Pharmaceutical Analysis 2008PHM Laboratory Manual

General information and background:

Pharmaceutical analysis is a course intended to describe both theoretical and
practical aspects of method validation and spectroscopy. The three laboratory
experiments you will be undertaking during the course aim to give you a hands
on experience in such validations as well as measuring and interpreting
spectroscopic results.
Laboratory Safety Information: Please refer to provided notes in the Laboratory.
Method Validation
Method validation pertains to measuring and controlling the quality of analytical
methods and seeks to address such questions as correct identity of drug in
formulation, whether the % stated composition of a drug present in a formulation
is correct and what concentrations of impurities are in drugs to name a few.
Criteria are used to judge the quality of an analysis method used to test
a drug and include

Identification tests,

Qualitative tests for impurities

Quantitative tests of the active moiety in samples of a drug,

substance or drug product.
Please see the printouts in the laboratory for a description of the United States
Pharmacopoeia (USP) and British Pharmacopoeia (BP) standards applying to
method validation applicable to the experiments here.
The laboratory aspect of 3026/2008PHM aims to introduce you to the physical
techniques of method validation and the types of equipment used in this process.
Structure of laboratory course:
Students will work in groups of 4 and perform 3 experiments, to be undertaken
in 3 single sessions.
1

Overview

of

Experimental

Stations: (1) Station 1

Concepts of measurement and reporting. This workstation introduces you some of
the physical techniques used in method validation/quality control relating to British
Pharmacopoeia (BP) standards.

Parameters such as disintegration, friability,

tablet hardness and dimensions will be determined on commercial caffeine

tables.
(2) Station
2
Using analytical/spectroscopic method to analyse drug dissolution. This station will
examine caffeine tablet properties in relation to dissolution. Dissolution
experiments will be performed under various conditions and temperatures using
different pH conditions and temperatures. Dissolution rates of caffeine from tablets
will be determined by using UV-VIS spectroscopy.
(3) Station
3
Overview of analytical/spectroscopic measurements. Determination of compound
stability, concentration and reproducibility between drug batches is an important
consideration in BP standards. Typically a combination of spectroscopic methods
including UV-VIS, IR, NMR, Mass Spectroscopy (MS), High Performance
Liquid Chromatography (HPLC) and Gas Chromatography (GC) are used in
tandem to determine the above. You will be introduced to IR, UV-VIS and HPLC as
methods for such measurement of the caffeine content in your tablets. This is
unique

from

your

previous

laboratory

sessions

involving

spectroscopic

measurements in that we are going to use the methods for validation purposes as
determined by BP standards.

Workstation 1
Disintegration, Friability of a
tablet
Tablets consist mainly of filler, except those in high-dose formulations. The most
common filler is lactose, while the lubricant is typically magnesium stearate or
steric acid.
You will be given a tablet containing approximately 100mg caffeine, and you will
undertake tests on several aspects of method validation procedures on these
tablets. Concepts such as accurate measurement and reporting, the mean and
Standard Deviation (SD), precision and
%relative SD
measured.

(%RSD)

will

be

You will be given three tablets, caffeine being the active

ingredient.
1. Tablet dimensions and hardness.
Part A: Uniformity of tablets under industrial production is very important.
Between batches great care must be taken to employ the same factors of
fill, die, and pressure. Therefore measuring tablet thickness which will be
influenced by the factors above is an important consideration for BP
standards
Using the Tablet thickness tester (Fig 1) measure the thickness of your three
tablets
(repeat

each

measurement

in

triplicate) (1) Zero the machine

(2) Depress the Lever (A on Fig 1)
(3) Place tablet under the elevated nut (B) on the stand
(4) Measure thickness
Q. Calculate the mean, SD and %RSD for the obtained results (2
Marks)

We did not measure it due to the machine being out of service.

Part B: After measurement of the three tablets place one of the tablets
in the tablet hardness tester (Fig 2) for measurement of its hardness
Repeat this for the other 2 tablets

Q. Record these values and report the mean SD and %RSD for
these. (2 Marks)
Values:
1) 16.6N

Mean = 16, 8 N

2) 18.1N

SD = 1, 21%

3) 15.7N

%RSD= 7, 20%

Q. Did your results match the BP standards? Explain. (1 Mark)

According to British Pharmacopoeia standards:
HARDNESS= 7kg
Group results:
HARDNESS = 1,71kg
Yes, our results matched with BP standards because its within the standard
established.

