.
1 -Pentanesulfonic acid sodium salt R. C5HnNa03S,H20.

Clarity and colour of solution. A solution of Ig in 25 ml of water is clear and
colourless.
Water. Determined by the Karl Fischer method; not more than 20mg/g.
1 -Pentanesulfonic acid TS
Procedure. Dissolve 0.960g of Tpentanesulfonic acid sodium salt R in 1000ml
of deaerated acetic acid (5.0 g/1) TS and adjust the pH to 4.3 with ammonia
(-260 g/1) TS. Filter and deaerate before use.
1377
Peptone (lg/1) TSl

Procedure. Dissolve 1 .0 g of peptone R1 (or similar peptic digest of animal tissue)
in sufficientwater to produce 1000ml, filter or centrifuge to clarify, adjust

the pH to 7.1 + 0,2, place 100ml portions into individual vessels, and ster-
ilize by maintaining at 121 °C for 18-20 minutes.
Peptone (1 g/1) TS2

Procedure. Dissolve in water,while heating, l.Og of dried peptone R and 9g of
sodium chloride R and dilute with sufficient water to produce 1000 ml.
Adjust to pH 8. 0-8. 4 and boil for 20 minutes. Filter, adjust to pH 7. 2-7. 4,
and sterilize by maintaining at 115°C for 30 minutes.
Peptone (5 g/1) TS
Procedure. Dissolve in water,while heating, 5.0 g of dried peptone R and 7g of
sodium chloride R and dilute with sufficient water to produce 1000 ml.
Adjust to pH 8. 0-8. 4 and boil for 20 minutes. Filter, adjust to pH 7. 2-7. 4,
and sterilize by maintaining at 115°C for 30 minutes.
Peptone Rl. Dried peptone R that conforms to the following requirement:

An autoclaved solution containing 0.02 g/ml is clear and neutral or almost
neutral.
Peptone, dried, R. (SRIP, 1963, p. 137).
Perchloric acid (~1170g/l) TS [perchloric acid (70 per cent, w/w) R]. (SRIP,
1963, p. 137); 1.67.
Perchloric acid (~140g/l) TS. Perchloric acid (~1170g/l) TS, diluted with
water to contain 141 g/1 of HCIO4; d - 1.09.
Perchloric acid (0.02mol/l) VS

Procedure. Dilute 20 ml of perchloric acid (0.1 mol/1) VS with sufficient glacial
acetic acid Rl to produce 100 ml.
Water and method of standardization. Immediately before use determine the
content of water and ascertain the exact concentration of the solution fol-
lowing the methods described under perchloric acid (0,1 mol/1) VS.
Perchloric acid (0.05 mol/1) VS

Procedure. To 900ml of glacial acetic acid Rl at about 25 °C, add 4.2 ml of per-
chloric acid (-1170 g/1) TS, mix, add 15 ml of acetic anhydride R, and mix
again. Cool to room temperature, add sufficient glacial acetic acid Rl to
produce 1000ml, and allow to stand for 24 hours.
Water and method of standardization. Determine the content of water and ascer-
tain the exact concentration of the solution following the method described
under perchloric acid (0.1 mol/1) VS.
1378
Perchloric acid (0.1 mol/1) VS

Procedure. To 900ml of glacial acetic acid Rl, at about 25 °C, add 8.2 ml of per-
chloric acid (~1170g/l) TS, mix, add 32 ml of acetic anhydride R, and mix
again. Cool to room temperature, add sufficient glacial acetic acid Rl, to
produce 1000ml, and allow to stand for 24 hours.
Water. Determine the content by the Karl Fischer method. If necessary, add suf-
ficient water or acetic anhydride R to adjust the content of water to between
0.1 and 2.0mg/ml, and allow to stand for a further 24 hours.
Method of standardization. Ascertain the exact concentration by titrating 0.5 g,
accurately weighed, of potassium hydrogen phthalate R, previously dried at
120 °C for 2 hours, using method A, as described under 2.6 Non-aqueous
titration. Each ml of perchloric acid (0.1 mol/1) VS is equivalent to 20.42 mg
of C8H5KO4. Record the temperature at which the standardization is carried

out.
Perchloric acid TS
Procedure. Dilute 82ml of perchloric acid (~1170g/l) TS with sufficient water to
produce 1000ml (approximately 1 mol/1).
Perchloric acid/dioxan (0.1 mol/1) VS

Procedure. Mix 8.5 ml of perchloric acid (~1170g/l) TS with sufficient dioxan R,
which has been especially purified by adsorption, to produce 1000ml.
titrating 0.7 g of potassium hydrogen phthalate R, accurately weighed and
previously dried at 120°C for 2 hours, using Method A, as described under
2.6 Non-aqueous titration. Each ml of perchloric acid/dioxan (0.1 mol/1) VS
is equivalent to 20.42 mg of C8H5KO4.
Periodic -acetic acid TS

Procedure. Dissolve 0.446g of sodium metaperiodate R in 2.5 ml of sulfuric acid
(-570 g/1) TS and dilute to 100 ml with glacial acetic acid R.
Petroleum, light, R [light petroleum R]. (SRIP, 1963, p. 108).
Petroleum, light, Rl
Description. A colourless, very volatile, highly inflammable liquid.
Boiling range. 40-60 °C.
Mass density. p 2o
= 0.630-0.650kg/l.
o-PhenanthroIine (1 g/1) TS
Procedure. Dissolve 0.11 g of o-phenanthroline R in sufficient water to produce
100 ml.
o-Phenanthroline R. 1,10-Phenanthroline, Ci2H8N2,H20 (SRIP, 1963, p. 138).
1379
o-PhenanthroIine TS
Procedure. Dissolve 0.7 g of ferrous sulfate R in about 70 ml of water, add about
1.5g of o-phenanthroline R and sufficient water to produce 100 ml.
Phenobarbital RS. International Chemical Reference Substance.
Pbenol (50g/l) TS. A solution of phenol R containing about 50 g of CgHsO

per litre.
Phenol R. CgHeO.
Description. Colourless, or at most faintly pink, cohering or separate acicular
crystals, or crystalline masses; odour, characteristic. Corrosive, and blanches
the skin and mucous membranes.
Solubility. Soluble in about 15 parts of water and in about 100 parts of liquid
paraffin R; freely soluble in ethanol (~750g/l) TS, and ether R.
Completeness of solution. l.Og dissolves completely in 15 ml of water at 15 °C.
Congealing temperature. Not below 40.5 °C.
at 105 °C; leaves not more than 0.5mg/g of residue.
Phenol red R. Phenolsulfonphthalein, C19H14O5S (SRIP, 1963, p. 139).
Phenol red/ethanol TS
Procedure. Dissolve 0.05 g ofphenol red R in a mixture of 2.85ml of sodium
hydroxide (0.05mol/l) VS and 5ml of ethanol (-710 g/1) TS. Warm the solu-
tion slightly and after cooling dilute with sufficient ethanol (-150 g/1) TS to
produce 250 ml.
Phenoldisulfonic acid (250 g/1) TS. A commercially available reagent of

suitable grade.
Phenoldisulfonic acid TS
Description. A clear liquid which may develop a pale brown colour on storage.
Procedure. Either of the following methods of preparation can be used: (1) To
3g of phenol R add 20 ml of sulfuric acid (-1760g/l) TS and heat on a water-
bath for 6 hours; store in a stoppered vessel. (2) Dilute phenoldisulfonic acid
(250 g/1) TS with sulfuric acid (-1760g/l) TS to contain 150 g of phenol per
litre.
Evaporate a solution containing 0.1 mg of potassium nitrate

Sensitivity to nitrate.
R to dryness in a porcelain dish on a water-bath. Cool, add 1.0 ml of the

solution to be examined, and allow to stand for 10 minutes. Add 10ml of
water, cool, add 10 ml of ammonia (-100 g/1) TS, and dilute to 25 ml with
water; a distinct yellow colour is produced when compared with a solution
prepared similarly but omitting the potassium nitrate R.
Phenolphthalein R. C20H14O4 (SRIP, 1963, p. 139).
1380
Phenolphthalein/ethanol TS
Procedure. Dissolve l.Og of phenolphthalein R in sufficient ethanol (-750 g/1) TS
to produce 100 ml.
Phenolphthalein/pyridine TS
Procedure. Dissolve l.Og of phenolphthalein R in sufficient pyridine R to
produce 100 ml.
Phenoxyacetic acid R. Phenoxyethanoic acid; CsHsOj.

Description. White or almost white crystals.
Melting temperature.About 98°C.
2-PhenoxyethanoI R. CbHioOj.
Description. A clear, colourless, oily liquid; odour, faintly aromatic.
Miscibility. Slighdy miscible with water; freely miscible with ethanol (-750 g/1)
TS and ether R.
Refractive index, ng* = about 1.537.
Freezing point. Not below 12.0 °C.
Phenoxymethylpenicillin calcium RS. International Chemical Reference

Substance.
Phenoxymethylpenicillin potassium RS. International Chemical Reference

Substance.
Phenoxymethylpenicillin RS. International Chemical Reference Substance.
1,4-Phenylenediamine dihydrochloride R. C6H8N2,2HC1.

Description. A white to pale tan, crystalline powder, turning pink on exposure
to air.
Solubility. Freely soluble in water; slightly soluble in ethanol (-750 g/1) TS and
ether R.
Storage. Keep in a well-closed container, protected from light.
2-PhenyIethanol R. CgHioO. A suitable grade to be used in gas-liquid chro-

matography.
2-PhenyIethanol TS
Procedure. Dissolve 1 g of 2-phenylethanol R in sufficient methanol R to produce
50 ml.
Phenylhydrazine hydrochloride (lOg/1) TS. A solution of phenylhydrazine

hydrochloride R containing lOg of C 6 HgN 2 ,HCl in 1000ml.
Phenylhydrazine hydrochloride R. C 6 HaN 2 ,HCl (SRIP, 1963, p. 140).
1381
Phenylhydrazine R. C^HaNs (SKIP, 1963, p. 140).
Phenylhydrazine/hydrochloric acid TS
Procedure. Dissolve 0.75 g of phenylhydrazine hydrochloride R in 50 ml of
water and shake with 2 g of charcoal R. Filter, add 25 ml of hydrochloric acid
(-420 g/1) TS and sufficient water to produce 200 ml.
Phenylhydrazine/sulfiiric acid TS
Procedure. Dissolve 65 mg of phenylhydrazine hydrochloride R, previously
recrystaUized from ethanol (-710 g/1) TS, in a sufficient quantity of a mixture
of 170ml of sulfuric acid (~1760g/l) TS and 80ml of water to produce
100 ml.
Note: Phenylhydrazine/sulfuric acid TS must be freshly prepared.
Phenyl/methylpolysUoxane R. A suitable grade of a mixture to be used in

gas chromatography composed of 5 g of phenylpolysiloxane and 95 g of
methylpolysiloxane per lOOg.
Phenytoin RS. International Chemical Reference Substance.
pH-Indicator paper R. A paper impregnated with a suitable mixture of colour

indicators such that the changes in colour permit estimation of the pH of a
solution with an adequate sensitivity (usually 1 pH unit), at least in the pH
range 1-10.
Phloroglucinol R. Benzene-1,3, 5-triol dihydrate; C6H603,2H20.

Description. White or pale cream coloured crystals.
Phosphate buffer, pH 4.0, TS

Procedure. Dissolve5.04g of disodium hydrogen phosphate R and 3.01 g of
potassium dihydrogen phosphate R in sufficient water to produce 1000ml
and adjust the pH to 4.0 with glacial acetic acid R.

Procedure. Dissolve 1.36g of potassium dihydrogen phosphate R in 50 ml of
carbon-dioxide-free water R, add 12.60ml of carbonate-free sodium hydrox-
ide (0.2 mol/1) VS, and dilute with sufficient carbon-dioxide-free water R to
produce 200 ml.

potassium dihydrogen phosphate R and 3.55 g of
Procedure. Dissolve 3.40 g of
disodium hydrogen phosphate R in sufficient carbon-dioxide-free water R
to produce 1000ml.
1382
Phosphate buffer, pH
7.0 (0.067mol/I), TS
Procedure. Dissolve 0.908 g of potassium dihydrogen phosphate R in sufficient
water to produce 100ml. Separately dissolve 2,38 g of disodium hydrogen
phosphate R in sufficient water to produce 100 ml. Mix 38,9 ml of the potas-
sium phosphate solution with 61.1ml of the sodium phosphate solution.

Procedure. Dissolve 5.76g of anhydrous disodium hydrogen phosphate R and
3.55 g of potassium dihydrogen phosphate R in sufficient water to produce
1000 ml.

Procedure. Dissolve 6.80 g of potassium dihydrogen phosphate R and 1.40 g of
sodium hydroxide R in sufficient water to produce 1000 ml.

Procedure. Dissolve 6.8 g ofpotassium dihydrogen phosphate R in 250 ml of
water and add 393.4ml of sodium hydroxide (0.1 mol/1) VS.

Procedure. Place 1.36g of potassium dihydrogen phosphate R in a 200-ml
volumetric flask,dissolve it in water, add 42.4 ml of sodium hydroxide
(0.2 mol/1) VS, and dilute to volume with water.

Procedure. Dissolve 8.95 g of anhydrous disodium hydrogen phosphate R and
1000 ml.
Phosphate buffer, sterile*, pH 10.5, TSl

Procedure. Dissolve 35.0 g of dipotassium hydrogen phosphate R in water, add
20ml of sodium hydroxide (1 mol/1) VS and sufficient water to produce
1000ml. If necessary, adjust the pH to 10.5 with phosphoric acid (~1440g/l)
TS or potassium hydroxide (~110g/l) TS and then sterilize the solution for
20 minutes in an autoclave at 120 °C.
Phosphate buffer, sterile*, pH 4.5, TS

Procedure. Dissolve 13. 6g of potassium dihydrogen phosphate R in sufficient
water to produce 1000 ml. If necessary, adjust the pH to 4.45-4.55 with
phosphoric acid (~1440g/l) TS or potassium hydroxide (-llOg/1) TS and
then sterilize the solution for 20 minutes in an autoclave at 120 °C.
’
The adjustment of the pH, if necessary, should be effected before sterilization of the solution.
1383

Procedure. Dissolve 2.0g of dipotassium hydrogen phosphate R and 8.0g of
potassium dihydrogen phosphate R water to produce 1000 ml.
in sufficient
If necessary, adjust the pH to 5.95-6.05 with phosphoric acid (-1440 g/1) TS
or potassium hydroxide (-110 g/1) TS and then sterilize the solution for 20
minutes in an autoclave at 120 °C.
Phosphate buffer, sterile’, pH 6.0, TS2

1000 ml. If necessary, adjust the pH to 5.95-6.05 with phosphoric acid
(-1440 g/1) TS or potassium hydroxide (~110g/l) TS and then sterilize the
solution for 20 minutes in an autoclave at 120 °C.

Procedure. Dissolve 20. Og of dipotassium hydrogen phosphate R and 80. Og of
potassium dihydrogen phosphate R in sufficient water to produce 1000 ml.
If necessary, adjust the pH to 5.95-6.05 with phosphoric acid (-1440 g/1) TS
or potassium hydroxide (-110 g/1) TS and then sterilize the solution for 20
Phosphate buffer, sterile’, pH 7.0, TS

1000ml. If necessary, adjust the pH to 6.95-7.05 with phosphoric acid
(-1440 g/1) TS or potassium hydroxide (-llOg/1) TS and then sterilize the

Procedure. Dissolve 6.80g of potassium dihydrogen phosphate R and 1.4 g of
sodium hydroxide R in sufficient water to produce 1000 ml. If necessary,
adjust the pH to 7. 1-7.3 with phosphoric acid (-1440 g/1) TS or potassium
hydroxide (~110g/l) TS and then sterilize the solution for 20 minutes in an
autoclave at 120 °C.

Procedure. Dissolve 6.8 g of potassium dihydrogen phosphate R in water, add
45.2 ml of (1 mol/1) VS and sufficient water to produce
sodium hydroxide
1000ml. If necessary, adjust the pH to 7.8 with phosphoric acid (-1440g/l)
TS or potassium hydroxide (-llOg/1) TS and then sterilize the solution for
20 minutes in an autoclave at 120 °C.
’
1384

Procedure. Dissolve 16.73g of dipotassium hydrogen phosphate R and 0.52 g of
potassium dihydrogen phosphate R in sufficient water to produce 1000 ml.
If necessary, adjust the pH to 7. 9-8.1 with phosphoric acid (~1440g/l) TS or
potassium hydroxide (~110g/l) TS and then sterilize the solution for 20


Procedure. Dissolve 8.95 g of anhydrous disodium hydrogen phosphate R and
1000 ml. If necessary, adjust the pH to 7. 9-8.1 with phosphoric acid
(-1440 g/1) TS or potassium hydroxide (~110g/l) TS and then sterilize the

Phosphate standard (5 pg/ml) TS

Procedure. Dissolve 0.716g of potassium dihydrogen phosphate R in sufficient
water to produce 1000 ml. Immediately before use dilute 1 ml to 100ml with
water.
Phosphate standard buffer, pH 6.8, TS

Procedure. Dissolve 3.40 g of potassium dihydrogen phosphate R and 3.53 g of
anhydrous disodium hydrogen phosphate R in sufficient carbon-dioxide-
free water R to produce 1000 ml.
Phosphate standard buffer, pH 7.4, TS

Procedure. Dissolve 1.18g of potassium dihydrogen phosphate R and 4.30 g of
anhydrous disodium hydrogen phosphate R in sufficient carbon-dioxide-
Phosphate/citrate buffer pH 4.5, TS

Procedure. Dissolve 2. 15 g of disodium hydrogen phosphate R in 30 ml of water
and adjust the pH of the solution to 4.5 with citric acid (20 g/1) TS.
Phosphate/citrate buffer pH 6.0, TS

Procedure. Dissolve 4.52 g of disodium hydrogen phosphate R in 60 ml of water,
add 35 ml of citric acid (20 g/1) TS, and, if necessary, adjust the pH of the
solution to 6.0.
Phosphomolybdic acid (80g/l) TS. A solution of phosphomolybdic acid R

containing about lOOg of H3P04,12Mo03,24H20 per litre.
Phosphomolybdic acid R. H3P04,12Mo03,24H20 (SRIP, 1963, p. 141).
’
1385
Phosphomolybdic acid/ethanol TS
Procedure. Dissolve 5g of phosphomolybdic acid R in sufficient dehydrated
ethanol R to produce 100 ml.
Phosphoric acid (-105 g/1) TS

Mix about 115g of phosphoric
Procedure. acid (~1440g/l) TS with 885g of
water.
Phosphoric acid (~1440g/l) TS [phosphoric acid R]. (SRIP, 1963, p. 141); d

~ 1.7.
Phosphoric acid (~20g/l) TS

Procedure. To 23 g of phosphoric acid (-1440 g/1) TS add 987 g of water and mix.
Phosphoric acid (-2.8 g/1) TS

Procedure. Dilute 2 ml of phosphoric acid (-1440 g/1) TS with sufficient water to
produce 100 ml.
Phosphorus pentoxide R. P2O5 (SRIP, 1963, p. 142).
Phosphorus, red R
Description. A dark red powder.
Solubility. Insoluble in water and dilute acids.
Soluble matter. Heat 2.0g with 30ml of acetic acid (-300 g/1) TS on a water-bath
for 15 minutes, cool, dilute to 50 ml, filter, evaporate 25 ml of the filtrate on
a water- bath and dry at 110°C for 2 hours; the residue weighs not more
than 50 mg.
Yellow phosphorus. Shake 5.0 g with 20 ml of carbon disulfide R in a glass-
stoppered cylinder, filter, and immerse in the filtrate a strip of filter-paper,
10 cm bycm, previously immersed in copper(ll) sulfate (80 g/1) TS, and

0.5
allow to dry in the air; no stain is produced.
Loss on drying. Dry to constant weight over sulfuric acid (~1760g/l) TS; it loses
not more than lOmg/g.
Phosphotungstic acid TS
Procedure. Dissolve 25 g of sodium tungstate R in 175 ml of water and add 18.75
ml of phosphoric acid (-1440 g/1) TS. Heat under a reflux condenser for 6
hours, filter, and add sufficient water to produce 250 ml.
Storage. Store at a temperature between 2 and 8 °C, protected from light.
Phthalate buffer, pH 3.4, TS

Procedure. Dissolve 2.04 g of potassium hydrogen phthalate R in 50 ml of carbon-
dioxide-free water R, add 10.40ml of hydrochloric acid (0.2mol/l) VS, and
dilute with sufficient carbon-dioxide-free water R to produce 200 ml.
1386

Procedure. Dissolve 2.04 g of potassium hydrogen phthalate R in 50ml of
carbon-dioxide-free water R, add 8.40 ml of hydrochloric acid (0.2 mol/1) VS,
and dilute with sufficient carbon-dioxide-free water R to produce 200 ml.

Procedure. Dissolve 2.042 g of potassium hydrogen phthalate R in 50ml of
water, add 0.40 ml of sodium hydroxide (0.2 mol/1) VS, and dilute to 200ml
with water.
Phthalic anhydride R. CgH 403 .
Description. White lustrous needles.

Solubility. Slightly soluble in water, more soluble in hot water; soluble in
Phthalic anhydride/pyridine TS
Procedure. Add 42 g of phthalic anhydride R, accurately weighed, to 300ml of
freshly distilled pyridine R (refluxed with barium oxide R) containing less
than 1 mg/ml of water in a glass-stoppered 1000 ml flask. Use a dark flask or
otherwise prevent exposure to light. Shake vigorously until complete solu-
tion is effected, and allow to stand overnight for completion of the reaction.
Note: Phthalic anhydride/pyridine TS must be freshly prepared.
Piperazine adipate RS. International Chemical Reference Substance.
Piperazine citrate RS. International Chemical Reference Substance.
Piperazine hydrate R. Piperazine hexahydrate; C4HioN2,6H20.

Description. Colourless, glossy, deliquescent crystals.
Melting point. 44 °C.
Piperidine R. CsHuN.
Description. A colourless to yellowish liquid; odour, characteristic.
Refractive index, tin = about 1.454.
Congealing temperature. Between 12 and 15 °C.
Plasma substrate R
Note-. Use water-repellent equipment (made from materials such as suitable
and handling blood.
plastics or suitably silicone-treated glass) for taking
Procedure. Collect a sufficient volume of blood from each of at least 5 sheep. A
285-ml volume of blood collected into 15 ml of anticoagulant solution is rec-
ommended but smaller volumes may be collected. The blood should be
1387
taken either from a live animal or at the time of slaughter, using a needle
attached to a cannula that is long enough to reach the bottom of the col-
lecting flask. Discard the first few ml and collect only free-flowing blood.
Collect the blood in a sufficient quantity of an anticoagulant solution con-
taining 8.7 g of sodium citrate R and 4 mg of aprotinin R in 100 ml of water
to give a final ratio of blood to anticoagulant solution of 19 to 1. During and
immediately after collection, swirl the flask gently to ensure mixing but do
not allow frothing to occur. When collection is complete, close the flasks
and cool to a temperature between 10 and 15°C. Then pool the contents of
all the flasks, with the exception of any that shows obvious haemolysis or
clots, and keep the pooled blood at 10-15 °C. Within 4 hours of collection,
centrifuge the pooled and cooled blood at a speed of 1000-2000g' for 30

minutes. Separate the supernatant liquid and centrifuge again at 5000g for
30 minutes {Note-. Faster centrifugation (20000g) may be necessary to clarify
the plasma, but filtration procedures should not be used.) Separate the super-
natant liquid and immediately mix thoroughly and distribute the plasma
substrateR into small stoppered containers in portions sufficient for a com-
Without delay, rapidly cool to a temperature
plete heparin assay (10-30 ml).
below -70 °C by immersing the containers in liquid nitrogen and store at a
temperature below -30 °C. The plasma is suitable for use as plasma sub-
strate R in the assay for heparin if, under the conditions of the assay, it gives
a clotting time appropriate to the method of detection used, and if it pro-
vides reproducible, steep, log dose - response curves. Just before use thaw
the quantity of plasma substrate R required in a water-bath at 37 °C and
gently swirl until thawing is complete; once thawed it should be kept
between 10 and 20 °C and used without delay. The thawed plasma sub-
strate R may be slightly centrifuged if necessary, but do not use any filtra-
tion procedures.
Platinic chloride (60g/l) TS. A solution of platinic chloride R containing

about 63 g of H 2 PtCl 6 per Htre.
,
Platinic chloride R. H2PtCl6,6H20 (SRIP, 1963, p. 144).
Polydimethylsiloxane R. A commercially available reagent of suitable grade

for use in gas chromatography.
Polysorbate 80R. The mono ester of oleic acid and tripolye thylene glycol 300-
sorbitan ether.
Description. Lemon to amber coloured, oily liquid.
Miscibility. Miscible with water, producing an odourless and nearly colourless
solution. Miscible with ethanol (-750 g/1) TS, ethyl acetate R, and vegetable
oils; immiscible with mineral oils.
^
Acceleration due to gravity = 9.81 m/s^.
1388
Potassio -cupric tartrate TS

Procedure. Dissolve 7 g of copper(II) sulfateR in sufficient water to produce
100 ml. Separately dissolve 35 g of potassium sodium tartrate R and 10 g of
sodium hydroxide R in 100ml of water. Shortly before use, mix together
equal volumes of both solutions.
Potassio -mercuric iodide TS

Procedure. Dissolve 1.355g of mercuric chloride R in 60 ml of water; separately
dissolve 5g of potassium iodide R in 20 ml of water; mix the two solutions
Potassio -mercuric iodide, alkaline, TS

Procedure. Dissolve 3.5 g of potassium iodide R and 1.25g of mercuric chloride
R in 80 ml of water, add while stirring a cold saturated solution of mercuric
chloride R in water until a slight red precipitate remains. Then add 12 g of
sodium hydroxide R and mix to dissolve, add a litde more of the saturated
solution of mercuric chloride Rand sufficient water to produce 100 ml; allow
to stand for 24 hours and decant the clear liquid.
Potassium acetate R. C2H3KO2 (SRIP, 1963, p. 144).
Potassium acetate TS
Procedure. Dissolve 100 g of potassium acetate R in sufficient glacial acetic acid
R to produce 1000ml.
Potassium antimonate R. KSbOj (SRIP, 1963, p. 145).
Potassium antimonate TS
Procedure. Boil 2 g of potassium antimonate R with 95 ml of water until it has
dissolved. Cool rapidly and add 50ml of potassium hydroxide (1 mol/1) VS
and 5 ml of sodium hydroxide (1 mol/1) VS. Allow to stand for 24 hours and
dilute with sufficient water to produce 150 ml.
Sensitivity to sodium. To 10ml add 7ml of sodium hydroxide (O.lmol/1) VS; a
white, crystalline precipitate is formed within 15 minutes.

Note. Potassium antimonate TS must be freshly prepared.
Potassium bicarbonate R. KHCO3 (SRIP, 1963, p. 145).
Potassium bromate (0.00833 moI/1) VS. Potassium bromate R, dissolved in

water to contain 1.392g of KBrOg in 1000 ml.
Potassium bromate (0.01 67 mol/1) VS. Potassium bromate R, dissolved in

water to contain 2.784 g of KBrOs in 1000 ml.
Potassium bromate (0.0333 moI/1) VS. Potassium bromate R, dissolved in

water to contain 5.562 g of KBrOs in 1000 ml.
1389
Potassium bromate (50g/l) TS. A solution of potassium bromate R con-

taining about 50g of KBrOg per litre.
Potassium bromate R. KBrOs (SKIP, 1963, p. 147).
Potassium bromide (0.119g/l) TS. A solution of potassium bromide R con-

taining about 0.1190g of KBr per litre.
Potassium bromide (lOOg/1) TS. A solution of potassium bromide R con-

taining about lOOg of KBr per litre.
Potassium bromide (125g/I) TS. A solution of potassium bromide R con-

taining about 125 g of KBr per litre.
Potassium bromide IR. Potassium bromide R that complies with the fol-
lowing test: The infrared absorption spectrum of a disc prepared as

described in Method 3 under 1.7 Spectrophotometry in the infrared region,
from potassium bromide R, previously dried at 250 °C for 1 hour, has a sub-
stantially flat baseline over the range 4000-670 cm“'; it exhibits no maxima
with an absorbance greater than 0.1 above the baseline, with the exception
of maxima due to water at 3440 and 1630 cm“'.
Potassium bromide R. KBr (SRIP, 1963, p. 148).
Potassium carbonate R. K 2 C 03 ,
1 |H 20 .
Description. Small granular crystals.

Solubility. Very soluble in water; practically insoluble in ethanol (~750g/l) TS.
Potassium carbonate, anhydrous, R. K 2 CO 3 .
Description. Granules or a granular powder; hygroscopic.

Solubility. Soluble in 1 part of water; practically insoluble in ethanol (-750 g/1)
TS.
Potassium chloride (100 g/1) TS. A solution of potassium chloride R con-

taining about lOOg of KCl per litre.
Potassium chloride (350g/l) TS. A saturated solution of potassium chloride

R containing about 350 g/1 of KCl.
Potassium chloride IR. Potassium chloride R that complies with the follow-
ing test: The infrared absorption spectrum of a disc prepared as described
in Method 3 under 1.7 Spectrophotometry in the infrared region, from
potassium chloride R, previously dried at 250 °C for 1 hour, has a substan-
tially flat baseline over the range 4000 —
670 cm“'; it exhibits no maxima
with an absorbance greater than 0.1 above the baseline, with the exception
of maxima due to water at 3440 and 1630 cm"'.
1390
Potassium chloride R. KCl (SKIP, 1963, p. 151).
Potassium chromate (lOOg/1) TS. A solution of potassium chromate R con-

taining about 97 g of K2Cr04 per litre (approximately 0.5 mol/1).
Potassium chromate R. K2Cr04 (SKIP, 1963, p. 152).
Potassium cyanide (lOOg/1) TS. A solution of potassium cyanide R con-

taining about lOOg of KCN per litre.
Potassium cyanide (50g/l) TS. A solution of potassium cyanide R contain-

ing about 50 g of KCN per litre.
Potassium cyanide PbTS

Procedure. Dissolve lOg of potassium cyanide R in 90ml of water, add 2 ml of
hydrogen peroxide (~60g/l) TS, allow to stand for 24 hours, dilute with
water to 100 ml, and filter.
Potassium cyanide R. KCN (SRIP, 1963, p. 153).
Potassium dichromate (0.0167 moI/1) VS. Potassium dichromate R, dis-

solved in water to contain 4.904 g of K2Cr20/ in 1000 ml.
Potassium dichromate (lOOg/1) TS. A solution of potassium dichromate R

containing about 98 g of K2Cr20/ per litre (approximately 0.4 mol/1).
Potassium dichromate R. K2Cr20/ (SRIP, 1963, p. 154).
Potassium dichromate Rl. Potassium dichromate R containing not less than

99.9% of K2Cr207.
Potassium dichromate TS
Procedure. Dissolveabout 60 mg, accurately weighed and previously dried at
130 °C, of potassium dichromate Rl in sufficient sulfuric acid (0.005mol/l)
VS to produce 1000.0ml.
Potassium dichromate TS2

Procedure. Dissolve 1 g of potassium dichromate R in 60 ml of water and cau-
tiously add 7.5 ml of sulfuric acid (-1760 g/1) TS.
Potassium dichromate TS3

Procedure. Dissolve 0.5 g of potassium dichromate R in sufficient sulfuric acid
Potassium dihydrogen phosphate (lOOg/1) TS. A solution of potassium

dihydrogen phosphate R containing about lOOg of KH2PO4 per litre.
1391
Potassium dihydrogen phosphate (13.6g/l) TS. A solution of potassium

dihydrogen phosphate R containing 13. 6g of KH2PO4 per litre (0.1 mol/1).
Potassium dihydrogen phosphate (27.2g/l) TS. A solution of potassium

dihydrogen phosphate R containing 27.2 g of KH2PO4 per litre (0.2 mol/1).
Potassium dihydrogen phosphate (70g/l) TS. A solution of potassium

dihydrogen phosphate R containing about 70g of KH2PO4 per litre.
Potassium dihydrogen phosphate R. KH2PO4 (SPRl, 1963, p. 155).
Potassium ferricyanide (lOg/1) TS

Wash about 1 g of crystalline potassium ferricyanide R with a little
Procedure.
water and dissolve the washed crystals in sufficient water to produce
100 ml.
Note: Potassium ferricyanide (lOg/1) TS must be freshly prepared.
Potassium ferricyanide (50g/l) TS

Wash about 5 g of crystalline potassium ferricyanide R with a little
Procedure.
water and dissolve the washed crystals in sufficient water to produce
100 ml.
Note: Potassium ferricyanide (50g/l) TS must be freshly prepared.
Potassium ferricyanide R. K3pe(CN)6 (SRIP, 1963, p. 156).
Potassium ferrocyanide (45g/l) TS. A solution of potassium ferrocyanide R

containing about 50 g of K4Fe(CN)5 per litre.
Potassium ferrocyanide R. K4pe(CN)6,3H20 (SRIP, 1963, p. 156).
Potassium hydrogen phthalate R. C8H5KO4 (SRIP, 1963, p. 157).
Potassium hydrogen phthalate standard TS

Procedure. Dissolve 10.21 g of potassium hydrogen phthalate R, previously
dried at 120 °C, in sufficient carbon-dioxide-free water R to produce
1000 ml. The pH of this solution is defined as having the value of 4.000 at
15°C.
Potassium hydrogen sulfate R. KHSO4 (SRIP, 1963, p. 146).
Potassium hydrogen tartrate R. C4H5KO6 (SRIP, 1963, p. 158).
Potassium hydrogen tartrate standard TS

Procedure. Add 2g of potassium hydrogen tartrate R to 100 ml of carbon-
dioxide-free water R contained in a glass-stoppered flask and shake the flask
1392
vigorously. Let the temperature of the solution reach room temperature,

allow the solid to settle, and remove it by filtration or decantation,
Note: Potassium hydrogen tartrate standard TS must be freshly prepared.
Potassium hydroxide (~110g/I) TS. A solution of potassium hydroxide R

containing about 112g/l of KOH (approximately 2mol/l).
Potassium hydroxide (~400g/l) TS. A solution of potassium hydroxide R

containing about 400 g of KOH per litre.
Potassium hydroxide (~560g/l) TS. A solution of potassium hydroxide R

containing about 560 g of KOH per litre.
Potassium hydroxide (O.Olmol/I) VS. Potassium hydroxide R, dissolved in

water to contain 0.5610g of KOH in 1000ml.
lowing the method described under potassium hydroxide (1 mol/1) VS.
Potassium hydroxide (0.1 mol/1) VS. Potassium hydroxide R, dissolved in

Potassium hydroxide (0.5 mol/1) VS. Potassium hydroxide R, dissolved in

Potassium hydroxide (1 mol/1) VS. Potassium hydroxide R, dissolved in

tion in the following manner: Dry about 5 g of potassium hydrogen phtha-
late R at 105°C for 3 hours and weigh accurately. If the potassium hydrogen
phthalate is in the form of large crystals, they should be crushed before
drying. Dissolve in 75 ml of carbon-dioxide-free water R and titrate with the
potassium hydroxide solution, using phenolphthalein/ethanol TS as indica-
tor. Each 0.2042 g of potassium hydrogen phthalate is equivalent to 1ml of
potassium hydroxide (1 mol/1) VS. Standard solutions of potassium hydrox-

ide should be restandardized frequently.
Storage. Solutions of alkali hydroxides absorb carbon dioxide when exposed to
air. They should, therefore, be stored in bottles with suitable non-glass,
weU-fitting stoppers, provided with a tube filled with soda lime R.
Potassium hydroxide R. KOH (SRIP, 1963, p. 159).
1393
Potassium hydroxide/ethanol (0.02mol/l) VS. Potassium hydroxide R, dis-

solved in ethanol (~710g/l) TS to contain 1.122g of KOH in 1000ml,
lowing the method described under potassium hydroxide/ethanol (0.5 mol/1)

VS.
Potassium hydroxide/ethanol (O.lmol/I) VS. Potassium hydroxide R, dis-

solved in ethanol (~710g/l) TS to contain 5.610g of KOH in 1000ml.
Potassium hydroxide/ethanol (0.5moI/I) VS. Potassium hydroxide R, dis-

solved in ethanol (~710g/l) TS to contain 28.05g of KOH in 1000ml,
solution in the following manner: Dilute 25.0ml of hydrochloric acid
(0.5 mol/1) VS with 50 ml of water and titrate with the potassium hydrox-
ide/ethanol solution, using phenolphthalein/ethanol TS as indicator.
Potassium hydroxide/ethanol (1 mol/1) VS. Potassium hydroxide R, dis-
solved in ethanol (~710g/l) TS to contain 56.10g of KOH in 1000ml.

lowing the method described under potassium hydroxide/ethanol (0.5 mol/1)

VS.
Potassium hydroxide/ethanol TSl

Procedure. Dissolve 40 g of potassium hydroxide R in 20 ml of water and add
sufficient ethanol (-750 g/1) TS to produce 1000 ml. Allow to stand
overnight, and pour off the clear liquid.
Potassium hydroxide/ethanol TS2

Procedure. Dissolve 112g of potassium hydroxide R in sufficient ethanol
(~710g/l) TS to produce 1000ml (approximately 2mol/l).
Potassium hydroxide/methanol TS
Procedure. Dissolve30 g of potassium hydroxide R in sufficient methanol R to
produce 1000 ml.
Potassium iodate (0.01 moI/1) VS. Potassium iodate R, dissolved in water to

contain 2.140g of KIO3 in 1000ml.
lowing the method described under potassium iodate (0.05 mol/1) VS.
Potassium iodate (0.05 mol/1) VS. Potassium iodate R, dissolved in water to

contain 10.70g of KIO3 in 1000ml.
solution in the following manner. Place 10.0ml of the potassium iodate solu-
1394
with 200 ml of water, add 2g of potas-

tion in a glass-stoppered flask, dilute
sium iodide R, and 25ml of sulfuric acid (~100g/l) TS. Allow the solution
to stand for 10 minutes and titrate the liberated iodine with sodium thio-
sulfate (0.1 mol/1) VS, adding 3ml of starch TS as the end-point is
approached. Correct for a blank determined on the same quantities of the
same reagents.
Potassium iodate (3.6mg/l) TS. A freshly prepared solution of potassium

iodate R containing 3.6mg of KIO3 per litre.
Potassium iodate R. KIO3 (SRIP, 1963, p. 160).
Potassium iodide (lOOg/I) TS. A solution of potassium iodide R containing

about lOOg of KI per litre.
Potassium iodide (160g/I) TS. A solution of potassium iodide R containing

about 160g of KI per litre.
Potassium iodide (300g/l) TS. A solution of potassium iodide R containing

about 300 g of KI per litre.

