KP 9 21

Monographs, Part I

22 Monographs, Part I

KP 9 23 graphy. Develop the plate with the upper layer of a mixture of water, n-butanol and glacial acetic acid (5 : 4 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 365 nm). Any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Loss on Drying Not more than 1.0% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.25 g of Acebutolol Hydrochloride, previously dried, dissolve in 20 mL of glacial acetic acid, add 80 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 37.289 mg of C18H28N2O4HCl Packaging and Storage Preserve in well-closed containers.

Acebutolol Hydrochloride
O C O CH3CH2CH2 C NH CH3 OH OCH2CCH2NHCH(CH3)2 H HCl

and enantiomer

C18H28N2O4HCl: 372.89 Acebutolol Hydrochloride, when dried, contains not less than 98.0% and not more than 102.0% of acebutolol hydrochloride (C18H28N2O4 HCl). Description Acebutolol Hydrochloride is white to pale yellowish white crystal or crystalline powder. Acebutolol Hydrochloride is freely soluble in water, in methanol, in ethanol or in glacial acetic acid and practically insoluble in ether. A solution of Acebutolol Hydrochloride (1 in 20) shows no optical rotation. Identification (1) Determine the absorption spectra of solutions of Acebutolol Hydrochloride and Acebutolol Hydrochloride RS, in 0.01mol/L hydrochloric acid TS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Acebutolol Hydrochloride and Acebutolol Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Acebutolol Hydrochloride (1 in 100) responds to the Qualitative Tests for chloride. Melting Point Between 141 C and 145 C. Purity (1) Heavy metalsProceed with 1.0 g of Acebutolol Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Acebutolol Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (3) Related substancesDissolve 40 mg of Acebutolol Hydrochloride in 2 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add methanol to make exactly 25 mL. Pipet exactly 1 mL of this solution, add methanol to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromato-

Aceclofenac
O Cl H N O COOH

Cl

C16H13Cl2NO4: 354.19 Aceclofenac, when dried, contains not less than 99.0% and not more than 101.0% of aceclofenac (C16H13Cl2NO4). Description Aceclofenac is a white crystalline powder. Aceclofenac is freely soluble in acetone or in dimethylformamide, soluble in ethanol or in methanol, and practically insoluble in water. Identification (1) Dissolve 10 mg of Aceclofenac in 10 mL of ethanol, and to 1 mL of this solution, add 0.2 mL of a mixture of equal volume of a solution of potassium ferricyamide (6 in 1000) and a solution of ferric chloride (9 in 1000). Allow to stand in the dark for 5 minutes, add 3 mL of a solution of hydrochloric acid (10 in 1000), and allow to stand in the dark for 15 minutes again: A blue color develops and a precipitate is formed. (2) Dissolve 50 mg of Aceclofenac in 100 mL of methanol, and to 2 mL of this solution, add methanol to

24 Monographs, Part I make 50 mL, and determine the absorption spectrum of this solution between 220 nm and 370 nm as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum at 275 nm. The specific absorbance at the maximum is 320 to 350. (3) Determine the infrared spectra of Aceclofenac and Aceclofenac RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Heavy metalsProceed with 2.0 g of Aceclofenac according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesDissolve 0.1 g of Aceclofenac, accurately weighed, in 50 mL of the mobile phase, and use this solution as the test solution. Dissolve diclofenac sodium RS, equivalent to 5 mg of diclofenac, in 50 mL of the mobile phase, and use this solution as the standard solution (1). Pipet 2 mL of the standard solution (1), add the mobile phase to make 50 mL, and use this solution as the standard solution (2). Pipet 5 mL of the standard solution (1), add 0.25 mL of the test solution, add the mobile phase to make 50 mL, and use this solution as the standard solution (3). Perform the test with 10 L each of the test solution and the standard solution (2) as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of these solutions: the area of each peak other than the peak of Aceclofenac from the test solution is not larger than the peak area of Aceclofenac from the standard solution (2) (not more than 0.2%), and the total area of each peak other than the peak of Aceclofenac from the test solution is not more than 2.5 times of the peak area of Aceclofenac from the standard solution (2) (not more than 0.5%). Omit the peak from the test solution, which peak area is less than 0.2 times of the peak area of the main peak from the standard solution (2). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 275 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: Adjust the pH of a mixture of acetonitile, tetrahydrofuran, and glacial acetic acid (1.2 in 1000) (225 : 225 : 550) to 3.5 with sodium hydroxide TS. Flow rate: 1 mL/minute System suitability: System performance: When the procedure is run with 10 L of the standard solution (3) under the above operating conditions, Aceclofenac and diclofenac are eluted in this order with the resolution between their peaks being not less than 8.0. Detection sensitivity: Adjust the detection sensitivity so that the peak height of Aceclofenac and diclofenac obtained from the standard solution (3) composes 50% of the full scale. Time span of measurement: About 10 times as long as the retention time of Aceclofenac. Loss on Drying Not more than 0.5% (1.0 g, between 100 C and 105 C) Residue on Ignition Not more than 0.1% (1.0 g). Assay Weigh accurately about 0.3 g of Aceclofenac, previously dried, dissolve in exact 40 mL of methanol, and titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction. Each mL of 0.1 mol/L sodium hydroxide VS = 35.419 mg of C16H13Cl2NO4 Packaging and Storage well-closed containers Preserve in light-resistant,

Acetaminophen
O CH3 C NH OH

Paracetamol

C8H9NO2: 151.16

Acetaminophen, when dried, contains not less than 98.0% and not more than 101.0% of acetaminophen (C8H9NO2). Description Acetaminophen is a white crystal or crystalline powder. Acetaminophen is freely soluble in methanol or in ethanol, sparingly soluble in water, and very slightly soluble in ether. Acetaminophen dissolves in sodium hydroxide TS. Identification Determine the infrared spectra of Acetaminophen and Acetaminophen RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 169 C and 172 C.

Purity (1) ChlorideDissolve 4.0 g of Acetaminophen in 100 mL of water by heating, cool with shaking in ice-water, allow to stand until ordinary temperature is attained, add water to make 100 mL and filter. To 25 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.40

KP 9 25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.014%). (2) SulfateTo 25 mL of the filtrate, obtained in (1), add 1 mL of dilute hydrochloric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.019%). (3) Heavy metalsProceed with 2.0 g of Acetaminophen according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Acetaminophen according to Method 3 and perform the test using Apparatus B (not more than 2 ppm). (5) Related substancesDissolve 50 mg of Acetaminophen in 1 mL of methanol, add the mobile phase to make 50 mL and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add the mobile phase to make exactly 200 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of both solutions by the automatic integration method: the total area of all peaks other than the peak area of Acetaminophen from the test solution is not larger than the peak area of Acetaminophen from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 225 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of 0.05 mol/L monobasic potassium phosphate TS, pH 4.7 and methanol (4:1). Flow rate: Adjust the flow rate so that the retention time of Acetaminophen is about 5 minutes. System Suitability System performance: Dissolve 10 mg each of Acetaminophen and p-aminophenol in 1 mL of methanol, add the mobile phase to make 50 mL. To 1 mL of this solution add the mobile phase to make 10 mL. When the procedure is run with 10 L of this solution under the above operating conditions. p-Aminophenol and Acetaminophen are eluted in this order with the resolution between their peaks being not less than 7. Detection sensitivity: Adjust the detection sensitivity so that the peak height of Acetaminophen obtained from 10 L of the standard solution is about 15% of the full scale. Time span of measurement: About 6 times as long as the retention time of Acetaminophen after the solvent peak. Loss on Drying Not more than 0.3% (0.5 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 20 mg each of Acetaminophen and Acetaminophen RS, previously dried, dissolve in 2 mL of methanol and add water to make exactly 100 mL. Pipet exactly 3 mL each of these solutions, add water to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Determine the absorbance, AT and AS , of the test solution and the standard solution, respectively, at the wavelength of maximum absorption at about 244 nm as directed under the Ultraviolet-visible Spectrophotometry, using water as the blank. Amount (mg) of acetaminophen (C8H9NO2) = amount (mg) of Acetaminophen RS Packaging and Storage tight containers
AT AS

Preserve in light-resistant,

Acetaminophen Tablets
Paracetamol Tablets Acetaminophen Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of acetaminophen (C8H9NO2: 151.17). Method of Preparation Prepare as directed under Tablets, with Acetaminophen. Identification (1) The retention time of main peak of the test solution and the standard solution for Assay is the same. (2) Weigh a portion of powdered Acetaminophen Tablets, equivalent to 50 mg of Acetaminophen according to labeled amount, add 50 mL of methanol and mix, filter and use this filtrate as the test solution. Separately, weigh 5 mg of Acetaminophen RS, add methanol to make 5 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with the upper layer of a mixture of dichloromethane and methanol (4 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm). The Rf values of the spots from the test solution and the standard solution are the same. Dissolution Test Perform the test with 1 tablet of Acetaminophen Tablets at 50 revolutions per minute accord-

26 Monographs, Part I ing to Method 2 under the Dissolution Test, using 900 mL of diluted phosphate buffer solution, pH 5.8, as the dissolution solution. Take the dissolution solution 30 minutes after starting the test, make any necessary dilution of the filtrate by adding the dissolution solution and use this solution as the test solution. Separately, weigh accurately about sufficient quantity of Acetaminophen RS, previously dried in a dessicator (silica gel for 18 hours) and dissolve in the test solution, to make the same concentration as the test solution and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution at 243 nm as directed under Ultraviolet-visible Spectrophotometry. The dissolution rate of Acetaminophen Tablets in 30 minutes should be not less than 80%. Phosphate buffer solution, pH 5.8To 50 mL of 0.2 mol/L dibasic potassium phosphate TS, add 3.6 mL of 0.2 mol/L of sodium hydroxide TS and add water to make 200 mL. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Acetaminophen Tablets. Weigh accurately equivalent to about 0.1 g of Acetaminophen, add 100 mL of mobile phase, shake for 10 minutes, then shake strongly for 5 minutes, add mobile phase to make 200 mL, pipet exactly 5 mL of this solution, add mobile phase to make 250 mL and filter through a membrane filter with pore size of not more than 0.5 m. Discard the first 10 mL of the filtrate, and use the subsequent filtrate as the test solution. Separately, weigh accurately about 20 mg of Acetaminophen RS, previously dried in 105 C for 2 hours, dissolves in mobile phase to make exactly 100 mL, pipet exactly 5 mL of this solution, add mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine, AT and AS , of the peak area of Acetaminophen. Amount (mg) of acetaminophen (C8H9NO2) = amount (mg) of Acetaminophen RS AT 5
AS

System performance: When the procedure is run with 10 L of the standard solution under the above operating condition, the symmetry factor of the peak of acetaminphen is not more than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak areas is not more than 2.0%. Packaging and Storage Preserve in tight containers.