2. Tablet Friability
Friability is a measurement of the means of reducing a solid object (a
tablet) into smaller objects.
Procedure;
Weigh and accurately record the mass of 5 tables. Place the tablets into the
Friabilator (Fig 3). Measure change in weight in 5 minutes intervals
for 30 minutes. (i) Remove tablets from Friabilator, remove surface powder
by lightly brushing the tablet, (ii) Place into a weighing boat and record
mass.
Record your data in the space provided below.
Initial weight =
1.640g

After 20 min = 1.626g

After 5 min = 1.638g

After 25 min = 1.622g

After 10 min =
1.634g

After 30 min = 1.614g

After 15 min =
1.631g

Plot this data using Microsoft Excel and paste this graph in the space
provided.
1.65
1.64
1.63
1.62
Mass (g)

1.61
1.6
0

10

15

20

Time (min)

25

30

35

Q. Did your results conform to compendial standards? Explain. (2

Marks)
There is not a compedial test, meaning that there is no BP standard for them.
However, we think these tablets can be sold because the loss of weight was minimal.

Figure 1. Tablet thickness test

Figure 2 Tablet Hardness tester.

Fig 3- Friabilator

3. Tablet Disintegration.
You will be given 3 tablets. Place these into the Tablet Disintegration testing
apparatus (Fig
3b).
Using the stopwatch supplied measure the time taken for complete
disintegration of each of your 3 tablets.
Record the mean, SD and %RSD for the obtained results (2 Marks)
Values:
1) 14.36 min
2) 13.37 min
3) 15.16 min
Mean =14.29 min
SD = 0.896%
%RSD=6.27%

Fig 3a. Tablet disintegration testing apparatus.

Workstation 2
Tablet Dissolution
Please see appendix for the USP NF standards on dissolution. The rate of dissolution
of a drug from a compounded product is an important consideration when designing
tablets for various release rates. Many factors such as pH, drug stability to light,
heat, storage, level of impurities
in the sample all affect the rate of release of drugs from compounded tablets.
Please refer to the standard operating procedures for operation of the dissolution
apparatus.
Method:
(1) Place the tablet in a cell in the dissolution apparatus (Fig 4) (write down cell
number).
Please note the clear plastic covers are to be inserted fat size facing
above the tablet.
(2) Initiate stirring at 37C at 100prm (start your timer)
(3) After exactly five minutes of stirring using the syringe provided withdraw
1.5 mL of solution from the dissolution vessel. IMPORTANT: Before
sucking liquid into syringe please cover the tip of the syringe with the
filters provided
(4) Syringe out this volume into a UV cuvette for measurement of UV-VIS
absorbance.
Record your absorbance maximum (See supplied standard operating
procedure and ask your demonstrator if unsure). The UV-VIS spectrum of
caffeine in water is shown in Figure 4
(5) Suck up the solution from the cuvette into the syringe and return this
to the bulk solution in the cell.
(6) Repeat the above measurement steps exactly every 5 minutes (up to
30 minutes)
You will also be given a solution containing 100 mg/ 900 mL of caffeine (represents
100%
dissolution).
Q. Measure the absorbance of this solution and record its value in the
space provided below.
A275 = 3.22

Using your absorbance maximum results complete the following table:

Time (min)

Absorbance @ 275 nm

% Dissolution

5
10

2.93/ 2.333/ 2.19/ 2.745

Average = 2.5495

80.94%

3.126/ 3.126/ 3.126/ 3.222

Average = 3.15

100%

3.126/ 3.126/ 3.126/ 3.222

Average = 3.15

100%

15
20
25
30
35
40
45
150
100

50

Dissolution

55
60

50

Dissolution (%)

Q.0 Prepare a dissolution profile (% dissolution vs. time) graph from the data
0
5 using
10 15 20 25 30 35
obtained
Time
(Min)
Microsoft Excel.
Past
your graph in the space provided. (4 Marks)

Q. What information can be obtained from this graph? Explain your answer
with reference to BP standards. (2 Marks)
In physiologic conditions the tablet disintegrates 100% in less than 20
min and can so have its action in the body. No BP standard was found to
make reference.