Potassium iodide (60g/I) TS. A solution of potassium iodide R containing


about 83g/l of KI (approximately 0.5mol/l).
Potassium iodide AsR. Potassium iodide R that complies with the following
test: Dissolve lOg of potassium iodide R in 25ml of hydrochloric acid
(-250 g/1) AsTS and 35 ml of water, add 2 drops of stannous chloride AsTS
and apply the general test for arsenic; no visible stain is produced.
Potassium iodide R. KI (SRIP, 1963, p. 161).
Potassium iodide/starch TSl

Procedure. Dissolve lOg of potassium iodide R in 95ml of water and add to it
5 ml of starch TS.
Note: Potassium iodide/starch TSl must be freshly prepared.
Potassium iodobismuthate TSl

Procedure. Dissolve lOOg of tartaricacid R in 400 ml of water and add 8.5 g of
bismuth oxynitrate R. Shake the solution for 1 hour, add 200ml of potas-
sium iodide (400 g/1) TS, and mix. Allow to stand for 24 hours and filter.
1395
Potassium iodobismuthate TS2

Procedure. Dissolve lOOg of tartaric acid R in 500ml of water and add 50ml of
potassium iodobismuthate TSl.
Potassium iodobismuthate/acetic acid TS

Procedure. Dissolve 8g of potassium iodide R in 20 ml of water and add to it a
solution composed of 0.85 g of bismuth oxynitrate R dissolved in 40 ml of
water and 10 ml of glacial acetic acid R.
Potassium iodoplatinate TS
Procedure. Dissolve 2.5 g of platinic chloride R in 50 ml of water, add 45 ml of a
O.lg/ml solution of potassium iodide R, and dilute to 100 ml with water.
Storage. Store in amber glass containers.
Potassium iodoplatinate TS2

Procedure. Dissolve 0.25 g of platinic chloride R in 2.5 ml of water, add 45 ml of
potassium iodide (100 g/1) TS, and dilute with sufficient acetone R to
produce 100 ml.
Potassium nitrate R. KNO3 (SRIP, 1963, p. 162).
Potassium nitrite (lOOg/1) TS. A solution of potassium nitrite R containing

about 100 g/1 of KNO2.
Potassium nitrite R. KNOj.

Description. White or slightly yellow, deliquescent granules or rods.
Solubility. Soluble in 0.35 part of water; slightly soluble in ethanol (-750 g/1) TS.
Potassium periodate R. KIO4 (SRIP, 1963, p. 164).
Potassium periodate TS
Procedure. To 2.8 g of potassium periodate R add 200ml of water followed by
20ml of sulfuric acid (-1760 g/1) TS added drop by drop while shaking to
effect solution; cool and add sufficient water to produce 1000ml.
Potassium permanganate (0.0002 mol/1) VS. Potassium permanganate R,

dissolved in water to contain 31.61 mg of KMn04 in 1000ml.
lowing the method described under “Potassium permanganate (0.02 mol/1)
VS”.
Potassium permanganate (0.002mol/l) VS. Potassium permanganate R,

dissolved in water to contain 0.3161 g of KMn04 in 1000 ml.
lowing the method described under potassium permanganate (0.02 mol/1) VS.
1396
Potassium permanganate (0.02mol/l) VS. Potassium permanganate R, dis-

solved in water to contain 3.161 g of KMnO^ in 1000 ml.
solution in the following manner; Dissolve about 0.2 g, accurately weighed,
of sodium oxalate at 110°C, in
R, previously dried to constant weight
250ml of water. Add 7ml TS, heat to about
of sulfuric acid (-1760 g/1)
70 °C and then slowly add the permanganate solution from a burette, with
constant stirring, until a pale pink colour, which persists for 15 seconds, is
produced. The temperature at the conclusion of the titration should be not

less than 60°C. Every 6.7mg of sodium oxalate are equivalent to 1ml of
potassium permanganate (0.02 mol/1) VS. Potassium permanganate solutions
should be restandardized frequently.
Storage. Store the solution in tightly closed containers, protected from light.
Potassium permanganate (~25g/l) TS. A solution of potassium perman-

ganate R containing about 25 g of KMn04 per litre.
Potassium permanganate (~10g/l) TS. A solution of potassium perman-

ganate R containing about 10 g/1 of KMn04.
Potassium permanganate (~lg/l) TS. A solution of potassium perman-

ganate R containing about 1 g of KMn04 per litre.
Potassium permanganate R. KMn04 (SRIP, 1963, p. 165).
Potassium permanganate, basic (~5g/l) TS

A solution of potassium permanganate R containing about 5g of KMn04 per
litre of sodium hydroxide (-40 g/1) TS.
Potassium permanganate, basic (~lg/l) TS

A solution of potassium permanganate R containing about 1 g of KMn04 per
litre of sodium hydroxide (-40 g/1) TS.
Potassium permanganate/phosphoric acid TS

Procedure. Dissolve 3g of potassium permanganate R in a mixture of 15ml of
phosphoric acid (-1440 g/1) TS and 70 ml of water, and dilute to 100ml with
water.
Potassium sodium tartrate R [sodium potassium tartrate R].
C4H4KNa0ei,4H20 (SRIP, 1963, p. 193).
Potassium sulfate (0.1 g/1) TS. A solution of potassium sulfate R containing

about 0.1 g of K2SO4 per litre.
1397
Potassium sulfate (174 mg/I) TS

Procedure. Dissolve 174 mg, accurately weighed, of potassium sulfate R in suf-
ficient water to produce 1000 ml.
Potassium sulfate R. K2SO4 (SKIP, 1963, p. 165).
Potassium tetraoxalate R. C4H3K0s,2H20 (SKIP, 1963, p. 166).
Potassium tetraoxalate standard TS

Procedure. Dissolve 25.42 g of potassium tetraoxalate R in sufficient carbon-
dioxide-free water R to produce 1000 ml.
Potassium thiocyanate (200 g/1) TS. A solution of potassium thiocyanate R

containing 200 g of KCNS per litre.
Potassium thiocyanate R. KCNS. Contains not less than 99.0% of KCNS,

Solubility. Soluble in 0.5 part of water and in 15 parts of dehydrated ethanol R.
Alkalinity. A 0.1 g/ml solution in carbon-dioxide-free water R is not alkaline to
bromothymol blue/ethanol TS.
Ammonia. Boil 1.0 g with 5 ml of sodium hydroxide (~80g/l) TS; no ammonia
is evolved.
Chlorides. Dissolve l.Og in a solution of g of ammonium nitrate R in 30ml of
1
hydrogen peroxide (-60 g/1) TS containing not more than 1 pg/g of chlorides,
add 1 g of sodium hydroxide R, and warm gently; when the vigorous reac-
tion subsides add a further 30ml of hydrogen peroxide (-60 g/1) TS and boil
for 2 minutes. Cool, add 5 ml of nitric acid (-1000 g/1) TS and 1 ml of silver
nitrate (40 g/1) TS; any opalescence produced is not greater than that pro-
duced by treating 1ml of hydrochloric acid (0.01 mol/1) VS in the same
manner.
Limit test for sulfates; not more than l.Omg/g.
Other sulfur compounds. Dissolve l.Og in 50ml of water, add 2 ml of hydrochlo-
ric acid (-70 g/1) TS, and titrate with iodine (0.05 mol/1) VS; not more than
0.5ml of iodine (0.05 mol/1) VS is required.
Loss on drying. Dry to constant weight at 1 05 °C; it loses not more than 20 mg/g.
Assay. Dissolve about 0.4 g, accurately weighed, in 50ml of water, add 5 ml of
nitric acid (-1000 g/1) TS, 50ml of silver nitrate (0.1 mol/1) VS, and 5ml of
ferric ammonium sulfate (45 g/1) TS, and titrate the excess of silver nitrate
with ammonium thiocyanate (0.1 mol/1) VS. Each ml of silver nitrate (0.1
mol/1) VS is equivalent to 9.7 18 mg of KCNS.
Note: Potassium thiocyanate R is deliquescent.
Praziquantel RS. International Chemical Reference Substance.
1398
Prednisolone acetate RS. International Chemical Reference Substance.
Prednisolone RS. International Chemical Reference Substance.
Prednisolone sodium phosphate RS. International Chemical Reference

Substance.
Prednisolone succinate RS. International Chemical Reference Substance.
Primaquine diphosphate RS. International Chemical Reference Substance.
Probenecid RS. International Chemical Reference Substance.
Procaine hydrochloride RS. International Chemical Reference Substance.
Procarbazine hydrochloride RS. International Chemical Reference

Substance.
Progesterone RS. International Chemical Reference Substance.
Proguanil hydrochloride RS. International Chemical Reference Substance.
2-Propanol R [/so-propanol R]; isopropyl alcohol; C 3 H 5O (SRIP, 1963, p. 167).
1-Propanol R. w-Propanol; propan-l-ol, CsHgO.



at 105 °C; it leaves a residue of not more than 0.1 mg/g.
Propranolol hydrochloride RS. International Chemical Reference

Substance.
Propylene glycol R. CgHgOg (SRIP, 1963, p. 168).
Protionamide RS. International Chemical Reference Substance.
Pyrantel embonate RS. International Chemical Reference Substance.
Pyrazinamide RS. International Chemical Reference Substance.
Pyridine R. C 5H N5 (SRIP, 1963, p. 169).
1399
Pyridine, anhydrous, R. Pyridine R that has been dried by allowing it to

stand over sodium hydroxide R.
Pyridine/acetic anhydride TS
Procedure. Mix 3 volumes of freshly distilled pyridine R with 1 volume of
freshly distilled acetic anhydride R.
Note: Pyridine/acetic anhydride TS must be freshly prepared.
Pyridostigmine bromide RS. International Chemical Reference Substance.
Pyrimethamine RS. International Chemical Reference Substance.
Pyrogallol R. Pyrogallic acid; 1,2,3-trihydroxybenzene, CeHgOj (SRIP, 1963,

p. 170).
Pyrogallol, alkaline, TS
Procedure. Dissolve 0.5 g of pyrogallol R in 2 ml of water. Dissolve separately
12 g of potassium hydroxide R in 8 ml of water. Immediately before use mix
the two solutions.
Quinaldine red R. 2-(/?-Dimethylaminostyryl)quinoline ethiodide; C21H23IN2.

Description. A dark blue-black powder.
Solubility. Sparingly soluble in water; freely soluble in ethanol (-750 g/1) TS.
Quinaldine red/ethanol TS
Procedure. Dissolve O.lg of quinaldine red R in 100ml of ethanol (~750g/l) TS.
Quinaldine red/methanol TS
Procedure. Dissolve l.Og of quinaldine red R in sufficient methanol R to produce
100 ml.
Quinhydrone R. ^-Benzoquinone compound with hydroquinone (1:1);

C12H10O4.
Description. Dark green, lustrous crystals or a crystalline powder.
Quinhydrone/methanol TS
Procedure. Dissolve 2.5 g of quinhydrone R in sufficient methanol R to produce
100 ml.
Quinine R. C20H24N2O2.
Description. A white, microcrystalline powder; odourless.
Solubility. Very slightly soluble in water; slightly soluble in boiling water; very
soluble in ethanol (-750 g/1) TS; soluble in ether R and benzene R.
1400
Identification. Very dilute solutions containing sulfuric acid (-100 g/1) TS show
Acid solutions are levorotatory. Dissolve about 5 mg in
a mixture of 5ml and 0.3ml of hydrochloric acid (~70g/l) TS. Mix
of water
the solution with 0.2 ml of bromine TSl and add 1 ml of ammonia (-35 g/1)
TS; an emerald green colour is produced.
Red stock standard TS

Procedure.To 40.5ml of cobalt colour TS, add 6.1ml of copper colour TS,
6.3ml of dichromate colour TS, 12.0ml of iron colour TS, dilute to 100.0ml
with sulfuric acid (-lOg/1) TS, and mix.
Resazurin sodium (lg/1) TS. A solution of resazurin sodium R containing

about lg/1 of Ci 2 H 6 NNa 04 .
Note: Resazurin sodium (1 g/1) TS must be freshly prepared.
Resazurin sodium R. Ci 2 H 6NNa 04 (SRIP, 1963, p. 170).
Reserpine RS. International Chemical Reference Substance.
Resorcinol (20g/l) TS. A solution of resorcinol R containing 20 g/1 of C(;H602.
Resorcinol R. 1,3-Dihydroxybenzene, C 5H 6 O 2 (SRIP, 1963, p. 171).
Resorcinol/toluene TS
Procedure. Shake 0.2 g of resorcinol R with 100 ml of toluene R until saturated,
and decant.
Note: Resorcinol/toluene TS should be prepared immediately before use.
Retinol acetate RS. International Chemical Reference Substance.
Retinol palmitate RS. International Chemical Reference Substance.
Retinol propionate RS. International Chemical Reference Substance.
Riboflavin RS. International Chemical Reference Substance.
Rifampicin quinone RS. International Chemical Reference Substance.
Rifampicin RS. International Chemical Reference Substance.
Ritonavir RS. International Chemical Reference Substance.
Salbutamol RS. International Chemical Reference Substance.
Salbutamol sulfate RS. International Chemical Reference Substance.
1401
Salicylaldehyde R. C/HsOj.
Description. A clear, colourless, oily liquid; odour, bitter, almond-like.
Solubility. Slightly soluble in water; soluble in ethanol (-750 g/1) TS and ether
R,
Relative density, if = 1.17.
Salicylaldehyde TS
Procedure. Mix 2g of salicylaldehyde R with 100 ml of methanol R and add
0.1ml of hydrocloric acid (~420g/l) TS.
Salicylic acid R. C/HÔs. Salicylic acid as described in the monograph for

“Salicylic acid”.
Saline TS. A sterile solution of sodium chloride R containing about 9 g/1 of

NaCl. Sterilization by heating in a steam autoclave at 120 °C for 30 minutes
is suitable.
Saquinavir mesilate RS. International Chemical Reference Substance.
Saquinavir RS. International Chemical Reference Substance.
Selenious acid R. H 2 Se 03 Contains not less than

. 93% of H 2 Se 03 .
Description. Colourless or white crystals.

Assay. Transfer about 0.1 g, accurately weighed, to a glass-stoppered flask, and
dissolve in 50ml of water. Add 10ml of potassium iodide (300 g/1) TS and
5 ml of hydrochloric acid (-420 g/1) TS, mix, insert the stopper into the flask,
and allow to stand for 10 minutes. Dilute with 50ml of water, add 3ml of
starch TS, and titrate with sodium thiosulfate (0.1 mol/1) VS until the colour
is no longer diminished, then titrate with iodine (0.05 mol/1) VS to a blue
colour. Subtract the volume of iodine (0.05 mol/1) VS from the volume of
sodium thiosulfate (0.1 mol/1) VS equivalent to selenious acid. Each ml of
sodium thiosulfate (0.1 mol/1) VS is equivalent to 3.225mg of H 2 Se 03 .
Note: Selenious acid R effloresces in dry air and is hygroscopic in moist air.
Selenious acid/sulfuric acid TS

Procedure. Dissolve lOmg of selenious acid R in 2ml of sulfuric acid (-1760g/l)
TS.
Selenium R. Se (SRIP, 1963, p. 172).

Caution. Selenium vapours are toxic.
Silica gel for chromatography R. A very finely divided (3-1 0pm) silica gel.
The particle size is indicated after the name of the reagent in the tests where
it is used.
1402
Description. A fine, white, homogeneous powder.

Solubility. Practically insoluble in water and ethanol (-750 g/1) TS.
Silica gel Rl. Silica gel G.

Description. A white, homogeneous powder.
Composition.A mixture of silica gel (particle size 10-40 pm) and calcium sulfate,
hemihydrate (about 130g/kg).
Silica gel R2. Silica gel HF(UV254).

Composition. Silica gel (particle size 10-40 pm) containing a fluorescent indica-
tor having an optimal intensity at 254 nm (about 15g/kg).
Silica gel R3. Silica gel H.

Particle size. 10-40 pm.
Silica gel R4. Silica gel GF (UV 254).

Composition. A mixture of silica gel (particle size
10-40 pm) and calcium sulfate,
hemihydrate (about 130g/kg) containing a fluorescent indicator having an
optimal intensity at 254 nm (about 15g/kg).
SUica gel R5. Silica gel 60.

Average pore size. 6nm.
Silica gel R6. 60 (UV 254).

Silica gel
Average pore size. 6nm.
Composition. Silica gel (average particle size 15 pm) containing a fluorescent
indicator having an optimal intensity at 254 nm (about 15g/kg).
SHica gel for chromatography, hexadecylamidylsilyl

which has been modified with chemically
Particles of silica gel, the surface of
bonded hexadecylamidylsilyl groups.
Silica gel for chromatography, octadecylsilyl, base deactivated

A very finely divided silica gel, pretreated before the bonding of octadecylsilyl
groups to minimize the interaction with basic compounds.
Silica gel, desiccant, R

Description. An amorphous, pardy hydrated Si02, occurring in glassy granules
of varying sizes. It is frequently coated with a substance that changes colour
when the capacity to absorb water is exhausted. Such coloured products
1403
may be regenerated (i.e. may regain their capacity to absorb water) by

heating at 110°C until the gel assumes the original colour.
Loss on ignition. Ignite 2g, accurately weighed, at 950 + 50 °C to constant
weight; the loss is not more than 60mg/g.
Water absorption. Place about 10 g in a tared weighing-bottle, and weigh. Then
place the bottle, with the cover removed, for 24 hours in a closed container
in which the atmosphere is maintained at 80% relative humidity by being
in equilibrium with sulfuric acid having a relative density of 1.19. Weigh
again; the increase in weight is not less than 310mg/g.
Silver nitrate (0.001 mol/1) VS. Silver nitrate R, dissolved in water to contain
0.1699g of AgNOs in 1000ml.
Method of standardization. Ascertain the exact concentration of the solution
following the method described under “Silver nitrate (0.1 mol/1) VS”.
Silver nitrate (O.Olmol/I) VS. Silver nitrate R, dissolved in water to contain

following the method described under silver nitrate (0.1 mol/1) VS.
8.494g of AgNOg in 1000ml.
following the method described under “Silver nitrate (0.1 mol/1) VS”.
solution in the following manner: Dilute 40.0 ml of the silver nitrate solu-
tion with 100 ml of water. Heat the solution and add slowly, with continu-
ous stirring, hydrochloric acid (~70g/l) TS until precipitation of the silver is
complete. Boil the mixture cautiously for about 5 minutes, then allow it to
stand in the dark until the precipitate has setded and the supernatant liquid
has become clear. Transfer the precipitate completely to a tared filtering cru-
cible and wash it with small portions of water that has been slightly acidi-
fied with nitric acid (~1000g/l) TS. Dry the precipitate to constant weight
at 110°C. From the weight of silver chloride calculate the concentration of
the silver nitrate solution in mol/1. Protect the silver chloride from light as
much as possible during the determination.
Silver nitrate (lOOg/1) TS. A solution of silver nitrate R containing lOOg of

AgNOa per litre.
Silver nitrate (40g/l) TS. A solution of silver nitrate R containing about

42.5 g/1 of AgNOj (approximately 0.25 mol/1).
1404
Silver nitrate R. AgNOg (SKIP, 1963, p. 173).
Silver nitrate/methanol TS
Procedure. Prepare a saturated solution of silver nitrate R in methanol R.
Silver oxide R. AggO.

Description. A brownish- black, heavy powder; odourless.
Solubility. Practically insoluble in water; freely soluble in nitric acid (~130g/l)
TS and ammonia (-260 g/1) TS.
Substances insoluble in nitric acid. Dissolve 5g in a mixture of 5 ml of nitric acid
(-1000 g/1) TS and 10ml of water, dilute to about65mlwith water, and filter
any undissolved residue on a tared filtering crucible (retain the filtrate for
the test for substances not precipitated by hydrochloric acid). Wash the cru-
cible with water until the last washing shows no opalescence with 1 drop
of hydrochloric acid (-250 g/1) TS, and dry to constant weight at 105 °C; not
more than 0.2mg/g.
Substances not precipitated by hydrochloric acid. Dilute the filtrate obtained in the
test for substances insoluble in nitric acid to 250 ml with water, heat to
boiling, and add dropwise sufficient hydrochloric acid (-250 g/1) TS to pre-
cipitate all of the silver (about 5 ml), avoiding any great excess. Cool, dilute
to 300 ml with water, and allow to stand overnight. Filter, evaporate 200 ml
of the filtrate to dryness in a suitable tared porcelain dish, and ignite; not
more than 0.5mg/g.
Alkalinity.Heat 2g with 40 ml of water on a water-bath for 15 minutes, cool,
and dilute to 50ml with water. Filter, discarding the first 10ml of the filtrate.
To 25 ml of the subsequent filtrate add 2 drops of phenolphthalein/ethanol
TS, and titrate with hydrochloric acid (0.02 mol/1) VS to the disappearance
of any pink colour; not more than 0.20 ml is required.
Silver standard (5 |ig Ag/ml) TS

Procedure. Dissolve 39.5 mg of silver nitrate R in sufficient water to produce 100
ml. Dilute 1.0 ml of this solution to 100 ml with water.
Soda lime R. (SRIP, 1963, p. 174).
Sodium l,2-naphthoquinone-4-sulfonate (5 g/1) TS.A solution of sodium

l,2-naphthoquinone-4-sulfonate R containing about 5g of CioHsNaOjS per
litre.
Sodium l,2-naphthoquinone-4-sulfonate R. CioHsNaOjS.

Description. A yellow or orange, crystalline powder.
Solubility. Soluble in water; insoluble in ethanol (-750 g/1) TS.
Sodium acetate (0.04mol/l) VS. Sodium acetate R, dissolved in water to

contain 3.281 g of CgHgNaOj in 1000ml.
1405
Sodium acetate (150g/l) TS. A solution of sodium acetate R containing

about 150g/l of C 2 H 3 Na 02 .
Sodium acetate (50g/l) TS. A solution of sodium acetate R containing about

50g of C2H3Na02 per litre.
Sodium acetate (60g/l) TS. A solution of sodium acetate R containing about

60g of C H 3Na 02
2 per litre.
Sodium acetate R. C2H3Na02,3H20. Contains not less than 99.0% of

C2H3Na02,3H20.
Solubility. Very soluble in water; sparingly soluble in ethanol (-750 g/1) TS.
Clarity and colour of solution. A 0.1 g/ml solution is clear and colourless.
Iron. Use 8g; the solution complies with the 2.2.4 Limit test for iron; not more
than 5.0pg/g.
Heavy metals. Use 1.0 g for the preparation of the test solution as described in
2.2.3 Limit test for heavy metals, Procedure 1; determine the heavy metals
content according to Method A; not more than lOpg/g.

Substances reducing permanganate. Dissolve 1 g in 100ml of boiling water, add
2 ml of sulfuric acid (-100 g/1) TS and 0.05 ml of potassium permanganate

(0.02 mol/1) VS, and boil for 5 minutes; the pink colour does not entirely
disappear.
R and 5 ml of acetic anhydride R. After 5 minutes add 10 drops of 1-naph-
tholbenzein/acetic acid TS, and titrate to a green end-point with perchloric
acid (0.1 mol/1) VS as described under 2.6 Non-aqueous titration, Method A.
C2H3Na02,3H20.
Sodium acetate/glacial acetic acid (0.1 mol/1) VS

Procedure. Dissolve 5.3 g of anhydrous sodium carbonate R in small portions in
100 ml of glacial acetic acid Rl, stirring well after each addition, and add
sufficient glacial acetic acid Rl to produce 1000 ml.
Method of standardization. Ascertain the exact concentration of the solution in
the following manner: Titrate the solution against 15.0ml of perchloric acid
(0.1 mol/1) VS using 2-3 drops of crystal violet/acetic acid TS. Each ml of
perchloric acid (0.1 mol/1) VS is equivalent to 8.203 mg of C2H3Na02.
Sodium alizarinsulfonate (1 g/1) TS

Procedure. Dissolve 0.11 g of sodium alizarinsulfonate R in sufficient water to
produce 100 ml.
1406
Sodium alizarinsulfonate (lOg/1) TS. A solution of sodium alizarinsulfonate

R containing about lOg of C],(H7Na07S per litre.
Sodium alizarinsulfonate R. Alizarin Red S, sodium salt of 3,4-dihydroxy-

9,10-anthraquinone-2-sulfonic acid; Ci4H7Na07S,H20.
Description. A yellow-brown or orange-yeUow powder.
Solubility. Freely soluble in water, producing a yellow solution; sparingly soluble
in ethanol (-750 g/1) TS.
Sodium amidotrizoate RS. International Chemical Reference Substance.
Sodium arsenite (0.05 mol/1) VS

Procedure. Dissolve 5g of arsenic trioxide R in a mixture of 20ml of sodium
hydroxide (-80 g/1) TS and 20 ml of water, dilute to 400 ml with water, and
add hydrochloric acid (-7 0 g/1) TS until the solution is neutral to litmus paper
R. Dissolve 4g of sodium hydrogen carbonate R in the prepared solution
solution in the following manner; dilute 25 ml with 50 ml of water, add 5g
of sodium hydrogen carbonate R, and titrate with iodine (0.05 mol/1) VS,
using starch TS as indicator.
Storage. Add 1 drop of mercury R for the preservation of the solution.
Sodium arsenite (O.lmol/I) VS

Procedure. Dissolve 5g of arsenic trioxide R in a mixture of 20ml of sodium
hydroxide (-80 g/1) TS and 20 ml of water, dilute to 400 ml with water and
add hydrochloric acid (-70 g/1) TS until the solution is neutral to litmus paper
R. Dissolve 2g of sodium hydrogen carbonate R in the prepared solution
lowing the method described under sodium arsenite (0.05mol/l) VS.

Storage. Add 1 drop of mercury R for the preservation of the solution.
Sodium carbonate (lOg/1) TS. A solution of sodium carbonate R containing

about 10. 6g of Na2C03 per litre (approximately 0.1 mol/1).
Sodium carbonate (200 g/1) TS. A solution of sodium carbonate R contain-

ing 200 g of Na2C03 per litre.
Sodium carbonate (50g/l) TS. A solution of sodium carbonate R containing

about 50 g/1 of Na2C03 (approximately 0.5 mol/1).
Sodium carbonate (75 g/1) TS. A solution of sodium carbonate R containing

about 75 g of Na2C03 per litre.
Sodium carbonate R. Na2CO3,10H2O (SRIP, 1963, p. 179).
1407
Sodium carbonate standard TS

Procedure. Dissolve 2.64 g of sodium carbonate R and 2.093 g of sodium hydro-
gen carbonate R in water R to produce
sufficient carbon-dioxide-free
1000 ml.
Sodium carbonate, anhydrous, FeR. Anhydrous sodium carbonate R that

complies with the following additional test: Dissolve 4.0 g in 25 ml of water,
add 8 ml of hydrochloric acid (-250 g/1) FeTS, and proceed with the 2.2.4
Limit test for iron, using 2 ml of iron standard FeTS; not more than lOpg/g.
Sodium carbonate, anhydrous, R. Na2C03 (SRIP, 1963, p. 179).
Sodium chloride (lOg/I) TS. A solution of sodium chloride R containing

about lOg of NaCl per litre.
Sodium chloride (300g/l) TS. A solution of sodium chloride R containing

about 300 g of NaCl per litre.
Sodium chloride (400 g/1) TS. A saturated solution of sodium chloride R con-
taining about 400 g of NaCl per litre.
Sodium chloride (9 g/1) TS. A solution of sodium chloride R containing about

9g of NaCl per litre.
Sodium chloride R. NaCl (SRIP, 1963, p. 181).
Sodium chloride, pyrogen-free, R. Sodium chloride R which complies with

the following additional test:
Pyrogens. Carry out the test as described under 3.5 Test for pyrogens injecting,
per kg of the rabbit’s weight, a solution containing 9 mg in 10 ml of sterile
water R.
Sodium citrate (250g/l) TS. A solution of sodium citrate R containing about

294g of C6H5Na307,2H20 in 1000ml.
Sodium citrate R. C 6 H 5 Na 30 j, 2 H 20 .
Contains not less than 99.0% of CgHsNasO/, calculated with reference to the
Description. White, granular crystals or a crystalline powder; odourless. Slightly
deliquescent in moist air.
Solubility. Soluble in less than 2 parts of water, practically insoluble in ethanol
(-750 g/1) TS.

A 100 g/1 solution is clear and colourless.
Appearance of solution.
Water. Determined by the Karl Fischer method, keeping the substance in
contact with the dehydrated methanol R for 15 minutes; not less than
llOmg/g and not more than 130mg/g.
1408
Assay. Dissolve about O.lSg, accurately weighed, in 20ml of glacial acetic acid
R, and titrate with perchloric acid (0.1 mol/1) VS as described in 2.6 Non-
aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/1) VS is
equivalent to 8.603mg of CeHsNasOj.
Sodium cobaltinitrite (lOOg/1) TS. A solution of sodium cobaltinitrite R

containing about lOOg/1 of Na3Co(N02)6.
Sodium cobaltinitrite R. NasCo(N02)6 (SRIP, 1963, p. 182).
Sodium cromoglicate RS. International Chemical Reference Substance.
Sodium diethyldithio carbamate (0.8 g/1) TS. A solution of sodium

diethyldithiocarbamate R containing about 0.8 g of C5HioNNaS2 per litre.
Sodium diethyldithiocarbamate R. C5HioNNaS2,3H20 (SRIP, 1963, p. 183).
Sodium dihydrogen phosphate (275 g/1) TS. A solution of sodium dihy-

drogen phosphate R containing about 275 g of NaH2P04 per litre.
Sodium dihydrogen phosphate (45 g/1) TS. A solution of sodium dihydro-

gen phosphate R containing about 47 g of NaH2P04 per litre.
Sodium dihydrogen phosphate, anhydrous R. [sodium biphosphmate];

sodium-phosphate, monobasic; NaH2P04.
Sodium dihydrogen phosphate dihydrate R. [sodium biphosphate];

sodium phosphate, monobasic; NaH2 PO4, 2 H2O.
Sodium dihydrogen phosphate R [sodium biphosphate]; sodium phos-

phate, monobasic; NaH2P04,H20 (SRIP, 1963, p. 178).
Sodium dithionite R. Sodium hydrosulfite, sodium sulfoxylate; Na204S2.

Description. A white or greyish white, crystalline powder.
Solubility. Very soluble in water, slightly soluble in ethanol (-750 g/1) TS.
Note: Sodium dithionite R oxidizes in air.
Sodium dithionite (200g/l) TS. A solution of sodium dithionite R contain-

ing about 200 g of Na204S2 per litre.
Sodium fluoride R. NaP (SRIP, 1963, p. 183).
Sodium formate R. CHNa02.

Description. White, deliquescent granules or a crystalline powder; slight odour
of formic acid.
1409
Sodium hexanesulfonate R. CgHisNaOsS.

Sodium hydrogen carbonate (lOOg/1) TS. A solution of sodium hydrogen

carbonate R containing about lOOg of NaHCOg in 1000 ml.
Sodium hydrogen carbonate (40g/l) TS. A solution of sodium hydrogen

carbonate R containing about 42 g of NaHCOj per litre (approximately
0.5 mol/1).
Sodium hydrogen carbonate R [sodium bicarbonate]; NaHCOg (SRIP, 1963,

p. 177).
Sodium hydrogen carbonate R. NaHCOj (SRIP, 1963, p. 177).
Sodium hydroxide (~150g/l) TS. A solution of sodium hydroxide R con-

taining about 150 g of NaOH per litre.

taining about 200 g/1 of NaOH.
Sodium hydroxide (~300g/I) TS. A solution of sodium hydroxide R con-


Sodium hydroxide (~80g/I) TS. A solution of sodium hydroxide R contain-

ing about 80g/l of NaOH (approximately 2mol/l).
Sodium hydroxide (~40g/l) TS. A solution of sodium hydroxide R contain-

ing about 40g/l of NaOH (approximately 1 mol/1).
Sodium hydroxide (0.001 moI/1) VS. Sodium hydroxide R, dissolved in

water to contain 40.01 mg of NaOH in 1000ml.
lowing the method described under sodium hydroxide (1 mol/1) VS.
Sodium hydroxide (O.OlmoI/1) VS. Sodium hydroxide R, dissolved in water

to contain 0.4001 g of NaOH in 1000 ml.
Sodium hydroxide (0.01 mol/1), carbonate-free, VS. Sodium hydroxide R,

dissolved in water to contain 0.4001 g of NaOH in 1000ml.
1410
Procedure, test for carbonates and method of standardization. Prepare the solution,
carry out the test, and ascertain the exact concentration following the
method described under carbonate-free sodium hydroxide (1 mol/1) VS.
Sodium hydroxide (0.02mol/l) VS. Sodium hydroxide R, dissolved in water

Sodium hydroxide (0.02mol/l), carbonate-free, VS. Sodium hydroxide R,

dissolved in water to contain 0.8001 g of NaOH
in 1000ml.
and method of standardization. Prepare the solution,
Procedure, test for carbonates
Sodium hydroxide (O.OSmoI/l) VS. Sodium hydroxide R, dissolved in water

to contain 2.000g of NaOH in 1000ml.
Sodium hydroxide (0.1 mol/1) BET. Prepare from sodium hydroxide R

and water BET. It is suitable if, after adjustment to pH 6.5-7 .5, it gives a
negative result under the conditions prescribed in the 3.4 Test for bacterial
endotoxins.
Sodium hydroxide (0.1 mol/1) VS. Sodium hydroxide R, dissolved in water

following the method described under sodium hydroxide (1 mol/1) VS.
Sodium hydroxide (0.1 mol/1), carbonate-free, VS. Sodium hydroxide R,

in 1000 ml.
Sodium hydroxide (0.2 mol/1) VS. Sodium hydroxide R, dissolved in water

following the method described under sodium hydroxide (1 mol/1) VS.
Sodium hydroxide (0.2mol/l), carbonate-free, VS. Sodium hydroxide R,

in 1000 ml.
1411
Sodium hydroxide (0.5mol/l) VS. Sodium hydroxide R, dissolved in water

to contain 20.00g of NaOH in 1000ml.
following the method described under sodium hydroxide (1 mol/1) VS,
Sodium hydroxide carbonate -free, VS. Sodium hydroxide R,

(0.5 mol/1),
dissolved in water to contain 20.00g of NaOH in 1000ml.
Procedure, test for carbonates and method of standardization. Prepare the solution,
Sodium hydroxide (1 mol/1) VS. Sodium hydroxide R, dissolved in water to

contain 40,01 g of NaOH in 1000 ml.
tion in the following manner; Dry about 5 g of potassium hydrogen phtha-
late Rat 105°C for 3 hours and weigh accurately. If the potassium hydrogen
sodium hydroxide solution, using phenolphthalein/ethanol TS as indicator.
Each 0.2042 g of potassium hydrogen phthalate is equivalent to 1ml of
sodium hydroxide (1 mol/1) VS. Standard solutions of sodium hydroxide
should be restandardized frequently.
air. They should, therefore, be stored in bottles with suitable non-glass,
well-fitting stoppers, provided with a tube filled with soda lime R.
Sodium hydroxide (1 mol/1), carbonate-free, VS. Sodium hydroxide R, dis-

solved in water to contain 40.01 g of NaOH in 1000 ml.
Procedure. Dissolve sodium hydroxide R in water to produce a 400-600 g/1 solu-
tion and allow to stand. Taking precautions to avoid absorption of carbon
dioxide, siphon off the clear supernatant liquid and dilute as required with
carbon-dioxide-free water R.
Test for carbonates. Titrate 45 ml of hydrochloric acid (1 mol/1) VS with the car-
bonate-free sodium hydroxide solution, using phenolphthalein/ethanol TS
as indicator. At the end-point add just sufficient acid to discharge the pink
colour and boil to reduce the volume to 20ml. Add, whilst boiling, suffi-
cient acid again to discharge the pink colour and prevent its reappearance
on continued boiling; not more than 0.1 ml of the acid is required.
tion in the following manner; Dry about 5 g of potassium hydrogen phtha-
late R at 105°C for 3 hours and weigh accurately. If the potassium hydrogen
carbonate-free sodium hydroxide solution, using phenolphthalein/ethanol
TS as indicator. Each 0.2042 g of potassium hydrogen phthalate is equiva-
1412
lent to 1 ml of carbonate-free sodium hydroxide (1 mol/1) VS. Standard solu-

tions of sodium hydroxide should be restandardized frequently.
air They should therefore be stored in bottles with suitable non-glass, well-
fitting stoppers, provided with a tube filled with soda lime R.
Sodium hydroxide (lOg/I) TS. A solution of sodium hydroxide R contain-

ing about lOg of NaOH per litre (approximately 0.25mol/l).
Sodium hydroxide (50g/l) TS. A solution of sodium hydroxide R contain-

ing about 50 g of NaOH per litre.
Sodium hydroxide R. NaOH (SRIP, 1963, p. 185).
Sodium hydro xide/ethanol TS

Procedure. Dissolve 50 g of sodium hydroxide R in sufficient ethanol (-750 g/1)
TS to produce 1000 ml.
Sodium hydroxide/methanol TS
Procedure. Dissolve 40 g of sodium hydroxide R in sufficient methanol R to
produce 1000ml.
Sodium hypobromite TS
Procedure. Dissolve 2.5 g of sodium hydroxide R in 7.5 ml of water, add 0.5ml
of bromine R and a sufficient quantity ofwater to produce 10 ml.
Note: Sodium hypobromite TS must be freshly prepared.
Sodium hypochlorite (~40g/l) TS

Description. A pale, greenish yellow, clear liquid; odour, resembling that of
chlorine.
Assay. Introduce 3 ml weigh accurately, and add
into a glass-stoppered flask,
50 ml of water. Add 2g of potassium iodide R and 10 ml of acetic acid
(-300 g/1) TS, and titrate the liberated iodine with sodium thiosulfate
(0.1 mol/1) VS, adding 3 ml of starch TS as the endpoint is approached. Each
ml of sodium thiosulfate (0.1 mol/1) VS is equivalent to 3.723 mg of NaOCl.