Acetazolamide
O CH3 C H N S O S O N N NH2

C4H6N4O3S2: 222.25 Acetazolamide contains not less than 98.0% and not more than 102.0% of acetazolamide (C4H6N4O3S2), calculated on the dried basis. Description Acetazolamide is a white to pale yellowish white, crystalline powder, is odorless and has a slight bitter taste. Acetazolamide is slightly soluble in ethanol, very slightly soluble in water, and practically insoluble in ether. Melting pointAbout 255 C (with decomposition) Identification (1) To 0.1 g of Acetazolamide, add 5 mL of sodium hydroxide TS, then add 5 mL of a solution of 0.1 g of hydroxylamine hydrochloride and 50 mg of cupric sulfate in 10 mL of water: a pale yellow color develops. Then heat this solution for 5 minutes: a deep yellow color is produced gradually. (2) To 20 mg of Acetazolamide, add 2 mL of dilute hydrochloric acid, boil for 10 minutes, cool and add 8 mL of water: this solution responds to the Qualitative Tests for primary aromatic amines. (3) To 0.2 g of Acetazolamide, add 0.5 g of granulated zinc and 5 mL of diluted hydrochloric acid (1 in 2): the gas evolved darkens moistened lead acetate paper. Purity (1) Clarity and color of solutionDissolve 1.0 g of Acetazolamide in 10 mL of sodium hydroxide TS: the solution is clear and colorless to pale yellow. (2) ChlorideTo 1.5 g of Acetazolamide, add 75 mL of water and warm at 70 C for 20 minutes with occasional shaking. After cooling, filter and to 25 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.20 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.014%). (3) SulfateTo 25 mL of the filtrate obtained in (2),

Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 243 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Mobile phase: A mixture of water and methanol (3 : 1). Flow rate: 1.5 mL/minute. System suitability

KP 9 27 add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.038%). (4) Heavy metalsProceed with 1.0 g of Acetazolamide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) Silver-reducing substancesWet 5 g of Acetazolamide with 5 mL of aldehyde-free ethanol and add 125 mL of water, 10 mL of nitric acid and exactly 5 mL of 0.1 mol/L silver nitrate VS. Stir for 30 minutes while protected from light, filter through a glass filter (G3) and wash the residue on the glass filter with two 10 mL volumes of water. Combine the filtrate with the washings, to the solution, add 5 mL of ferric ammonium sulfate TS and titrate with 0.1 mol/L ammonium thiocyanate VS: not less than 4.8 mL of 0.1 mol/L ammonium thiocyanate VS is consumed. Loss on Drying Not more than 0.5% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately about 0.15 g of Acetazolamide and dissolve in 400 mL of water in a water-bath. After cooling, add water to make exactly 1000 mL. Pipet exactly 5 mL of the solution, add 10 mL of 1 mol/L hydrochloric acid TS and then add water to make exactly 100 mL. Determine the absorbance A of this solution at the wavelength of maximum absorption at about 265 nm as directed under the Ultraviolet-visible Spectrophotometry. Amount (mg) of acetazolamide (C4H6N4O3S2) A = 474 200000 Packaging and Storage well-closed containers. Preserve in light-resistant, sparingly soluble in acetone, slightly soluble in methanol or in ethanol, and practically insoluble in water. Melting pointAbout 185 C (with decomposition) Identification (1) Dissolve 0.10 g each of Acetohexamide and Acetohexamide RS in 100 mL of methanol. To 5 mL each of these solutions, add 20 mL of 0.5 mol/L hydrochloric acid TS and 75 mL of methanol and pipet exactly 10 mL each of these solutions and dilute with methanol to exactly 50 mL. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry, using methanol as the blank, respectively: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Acetohexamide and Acetohexamide RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) ChlorideDissolve 1.5 g of Acetohexamide in 40 mL of dimethylformamide, add 6 mL of dilute nitric acid and dimethylformamide to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.45 mL of 0.01 mol/L hydrochloric acid VS, add 6 mL of dilute nitric acid and dimethylformamide to make 50 mL (not more than 0.011%). (2) SulfateDissolve 2.0 g of Acetohexamide in 40 mL of dimethylformamide and add 1 mL of dilute hydrochloric acid and dimethylformamide to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.40 mL of 0.005 mol/L sulfuric acid VS, add 1 mL of dilute hydrochloric acid and dimethylformamide to make 50 mL (not more than 0.010%). (3) Heavy metalsProceed with 1.0 g of Acetohexamide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Related substances (i) Cyclohexylamine Dissolve exactly 1.0 g of Acetohexamide in exactly 30 mL of 0.5 mol/L sodium hydroxide TS, add exactly 5 mL of hexane, shake vigorously for 60 minutes, allow to stand for 5 minutes, and use the upper layer as the test solution. Separately, dissolve about 50 mg of Cyclohexylamine RS, accurately weighed, in 0.5 mol/L sodium hydroxide TS to make exactly 50 mL. Pipet exactly 2 mL of this solution, and add 0.5 mol/L sodium hydroxide TS to make exactly 300 mL. Pipet 30 mL of this solution, add exactly 5 mL of hexane, shake vigorously for 60 minutes, allow to stand for 5 minutes, and use the upper layer as the standard solution. Perform the test with exactly 2 L each of the test solution and standard solution as directed under Gas Chromatography according to the following conditions, and determine the peak area of cyclohexylamine by the automatic integration method: the peak area of cyclohexylamine is not more than that with the standard solution.

Acetohexamide
O CH3 C O S O NH O C NH

C15H20N2O4S: 324.40 Acetohexamide, when dried, contains not less than 98.0% and not more than 101.0% of acetohexamide (C15H20N2O4S). Description Acetohexamide is a white to yellowish white powder. Acetohexamide is freely soluble in dimethylformamide,

28 Monographs, Part I 1000 mL of 0.05 mol/L sodium dihydrogen phosphate TS, and adjust to pH 6.5 with 0.5 mol/L sodium hydroxide TS. To 500 mL of this solution add 500 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of dicyclohexylurea is about 10 minutes. System suitability System performance: When the procedure is run with 50 L of the standard solution under the above operating conditions, the number of theoretical plates of the peak of dicyclohexylurea is not less than 10000. System repeatability: When the test is repeated 6 times with 50 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of dicylohexylurea is not more than 2.0%. (iii) Other related substancesDissolve 0.1 g of Acetohexamide in 10 mL of acetone and use this solution as the test solution. Pipet exactly 1 mL of this solution, add acetone to make exactly 20 mL. Pipet exactly two 1-mL portions of this solution, add acetone to make exactly 10 mL and 25 mL, respectively, and use these solutions as the standard solutions (1) and (2). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for thin-layer chromatography and dry the plate. Then, develop the plate with a mixture of ethyl acetate, methanol, cyclohexane and strong ammonia water (6 : 2 : 1 : 1) to a distance of about 10 cm and dry the plate with air. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than that from the standard solution (1) and the number of spots being more intense than that from the standard solution (2) is not more than 4. Loss on Drying Not more than 1.0% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Acetohexamide, previously dried, dissolve in 30 mL of dimethylformamide, add 10 mL of water and titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination using a solution prepared by adding 19 mL of water to 30 mL of dimethylformamide and make any necessary correction. Each mL of 0.1 mol/L sodium hydroxide VS = 32.440 mg of C15H20N2O4S Packaging and Storage Preserve in well-closed containers.

Operating conditions Detector: A hydrogen flame-ionization detector. Column: A fused-silica column 0.53 mm in inside diameter and 30 cm in length, coated the inner surface with methylsilicone polymer for Gas Chromatography 1.5 m in thickness. Column temperature: A constant temperature of about 90 C. Injection port temperature: A constant temperature of about 150 C. Detector temperature: A constant temperature of about 210 C. Carrier gas: Helium Flow rate: Adjust the flow rate so that the retention time of cyclohexylamine is about 4 minutes. System suitability System performance: When the procedure is run with 2 L of the standard solution under the above operating conditions, the number of theoretical plates of the peak of cyclohexylamine is not less than 8000. System repeatability: When the test is repeated 6 times with 2 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of cyclohexylamine is not more than 5.0%. (ii) DicyclohexylureaDissolve 1.0 g of Acetohexamide, accurately weighed, in exactly 10 mL of 0.5 mol/L sodium hydroxide TS, add exactly 20 mL of methanol, shake, then add exactly 5 mL of diluted hydrochloric acid (1 in 10), shake vigorously for 15 minutes, and centrifuge. Filter 10 mL or more of the supernatant liquid through a membrane filter with pore size of not larger than 0.5 m. Discard the first 5 mL of the filtrate, and use the subsequent filtrate as the test solution. Separately, weigh accurately about 50 mg of dicyclohexylurea RS, dissolve with methanol to make exactly 100 mL. Pipet exactly 2 mL of this solution, add methanol to make exactly 100 mL, pipet exactly 20 mL of this solution, add exactly 10 mL of 0.5 mol/L sodium hydroxide TS, shake, then add exactly 5 mL of diluted hydrochloric acid (1 in 10), shake, and use this solution as the standard solution. Perform the test with exactly 50 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine the peak area of dicyclohexylurea by the automatic integration method: the peak area of dicyclohexylurea obtained with the test solution is not more than that with the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 0.5 g of sodium hydroxide in

KP 9 29 line Chloride for Injection according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement. It