Q. What effect on would repeating this experiment have on both

absorbance and dissolution properties at pH 1, pH 4, and pH 6.8? (2 Marks)
When we change the pH, the solubility changes as well. Therefore,
pH 1 increase the solubility of the caffeine more than pH 4 and Ph6.8
does because of protonation of nitrogen. The absorbance also
changes because the measure of .

Q. What other spectroscopic method(s) could you use to measure caffeine

dissolution rates? Explain any advantages and/or disadvantages over UVVIS. (2 Marks)
Another spectroscopic method that could be used is HPLC.
One of advantages of HPLC over UV-VIS spectrometry is the separation
capabilities, providing higher specificity and sensitivity. In addition this method is more
accurate and precise than UV-VIS method.
Some disadvantages of HPLC are that this method requires elaborate treatment
and procedures usually associated with chromatographic method. This way the method
consumes long time and its expensive.

Figure 4 Tablet dissolution system

Fig 5 (A) UV-VIS Spectrum of Caffeine in water (B) Chemical structure of caffeine.

Figure 6 IR-spectrum of caffeine

Workstation 3
Overview
of
analytical/spectroscopic
pharmaceutical analysis.

measurements

in

In this workstation you will use spectroscopic techniques such as UV-VIS to

gain an understanding of the methods used for measurement of accuracy, precision,
range, robustness and specificity of the method.
Metho
d:
You will be given two samples containing caffeine at a concentration of 0.1mg/mL.
For the first sample:
(1) Prepare a standard solution containing 10mg caffeine in 100mL.
(2) Read the absorbance using a UV-VIS spectrometer and record the reading
(3) Progressively dilute the sample in 1/2 dilution steps, then record the
readings after each dilution.
(4) Prepare a standard curve during the practical using Microsoft Excel on the
provided.
NOTE: Please send your file to your demonstrator after you have
completed this
(5) Calculate the mean, the SD, and the %RSD for the data. (2 Marks)
Abs.273n
m
2500uL
1250uL
625uL
312uL
156uL
365uL
300uL
235uL

Sample
1
2.761
1.618
0.695
0.375
0.285
0.403
0.374
0.301

Sample
2
2.61
1.514
0.675
0.556
0.305
0.422
0.37
0.385

Sample
3
2.243
1.479
0.813
0.547
0.258
0.443
0.376
0.287

Mean
2.5380
1.5370
0.7277
0.4927
0.2827
0.4227
0.3733
0.3243

SD
0.2664
0.0723
0.0746
0.1020
0.0236
0.0200
0.0031
0.0530

%RSD
10.4965
4.7038
10.2484
20.7040
8.3444
4.7338
0.8183
16.3422

For the second sample: Choose three absorbance points on your graph, then
prepare a solution which corresponds to the concentration of caffeine at this point.
Measure this solution in the UV-VIS spectrometer, and record the absorbance.
13

Q. What is the % recovery of your

sample? (1Mark)
Q. Define the linearity of the method (1 Marks)
It is a form of internal or relative accuracy; for a specific focal
point, it reflects how well a system responds to a sequence of
dilutions in the proper matrix. When the experiment was done with
caffeine, it was diluted and it was possible to see how well the
system was responding.
Q. What is the accuracy of the method? (1 Mark)
Accuracy is a measure of the agreement of a particular
measurement with the true (or accepted) value of the parameter
under the conditions.
This analysis was accurate
Q. What is the precision of the method? (1 Mark)
The precision of a method is generally approached in terms of
either repeatability or reproducibility. Precision is the closeness of
agreement among test results obtained under prescribed conditions
The analysis showed itself imprecise
Q. What is the Limit of Detection? (1 Mark)
The limit of detection, expressed as the concentration, or the
quantity, is derived from the smallest measure, that can be detected
with a reasonable certainty for a given analytical procedure.
Q. What is the Limit of Quantification? (1 mark)

13

It is the lowest concentration at which the analyte can not only

be reliably detected but at which some predefined goals for bias and
imprecision are met.

Your demonstrator will show you the operation of an HPLC.

Q. Comment on the accuracy and specificity of these devices and relate
this to your UV-Vis. (2Marks)
The advantage of HPLC over UV spectrometry is its separation
capabilities, providing higher specificity and sensitivity as well as its
applicability in formulations with multiple API's or very low dose.