Storage. Sodium hypochlorite (-40 g/1) TS must be kept in a light-resistant con-
tainer at a temperature not exceeding 25 °C.
Sodium hypochlorite TSl

Procedure. Dilute 10ml of sodium hypochlorite (-40g/l) TS to 100ml with
water (contains approximately 0.5% of chlorine).
Sodium laurilsulfate (lOg/1) TS. A solution of sodium laurilsulfate R con-

taining about lOg of Ci2H25Na04S per litre.
1413
Sodium laurilsulfate R. Sodium lauryl sulfate; a mixture of sodium alkyl sul-

fates, consisting mainly of sodium dodecyl sulfates, Ci2H25Na04S.
Description. A white or pale yellow powder, crystals, or flakes; odour, faint, but
characteristic.
Solubility, Very soluble in water giving an opalescent solution; partly soluble in
Sodium mercaptoacetate R (Sodium thioglycolate R.) C2H3Na02S.

Description. Hygroscopic crystals.
Sodium metabisulfite (50g/l) TS. A solution of sodium metabisulfite R con-

taining about 50 g of Na20sS2 per litre.
Sodium metabisulfite R. Na20sS2 (SRIP, 1963, p. 187).
Sodium metaperiodate R. Sodium periodate Nal04. Contains not less than

98.0% of Nal04.
Description. White crystals or a white, crystalline powder.
Assay. Dissolve 0.5g in 100ml of water. Add 3g of sodium hydrogen carbon-
ate R and 3g of potassium iodide R, and titrate the liberated iodine with
sodium Each ml of sodium arsenite (0.05mol/l) VS
arsenite (0.05 mol/1) VS.
is equivalent to 10.69mg of NaI04.
Sodium metaperiodate TS
Procedure. Dissolve 60g of sodium metaperiodate R in 120ml of sulfuric acid
(0.05mol/l) VS and dilute to 1000ml with water. Do not heat to dissolve the
periodate. If the solution is not clear, filter through a sintered-glass filter. Store
the solution in a glass-stoppered, light-resistant container.
Suitability test. ml into a 250-ml volumetric flask, dilute to volume
Pipette 10
with water, and mix. To about 550 mg of glycerol R dissolved in 50ml of
water add, using a pipette, 50ml of the diluted sodium metaperiodate. For
a blank, transfer 50 ml of the diluted sodium metaperiodate solution to a
flask containing 50 ml of water. Allow the solutions to stand for 30 minutes,
then to each add 5 ml of hydrochloric acid (-420 g/1) TS and 10 ml of potas-
sium iodide (80 g/1) TS, and swirl to mix. Allow to stand for 5 minutes, add
100 ml of water, and titrate with sodium thiosulfate (0.1 mol/1) VS, shaking
continuously and adding 3 ml of starch TS as the endpoint is approached.
The ratio of the volume of sodium thiosulfate (0.1 mol/1) VS required for
sodium metaperiodate TS to that required for the blank should be between
0.750 and 0.765.
1414
Sodium methoxide (0.1 mol/1) VS

Procedure. Cool in ice-water 150ml of dehydrated methanol R and add in small
portions 2.5 g of freshly cut sodium R. When the metal has dissolved, add
sufficient toluene R to produce 1000ml.
solution in the following manner: Titrate 0. 1 0 g of benzoic acid R, accurately
weighed, as described under 2.6 Non-aqueous titration. Method B. Each
12.21 mg of C 7 HsOs is equivalent to 1 ml of sodium methoxide (0.1 mol/1) VS.
Note: Sodium methoxide (0.1 mol/1) VS must be standardized immediately
before use.
Sodium molybdotungstophosphate TS
Procedure. Boil under a reflux condenser for 2 hours 350 ml of water with 50 g
of sodium tungstate R, 12 g of phosphomolybdic acid R, and 25ml of phos-
phoric acid (-1440 g/1) TS; cool and add sufficient water to produce 500 ml.
Sodium nitrate R. NaNOj.

Sodium nitrite (0.1 mol/1) VS. Sodium nitrite R, dissolved in water to contain
6.900g of NaN02 in 1000ml.
solution in the following manner: Place 50.0 ml of potassium permanganate
(0.02 mol/1) VS in a glass-stoppered flask, dilute with 300 ml of water, add
25ml of sulfuric acid (-100 g/1) TS and 20.0 ml of the sodium nitrite solu-
tion.Allow the solution to stand for 10 minutes. Then add 2g of potassium
iodide R and titrate with sodium thiosulfate (0.1 mol/1) VS, using starch TS
as indicator. Perform a blank determination and make any necessary
corrections.
Sodium nitrite (lg/1) TS. A solution of sodium nitrite R containing about

1 g of NaN 02 per litre.
Note: Sodium nitrite (1 g/1) TS must be freshly prepared.
Sodium nitrite (lOg/1) TS. A solution of sodium nitrite R containing about

10 g/1 of NaN 02 .
Sodium nitrite (100 g/1) TS. A solution of sodium nitrite R containing about
100 g of NfaN 02 per litre.
20 g of NaN 02 per litre.

3g of NaNOj per litre.
Note: Sodium nitrite (3 g/1) TS must be freshly prepared.
1415
Sodium nitrite (35g/l) TS. A solution of sodium nitrite R containing about

35 g of NaNOj per litre (approximately 0.5 mol/1).
Sodium nitrite (50g/l) TS. A solution of sodium nitrite R containing about

50 g of NaNOj per litre.
Sodium nitrite R. NaNOj (SRIP, 1963, p. 189).
Sodium nitrite/hydrochloric acid TS

Procedure. Dissolve 0.5 g of sodium nitrite R in sufficient hydrochloric acid
(0.1 mol/1) VS to produce 100ml.
Sodium nitroprusside (45 g/I) TS. A solution of sodium nitroprusside R con-

taining about 45 g of Na 2 pe(NO)(CN )5 per litre.
Note: Sodium nitroprusside (45g/l) TS must be freshly prepared.
Sodium nitroprusside (8.5 g/1) TS. A solution of sodium nitroprusside R

containing about 8.5g of Na 2 pe(NO)(CN )5 per litre.
Sodium nitroprusside R. Na2pe(N0)(CN)5,2H20 (SRIP, 1963, p. 190).
Sodium nitroprusside, alkaline, TS

Procedure. Dissolve 1 g of sodium nitroprusside R and 1 g of sodium carbonate
R in sufficient water to produce 100 ml.
Sodium octanesulfonate R. CsHi/NaOgS.

Contains not less than 98.0% of CaHuNaOgS.

Absorbance. A 0.05 g/ml solution of a 1-cm layer measured at a wavelength of
about 250 nm has an absorbance of not greater than 0.01.
Sodium oxalate R. C Na 204

2 (SRIP, 1963, p. 190).
Sodium peroxide R. Na202 (SRIP, 1963, p. 191).
Sodium R. Na (SRIP, 1963, p. 175).
Sodium salicyclate R. C/HsNaOj. Use sodium salicylate as described in the

monograph for “Sodium salicylate”.
Sodium salicylate (11.5 g/1) TS. A solution of sodium salicylate R contain-

ing about 11.5g of C/HsNaOa per litre.
Sodium standard (200 pg Na/ml) TS

Procedure. Dissolve 0.5084 g of sodium chloride R, previously dried at 100-
105 °C for 3 hours, in sufficient water to produce 1000 ml.
1416
Sodium sulfate, anhydrous, R. Na 2 S 04 (SKIP, 1963, p. 195).
Sodium sulfide R. Na2S,9H20 (SKIP, 1963, p. 195).
Sodium sulfide TS
Procedure. Dissolve 12 g of sodium sulfide R in 25 ml of water and add sufficient
glycerol R to produce 100 ml.
Sodium sulfite R. Na 2 S 03 7 H 20
,
(SRIP, 1963, p. 196).
Sodium tetraborate (lOg/1) TS. A solution of sodium tetraborate R con-

taining about lOg of Na 2 B 407 per litre.
Sodium tetraborate R. Borax, Na2B4O7,10H2O.

Description. Transparent, colourless crystals, or a white, crystalline powder;
odourless.
Solubility. Soluble in 20 parts of water, and in 0.6 part of boiling water; very
slightly soluble in ethanol (-750 g/1) TS.
pH Value of a O.O'tmol/l solution. Dissolve 0.3814g in water and dilute to 100ml,
using water having a pH of 6.5-7. 4. The pH should be from 9.15-9.20 at 25°C.
Chlorides. Dissolve 1.0 g in 20 ml of water, filter if necessary through a chloride-
free filter, add 1 ml of nitric acid (-1000 g/1) TS, and proceed as described in
2.2.1 Limit test for chlorides. Sodium tetraborate R contains not more than
250pg/g.
Sulfates. Dissolve 0.5 g in 20 ml of water, add 2 ml of hydrochloric acid (-70 g/1)
TS, and filter. Proceed as described in 2.2.2 Limit test for sulfates. Sodium
tetraborate R contains not more than l.Omg/g.
Sodium tetraborate standard TS

Procedure. Dissolve 3.81 g of sodium tetraborate R in sufficient carbon-dioxide-
Storage. Store the solution protected from atmospheric carbon dioxide and
keep it stoppered at all times except when actually in use.
Sodium tetraphenylborate (30g/l) TS. A solution of sodium tetraphenylb-

orate R containing about 30 g/1 of C 24 H 2 oBNa.
Note: If necessary, stir for 5 minutes with 1 g of aluminium hydroxide R or
charcoal R, and filter to clarify.
Sodium thioglycolate R. See Sodium mercaptoacetate R.
Sodium tetraphenylborate R. C 24 H 2 oBNa.

Description. A fluffy, white or almost white powder.
Solubility. Freely soluble in water and acetone R; insoluble in light petroleum
R.
pH Value. pH of a 20 g/1 solution, not less than 7.5.
1417
Sodium thiosulfate (0.002 mol/1) VS. Sodium thiosulfate R, dissolved in

water to contain 0.31 6g of Na2S203 in 1000 ml.
following the method described under sodium thiosulfate (0.1 mol/1) VS.

water to contain 1.582g of Na2S203 in 1000 ml.

water to contain 3.164g of Na2S203 in 1000 ml.
lowing the method described under sodium thiosulfate (0.1 mol/1) VS.

water to contain 7.91 Og of Na2S203 in 1000 ml.
Sodium thiosulfate (0.1 mol/1) VS. Sodium thiosulfate R, dissolved in water

to contain 15.82g of Na2S203 in 1000ml.
Method of standardization (alternative procedure). Ascertain the exact concen-
manner; To about 40 ml of
tration of the 0.1 mol/1 solution in the following
water in a glass-stoppered conical flask, add 10.0 ml of potassium bromate
(0.0167mol/l) VS, Ig of potassium iodide R, and 3ml of sulfuric acid
(-1760 g/1) TS. AUow the solution to stand for 5 minutes, and titrate the
liberated iodine with the sodium thiosulfate solution, adding 3 ml of starch
TS as the end-point is approached. Perform a blank determination on the
same quantities of the reagents and make any necessary corrections.
Sodium thiosulfate (320g/l) TS. A solution of sodium thiosulfate R con-

taining about 320 g of Na2S203 per litre.
Sodium thiosulfate R. Na2S203,5H20 (SRIP, 1963, p. 197).
Sodium tungstate R. Na204W,2H20 (SRIP, 1963, p. 197).
Sorbitol R. CeHuO^. Contains not less than 97.0% of C^HMOe.

Description. White granules or powder or a white, crystalline mass.
Solubility. Very soluble in water; sparingly soluble in ethanol (-750 g/1) TS; prac-
Assay. Dissolve about 0.2 g, previously dried and accurately weighed, in suffi-
cient water to produce 100ml. Transfer 10.0ml to an iodine flask, add
50.0ml of potassium periodate TS, and heat for 15 minutes on a water-bath.
Cool, add 2.5 g of potassium iodide R, stopper tightly, and shake well. Allow
1418
to stand for 5 minutes protected from and titrate with sodium thio-
light
sulfate (0.1 mol/1) VS, using 3 ml of starch TS as an indicator. Perform a blank
titration and make any necessary corrections. Each ml of sodium thiosulfate
(0.1 mol/1) VS is equivalent to 1.822 mg of CsHÔs.
Spectinomycin hydrochloride RS. International Chemical Reference

Substance.
Spironolactone RS. International Chemical Reference Substance.
Squalane R. 2,6,10,15,19,23-Hexamethyltetracosane; C30H52.

Solubility. Freely soluble in ether R; slightly soluble in acetone R and ethanol
(-750 g/1) TS.
Relative density, dfo = 0.811-0.813.
Refractive index, tto = 1.451-1.453.
Stannous chloride AsTS

Procedure. Prepare from stannous chloride TS by adding an equal volume of
hydrochloric acid (-250 g/1) TS, boil down to the original volume, and filter
through a fine-grained filter-paper.

Test for arsenic. To 10ml add 6ml of water and 10ml of hydrochloric acid
(-250 g/1) AsTS, and distil 16ml. To the add 50 ml of water and 2
distillate
drops of stannous chloride AsTS; then apply the general test for arsenic. The
colour of the stain produced is not more intense than that produced from a
1-ml standard stain, showing that the amount of arsenic does not exceed
1 pg/ml.
Stannous chloride R. SnCl2,2H20 (SRIP, 1963, p. 198).
Stannous chloride TS
Procedure. Dissolve 330 g of stannous chloride R in 100 ml of hydrochloric acid
(-250 g/1) TS and sufficient water to produce 1000ml.
Stannous chloride/hydrochloric acid TSl

Procedure. Dissolve 10 g of stannous chloride R in sufficient hydrochloric acid
(-70g/l) TS to produce 100ml.
Starch iodide TS
Procedure. Dissolve 0.75g of potassium iodide R in 5ml of water and 2g of
zinc chloride R in 10 ml of water, mix the two solutions, and add 100 ml of
water. Heat the solution to boiling and add, with constant stirring, a sus-
pension of 5 g of corn or potato starch R in 35 ml of water. Boil for 2 minutes
and cool.
Storage. Store in a well-closed container and keep in a cool place.
1419
g
Starch R [potato starch R or corn starch R], (SRIP, 1963, p. 199).
Starch TS
Procedure. Mix 0,5 g of starch R or of soluble starch R with 5 ml of water, and
add this solution, with constant stirring, to sufficient water to produce about
100 ml; boil for a few minutes, cool, and filter.
Note: Starch TS must be freshly prepared.
Starch, soluble, R. (SRIP, 1963, p. 199).
Starch/iodide paper R [starch-iodide paper R]. (SRIP, 1963, p. 200).
Streptomycin sulfate RS. International Chemical Reference Substance.
Strychnine sulfate R. C42H44N404,H2S04,5H20 (SRIP, 1963, p. 200).
Sudan red G R. l-(4-Phenylazophenylazo)-2-naphthol; Sudan III; Solvent red

23; C.I. 26100; C22H16N4O.
Description. A reddish brown powder.
Solubility. Practically insoluble in water.
Sudan red TS
Procedure. Dissolve 0.5 of Sudan red GR in 100 ml of glacial acetic acid Rl.
Sulfacetamide RS. International Chemical Reference Substance,
Sulfadiazine RS. International Chemical Reference Substance.
Sulfadimidine RS. International Chemical Reference Substance.
Sulfadoxine RS. International Chemical Reference Substance.
Sulfamethoxazole RS. International Chemical Reference Substance.
Sulfamethoxypyridazine RS. International Chemical Reference Substance.
Sulfamic acid (5g/l) TS. A solution of sulfamic acid R containing about 5 g

of H3NO3S per litre.
Sulfamic acid (50g/l) TS. A solution of sulfamic acid R containing about

50g of H3NO3S per litre.
Note: Sulfamic acid (50g/l) TS must be freshly prepared.
Sulfamic acid (80g/l) TS. A solution of sulfamic acid R containing about

80g of H3NO3S per litre.
Note: Sulfamic acid (80g/l) TS must be freshly prepared.
1420
Sulfamic acid R. H3NO3S.

Description. Colourless or white crystals.
Solubility. Soluble in water; slightly soluble in ethanol (-750 g/1) TS.
4-Sulfamoylbenzoic acid R. /?-Sulfamoylbenzoic acid; C7H7NO4S.

Sulfanilamide RS. International Chemical Reference Substance.
Sulfanilic acid R. C6H7NO3S (SKIP, 1963, p. 201).
SulfanUic acid, diazotized, TS

Procedure. Dissolve 0.2 g of sulfanilic acid R in 20ml of hydrochloric acid
(1 mol/1) VS with warming, cool in ice, add drop by drop and with con-
tinuous stirring 2.5ml of sodium nitrite (35 g/1) TS, allow to stand in ice for
10 minutes and then add 1 ml of sulfamic acid (50 g/1) TS.
Sulfasalazine RS. International Chemical Reference Substance.
Sulfo salicylic acid (175g/l) TS. A solution of sulfosalicylic acid R contain-

ing about 175 g/1 of C7H6O6S.
Sulfosalicylic acid R. C7Hs06S,2H20.

Description. White or slightly pink coloured, needle-like crystals.
Dissolve 5.0 g in 50ml of water, heat to boiling and digest in a
Insoluble matter.
covered beaker on a water-bath for 1 hour. Filter through a tared filtering
crucible, wash thoroughly, and dry at 105 °C. The weight of the residue does
not exceed 1.0 mg.
Sulfated ask. Gently ignite l.Og in a tared crucible or dish, other than platinum,
until charred. Cool, moisten the residue with 1 ml of sulfuric acid (-1760 g/1)
TS, and ignite again; not more than l.Omg/g.
Sulfur dioxide R. SO^ (SRIP, 1963, p. 202).
Sulfuric acid (-10 g/1) TS

Procedure. Mix 100ml of sulfuric acid (-lOOg/1) TS with sufficient water to
produce 1000ml.

Procedure.Add 57 ml of sulfuric acid (-1760 g/1) TS to sufficient water to
produce 1000ml (approximately 1 mol/1); d - 1.065.
Sulfuric acid (-1125 g/1) TS. Sulfuric acid (-1760g/l) TS, diluted with water
to contain about 1125g of H2SO4 per litre; d~ 1.61.
1421
Sulfuric acid (~1760g/l) TS [sulfuric acid R], (SKIP, 1963, p. 202); d~ 1.84.
Sulfuric acid (-1760 g/1), nitrogen-free, TS. Sulfuric acid (-1760 g/1) TS con-
taining not less than 1760g/l of H2SO4 and complying with the test for
nitrates.
Nitrates. Mix 45ml with 5ml of water, cool, and add 8mg of diphenylbenzi-
dine R; the solution is colourless or not more than very pale blue.

Procedure. Mix 1 volume of sulfuric acid (-1760 g/1) TS with 9 volumes of water,
and cool.The resulting solution contains about 190 g/1 of H2SO4; d ~ 1.12.

Procedure. Dilute 485ml of sulfuric acid (-1760 g/1) TS to 1000ml with water
(~4.5mol/l); 1.25.

Procedure. To 50 ml of sulfuric acid (-100 g/1) TS add about 50 ml of water and
mix.

Procedure.Slowly add 3 volumes of sulfuric acid (~1760g/l) TS to 7 volumes of
water while gently stirring and cool; d ~ 1.33.
Sulfuric acid (-635g/l) TS. Sulfuric acid (-1760 g/1) TS, diluted with water
to contain about 635 g of H2SO4 per litre; d- 1.36.

Procedure.Slowly add sulfuric acid (-1760 g/1) TS to an equal weight of water
while gendy stirring and cool; d - 1.40.
Sulfuric acid (0.005mol/l) VS. Sulfuric acid (~1760g/l) TS, diluted with
water to contain 0.4904 g of H2SO4 in 1000 ml.
lowing the method described under sulfuric acid (0.5mol/l) VS.
Sulfuric acid (0.01 mol/1) VS. Sulfuric acid (-1760 g/1) TS, diluted with water
to contain 0.9808 g of H2SO4 in 1000 ml.
following the method described under sulfuric acid (0.5mol/l) VS.
Sulfuric acid (0.05mol/l) VS. Sulfuric acid (-1760 g/1) TS, diluted with water
to contain 4.904 g of H2SO4 in 1000ml.
1422
Sulfuric acid (O.lmol/1) VS. Sulfuric acid (~1760g/l) TS, diluted with water
to contain 9.808 g of H
2 SO 4 in 1000ml.

following the method described under sulfuric acid (0,5mol/l) VS.
Sulfuric acid (0.125mol/l) VS. Sulfuric acid (~1760g/l) TS, diluted with
water to contain 12.52g of H 2 SO 4 in 1000ml.
2 SO 4 in 1000ml.

2 SO 4 in 1000ml.

solution in the following manner: Dissolve about 1.5g, accurately weighed,
of anhydrous sodium carbonate R, previously dried at 270 °C for 1 hour, in
50ml of water and titrate with the sulfuric acid solution, using methyl
orange/ethanol TS as indicator. Each 52.99mg of anhydrous sodium car-
bonate is equivalent to 1ml of sulfuric acid (0.5mol/l) VS.
Sulfuric acid/ethanol TS
Procedure.Cool separately 10 ml of ethanol (-750 g/1) TS and 90 ml of sulfuric
acid (~1760g/l) TS to about -5°C. Carefully add the acid to the ethanol,
keeping the solution as cool as possible, and mix gently.
Sulfuric acid/methanol TS
Procedure. Cool separately 10ml of sulfuric acid (-1760g/l) TS and 90ml of
methanol R. Carefully add the acid to the methanol, keeping the solution
as cool as possible, and mix gently.
Sulfurous acid TS [sulfurous acid R]. (SRIP, 1963, p. 204).
Tamoxifen citrate E-isomer RS. International Chemical Reference

Substance.
Tamoxifen citrate RS. International Chemical Reference Substance.
Tannic acid (50g/l) TS. A solution of tannic acid R containing about 50 g of

CyMb2046 per litre.
Tannic acid R. C 76 H 52 O 46 (SRIP, 1963, p. 205).
1423
Tartaric acid (lOg/1) TS. A solution of tartaric acid R containing about lOg
of C4H6O5 per litre.
Tartaric acid (200g/l) TS. A solution of tartaric acid R containing about

200 g of C4H6O6 per litre.
Tartaric acid (5g/l) TS. A solution of tartaric acid R containing about 5g of

C4H6O6 per litre.
Tartaric acid R. C4H(;06 (SRIP, 1963, p. 205).
p-Terphenyl R. 1,4-Diphenylbenzene, CisHi4. Suitable for scintillation counting.
Thioglycolic acid R. See Mercaptoacetic acid R.
Testosterone enantate RS. International Chemical Reference Substance.
Testosterone propionate R. C22H32O3. Use testosterone propionate as

described in the monograph for "Testosterone propionate”.
Testosterone propionate RS. International Chemical Reference Substance.
Testosterone propionate/ethanol TS
Procedure. Dissolve 10 mg of testosterone propionate R in sufficient ethanol
Tetrabromophenolphthalein ethyl ester R. 3',3",5',5"-Tetrabromophe-

nolphthalein, ethyl ester; C22Hi4Br404. Use a suitable reagent grade.
Tetrabromophenolphthalein ethyl ester TS

Procedure. Dissolve O.lOg of tetrabromophenolphthalein ethyl ester R in suffi-
cient glacial acetic acid R to produce 100 ml.
Note: Tetrabromophenolphthalein ethyl ester TS should be freshly prepared.
Tetrabutylammonium hydrogen sulfate R. TBAHS; Ci<;H37N04S.

Solubility. Freely soluble in water and methanol R; soluble in ethanol (-750 g/1)
TS producing a slightly hazy, colourless solution.

Absorbance. A 0.05 g/ml solution of a 1-cm layer measured at a wavelength
between 240 nm and 300 nm has an absorbance of not greater than 0.05.
Tetrabutylammonium hydroxide (0.1 mol/1) VS

Procedure. Dissolve 40 g of tetrabutylammonium iodide R in 90 ml of dehy-
drated ethanol R, add 20 g of finely powdered, purified silver oxide R, and
1424
shake vigorously for 1 hour. Centrifuge a small volume of the mixture and
test the supernatant liquid for iodides. If a positive reaction is obtained, add
an additional 2 g of R and shake for a further 30 minutes. Repeat
silver oxide
this is free from iodides, filter the mixture through
procedure until the liquid
a fine sintered glass and rinse the reaction vessel and the filter with 3
filter,
quantities of dry benzene R, each of 50 ml. Add the washings to the filtrate
and dilute to 1000ml with dry benzene R. Pass dry carbon-dioxide-free
nitrogen R through the solution for 5 minutes.
Method of standardization. Titrate 10 ml of dimethylformamide R with the
tetrabutylammonium hydroxide solution, using 3 drops of thymol
blue/methanol TS as indicator, until a pure blue colour is obtained. Imme-
diately add about 0.06g of benzoic acid R, accurately weighed, stir to effect
solution, and titrate with the tetrabutylammonium hydroxide solution until
the full blue colour of the indicator is again obtained. The solution must be
protected from atmospheric carbon dioxide throughout the titration. From
the volume of the used in the second titration ascertain the exact
titrant
concentration of the 0.1 mol/1 solution. Each 12.21 mg of benzoic acid is
equivalent to 1ml of tetrabutylammonium hydroxide (0.1 mol/1) VS.
Note: Tetrabutylammonium hydroxide (0.1 mol/1) VS must be standardized
immediately before use.
Tetrabutylammonium hydroxide TS. C 15H 37NO. A solution in water con-

taining about 400 g of CigHa/NO per litre (-1.5 mol/1).
Tetrabutylammonium hydroxide/methanol TS
Procedure. Dilute a sufficient volume of tetrabutylammonium hydroxide TS
with methanol R to obtain a solution containing 0.25 g of C 16H 37NO per ml.
Tetrabutylammonium iodide R. CisHgelN. Contains not less than 98.0% of

CisHsJN.
White or
Descrifation. slightly cream-coloured crystals or a crystalline powder.
Solubility. Soluble in ethanol (-750 g/1) TS.
Assay. Dissolve about 1.2g, accurately weighed, in 30 ml of water. Add 50ml
of silver nitrate (0.1 mol/1) VS and 5ml of nitric acid (-130g/l) TS. Titrate the
excess of silver nitrate with ammonium thiocyanate (0.1 mol/1) VS, using
ferric ammonium sulfate (45g/l) TS as indicator. Each ml of silver nitrate
(0.1 mol/1) VS is equivalent to 36.94 mg of CieHssIN.
n-Tetradecane R. ChHsq.
Description. A clear and colourless liquid.
Miscibility. Miscible with ethanol (-750 g/1) TS.
Mass density. P 20 = about 0.76kg/l.
Refractive index, no* = 1.428-1.429.
1425
Tetrachloro ethane R. 1,1,2,2-Tetrachloroethane, C2H2CI4.

TS and ether R.
Refractive index. p2o = 1.493-1.495.
Mass density, n™ = 1. 590-1. 595kg/l.
Tetracycline hydrochloride RS. International Chemical Reference

Substance.
Tetrahydrofuran R. C^HgO.
Description. A colourless liquid; odour, characteristic, pungent.
Mass density.
p 2o
= 0. 884-0. 886kg/l.
Storage. Store in small, well-filled containers, protected from light.
Labelling. The name and concentration of any suitable preservative, not exceed-
ing 0.1%, should be stated on the label.
Tetramethylammonium hydroxide (-100 g/1) TS. Contains about 100 g/1

of (CH3)4N0H in water.
Description. A clear and colourless liquid; odour, strong, ammonia-like.
Residue on evaporation. Evaporate 5ml on a water-bath and dry at 105 °C for
1 hour; it leaves a residue of not more than l.Omg (0.2mg/g).
Ammonia and other amines. Weigh accurately a quantity of the solution, equiv-
alent to about 0.3 g of (CH3)4NOH, in a low-form weighing bottle tared with
5 ml of water. Add a slight excess of hydrochloric acid (1 mol/1) VS (about
4 ml), evaporate to dryness on a water-bath, and dry at 105°C for 2 hours.
The weight of the residue obtained, multiplied by 0.8317, represents the
quantity in mg of (CH3)4NOH corresponding to within + 0.2% of that found
in the assay.
Assay. Weigh accurately a glass-stoppered flask containing about 15ml
of water. Add a quantity of the solution equivalent to about 0.2 g of
(CH3)4N0H, and weigh again. Add methyl red/ethanol TS and titrate with
hydrochloric acid (0.1 mol/1) VS. Each ml of hydrochloric acid (0.1 mol/1) VS
is equivalent to 9.115mg of (CH3)4NOH.
Tetramethylammonium hydroxide/ethanol TS
Procedure. Dilute 10ml of tetramethylammonium hydroxide (-100 g/1) TS with
sufficient ethanol (-750 g/1) TS to produce 100ml.
Thioacetamide R. C2H5NS.
Note: Thioacetamide R is toxic.
Description. Colourless crystals or a white, crystalline powder; odour, faint, of
hydrogen sulfide.
1426

Melting p’oint. About 113 °C.
Thioacetamide, alkaline, TS
Procedure. Dissolve 0.4g of thioacetamide R in 10ml of water. Immediately
before use add 0.2 ml of this solution to
1 ml of a mixture of 15 ml of sodium
hydroxide (1 mol/1) VS, 5ml of water, and 20ml of glycerol R. Heat on a

water-bath for 20 seconds.
Thioacetazone RS. International Chemical Reference Substance.
4, 4'-Thio dianiline RS. International Chemical Reference Substance.

Note: 4,4'-Thiodianiline RS decomposes on storage, especially on exposure to
the air. If a slight decomposition is suspected, the substance can be purified
by treatment of a solution in methanol R with activated charcoal.
Thiopental RS. International Chemical Reference Substance.
Thiourea (0.1 g/1) TS. A solution of thiourea R containing 0.1 g of CH4N2S

per litre.
Thiourea R. CH4N2S (SRIP, 1963, p. 207).
Thorin (2 g/1) TS
Procedure. Dissolve 0.2g of thorin R in sufficient water to produce 100ml.
Storage. Store the solution protected from light.
Shelf-life. Use within 1 week of preparation.
Thorin R. 2,7-Disodium 4-[(o-arsonophenyl)azo]-3-hydroxy-2,7-naph-

thalenedisulfonate, C,6H]iAsN2Na20,oS2.
Thorium nitrate (0.005 mol/1) VS. Thorium nitrate R, dissolved in water to

contain 2.401 g of Th(N03)4 in 1000 ml.
solution in the following manner: Transfer 0.050 g, accurately weighed, of
sodium and dissolve in sufficient
fluoride R, previously dried, to a flask
water to produce 250ml. To 20.0ml of add 0.6ml of sodium
this solution
alizarinsulfonate (1 g/1) TS and then, by drops, sodium hydroxide (0.1 mol/1)
VS until the colour changes from pink to yellow. Add 5 ml of acetate buffer,
pH 3.0, TS and titrate with the thorium nitrate solution until the yellow
colour changes to pinkish yellow. Each 0.8398 mg of sodium fluoride is
equivalent to 1ml of thorium nitrate (0.005 mol/1) VS.
Thorium nitrate R. Th(N 03 ) 4 4 H 20 ,

.
Description. White, slightly deliquescent crystals.

Solubility. Very soluble in water and ethanol (-750 g/1) TS.
1427
Thymol blue R. Thymolsulfonphthalein, C27H30O5S (SKIP, 1963, p. 207).
Thymol blue/dimethylformamide TS
Procedure. Dissolve 0.3 g of thymol blue R in sufficient dimethylformamide R
to produce 100 ml.
Thymol blue/ethanol TS
Procedure. Dissolve 0.1 g of thymol blue R in sufficient ethanol (-750 g/1) TS to
produce 100 ml; filter if necessary.
Thymol blue/methanol TS
Procedure. Dissolve 0.3 g of thymol blue R in sufficient methanol R to produce
100 ml.
Thymol R. CiqHmO.
Description. Colourless, often large crystals, or a white, crystalline powder;
odour, aromatic, resembling that of thyme.
Solubility. Soluble in about 1000 parts of water, in 1 part of ethanol (-750 g/1)
TS, and in 1.5 parts of ether R.
Melting range. Between 48 and 51 °C; when the melted substance is cooled, it
remains liquid at a considerably lower temperature.

Residue on volatilization. Volatilize 2g on a water-bath and dry to constant
weight at 105 °C; it leaves a residue of not more than 0.5mg/g.
Storage. Store in tightly closed containers, protected from light.
Thymol TSl
Procedure. Dissolve 0.225 g of thymol R in sufficient carbon tetrachloride R to
produce 100 ml.
Thymol TS2
Procedure. Dilute 10 ml of thymol TSl to 100 ml with carbon tetrachloride R.
Thymol TS3
Procedure. Dilute 10ml of thymol TSl to 150ml with carbon tetrachloride R.
Thymolphthalein R. C28H30O4 (SRIP, 1963, p. 207).
Thymolphthalein/dimethylformamide TS
Procedure. Dissolve 0.1 g of thymolphthalein R in sufficient dimethylformamide
R to produce 100ml.
Thymolphthalein/ethanol TS
Procedure. Dissolve 0.1 g of thymolphthalein R in 100ml of ethanol (-750g/l)
TS, and filter if necessary.
Tiabendazole RS. International Chemical Reference Substance.
1428
Timolol maleate RS. International Chemical Reference Substance.
Titan yellow R. C2aHi9N5Na20,S4 (SKIP, 1963, p. 208).
Titan yellow TS
Procedure. Dissolve 0.05 g of titan yellow R in sufficient water to produce
100 ml.
Titanium dioxide R. TiOj.