Acetylcholine Chloride for Injection

CH3 H3C N CH3 CH2CH2OCOCH3 Cl

C7H16ClNO2: 181.66 Acetylcholine Chloride for Injection is a preparation for injection which is dissolved before use. Acetylcholine Chloride for Injection contains not less than 98.0% and not more than 102.0% of acetylcholine chloride (C7H16ClNO2) and not less than 19.3% and not more than 19.8% of chlorine (Cl: 35.45), calculated on the dried basis. Acetylcholine Chloride for Injection contains not less than 93.0% and not more than 107.0% of the labeled amount of acetylcholine chloride (C7H16ClNO2). Method of Preparation Prepare as directed under Injections. Description Acetylcholine Chloride for Injection is a white crystal or crystalline powder. Acetylcholine Chloride for Injection is very soluble in water, freely soluble in ethanol. Acetylcholine Chloride for Injection is extremely hygroscopic. Identification (1) Determine the infrared spectra of Acetylcholine Chloride for Injection and Acetylcholine Chloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) A solution of Acetylcholine Chloride for Injection in water (1 in 10) responds to the Qualitative Tests (2) for chloride. Melting Point Between 149 C and 152 C. Seal Acetylcholine Chloride for Injection in a capillary tube for melting point determination immediately after drying both the sample and the tube at 105 C for 3 hours and determine the melting point. Purity (1) Clarity and color of solutionDissolve 1.0 g of Acetylcholine Chloride for Injection in 10 mL of water: the solution is clear and colorless. (2) AcidityDissolve 1.0 g of Acetylcholine Chloride for Injection in 10 mL of freshly boiled and cooled water, and add 1 drop of bromthymol blue TS, and use this solution as the test solution. To the test solution add 0.30 mL of 0.01 mol/L sodium hydroxide VS: the solution is blue in color. (3) Heavy metalsProceed with 2.0 g of Acetylcho-

Uniformity of Dosage Units It meets the requirement. Loss on Drying Not more than 1.0% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay (1) Acetylcholine chlorideWeigh accurately the contents of not less than 10 Acetylcholine Chloride for Injections. Weigh accurately about 0.5 g of the contents, dissolve in 15 mL of water, then add exactly 40 mL of 0.1 mol/L sodium hydroxide VS, stopper loosely and heat on a water-bath for 30 minutes. Cool quickly and titrate the excess sodium hydroxide with 0.05 mol/L sulfuric acid VS (indicator: 3 drops of phenolphthalein TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L sodium hydroxide VS = 18.166 mg of C7H16ClNO2 (2) ChlorineTitrate the solution, which has been titrated in (1), with 0.1 mol/L silver nitrate VS (indicator: 3 drops of fluorescein sodium TS). Each mL of 0.1 mol/L silver nitrate VS = 3.5453 mg of Cl Packaging and Storage Preserve in hermetic containers.

Acetylcysteine
O HS H3C NH OH

C5H9NO3S: 163.20 Acetylcysteine, when dried, contains not less than 98.0% and not more than 102.0% of acetylcysteine (C5H9NO3S).

30 Monographs, Part I Description Acetylcysteine is a white, crystalline powder. Acetylcysteine is freely soluble in water or ethanol, and practically insoluble in dichloromethane. Identification (1) Determine the infrared spectra of Acetylcysteine and Acetylcysteine RS, respectively, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between +21 and +27 Weigh accurately about 1.25 g of Acetylcysteine, dissolve in 1 mL of disodium ethylenediaminetetraacetate solution (1 in 100) and 7.5 mL of sodium hydroxide solution (1 in 25), and add phosphate buffer solution, pH 7.0, to make 25 mL, and determine the optical rotation of this solution. pH 7.0 phosphate buffer solutionAdd 50 mL of 1 mol/L monobasic potassium phosphate TS and 29.5 mL of 1 mol/L sodium hydroxide solution to a volumetric flask, adjust to a pH of 7.0 by adding water, and add water again to make 100 mL. pH Dissolve 1.0 g of Acetylcysteine in 100 mL of freshly boiled and cooled water: the pH of this solution is between 2.0 and 2.8. Purity Heavy metals Proceed with 2.0 g of Acetylcysteine according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of lead standard solution (not more than 10 ppm). Loss on Drying Not more than 0.1% (1 g, in vacuum at the pressure not exceeding 0.67 kPa, 70 C, 4 hours). Residue on Ignition Not more than 0.5% (2 g, 600 C). Assay Weigh accurately about 1.0 g of Acetylcysteine, previously dried, dissolve in freshly prepared sodium metabisulfite solution (1 in 2000) to make exactly 100 mL. Pipet exactly 10 mL of this solution, add exactly 10 mL of the internal standard solution, then add sodium metabisulfite solution (1 in 2000) to make exactly 200 mL, and use this solution as the test solution. Separately, dissolve about 1.0 g of Acetylcysteine RS, accurately weighed, in sodium metabisulfite solution (1 in 2000) to make exactly 100 mL. Pipet exactly 10 mL of this solution, add exactly 10 mL of the internal standard solution, then add sodium metabisulfite solution (1 in 2000) to make exactly 200 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS , of the peak area of Acetylcysteine to that of the internal standard, respectively.

Amount (mg) of Acetylcysteine (C5H9NO3S) = amount (mg) of Acetylcysteine RS

QT QS

Internal standard solutionDissolve of 1 g of dlphenylalanine in freshly prepared sodium metabisulfite solution (1 in 2000) to make 200 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 214 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, having octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A solution of potassium dihydrogen phosphate, pH 3.0 (6.8 in 1000). Flow rate: 1.5 mL/minute. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, acetylcystein and dl-phenylalanine are eluted in this order with the resolution between their peaks being not less than 6.0. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area is not more than 2.0%. Packaging and Storage Preserve in tight containers.

Acrinol Hydrate
NH2 OCH2CH3 OH CH3CHCO2H H2N N H2O

Ethacridine Lactat

C15H15N3OC3H6O3H2O: 361.39

Acrinol Hydrate, contains not less than 98.5% and not more than 101.0% of acrinol (C15H15N3OC3H6O3 : 343.38), calculated on the anhydrous basis. Description Acrinol Hydrate is a yellow, crystalline powder. Acrinol Hydrate is freely soluble in hot water, sparingly soluble in water, and slightly soluble in ethanol. pHThe pH of an aqueous solution of Acrinol Hydrate (1 in 100) is between 5.5 and 7.0. Melting pointAbout 245 C (with decomposition) Identification (1) Determine the absorption spectra of solutions of Acrinol Hydrate and Acrinol Hydrate RS, in

KP 9 31 water (3 in 250000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Acrinol Hydrate and the Acrinol Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) To 5 mL of a solution of Acrinol Hydrate in water (1 in 100), add 5 mL of dilute sulfuric acid, shake well, allow to stand for about 10 minutes at room temperature and filter: the filtrate responds to the Qualitative Tests for lactate. Purity (1) ChlorideDissolve 1.0 g of Acrinol Hydrate in 80 mL of water by warming in a water-bath, cool and add 10 mL of sodium hydroxide TS and water to make 100 mL. Shake well, allow to stand for 30 minutes, filter, to 40 mL of the filtrate, add 7 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare 50 mL of the control solution with 4 mL of sodium hydroxide TS, 7 mL of dilute nitric acid, 0.30 mL of 0.01 mol/L hydrochloric acid VS and water (not more than 0.026%). (2) Heavy metalsProceed with 1.0 g of Acrinol Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Volatile fatty acidsDissolve 0.5 g of Acrinol Hydrate in a mixture of 20 mL of water and 5 mL of dilute sulfuric acid, shake well, filter and heat the filtrate: no odor of volatile fatty acids is perceptible. (4) Related substancesDissolve 10 mg of Acrinol Hydrate in 25 mL of the mobile phase, and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add the mobile phase to make exactly 100 mL, and use this solution as the standard solution (1). Pipet exactly 1 mL of standard solution (1), add the mobile phase to make exactly 10 mL, and use this solution as the standard solution (2). Perform the test with exactly 10 L each of the test solution and the standard solutions (1) and (2) as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of any peak other than acrinol obtained with the test solution is not larger than 3 times the peak area of acrinol obtained with the standard solution (2), and the total area of all peaks other than acrinol obtained with the test solution is not larger than the peak area of acrinol with the standard solution (1). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 268 nm). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 7.8 g of sodium dihydrogen phosphate in 900 mL of water, adjust to pH 2.8 with phosphoric acid, and add water to make 1000 mL. To 700 mL of this solution add 300 mL of acetonitrile for Liquid Chromatography, and add 1.0 g of sodium 1octanesulfonate to dissolve. Flow rate: Adjust the flow rate so that the retention time of acrinol is about 15 minutes. System suitability Test for required detectability: Confirm that the peak area of acrinol obtained with 10 L of the standard solution (2) is equivalent to 7 to 13% of that with 10 L of the standard solution (1). System performance: When the procedure is run with 10 L of the standard solution (2) under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of acrinol are not less than 5000 and not more than 2.0, respectively. System repeatability: When the test is repeated 6 times with 10 L of the standard solution (1) under the above operating conditions, the relative standard deviation of the peak area of acrinol is not more than 1.5%. Time span of measurement: About 3 times as long as the retention time of acrinol beginning after the solvent peak. Water Between 4.5and 5.5% (0.2 g, volumetric titration, direct titration) Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.27 g of Acrinol Hydrate, dissolve in 5 mL of formic acid, add 60 mL of a mixture of acetic anhydride and glacial acetic acid (1 : 1), and titrate immediately with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination in the same manner, and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 34.34 mg of acrinol (C15H15N3OC3H6O3) Packaging and Storage tight containers. Preserve in light-resistant,