13

Extra questions
Workstation 1
Tablet
Dimensions

Size

Tablet 1

Tablet 2

Tablet 3

Tablet 4

Tablet 5

4.21mm

4.26mm

4.28mm

4.17mm

4.32mm

Tablet 1

Tablet 2

Tablet 3

Tablet 4

Tablet 5

15.6N

18.9N

17.7N

16.5N

15.0N

Tablet 4

Tablet 5

Hardness

Force
Applied

Friability - N.B 5 Tablets used

Time
Tablets Weight
0 Mins

1.654

5 Min

1.651

10 Min

1.649

15 Min

1.646

20 Min

1.640

25 Min

1.636

30 Min

1.632

Disintegration
Tablet 1

Tablet 2

Disintegration 10min 37sec 12min 5sec

Time

Tablet 3

13min 52 sec 11min 25sec 12min 10sec

17

Shown above are the data results for some Quality Assurance tests
on uncoated 500mg Paracetamol tablets manufactured by Griffith
Pharmaceuticals. The procedures used conformed to the standards
set out by the British Pharmacopoeia.
1. Please determine the mean, SD, %RSD for the four methods above
(8 Marks)
TABLET DIMENSIONS:
MEAN: 4.25mm
SD: 0.06
%RSD: 1.39
HARDNESS
MEAN: 16.74N
SD: 1.58
%RSD: 9.43
FRIABILITY
MEAN: 1.644g
Loss of weight: Initial Weight Final Weight = 0.022g
DESINTEGRATION
MEAN: 12min 2 sec
SD: 72.05
%RSD: 9.98

2. Based on your understanding of British Pharmacopoeia standards for

tablet disintegration, friability, hardness and tablet dimensions, do you
feel these tablets would pass as viable tablets to be sold in Australia?
(7 Marks)
According to British Pharmacopoeia standards:
FRIABILITY=1%
HARDNESS= 7kg
DISINTEGRATION=10min
DIMENSION= ------

Our results:
FRIABILITY= 1.64g = 0.328%
HARDNESS= 16.74 = 1.70 kg
DISINTEGRATION= 12min 02sec
DIMENSION= 4.25 mm
As can be seen friability and hardness are within standards but disintegration is out.
Therefore, these tablets would not pass to be sold in Australia because not all
validation methods are following the standards.

Workstation 2
Q1. What are some of the major sources of error during this experiment?
Explain your answer.
(10 Marks)

Do not change the filter

Time
Becker (its not a precise instrument)
Equipament ( not calibrated properly)
Human error

Q2. You are undertaking a dissolution study of a sample containing the

sodium salt of aspirin. What effect would decreasing the pH to 3 have on
both the solubility rate and UV-VIS spectrum of aspirin?
(5 Marks)
The sodium aspirin, when in solution, dissociate in C 9H7O4 -- and Na+ having its
maximum solubility. If the pH decrease the charge of the molecule will change and it
will be neutral, reducing so the solubility. Thus, with the decrease of the pH the
solution will absorb in a smaller wavelength because more energy would be
necessary to an electronic excitation to occur.

pH

Workstation 3
Q1.
using

Briefly

describe

the

procedure

of

your

method

validation

UV-visible spectrophotometer.(3 Marks)

The method of validation includes verify the precision of the

method, the accuracy, and how is the graph and if it is showing the
result that was expected.
Q2. Discuss your results whether its within acceptable range or out of
range as per official pharmacopeia. Mention some common errors which
you have found during your experiment. (5Marks)
The method was imprecise and inaccurate. Because of this our results are not within
acceptable range.
Common errors: improper pipetting, different operators for the same process and
wrong reading of the absorbance.
Q3. What are the basic units of an analytical HPLC? What is PDA? What
is the difference between PDA of an HPLC and normal UVspectrophotometer? (4 Marks)
PDA (Diode array detectors) is used for obtaining spectral
profiles from molecular mixtures or chromatographically separated
samples. They can measure different wavelengths simultaneously at
once.
Normal UV-spectrophometer the compound can be analyzed at a
particular wavelength selected but in PDA the compound can be
analyzed

using

multiple

of

wavelength

due

to

presence

of

polychromator.
Q4. What is the importance of IR spectroscopy in pharmaceutical quality
control area?
(3 Marks)

It is used to determine functional groups in molecules. It is also

important to pharmaceutical quality control area because with IR
spectroscopy it is possible to determine functional groups in
molecules.