Solubility. Practically insoluble in water; slowly soluble, when heated, in sulfu-
ric acid (~1760g/l) TS.
Titanium dioxide/sulfuric acid TS

Procedure. To 0.1 g of titanium dioxide R add 100ml of sulfuric acid (~1760g/l)
TS. Heat cautiously with occasional stirring until a clear solution is effected
and fumes are evolved; cool.
Storage. Store in glass-stoppered bottles.
Titanium trichloride (0.1 mol/1) VS

Procedure. Dilute 100ml of titanium trichloride R with 200 ml of hydrochloric
acid (-250 g/1) TS and add sufficient carbon-dioxide-free water R to produce
1000 ml.
Method of standardization. Ascertain the exact concentration immediately before
use. With the solution titrate 25ml of ferric ammonium sulfate (0.1 mol/1)
VS acidified with sulfuric acid (-100 g/1) TS in an atmosphere of carbon
dioxide R, adding ammonium thiocyanate (75g/l) TS just before the end-
point as indicator. Each ml of ferric ammonium sulfate (0.1 mol/1) VS is
equivalent to 15.43 mg of TiCh.
Titanium trichloride R. A solution of titanium trichloride containing about

15% of TiCls (SRIP, 1963, p. 208).
Mass density. p 2o
= -1.2 kg/1.
Tolbutamide RS. International Chemical Reference Substance.
Toluene R. CyHs (SRIP, 1963, p. 209).
Toluene-2-sulfonamide RS. International Chemical Reference Substance.
4-Toluenesulfonamide R. C 7 H 9NO 2 S.
4-Toluenesulfonic acid R. C 7 Hg 03 S,H 20 . Contains not less than 98.0% of

C7H8O3S.
1429
Solubility. Soluble in water, ethanol (~750g/l) TS and ether R.

Assay. Dissolve 0.8g, accurately weighed, in 50ml of water and titrate with
sodium hydroxide (0.1 mol/1) VS, using phenolphthalein/ethanol TS as indi-
cator. Each ml of sodium hydroxide (0.1 mol/1) VS is equivalent to 19.02 mg
of C7H803S,H20.
4 -Toluenesulfonic acid/ethanol TS
Procedure. Dissolve 20 g of 4-toluenesulfonic acid R in sufficient ethanol
Tosylchloramide sodium (15 g/1 ) TS. A solution of tosylchloramide sodium

R containing about 1 fig of CjH/ClNNaOjS per litre.
Tosylchloramide sodium R. C7H7CLNNa02S,3H20. Contains not less than

98.0% of C7H7ClNNa02S,3H20.
Description. White crystals or a white, crystalline powder; odour, resembling
that of chlorine.
Solubility.Soluble in 7 parts of water and in 2 parts of boiling water; soluble in
ethanol (-750 g/1) TS; insoluble in ether R.
Sodium chloride. R without the aid
Treat l.Og with 15ml of dehydrated ethanol
of heat, and filter; it more than 15mg.
leaves a residue of not
Assay. Dissolve 0.4g, accuratelyweighed, in 50ml of water, placed in a glass-
stoppered vessel. Add 10ml of potassium iodide (80 g/1) TS and 5 ml of sul-
furic acid (-lOOg/1) TS. Allow to stand for 10 minutes and titrate the
liberated iodine with sodium thiosulfate (0.1 mol/1) VS. Each ml of sodium
thiosulfate (0.1 mol/1) VS is equivalent to 14.08mg of C7H7ClNNa02S,3H20.
Note: Tosylychloramide sodium R is efflorescent.
Tributyl phosphate R. C12H27O4R

Miscibility. Slighdy miscible with water; miscible with most organic solvents.
Note: Before use, wash the reagent three times as follows: Shake fiOml with
10ml of a solution containing 1 g of sodium chloride R and 0.1 g of disodium
hydrogen phosphate R.
Trichloroacetic acid R. C2HCI3O2. (SRIP, 19fi3, p. 209).
Trichloroethylene R. C2HCI3.
Description. A colourless or pale blue, clear, mobile liquid; odour, characteristic,
resembling that of chloroform.
Miscibility. Almost immiscible with water; miscible with dehydrated ethanol R,
and ether R.
1430
Trichlorotrifluoroethane R. l,l,2-Trichloro-l,2,2-trifluoroethane. C 2 CI 3F 3 .
Description. A colourless, volatile liquid.

Miscibility. Immiscible with water; miscible with acetone R and ether R,
Trichlorotrifluoroethane TS
Procedure. Mix 0.05 |il of trichlorotrifluoroethane R with 1.0 ml of
dichlorome thane R (other suitable solvents can be used).
Triethylamine R. C^HisN.
Description. A colourless liquid; odour, ammoniacal.
Boiling range. 89-90 °C.
Refractive index, no = 1.4003.
Triethylenediamine R. l,4-Diazabicyclo[2.2.2]octane; C 6H 12 N 2 .
Description. Hygroscopic crystals.

Trihexyphenidyl hydrochloride RS. International Chemical Reference

Substance.
Triketohydrindene hydrate (1 g/1) TS. A solution of triketohydrindene

hydrate R containing about 1 g of C9H4O3 per litre.
Triketohydrindene hydrate R. Ninhydrin, C 9 H 403 ,H 20 (SRIP, 1963, p. 210).
Triketohydrindene/butanol TS
Procedure. Dissolve 0.1 g of triketohydrindene hydrate R in sufficient 1 -butanol
R previously saturated with water to produce 100 ml.
Triketohydrindene/butanol/acetic acid TS
Procedure. Prepare a 20 mg/ml solution of triketohydrindene hydrate R in a
mixture of 95 volumes of 1-butanol R and 5 volumes of acetic acid
(-120 g/1) TS.
Triketohydrindene/cadmium TS
Procedure. Dissolve 0.050 g of cadmium acetate R in a mixture of 5 ml of water
and 1 ml of glacial acetic acid R and add sufficient ethyhnethylketone R to
produce 50ml. Dissolve 20 mg of triketohydrindene hydrate R in 10ml of
this solution.
Note: Prepare immediately before use.
Triketohydrindene/ethanol TS
Procedure. Prepare a saturated solution of triketohydrindene hydrate R in
1431
Triketohydrindene/methanol TS
Procedure. Dissolve 1.0 g of triketohydrindene hydrate R in sufficient methanol
R to produce 100ml.
Note. Triketohydrindene/methanol TS must be freshly prepared.
Triketohydrindene/pyridine/acetone TS
Procedure. Dissolve 0.25g of triketohydrindene hydrate R in 100 ml of a mixture
of equal volumes of pyridine R and acetone R.
Triketohydrindene/pyridine/butanol TS
Procedure. Dissolve 1 g of triketohydrindene hydrate R in 1 ml of pyridine R and
dilute with sufficient 1 -butanol R to produce 100 ml.
Note: It should be freshly prepared.
Triketohydrindene/sodium metabisulfite TS
Procedure. Dissolve 3g of triketohydrindene hydrate R in 100ml of a solution
containing 4.55 g of sodium metabisulfite R in 100 ml of water.
Triketobydrindene/stannous chloride TS
Procedure. Dissolve 4g of triketohydrindene hydrate R in 100 ml of ethylene
glycolmonomethyl ether R. Shake gently with 1 g of cation exchange resin
(300pm - 840pm) and filter (solution A). Dissolve 0.16g of stannous chlo-
ride R in 100ml of acetate buffer, pH 5.5, TS (solution B). Immediately
before use, mix equal volumes of the two solutions.
Trimetbadione RS. International Chemical Reference Substance.
Trimethoprim RS. International Chemical Reference Substance.
2,2,4-Trimethylpentane R. /so-Octane; CsHis (SRIP, 1963, p. 129).
Trimethylpyridine (50g/l) TS. A solution of trimethylpyridine R containing

about 50 g of CgHijN per litre.
Trimethylpyridine R. CsHnN. 2,4,6-Trimethylpyridine; sym. collidine.

Description. Liquid; odour, aromatic.
Miscibility. More miscible with cold water than with hot water; miscible with
ethanol (-750 g/1) TS; ether R, and methanol R.
Relative density, df = 0.914.
Refractive index, n™ = 1.498.
Trinitrophenol (7 g/1) TS. A solution of trinitrophenol R containing 7 g of

C 6 H 3N 3 O 7 per litre.
Trinitrophenol R. C 6H 3N 3 O 7 (SRIP, 1963, p. 211).
1432
Trinitrophenol, alkaline, TS
Procedure. Mix 20ml of a lOmg/ml solution of trinitrophenol R with 10ml of
a 50 mg/ml solution of sodium hydroxide R, dilute with water to 100 ml,
and mix.
Note: After preparation alkaKne trinitrophenol TS should be used for only 48
hours.
Trinitrophenol/ethanol TS
Procedure. Dissolve33 g of trinitrophenol R in sufficient ethanol (-750 g/1) TS to
produce 1000ml.
Triphenylantimony R. CigHisSb.
About 55°C.
Melting temperature.
Trisodium orthophosphate ( 2 g/1 ) TS. A solution of trisodium orthophos-

phate R containing about 2g of Na3P04 per litre.
Trisodium orthophosphate R. Na3P04,12H20.

Solubility. Freely soluble in water; practically insoluble in ethanol (-750 g/1) TS
and carbon disulfide R.
Tropicamide RS. International Chemical Reference Substance.
Tyrosine R. Cc,HuN03 (SRIP, 1963, p. 212).
Uranyl acetate R. C4H60eU,2H20 (SRIP, 1963, p. 213).
Uranyl/zinc acetate TS
lOg of uranyl acetate
Procedure. Dissolve R by heating with 50ml of water and
5 ml of acetic acid (-300 g/1) TS; dissolve 30 g of zinc acetate R by heating
with 30 ml of water and 3 ml of acetic acid (-300 g/1) TS. Mix the two solu-
tions, allow to cool to room temperature, and remove by filtration any solid
material that separates.
Urea R. CH4N2O (SRIP, 1963, p. 214).
Uridine R. l-|3-D-Ribofuranosyluracil; C9H12N2O6.

Storage. Store in a cool place.
Valproic acid RS. International Chemical Reference Substance.
Vanadium pentoxide R. V2O5.

Description. A yellow-brown to rust-brown powder.
1433
Solubility. Slightly soluble in water; soluble in concentrated acids and alkalis;
practically insoluble in ethanol (~750g/l) TS.
Vanadium/sulfuric acid TS
Procedure. Dissolve 0.20 g of vanadium pentoxide R in 4 ml of sulfuric acid
(-1760 g/1) TS and dilute carefully with water to 100 ml.
Vanillin (lOg/1) TS. A solution of vanillin R containing about lOg of CgHsOj

per litre.
Vanillin R. CsHsOj (SRIP, 1963, p. 214).
Vanillin/hydrochloric acid TS
Procedure. Dissolve 1.0 g of vanillin R in sufficient hydrochloric acid (-250 g/1)
TS to produce 100 ml.
Note-. Vanillin/hydrochloric acid TS must be freshly prepared.
Vanillin/sulfuric acid TSl

Procedure. Dissolve 5g of vanillin R in 100ml of sulfuric acid (-1760 g/1) TS.
Note-. Vanillin/sulfuric acid TSl should be freshly prepared.
Vanillin/sulfiiric acid TS2

Procedure. Dissolve 1 g of vanillin R in sufficient ethanol (-750 g/1) TS to produce
100ml. Carefully add, drop by drop, 2ml of sulfuric acid (-1760 g/1) TS.
Note-. Vanillin/sulfuric acid TS2 must be used within 48 hours.
Verapamil hydrochloride RS. International Chemical Reference Substance.
Vinblastine sulfate RS. International Chemical Reference Substance.
Vincristine sulfate RS. International Chemical Reference Substance.
Warfarin RS. International Chemical Reference Substance.
Water R. Purified water as defined in the monograph for Purified water.

Note-. Unless otherwise specified, all solutions indicated in the tests and assays
of The International Pharmacopoeia are prepared with water R.
Water, ammonia-free, R. Water that complies with the following additional

test: to 50 ml add 2 ml of alkaline potassio-mercuric iodide TS; no colour is
produced.
Water, carbon-dioxide-free and ammonia-free, R. Ammonia-free water R

that has been treated as described under carbon-dioxide-free water R.
1434
Water, carbon-dioxide-free, R. Water that has been boiled vigorously for a

few minutes and protected from the atmosphere during cooling and storage.
Water for injections R. Water for injections as described in the monograph

for "Water for injections”.
Water BET. Water is suitable if it gives a negative result under the conditions
prescribed in 3.4 Test for bacterial endotoxins. It may be prepared by dis-
water three times in an apparatus fitted with an effective device to

tilling
prevent the entrainment of droplets, or by other means which give water

of the requisite quality.
Water, sterile, R. Sterile water R that complies with the following additional
test:
Pyrogens. Carry out the test as described under 3.5 Test for pyrogens injecting,
per kg of the rabbit’s weight, 10 ml of water that has been rendered isotonic
by the addition of pyrogen-free sodium chloride R.
Water vapour detector tube. A cylindrical, sealed glass tube containing

adsorbent filters and suitable supports for the magnesium perchlorate indi-
cator. The minimum value indicated is dOpl/l or less, with a relative stan-
dard deviation of at most ±20%.
Xanthydrol R. C13H10O2 (SRIP, 1963, p. 210).
Xanthydrol TS
Procedure. Dissolve 20 mg of xanthydrol R in 1ml of hydrochloric acid
(-420 g/1) TS and 99 ml of acetic acid (-300 g/1) TS.
Xylene R. CsHjo (SRIP, 1963, p. 215).
Xylenol orange indicator mixture R

Procedure. Mix 0.1 g of xylenol orange R with lOg of potassium nitrate R.
Xylenol orange R. [3/7-2, l-Benzoxathiol-3-ylidene bis[(6-hydroxy-5-methyl-

m-phenylene) methylenenitriloj] tetraacetic acid, S, S-dioxide,
C31H32N2O13S.
Description. An orange powder.
Yeast extract, water-soluble, R. (SRIP, 1963, p. 215).
Yellow stock standard TS

Procedure. To 9.5ml of cobalt colour TS, add 1.9ml of copper colour TS,
10.7 ml of dichromate colour TS, 4.0ml of iron colour TS, dilute to 100.0ml
with sulfuric acid (-lOg/1) TS, and mix.
1435
Zinc acetate R. C4H604Zn,2H20 (SKIP, 1963, p. 216).
Zinc AsR, granulated. Granulated zinc R that complies with the following
tests:
Limit of arsenic. Add 10 ml of stannated hydrochloric acid (-250 g/1) AsTS to

50ml of water, and apply the general test for arsenic; use lOg of granulated
zinc R and allow the reaction to continue for 1 hour; no visible stain is
produced.
Repeat the test for arsenic with the addition of 0.1 ml of dilute
Test for sensitivity.
arsenic AsTS; a faint, but distinct yellow-coloured stain is produced.
Zinc bis(dibenzyldithiocarbamate) R. Zn(C6HioNS2)2-

Solubility. Soluble in chloroform R.
Zinc bis(dibenzyldithiocarbamate) TS
Procedure. Dissolve 10.0 mg of zinc bis(dibenzyldithiocarbamate) R in sufficient
carbon tetrachloride R to produce 100 ml.
Zinc chloride R. ZnClj (SRIP, 1963, p. 217).
Zinc R. Zn (SRIP, 1963, p. 216); granulated, powder, or dust.
Zinc standard ( 20 |Xg/inl Zn) TS

Procedure. To 4.398g of zinc sulfate R add 1 ml of acetic acid (~300g/l) TS and
dilute with sufficient water to produce 1000 ml. Dilute 1ml of this solution
Zinc sulfate R. ZnS04, 7H2O. Contains not less than 99.0% and not more
than 105.0% of ZnS04,7H20.
Description. Colourless crystals or a white, crystalline powder; odourless;
efflorescent.
Solubility. Very soluble in water; practically insoluble in ethanol (~750g/l) TS.
Clarity and colour of solution. A 0.05 g/ml solution is clear and colourless.
30 ml of water, and proceed as described under 2.2.1 Limit test for chlorides;
not more than 0.35 mg/g.
Iron. Use 0.4 g; the solution complies with the 2.2.4 Limit test for iron; not more
than 0.10 mg/g.

Assay. Dissolve about 0.2 g, accurately weighed, in 5 ml of acetic acid (-60 g/1)
TS, and proceed with the titration as described under 2.5 Complexometric
titrations. Each ml of disodium edetate (0.05 mol/1) VS is equivalent to 14.38
mg of ZnS04,7H20.
Storage. Store at a temperature below 35 °C, in a tightly closed container.
1436
Zirconyl nitrate R. Contains not less than 43.5% and not more than 45.5%
of ZrOj.
Description. A white powder.
Solubility. Soluble in water giving a solution that is clear or not more than faintly
turbid.
Assay. Dissolve about 0.1 g, accurately weighed, in 5ml of sulfuric acid
(-1760 g/1) TS and add carefully 50ml of water. Add, with stirring, 5ml of
hydrogen peroxide (-330 g/1) TS and 350 ml of diammonium hydrogen
phosphate (lOOg/1) TS. Add 40ml of sulfuric acid (-1760 g/1) TS and keep
the mixture at a temperature of 40-50 °C for 2 hours. Filter and wash with
not more than 200 ml of cold ammonium nitrate (50 g/1) TS until the wash-
ings no longer give the reaction A for orthophosphates described under 2.1
General identification tests. Dry and ignite to constant weight. Each g of
residue is equivalent to 0.4647 g of ZrOj.
Zirconyl nitrate TS
Procedure. Dissolve 0.1 g of zirconyl nitrate R in a mixture of 60ml of
hydrochloric acid (-420g/l) TS and 40ml of water.
Zuclomifene RS. Clomifene citrate Z-isomer. International Chemical Refer-

ence Substance.
1437
Supplementary information
Supplementary Information: Contents

International Nonproprietary Names and graphic formulae 1441
List of International Chemical Reference Substances 1443
List of International Infrared Reference Spectra 1450
General guidelines for the establishment, maintenance and
distribution of chemical reference substances 1452
Recommendations on Risk of Transmitting Animal Spongiform
Encephalopathy Agents via Medicinal Products 1465
1440
Supiplementary information
International Nonproprietary Names (INN) and

graphic formulae
International Nonproprietary Names identify pharmaceutical substances or
active pharmaceutical ingredients. Each INN is a uniquename that is globally
recognized and is public property. A nonproprietary name is also known as a
generic name.
Guidance concerning the use and selection of INN and the graphic repre-
sentation of chemical formulae can be found in the following references:
1. Procedure for the selection of recommended international nonproprietary

names and General principles for guidance in
for pharmaceutical substances
devising international nonproprietarynames for pharmaceutical substances.
The adopted by the
text WHO
Executive Board in December 2004 is avail-
able on the WHO
Medicines website (http://www.who.int/medicines/)
under “International Nonproprietary Names: Revised Procedure (EB115/11
- 2004)”.
2. International nonproiarietary names (INN) for pharmaceutical substances. Cumula-

tive list No. H. Geneva, World Health Organization, 2004, available on
CD-ROM.
The INN system as it exists today was initiated in 1950 by a World Health
Assembly resolution WHA3.11. The procedure for the selection of recom-
mended international nonproprietary names for pharmaceutical substances,
and the general principles for selecting international nonproprietary names
for pharmaceutical substances, have been updated regularly since the INN
programme began in 1950.
3. Guidelines for the graphic representation of chemical formulae. In: WHO

Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-fourth
report. Geneva, World Health Organization, 1996, Annex 1 (WHO Techni-
cal Report Series, No. 863, pp. 16-49).
These unique guidelines are intended to help scientists to portray chemical

names and structures correctly and unambiguously in pharmacopoeias and
other compendia. For details of chemical nomenclature conventions, readers
are referred to the recommendations of the International Union of Pure and
Applied Ghemistry.'
'
InternationalUnion of Pure and Applied Chemistry, Organic Chemistry Division, Commission
on the Nomenclature of Organic Chemistry. Nomenclature of organic chemistry, sections, A, B, C, D,
and H, 4'êd. Oxford, Pergamon, 1979.
E, F,
Leigh GJ, ed. Nomenclature of inorganic chemistry: recommendations 1990. Oxford, Blackwell
Scientific, 1990.
1441
The guidelines should be followed as closely as possible, although it should
be noted that unwavering adherence to these principles is not always prac-

ticable. Therefore, they may be adapted, with certain exceptions, where
necessary to produce accurately drawn structural formulae. Details of the
formulae, such as bond lengths, the position of subscripts and superscripts,
and the closeness of apposition of the individual atomic symbols, will
depend on the drawing method used, whether computer-based or manual.
The guidelines cover acyclic, cyclic, and ionic structures, isotopically mod-
ified and coordination compounds, stereochemistry, carbohydrates,
steroids, terpenoids, prostanoids, alkaloids, antibiotics, polypeptides, and
polymers.
1442
List of International Chemical Reference

Substances^
International Chemical Reference Substances (ICRS) are established on the
advice of the WHO Expert Committee on Specifications for Pharmaceutical
Preparations. They are supplied primarily for use in physical and chemical tests
and assays described in the specifications for quality control of drugs published
in The International Pharmacopoeia or proposed in draft monographs. The ICRS
are mainly intended to be used as primary standards to calibrate secondary
standards.
Directions for use, and analytical data required for the use described in the
relevant specifications of The International Pharmacopoeia, are given in the cer-
tificates enclosed with the substances when distributed. More detailed analyt-
ical reports on the substances may be obtained from the WHO Collaborating
Centre for Chemical Reference Substances.
ICRS may also be used in tests and assays not described in The International
Pharmacopoeia. However, the responsibility for assessing the suitability of the
substances then rests with the user or with the pharmacopoeia commission or
other authority that has prescribed this use.
It is generally recommended that the substances should be stored protected
from light at a temperature of about 5°C. When
and moisture and preferably
special storage conditions are required, this is stated on the label or in the
accompanying leaflet. It is recommended that the user purchase only an amount

sufficient for immediate use.
The stability of the ICRS kept at the Collaborating Centre is monitored by
regular re-examination, and any material that has deteriorated is replaced by
new batches as necessary. Lists giving control numbers for the current batches
are issued in the annual reports from the Centre and new yearly lists may be
obtained on request.
Orders for the ICRS should be sent to:
WHO Collaborating Centre for Chemical Reference Substances

Apoteket AB
Produktion & Laboratorier Centrallaboratoriet, ACL
Prismavagen 2
S-141 75 Kungens Kurva
Sweden
(Fax: -1-46 8 740 6040; email: who.apl@apoteket.se)
^
As updated at the thirty-ninth meeting of the WHO Expert Committee on Specifications for
Pharmaceutical Preparations, 24-28 October 2005. Consult the WHOMedicines website
(http://www.who.int/medicines/) for the current list.
1443
The International Chemical Reference Substances (ICRS) are only supplied in

standard packages as indicated in the following list.
Catalogue Reference substances Package Control

number size number
9930375 p-Acetamidobenzalazine 25mg 290042

9930202 Acetazolamide 100 mg 186128
9930204 Allopurinol 100 mg 287049
9930206 Amidotrizoic acid 100 mg 196205
9930191 2-Amino-5-nitrothiazole 25mg 186131
9930194 3-Aminopyrazole-4-carboxamide 100 mg 172050
hemisulfate
9930193 3-Amino-2,4,6-triiodobenzoic acid 100 mg 196206
9930208 Amitriptyline hydrochloride 100 mg 181101
9930209 Amodiaquine hydrochloride 200 mg 192160
9930210 Amphotericin B 400 mg 191153
9930211 Ampicillin (anhydrous) 200 mg 390001
9930212 Ampicillin sodium 200 mg 388002
9930213 Ampicillin trihydrate 200 mg 274003
9930214 Anhydrotetracycline hydrochloride 25 mg 180096
9931408 Artemether 100 mg 103225
9931406 Artemisinin 100 mg 103222
9931407 Artemotil lOOmg 103226
9931410 Artenimol 100 mg 103223
9931409 Artesunate 100 mg 103224
9930215 Atropine sulfate 100 mg 183111
9930216 Azathioprine 100 mg 172060
9930218 Bacitracin zinc 200 mg 192174

9930219 Beclometasone dipropionate 200 mg 192175
9930225 Benzylpenicillin potassium 200 mg 180099
9930226 sodium
Benzylpenicillin 200 mg 280047
9930227 Bephenium hydroxynaphthoate 100 mg 183112
9930228 Betamethasone 100 mg 183113
9930229 Betamethasone sodium phosphate 100 mg 196203
9930230 Betamethasone valerate 100 mg 190145
9930233 Bupivacaine hydrochloride 100 mg 289054
9930234 Caffeine 100 mg 181102

9930236 Calcium folinate (Leucovorin calcium) 100 mg 194188
9930237 Captopril 100 mg 197214
9930238 Captopril disulfide 25mg 198216
9930239 Carbamazepine 100 mg 189143
9930240 Carbenicillin monosodium 200 mg 383043
9930241 Chloramphenicol 200 mg 486004
1444

number size number
9930242 Chloramphenicol palmitate ig 286072

9930243 Chloramphenicol palmitate (Polymorph A) 200 mg 175073
9930199 5-Chloro-2-methylaminobenzophenone 100 mg 172061
9930245 Chloroquine sulfate 200 mg 195201
9930190 2-(4-Chloro-3-sul£amoylbenzoyl)benzoic 50 mg 181106
add
9930246 Chlorphenamine hydrogen maleate 100 mg 182109
9930247 Chlorpromazine hydrochloride 100 mg 178080
9930248 Chlortalidone 100 mg 183114
9930249 Chlortetracycline hydrochloride 200 mg 187138
9930250 Cimetidine 100 mg 190150
9930256 Ciprofloxacin hydrochloride 400 mg 197210
9930252 Ciprofloxacin by-compound A 20 mg 198220
9930253 Ciprofloxacin desfluoro-compound 20 mg 198219
9930254 Ciprofloxacin ethylenediamine-compound 20 mg 198218
9930255 Ciprofloxacin fluoroquinolonic acid 20 mg 198217
9930258 Cisplatin 100 mg 197207
9930259 Clomifene citrate 100 mg 187136
Clomifene citrate Z-isomer see
Zuclomifene
9930261 Cloxacillin sodium 200 mg 274005
9930262 Colecalciferol (Vitamin D3) 500 mg 190146
9930263 Cortisone acetate 100 mg 167006
9930265 Dapsone 100 mg 183115

9930266 Desoxycortone acetate 100 mg 167007
9930267 Dexamethasone 100 mg 388008
9930268 Dexamethasone acetate 100 mg 288009
9930269 Dexamethasone phosphoric acid 100 mg 192161
9930270 Dexamethasone sodium phosphate 100 mg 192158
9930282 Diazoxide 100 mg 181103
9930283 DicloxaciUin sodium 200 mg 174071
9930285 Dicoumarol 100 mg 178077
9931413 Didanosine lOmg 104228
9931414 Didanosine for System Suitability lOmg 104230
9930287 Diethylcarbamazine dihydrogen citrate 100 mg 181100
9930288 Digitoxin 100 mg 277010
9930289 Digoxin 100 mg 587011
9930290 Dopamine hydrochloride 100 mg 192159
9930292 Doxorubicin hydrochloride 100 mg 196202
9931411 Efavirenz 100 mg 104229

9930294 Emetine hydrochloride 100 mg 187134
1445

number size number
9930197 4-Epianhydrotetracycline hydrochloride 25 mg 288097

9930198 4-Epitetracycline hydrochloride 25 mg 293098
9930295 Ergocalciferol (Vitamin D2) 500 mg 190147
9930296 Ergometrine hydrogen maleate 50 mg 277012
9930297 Ergotamine tartrate 50mg 385013
9930298 Erythromycin 250 mg 191154
9930299 Erythromycin B 150 mg 194186
9930300 Erythromycin C 25mg 194187
9930301 Estradiol benzoate 100 mg 167014
9930302 Estrone 100 mg 279015
9930304 Ethambutol hydrochloride 100 mg 179081
9930305 Ethinylestradiol 100 mg 301016
9930306 Ethisterone 100 mg 167017
9930307 Ethosuximide 100 mg 179088
9930309 Flucloxacillin sodium 200 mg 195194

9930310 Flucytosine 100 mg 184121
9930311 Fludrocortisone acetate 200 mg 195199
9930312 Fluorouracil 100 mg 184122
9930313 Fluphenazine decanoate dihydrochloride 100 mg 182107
9930314 Fluphenazine enantate dihydrochloride 100 mg 182108
9930315 Fluphenazine hydrochloride 100 mg 176076
9930316 Folic acid lOOmg 388019
9930195 3-Formylrifamycin 200 mg 202149
9930355 Framycetin sulfate (Neomycin B sulfate) 200 mg 193178
9930318 Furosemide 100 mg 171044
9930319 Gentamicin sulfate 100 mg 194183

9930322 Griseofulvin 200 mg 280040
9930323 Haloperidol 100 mg 172063

9930324 Hydrochlorothiazide 100 mg 179087
9930325 Hydrocortisone 100 mg 283020
9930326 Hydrocortisone acetate 100 mg 280021
9930327 Hydrocortisone sodium succinate 200 mg 194184
9930188 (-)-3-(4-Hydroxy-3-methoxyphenyl)-2-
hydrazino-2-methylalanine
(3-o-Methylcarbidopa) 25 mg 193180
9930189 (-)-3-(4-Hydroxy-3-methoxyphenyl)-
2 -me thylalanine
(3-o-Methylmethyldopa) 25 mg 179085
1446
Sup’pkmentary information

number size number
9930328 Ibuprofen 100 mg 183117

9930329 Imipramine hydrochloride 100 mg 172064
9930330 Indometacin 100 mg 178078
9930331 Isoniazid 100 mg 185124
9930332 Kanamycin monosulfate 12mg 197211
9930333 Lanatoside C 100 mg 281022

9930334 Levodopa 100 mg 295065
9930335 Levonorgestrel 200 mg 194182
9930336 Levothyroxine sodium 100 mg 189144
9930337 Lidocaine 100 mg 181104
9930338 Lidocaine hydrochloride 100 mg 181105
9930339 Liothyronine sodium 50 mg 193179
9930340 Loperamide hydrochloride 100 mg 194185
9930341 Mebendazole 200 mg 195195
Melting Point Reference Substances

9930217 Azobenzene (69 °C) Ig 192168
9930438 Vanillin (83 °C) Ig 299169
9930222 Benzil (96°C) 4g 294170
9930201 Acetanilide (116°C) Ig 297171
9930380 Phenacetin (136 °C) Ig 297172
9930221 BenzaniKde (165 °C) Ig 192173
9930422 Sulfanilamide (166°C) Ig 192162
9930423 Sulfapyridine (193 °C) 4g 192163
9930286 Dicyanodiamide (210°C) Ig 192164
9930411 Saccharin (229 °C) Ig 192165
9930235 Caffeine (237 °C) Ig 299166
9930382 Phenolphthalein (263 °C) Ig 299167
9930345 Methotrexate 100 mg 194193
3-o-Methylcarbidopa see (-)-3-(4-
Hydroxy-3-methoxyphenyl)-2-
hydrazino-2-methylalanine 3-o-
Methylmethyldopa see (-)-3-(4-
Hydroxy-3-methoxyphenyl)-2-
methylalanine
9930346 Methyldopa 100 mg 179084
9930347 Methyltestosterone 100 mg 167023
9930348 Meticillin sodium 200 mg 274024
9930350 Metronidazole lOOmg 183118
1447

number size number
9930351 sodium
Nafcillin 200 mg 272025
9930354 Neamine hydrochloride
(Neomycin A hydrochloride) 0.5 mg 193177
Neomycin B sulphate see Framycetin
sulfate
9930356 Neostigmine metilsulfate 100 mg 187135
9931412 Nevirapine 100 mg 104227
9930357 Nicotinamide 100 mg 200090
9930358 Nicotinic acid 100 mg 179091
9930359 Nifurtimox lOOmg 194189
9930360 Niridazole 200 mg 186129
9930361 Niridazole-chlorethylcarboxamide 25 mg 186130
9930366 Norethisterone 100 mg 186132
9930367 Norethisterone acetate 100 mg 185123
9930369 Nystatin 200 mg 300152
9930371 Ouabain 100 mg 283026

9930372 Oxacillin sodium 200 mg 382027
9930373 Oxytetracycline dihydrate 200 mg 189142
9930374 Oxytetracycline hydrochloride 200 mg 189141
9930376 Papaverine hydrochloride 100 mg 185127

9930377 Paracetamol 100 mg 195198
9930378 Paromomycin sulfate 75mg 195197
9930383 Phenoxymethylpenicillin 200 mg 179082
9930384 Phenoxymethylpenicillin calcium 200 mg 179083
9930385 Phenoxymethylpenicillin potassium 200 mg 176075
9930387 Phenytoin 100 mg 179089
9930388 Piperazine adipate 100 mg 197212
9930389 Piperazine citrate 100 mg 197213
9930390 Praziquantel 100 mg 194191
9930391 Prednisolone 100 mg 389029
9930392 Prednisolone acetate 100 mg 289030
9930393 Prednisolone hemisuccinate 200 mg 195196
9930394 Prednisolone sodium phosphate 200 mg 194190
9930395 Prednisone 100 mg 167031
9930396 Prednisone acetate 100 mg 169032
9930397 Probenecid 100 mg 192156
9930398 Procaine hydrochloride 100 mg 183119
9930399 Procarbazine hydrochloride 100 mg 184120
9930400 Progesterone 100 mg 167033
9930402 Propranolol hydrochloride 100 mg 187139
9930403 Propylthiouracil 100 mg 185126
1448

number size number
9930404 Pyrantel embonate (Pyrantel pamoate) 500 mg 192157

9930405 Pyridostigmine bromide 100 mg 182110
9930406 Reserpine 100 mg 186133

9930407 Retinol acetate (solution) 5 caps (*) 898038
9930408 Riboflavin 250 mg 382035
9930409 Rifampicin 300 mg 191151
9930410 Rifampicin quinone 200 mg 202148
9930412 Sodium amidotrizoate 100 mg 198221

9930413 Sodium cromoglicate 100 mg 188140
9930415 Spectinomycin hydrochloride 200 mg 193176
9930416 Streptomycin sulfate 100 mg 197215
9930417 Sulfacetamide 100 mg 196200
9930419 Sulfamethoxazole 100 mg 179092
9930420 Sulfamethoxypyridazine 100 mg 178079
9930421 Sulfanilamide 100 mg 179094
9930424 Sulfasalazine 100 mg 191155
9930425 Tamoxifen citrate 100 mg 196208

9930426 Tamoxifen citrate £-isomer lOmg 196209
9930427 Testosterone enantate 200 mg 194192
9930428 Testosterone propionate 100 mg 167036
9930429 Tetracycline hydrochloride 200 mg 180095
9930430 Thioacetazone 100 mg 171046
9930196 4,4' - Thiodianiline 50mg 183116
Thyroxine sodium see Levothyroxine
sodium
9930431 Tolbutamide 100 mg 179086
9930432 Tolnaftate 100 mg 176074
9930433 Toluene-2-sulfonamide 100 mg 196204
9930434 Trimethadione 200 mg 185125
9930435 Trimethoprim 100 mg 179093
9930440 Vincristine sulfate 9.7mg/vial 193181

Vitamin A acetate (solution) see Retinol
acetate (solution)
9930439 Warfarin 100 mg 168041
9930260 Zuclomifene 50mg 187137
(*) About 8 mg in 230 mg oil per capsule
1449
List of International Infrared Reference Spectra^

International Infrared Reference Spectra are established on the advice of the
WHO Expert Committee on Specifications for Pharmaceutical Preparations.
Full-scale reproductions of spectra produced from authenticated material on a
suitable instrument are supplied for use in identification tests described in the
specifications for quality control of drugs, published in The International Phar-
macopoeia or proposed in draft monographs.
Precise instructions for the preparation of spectra are given on the label of
each reference spectrum. All International Infrared Reference Spectra are dis-
tributed together with a document giving further details on the use of such
spectra, entitled “General recommendations for the preparation and use of
infrared spectra in pharmaceutical analysis”.^
Orders for International Infrared Reference Spectra should be sent to:
WHO Collaborating Centre for Chemical Reference Substances