Acyclovir
O N OH H2N N N O HN

C8H11N5O3: 225.21 Acyclovir contains not less than 98.0% and not more

32 Monographs, Part I than 101.0% of acyclovir (C8H11N5O3), calculated on the anhydrous basis. Description Acyclovir is a white crystalline powder. Acyclovir is freely soluble in dimethylsulfoxide, slightly soluble in water, and very slightly soluble in ethanol. Acyclovir dissolves in dilute hydrochloric acid. Identification (1) Determine the infrared spectra of Acyclovir and Acyclovir RS, respectively, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The retention time of main peak of the test solution for Assay and the standard solution is the same. Water Not more than 6.0% (0.5 g, volumetric trtration, direct titration) Purity (1) Related substancesDissolve a certain amount of Acyclovir, accurately weighed, in dimethylsulfoxide to contain about 10 mg per mL of Acyclovir, and use this solution as the test solution. Pipet exactly 1 mL of this solution, add dimethylsulfoxide to make exactly 100 mL, and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, methanol, and strong ammonia water (80 : 20 : 2) to a distance of about 15 cm, and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 and 366 nm): the spots other than principal spot from the test solution are not more intense than the spot from the standard solution (not more than 1%) (2) GuanineDissolve about 0.1 g of Acyclovir, accurately weighed, in water to make exactly 200 mL. Pipet exactly 10 mL of this solution, add 0.01 mol/L sodium hydroxide TS to make exactly 50 mL, and use this solution as the test solution. Separately, dissolve about 8.8 mg of Guanine RS, accurately weighed, in 50 mL of 0.1 mol/L sodium hydroxide TS, add water to make exactly 500 mL. Pipet exactly 2 mL of this solution, add 0.01 mol/L sodium hydroxide TS to make exactly 50 mL, and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the peak areas of guanine, AT and AS , for the test solution and the standard solution, respectively (not more than 0.7%). Amount (g) of guanine (C5H5N5O) = 1000 C AT C: Concentration of the standard solution (g/mL),

Residue on Ignition Not more than 0.1% (1.0 g). Assay Dissolve about 0.1 g of Acyclovir, accurately weighed, in 20 mL of 0.1 mol/L sodium hydroxide TS, and add water to make exactly 200 mL. Pipet exactly 10 mL of this solution, add 0.01 mol/L sodium hydroxide TS to make exactly 50 mL, and use this solution as the test solution. Separately, dissolve about 25 mg of Acyclovir RS, accurately weighed, in 5 mL of 0.1 mol/L sodium hydroxide TS, add water to make exactly 50 mL. Pipet exactly 10 mL of this solution, add 0.01 mol/L sodium hydroxide TS to make exactly 50 mL, and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the peak areas of acyclovir, AT and AS , for the test solution and the standard solution, respectively. Amount (g) of acyclovir (C8H11N5O3) = 1000 C AT
AS

C: Concentration of Acyclovir RS in the standard solution (g/mL) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.2 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Mobile phase: Glacial acetic acid solution (1 in 1000). Flow rate: 3 mL/minute. System suitability System performance: When the procedure is run with 40 L of the mixture of equal volumes of the standard solution in the Purity (2) under Acyclovir and the standard solution in Assay Acyclovire RS under the above operating conditions, Acyclovir and guanine are eluted in this order with the resolution between their peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak areas is not more than 2.0%. Packaging and Storage Preserve in tight containers.

AS

KP 9 33 of water. Stir for 30 seconds, pass through a coarse filter, and use the filtrate as the test solution. Prepare a sulfate standard solution by adding 0.15 mL of 0.01 mol/L sulfuric acid to 15 mL of water. To the test solution and the standard solution add 2 mL of barium chloride TS and 1 mL of 3 mol/L hydrochloric acid, dilute each solution with water to 30 mL, and mix. Allow the solutions to stand for 5 minutes: the test solution is not more turbid than the standard solution (not more than 0.02%). (4) Ammonia Suspend 0.5 g of Adenosine in 10 mL of water. Stir for 30 seconds, pass through a coarse filter. Dilute the filtrate with water to 15 mL, mix, and use the filtrate as the test solution. Dilute 1 mL of ammonium chloride solution (314 mg in 1000 mL) with 100 mL of water. Mix 2 mL of this ammonia standard solution with 13 mL of water, and use this solution as the reference solution. To the test solution and reference solution add 0.3 mL of alkaline mercuric-potassium iodide TS, cap the test tubes, and allow to stand for about 5 minutes : the test solution does not exhibit a more intense yellow color than that of the reference solution (not more than 0.0004%). (5) Heavy metalsProceed with 1.0 g of Adenosine according to Method 2 and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (6) Related substancesWeigh accurately 25 mg of Adenosine and dissolve in mobile phase and add to make exactly 25 mL and use this solution as the test solution. Perform the test with exactly with 20 L of the test solution as directed under Liquid Chromatography according to the following conditions, and determine the percentage of each impurity in the portion of Adenosine taken by the peak area percentage method: not more than 0.2% of adenine is found; and not more than 0.5% of total impurities is found. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: A mixture of sulfate buffer and a solution (1 in 10000) of sodium azide (60:40) Flow rate: 1.5 mL/minute System suitability System performance: Weigh accurately each 20 mg of adenosine and 20 mg of inosine and dissolve in mobile phase to make 100 mL volume. When the procedure is run with 20 L of this solution under the above operating condition, the resolution between adenosine and inosine is not less than 9.0 and the symmetry factor is not more than 2.5. System repeatability: Weigh 20 mg each of adenosine and inosine, dissolve in mobile phase to make 100 mL. When the test is repeated 5 times with 20 L of this solution under the above operating conditions, the rela-

Adenosine
NH2 N N

N HO O H HO H OH

C10H13N5O4 : 267.24 Adenosine contains not less than 99.0% and not more than 101.0% of adenosine (C10H13N5O4), calculated on the dried basis. Description Adenosine is a colorless crystalline powder and produces adenine and D-ribose by hydrolysis. Adenosine is freely soluble in water, sparingly soluble in hot water, and slightly soluble in ethanol. Identification Determine the infrared spectra of Adenosine and Adenosine RS, previously dried, as directed in the paste method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 233 and 238 C

Specific Optical Rotation [ ]20 D : Between -68 to -72 (after drying, 0.4 g, sodium hydroxide solution (1 in 10), 20 mL, 100 mm). Purity (1) Acidity or alkalinitySuspend 1 g of Adenosine in 20 mL carbon dioxide free water. Stir for 30 seconds, and pass through a coarse filter. To each of two 10 mL portions of the filtrate add 0.1 mL of bromocresol purple TS. Not more than 0.3 mL of 0.01 N sodium hydroxide is required to produce a blue-violet color in one portion, and not more than 0.1 mL of 0.01 N hydrochloric acid is required to produce a yellow color in the other portion. (2) ChlorideSuspend 0.75 g of Adenosine in 15 mL of water. Stir for 30 seconds, pass through a coarse filter, and use the filtrate as the test solution. Prepare a chloride standard solution by diluting 1 mL of sodium chloride solution (231 mg in 1000 mL) with 100 mL of water. To the test solution and 10 mL of the chloride standard solution add 1 mLof nitric acid and 1 mL of silver nitrate TS, dilute each solution with water to 40 mL, and mix. Allow the solutions to stand for 5 minutes, protected from light. When viewed against a dark background, the test solution is not more turbid than the standard solution (not more than 0.007%). (3) SulfateSuspend 0.75 g of Adenosine in 15 mL

34 Monographs, Part I tive standard deviation of the peak area is not more than 2.0%. Time span of measurement: Adjust the run time to at least twice the retention time of the major peak. Sulfate bufferDissolve 6.8 g of potassium hydrogen sulfate and 3.4 g of tetrabutylammonium hydrogen sulfate in water, dilute with water to 1000 mL, and mix. Adjust with 2 N potassium hydroxide to a pH of 6.5. Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Adenosine, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 26.724 mg of C10H13N5O4 Packaging and Storage Preserve in tight containers.

1% Absorbance E1 cm (249 nm): Between 257 and 271 (after drying, 2 mg, ethanol, 100 mL). 1% E1 cm (292 nm): Between 85 and 95 (after drying, 2 mg, ethanol, 100 mL).

Specific Optical Rotation [ ]20 D : Between + 136 and + 151 (after drying, 0.5 g, chloroform, 50 mL, 100 mm). Purity Related substancesDissolve 0.1 g of Ajmaline in 10 mL of chloroform and use this solution as the test solution. Pipet exactly 1 mL of this solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescence indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, acetone and diethylamine (5 : 4 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Loss on Drying Not more than 1.0% (0.6 g, in vacuum, 80 C, 3 hours). Residue on Ignition Not more than 0.2% (0.5 g). Assay Weigh accurately about 0.3 g of Ajmaline, previously dried, dissolve in 50 mL of acetic anhydride and 50 mL of acetone for nonaqueous titration and titrate with 0.05 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L perchloric acid VS = 16.322 mg of C20H26N2O2 Packaging and Storage well-closed containers. Preserve in light-resistant,

Ajmaline
OH H H

H N CH3 H N

H OH CH2CH3 H

C20H26N2O2: 326.43 Ajmaline, when dried, contains not less than 96.0% and not more than 101.0% of ajmaline (C20H26N2O2). Description Ajmaline is a white to pale yellow, crystalline powder, is odorless and has a bitter taste. Ajmaline is freely soluble in acetic anhydride or in chloroform, sparingly soluble in methanol, in ethanol, in acetone or in ether, and very slightly soluble in water. Ajmaline dissolves in dilute hydrochloric acid. Melting pointAbout 195 C (with decomposition). Identification (1) Dissolve 50 mg of Ajmaline in 5 mL of methanol and use this solution as the test solution. Add 3 mL of nitric acid to 1 mL of the test solution: a deep red color develops. (2) Spot the test solution of (1) on filter paper and spray Dragendorff's TS: an orange color develops.