Apoteket AB
Produktion & Laboratorier Centrallaboratoriet, ACL
Prismavagen 2
S-141 75 Kungens Kurva
Sweden
(Fax: -1-46 8 740 6040; emailwho.apl@apoteket.se)
The following International Infrared Reference Spectra are currently avail-

able from the Centre:
aceclidine salicylate caffeine (anhydrous)

acetazolamide calcium folinate
allopurinol carbidopa
amiloride hydrochloride chlorphenamine hydrogen maleate
amitriptyline hydrochloride clofazimine
ampicillin trihydrate cloxacillin sodium
colchicine
beclometasone dipropionate cytarabine
benzylpenicillin potassium
biperiden dexamethasone
biperiden hydrochloride dexamethasone acetate,
bupivacaine hydrochloride mono-hydrate
’
As updated at the thirty-ninth meeting of the WHO Expert Committee on Specifications for
Pharmaceutical Preparations, 24-28 October 2005. Consult the WHO Medicines website
(http://www.who.int/medicines/) for the current list.
^
WHO Exfpert Committee on Specifications for Pharmaceutical Preparations. Thirty-fourth report. Geneva,
World Health Organization, 1996, Annex 4 (WHO Technical Report Series, No. 863).
1450
dextromethorphan hydrobromide niclosamide

diazepam nicotinamide
dicolinium iodide noscapine
dicoumarol
diethylcarbamazine dihydrogen oxamniquine
citrate
diphenoxylate hydrochloride papaverine hydrochloride
phenobarbital
erythromycin ethylsuccinate phenoxymethylpenicillin calcium
erythromycin stearate phenytoin
etacrynic acid primaquine phosphate
ethionamide propylthiouracil
ethosuximide protionamide
pyrimethamine
furosemide
salbutamol
gallamine triethiodide salbutamol sulfate
glibenclamide sulfadimidine
sulfadoxine
haloperidol sulfamethoxazole
hydrochlorothiazide sulfamethoxypyridazine
ibuprofen tiabendazole
imipramine hydrochloride trihexyphenidyl hydrochloride
indometacin trimethoprim
isoniazid
valproic acid
lidocaine verapamil hydrochloride
lidocaine hydrochloride
lindane
metronidazole
miconazole nitrate
1451
General guidelines for the establishment,

maintenance, and distribution of chemical
reference substances
Introduction
In 1975, the WHO
Expert Committee on Specifications for Pharmaceutical
Preparations recommended “General guidelines for the establishment, mainte-
nance and distribution of chemical reference substances” (-/).' At that time these
general guidelines were aimed at fostering greater collaboration and harmo-
nization among various national and regional authorities responsible for col-
lections of chemical reference substances. This aim is still relevant. The
guidelines initially drawn up for particular use by the
were Collaborat- WHO
ing Centre for Chemical Reference Substances in Sweden, which provides
InternationalChemical Reference Substances (ICRS). These substances are pri-
marily intended for use with pharmacopoeial monographs included in The Inter-
national Pharmacopoeia (2).
It became evident that in order to meet particular national or regional phar-

macopoeial requirements, it was necessary to establish chemical reference sub-
stances external to the WHO Collaborating Centre for Chemical Reference
Substances. Another difficulty was to ensure prompt dispatch of the substances.
Since the meticulous work of the WHO Collaborating Centre establishing the
international collection would have to be duplicated in local or regional labo-
ratories, guidelines were necessary to ensure the integrity of national or regional
collections. In order to clarify the need for national and regional collections, the
1975 guidelines were reviewed and modified in 1982 (3). In view of refinements
in pharmaceutical and analytical methods since then, the present revision was
considered essential.
The purpose of having chemical reference substances is to achieve accuracy
and reproducibility of the analytical results required by pharmacopoeial testing
and pharmaceutical control in general. These substances are normally prepared
and issued by the regional/national pharmacopoeial commission or the
regional/national quality control laboratory on behalf of the drug regulatory
authority. In the context of these guidelines, the general use of a chemical ref-
erence substance should be considered an integral part of a compliance-oriented
monograph or test procedure used to demonstrate the identity, purity and
content of pharmaceutical substances and preparations.
The establishment of chemical reference substances should be based on
reports in which the results of analytical testing have been evaluated. These
reports should subsequently be approved and adopted by a certifying body,
normally the relevant pharmacopoeial committee or the drug regulatory
^
The term chemical reference substances, as used in this text, refers to an authenticated uniform mate-
rial that is intended for use in specified chemical and physical tests, in which its properties are
compared with the properties of a product under examination, and which possesses a degree of
purity adequate for its intended use.
1452
authority. Such establishment can be on an international, national or regional

basis. Each substance is generally established for a specific analytical purpose,
defined by the issuing body. Its use for any other purpose becomes the respon-
sibility of the user and a suitable caution is included in the information sheet
accompanying a reference substance. The present guidelines are concerned

with both primary and secondary chemical reference substances as defined
below.
The preparation of a chemical reference substance should comply with the
requirements for quality assurance systems, including principles of good man-
ufacturing practices (GMP) and good control laboratory practices {4-6).
Adequate training programmes are also required. Both the WHO Collabo-
rating Centre and other laboratories concerned with the evaluation and estab-
lishment of chemical reference substances give assistance in training, subject to
the availability of resources.
Primary chemical reference substance

A designated primary chemical reference substance is one that is widely
acknowledged to have the appropriate qualities within a specified context, and
whose value is accepted without requiring comparison to another chemical
substance.
Secondary chemical reference substance

A secondary chemical reference substance is a substance whose characteristics
are assigned and/or calibrated by comparison with a primary chemical refer-
ence substance. The extent of characterization and testing of a secondary chem-
ical reference substance may be less than for a primary chemical reference
substance. This definition may apply inter alia to some substances termed
“working standards”.
Part A. Primary chemical reference substances

1. Assessment of need for the establishment
of chemical reference substances
The production, validation, maintenance and distribution of chemical reference
substances is a costly and time-consuming undertaking. It is, therefore, of great
importance to determine critically whether a need for a given substance exists.
Requests for new chemical reference substances usually arise when a particu-
lar approach to developing a specification for a new substance or product has
been adopted. Methods may have been proposed in a specification that require
the establishment of a chemical reference substance for use as a comparative
standard. Therefore, the first matter that should be assessed is whether an alter-
native, equally satisfactory, procedure could be adopted that does not require
a comparative standard.
Analytical procedures currently used in specifications for pharmaceutical
substances and products that may require a chemical reference substance are:
1453
(a) infrared (IR) spectrophotometry, whether for identification or quantitative

purposes;
(b) quantitative methods based on ultraviolet (UV) absorption spectro-
photometry;
(c) quantitativemethods based on the development of a colour and the mea-
surement of its intensity, whether by instrumental or visual comparison;
(d) methods based on chromatographic separation for identification or quanti-
tative purposes;
(e) quantitative methods (including automated methods) based on other
separation techniques that depend on partition of the substance to be
determined between solvent phases, where the precise efficiency of the
extraction procedure might depend upon ambient conditions that vary from
time to time and from laboratory to laboratory;
(f) quantitative methods, often titrimetric but sometimes gravimetric, that are
based on non-stoichiometric relationships;
(g) assay methods based on measurement of optical rotation; and
(h) methods that might require a chemical reference substance consisting of a
fixed ratio of known components (for example, cis/trans isomers, spiked
samples).
2. Obtaining source material

Source material of satisfactory quality can be selected from a batch (lot) of the
substance originating from the normal production process, if the purity is
acceptable. Further purification techniques may be needed to render the mate-

rial acceptable for use as a chemical reference substance.
The purity requirements for a chemical reference substance depend upon
its intended use. A chemical reference substance proposed for an identification
testdoes not require meticulous purification, since the presence of a small per-
centage of impurities in the substance often has no noticeable effect on the test.
On the other hand, chemical reference substances that are to be used in
assays should possess a high degree of purity. As a guiding principle, a purity
of 99.5% or higher is desirable, calculated on the basis of the material in its
anhydrous form or free of volatile substances. However, where the selectivity
of the analytical procedure for which the chemical reference substance is
required is low, such a degree of purity may not be making a deci-

necessary. In
sion about the suitability of a chemical reference substance, the most impor-
tant consideration is the influence of the impurity on the attribute measured in
the assay when used in a non-specific assay procedure. Impurities with physic-
ochemical characteristics similar to those of the main component will not
impair the usefulness of a chemical reference substance, whereas even traces
of impurities with significantly different properties may render a substance
unsuitable as a chemical reference substance.
When source material to be used as a chemical reference substance is
obtained from a supplier, the following should be supplied with the material:
1454
• Certificate with complete information as to test methods

of analysis
employed, values found and number of replicates used, where applicable,
and relevant spectra and/or chromatograms,
• Information on optimal storage conditions required for stability (tempera-

ture and humidity considerations).
• Results of any hygroscopicity study and/or statement of the hygroscopicity

of the source material.
• Results of any accelerated stability studies.
• Identification of detected impurities (by preference), and/or specific infor-

mation on the relative response factor as determined in compendial
methods concerning the principal component, and/or the percentage mass
of the impurity.
• Updated Material Safety Data Sheet outlining any health hazards associated
with the material.
For new drug substances, manufacturers should be aware that elaboration

of pharmacopoeial monographs will be needed and a batch of the new sub-
stance should be set aside to be used if necessary as the chemical reference sub-
stance. It is desirable for bodies that issue chemical reference substances to
provide each other with a sample of the same batch of material, even if the
substance will be employed for different test methods. This will require the
exchange of information concerning the establishment process, supplier(s),
availability and conditions of supply.
3. Evaluation of chemical reference substances

The suitability of a substance proposed for use as a chemical reference requires
careful evaluation by the issuing body. It is necessary to consider all data
obtained from testing the material by a wide variety of analytical methods.
When taken as a whole, this will ensure that the substance is suitable for its
intended use. The extent of the analyses required depends on the purpose(s)
for which the chemical reference substance is to be employed, and may involve
a number of independent laboratories.
3.1 Use in identification tests
For use in identification tests (infrared spectrophotometry and/or chromato-

graphic methods), a batch of good quality material selected from the normal
production process is satisfactory if it is of acceptable purity. Additional purifi-
cationby the supplier may be necessary. The most important check is the appli-
which the substance is intended. It is usual for at least
cation of the test(s) for
one laboratory to apply all the tests described in the relevant monograph.
1455
3.Z Use in purity tests
The characterization of a chemical reference substance used in the determina-

tion of a specific impurity is more extensive, especially when used in a limit
test. If the technique employed is thin-layer chromatography (TLC) an accept-

able minimum purity is recommended (normally at least 90%), but purer mate-
rial may be required for liquid chromatography (LC) or gas chromatography
(GC). If the proposed reference substance is being prepared or isolated for the
first time, appropriate chemical and physicochemical tests, such as nuclear mag-
netic resonance (NMR), mass spectrometry (MS) and elemental analysis, must
be applied to characterize it.
3.3 Use in assays
If the chemical reference substance is to be used in an assay (colorimetry,

LC, GC or UV spectrophotometry), the extent of testing is very much greater.
Several (a minimum of three) laboratories should collaborate in testing the pro-

posed substance, using a variety of established and validated techniques,
including the method used in the pharmacopoeial specification. The relative
reactivity or relative absorbance of the impurities present must be checked
when a non-specific assay method is employed, e.g. by colorimetry or UV spec-
trophotometry. When a selective assay method is employed, it is particularly
important to determine the quantity of impurities. In such a case, it is best to
examine the proposed reference substance by as many methods as practicable
including, where possible, absolute methods. For substances that are acidic or
basic a titration with alkali or acid is simple, but other reactions which are
known to be stoichiometric may be used. Phase solubility analysis and differ-
ential scanning calorimetry may also be employed in certain cases.
The total of the determinations of water content, organic solvents, mineral
impurities and organic components should amount to 100%. For most chemi-
cal reference substances intended for assays, the content may be expressed “as
is”. When establishing the chemical reference substance it is, therefore, essen-
tial to determine the content of water and residual solvents for a non-specific
assay, and also to determine the content of impurities for a selective assay.
3.4 Use in the calibration of an instrument
Where the chemical reference substance is to be employed as calibration mate-

the extent of testing is similar to that for a chemical reference substance
rial,
used in assays. Several laboratories should collaborate in testing the proposed

substance using a variety of techniques to check that its purity is adequate. An
appropriate number of collaborating laboratories should also participate, after
the reference substance has been deemed suitable, to establish a value for the
essential property of the substance using an appropriate instrument.
1456
4. Chemical and physical methods used in evaluating

chemical reference substances
important to establish by individual
It is testing that a substance proposed for
use as a chemical reference is suitable.
The methods used to establish the suitability of such a substance fall into
two broad groups: those intended primarily to identify the substance and those
used to establish its purity. With most methods, the percentage purity of a
chemical reference substance cannot be expressed as an absolute value if the
The quoted purity is then an estimate based
impurities have not been identified.
upon the data obtained by the various analytical methods.
4. ^ Methods used to verify the identity of chemical reference substances

Where proposed substance consists of a compound whose structure has been
a
satisfactorily defined,its identity may be confirmed by matching the IR spectra
of the substance to that of an authentic compound. Particular care should be

taken when polymorphism exists (7). Other highly specific techniques, such as
NMR spectroscopy, MS, or X-ray diffraction crystallography, may also be used
for such comparisons. The identity of a substance that is intended to replace
an established chemical reference substance of the same molecular constitution
must be verified, to determine that the characteristic properties of the two spec-
imens are identical. For this purpose it is often sufficient to compare their IR
absorption spectra.
However, where no authentic specimen of the proposed substance is avail-
able for comparison,and definitive data about its properties are lacking, it may
its identity by applying several analytical techniques cur-
be necessary to verify
rently used to characterize new compounds. Such analytical methods may
include elemental analyses, crystallographic studies, MS, NMR spectroscopy,
functional group analyses, and IR or UV spectrophotometry, as well as other
supplementary tests as required to establish that the proposed substance is fully
characterized.
4.-2 Methods used to determine the purity of chemical reference substances
The analytical methods to be employed in examining a substance should be

considered in relation to its intended use. These analytical methods may be
divided into three broad categories: those that require comparison with an
external chemical reference substance chromatographic or spectrophoto-
(e.g.
metric methods), those that depend solely on an intrinsic dynamic property

(e.g. phase solubility analysis and differential scanning calorimetry) and other
methods.
4 2.1 Separation technicjues

.
The methods used for the determination of purity should be established and
validated with system suitability requirements as appropriate.
1457
Chromatographic methods. Methods of

analysis based on chromatographic sepa-
and determining impurities in chemi-
ration are especially useful for detecting
High-performance liquid chromatography (HPLC) is
cal reference substances.
the most widely used chromatographic method, butTLC and GC are also used.
The individual components separated by chromatographic methods may some-
times be recovered for characterization.
The selectivity of HPLC and of GC usually exceeds that of TLC. Both the
firsttwo methods also have the advantage of being readily applicable on a
more complex equipment HPLC, employ-
quantitative basis, but they require
ing a spectrophotometric method of detection, is of particular value in the
examination of chemical reference substances intended for use in UV spec-
trophotometric assays. The UV wavelength of detection employed for deter-
mining the impurity content of the chemical reference substance should be
chosen so that the detection responses of the substance and its known impu-
rities are similar. When the response factors are significantly different at the
optimal wavelength of detection, appropriate corrections must be made to esti-
mate the content of impurities. LC with diode-array detection is very useful for
recording the UV spectra of both the main peak and the impurities. LC with
MS detection is used for identification of separated impurities as well as for
the main component, and is particularly important for chemical reference
substances where no other reference standards or IR reference spectra are

available.
In a GC method used for an assay, as with LC, the detection responses of
the known impurities are determined. Generally, GC monograph methods are
of particular value in detecting and determining volatile impurities, including
solvent residues, in chemical reference substances.
TLC uses apparatus that is simple and cheap; the technique is easy to carry
out and is even in the microgram range. It can separate closely
readily applicable
related compounds, such and the members of a homolo-
as geometric isomers
gous substance submitted to chromatography
series. All the constituents of a
appear somewhere on the chromatogram. However, some constituents may

remain on the starting line, some may move with the solvent front, some may
migrate at the same rate as the main component, and some may remain unde-
tected. For this reason, the usefulness of the method may be greatly enhanced
by means of two-dimensional chromatography and by using a number of dif-
ferent solvent systems and a variety of detection methods. In some cases the
method may be used quantitatively with acceptable accuracy by using a
densitometer.
Capillary electrophoresis. Capillary electrophoresis is an increasingly common
method. It may be considered as complementary to LC for detecting

impurities.
4.2.2 Methods based on intrinsic thermodynamic properties

Methods in this group measure total impurity levels in absolute terms.
1458
Differential scanning calorimetry. This technique is used to check the presence of

different polymorphic forms and to determine the total amount of solid impu-
rities. Purity estimation is based on determination of the heat of fusion of the
sample and of the change in its melting point caused by the presence of impu-
rities. This analytical method can be performed rapidly and with high preci-
sion. However, it is not applicable if the substance decomposes on melting.

This limits its value as a general procedure for purity estimation of chemical
reference substances. It is also inapplicable if solid solutions are formed.
Phase solubility analysis. The method has occasionally been used, but its value
is limited and the procedure is time consuming. It may be employed to detect
contaminating substances, including isomeric species, and to estimate their con-
centration. Some factors that may make the method inapplicable are degrada-
tion of the substance during the course of analysis, formation of a solid solution,
and polymorphism in the main component.
4.2.3 Other methods

Spearophotometric methods. UV spectrophotometry is occasionally used to deter-
mine purity. Since it depends upon the presence of a characteristic chro-
mophore, it can detect impurities that contribute excessively to the absorbance
value and may indicate the presence of impurities that have a negligible or
distinctive absorbance.
However, the utility of the method is by the small number of absorp-
limited
tion maxima in the UV range, the large numbers of compounds containing
similar characteristic chromophores, and the need for an external chemical
reference substance.
IR spectrophotometry may be used to identify and determine the propor-
tions of geometric isomers. NMR spectroscopy, a powerful spectroscopic iden-
tification tool, is also occasionally useful in the determination of purity.
Titrimetric methods. Titrimetric methods provide a valuable means of confirming

the identity and purity of a proposed chemical reference substance and are
useful in confirming purity values obtained by other methods.
Optical rotation methods. Many chemical reference substances are optically active
and the relative proportion of optical isomers can sometimes be determined by
an optical rotation method, but generally such methods lack sensitivity.
However, the quantitative use of these techniques is well established and can
yield results of high precision, depending on the solvent and the wavelength
chosen for measurement. Chiral chromatography and NMR are becoming
increasingly important.
Determination of water and organic volatiles. It is essential that an accurate assess-

ment of the moisture content and the content of volatile contaminants be made.
These total values may often be obtained by drying under defined conditions
that are appropriate to the proposed substance. Sometimes this may not be
1459
possible or may yield misleading results. In such cases, thermogravimetric

analysis may be used to determine the water and volatile content. Alternatively,
the water content may be determined by Karl Fischer titration and the content
of volatile solvents by GC. Without an accurate assessment of these values at
the time that other determinations are being made, judgements of the accept-
ability of the proposed chemical reference substance will be invalid.
5. Assignment of content
If a content is to be assigned to a chemical reference substance, it should be
borne in mind that the value is based on the results of a collaborative inter-
laboratory programme using different analytical methods. This experimentally
obtained value represents the best estimate of the true value. In general, the
assignment of content for a chemical reference substance is 100% minus the
content of water and volatiles, and when a substance is intended for use as an
assay standard based on a separation technique the impurity content, as deter-
mined by that method, must also be subtracted. Sometimes the chemical ref-
erence substances must be dried before use, in which case the content is
expressed on the basis of the dried material.
6. Handling and distribution of chemical reference substances

The handling, distribution and use of established chemical reference substances
must ensure that their integrity is safeguarded and maintained throughout their
period of use.
6. 1 Packaging operations
Current GMP requirements (5) should be observed. The various stages in pack-
aging chemical reference substances should be clearly defined and controlled,
to avoidcontamination of the sample, mislabelling of containers, or any other
event which might result in mishandling or mismanagement.
Containers for chemical reference substances should protect their contents
from moisture, light and oxygen and must be tested for moisture permeability.
Additional measures may be necessary to ensure long-term integrity and
stability. The best containers for chemical reference substances from the point
of view of stability are sealed glass ampoules, but these have certain disad-
vantages. There is the risk of contaminating the substance with glass particles
when the ampoules are opened, and reclosure is difficult. Sealable glass
ampoules are therefore principally used for substances that must be kept in an
oxygen-free atmosphere. Certain other substances may require even more elab-
orate protection. Most chemical reference substances, however, are conve-
niently supplied in reclosable containers which should be uniform in type and
size to facilitate distribution. The lack of permeability to moisture is an impor-
tant factor in determining the suitability of container closure systems.
Before undertaking any packaging operations, the health hazards of the item
to be packaged should be assessed through information sources, e.g. the
1460
Material Safety Data Sheet. Appropriate precautions should be taken to protect

the person handling the chemical reference substance.
The packaging of a batch of a chemical reference substance into containers
is a small-scale operation for which suitable equipment is now always avail-
able to the manufacturer of the material. Therefore, the packaging of chemical
reference substances is usually undertaken by the responsible issuing body.
Screw-type feeders have been constructed, but generally the packaging of
chemical reference substances is carried out manually. Substances which are
expensive or only available in very small quantities may have to be divided
between containers and then lyophilized, or evaporated to dryness.
in solution
Some chemical must be packaged under an inert gas
reference substances
or in conditions of controlled humidity. Therefore, the use of a glove-box or an
airtight cabinet is necessary.
6.Z Storage
Information about suitable storage conditions can often be obtained from the
manufacturer of the source material and should be requested routinely when
a new chemical reference substance is established. Theoretically, the stability
of the substances should be enhanced by keeping them at low temperatures
but, for substances that contain water, storage below 0 °C may impair the sta-
bility. It should also be remembered that the relative humidity in normal refrig-
erators or cold-rooms may be high and, unless ampoules or other tightly closed
containers are used, the improvement in stability may be more than offset by
degradation due to the absorption of moisture. Storage at about -t5°C, with
precautions to prevent such absorption, has proved satisfactory for most chem-
ical reference substances.
6.i Stability
A chemical reference substance is an integral part of the drug specification.

Thus, if the reference substance deteriorates, this will change the specification
of the drug. It is, therefore, of the utmost importance that the stability of chem-
ical reference substances should be monitored by regular re-examination and
that they should be replaced as soon as a significant change in a property is

noted.
The definition of what is a “significant change” differs according to the
intended use of the chemical reference substance. Several per cent of degrada-
tion products found in a substance may not impair the usefulness of the mate-
rial inidentification tests. For chemical reference substances that are used in
chromatographic assays, however, even small amounts of impurities may be
unacceptable. When establishing a chemical reference substance, consideration
must be given to its intended use and to the performance characteristics of the
analytical methods in which it will be used. The tolerable degree of degrada-
tion will be different from case to case.
1461
Laboratories in charge of collections of chemical reference substances should

have a system for regular re-examination of the materials in stock. The fre-
quency of re-testing may be modified according to the need. It must be borne
in mind that the stability of a specially prepared chemical reference substance
may not always be the same as that of commercial samples of the same
material.
The selection of suitable analytical methods for monitoring the stability of
chemical reference substances depends on the nature and intended use of the
substance. A substance used solely for identification purposes will normally
only require demonstration that it is still suitable for this use, e.g. that the IR
spectrum is identical to that obtained during establishment. If substances are
employed for other purposes, the must be more extensive but should
testing
use methods which are rapid and consume too much of
sensitive so as not to
the existing stock. It is important to check that there has been no significant
uptake of moisture, which could result in degradation by hydrolysis and/or
a decrease in the assigned content of the substance. Chromatography is
employed extensively, as well as absolute methods such as differential scan-
ning calorimetry where applicable. Changes in the impurity profile or purity
determination usually mean that the batch must be replaced. Changes which
compromise the integrity of the batch indicate it should immediately be with-
drawn from use. Sometimes a batch of a chemical reference substance will dis-
colour or otherwise change in appearance. Steps should be taken to replace this
substance whether or not the results of subsequent analyses indicate significant
degradation. Such changes in physical appearance reduce the confidence of the
user in the suitability of the chemical reference substance. Appropriate testing
of active bulk substance should be carried out before further dispensing into
vials or ampoules.
6.4 Information to be supplied with chemical reference substances
Labels on chemical reference substances should give the following information:
— the appropriate name of the substance: the international nonproprietary

name (INN) should be used wherever possible;
— name and address of the issuing body;
— approximate quantity of material in the container; and
— batch or control number.
Where associated documents are provided they should incorporate relevant

items from the list above. The following information should be given, as nec-
essary, on the labels and/or in associated documents:
— recommended storage conditions (if special conditions apply);

— intended use of the chemical reference substance;
— directions for use (e.g. storage and handling);
1462
— information about assigned analytical value of the chemical reference sub-

stance (needed for calculation of the results of tests in which the substance
will be used);
— a disclaimer of responsibility when chemical reference substances are
misused, or stored under inappropriate conditions, or used for other pur-
poses than those intended by the issuing body; and
— health hazard information or warning in conformity with national and
regional regulations or international agreements.
If analytical data are to be supplied with the chemical reference substances,

it is recommended that the data provided be limited to what is necessary for
the proper use of the substances in the tests and assays.
6.5 Distribution and supfly

Distribution of chemical reference substances within the same country usually
does not present problems. However, when samples are to be sent to other
countries, both the sender and the receiver of the goods may encounter diffi-
culties because of the vagaries of postal and customs regulations, e.g. the appli-
cation of special procedural requirements applicable to substances under
international control. Distributors of chemical reference substances waste con-
siderable resources in seeking information on different international import
regulations, and in completing the required forms. A way of reducing such
difficulties and barriers to effective distribution of chemical reference sub-
stances should be sought. There should be the minimum delay in providing the
chemical reference substances to the users, and the most speedy means of
transport should be chosen.
6.6 Period of use
Chemical reference substances do not carry an “expiry date” in the conven-

tional sense. To avoid the unnecessary discarding of satisfactory substances, a
mechanism for general control of the batch of a chemical reference substance
may be used by the issuing body. If the issuing body applies stability consid-
erations and a monitoring procedure based on its experience to its collection,
thisshould guarantee the user of the acceptability of the chemical reference
substance for its intended use.
If it is considered necessary to specify a beyond-use date, it should be stated
on the label and/or on a document accompanying the chemical reference sub-
stances. Adequate shipping records should exist to enable contact with the pur-
chaser of a batch for recall or other notification.
The storage and maintenance of unopened containers of the chemical ref-
erence substance in accordance with information provided are integral to its
suitability of use. To avoid potential doubts concerning the integrity of opened

containers, it is suggested that potential users obtain only the quantities of
1463
.
substances necessary for short-term need and obtain fresh stocks (held under
controlled and known conditions) when needed. Long-term storage of sub-
stances in opened containers is to be avoided. Similarly, efforts should be made
to avoid possible degradation, contamination and/or introduction of moisture
during the repeated use of a substance.
Part B. Secondary chemical reference substances

The establishment of secondary chemical reference substances calibrated
against a primary chemical reference substance may be desirable for various
practical reasons, e.g. the latter may
not be available in adequate quantities to
supply all Moreover, the availability of such secondary chemical
local needs.
reference substances (for example, on a regional basis) would reduce the delay
in receiving the reference material.
The body which establishes a secondary chemical reference substance for
national/regional use should be clearly defined by the competent drug regula-
tory authority. Clear documentation must exist to establish the relationship
between the secondary and the primary chemical reference substance.
References
The references below are those quoted in the guidelines published as Annex 3 to
the Thirty-fifth Report of the Expert Committee on Specifications. Geneva, World
Health Organization, 1999 (WHO Technical Report Series, No. 885). For current
WHO guidelines on, for example, GMP, consult the WHO Medicines website
(http :/ /www.who .int/ me dicines)
1. WHO Expert Committee on Specifications for Pharmaceutical Preparations. Twenty-fifth

report. Geneva, World Health Organization, 1975, Annex 3 (WHO Technical Report
Series, No. 567).
2. The International Pharmacopoeia, 3rd ed. Vol. i. General methods of analysis; Vol. Z. Quality
specifications; Vol. 3. Quality specifications; Vol. 4- Tests, methods,and general recjuirements.
Quality specifications for pharmaceutical substances, excipients, and dosage forms. Geneva,
World Health Organization, 1979-1994.
3. WHO Expert Committee on Specifications for Pharmaceutical Preparations. Twenty-eighth
report. Geneva, World Health Organization, 1982, Annex 1 (WHO Technical Report
Series, No. 681).
4. Good laboratory practices in governmental drug control laboratories. In: WHO Expert
Committee on Specifications for Pharmaceutical Preparations. Thirtieth report. Geneva, World
Health Organization, 1987, Annex 1 (WHO Technical Report Series, No. 748).
5. Good manufacturing practices for pharmaceutical products. In: Expert Commit- WHO
tee on Specifications for Pharmaceutical Preparations. Thirty-second report. Geneva, World
Health Organization, 1992, Annex 1 (WHO Technical Report Series, No. 823).
6. Good manufacturing practices for biological products. In: Expert Committee on WHO
Specifications for Pharmaceutical Preparations. Thirty-third report. Geneva, World Health
Annex 3 (WHO Technical Report Series, No. 834).
Organization, 1993,
7. General recommendations for the preparation and use of infrared spectra in phar-
maceutical analysis. In: WHO Expert Committee on Specifications for Pharmaceutical
1464
^
Suf?p>lementary information
Geneva, World Health Organization, 1996, Annex 4

Prep>arations. Thirty-fourth refort.
(WHO Technical Report Series, No. 863).
Recommendations on Risk of Transmitting

Animal Spongiform Encephalopathy Agents via
Medicinal Products
Products with risk o£ transmitting agents of animal spongiform
encephalopathies are those derived from tissues or secretions of animals sus-
ceptible to transmissible spongiform encephalopathies other than by experi-
mental challenge. This definition applies to all substances or preparations
obtained from such animals and to all substances or preparations where prod-
ucts obtained from such animals are included as active substances or excipients
or have been used during production, e.g. as raw or source materials, starting
materials or reagents.
Materials of animal origin should be avoided whenever possible. However,
if used, manufacturers should be aware of the risk and have a system in place
to minimize it, especially since international trading patterns often include the
processing and re-export of products, so that their origin may not be traceable.
In order to minimize the risk of transmitting animal spongiform encephalopa-
thy agents via medicinal products, manufacturers should follow the current
WHO Guidelines on Transmissible Spongiform Encephalopathies in Relation
to Biologicaland Pharmaceutical Products' and the recommendations of the
Joint Technical Consultation on Bovine Spongiform Encephalopathy, public
health, animal health and trade, convened by the WHO, the Food and Agri-
culture Organization of the United Nations and the Office International des
Epizooties 11-14 June 2001 in Paris.
'
Note: the current guidelines are published on the WHO website [www.who.int/bloodprod-
ucts/tse]. They were adopted by ECBS in 2003; they were reviewed in 2005 and are likely to be
updated.
^
Proceedings - Joint WHO/FAO/OIE Technical Consultation on BSE: public health, animal health
and trade. Paris: Office International des Epizooties, World Health Organization, Food and Agri-
culture Organization, World Organisation for Animal Health, 2002.
1465
CopyrigfiJed i
Index
Pages Volume 1 i-xviii
1-712
Pages Volume 2 xix-xxi
713-1499
Abbreviations used:
AsR reagent for arsenic limit test

AsTS test solution for arsenic limit test
BET bacterial endotoxin test
CITS test solution for chloride limit test
Cm culture medium
FeTS test solution for iron limit test
IR reagent for infrared spectrophotometry
PbTS test solution for lead limit test
R reagent
RS International Chemical Reference Substance
TS test solution
VS volumetric solution
2 1
Index
Abbreviations, 13 Acidum lacticum, 43

Absolute alcohol, see Ethanol Acidum nicotinicum, 51
Absorbance (A), 13,. 1 155 Acidum salicylicum, 53
specific, 13 Acknowledgements, xiii
Absorption spectrometry, atomic, 1164 Activated charcoal, 21

Absorption spectrophotometry, 1 154 Active ingredient, 4
Absorption spectrum, 1 156 Additional information, 6
Absorptivity, 11 55. Adeps lanae, 55
Acacia, 460 cum aqua, 55
R, 1233 Adeps solidus, 52
TS (5g/i), 1283 Adrenaline, see Epinephrine
Accuracy, 9. Adrenaline tartrate, see Epinephrine hydrogen
expression of concentration, 9. tartrate
pH values, 9 Adsorption column chromatography, 1182
quantities, 9 Agar R, 1 286
temperature measurement, 13 Agreement for the Unification of the
Acetaldehyde R, 1284 Formulae of Potent Drugs (1902), ix
^-Acetamidobenzalazine RS, 1 284, 1444 Albendazole, 53
Acetaminophen, see Paracetamol RS, 1236
Acetanilide, 1447 Albendazolum, 53
Acetate buffers TS Alcohol, 64
pH 3H to pH 1284 absolute see Ethanol
standard, 1285 dehydrated see Ethanol
Acetazolamide, 23 Alcohol benzylicus, 63
RS, 1285, 1444 Alcohol cetylicus, 61
Acetazolamidum, 23 Alcohol cetylstearylicus, 63
Acetic acid, 24 Alcoholum, 64
dilute, 24 Alcuronii chloridum, 66
PbTS (~60g/l), 1285 Alcuronium chloride, 66
TS(2Mto -300 p/1), 1285 RS, 1286
VS (0.07mol/lX 1235 Alcuronium dichloride, see Alcuronium
Acetic acid, glacial, 24 chloride
R, 1235 Alexandrian senna
R1, 1285 fruit, 858
Acetic anhydride R, 1286 leaf, 856

Acetic anhydride/dioxan TS, 1286 Alginic acid, 29
Acetone R, 1286 Alizarin Red S, see Sodium alizarinsulfonate
Acetonitrile Allopurinol, 69
R, 1286 RS, 1 86,1444
TS (400 g/1), 1236 tablets, 978
Acetyl chloride R, 1 286 Allopurinoli compressi, 978
Acetylated substances, 1206 Allopurinolum, 69
Acetylsalicylic acid, 2Z Alum R, 1286
tablets, 977 Aluminii hydroxidum, 70
Acid value, determination, 1 254 Aluminii magnesii silicas, 22.
Acidi acetylsalicylic! compress!, 977 Aluminii sulfas, 74
Acids Aluminium
titration, 1 223 complexometric titration, 1 222
see also specific named acids R, 1282
Acidum aceticum, 24 standard TS (10 [igAl/ml), 1 287
Acidum acetylsalicylicum, 22 Aluminium chloride
Acidum alginicum, 29. R,12S6
Acidum amidotrizoicum, 30. TS,1282
Acidum ascorbicum, 33 Aluminium hydroxide, 23
Acidum benzoicum, 35 R, 1287
Acidum citricum, 37 Aluminium magnesium silicate, 72
Acidum folicum, 89 Aluminium oxide R, 1 287
Acidum hydrochloricum, 41 Aluminium sulfate, 74
dilutum, 43 Aluminum, see Aluminium
Acidum iopanoicum, 44 American Type Culture Collection (ATCC),
Acidum iotroxicum, 46 18, 1282
Pages Vol 1 i-xviii, 1-712 : Vol 2 xix-xxi, 713-1499 1469

Amethocaine hydrochloride, see Tetracaine buffer TS, pH 18l5, 1221

hydrochloride R, 1291
Amidotrizoic acid, 3Q TS (10 p/ml to 100 g/1 4), NH 1228
anhydrous, TS (Nessler’s reagent), 1221
dihydrate, HQ TS dilute, 1 291
RS, 1287, 1444 Ammonium dihydrogen phosphate, 1 221
Amikacin, Zfi Ammonium mercurithiocyanate TS, 1291

Amikacin sulfate, Z5 Ammonium molybdate
non-injectable, 25 R, 1291
sterile, 25 TS(45 and 25 g/1), 1221
Amikacini sulfas, 25
2-
Ammonium molybdate/nitric acid TS, 1291
Amikacinum, 78 Ammonium molybdate/sulfuric acid TS,
Amiloride hydrochloride, SO. 1291
anhydrous, SQ Ammonium molybdate/vanadate TS, 1291
dihydrate, SQ Ammonium nitrate
RS, 1287 R, 1222
Amiloridi hydrochloridum, SH TS and TS (50 g/1), 1222
Amines Ammonium oxalate
primary aromatic, 1207 R, 1 292
1-
2- volatile aliphatic, 1207 TS(ând 50 g/1), 1222
Amino-5-nitrothiazole Ammonium persulfate R, 1292
R, 12S2 Ammonium persulfate/phosphate buffer TS,
RS, 1444 1292
3- Amino-2.4,6-triiodobenzoic acid RS, 1287, Ammonium pyrrolidinedithiocarbamate
3- 1444 R, 1292
4- Amino-6-chloro-l,3-benzenedisulfonamide TS (10 g/1), 1222
R, 1282 Ammonium reineckate
Aminoacetic acid, see Glycine R, 1292
4-Aminoantipyrine TS (10 g/1), 1222
R, 1282 Ammonium sulfamate
TSl and TS2, 1288 R, 1293
4-Aminobenzoic acid R, 1 288 TS(5to 50 g/1), 1222
Aminobutane, see 1-Butylamine Ammonium sulfate R, 1 293
Aminobutanol R, 288 1 Ammonium sulfide TS, 1 293
7-[(2-Aminoethyl)amino]-l-cyclopropyl-6- Ammonium thiocyanate

fluoro-l,4-dihydro-4-oxoquinoline-3- R,1223
carboxylic acid RS, 1288 TS (10 and 75 g/1), 1228
4-Aminophenol R, 1 289 VS (8111 to 0.1 mol/1), 1223
Aminophylline, 82 Ammonium thiocyanate/cobalt(II) nitrate TS,
Aminophyllinum, 82 1223
Aminopyrazole-4-carboxamide hemisulfate Ammonium vanadate R, 1 293
RS, 1289, 1444 Amodiaquine, 88
Aminopyrazolone, see 4-Aminoantipyrine Amodiaquine hydrochloride, 86
Amitriptyline hydrochloride, 84 RS, 1293, 1444
RS, 12Rg, 1444 Amodiaquini hydrochloridum, 86
Amitriptylini hydrochloridum, 84 Amodiaquinum, 88
Ammonia Amoxicillin trihydrate, 28
buffers TS and TS2, 289 1 RS, 1294
FeTS C~100g/1), 1282 Amoxicillinum trihydricum, 28
identification, 207 1 Amphotericin B, 23
PbTS (~100g/l), 1282 powder for injections, 979
TS (~J7 to -260 g/1), 1282 RS, 1294, 1444
Ammonium, identification, 1207 Amphotericini B pulvis ad injectionem, 979
Ammonium acetate Amphotericinum B, 23
buffer TS, pH 4.62, 1290 26
Ampicillin,
R, 1290 anhydrous, 26
TS, 1290 capsules, 981
TS (2 to 100 g/1), 1220 RS, 1294, 1444
Ammonium carbonate R, 1 290 Ampicillin sodium, 28
Ammonium chloride non-injectable, 28
buffer TS, pH 1811,1228 powder for injections, 983
1470 Pages Vol 1 i-xviii, 1-712 : Vol 2 xix-xxi, 713-1499