Ajmaline Tablets
Ajmaline Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of ajmaline (C20H26N2O2: 326.43). Method of Preparation Prepare as directed under Tablets, with Ajmaline. Identification (1) Shake a quantity of powdered Ajmaline Tablets, equivalent to 0.10 g of Ajmaline according to the labeled amount, with 30 mL of chloroform and fil-

KP 9 35 ter. Evaporate the filtrate in a water-bath to dryness. With the residue, proceed as directed in the Identification under Ajmaline. (2) Dissolve 10 mg of the residue of (1) in 100 mL of ethanol. To 10 mL of this solution, add ethanol to make 50 mL and determine the absorption spectrum of the solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 247 nm and 251 nm and between 291 nm and 294 nm and a minimum between 269 nm and 273 nm. Dissolution Test Perform the test with 1 tablet of Ajmaline Tablets at 100 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of diluted phosphate buffer solution, pH 6.8, (1 in 2) as the dissolution solution. Take 20 mL or more of the dissolved solution 60 minutes after start of the test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately about 28 mg of Ajmaline RS, previously dried in vacuum at 80 C for 3 hours, dissolve in diluted phosphate buffer solution, pH 6.8, (1 in 2) to make exactly 500 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 288 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Ajmaline Tablets in 60 minutes is not less than 75%. Dissolution rate (%) with respect to the labeled amount of ajmaline (C20H26N2O2) = W S AT 1 180
AS C

= 16.322 mg of C20H26N2O2 Packaging and Storage well-closed containers. Preserve in light-resistant,

Albendazole
N H3C S H N OCH 3 O N

C12H15N3O2S: 265.33 Albendazole, when dried, contains not less than 98.0% and not more than 102.0% of albendazole (C12H15N3O2S). Description Albendazole is white to pale yellow powder. Albendazole is freely soluble in anhydrous formic acid, very slightly soluble in ether or methylene chloride and practically insoluble in ethanol or water. Identification (1) Perform the test as directed in the Related substances: the principal spots from the test solution and the standard solution show the same Rf value. (2) Determine the infrared spectra of Albendazole and Albendazole RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity Related substancesDissolve 50 mg of Albendazole in 3 mL of glacial acetic acid, add glacial acetic acid to make 5 mL and use this solution as the test solution. Separately, weigh accurately a portion of Albendazole RS, dissolve in the glacial acetic acid to contain 5 mg per mL and use this solution as the standard solution. Pipet 1.0 mL of the standard solution, dilute with glacial acetic acid to make exactly 100 mL and use this solution as the diluted standard solution. Perform the test with the these solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution, the standard solution and the diluted standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform, glacial actic acid and ether (60 : 10 : 10) to a distance of about 15 cm and airdry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not larger and not more intense than the spot from the standard solution. (not more than 0.5%) Loss on Drying Not more than 0.5% (105 C, 4 hours). Residue on Ignition Not more than 0.2% (1 g).

WS : Amount (mg) of Ajmaline RS, C: Labeled amount (mg) of ajmaline (C20H26N2O2) in 1 tablet.
Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder, not less than 20 Ajmaline Tablets. Weigh accurately a portion of the powder, equivalent to about 0.3 g of ajmaline (C20H26N2O2), add 15 mL of strong ammonia water and extract with four 25 mL volumes of chloroform. Combine the chloroform extracts, wash with 10 mL of water, add 5 g of anhydrous sodium sulfate, shake well and filter, wash the container and the residue with two 10 mL volumes of chloroform and filter. Evaporate the combined filtrate in a water-bath to dryness, dissolve the residue in 50 mL of acetic anhydride and 50 mL of acetone for nonaqueous titration and titrate with 0.05 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L perchloric acid VS

36 Monographs, Part I Assay Weigh accurately about 0.25 g of Albendazole, previously dried, dissolve in 100 mL of glacial acetic acid, and warm if necessary. After cooling, add 1 drop of solvent blue 19 in glacial acetic acid solution (1 in 200), and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes to violet. Perform a blank determination, and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 26.533 mg of C12H15N3O2S. Packaging and Storage Preserve in well-closed containers. well and allow to stand for 20 minutes at 40 1 C in a water-bath. Add 1.0 mL of dilute hydrochloric acid, shake and allow to stand for 3 hours at 40 1 C. Cool rapidly to ordinary temperature and filter. Wash the residue with three 10 mL volumes of water, dry in a desiccator (silica gel) for 18 hours and dry at 105 C for 5 hours: the weight of the residue is 0.50 g to 0.58 g. Packaging and Storage tight containers. Preserve in light-resistant,

Aldioxa
H N O O N HN OAl(OH)2

Albumin Tannate
Tannalubin Albumin Tannate is a compound of tannic acid and a protein. The label states the origin of the protein of Albumin Tannate. Description Albumin Tannate is a pale brown powder. Albumin Tannate is odorless, or has a faint, characteristic odor. Albumin Tannate is practically insoluble in water or in ethanol. Albumin Tannate dissolves in sodium hydroxide TS or sodium carbonate TS with turbidity. Identification (1) To 0.1 g of Albumin Tannate, add 10 mL of ethanol and heat in a water-bath for 3 minutes with shaking. After cooling, filter and to 5 mL of the filtrate, add 1 drop of ferric chloride TS: a blue-purple to bluish black color is produced. On standing, a bluish black precipitate is produced. (2) To 0.1 g of Albumin Tannate, add 5 mL of nitric acid: an orange-yellow color develops. Purity (1) AcidShake 1.0 g of Albumin Tannate with 50 mL of water for 5 minutes and filter. To 25 mL of the filtrate, add 1.0 mL of 0.1 mol/L sodium hydroxide VS and 2 drops of phenolphthalein TS: a red color develops. (2) FatsTo 2.0 g of Albumin Tannate, add 20 mL of petroleum benzine, shake vigorously for 15 minutes and filter. Evaporate 10 mL of the filtrate on a waterbath: the weight of the residue is not more than 50 mg. Loss on Drying Not more than 6.0% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 1.0% (0.5 g). Digestion Test To 1.00 g of Albumin Tannate, add 0.25 g of saccharated pepsin and 100 mL of water, shake
H2N C

Dihydroxyaluminum Allantoinate C4H7AlN4O5: 218.10 Aldioxa is a condensation product of allantoin and aluminum hydroxide. When dried, Aldioxa contains not less than 65.3% and not more than 74.3% of allantoin (C4H6N4O3: 158.12) and not less than 11.1% and not more than 13.0% of aluminum (Al: 26.98). Description Aldioxa is a white powder, is odorless and tasteless. Aldioxa is practically insoluble in water, in ethanol or in ether. Aldioxa dissolves in dilute hydrochloric acid or in dilute nitric acid. Melting pointAbout 230 C (with decomposition). Identification (1) To 0.2 g of Aldioxa, add 10 mL of dilute hydrochloric acid, boil for 5 minutes and add 10 mL of a solution of phenylhydrazine hydrochloride (1 in 100). After cooling, mix well with 0.5 mL of potassium ferricyanide TS and shake with 1 mL of hydrochloric acid: a red color develops. (2) To 0.2 g of Aldioxa, add 10 mL of dilute hydrochloric acid, dissolve by warming and cool: the solution responds to the Qualitative Tests for aluminum salt. Purity (1) ChlorideTo 0.10 g of Aldioxa, add 6 mL of dilute nitric acid, boil to dissolve with shaking for 5 minutes, cool and add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.142%). (2) SulfateTo 0.20 g of Aldioxa, add 6 mL of dilute hydrochloric acid, boil to dissolve with shaking for 5 minutes, cool and add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.240%). (3) NitrateTo 0.10 g of Aldioxa, add carefully 5

KP 9 37 mL of water and 5 mL of sulfuric acid, dissolve by shaking, cool and superimpose 2 mL of ferrous sulfate TS: no brown ring is produced at the zone of contact. (4) Heavy metalsTo 1.0 g of Aldioxa, add 3mL of hydrochloric acid and 3 mL of water, heat gently to boil with shaking and evaporate in a water-bath to dryness. To the residue, add 30 mL of water, shake under warming, cool, filter and to the filtrate, add 2 mL of dilute acetic acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 3 mL of hydrochloric acid add 3 mL of water, evaporate in a water-bath to dryness and add 2.0 mL of standard lead solution, 2 mL of dilute acetic acid and water to make 50 mL (not more than 20 ppm). (5) ArsenicPrepare the test solution with 1.0 g of Aldioxa according to Method 2 and perform the test (not more than 2 ppm). Loss on Drying Not more than 4.0% (1 g, 105 C, 2 hours). Assay (1) AllantoinWeigh accurately about 0.1 g of Aldioxa, previously dried, dissolve in 50 mL of dilute sulfuric acid by heating, cool and add water to make exactly 100 mL. Pipet exactly 10 mL of this solution and perform the test as directed under the Nitrogen Determination. Each mL of 0.005 mol/L sulfuric acid VS = 0.39529 mg of C4H6N4O3 (2) AluminumWeigh accurately about 0.2 g of Aldioxa, previously dried, dissolve carefully in 50 mL of dilute hydrochloric acid by heating, cool and add dilute hydrochloric acid to make exactly 100 mL. Pipet exactly 4 mL of this solution, add water to make exactly 25 mL and use this solution as the test solution. Separately, pipet a suitable quantity of aluminum standard stock solution, dilute with water so that each mL of the solution contains not less than 16.0 g and not more than 64.0 g of aluminum (Al: 26.98) and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Atomic Absorption Spectrophotometry according to the following conditions and calculate the aluminum content of the test solution from the calibration curve obtained from the absorbance of the standard solution. Gas: Combustible gas- Acetylene. Supporting gas- Nitrous oxide. Lamp: An aluminum hollow cathode lamp. Wavelength: 309.2 nm. Packaging and Storage Preserve in well-closed containers.

Alfacalcidol
H H3C CH3 HO CH2 H OH H CH3 CH3

C27H44O2 : 400.64 Alfacalcidol contains not less than 97.0% and not more than 102.0% of alfacalcidol (C27H44O2). Description Alfacalcidol is a white crystals. Alfacalcidol is freely soluble in ethanol, soluble in fatty oils, and practically insoluble in water. Alfacalcidol is sensitive to air, heat and light. A reversible isomerisation to pre-alfacalcidol takes place in solution, and the activity is due to both compounds. Identification (1) Determine the infrared spectra of Alfacalcidol and Alfacalcidol RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Examine the chromatograms obtained in the assay. The principal peak in the chromatogram obtained with the test solution is same in retention time as the principal peak in the chromatogram obtained with standard solution (1). Purity Related substancesExamine by liquid chromatography as described under Assay. Calculate the percentage content of related substances, other than prealfacalcidol, that are eluted within twice the retention time of alfacalcidol from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure. The content of any individual related substance is not more than 0.5% and the total area of all the related substances is not more than 1.0%. Disregard any peak not more than 0.1%. Assay Carry out the assay as rapidly as possible, avoiding exposure to light and air. Weigh accurately about 1.0 mg of Alfacalcidol, dissolve in mobile phase to make exactly 10 mL and use this solution as the test solution. Separately, weigh accurately 1.0 mg of Alfacalcidol RS and dissolve in mobile phase without heating to make exactly 10 mL and use this solution as the standard solution (1). To 1.0 mL of the standard solution (1) add mobile phase to make exactly 100 mL and use this solution as the standard solution (2). Heat 2 ml of the standard solution (1) in a water-bath at 80 C under a reflux condenser for 2 hours and cool and use this solution as the

38 Monographs, Part I standard solution (3). Perform the test with 100 L each of the test solution and the standard solutions (1) and (2) as directed under Liquid Chromatography according to the following conditions, and determine each peak area, AT and AS of alfacalcidol, by the automatic integration method. Amount (mg) of alfacalcidol (C27H44O2) = amount (mg) of Alfacalcidol RS
AT AS

Description Alfuzosin Hydrochloride is a white crystalline powder. Alfuzosin Hydrochloride is freely soluble in water, sparingly soluble in ethanol, and practically insoluble in dichloromethane. Alfuzosin Hydrochloride is hygroscopic. Identification (1) Determine the infrared spectra of Alfuzosin Hydrochloride and Alfuzosin Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) To 0.5 g of Alfuzosin Hydrochloride add 25 mL of water. The diluted solution of 1 mL of this solution by 1 mL of water responds to the Qualitative Tests (2) for chlorides. pH The pH of the solution of 0.5 g of Alfuzosin Hydrochloride in 25 mL of water is between 4.0 and 6.0. Specific Optical Rotation [ ]20 D : Between -0.10 and +0.10 (calculated on the anhydrous basis, 0.2 g, water, 10 mL, 100 mm) Purity Related substancesWeigh accurately 20.0 mg of Alfuzosin Hydrochloride, dissolve in mobile phase to make exactly 100 mL and use this solution as test solution. Pipet exactly 1.0 mL of the test solution, add mobile phase to make exactly 50 mL, pipet 5.0 mL of this solution, add mobile phase to make exactly 20 mL and use this solution as the standard solution (1). Separately, weigh accurately 5 mg of Alfuzosin Impurity I {N-[3[(4-Amino-6,7-dimethoxyquinolin-2-yl)(methyl)amino] propyl]furan-2-carboxamide} RS, dissolve in mobile phase to make 25 mL, pipet exactly 1 mL of this solution, add 1 mL of the test solution and then add mobile phase to make exactly 100 mL and use this solution as the standard solution (2). Perform the test with exactly 20 L each of the test and the standard solution (2) as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of any peak other than the principal peak from the test solution is not greater than 0.6 times the area of the principal peak from the standard solution (1) (0.3%); the total area of all peaks other than the principal peak is not greater than the area of the principal peak with the standard solution (1) (0.5%). Disregard any peak with an area less than 0.025 times that of the principal peak from the standard solution (1). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter).

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 265 nm). Column: A stainless steel column about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: the mixture of acetonitrile, water and 9 mol/L ammonia water (800 : 200 : 1). Flow rate: 2.0 mL/minute. System suitability System performance: When the procedure is run with 100 L of the standard solution (3) under the above operating condition, the retention time for prealfacalcidol, relative to alfacalcidol, is about 1.3 with the resolution between the peaks due to pre-alfacalcidol and alfacalcidol being not less than 4.0 System repeatability: When the test is repeated 6 times with 100 L of the standard solution (3) under the above operating conditions, the relative standard deviation of the peak areas of alfacalcidol is not more than 1.0%. Packaging and Storage Preserve in a tight container, under nitrogen, protected from light, at a temperature of 2 C to 8 C. The contents of an opened container are to be used immediately.

Alfuzosin Hydrochloride
CH3 H3CO N N H N O H

HCl
N H3CO NH2 O

and enantiomer C19H27N5O4HCl : 425.91 Alfuzosin Hydrochloride contains not less than 98.5% and not more than 101.0% of alfuzosin hydrochloride (C19H27N5O4HCl), calculated on the anhydrous basis.

KP 9 39 Mobile phase: The mixture of sodium perchlorate solution, acetonitrile and tetrahydrofuran (80 : 20 : 1). Flow rate: 1.5 mL/minute System suitability System performance: Perform the test with 20 l of standard solution (2) according to the above conditions. Adjust the sensitivity of the system so that the height of the two peaks in the chromatogram obtained is at least 50% of the full scale of the recorder. The test is not valid unless the resolution between the peaks corresponding to alfuzosin and alfuzosin related substance I is at least 3.0. Sodium perchlorate solutionMix 5 mL of perchloric acid and 900 mL of water, adjust pH to 3.5 with 8.5w/v% sodium hydroxide VS and add water to make 1000 mL. Water Not more than 2.0% (0.5 g, volumetric titration, direct titration) Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Alfuzosin Hydrochloride, dissolve in the mixture of 40 mL of glacial acetic acid and 40 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 42.59 mg of C19H28ClN5O4. Packaging and Storage tight containers. Preserve in light-resistent, (1 in 50) is between 5.0 and 6.5. Alimemazine Tartrate is gradually colored by light. Identification (1) To 2 mL of a solution of Alimemazine Tartrate (1 in 100), add 1 drop of ferric chloride TS: a red-brown color is produced and immediately a yellow precipitate is formed. (2) Dissolve 1 g of Alimemazine Tartrate in 5 mL of water, add 3 mL of sodium hydroxide TS, extract with two 10 mL volumes of ether [use the aqueous layer obtained in the Identification (4)]. Shake the combined ether extracts with 3 g of anhydrous sodium sulfate, filter and evaporate the ether with the aid of a current of air. Dry the residue in a desiccator (in vacuum, P2O5) for 16 hours: it melts between 66 C and 70 C. (3) Determine absorption spectra of solutions of Alimemazine Tartrate and Alimemazine Tartrate RS, in water (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) The aqueous layer, obtained in the Identification (2), when neutralized with dilute acetic acid, responds to the Qualitative Tests (1) and (2) for tartrate. Melting Point Between 159 C and 163 C.

Alimemazine Tartrate
CH3 CH3 CH2CHCH2N CH3 N CH(OH)CO 2H CH(OH)CO 2H

Purity (1) Clarity and color of solution Dissolve 1.0 g of Alimemazine Tartrate in 20 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Alimemazine Tartrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Alimemazine Tartrate according to Method 3 and perform the test. Use a solution of magnesium nitrate in ethanol (1 in 5) (not more than 2 ppm). Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.8 g of Alimemazine Tartrate, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from red through brown to green-brown (indicator: 2 mL of naphtholbenzeine TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 37.349 mg of (C18H22N2S)2C4H6O6 Packaging and Storage tight containers. Preserve in light-resistant,

(C18H22N2S)2C4H6O6 : 746.98 Alimemazine Tartrate, when dried, contains not less than 98.0% and not more than 101.0% of alimemazine tartrate (C18H22N2S)2 C4H6O6). Description Alimemazine Tartrate is a white powder, is odorless and has a bitter taste. Alimemazine Tartrate is freely soluble in water or in glacial acetic acid, sparingly soluble in ethanol, and practically insoluble in ether. pH The pH of a solution of Alimemazine Tartrate

40 Monographs, Part I make exactly 10 mL and use this solution as test solution (1). Pipet 1 mL of the test solution (1), add a mixture of methanol and water (1:1) to make exactly 10 mL and use this solution as the test solution (2). Separately, weigh accurately about 10 mg of Allantoin RS, add a mixture of methanol and water (1 : 1) to make exactly 10 mL and use this solution as the standard solution (1). Weigh accurately about 10 mg of urea RS, add water to make exactly 10 mL, pipet 1 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution (2). Mix 1 mL each of the standard solution (1) and the standard solution (2) and use this solution as the standard solution (3). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 L of the test solution (1) and 5 L each of the test solution (2), the standard solution (1), the standard solution (2) and the standard solution (3) on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of 1-butanol, water and glacial acetic acid (60 : 25 : 15) to a distance of about 10 cm and air-dry the plate. Spray evenly the plate with a solution of 0.1 g dimethylaminobenzaldehyde in 20 mL of a mixture of methanol and hydrochloric acid (3 : 1). Dry the plate in a current of hot air. Examine under daylight after 30 min: any spot frorm the test solution (1), other than the principal spot, is not more intense than the spot from standard solution (2) (0.5%). The test is not valid unless the chromatogram from standard solution (3) shows two clearly separated principal spots. Loss on Drying Not more than 0.1% (2 g, 105 C, constant mass). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 1.2 g of Allantoin, previously dried, add 40 mL of water to dissolve. Titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L sodium hydroxide VS = 15.812 mg of C4H6N4O3 Packaging and Storage Preserve in well-closed containers