2
Index
RS, 1294, UM injection, 991

Sterile, 98 RS, 1296, 1444
Ampidllin trihydrate, 96. tablets, 988
RS, 1294, 1444 Artemetheri
Ampicillini capsulae, 9_81 capsulae, 986
Ampicillini natrici pulvis ad injectionem, 988 compressi, 988
Ampicillinum, 96 injectio, 991
Ampicillinum natricum, 92. Artemetherum, 109
Ampyrone, see 4-Aminoantipyrine Artemisinin, 1 18
Amyl alcohol R, 1294 capsules, 994
Amyla, 101 RS, 1 96,1444
Anaesthetic ether, see Ether, anaesthetic tablets, 998
Analysis, methods of, see Methods of Artemisinini
analysis capsulae, 994
Angle of rotation ([c(]), 13 compressi, 998
Anhydrotetracycline hydrochloride RS, 1294, Artemisininum, 1 18
1444 Artemotil, 117
Aniline injection, 1 001
R, 1294 RS, 1296, 1444
TS (25g/l), 1294 Artemotili injectio, 1 001
Animal spongiform encephalopathies, risk of Artemotilum, 117
transmitting, 465 1 Artenimol, 1 20
Anisaldehyde R, 1294 RS, 1297, 1444
Anisaldehyde/methanol TS, 1294 tablets, 1004
Anisaldehyde/sulfuric acid TS, 1294 Artenimoli compressi, 1 004
Anthralin, see Dithranol Artenimolum, 120
Anthrone Artesunate, 1 28
R, 1294 RS, 1297, 1444
TS and TS2, 1295 tablets, 1 007
Antibiotics Artesunati compressi, 1007
microbiological assay, 1 280 Artesunatum, 128
sterility testing, 1 241 Ascorbic acid, 88
undue toxicity test, 1251) Ash
see also individual antibiotics acid-insoluble, determination, 1 251
Antimony sodium tartrate, 864 determination, 1251
R, 1295 sulfated, 1218
TS (50g/l), 1295 Assays, see Tests and assays
Antimony trichloride ATCC, see American Type Culture Collection
R, 1295 Atenolol, 12
TS,1295 for column validation RS, 1297
Antiretroviral substances,v RS, 129Z
Appendices to general notices, 13 Atenololum, 127
Apressinum, see Hydralazine hydrochloride Atomic mass, relative, 19
Aprotinin R, 1295 Atomic spectrometry, 1164
Aqua pro injectione, 1 08 Atropine sulfate, 1 29
Aqua purificata, 105 RS, 1297, 1444
Aqua sterilisata pro injectione, 106 tablets, 1010
Arachis oil, 698 Atropini sulfas, 1 29
R, 1295 Atropini sulfatis compressi, 1010
Argenti nitras, 1 08 Autoclaves, 1 277
Argon R, 1296 Azathioprine, 182
Arsenic RS, 1297, 1444
dilute, AsTS, 1296 Azathioprinum, 182
limit test, 1216 Azo violet
strong, AsTS, 1296 R, 1297
Arsenic trioxide TS, 1297
R, 1296 Azobenzene, 1447
R1, 1296
Arteether, see Artemether Bacillus cereus, 1 288, 1 280
Artemether, 109 Bacillus pumilis, 1 288, 1 280
capsules, 986 Bacillus sphaericus, 1 280

Bacillus stearothermoyhilus, 1 ^7R Benzoylis peroxidum cum aqua, 1 52

Bacillus suhtilis, 1233, 1278, 1280 Benzyl alcohol, 69
Bacitracin, 134, 1234 R, 1299
Bacitracin zinc, 1 36 Benzyl benzoate, 154
non-injectable, 126. R, 1300
RS, 1297, 1444 Benzyl hydroxybenzoate, 156
sterile, 136 Benzylis benzoas, 154
Bacitracinum, 134 Benzylis hydroxybenzoas, 15.6
non-injectable, 134 Benzylpenicillin potassium, 158
sterile,134 non-injectable, 158
Bacitracinum zincum, 136 powder for injections, 1012
Bacterial endotoxin test (BET), 957, 1245 RS, 1300, 1444
Bacteriostatic agents, radiopharmaceuticals, sterile, 1.58
1134 Benzylpenicillin sodium, 160
Barii sulfas, 138 non-injectable, 160
Barium chloride R, 1.300
R, mz RS, 1.300, 1444
TS (50g/l), 122Z sterile, 160
VS (0.5 mol/1), mi T.S, 1300
Barium hydroxide Benzylpenicillini kalici pulvis ad injectionem,
R, 122Z 1012
TS (15g/l), mi Benzylpenicillinum kalicum, 158
vs (0.15mol/l), mi Benzylpenicillinum natricum, 160
Barium nitrate Bephenii hydroxynaphthoas, 163
R, 1298 Bephenium hydroxynaphthoate, 163
VS (0.01 mol/1), 1297 RS, 1300, 1444
Barium oxide R, 298 1 BET, see Bacterial endotoxin test
Barium sulfate, 138 Betamethasone, 167
suspension TS, 1 298 RS, 1.300, 1444
Bases, titration, 1223 Betamethasone sodium phosphate RS, 1300,
Basic blue 2£if see Methyl green 1444
Beclometasone dipropionate, 140 Betamethasone valerate, 1 65
KS, 1292, 1AM RS, 1.300, 1444
Beclometasoni dipropionas, 140 Betamethasoni valeras, 165
Beef extract R, 129.8 Betamethasonum, 167
Bentonite, 142 BHA, see Butylated hydroxyanisole
Bentonitum, 142 BHT, see Butylated hydroxytoluene
Benzalkonii chloridum, 144 Biological methods of analysis, 1 230
Benzalkonium chloride, 1 44 Biological standards, 12
T.S, 1298 Biological substances and preparations,
rSl, 1299 general requirements, Z
Benzanilide, 1447 Biperiden, 171
Benzathine benzylpenicillin, 1 45 RS, IROQ
non-injectable, 145 Biperiden hydrochloride, 170
sterile, 145 RS, 13QQ
Benzathini benzylpenicillinum, 145 Biperideni hydrochloridum, 170
Benzene R, 1299 Biperidenum, 171
Benzhexol hydrochloride, see BIPM, see Bureau International des Poids et
Trihexyphenidyl hydrochloride Mesures
Benzil, 1 447 9,9^-Bisanthracene-10,10^(7H, 9'H)-dione RS,
Benznidazole, 148 1.300
RS, 1299 4,4^-Bis(dimethvlamino)benzophenone R,
Benznidazolum, 148 1.300
Benzocaine, 150 Bismuth
Benzocainum, 150 complexometric titration, 1 222
Benzoic acid, 35 identification, 1207
R, 1299 Bismuth oxynitrate R, 1300
Benzophenone R, 1299 Bismuth subnitrate R, 1301
Benzoyl chloride R, 1 299 Bleomycin hydrochloride, 73 1
Benzoyl peroxide, hydrous, 152 non-injectable, 1 73

R, 1299 sterile, 173

-
Index
Bleomycin 77
sulfate, 1 Butylated hydroxytoluene, 1 87
non-injectable, 177 R, 1305
sterile, 177 Butylhydroxyanisolum, 1 85
Bleomycini hydrochloridum, 1 73 Butylhydroxytoluenum, 1 87
Bleomycini sulfas, 177
Blue tetrazolium R, 1301 Cadmium acetate R, 1 305
Blue tetrazolium/ethanol TS, 1301 Caephaelis acuminata, 521
Blue tetrazolium/sodium hydroxide TS, Caephaelis if?ecacuanha, 521
1301 Caesium chloride R, 1305
Boiling point, determination, 1143 Caffeine, 288
Boiling range, determination, 1143 anhydrous, 288
Bolus alba, see Kaolin monohydrate, 2fiR
Borate buffers TS (pH Ril to 9.6), 1301 R, 1305
Borax, see Sodium tetraborate RS, 1444, 1447
Bordetella bronchisef^tica, 1 233 Calamine, 1 88
Boric acid Calaminum, 188
R, 1302 Calcii carbonas, 190
TS (50g/l), 1302 Calcii folinas, 192
Bovine spongiform encephalopathy, 1465 Calcii gluconas, 194
Brilliant green R, 1 302 Calcii hydrogenophosphas, 197
Brilliant green/acetic acidTS, 1302 Calcii phosphas, 199
Bromides, identification, 1 207 202
Calcii stearas,
Bromine 203
Calcii sulfas,
AsTS, 1302 Calcium
oxygen flask method, 1219 complexometric titration, 1 222
R, 1303 identification, 1208
TS1, 1303 standard, ethanolic, TS (100|ig/ml Ca),
Bromocresol green 1306
R, 1303 standard, TS (lOpg/ml Ca), 306 1
TS1, 1303 Calcium acetate

Bromocresol green/ethanol TS, 1303 R, 1305
Bromocresol purple R, 1303 VS (0.25 mol/1), 1305
Bromocresol purple/ethanol TS, 1303 Calcium carbonate, 19_0
Bromophenol blue
2- R1, 1305
R, laoa R2, 1305
TS, 1303 Calcium chloride
TS (1 g/1), 1303 anhydrous, R, 1306
Bromophenol blue/ethanol TS, 303 1 hydrated, R, 1306
Bromothymol blue R, 1303 TS(3Zand 55 g/1), 1305
Bromothymol blue/dimethylformamide TS, Calcium fluoride R, 1306
1304 Calcium folinate, 192
Bromothymol blue/ethanol TS, 1304 RS, 1306, 1444
Brown stock standard TS, 1171, 1304 Calcium gluconate, 94 1
Brussels Agreement (1925), ix Calcium hydrogen phosphate, 197

Buccal tablets, 967 anhydrous, 197
Buffers, see sprecific buffers dihydrate, 197
Bupivacaine hydrochloride, 181 Calcium hydroxide
RS, 1304, 1444 R, 1306
Bupivacaini hydrochloridum, 181 TS, 1306
Bureau International des Folds et Mesures Calcium phosphate, 199
(BIPM), 16 dibasic see Calcium hydrogen
Busulfan, 184 phosphate
Busulfanum, 184 tribasic see Calcium phosphate
1 Butanol R (w-Butanol R), 1304 Calcium stearate, 202
Butanol R, 1304 Calcium sulfate, 203
rerr-Butanol R, 1304 hemihydrate R, 1306
Butyl acetate R, 1304 R, 1306
teri-Outyl methyl ether R, 1304 TS, 1306
1-Butylamine R, 1 304 Calcon, 1222
Butylated hydroxyanisole, 1 85 indicator mixture, 1307
R, 1305 R, 130Z

1 5 0
Calcon carboxylic acid, 1 Cellulose

indicator mixture R, 1306 microcrystalline, 219
R, 13M Ri to R3, mm
Calculation of results, 111 Cellulose acetate phthalate, see Cellacefate
Calibration, instrument, use of chemical Cellulose acetate strips, electrophoresis on,
reference substances, 1 456 1201
Capsules, 947 Cellulose ethyl ether, see Ethylcellulose
delayed-release (enteric), 951 Cellulose 2-hydroxyethyl ether, see
disintegration test, 949, 950, 951, 1258 Hydroxyethylcellulose
dissolution test, 948, 1262 Cellulose 2-hydroxypropyl ether, see
extended-release, 950 Hydroxypropylcellulose
hard, 948 Cellulose methyl ether, see Methylcellulose
modified-release, 950 Cellulosum microcrystallinum, 219
soft, 949 Cephaeline hydrochloride R, 1308
uniformity of content, 948, 1 256 Cephalin TS, 1 308
uniformity of mass, 947, 1257 Cera carnauba, 221
Captopril, 204 Cera cetyla, 221
RS, 1307, 1444 Ceric ammonium nitrate
Captopril disulfide RS, 1307, 1444 R, 1309
Captoprilum, 204 TS, 1310
Carbamazepine, 207 VS (0.01 mol/1), 15fl9
RS, 1307, 1444 Ceric ammonium sulfate
tablets, 1015 R, 1310
Carbamazepini compress!, 1015 VS (0.1 mol/1), 1BQ9
Carbamazepinum, 207 Ceric ammonium sulfate/nitric acid TS, 1310
Carbenicillin monosodium RS, 1444 Ceric sulfate
Carbidopa, 209 R, 1310
Ks,mi TS (35g/l),
Carbidopum, 209 VS (0.1 mol/1), 13111
Carbo activatus, 21 Cesium-137, 1 120
Carbomer, 214 Cetomacrogol 1 000, 222
R, 1307 Cetomacrogolum 1 000, 222
Carbomerum, 214 Cetostearyl alcohol, 63

Carbon dioxide Cetrimide, 224
detector tube, 13.07 Cetrimidum, 224
in medicinal gases, 354, 703 Cetyl alcohol, 61
R, laoz Cetyl esters wax, 221
Carbon disulfide Charcoal
IR, 1307 activated, 211
R, mm R, 1310
Carbon monoxide Chemical Abstracts Service Registration
detector tube, 1308 Number (CAS Reg. No.), 3,. 13
in medicinal gases, 351 ,
702 Chemical formulae, 3^ 1441
R, 1308 Chemical methods of analysis, 1206
Carbon tetrachloride R, 1 308 Chemical names, 3
Carboxymethylcellulose R, 1308 Chemical reference substances, 11
Carboxymethylcellulose sodium, see defined, 1452
Carmellose sodium guidelines for establishment, maintenance
Carmellose sodium, 215 and distribution, 1452
Carmellosum natricum, 21 international see International Chemical
Camauba wax, 221 Reference Substances
CAS, see Chemical Abstracts Service primary, 1453
Cassia angustifolia, 856, 858 secondary, 1 453, 1464
Cassia senna, 856, 858 Chewable tablets, 967
Category, 5 Chloral hydrate, 225
CD-ROM, vi Chloral! hydras, 225
Cefalexin, 1234 Chloralose R, 1 31
Cefalotin, 1234 Chlorambucil, 227
Cellacefate, 217 Chlorambucilum, 2.27
Cellacefatum, 217 Chloramphenicol, 235
Cellacephate, see Cellacefate RS, 1311, 1444

1 1
Index
Chloramphenicol disodium disuccinate RS, Chlorpromazine hydrochloride, 252

1311 RS, 1312, 1445
Chloramphenicol palmitate, 232 Chlorpromazini hydrochloridum, 252
RS, 1311, 1445 Chlortalidone, 254
Chloramphenicol sodium succinate, 229. RS, 1312, 1445
RS, 1311 Chlortalidonum, 254
Chloramphenicoli natrii succinas, 229 Chlortetracycline, 1234
Chloramphenicoli palmitas, 232 Chlortetracycline hydrochloride, 256.
Chloramphenicolum, 235 non-injectable, 256
Chloraniline R, 1311 RS, 1312, 1445
Chlorhexidine diacetate, 237 sterile, 256
Chlorhexidine dihydrochloride, 239 Chlortetracyclini hydrochloridum, 256
Chlorhexidini diacetas, 237 Cholecalciferol, see Colecalciferol
Chlorhexidini dihydrochloridum, 239 Chromatography, 1 175
Chloride(s) column, 1182
2-
identification, 1208 gas, 1193
limit test, 1211 high-performance liquid, vi, 1 184
standard (Spg/l) TS, 131 impurities, 10,. 1 178
Chlorine ion-exchange, 1183
oxygen flask method, 1219 paper, 1180
R, im symbols, 15
TS, 1311 thin-layer, 1 175
Chlormethine hydrochloride, 241 Chromic acid TS, 1312
Chlormethini hydrochloridum, 241 Chromium-51, 1120
l-Chloro-2,4 dinitrobenzene R, 131 Chromium trioxide R, 1312
1- Chloro-2,4 dinitrobenzene/ethanol TS, 1311 Chromotropic acid sodium salt, see Disodium
Chloro-4-nitroaniline R, 1312 chromotropate
2-(4-Chloro-3-sulfamoyl)benzoic acid RS, C.I. 14645, see Mordant black 11
1311^ 1445 C T 25100, see Sudan red
5-Chloro-2-methylaminobenzophenone RS, C.T. 42040, see Brilliant green
1311, 1445 C.I. 42535, see Methyl violet 2B
7-Chloro-l-cyclopropyl-l,4-dihydro-4-oxo-6- C.T. 42585, see Methyl green
(piperazin-l-yl) quinoline-3-carboxylic C,L 50040, see Neutral red
acid RS, 1311 C.T. Basic blue 9^ see Methylthioninium
4-Chloroacetanilide R, 1312 chloride
Chlorobutanol, 243 C.T. Basic green, see Brilliant green
anhydrous, 243 C.T. Basic red, see Neutral red
hemihydrate, 243 Basic violet, see Methyl violet 2B
Chlorobutanolum, 243 C.T. Basic violet 3,. see Methylrosanilinium
Chlorocresol, 245 chloride
Chlorocresolum, 245 C.T. Mordant black 11, see Mordant black
Chloroform 11
ethanol-free R, 1312 C.T. Mordant black IZ, see Calcon
R, 1312 Ciclosporin, 260
Chloroquine phosphate, 245 RS, iai2
tablets, 1Q15 URS, 1312
Chloroquine sulfate, 248 Ciclosporinum, 260
RS, 1312, 1445 Cimetidine, 252
tablets, 1018 RS^ 1313 1445
Chloroquini phosphas, 245 Cimetidinum, 252
Chloroquini phosphatis compress!, 1016 Cinchonidine R, 1313
Chloroquini sulfas, 248 Cinchonine R, 1313
Chloroquini sulfatis compressi, 1018 Ciprofloxacin, 258
Chlorphenamine hydrogen maleate, 250 by-compound A, 1311, 1445
RS, 1312, 1445 desfluoro-compound, 1321, 1445
tablets, 1019 ethylenediamine compound, 1288, 1445
Chlorphenamini hydrogenmaleas, 250 fluoroquinolonic acid, 1445
Chlorphenamini hydrogenomaleatis RS, 1313
compressi, 1 019 Ciprofloxacin hydrochloride, 254
Chlorpheniramine hydrogen maleate, see RS, 1313, 1445
Chlorphenamine hydrogen maleate Ciprofloxacini hydrochloridum, 254

7
The Internatmial Pharmacopoeia
Ciprofloxacinum, Codeine phosphate, 283

Cisplatin, 271 hemihydrate, 283
1.21.2 1445 sesquihydrate, 283
Cisplatinum, 271 tablets, 1 026
Citrate Codeini phosphas, 283
buffers, pH 4.0 and 5.4, TS, 1313 Codeini phosphatis compressi, 1 026
identification, 1208 Codeinum monohydricum, 286
Citric acid, 3Z Coffeinum, 2S8
anhydrous, 3Z Colchicine, 290
copper-free R, 1314 RS, 1315
FeR, 1313 tablets, 1028
FeTS C180g/1), IhlB Colchicini compressi, 1028
monohydrate, 3Z Colchicinum, 290
PbR, 1313 Colecalciferol, 292
R, 1313 RS, 131.5, 1445
TS (20g/l), 1313 Colecalciferolum, 292
Clarity of solution, B Collection de Bacteries de I’Institut Pasteur,
Clindamycin hydrochloride RS, 1314 13
Clindamycin phosphate, 273 Colour Index Number, see C.I.
RS, 1314 Colour of liquids, 1 170
Clindamycini phosphas, 273 Colourless solution, S
Clofazimine, 277 Column chromatography, 1 1 82
RS, 1314 adsorption, 1182
Clofaziminum, 277 partition, 1183
Clomifene citrate, 279 Complexometric titrations, 1 220
R.S. 1314, 1445 Concentration, expression of, 9.
Z-isomer RS, 1314, 1445 Congealing point, determination, 1 1 43
Clomifeni citras, 279 Congo red paper R, 1315
Closures, parenteral preparations, 958 Consistency, uniform, see Uniform
Cloxacillin, 1234 consistency
Cloxacillin sodium, 281 Containers, 5
capsules, 1 021 hermetically closed, 5
non-injectable, 281 ophthalmic preparations, 9.53, 9.54, 955
powder for injections, 1 024 parenteral preparations, 958
R.S, 1314, 1445 suppositories, 962
sterile, 281 tamper evident, 5
Cloxacillini natrici tightly closed, 5
capsulae, 1021 topical semi-solid dosage forms, 970
pulvis ad injectionem, 1024 well-closed, 5
Cloxacillinum natricum, 281 Content, uniformity of, see Uniformity of
Coal tar, 757 content
Coated tablets, 967 Copperfll) acetate
Cobalt-57. 112Q R, 1316
Cobalt-58, 1120 TS (45g/l), 1316
Cobalt-60, imi Copperfll) chloride/ammonia, 1317
Cobalt(II) chloride Copperfll) chloride R, 1317
R, 12dA Copper colour
TS, 1315 strong, TS, 1316
TS{5and 30g/l), 1314 TS, 1315
Cobalt colour Copper edetate TS, 1316
strong, TS, 1314 Copper standard
TS, 1314 TS (lOpg/l Cu),1316
Cobalt(Il) nitrate TS1,1316
R, 1315 TS2, 1316
TS(10and 100g/l),1315 Copperfll) sulfate
Cobaltous chloride R, 1312
R, 1315 TS(1 to 16Qg/l), 1317
TS,1315 Copper(II) sulfate/ammonia TS, 1317
Cobaltous thiocyanate TS, 1315 CopperÎ) sulfate/pyridine TS, 1 31
Codeine R, 1315 Copper tetramine hydroxide TS, 1316
Codeine monohydrate, 286 Com starch, 101

7
Index
Cortisol, see Hydrocortisone Desferrioxamine mesylate, see Deferoxamine

Cortisone acetate RS, 1445 mesilate
Creams, 971 Desoxycortone acetate, 1445
hydrophilic (o/w), 972 Dexamethasone, 316
hydrophobic (w/o), 9Z1 RS, 1321, 1445
o-Cresol R, 1 31 tablets, 1 031
Cresol red R, 1318 Dexamethasone acetate, 310
Cresol red/eânol TS, 1318 anhydrous, 310
Crystal violet monohydrate, 310
R, laiB RS, 1321, 1445
see also Methylrosanilinium chloride Dexamethasone phosphoric acid, 1 44 5
Crystal violet/acetic acid TS, 1318 Dexamethasone sodium phosphate, 313
Culture medium (Cm) RS, 1321, 1445
antibiotic assays, 1233 Dexamethasoni acetas, 310
Cml to CmlTlTld Dexamethasoni compressi, 031 1
sterility tests, 1237 Dexamethasoni natrii phosphas, 313

Cyanide/oxalate/thiosulfate TS, 1320 Dexamethasonum, 316
Cyanocobalamin, 294 Dextromethorphan hydrobromide, 319
Cyanocobalaminum, 294 RS, 1321
Cyanoethylmethyl silicone gum R, 1 320 Dextromethorphani hydrobromidum, 319
Cyanogen bromide TS, 1320 Dextrose, see Glucose
Cyclodolum, see Trihexyphenidyl l,4-Di|'2-(methyl-5-phenyloxazole)]benzene
hydrochloride R, 1321
Cyclohexane R and Rl, 1320 3,3^-Diaminobenzidine tetrahydrochloride
Cyclophosphamide, 296 R, 1321
Cyclophosphamidum, 296 TS (5g/l),1322
Cyclophosphanum, see Cyclophosphamide Diammonium hydrogen phosphate
1 -Cyclopropyl-1,4-dihydrQ-4-oxQ-7- R, 1322
(piperazin-l-yl)quinoline-3-carboxylic TS (lOOg/1), 1322
acid RS, 1321 Diatomaceous support R, 1322
Cyclosporin, see Ciclosporin Diatrizoate sodium, see Sodium
L-Cystine R, 1321 amidotrizoate
Cytarabine, 298 Diatrizoic acid, see Amidotrizoic acid
RS, 1321 Diazepam, 321
Cytarabinum, 298 RS, 1322
Diazepamum, 321
Dacarbazine, 300 Diazobenzenesulfonic acid TS, 1322
related compound A RS, 1321 Diazomethane TS, 1322
related compound B RS, 1321 Diazoxide, 324
1B21
RS, RS, 1322, 1445
Dacarbazinum, 300 Diazoxidum, 324
Dactinomycin, 302 Dibasic calcium phosphate, see Calcium
RS, 1321 hydrogen phosphate
Dactinomycinum, 302 Dibromomethane R, 1323
Dantron R, 1321 Dibutyl ether R, 1323
Dapsone, 304 Dibutyl phthalate R, 1323
RS, 1321, 1445 Dicainum, see Tetracaine hydrochloride
tablets, 1 030 Dichloroethane R, 1323
Dapsoni compress!, 1030 Dichlorofluorescein
Dapsonum, 304 R, 1323
Deferoxamine mesilate, 306 TS, 1323
Deferoxamini mesilas, 306 Dichloromethane R, 1323
Definition, 4 2,6-Dichloroquinone chlorimide R, 1323
new general notice, iv 2,6-Dichloroquinone chlorimide/ethanol TS,
Dehydrated alcohol, see Ethanol 1323
Dehydroemetine dihydrochloride, 308 Dichromate colour
Dehydroemetini dihydrochloridum, strong, TS, 1324
308 TS, 1324
Delayed-release capsules, 951 Dicloxacillin, 234 1
Delayed-release tablets, 968 Dicloxacillin sodium, 325

Description, 4 RS, 1 324 1445

Dicloxacillinum natricum, 4-Dimethylaminocinnamaldehyde

Dicoumarol, 327 R, 1327
1324 1445 TSl and TS2, 1322
Dicoumarolum, 327 A,A/-Dimethylaniline R, 1327
Dicyanodiamide, 1447 1.1- Dimethylethyl methyl ether, see zerr-Butyl
Didanosine, 329 methyl ether R
RS, 1324, 1445 Dimethylformamide R, 1328
for system suitability RS, 1324^ 1 44 5 N,A/-Dimethyloctylamine R, 1328
Didanosinum, 329 Dinatrii edetas, 348
Diethoxytetrahydrofuran R, 1324 Dinitrobenzene R, 1328
Diethoxytetrahydrofuran/acetic acid TS, 1324 Dinitrobenzene/ethanol TS, 1328
Diethyl ether, see Ether, anaesthetic 2.4- Dinitrochlorobenzene R, 1328
N,A^-Diethyl-/?-phenylenediamine sulfate R, Dinitrogen oxide, 350
1325 R, 1328
Diethyl phthalate R, 1 324 Dinitrogenii oxidum, 350
Diethylamine R, 1324 Dinonyi phthalate R, 1328
Diethylaminocellulose R, 325 1 Dioxan R, 1328
Diethylcarbamazine dihydrogen citrate, 335 1.4- Dioxane, 1328
RS, 1325, 1445 Diphenoxylate hydrochloride, 357
tablets, 1034 RS, 1329
Diethylcarbamazini dihydrogenocitras, 335 Diphenoxylati hydrochloridum, 357
Diethylcarbamazini dihydrogenocitratis Diphenyl ether R, 1329
compressi, 1034 Diphenylamine R, 1329
Diethylene glycol R, 1325 Diphenylamine/sulfuric acid TS, 1329
Diethylene glycol succinate R, 1325 Diphenylbenzidine R, 1329
Diethylphenylenediamine sulfate TS, 1325 1 .5- Diphenylcarbazide R, 1329
Diethyltoluamide, 338 Diphenylcarbazide TS, 1 329

RS,1325 Diphenylcarbazone R, 1330
Diethyltoluamidum, 338 Diphenylcarbazone/ethanol TS, 1330
Digitonin 1.2- Diphenylethylammonium 3-mercapto-2-
R, 1^ methylpropanoate RS, 1330
TS, 1325 5.5- Diphenylhydantoin, see Phenytoin
Digitoxin, 340 2.5- Diphenyloxazole R, 1330
RS, 1326, 1445 Dipotassium hydrogen phosphate R, 1 330
Digitoxinum, 34Q Diprazinum, see Promethazine
Digoxin, 342 hydrochloride
RS, 1326, 1445 Disintegration tests
Digoxinum, 342 capsules, 249, 250, 931, 1258
Dihydroartemisinin, see Artenimol suppositories, 962, 1259
1,8-Dihvdroxyanthraquinone, 1321 tablets, 266, 264 268, 1238
Diisopropyl ether R, 1326 Disodium chromotropate
Diloxanide furoate, 345 R, 1330
RS,1326 TS and TS (lOg/1), 1880
tablets, 1036 Disodium edetate, 348
Diloxanidi furoas, 345 R, 1881
Diloxanidi furoatis compressi, 1036 RS, 1331
Dimercaprol, 347 TS (lOto 50g/l),1881
Dimercaprolum, 347 VS (0.01 to 0.1 mol/1), 1880
Dimethyl 2,6-dimethyl-4-(2- Disodium hydrogen phosphate
nitrophenyl)pyridine-3,5-dicarboxylate anhydrous, R, 1 332
KS, 1326 R, 1881
Dimethyl 2, 6-dimethyl -4-(2- TSC28^tol00g/l), 1881
nitrosophenyl)pyridine-3,5-dicarboxylate TS (pH
Dissolution buffers, 18 to pH 7.2),
RS, 1326 1267
Dimethyl sulfoxide R, 1326 Dissolution media, 1 266
Dimethylacetamide R, 1326 Dissolution tests
Dimethylamine R, 326 1 capsules, 948, 1 262
Dimethylamine/ethanol TS, 1326 solid oral dosage forms, 1262
4-Dimethylaminobenzaldehyde tablets, 965, 1 262
R, 1326 Distribution, chemical reference substances,

TSl to TS6, 1326 1463

9
Index
5,5^-Dithiobis(2-nitrobenzoic acid) R, 1 ?<?>') Eosin Y

5,5'-Dithiobis-2-nitrobenzoic acid/methanol R, 1333
TS, 1332 TS (5g/l), 1333
Dithizone Ephedrine, 377
R, 1332 Ephedrine hydrochloride, 373
standard TS, 1332 Ephedrine sulfate, 37.5
TS, 1332 injection, 1040
Dithranoi, 359 RS, 1333
RS, 1332 Ephedrini hydrochloridum, 373
Dithranolum, 359 Ephedrini sulfas, 375
Domiphen bromide Ephedrini sulfatis injectio, 1040
R, 1332 Ephedrinum, 377
TS (lOg/1), 1332 4-Epianhydrotetracycline hydrochloride RS,
Dopamine hydrochloride, 363 1333, 1446
RS, 1445 6-Epidoxycycline hydrochloride RS, 1333
Dopamini hydrochloridum, 363 Epinephrine, 381
Dosage forms Epinephrine hydrogen tartrate, .379
general monographs, 947 R, 1333
Latin nomenclature, 3 Epinephrini hydrogenotartras, 379
specific monographs, 977 Epinephrinum, 381
see also sf>ecific dosage forms 4-Epitetracycline hydrochloride RS, 1334,
Doxorubicin hydrochloride, 365 1446
RS, 1 332, 1 445 Epsom salt, see Magnesium sulfate,
Doxorubicini hydrochloridum, 365 heptahydrate
Doxycycline hyclate, 367 Ergocalciferol, 384
RS. 1332 RS, 1334, 1446
tablets, 1 037 Ergocalciferolum, 384
Doxycyclini hyclas, 367 Ergometrine hydrogen maleate, 386
Doxycyclini hyclatis compressi, 1 037 injection, 1 041
Dragendorff reagent TS, 1332 RS, 1334, 1446
modified, 1 333 tablets, 1043
Dry-heat sterilization, 1 278 Ergometrine tartrate RS, 1446
Dry to constant mass, 3 Ergometrini hydrogenomaleas, 386
Drying, loss on, 3 Ergometrini hydrogenomaleatis
compressi, 1.043
Econazole nitrate RS, 1333 injectio, 1041
Edetate disodium, see Disodium Ergosterol R, 1334
edetate Ergotamine tartrate, 388
Edetic acid, 1220 RS, 1334
Edrophonii chloridum, 369 Ergotamini tartras, 388
Edrophonium chloride, 369 Eriochrome black, see Mordant black 11
Efavirenz RS, 1445 Eriochrome blue black, see Calcon
Effervescent tablets, 966 Erythromycin, 398, 1 234
Electrophoresis, 1199 B RS, 1446
cellulose acetate strips, 1 201 CRS, 1446
gel, 1201 RS, 1.334, 1446
moving boundary (free-flow), 1199 Erythromycin ethylsuccinate, 391
paper, 200
1 RS, 1.3.34
zone, 1199 tablets, 1045

Elements, 1 Erythromycin lactobionate, 393
Emetine hydrochloride, 371 RS, 1334
heptahydrate, 371 Erythromycin stearate, 395
pentahydrate, 371 RS, 1.3.34
RS, 1333, 1445 tablets, 1047

Emetini hydrochloridum, 371 Erythromycini ethylsuccinas, 391
Emission spectrometry, atomic, 1164 Erythromycini ethylsuccinatis compressi,
Endotoxin 1045
bacterial, test for (BET), 957, 1245 Erythromycini lactobionas, 393
RS,I333 Erythromycini stearas, 395
Enteric capsules, 951 Erythromycini stearatis compressi, 1047
Enteric-coated tablets, 968 Erythromycinum, .398

0
2 0 88
Eserine salicylate, see Physostigmine salicylate suppositories, 961

Estradiol benzoate RS, 1446 tablets, 963
Estrone RS, 1 334^ 1446 topical semi-solid dosage forms, 969
Ethambutol hydrochloride, 400 Expert Committee on Specifications for
RS, 1SS4, 1446 Pharmaceutical Preparations, ix
tablets, 1049 Expiry date, radiopharmaceuticals, 1 134
Ethambutoli hydrochloridi compressi, 1049 Extended-release capsules, 950
Ethambutoli hydrochloridum, 400 Extended-release tablets, 968
Ethanal, see Acetaldehyde Extractable volume for parenteral
Ethanol, 402 preparations, test for, 1273
aldehyde-free TS, 1335 Eye preparations, see Ophthalmic
dehydrated R, 1335 preparations
neutralized TS, 1335
sulfate-free TS, 1335 Fats
TS (-150 to -750 g/1), 1334 hard, 5Z
Ethanolum, 402 melting point, 1146
Ether sterilization, 1 1ll
anaesthetic, 404 Ferric ammonium sulfate
peroxide-free R, 1335 R, 1338
R, 1335 TS (45 g/1), 133Z
Ether anaesthesicus, 404 TSl and TS2, 1338
Ethinylestradiol, 406 VS (0.1 mol/1), 1337
RS, 1335, 1446 Ferric chloride
Ethinylestradiolum, 406 R, 1338
Ethionamide, 408 TS(25to 65 g/1), 1338
RS, 1335 Ferric chloride/ferricyanide/arsenite TS, 1338
Ethionamidum, 408 Ferric chloride/potassium ferricyanide TS,
Ethisterone RS, 1446 1338
Ethosuximide, 41 Ferricyanide standards (50pg/ml and
RS, 1335, 1446 100pg/ml)TS, 1338
Ethosuximidum, 41 Ferroin TS, 1339
Ethyl acetate R, 1 335 Ferrosi fumaras, 41
Ethyl alcohol, see Ethanol Ferrosi sulfas, 420
Ethyl aminobenzoate, 150 Ferrous ammonium sulfate
Ethyl ether, see Ether, anaesthetic R, 1339
Ethyl hydroxybenzoate, 413 TS (1 g/1), 1339
Ethyl iodide R, 1 336 VS (0.1 moM), 1339
Ethylcellulose, 41 Ferrous fumarate, 41
Ethylcellulosum, 412 Ferrous salts, identification, 1208
Ethylene glycol monoethyl ether R, 1336 Ferrous sulfate, 420
Ethylene glycol monomethyl ether R, 1336 exsiccated, 420
Ethylene oxide heptahydrate, 420
R, 1336 R, 134Q
sterilization, 1280 TS (Zand 15 g/1), 1339
stock solution R, 1336 VS (0.1 mol/1), 1339
TS. 1336 Ferrous sulfate/hydrochloric acid TS, 1340
Ethylenediamine R, 1337 Film-coated tablets, 967
Ethylenediaminetetraacetic acid, see Edetic Filtration, sterilization by, 1 279
acid Firebrick, pink, R, 1340
Ethylis hydroxybenzoas, 413 Flame photometry, 1 164
Ethylmethylketone R, 1337 Flucloxacillin sodium RS, 1340, 1446
Ethylparaben, see Ethyl hydroxybenzoate Flucytosine, 422
1-Ethylquinaldinium iodide RS, 1340, 1446
R, 133Z Flucytosinum, 422
TS (15g/l), 133Z Fludrocortisone acetate, 424
Etoposide, 415 RS, 1340, 1446
RS, 1337 Fludrocortisoni acetas, 424
Etoposidum, 415 Fluid mercaptoacetate (thioglycolate)
Excipients, 4 medium, 1319
capsules, 947 with penicillinase, 1319
parenteral preparations, 956 Fluorescein sodium, 426

Index
Fluoresceinum natricum, Gels, 922

Fluorescence emission spectrum, 1 1 66 hydrophilic, 972
Fluorescence excitation spectrum, 1 166 hydrophobic, 972
Fluorescence intensity, 1 166 General notices, iv, 1
Fluorescence spectrophotometry, 1 166 appendices, 13
Fluorine, oxygen flask method, 1219 General requirements, v, 6
5-Fluorocytosine, see Flucytosine Generic names, see International
Fluoroquinolonic add RS, 1.^40 Nonproprietary Names
Fluorouracil, 430 Gentamicin sulfate, 445
RS, 1340, 1446 non-injectable, 445
Fluorouracilum, 430 RS, 1342 ,1446
Fluphenazine decanoate, 432 sterile, 445
RS, IMQ Gentamicini sulfas, 445
Fluphenazine decanoate dihydrochloride RS, Gentian violet, see Methylrosanilinium
1446 chloride
Fluphenazine enantate, 434 Glacial acetic acid, see Acetic acid, glacial
RS, 1340 Glassware, tests and assays, S
Fluphenazine enantate dihydrochloride RS, Glauber's salt, see Sodium sulfate
1446 Glibenclamide, 447
Fluphenazine hydrochloride, 436 RS, 1342
RS, 1 340, 1 446 Glibenclamidum, 447
Fluphenazini decanoas, 432 Glucose, 449
Fluphenazini enantas, 434 anhydrous, 449
Fluphenazini hydrochloridum, 436 anhydrous R, 1343
Folic acid, 39. monohydrate, 449
RS. 1340, 1446 Glucose hydrate R, 1342
Formaldehyde TS, 1340 Glucosum, 449
Formaldehyde/sulfuric acid TS, 1340 Glyburide, see Glibenclamide
Formamide R, 1 340 Glycerin, see Glycerol
Formic acid Glycerol, 453
anhydrous R, 1 340 35% m/m, 456
TS (~1080g/l), 1340 dilute, 456
Formulae, chemical, 3^ 1441 R, 1343
3-Formylrifamycin RS, 1341, 1 446 Glyceroli monostearas, 451
Framycetin sulfate RS, 1341, 1446 Glycerolum, 453
Fuchsin 85% m/m, 456
basic, R, 1341 Glyceryl monostearate, 451
decolorized, TS, 1341 Glyceryl trinitrate tablets, 1 051
TS, 1341 Glycerylis trinitratis compressi, 1051
Fuchsin/sulfurous acid, 1342 Glycine R, 1343
Furadoninum, see Nitrofurantoin Glyoxal bis(2-hyroxyanil)
Furosemide, 438 R, 1343
RS, 1342, 1446 TS, 1343
Furosemidum, 438 Glyoxaline, see Imidazole
Gold-198, 1121
Gallamine triethiodide, 441 Gold-199, 1121
RS, 1342 Graphic representation of chemical formulae,
Gallamini triethiodidum, 441 1441
Gallium-67, 1121 Green stock standard TS, 1170, 1 344
Gamma benzene hexachloride, see Lindane Griseofulvin, 458
Gammahexachlorcyclohexane, see Lindane RS, 1344, 1446
Gas chromatography, 1193 tablets, 1 053
recommended procedure, 1 196 Griseofulvini compressi, 1053
static head-space, 1197 Griseofulvinum, 458
Gas sterilization, 1 280 Gum arable, see Acacia
Gastric fluid, simulated, TS, 1267 Gummi arabicum, 460
Gel electrophoresis, 1201
Gelatin, 443 Half-life period, radioactivity, 1115
R, 1342 Halogens
TS, 1342 in medicinal gases, 355
Gelatina, 443 oxygen flask method, 1218

Haloperidol, 462 VS (0 0001 to 0.05 mol/1), 1347

RS, 1344, 1446 VS (OIL to 5 mol/1), 1348
Haloperidolum, 462 Hydrochloric acid/ethanol (01 and 1 mol/l)
Halothane, 464 VS, 1349
Halothanum, 46^ Hydrochloric acid/methanol (0.01 mol/l) VS,
Hard capsules, 948 1349
Hard fat, 57 Hydrochlorothiazide, 480
Hard paraffin, 718 RS, 1.349, 1446
Health Organization of the League of Hydrochlorothiazidum, 480
Nations, ix Hydrocortisone, 487
Heavy metals, limit test, 1212 R, 1 349
Helium R, 1344 RS, 1349, 1446
Heparin RS, 1344 Hydrocortisone acetate, 482
Heparin calcium, 467 RS, 1349, 1446
Heparin sodium, 470 Hydrocortisone sodium succinate, 485
Heparinized saline TS, 1344 RS, 1349, 1446
Heparinum calcicum, 467 Hydrocortisoni acetas, 482
Heparinum natricum, 470 Hydrocortisoni natrii succinas, 485
Heptane R, 1344 Hydrocortisonum, 487
Hermetically closed containers, 5 Hydrogel, 972
Hexamethyldisilazine R, 1344 Hydrogen peroxide (-60 and -330 g/1) TS,
Hexane R, 1344 1349
Hexaneamine, see Hexylamine R Hydrogen sulfide
Hexylamine R, 1 344 in medicinal gases, 355
High-performance liquid chromatography R, 1349
(HPLC), 1184 TS. 1349
apparatus, 1 1 84 Hydrophilic creams (o/w), 972
recommended procedure, 1192 Hydrophilic gels, 972
reverse-phase, 1 1 86 Hydrophilic ointments, 97.3
revision, vi Hydrophobic creams (w/o), 971
system suitability, 1188 Hydrophobic gels, 972
Histamine Hydrophobic ointments, 972
strong, TS, 1345 Hydroquinone R, 1 350
TS, 1345 Hydroxocobalamin, 493
Histamine dihydrochloride R, 1344 anhydrous, 493
Histamine-like substances, test for, 1249 hydrate, 493
Histamine phosphate R, 1345 Hydroxocobalamin chloride, 489
History of Imermtional Pharmacopoeia, ix Hydroxocobalamin sulfate, 489
Holmium oxide R, 1345 Hydroxocobalamini chloridum, 489
Holmium perchlorate TS, 1345 Hydroxocobalamin! sulfas, 489
Homoatropine hydrobromide, 473 Hydroxocobalaminum, 493
Homoatropine methylbromide, 475 (-)-3-(4-Hydroxy-3-methoxyphenyl)-2-
Homoatropini hydrobromidum, 473 hydrazino-2-methylalanine RS, 1 350,
Homoatropini methylbromidum, 475 1446
Hydralazine hydrochloride, 477 (— )-3-(4-Hydroxy-3-methoxyphenyl)-2-
Hydralazini hydrochloridum, 477 methylalanine RS, 1350, 1446
Hydrargyri oxycyanidum, 479 1-Hydroxy -9-anthrone RS, 1350
Hydrazine hydrate R, 1345 Hydroxyethylcellulose, 496
Hydrazine sulfate R, 1346 R, 1550
Hydriodic acid (-970 g/1) TS, 1 346 TS, 1350
Hydrochloric acid, 41 Hydroxyethylcellulosum, 496
AsTS (-250 g/1), 1546 Hydroxyl value, determination, 1254
BET (01mol/i),1547 Hydroxylamine hydrochloride
brominated, AsTS, 1349 R, 1551
CITS, 1548 TS(70and 200 g/I), 1551
dilute, 45 TS and TS2, 1351
FeTS (-250 g/1), 1346 Hydroxypropylcellulose, 497
stannated, AsTS (-250 g/1), 1347 Hydroxypropylcellulosum, 497
TS (~70g/l),I54Z 8-Hydroxyquinoline R, 13.51
TS (-250 g/1), 1548 8-Hydroxyquinoline/chloroform TS,
TS (-330 and -420 g/1), 1546 1.351

2
Index
Hypophosphorous acid INN, see International Nonproprietary

dilute, TS, 1351 Names
R, 1351 Insulin, 515
Hypoxanthine R, 1351 beef, 515
Hypromellose, 499 pork, 515.
Hypromellosum, 499 Insulinum, 515
International Biological Standards, 12
Ibuprofen, 500 International Chemical Reference Substances
RS, 1352,1442 (ICRS), 11^ 1452
tablets, 1055 list of, 1443
Ibuprofeni compressi, 1055 see also Chemical reference substances
Ibuprofenum, 500 International Infrared Reference Spectra, 12,
ICRS, see International Chemical Reference 1450
Substances International Laboratory for Biological
Identity tests, Z Standards, 12
for chemical reference substances, 1457 International Nonproprietary Names (INN),
general, 1 205 1441
use of chemical reference substances, 1 455 graphic formulae, 1441
Idoxuridine, 503 Latin forms, 3
RS, 1352 Programme, x
Idoxuridinum, 503 International Pharmaceutical Federation, x
Ignite to constant mass, S International Pharmacoj?oeia (Ph. Int.)
Imidazole history, ix
R,1352 scope and function, vi
recrystallized, R, 1 352 edition, x
first
Imidazole/mercuric chloride TS, 1352 second edition, x

Iminodibenzyl R, 1 352 third edition, xi
Imipramine hydrochloride, 505 fourth edition, iv, xi
RS, 1352, 1442 International System of Units (SI), 16
Imipramini hydrochloridum, 505 International Union of Pure and Applied
Imizine, see Imipramine hydrochloride Chemistry (lUPAC), 3^ 1441
Implants, 960 International Units (lU), 7, 1 230
Impurities, IQ Intestinal fluid, simulated, TS, 1267
chromatography, Ifl, 1 178 Intravenous infusions, 959
new general notice, v extractable volume, .1 274
scope of monograph requirements, vii visual inspection, 1274
Indicators, visual determination of pH values, lodide(s)
2 identification, 1 209
Indinavir RS, 1352 standard (20pgl/ml) TS, 1353
Indinavir sulfate, 508 516
Iodine,
Indinavir! sulfas, 508 oxygen flask method, 1219
Indium-111, 1121 R, 1354
Indium-1 13m, 1121 TS, 1354
Indometacin, 512 value, determination, 1251
RS, 1352, 1447 VS (0.0001 to 0.1 mol/1), 1353
tablets, 1055 Iodine-123, 1121
Indometacini compressi, 1056 Iodine-125, 1122
Indometacinum, 51 Iodine-126. 1122
Infrared spectra, Z Iodine-131, 1122
reference, 1 2, 1 450 Iodine-132, 1 122
Infrared spectrophotometry, 1 160 Iodine bromide
Infusions, parenteral R, 1353
extractable volume, 1274 TS,1354
intravenous, 959 Iodine/ chloroform TS, 1354
visual inspection, 1274 lodine/ethanol TS, 1 354
Injections, 959 Iodine pentoxide R, 1354
extractable volume, 1 273 lodinum, 516
multidose, 959 lohexol, 518
powders for see Powders for injections RS, 1354
single-dose, 959 lohexolum, 518
visual inspection of particulate matter, 1274 Ion-exchange chromatography, 1 183

11
Ionizing radiation, sterilization by, 1 779 Labelling, 6

lopanoic acid, 44 capsules, 948
RS, 1R54 chemical reference substances, 1462
lotroxic acid, 46 ophthalmic preparations, 953
KS, 135A parenteral preparations, 95.8
Ipecacuanha root, 520 radiopharmaceuticals, 1 1 35
Ipecacuanhae radix, 520 suppositories, 962
Iron tablets, 965, 966
1214
limit test, topical semi-solid dosage forms, 970
reduced, R, 1355 Lactic acid, 4S
standard FeTS, 1355 Lactobionic acid R, 1357
Iron-55, 123 1 Lactose, 544
Iron-59. 1123 anhydrous, 544
Iron colour monohydrate, 544
strong, TS, 1 354 Lactosum, 544
TS, 1354 Lanatoside C RS, 1447
Iron(2+) fumarate, see Ferrous fumarate Lanolin, 55
Iron salicylate TS, 1 355 anhydrous, 55
Iron(2+) sulfate, see Ferrous sulfate Lanthananum nitrate
Isobutyl methyl ketone R, 1 355 R, 135Z
Isoniazid, 523 TS (30g/l), 1357
R, 1355 Latin names, x, 3
RS, 1355, 1447 Lead
tablets, 1058 complexometric titration, 1223
TS, 1 355 dilute, PbTS, 1353
Isoniazidi compressi, 1058 strong, PbTS, 1358
Isoniazidum, 523 Lead acetate
Isonicotinic acid hydrazide, see Isoniazid paper R, 1357
Isoprenaline hydrochloride, 525 TS (80g/l), 1357
Isoprenaline sulfate, 527 Lead nitrate
Isoprenalini hydrochloridum, 525 paper R, 1358
Isoprenalini sulfas, 527 VS (0.05 to 100 mol/1), 1357
Isopropyl alcohol, see 2-Propanol Lead(IV) oxide R, 1 358
Isopropylamine R, 1 355 Lead subacetate TS, 1358
Isosorbide dinitrate, diluted, 529 League of Nations, ix
Isosorbidi dinitras dilutus, 529 Leucovorin calcium, see Calcium folinate
lUPAC, see International Union of Pure and Levamisole hydrochloride, 546
Applied Chemistry RS, 1353
Levamisoli hydrochloridum, 546
K,1192i Levarterenol hydrogen tartrate R, 1358
Kalii chloridum, 532 Levodopa, 548
Kalii citras, 534 RS, 1358, 1447
Kalii iodidum, 536 Levodopum, 548
Kanamycin monosulfate RS, 1355, 1447 Levonorgestrel, 551
Kaolin, v538. RS, 1359,1447
light, R, 1355 Levonorgestrelum, 551
suspension TS, 1355 Levothyroxine sodium, 553
Kaolinum, 538 RS, 1359, 1447
Karl Fischer method, titrimetric determination Levothyroxinum natricum, 553
of water, 1 227 Lidocaine, 558
Karl Fischer reagent TS, 1355 RS, 1359, 1447
Ketamine hydrochloride, 540 Lidocaine hydrochloride, 555
RS, 1356 RS, 1447
Ketamini hydrochloridum, 540 Lidocaini hydrochloridum, 555
Ketoconazole, 542 Lidocainum, 558
RS. 1356 Light, protection from, 5
Ketoconazolum, 542 Limit tests, 121
Khartoum senna leaf, see Alexandrian senna arsenic, 1216
leaf chlorides, 1 2,1
Kieselguhr R1 to R5, 1356 heavy metals, 1212

1
Index
iron, 1 0A 4 Magnesium sulfate

sulfates, 121 heptahydrate, 571
Limulus amoebocyte lysate, 1.259 R, 1362
Lincomycin hydrochloride RS, 1359 TS (50g/l), 1.362
Lindane, 56.(1 Magnesium sulfate/sulfuric acid TS, 1362
RS, 1359 Malachite green G, see Brilliant green
Lindanum, 56Q Maleic acid R, 1 363
Liothyronine RS, 1359 Manganese dioxide R, 1 36.3
Liothyronine sodium RS, 1447 Manganese/silver paper R, 1363
Lipophilic ointments, 972 Manganese sulfate
Liquid paraffin, R, 1376 R, 1363
Liquids, colour of, 1170 TS n5g/l), 1363
Lithii carbonas, 562 Mannitol, 573
Lithium R, 1360 Mannitolum, 573
Lithium carbonate, 562 Manufacture, 4
R, 1359 capsules, 949, 950, 951
Lithium carbonate/trinitrophenol TS, new general notice, v
1 359 ophthalmic preparations, 951
Lithium chloride parenteral preparations, 956
R, 1359 quality assurance, vii
TS (lOg/1), 155R suppositories, 961
Lithium methoxide (0.1 mol/1) VS, 1 359 tablets, 963
Lithium perchlorate R, 1360 topical semi-solid dosage forms, 969
Lithium perchlorate/acetic acid TS, 1360 Manufacturers' release specifications, vii
Litmus Mass
paper R, 1360 measurement, 1 141
R, 1360 uniformity of see Uniformity of mass
T.S, 1360 Mass density, 14
Loperamide hydrochloride, -563 determination, 1 1 50
RS, 1360, 1447 Mebendazole, 575
Loperamidi hydrochloridum, 563 RS, 1 363, 1447
Loss on drying, S tablets, 1 059
Mebendazoli compressi, 10.59
Macrogol 2fl R, 1360M Mebendazolum, 575
Macrogol 20.0 Medicines website, WHO, see World Health
R, 1360 Organization, Medicines website
TS, 1360 Medroxyprogesterone acetate, 577
Macrogol 400 R, 1361 RS, 1363
Macrogol 1000 R, 1360 Medroxyprogesteroni acetas, 577
Macrogol /^-isooctylphenyl ether R, 1362 Mefloquine hydrochloride, 580
Magnesii hydroxidum, 565 RS, 1363
Magnesii oxidum, 567 Mefloquini hydrochloridum, 580
Magnesii stearas, 569 Meglumine, .583
Magnesii sulfas heptahydras, 571 R, 1.363
Magnesium TS (100 g/1), 1363
complexometric 1223titration, Megluminum, 583
standard (lOpg/mlMg) TS, 1362 Melarsoprol injection, 1061
TS (0.1 mg/ml Mg), 1M2 Melarsoproli injectio, 1 061
Magnesium acetate R, 1362 Melting point reference substances, 1 447
Magnesium aluminosilicate, see Aluminium Melting range, determination, 1 143
magnesium silicate Melting temperature, 2
Magnesium chloride determination, 1 1 43
R 1 367. Menadione R, 1.364
VS (0.1 mol/1), mi Meperidine hydrochloride, see Pethidine
Magnesium hydroxide, 565 hydrochloride
Magnesium oxide, 567 Mercaptoacetate medium, fluid, see Fluid
heavy, 567 mercaptoacetate medium
light, 567 Mercaptoacetic acid R, 1364
Magnesium oxide R, 1362 Mercaptopurine, 585
Magnesium stearate, 569 Mercaptopurinum, 585

1
Mercuric acetate R, 1 ^(^4 Methylamine hydrochloride

Mercuric acetate/acetic acid TS, 1364 R, 1368
Mercuric bromide TS (20g/l), 1362
AsTS, 1364 Methylcellulose, 591
paper AsR, 12£A Methylcellulosum, 59T
R, 1364 Methyldopa, 592
Mercuric chloride RS, 1368, 1447
R, 1B64 Methyldopum, 592
TS{ând 65 p/1V 1364 Methylene blue, see Methylthioninium
Mercuric chloride/ethanol TS, 1364 chloride
Mercuric iodide R, 1364 Methylene chloride, 1323
Mercuric nitrate Methylis hydroxybenzoas, 595
R, IMS Methylisobutylketone R, 1368
TS, 1365 Methylparaben, see Methyl
VS (Qill and 0.02 mol/1), IMS hydroxybenzoate
Mercuric oxide, yellow, R, 1365 2-Methylphenol, 1317
Mercuric oxycyanide, 479 A/-Methylpiperazine R, 1367
Mercuric sulfate TS, 1 365 2-Methylpropan-2-ol, see /er/-Butanol
Mercury R, 1365 Methylrosanilinii chloridum, 596
Mercury-197, 1123 Methylrosanilinium chloride, 596
Mercury-197m, 1124 see also Crystal violet
Mercurv-203, 1124 Methyltestosterone, 599
Mercury/nitric acid TS, 1365 RS, 1368, 1447
Metacycline hydrochloride RS, 1366 Methyltestosteronum, 599
Metals, heavy, limit test, 1212 Methylthioninii chloridum, 601
Methanesulfonic acid R, 1366 Methylthioninium chloride, 601
Methanol R, 1368
dehydrated, R, 1366 TS (0.2 g/1 and lg/1), 1368
R, 1266. Methylthymol blue
Methenamine R, 1366 mixture R, 1368
Dl-Methionine, 587 R, 1362
RS, 1366 Meticillin sodium RS, 1447
DTMethioninum, S8Z Metoclopramide hydrochloride, 604
Methods of analysis, v, T_^ 1 137 RS, 1368
biological, 123.0 Metoclopramidi hydrochloridum, 604
chemical, 1206 Metrifonate, 606
materials of plant origin, 1251 Metrifonatum, 606
pharmaceutical technical procedures, Metronidazole, 609
1 2.55 injection, 1063
physical and physicochemical, 1141 RS, 1369, 1447
Methotrexate, 589 tablets, 1065
RS, 1366, 1447 Metronidazole benzoate, 607
Methotrexatum, 589 RS, 1368
4-Methoxybenzaldehyde, 1294 Metronidazoli
Methoxyl, determination, 1 228 compressi, 1.065
2-Methyl-5-nitroimidazole R, 1368 injectio, 1063
Methyl green R, 1366 Metronidazoli benzoas, 607
Methyl green/iodomercurate paper R, 1367 Metronidazolum, 609
Methyl hydroxybenzoate, 595 Michler's ketone, 1300
W-Methyl-N-nitrosotoluene-4-sulfonamide R, Miconazole nitrate, 61
1367 RS, 1369
Methyl orange Miconazoli nitras, 611
ethanol TS, 1367 Microbial purity of pharmaceutical
R, IMZ preparations, 1243
Methyl orange/acetone TS, 1 367 Microbiological assay of antibiotics, 1 230
Methyl red R, 1367 Micrococcus luteus, 1233
Methyl red/ethanol TS, 367 1 Microcrystalline cellulose, 219
Methyl red/methylthioninium chloride TS, Modified-release capsules, 950
136Z Modified-release tablets, 968
Methyl silicone gum R, 1367 Mol/1,9,14
Methyl violet 2B R, 1368 Molar absorptivity, 1 1 55

8
Index
Molecular mass, relative, 3 W-(l-Naphthyl)ethylenediamine/propylene

Molybdenum-99, 1124 glycol TS, 1370
Molybdenum trioxide R, 1369 National Collection of Type Cultures
Monoethanolamine (NCTC), 14, 1232
R, 1362 National Collection of Yeast Cultures
VS (0.1 mol/1), 1362 (NCYC), 14, 1232
Monographs National Collections of Industrial and Marine
dosage forms, 947 Bacteria Ltd (NCIMB), 14
future revisions, vi Natrii amidotrizoas, 621
new, V Natrii calcii edetas, 623
nomenclature, 3 Natrii chloridum, 626
pharmaceutical substances, 23 Natrii citras, 628
radiopharmaceuticals, 1115 Natrii cromoglicas, 630
revisions, v Natrii fluoridum, 632
Monostearin, see Glyceryl monostearate Natrii hydrogenocarbonas, 633
Mordant black 11,. 1221 Natrii hydroxydum, 635
1362
indicator mixture R, Natrii nitris, 637
1362
R, Natrii nitroprussidum, 638
Morphine hydrochloride, 613 Natrii salicylas, 641
Morphine sulfate, 615 Natrii stibogluconas, 642
tablets, 1066 Natrii sulfas, 644
Morphini hydrochloridum, 613 anhydricus, 646
Morphini sulfas, 615 Natrii thiosulfas,648
Morphini sulfatis compressi, 1066 Natrii valproas, 650
Morpholine R, 1369 Neamine hydrochloride RS, 1371, 1 448
Myelosanum, see Busulfan Nelfinavir mesilate, 653
RS, 1371
n 1154
(refractive index), 14,. Nelfinaviri mesilas, 653
Nafcillin sodium RS, 1 443 Neomycin, 1 234
1-
Nagananinum, see
2- Suraminum natricum Neomycin B sulfate, see Framycetin
Naloxone hydrochloride, 618 sulfate
anhydrous, 61 Neomycin sulfate, 656
dihydrate, 618 Neomycini sulfas, 656
RS, 1370 Neostigmine bromide, 659
Naloxoni hydrochloridum, 618 Neostigmine metilsulfate, 660
Names RS, 1371, 1448
chemical, 3 Neostigmini bromidum, 659
elements, 12 Neostigmini metilsulfas, 660
international nonproprietary see Nephelometry, 1169
International Nonproprietary Names Nessler's reagent, 1291
Latin, x, 3 Neutral red R, 13Z1
other, 4 Neutral red/ethanol TS, 1371
Naphthalene- 1 ,3-diol R, 1 370 Nevirapine, 663
Naphthalene-1, 3-diol/ethanol TS, 1370 anhydrous, 663
1-NaphthoI anhydrous RS, 1 37 1.
R; 1370 hemihydrate, 663

TS1,13ZQ impurity B RS, 1371
Naphthol/ethanol TS, 1371 RS, 1448
Naphthol Nevirapinum, 663
R, 1370 Niclosamide, 667
TS1,13ZQ anhydrous, 667
1-Naphtholbenzein R, 1 370 monohydrate, 667
1-Naphtholbenzein/acetic acid TS, 1370 RS, 1371
N-(l-Naphthyl)ethylenediamine/ethanol TS, tablets, 1067
1369 Niclosamidi compressi, 1067
N-{\ -Naphthyl)ethylenediamine Niclosamidum, 667
hydrochloride Nicotinamide, 669
R, 1362 RS, 1371, 1448
TS(land5g/l), 1362 Nicotinamidum, 669
A/-(l -Naphthyl)ethylenediamine Nicotinic acid, 51
hydrochloride/1 -propanol TS, 1369 RS, 1371, 1448

Nifedipine, £Z1 Nonoxinol % 683

RS, 1371 RS, 1373
Nifedipinum, 671 Nonoxinolum % 683
Nifurtimox, 674 Norethindrone enantate, see Norethisterone
RS, 1^144a enantate
Nifurtimoxum, 674 Norethisterone, 690
Ninhydrin, see Triketohydrindene hydrate RS, 1373, 1448
Niridazole, 676 Norethisterone acetate, 686
RS, 1371, 1448 RS, 1373, 1448
Niridazole-chlorethylcarboxamide RS, 1371, Norethisterone enantate, 688
144R RS, 13 73
Niridazolum, 676 Norethisteroni acetas, 686
209
Nitrates, identification, 1 Norethisteroni enantas, 688
Nitratophenylmercury, see Phenylmercuric Norethisteronum, 690
nitrate Noscapine, 694
Nitrazepam, 678 RS, 1373
Nitrazepamum, 678 Noscapine hydrochloride, 692
Nitric acid Noscapini hydrochloridum, 692
fuming, R, 1372 Noscapinum, 694
TS (-130 and -1000 g/1), 1371 Novobiocin, 1 234
TS(3andl5g/l), 13Z2 Nuclide, 11 15
VS (Oils and 1 mol/1), 1371 Nystatin, 696, 1 235
1226
Nitrite titration, RS, 1373, 1448
4-Nitroaniline tablets, 1071
R, 1372 Nystatini compressi, 1071
TSl and TS2, 1372 Nystatinum, 696
Nitrobenzene R, 1 372
4-Nitrobenzoyl chloride R, 1372 /so-Octane, see 2,2,4-Trimethylpentane
Nitrofurantoin, 680 Octanoic acid R, 1 374
anhydrous, 680 Oil detector tube, 1374
monohydrate, 680 Oils
RS, 1372 peroxides in fixed, 1 252
tablets, 1Q6.S sterilization, 1 277
Nitrofurantoin! compress!, 1 069 see also individual named oils
Nitrofurantoinum, 680 Ointments, 972

Nitrogen hydrophilic, 973
for chromatography R, 1373 hydrophobic (lipophilic), 972
determination, 1229 ophthalmic, 955
oxygen-free, R, 1373 water-emulsifying, 973
R, 13Z3 Oleogel, 972
Nitrogen dioxide Oleum arachidis, 698
detector tube, 1372 Olive oil R, 1374
in medicinal gases, 353 Opalescence standards TSl to TS3, 1374
Nitrogen monoxide Opalescence stock standard TS, 1374
detector tube, 1.>3.7.2 Ophthalmic preparations, 951
in medicinal gases, 353 drops, 953
R, ldZ2 emulsions, 954
Nitroglycerin tablets, see Glyceryl trinitrate ointments, 955
tablets suspensions, 955
Nitromethane R, 1 373 Optical rotation
Nitrophenanthroline determination, 1151
R, 1373 specific, 13,. 1151
TS,1R23 Oracet blue B R, 1374
l-Nitroso-2-naphthol-3, 6-disodium Oracet blue B/acetic acid TS, 1374
disulfonate Oral dosage forms, solid
R, 1373 dissolution test, 1262
TS (2 g/1), 1373 see also Capsules; Tablets
Nitrous acid, sodium salt, see Sodium Oral powders
nitrite uniformity of content, 1 256
Nitrous oxide, see Dinitrogen oxide uniformity of mass, 1257
Nomenclature, monograph, 3 Oral rehydration salts (ORS), v, 1072

R
Index
Organoleptic inspection Paraldehyde R, 1 376

ophthalmic preparations, 955 Parenteral infusions, see Infusions,
topical semi-solid dosage forms, 970 parenteral
Orthophosphates, identification, 1209 Parenteral preparations, 956
ORS, see Oral rehydration salts extractable volume, test for, 1273
Osmium tetroxide R, 1 375 implants, 960
Other names, 4 injections, 959
Ouabain RS, 1448 intravenous infusions, 95.9
Ox brain, acetone-dried, R, 1375 powders for injections, 960
Oxacillin, 1235 revision, vi
Oxacillin sodium RS, 1 44 8 sterility test, 957, 1236
Oxalic acid visual inspection, 957, 1274
R 1 !875 Paromomycin sulfate, 719
TS (0.05 g/1), 1375 RS, 1376, 1448
Oxalic acid/sulfuric acid TS, 1375 Paromomycini sulfas, 719
Oxamniquine, 699 Particle size, ophthalmic preparations,
RS, 13Z5 952
Oxamniquinum, 699 Particulate matter, visual inspection of
Oxygen, 701 injectable preparations, 1274
Oxygen flask method, 1218 Partition coefficient (K), 1 1 93
Oxygenium, 701 Partition column chromatography, 1 183
Oxytetracycline, 1 23.5 Parts per million by weight (ppm), lH
Oxytetracycline dihydrate, 707 Pastes, 973
non-injectable, 707 Patents, IQ
RS, 1375, 1448 Peanut oil, see Arachis oil
sterile, 707 Penicillamine, 721
Oxytetracycline hydrochloride, 710 Penicillaminum, 721
non-injectable, 710 Penicillin G benzathine, see Benzathine
RS, 137.5, 1448 benzylpenicillin
sterile, 710 Penicillin G procaine, see Procaine
Oxytetracyclini dihydras, 707 benzylpenicillin
Oxytetracyclini hydrochloridum, 710 Penicillinase
R, 1326
Packaging, chemical reference substances, TS, 1377
1460 Pentamidine isetionate, 723
Pancreatic digest of casein R, 1375 powder for injections, 1078
Papaic digest of soybean meal R, 1375 RS, IhZZ
Papaverine hydrochloride, 713 Pentamidine mesilate, 725
RS, 1375,1448 Pentamidini isetionas, 723
Papaverini hydrochloridum, 713 Pentamidini isetionatis pulvis ad injectionem,
Paper chromatography, 1 1 80 1078
ascending, 1181 Pentamidini mesilas, 725
descending, 1 181 H-Pentane R, 1377
Paper electrophoresis, 1200 1-Pentanesulfonic acid TS, 1377
Paracetamol, 715 1-Pentanesulfonic acid sodium salt R,
4-aminophenol-free, R, 1375 1377
R, iaZ5 Peptone
RS, 1448 dried, R, 13ZS
tablets, 1075 Rl,137a
Paracetamol! compressi, 1 075 TS (5 g/1), 1378
Paracetamolum, 715 TSl and TS2 (lg/1), 13Z8
Paraffin Percentage (%)
hard, 718 expression of concentration, 9
liquid, R, 1376 impurities, IQ
wax see Paraffin, hard Perchloric acid
white, 717
soft, TS. 1379
yellow, soft, 717 TS (-140 and -11 70 g/1), 13ZS
Paraffinum album, 717 VS (0112 to 0.1 mol/1), 1378
Paraffinum durum, 71 Perchloric acid/dioxan (0.1 mol/1) VS, 1 379
Paraffinum flavum, 717 Period of use, chemical reference substances,
Paraformaldehyde R, 1376 1463

Periodic-acetic acid TS, 1 .^79 2-Phenylethanol

Peroxides in fixed oils, determination, 1 252 R, 1381
Pethidine hydrochloride, 727 js,mi
tablets, 1 080 Phenylhydrargyri nitras, 740
Pethidini hydrochloridi compress!, 1Q8.Q Phenylhydrazine R, 1382
Pethidini hydrochloridum, 727 Phenylhydrazine hydrochloride
Petrolatum, see Paraffin R, 1381
Petroleum, light, R and Rl, 1379 TS (10 g/1), 1381
pH, 14, 1172 Phenylhydrazine/hydrochloric acid TS, 1382
accuracy and precision, 9. Phenylhydrazine/sulfuric acid TS, 1382
determination, 1172 Phenylmercuric nitrate, 740
indicator paper R, 1382 Phenytoin, 741
indicators for visual determination, 9 RS, 1382, 1448
Pharmaceutical preparations, microbial purity, Phenytoin sodium, 743
1 243 tablets, 1 085
Pharmaceutical substances, 23 Phenytoin! natrici compress!, 1085
names see Names Phenytoinum, 741
Pharmaceutical technical procedures, 1 255 Phenytoinum natricum, 743
Pharmacofpoea Internationalis, x Phloroglucinol R, 1382
Phase solubility analysis, 1 202 Phosphate buffers
Phenacetin, 1447 pH 4il to Mi TS, 1882
c-Phenanthroline sterile, pH 4*5 to 1 0.5, TS (and TSl to TS3),
R, 1BZ9 1383
TS and TS (lg/1), iaZ9 Phosphate/citrate buffers, pH 4*5 and 5.0, TS,
Phenobarbital, 729 1385
RS, 1380 Phosphate standard
tablets, 1 082 buffers, pH 5.8 and 7.4, TS, 1385
Phenobarbital sodium, 731 TS (5 pg/ml), 1385
Phenobarbital! compress!, 1 082 Phosphomolybdic acid
Phenobarbitalum, 729 R, 1885
Phenobarbitalum natricum, 731 TS (80 g/1), 1885
Phenol Phosphomolybdic acid/ethanol TS, 1386
R, lasn Phosphoric acid (-2.8 to -1440 g/1) TS, 1386
TS (50g/l), 1380 Phosphorus, red, R, 1386
Phenol red R, 13.8Q Phosphorus-32, 1125
Phenol red/ethanol TS, 1380 Phosphorus pentoxide R, 1386
Phenoldisulfonic acid TS and (250 g/1) TS, Phosphotungstic acid TS, 1386
1380 Phthalate buffers, pH 8*4 to 4.0, TS, 1386
Phenolphthalein Fhthalic anhydride R, 1387
R, mi Phthalic anhydride/pyridine TS, 1387
RS, 1447 Phylloquinone, see Phytomenadione
Phenolphthalein/ ethanol TS, 1381 Physical and physicochemical methods of
Phenolphthalein/pyridine TS, 1381 analysis, 1 141
Phenoxyacetic acid R, 1381 Physostigmine salicylate, 745
2-Phenoxyethanol R, 1381. Physostigmini salicylas, 745
Phenoxymethylpenicillin, 733 Phytomenadione, 747
RS, 1381, 1448 Phytomenadionum, 747
Phenoxymethylpenicillin calcium, 735 Phytonadione, see Phytomenadione
R.S, 1381, 1448 Pilocarpine hydrochloride, 749
Phenoxymethylpenicillin potassium, 738 Pilocarpine nitrate, 751
RS, 1381, 1448 Pilocarpini hydrochloridum, 749
tablets, 1083 Pilocarpini nitras, 751
Phenoxymethylpenicillini kalici compress!, Piperazine adipate, 753
1083 RS, 1387, 1448
Phenoxymethylpenicillinum, 733 tablets, 1 087
Phenoxymethylpenicillinum calcicum, 735 Piperazine citrate, 755
Phenoxymethylpenicillinum kalicum, 738 RS, 1387, 1448
Phenyl ether, see Diphenyl ether tablets, 1089
Phenyl/methylpolysiloxane R, 1 382 Piperazine hydrate R, 1 387
1 ,4-Phenylenediamine dihydrochloride R, Piperazini adipas, 753
1381 Piperazini adipatis compressi, 1 087