Allantoin
O NH2 NH O

HN

NH

C4H6N4O3 : 158.12 Allantoin, when dried, contains not less than 98.5% and not more than 101.0% of allantoin (C4H6N4O3). Description Allantoin is a white crystalline powder. Allantoin is slightly soluble in water and very slightly soluble in ethanol. Allantoin dissolves in sodium hydroxide TS. Melting Point About 225 C (decomposition) Identification (1) Boil 20 mg with a mixture of 1 mL of 2 mol/L sodium hydroxide TS and 1 mL of water. Allow to cool. Add 1 mL of 2 mol/L hydrochloric acid TS. To 0.1 mL of the solution add 0.1 mL of a solution of potassium bromide (1 in 10), 0.1 mL of a solution of resorcinol (1 in 50) and 3 mL of sulfuric acid. Heat for 5 min to 10 min on a water-bath. A dark blue color develops, which becomes red after cooling and pouring into about 10 mL of water. (2) Determine the infrared spectra of Allantoin and Allantoin RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) The principal spots in the chromatograms obtained with test solution (2) and the standard solution (1) in Related substances under Purity have the same Rf values. Specific Optical Rotation [ ]20 D : Between -0.10 and +0.10 (0.2 g, water, 20 mL, 100 mm) Purity (1) Acidity or alkalinityTo a 10 mL solution of about 50 mg of Allantoin in carbon dioxide-free water, add 0.1 mL of methyl red TS and 0.2 mL of 0.01 mol/L sodium hydroxide TS: the solution is yellow. Add 0.4 mL of 0.01 mol/L hydrochloric acid TS: the solution is red. (2) Reducing substancesMix 1.0 g of Allantoin with 10 mL of water by shaking for 2 minutes and filter. Add 1.5 mL of 0.02 mol/L potassium permanganate TS: the solution must remain violet for at least 10 min. (3) Potassium permangate Related substances Weigh accurately about 0.10 g of Allantoin, dissolve in 5 mL of water with heating. After cool add methanol to

Allopurinol
N H N N N

OH

C5H4N4O: 136.11

KP 9 41 nm): any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.16 g of Allopurinol, previously dried, dissolve in 70 mL of dimethylformamide by warming. Cool and titrate with 0.1 mol/L tetramethylammonium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Separately to 70 mL of dimethylformamide, add 12 mL of water, perform a blank determination with this solution and make any necessary correction. Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 13.611 mg of C5H4N4O Packaging and Storage Preserve in tight containers.

Allopurinol, when dried, contains not less than 98.0% and not more than 101.0% of allopurinol (C5H4N4O). Description Allopurinol is a white to pale yellowish white crystal or crystalline powder and is odorless. Allopurinol is slightly soluble in dimethylformamide, very slightly soluble in water, and practically insoluble in ethanol or in ether. Allopurinol dissolves in sodium hydroxide TS or in ammonia TS. Melting pointNot lower than 320 C (with decomposition) Identification (1) Dissolve 0.1 g of Allopurinol in 50 mL of water by warming. To 5 mL of this solution, add 1 mL of ammonia TS and 1 mL of silver nitrate TS: a white precipitate is produced. (2) Determine the absorption spectra of solutions of Allopurinol and Allopurinol RS, in water (1 in 200000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Allopurinol and Allopurinol RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Clarity and color of solutionDissolve 0.5 g of Allopurinol in 10 mL of sodium hydroxide TS: the solution is clear and has no more color than Color Matching Fluid D. (2) SulfateTo 2.0 g of Allopurinol, add 100 mL of water and boil for 5 minutes. Cool, add water to make 100 mL and filter. To 25 mL of the filtrate, add 1 mL of dilute hydrochloric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.038%). (3) Heavy metalsProceed with 1.0 g of Allopurinol according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Allopurinol according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 50 mg of Allopurinol in 10 mL of ammonia TS and use this solution as the test solution. Pipet exactly 1 mL of this solution, add ammonia TS to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of cellulose with fluorescent indicator for thin-layer chromatography. Develop the plate with ammonia TS saturated with nbutanol to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254

Allopurinol Tablets
Allpurinol Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of allopurinol (C5H4N4O : 136.11). Method of Preparation Prepare as directed under Tablets, with Allopurinol. Identification To a quantity of powdered Allopurinol Tablets, equivalent to 50 mg of Allopurinol according to the labeled amount, add 10 mL of 0.1 mol/L sodium hydroxide VS, mix thoroughly and extract. After filtration, make the filtrate acidic with 1 mol/L acetic acid. Collect the precipitate, wash few times with 3 mL of dehydrated ethanol, 4 mL of anhydrous ether, dry in air for 15 minutes and dry at 105 C for 3 hours. Determine the infrared spectra of the residue and Allopurinol RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Dissolution Test Perform the test with 1 tablet of Allopurinol Tablets at 75 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of 0.1 mol/L hydrochloric acid as the dissolution solution. Take a volume of the dissolved solution 45 minutes after starting the test and filter through a membrane filter, dilute with dissolution solution and use this solution as the test solution. Separately, weigh accurately a portion of Allopurinol RS, dissolve in the dissolution solution to make the same concentration with the test solution and use this solution as the standard solution. Determine the

42 Monographs, Part I 15 L of the standard solution under the above operating conditions. Hypoxanthin and allopurinol are eluted in this order with the resolution between their peaks being not less than 5. System repeatability: When the test is repeated 6 times with 15 L of the standard solution, the relative standard deviation of the ratios of the peak area of alloprinol to that of internal standard is not more than 3.0%. Packaging and Storage well-closed containers. Preserve in light-resistant,

absorbances, AT and AS , of the test solution and the standard solution, respectively, at approximately 250 nm as directed under the Ultrviolet-visible Spectrophotometry. The dissolution rate of Allopurinol Tablets in 45 minutes should be not less than 75%. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 tablets of Allopurinol Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of allopurinol (C5H4N4O), add exactly 10 mL of 0.1 mol/L sodium hydroxide VS, shake for 10 minutes and add water to make exactly 50 mL (perform the following assay as soon as possible). After centrifugation, discard 10 mL of the first filtrate, collect exactly 4 mL of the subsequent filtrate, add exactly 2.0 mL of the internal standard solution and the mobile phase to make exactly 200 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Allopurinol RS, previously dried at 105 C in vacuum for 5 hours, dissolve in 10 mL of 0.1 mol/L sodium hydroxide VS, mix in shaker for 10 minutes and add water to make exactly 50 mL. To exactly 4 mL of this solution, add exactly 2 mL of the internal standard solution and the mobile phase to make 200 mL and use this solution as the standard solution (prepare freshly when used). Perform the test with 15 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Allopurinol to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of allopurinol (C5H4N4O) = amount (mg) of Allopurinol RS QT
QS

Alprazolam
H3C N N N

Cl

C17H13ClN4: 308.77 Alprazolam, when dried, contains not less than 98.5% and not more than 101.0% of alprazolam (C17H13ClN4). Description Alprazolam is a white crystal or crystalline powder. Alprazolam is freely soluble in chloroform, soluble in methanol or in ethanol, sparingly soluble in acetic anhydride, and practically insoluble in water. Alprazolam dissolves in dilute nitric acid. Identification (1) Determine the absorption spectrum of a solution of Alprazolam in ethanol (1 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maximum at around 222 nm. (2) Dissolve 50 mg of Alprazolam in 0.7 mL of deuterochloroform for nuclear magnetic resonance spectroscopy, and determine the spectrum of this using tetramethylsilane for nuclear magnetic resonance spectroscopy as an internal reference compound, as directed under the Nuclear Magnetic Resonance Spectroscopy (1H): it exhibits singlet signal A at around 2.6 ppm, doublet signals B and C at around 4.0 ppm and 5.4 ppm, and a broad signal D between 7.1 ppm and 7.96 ppm. The ratio of integrated intensity of each signal, A : B : C : D, is about 3 : 1 : 1 : 8. (3) Perform the test with Alprazolam as directed under the Flame Coloration Test (2): a green color appears. Melting Point Between 228 C and 232 C. Purity (1) ChlorideDissolve 0.5 g of Alprazolam in 10 mL of dilute nitric acid, add water to make 50 mL,

Internal standard solutionA solution of 50 mg of hypoxantin and 10 mL of 0.1 mol/L sodium hydroxide VS, mix in shaker, add water to make exactly 50 mL. Prepare freshly when used. Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 - 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: 0.05 mol/L dibasic ammonium phosphate solution. Flow rate: 1.5 mL/minute. System suitability System performance: When procedure is run with

KP 9 43 and use this solution as the test solution. Prepare the control solution with 0.20 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.014%). (2) Heavy metalsProceed with 2.0 g of Alprazolam according to Method 4, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve 50 mg of Alprazolam in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of this solution, add methanol to make exactly 100 mL, then pipet exactly 1 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with these solutions as directed under the Thinlayer Chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plates with an upper layer of a mixture of acetone, hexane, ethyl acetate and ethanol (4 : 2 : 2 : 1) to a distance of about 10 cm, and air-dry the plates. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than principal spot from the test solution is not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, in vacuum at the pressure not exceeding 0.67 kPa, 60 C, 4 hours). Residue on Ignition Not more than 0.1% (1.0 g). Assay Weigh accurately about 0.25 g of Alprazolam, previously dried, dissolve in 100 mL of acetic anhydride, and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 15.438 mg of C17H13ClN4 Packaging and Storage Preserve in well-closed containers. tal or crystalline powder. Alprenolol Hydrochloride is freely soluble in water, in ethanol, or in glacial acetic acid, slightly soluble in acetic anhydride, and practically insoluble in ether. Identification (1) To 2 mL of a solution of Alprenolol Hydrochloride (1 in 100), add 50 L of cupric sulfate TS and 2 mL of sodium hydroxide TS: a blue-purple color is observed. To this solution, add 1 mL of ether, shake well and allow to stand: a red-purple color is observed in the ether layer. (2) Dissolve 50 mg of Alprenolol Hydrochloride in 5 mL of water, add 1 to 2 drops of bromine TS and shake: the color of the test solution disappears. (3) Determine absorption spectra of solutions of Alprenolol Hydrochloride, and Alprenolol Hydrochloride RS, in ethanol (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Alprenolol Hydrochloride and Alprenolol Hydrochloride RS, previously dried, as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (5) A solution of Alprenolol Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride. pH Dissolve 1.0 g of Alprenolol Hydrochloride in 10 mL of water: the pH of the solution is between 4.5 and 6.0. Melting Point Between 108 C and 112 C. Purity (1) Clarity and color of solutionDissolve 1.0 g of Alprenolol Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Alprenolol Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Alprenolol Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.10 g of Alprenolol Hydrochloride in 10 mL of ethanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add ethanol to make exactly 100 mL. Pipet exactly 2.5 mL of this solution, add ethanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of dichloromethane, acetone, glacial acetic acid and water (60 : 42 : 5 : 3) to a distance of about 10 cm, air-dry the plate and then dry at 80 C for 30 minutes. After cooling, allow the plate to stand in iodine vapor for 30 minutes: any spot other than the principal spot and the spot on the

Alprenolol Hydrochloride
OH OCH2CCH2NHCH H CH2 CH3 CH3 HCl

CH2CH

and enantiomer C15H23NO2HCl: 285.81 Alprenolol Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of alprenolol hydrochloride (C15H23NO2HCl). Description Alprenolol Hydrochloride is a white crys-

44 Monographs, Part I starting point from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, silica gel, in vacuum, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Alprenolol Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 28.581 mg of C15H23NO2.HCl Packaging and Storage Preserve in well-closed containers.