Index
Piperazini citras, 75.S Potassium dichromate

Piperazini citratis compressi, 1089 Rand Rl, mi
Piperidine R, 1887 TS,TS2 and TS3, mi
Pix lithanthracis, 757 TS (lOOg^, mi
Plant origin, materials of, 1251 VS (0.0167 mol/1), mi
acid value,1 254 Potassium dihydrogen phosphate
ash and acid-insoluble ash, 1251 R, m2
hydroxyl value, 1254 TS (iMto loog^, mi
iodine value, 1251 Potassium ferricyanide
peroxides in fixed 1252 oils, R, 1392
saponification value, 1252 TS(10and5Dg/l), 1392
unsaponifiable matter, 1253 Potassium ferrocyanide
Plasma substrate R, 1387 p 1 QQO
Platinic chloride TS (45g/l), 1322
R, IRBS Potassium hydrogen phthalate
TS (60g^ IRSS R, 1322
Podophylli resina, 757 standard TS, 1392
Podophyliinum, see Podophyllum resin Potassium hydrogen sulfate R, 1 392.
Podophyllum resin, 757 Potassium hydrogen tartrate
Polydimethylsiloxane R, 1 388 R, 1392
Polyethylene glycol 400, see Macrogol 400 standard TS, 1392
Polymyxin B, 1 235 Potassium hydroxide
Polysorbata ^^^
Poiysorbate SO R, 1388
759 R, 1323
TS (-110 to -560 1323
Polysorbates (20, 60, 80), 759
Polyvinylpyrrolidone, see Povidone
VS
^ to I moVl), 1323
Potassium hydroxide/ethanol
Potassio-cupric tartrate TS, 1389 TSl and TS2, 1324
Potassio-mercuric iodide
alkaline, TS,
VS
^ to 1 mol/1), 1324
Potassium hydroxide/methanol TS, 1394
TS, 1389 Potassium iodate
Potassium, identification, 1210 R, 1325
Potassium acetate TS (3.6mg/l), 1325
R, lass VS (OOl and 0.05 mol/1), 1324
TS, 1389 Potassium iodide, 536
Potassium antimonate AsR, 1325
R, 1T89 R, 1325
TS, 1389 TS to 400 1325
Potassium bicarbonate R, 1389 Potassium iodide/starch TSl, 1 395
Potassium bromate Potassium iodobismuthate TSl and TS2,
R, mo 1.395
TS (^g/lL IMQ Potassium iodobismuthate/acetic acid TS,
VS (0.00833 to 0.0333 mol/1), 1BS9 1.396
Potassium bromide Potassium iodoplatinate TS and TS2, 1 396
IR, IMD Potassium nitrate
R, 1S9Q R, 1326
TS (0.119 to 125 mo TS (ITOg^ 1326
Potassium carbonate, anhydrous, R, 1390 Potassium nitrite R, 1396
Potassium chloride, 532 Potassium periodate
IR, 1390 R, 1326
non-injectable, 532 TS, 1396
for parenteral use, 532 Potassium permanganate
R, mi basic TS (-1 and -5 g/l), 1397
TS
^ and 350
Potassium chromate
mO R, 1322
TS (-1 to -25g^ 1322
R, mi VS (0.0002 to 0.02 mol/1), 1326
TS mi Potassium permanganate/phosphoric acid TS,
Potassium citrate, 534 1.397
Potassium cyanide Potassium sodium tartrate R, 1397
PbTS, mi Potassium sulfate
R, 1391 R, 132B
TS (50 and mOg/I), mi TS ((M and 174mg/l), 1322

-
Potassium tetraoxalate Procaine benzylpenicillin, 779

R, 1398 non-injectable, 779
standard TS, 1398 powder for injections, 1 102
Potassium thiocyanate sterile, 779
R, la^ Procaine hydrochloride, Z82
TS (200 g/1), 1398 RS, 1399, 1448
Potato starch, 101 Procaine penicillin, see Procaine
Povidone, Z62 benzylpenicillin
Povidonum, 762 Procaini benzylpenicillin! pulvis ad
Powders injectionem, 1102
fineness and sieves, 1171 Procaini benzylpenicillinum, 779
oral see Oral powders Procaini hydrochloridum, 782
Powders for injections, 960 Procarbazine hydrochloride, 784
uniformity of content, 960, 1256 RS, 1399, 1448
uniformity of mass, 960, 1 257 2-
Procarbazini hydrochloridum, 784
Ppm (parts per million by weight), lU Progesterone, 785
Praziquantel, 764 RS, 1 399 1448
RS, 1398, 1448 Progesteronum, 785
tablets,1091 Proguanil hydrochloride, 788
Praziquantel! compress!, 1 091 RS, 1399
Praziquantelum, 764 Proguanili hydrochloridum, 788
Precision, 2 Promethazine hydrochloride, 790
expression of concentration, 2 Promethazini hydrochloridum, 790
pH values, 2 1,2-Propanediol, see Propylene glycol
quantities, 2 1 Propanol R, 1 399
temperature measurement, 10 Propanol, 792

Prednisolone, 771 R, 1399
RS, 1 399, 1 448 2-Propanolum, 792
tablets, 1092 Propranolol hydrochloride, 793
Prednisolone acetate, 765 RS, 1399, 1448
RS, 1399, 1448 Propranolol! hydrochloridum, 793
Prednisolone hemisuccinate RS, 1448 Propyl hydroxybenzoate, 799
Prednisolone sodium phosphate, 768 Propylene glycol, 795
injection, 1 094 R, 1399
RS, 1 399, 1 448 Propyleneglycolum, 795
Prednisolone sodium succinate powder for Propyliodone, 797
injections, 1096 Propyliodonum, 797
Prednisolone succinate RS, 1399 Propylis hydroxybenzoas, 799
Prednisoloni acetas, 765 Propylparaben, see Propyl
Prednisoioni compressi, 1092 hy d roxy b enz oa te
Prednisoloni et natrii phosphatis injectio, Propylthiouracil, 800
1094 RS, 1448
Prednisoloni et natrii succinatis pulvis ad Propylthiouracilum, 800
injectionem, 1096 Protamine sulfate, 802
Prednisoloni natrii phosphas, 768 Protamini sulfas, 802
Prednisolonum, 771 Protionamide, 804
Prednisone acetate RS, 1448 RS, 1222
Prednisone RS, 1448 Protionamidum, 804
Primaquine diphosphate, 774 Purified water, 105
RS, 1399 Purity
tablets, 1099 of chemical reference substances, 1454,
Primaquini diphosphas, 774 1457
Primaquini diphosphatis compressi, 1099 tests, use of chemical reference substances,
Probability (P), lA 1456
Probenecid, 775 Pycnometer, 1 1 51
RS, 1399, 1448 Pyrantel embonate, 806
tablets, 1 100 RS, 1399, 1449
Probenecid! compressi, 1100 tablets, 1104
Probenecidum, 775 Pyrantel pamoate, see Pyrantel
Procainamide hydrochloride, 777 embonate
Procainamidi hydrochloridum, 777 Pyranteli embonas, 806

2
Index
Pyranteli embonatis compressi, 1104 Radionuclidic purity, 1117

Pyrazinamide, 808 determination, 1131
RS, 12 m Radiopharmaceuticals, 1115
tablets, 1106 Reagents (R), 1 283
Pyrazinamidi compressi, IIQ^ accuracy and precision of measurement, 9
Pyrazinamidum, 808 tests and assays, 11
Pyrazinecarboxamide, see Pyrazinamide Red stock standard TS, 1170, 1401
Pyridine Reference spectra, 12, 1450
anhydrous, R, 1400 Reference substances (RS), 11
R, im. biological, 12
Pyridine/acetic anhydride TS, 1400 chemical see Chemical reference substances
Pyridostigmine bromide, 810 melting point, 1447
RS, 1400, 1449 Refractive index («), 14
Pyridostigmini bromidum, 810 determination, 1154
Pyridoxine hydrochloride, 81 Relative atomic mass, 19
Pyridoxini hydrochloridum, 812 Relative density {d), 13.
Pyrimethamine, 814 determination, 1 150
RS, 1400 Relative molecular mass, 3
Pyrimethaminum, 814 Resazurin sodium
Pyrogallol R, 1401
alkaline, TS, 1400 TS (lg/1), 14(11
R, 1400 Reserpine, 828
Pyrogen test, 1247 RS, 1401, 1449
parenteral preparations, 957 Reserpinum, 828
radiopharmaceuticals, 1 183 Resorcinol
R, 1401
Quality assurance, vii TS (20g/l), 14(11
Quantities, accuracy and precision, 9 Resorcinol/toluene TS, 1401
Quinaldine red R, 1 400 Results, calculation of, IQ
Quinaldine red/ethanol TS, 1400 Retinol acetate RS, 1401, 1449
Quinaldine red/methanol TS, 1400 Retinol concentrate, oily form, 830
Quinhydrone R, 1 400 Retinol palmitate RS, 1401
Quinhydrone/methanol TS, 1400 Retinol propionate RS, 1401
Quinidine sulfate, 816 Retinolum densatum oleosum, 830
Quinidini sulfas, 816 Reverse-phase partition chromatography,
Quinine R, 1400 1183
Quinine bisulfate, 818 Rf, 15, 1177
Quinine dihydrochloride, 821 Riboflavin, 832
injection, 1107 RS, 1401, 1449
Quinine hydrochloride, 823 Riboflavinum, 832
Quinine sulfate, 825 Rice starch, 101
Quinini bisulfas, 818 Pdfampicin, 834
Quinini dihydrochloridi injectio, 1 1 07 RS, 1401, 1449
Quinini dihydrochloridum, 821 Rifampicin quinone RS, 1401, 1449
Quinini hydrochloridum, 823 Rafampicinum, 834
Quinini sulfas, 825 Rifampin, see Rifampicin
8-Quinolinol, see 8-Hydroxyquinoline Ritonavir, 837
RS, 14(11
R, see Reagents Ritonavirum, 837
Radiation R„ 15, 1177
ionizing, sterilization by, 1279 RS, see Reference substances
shielding, 1131
Radiation spectrometry, 1 130 Saccharimidum natricum, see Saccharinum
Radioactive concentration, 1116 natricum
Radioactivity, 1115 Saccharin, 1447
half-life period, 1115 Saccharin sodium, 840
specific, 1116 anhydrous, 840
units, 1 1 15 dihydrate, 840
Radiochemical purity, 1118 Saccharinum natricum, 840
determination, 1 133 Saccharomyces cerevisiae, 1236
Radionuclide, 1 115 Salazosulfapyridine, see Sulfasalazine

Salbutamol, R4.S Sodium acetate/glacial acetic acid (0.1 mol/1)

KS, 1401 VS, 1406
Salbutamol sulfate, 843 Sodium alizarinsulfonate
RS, MQl R, 140Z
Salbutamoli sulfas, 843 TS (1 and 10^ 1406
Salbutamolum, 845 Sodium amidotrizoate, 621
Sales perorales ad rehydratationem, 1072 RS, 140Z, 1449
Salicylaldehyde Sodium arsenite (0.05 and 0.1 mol/1) VS, 1407
R, 1402 Sodium bicarbonate, see Sodium hydrogen
TS, 1402 carbonate
Salicylates, identification, 1210 Sodium biphosphate, see Sodium dihydrogen
Salicylic add, 53 phosphate
R, 1402 Sodium calcium edetate, 623
Saline Sodium carbonate
heparinized, TS, 1344 anhydrous, FeR, 1 408
TS, 1402 anhydrous, R, 1408
Saponification value, detemiination, 1 252 R, 14QZ
Saquinavir, 851 standard TS, 1 408
RS, 1402 TSflOto 200 g/I), 140Z
Saquinavir mesilate, 847 Sodium chloride, 626
RS, 1402 pyrogen-free, R, 1408
Saquinaviri mesilas, 847 R, 1408
Saquinavirum, 851 TS(9to40Dg^ 1408
Selenii disulfidum, 854 Sodium citrate, 628
Selenious acid R, 1402 anhydrous, 628
Selenious acid/sulfuric acid TS, 1402 dihydrate, 628
Selenium R, 1 402 R, 1408
Selenium-75, 1 125 TS(^g^ 1408
Selenium disulfide, 854 Sodium cobaltinitrite
Senna fruit, 858 R, 1409
Senna leaf, 85ri TS 1409
Sennae folium, 856 Sodium cromoglicate, 630
Sennae fructus, 853 RS, 14^ 1449
SI units, 16 Sodium diatrizoate, see Sodium amidotrizoate
Sieves, 1171 Sodium diethyldithiocarbamate
Silica gel R, 1409
desiccant, R, 1403 TS (0.8^ 1409
R1 to R6, 1403 Sodium dihydrogen phosphate
chromatography
Silica gel for anhydrous, R, 1409
hexadecylamidylsilyl, 1403 dihydrate, 1409
octadecylsilyl, base deactivated, 1 403 R, 1409
R, 1402 TS (45 and 275 g/1), 1409
Silver nitrate, 108 Sodium dithionite
R,1405 R, 1409
TS (40 and lOOg/1), 1404 TS (200 MQ9
VS fO.QOl to 0.1 mol/1), 1404 Sodium fluoride, 632
Silver nitrate/methanol TS, 1405 R, 1409
Silver oxide R, 1405 Sodium formate R, 1409
Silver standard (5ia.gAg/ml) TS, 1405 Sodium hexanesulfonate R, 1410
Soda lime R, 1 405 Sodium hydrogen carbonate, 633
Sodium R, 1410
identification, 1210 TS (40 and 100 1410
R, 1416 Sodium hydroxide, 635
standard (200pgNa/ml) TS, 1416 BET (0.1 mol/1), 1414
Sodium l,2-naphthoquinone-4-suIfonate carbonate-free VS (0.01 to 1 mol/1), 1410
R, 1405 R, 1413
TS (5g/l), 1405 TS (~40 to -400 g^ 1410
Sodium acetate TS(10and50g^ 1413
R (volume 1 and
TS
406
to 150g/I), 1406
^ 1 VS (0.001 to 1 mol/l), 1410
Sodium hydroxide/ethanol TS, 1413
VS (0.04moi/l), 1405 Sodium hydroxide/methanol TS, 1413

8 S
Index
Sodium hypobromite TS, 1 41 ^ Solochrome black, see Mordant black 11

Sodium hypochlorite Solochrome dark blue, see Calcon
TS (~40g/l),141B Solubility, 4
TS1, 1413 Soluble tablets, 966
Sodium laurilsulfate Solution
R, 1414 clarity of, 3
TS (lOg/1), 141h colourless, 3
Sodium mercaptoacetate R, 1414 tests and assays, 9
Sodium metabisulfite Solvent blue 19^ see Oracet blue B
R, 1414 Solvent red 23,. see Sudan red
TS (50g/l), 1414 Sorbitol R, 1418
Sodium metaperiodate Soybean-casein digest medium, 1319
R, 1414 with penicillinase, 1319
TS, 1414 Specific optical rotation, 13
Sodium methoxide (0.1 mol/1) VS, 1 41 determination, 1151
Sodium molybdotungstophosphate TS, 1415 Specific radioactivity (specific activity), 1116
Sodium nitrate R, 1415 Sji’eciflcations for reagents mentioned in the
Sodium nitrite, 637 International Pharmacopoeia (SRIP), 1 283
R, 141^ Specifications for the Quality Control of
TS n to in0g/1), 1415 Pharmaceutical Preparations, x
VS (0.1 mol/1), 1415 Spectinomycin hydrochloride, 860
Sodium nitrite/hydrochloric acid TS, 1415 RS, 1419, 1449
Sodium nitroferricyanide, see Sodium Spectinomycini hydrochloridum, 860
nitroprusside Spectrometry, atomic, 1 164
Sodium nitroprussiate, see Sodium nitroprusside Spectrophotometry
Sodium nitroprusside, 638 absorption, 1154
alkaline, TS, 1416 fluorescence, 1 166
R, 1416 infrared, 1160
TSfând 45g/l),14M visible and ultraviolet region, 1 54 1
Sodium octanesulfonate R, 1416 Spermaceti, synthetic, see Cetyl esters wax

Sodium oxalate R, 1416 Spironolactone, 862
Sodium peroxide R, 1416 RS, 1419
Sodium salicylate, 641 Spironolactonum, 862
R, 1416 Spongiform encephalopathies, animal, risk of
TS (11.5g/l), 1416 transmitting, 1465
Sodium stibogluconate, 642 Squalene R, 1419
Sodium sulfate, 644 Stability
anhydrous, 646 chemical reference substances, 11,. 1461
anhydrous, R, 1417 information, 6
Sodium sulfide Stannous chloride
R, 1417 AsTS, 1412
T.S, 1417 R, 1419
Sodium sulfite R, 1417 TS, 1412
Sodium tetraborate Stannous chloride/hydrochloric acid TSl, 1419
R, 141Z Staphylococcus aureus, 1 236
standard TS, 1417 Starch(es), 101
TS (lOg/1), 141Z R, 1423
Sodium tetraphenylborate soluble, R, 1420
R, 1417 TS, 142Q
TS (30g/l), 1417 Starch iodide TS, 1419
Sodium thiosulfate, 648 Starch/iodide paper R, 1420
R, 1413 Static head-space gas chromatography, 1197
TS (320 g/1), 1413 Steam sterilization, 1277
VS (Q.QQ2 to 0.1 mol/1), 1413 Sterile water for injections, 106
Sodium tungstate R, 1 41 Sterility tests, 1 236
Sodium valproate, 650 antibiotics, 1241
Soft capsules, 949 non-injectable preparations, 1 240
Soft paraffin ophthalmic preparations, 952
white, 717 parenteral preparations, 957^ 1 2.36
yellow, 717 radiopharmaceuticals, 33 1 1
Solid oral dosage forms, dissolution test, 1262 topical semi-solid dosage forms, 970

Sterilization, 1 Sulfamethoxypyridazine, 884

dry-heat, 1278 RS, 1420, 1449
filtration, 1279 Sulfamethoxypyridazinum, 884
gas, 1 280 Sulfamic acid
ionizing radiation, 1279. R, 1421
steam, 1 277 TS(5to 80g/l), 1420
Stibii natrii tartras, 864 4-Sulfamoylbenzoic acid R, 1421
Storage, 5
viii, Sulfanilamide RS, 1421, 1447, 1442
capsules, 948 Sulfanilic acid
chemical reference substances, 11^ diazotized, TS, 1421
1461 R, 142 1
containers, 5 Sulfapyridine, 1447
InternationalChemical Reference Sulfasalazine, 886
Substances, 1443 RS, 1421, 1449
ophthalmic preparations, 953 Sulfasalazinum, 886
protection from light, 5 Sulfated ash, 1218
radiopharmaceuticals, 1 135 Sulfates
suppositories, 963 identification, 1210
tablets, 965 limit test, 1211
temperature, JH Sulfosalicylic acid
topical semi-solid dosage forms, 971 R, 1421
Streptomycin, 1 235 TS (175 g/1), 1421
Streptomycin sulfate, 866 Sulfur, oxygen flask method, 1218, 1220
non-injectable, 866 Sulfur dioxide R, 1421
powder for injections, 1 1 09 Sulfuric acid
RS. 1420, 1449 nitrogen-free, TS (~1760g/l), 1422
sterile, 866 TS (rin to ~1760g/l), 1421
Streptomycini sulfas, 866 VS (0.005 to 0.5 mol/1), 1422
Streptomycini sulfatis pulvis ad injectionem, Sulfuric acid/ethanol TS, 1 423
1109 Sulfuric acid/methanol TS, 1423
Strychnine sulfate R, 1420 Sulfurous acid TS, 1 423
Sublingual tablets, 967 Supplementary information, 1439
Succinylcholine chloride, see Suxamethonium Supply, chemical reference substances,
chloride 1463
Sudan red Suppositories, 960
GR, 1420 disintegration test, 962, 1259
TS,1420 uniformity of content, 962, 1256
Sugar-coated tablets, 967 uniformity of mass, 962, 1 258
Sulfacetamide, 870 Suramin sodium, 889
RS, 1420, 1449 Suraminum natricum, 889
Sulfacetamide sodium, 872 Suxamethonii chloridum, 892
Sulfacetamidum, 870 Suxamethonium chloride, 892
Sulfacetamidum natricum, 872 Symbols, 13
Sulfacylum-natrium, see Sulfacetamidum elements, 19
natricum Synonyms, 4
Sulfadiazine RS, 1420 Systeme international d'Unites (SI),
Sulfadiazine silver, 874

Sulfadiazinum argentum, 874 Tablets, 963
Sulfadimezinum, see Sulfadimidinum buccal, 967
Sulfadimidine, 876 chewable, 967
RS,142Q coated, 967
Sulfadimidine sodium, 878 delayed-release (enteric-coated), 968
Sulfadimidinum, 876 disintegration test, 966, 967, 968, 1258
Sulfadimidinum natricum, 878 dissolution test, 965, 1262
Sulfadoxine, 880 effervescent, 966
RS, 1420 extended-release, 968
Sulfadoxinum, 880 film-coated, 967
Sulfamethazine, see Sulfadimidine modified-release, 968
Sulfamethoxazole, 882 soluble, 966
RS, 1420, 1449 sublingual, 967
Sulfamethoxazolum, 882 sugar-coated, 967

2
Index
uncoated, 9^^ Tetracycline hydrochloride, 204

uniformity of content, 965, 1256 non-injectable, 904
uniformity of mass, 964, 967, 968, 1 256 RS, 14k 1449
for use in mouth, 967 sterile, 904
Talc, 8M Tetracyclini hydrochloridum, 204
Talcum, 894 A/-Tetradecane R, 1425
Tamoxifen citrate, 896 Tetrahydrofuran R, 1426
E-isomer RS, 1423,1442 Tetramethylammonium hydroxide (-100 g/1)
RS, 1423, 1449 TS, 1426
Tamoxifen! citras, 896 Tetramethylammonium hydroxide/ethanol
Tamper evident containers, 5 TS, 142 6
Tannic acid Tetramethyldiaminobenzophenone, see
R, 1428 Bis(dimethylamino)benzophenone
TS (50g/l), 1428 Thallium-201, 1125
Tartaric acid Thiamine hydrobromide, 909
R, 1424 anhydrous, 909
TS (5 to 200 g/1), 1424 hemihydrate, 909
1210
Tartrates, identification, Thiamine hydrochloride, 910
Technetium-99m, 1 125 Thiamine mononitrate, 912
Temperature Thiamini hydrobromidum, 909
accuracy and precision of measurement, Thiamini hydrochloridum, 910
in Thiamini mononitras, 91
boiling point, 1148 Thin-layer chromatography, 1176
congealing point, 1143 Thioacetamide
melting, Z, 1 148 alkaline, TS, 142Z
storage, 6^ 13 R, 1426
tests and assays, S Thioacetazone, 914
/^-Terphenyl R, 1424 RS, 1427, 1449
Test solutions (TS), 11, 1 288 Thioacetazonum, 914
Testosterone enantate, 898 4,4''-Thiodianiline RS, 1427, 1449
RS, 1424,1442 Thioglycolate medium, fluid, see Fluid
Testosterone propionate, 901 mercaptoacetate medium
R, 1424 Thioglycolic acid R, see Mercaptoacetic acid R
RS, 1424, 1449 Thiopental RS, 1427
Testosterone propionate/ethanol TS, 1424 Thiopental sodium, 916
Testosteroni enantas, 898 Thiopentalum natricum, 916
Testosteroni propionas, 901 Thiourea
Tests and assays, 8 R, 142Z
accuracy and precision, 2 TS (0.1 g/1), 1427
calculation of results, 13 Thorin
chemical reference substances, 1 455 R, 1427
impurities, 13 TS (2 g/1), 1427
indicators for pH values, 2 Thorium nitrate
patents and trademarks, 13 R, 1427
reagents, reference substances, test VS (0.005 mol/1), 1427
and volumetric solutions, 11
solutions, Thymol
Tetrabromophenolphthalein ethyl ester R,1428
R, 1424 TSl to TS3, 1428
TS,1424 Thymol blue R, 1428
Tetrabutylammonium hydrogen sulfate R, Thymol blue/dimethylformamide, 428 1
1424 Thymol blue/ethanol TS, 1428

Tetrabutylammonium hydroxide Thymol blue/methanol TS, 1428
TS,1425 Thymolphthalein R, 1428
VS (0.1 mol/1), 1424 Thymolphthalein/dimethylformamide TS,
Tetrabutylammonium hydroxide/methanol 1428
TS, 1425 Thymolphthalein/ethanol TS, 1428
Tetrabutylammonium iodide R, 425 1 Thyroxine sodium, see Levothyroxine sodium
Tetracaine hydrochloride, 902 Tiabendazole, 918
Tetracaini hydrochloridum, 902 RS, 1428
Tetrachloroethane R, 1426 Tiabendazolum, 918
Tetracycline, 1 285 Tightly closed containers, 5

Timolol maleate, 920 Triketohydrindene/stannous chloride TS,

RS, 1429 1432
Timololi maleas, 920 Trimethadione, 926
Tin-113, 1125 RS, 1432, 1449
Tinnevelly senna leaf, 856, 858 Trimethadionum, 926
Titan yellow Trimethoprim, 928
R, 1429 RS, 1432, 1449
TS, 1429 Trimethoprimum, 928
Titanium dioxide R, 1429 2,2,4-Trimethylpentane R, 1432
Titanium dioxide/sulfuric acid TS, 1429 Trimethylpyridine
Titanium trichloride R, 1432
R, 1429 TS (50g/l), 1432
VS (0.1 mol/1), 1429 Trinitrophenol
Titration alkaline, TS, 1433
complexometric, 1220 R, 1432
1226
nitrite, TS (7g/l), 1432
non-aqueous, 1223 Trinitrophenol/ethanol TS, 1433
water, by Karl Fischer method, 1227 Triphenylantimony R, 1433
Tolbutamide, 922 Trisodium citrate, see Sodium citrate
RS, 1429, 1449 Trisodium orthophosphate
Tolbutamidum, 922 R, 1433
Tolnaftate RS, 1449 TS (2g/l), 1433
Toluene R, 1429 Tritium, 1126
Toluene-2-sulfonamide RS, 1429, 1449 Tropicamide, 930
4-Toluenesulfonamide R, 1429 RS, 1433
4-Toluenesulfonic acid R, 1429 Tropicamidum, 930
4-Toluenesulfonic acid/ethanol R, 1430 TS, see Test solutions
Topical semi-solid dosage forms, 969 Tubocurarine chloride, 932
Tosylchloramide sodium Tubocurarin! chloridum, 932
R, 1430 Turbidance, 1169
TS (15g/l), 1430 Turbidimetry, 1169
Toxicity, undue, test for, 1250 Turbidity, 1169
Trademarks, 10 Tyrosine R, 1433
Transmissible spongiform encephalopathies,
1465 Ultraviolet light, examination in, 7
Transmittance, 1155, 1169 Ultraviolet spectrophotometry, 1154
Tribasic calcium phosphate, see Calcium Uncoated tablets, 966
phosphate Under reduced pressure, 8
Tributyl phosphate R, 1430 Undue toxicity test, 1250
Trichloroacetic acid R, 1430 Uniform consistency
Trichloroethylene R, 1430 ophthalmic preparations, 955
Trichlorotrifluoroethane topical semi-solid dosage forms, 970
R, 1431 Uniformity of content
TS, 1431 capsules, 948, 1256
Triethylamine R, 1431 powders for injections, 960, 1256
Triethylenediamine R, 1431 single-dose preparations, 1255
Trihexyphenidyl hydrochloride, 924 suppositories, 962, 1256
RS, 1431 tablets, 965, 1256
Trihexyphenidyl! hydrochloridum, 924 Uniformity of mass
Triketohydrindene hydrate capsules, 947, 1257
R, 1431 powders for injections, 960, 1257
TS (lg/1), 1431 single-dose preparations, 1256
Triketohydrindene/butanol TS, 1431 suppositories, 962, 1258
Triketohydrindene/butanol/acetic acid TS, 1431 tablets, 964, 967, 968, 1256
Triketohydrindene/cadmium TS, 1431 Units of measurement, 16
Triketohydrindene/ethanol TS, 1431 Unsaponifiable matter, determination,
Triketohydrindene/methanol TS, 1432 1253
Triketohydrindene/pyridine/acetone TS, 1432 Uranyl acetate R, 1433
Triketohydrindene/pyridine/butanol TS, 1432 Uranyl/zinc acetate TS, 1433
Triketohydrindene/ sodium meta bisulfite TS, Urea R, 1433
1432 Uridine R, 1433

Index
Valproic acid RS, 1433 Water-baths, 8

Vanadium pentoxide R, 1433 Water-emulsifying ointments, 973
Vanadium/sulfuric acid TS, 1434 Wax(es)
Vanillin, 1447 camauba, 221
R, 1434 cetyl esters, 221
TS (lOg/1), 1434 melting point, 1146
Vanillin/hydrochloric acid TS, 1434 Weighing, 1141
Vanillin/sulfuric acid TSl and TS2, 1434 accuracy and precision, 9
Vaselinum album, see White soft paraffin apparatus, 1141
Vaselinum flavum, see Yellow soft paraffin recommended procedure, 1142
Vasodepressor substances, test for, 1249 Well-closed containers, 5
Verapamil hydrochloride, 934 Wheat starch, 101
RS, 1434 White soft paraffin, 717
Verapamili hydrochloridum, 934 WHO, see World Health Organization
Vinblastine sulfate, 937 Wool fat, hydrous, 55
RS, 1434 World Health Organization (WHO)
Vinblastini sulfas, 937 Collaborating Centre for Chemical
Vincristine sulfate, 939 Reference Substances, 11, 12, 1443, 1450,
RS, 1434, 1449 1452
Vincristini sulfas, 939 Medicines website, vi
Visible spectrophotometry, 1154 Model List of Essential Medicines, vii, xi
Visual inspection responsibility for International
capsules, 947 Pharmacop>oeia, ix
ophthalmic preparations, 952, 954, 955
parenteral preparations, 957, 1274 Xanthydrol
suppositories, 961 R, 1435
tablets, 964 TS, 1435
Vitamin A, see Retinol Xenon-131m, 1122, 1126
Vitamin B] 2 see Cyanocobalamin
,
Xenon-133, 1126
Vitamin B, 2 a and Bi 2 b, see Xenon-133m, 1126
Hydroxocobalamin Xylene R, 1435
Vitamin C, see Ascorbic acid Xylenol orange, 1222
Vitamin D 2 see Ergocalciferol
,
indicator mixture R, 1435
Vitamin D3, see Colecalciferol R, 1435
Vitamin Ki, see Phytomenadione
Volumes, accuracy and precision, 9 Yeast extract, water-soluble, R, 1435
Volumetric solutions (VS), 11, 1283 Yellow soft paraffin, 717
VS, see Volumetric solutions Yellow stock standard TS, 1170, 1435
Ytterbium-169, 1126
Warfarin RS, 1434, 1449
Warfarin sodium, 941 Zinc
Warfarinum natricum, 941 AsR, granulated, 1436
Water complexometric titration, 1223
ammonia-free, R, 1434 R, 1436
BET, 1435 standard (20|j.g/mlZn) TS, 1436
carbon-dioxide-free, R, 1435 Zinc acetate, R, 1436
carbon-dioxide-free and ammonia-free, R, Zinc bis(dibenzyldithiocarbamate)
1434 R, 1436
determination by Karl Fischer method, TS, 1436
1227 Zinc chloride R, 1436
for injections, 103 Zinc oxide, 943
for injections, R, 1435 Zinc sulfate R, 1436
purified, 105 Zinci oxydum, 943
R, 1434 Zirconyl nitrate
106
sterile, for injections, R, 1437
1435
sterile, R, TS, 1437
vapour detector tube, 1435 Zuclomifene RS, 1437, 1449

SELECTED WHO PUBLICATIONS OF RELATED INTEREST
Basic tests for drugs:

pharmaceutical substances, medicinal plant materials
and dosage forms
1998 (94 pages)
Basic tests for pharmaceutical dosage forms

1991 (134 pages)
Quality Assurance of Pharmaceuticals: a compendium of guidelines

and related materials
Volume 1: 1997 (244 pages)
Volume 2: good manufacturing practices and inspection. 2004 (236 pages)
WHO Expert Committee on Specifications for

Pharmaceutical Preparations
Fortieth report
2006 (461 pages)
International nonproprietary names (INN) for

pharmaceutical substances
Cumulative list no. 11
2004 (available in CD-ROM format only)
The use of essential medicines

Report of the WHO Expert Committee
(including the 13th Model List of Essential Medicines)
2004 (133 pages)
WHO Expert Committee on Biological Standardization

Fifty-fourth report
2005 (160 pages)
Further information on these or other Wt-iO publications can be obtained from

WFfO Press, World Health Organization, 1211 Geneva 27, Switzerland.
Attention is also drawn to the WHO Medicines web site
(http://www.who.int/medicines)
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International Pharmacopoeia 3rd Ed Vol 1 &amp; 2

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International Pharmacopoeia 3rd Ed Vol 1 &amp; 2

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The

World Health Organization

1. Pharmacopoeias 2. Pharmaceutical preparations - standards 3. Pharmaceutical preparations -

ISBN 92 4 155301 X (LC/NLM classification: QV 738.1)

© World Health Organization 2006

World Health Organization can be obtained from WHO

The International Pharmacop’oeial' (Ph. Int.) comprises a collection of recom-

ments in monographs. The newon Manufacture governs the interpre-

to Spectrometry in the infrared region, High performance liquid chromatogra-

a reduced sodium chloride and glucose content providing a solution with a

cholera diarrhoea, WHO

Scope and function

WHO Technical Report Series, No. 908, 2003, Annex 2.

it is based primarily on those substances included in the current WHO Model

element of the quality assurance of medicines. Pharmaceutical substances and

standards and manufacturers' release specifications. Pharmacopoeial standards

tests, limits are indicated additionally in brackets in percentage terms: as stated

When pharmacopoeial standards are used to establish the compliance

• The interpretation of a monograph must be in accordance with all

’ Quality assurance of pharmaceuticals: a compendium of guidelines and related materials. Volume 1.

tional pharmacopoeia, the Health Organization of the League of Nations set up

of Pharmaceutical Preparations, with a subtitle classifying it as the second edition

national pharmacopoeias and in other volumes of specifications for pharma-

The specifications included in the fourth edition were developed in collabora-

Ghana, Legon, Accra, Ghana; Professor J.-M. Aiache, Laboratory of Biophar-

of Medicines, Council of Europe, Strasbourg, France; Ms Cheah Nuan Ping,

for Pharmaceutical Ghemistry and Drug Analysis, Leuven, Belgium; Mr R.K.

Public Health, Nonthaburi, Thailand;Dr A. Kaukinen, Helsinki, Finland; Dr E.

Woden, ACT, Australia; Mr Keuser, Novartis Pharma Ltd, Basel, Switzerland;

Dr J. Kincaid, Merck, Manufacturing Division, Westpoint, PA, USA; Mr R.H.

cal Manufacturers Associations, Geneva, Switzerland; Dr H. Muller, Messer

Zurich, Switzerland; Dr Pham Hoang Ngoc, National Center for Scientific

Bandung, Indonesia; Dr M. Scheiwe, Mepha AG, Aesch, Basel, Switzerland;

Control Division, National Institute of Pharmacy, Budapest, Hungary; Mrs A.M.

Uziely, Institute for the Standardization and Control of Pharmaceuticals, Min-

Basel, Switzerland; Mr Wang Cunzhi, Kunming Pharmaceutical Corporation,

ment of Community Services and Health, Woden, ACT, Australia; Dr C. Wong-

Academy of Sciences, Shanghai, People’s Republic of China; Professor Yang

Control of Drugs, Ministry of Health, Moscow, Russian Federation; WHO Col-

The International Pharmacopoeia

General Notices: Contents

Chemical formula and relative molecular mass

of solvent represented by the stated number of parts in which 1 gram (g) of

Very soluble Less than 1 part

or use. The statements are in no way intended to be binding.

airtight after reclosure.

(b) Protection from light

of light. Alternatively, the container may be placed inside a suitable light-

given in the section "Additional information”.

Examination in ultraviolet light

carried out over phosphorus pentoxide, at a pressure not exceeding O.dkPa at

Tests and assays

Indicators for visual determination of pH values

Accuracy and Precision

% nt/m for the number of grams of solute in lOOg of product

The concentration expressed as “mol/1” refers to the number of moles of the

(d) Temperature measurement

— if the last figure calculated is 5 to 9, the preceding figure is increased by 1;

Other calculations - for example, in the standardization of volumetric solutions

In all cases where an impurity

Patents and trademarks

Reference to a particular trade name in the description of certain materials

Reagents, reference substances, test solutions,

from the centre.

International Pharmacopoeia 3rd Ed Vol 1 & 2

International Pharmacopoeia 3rd Ed Vol 1 & 2