Specific Optical Rotation [ ]20 D : Between -53 and 61 (after drying, 25 mg, tetrahydrofuran, 5 mL, 100 mm) Melting Point Between 114 C and 118 C.

Alprostadil
O O H OH CH3 H OH H H OH

Purity Related substancesDissolve 4 mg of Alprostadil in 2 mL of a mixture of acetonitrile and water (9 : 1) and use this solution as the test solution. Pipet exactly 0.5 mL of this solution, add a mixture of acetonitrile and water (9 : 1) to make exactly 10 mL, pipet exactly 2.0 mL of this solution, add a mixture of acetonitrile and water (9 : 1) to make exactly 10 mL and use this solution as the standard solution. Perform the test with exactly 5 L each of the test solution and the standard solutions as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the areas of the peaks being about 0.70 and about 1.26 in relative retention time reference to the peak of alprostadil in the test solution are not larger than 0.5 times the peak area of alprostadil obtained with the standard solution; the areas of the peaks being about 0.88 and about 1.18 in relative retention time reference to alprostadil in test solution are not larger than the peak area of alprostadil obtained with the standard solution; the area of the peaks other than alprostadil and above mentioned peaks in the test solution is not larger than 0.1 times the peak area of alprostadil in standard solution. The total area of all peaks other than alprostadil is not larger than 2 times the peak area of alprostadil from the standard solution. Operating conditions Detector, column, column temperature, mobile phase, flow rate: Perform as directed in the operating conditions in the Assay. System suitability Test for required detectability: Pipet 2 mL of the standard solution, add a mixture of acetonitrile and water (9 : 1) to make 20 mL. Confirm that the peak area of alprostadil obtained with 5 L of this solution is equivalent to 7 to 13% of that with 5 L of the standard solution. System performance: Perform as directed in the system suitability in the Assay. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of alprostadil is not more than 1.5%. Time span of measurement: About 3 times as long as the retention time of alprostadil beginning after the solvent peak. Loss on Drying Not more than 1.0% (0.1 g, in vacuum, phosphorus (V) oxide, 4 hours). Assay Weigh accurately about 5 mg each of Alprostadil and Alprostadil RS, previously dried, to each add exactly 5 mL of internal standard solution to dissolve, add a mixture of acetonitrile and water (9 : 1) to make exactly

Prostaglandin E1

C20H34O5 : 354.48

Alprostadil, when dried, contains not less than 97.0% and not more than 103.0% of alprostadil (C20H34O5). Description Alprostadil is a white crystals or crystalline powder. Alprostadil is freely soluble in dehydrated ethanol or tetrahydrofuran, slightly soluble in acetonitrile, and practically insoluble in water. Identification (1) To 10 mL each of the solutions of Alprostadil and Alprostadil RS, respectively, in dehydrated ethanol (1 in 100000) add 1 mL of potassium hydroxideethanol TS and allow to stand for 15 minutes. Determine the absorption spectra of the solutions as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Alprostadil and Alprostadil RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers.

KP 9 45 50 mL, and use the solutions as test solution and standard solution, respectively. Perform the test with 5 L of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of alprostadil to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of alprostadil (C20H34O5) = amount (mg) of Alprostadil RS
QT QS

hydroxide. Aluminum Hydroxide Gel contains not less than 3.6% and not more than 4.4% of the labeled amount of aluminum oxide (Al2O3: 101.96) when analyzed as aluminum hydroxide suspension. Aluminum Hydroxide Gel may contain peppermint oil, glycerin, sorbitol, sucrose, saccharin, or other suitable flavors and suitable antimicrobial agents. Description Aluminum Hydroxide Gel is a white, viscous suspension. A volume of water layer is separated on standing. Identification Dissolve approximately 5 mL of Aluminum Hydroxide Gel in 10 mL of dilute hydrochloride acid and warm: the solution responds to Qualitative Tests for aluminum salt. pH Between 5.5 and 8.0. Purity (1) ChlorideTransfer an accurately measured volume of Aluminum Hydroxide Gel, equivalent to 10 g of Al(OH)3, to a porcelain dish. Add 0.1 mL of potassium chromate TS and 25 mL of water. Stir and add 0.10 mol/L of silver nitrate until a faint, persistent pink color is obtained: not more than 8.0 mL of 0.10 mol/L nitrate is required (not more than 0.28%). (2) SulfateAdd 5.0 mL of 3 mol/L of hydrochloric acid to an accurately measured volume of Aluminum Hydroxide Gel, equivalent to 5 g of Aluminum Hydroxide Gel and heat to dissolve. Cool, dilute with water to make 250 mL and filter, if necessary. Add 1 mL of dilute hydrochloric acid to a 20 mL portion of the filtrate, dilute with water to make 50 mL and use this solution as the test solution. 0.40 mL of 0.005 mol/L sulfuric acid are added to reference standard (not more than 0.05%). (3) ArsenicAdd 10 mL of dilute sulfuric acid to 1.2 g of Aluminum Hydroxide Gel, shake, heat to boiling and cool. Dilute with water to make 20 mL and filter. Use 10 mL of the filtrate as the test solution. Perform the test (not more than 0.6 ppm). (4) Heavy metalAdd 5 mL of dilute hydrochloric acid to 5.0 g of Aluminum Hydroxide Gel, shake, heat to boiling and evaporate in a water-bath to dryness. Add 30 mL of water to the residue, warm, shake, cool and filter. Add 2 mL of dilute acetic acid and dilute with water to make 50 mL. Use this solution as the test solution and perform the test. Evaporate 5 mL of dilute hydrochloric acid in a water-bath. Add 2.5 mL of the standard lead solution and 2 mL of diluted acetic acid. Dilute with water to make 50 mL and use this solution as the control solution (not more than 5 ppm). Test for Acid-Neutralizing Capacity Add about 1.5 mL of Aluminum Hydroxide Gel in a flask equipped with a stopper, previously weighed. Weigh accurately and perform the test: The volume of 0.1 mol/L of hydrochloric acid VS consumed is 12.5 to 25.0 mL per g of Aluminum Hydroxide Gel.

Internal standard solutionA solution of dimethyl phthalate in a mixture of acetonitrile and water (9 : 1) (1 in 10000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 196 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 o C . Mobile phase: Dissolve 9.07 g of potassium dihydrogen phosphate in water to make 1000 mL, add the solution prepared by dissolving 9.46 g of potassium monohydrogen phosphate in water to make 1000 mL to adjust pH to 6.3. Dilute this solution 10 times with water. To 360 mL of this solution add 110 mL of acetonitrile and 30 mL of methanol and mix. Flow rate: Adjust the flow rate so that the retention time of alprostadil is about 10 minutes. System suitability System performance: When the prodedure is run with 5 L of the standard solution, as diredcted under the above operating, alprostadil and the internal standard are eluted in this order with the resolution between their peaks being not less than 9. System repeatability: When the test is repeated 6 times with 5 L of the standard solution, as directed under the above conditions, relative standard deviation of the ratios of the peak area of alprostadil reference to the internal standard is not more than 1.0%. Packaging and Storage Preserve in light-resistant, tight containers. Store at below 5 C.

Aluminum Hydroxide Gel

AI(OH)3: 78.00 Aluminum Hydroxide Gel is a suspension of aluminum

46 Monographs, Part I Microbial Limits Its total viable aerobic microbial count does not exceed 100 per mL and it meets the requirement of the test for the absence of Escherichia coli. Assay Transfer an accurately measured volume of Aluminum Hydroxide Gel, equivalent to about 25 g of Aluminum Hydroxide Gel, to a beaker, add 15 mL of hydrochloric acid and heat gently until solution is clear. Cool, transfer to a volumetric flask, dilute with water to make 500 mL and mix. Pipet 20 mL of this solution into a beaker and add 25.0 mL of disodium ethylenediaminetetraacetate VS and 20 mL of acetic acid ammonium acetate buffer TS, with continuous stirring then heat near the boiling point for 5 minutes. Cool and add 50 mL of ethanol and 2 mL of dithizone TS. Titrate the solution with 0.05 mol/L of zinc sulfate VS until the color changes from green-violet to rose-pink. Perform a blank determination, substituting 20 mL of water for the sample and make any necessary correction. Each mL of 0.05 mol/L of disodium ethylenediaminetetraacetate VS = 2.5490 mg of Al2O3 Packaging and Storage Preserve in tight containers, avoiding freezing. heat gently to boiling. Cool, dilute with water to make 250 mL and centrifuge. Add 1 mL of dilute hydrochloric acid to 25 mL of the supernatant and dilute with water to make 50 mL. Use this solution as the test solution. Prepare the control solution with 1.0 mL of 0.005 mol/L of hydrochloric acid (not more than 0.480%). (4) NitrateAdd 0.1 g of Dried Aluminum Hydroxide Gel to 5 mL of water. Add 5 mL of sulfuric acid carefully to dissolve, shake and cool. No brown ring is produced when ferric chloride is added drop-wise. (5) Heavy metalAdd 0.5 g of Dried Aluminum Hydroxide Gel to 5 mL of dilute hydrochloric acid and dilute with water to make 50 mL. Use this solution as the test solution and perform the test. Evaporate 5 mL of dilute hydrochloric acid to dryness. Add 3.0 mL of the standard lead solution and 2 mL of dilute acetic acid. Dilute with water to make 50 mL (not more than 60 ppm). (6) ArsenicTo 0.8 g of Dried Aluminum Hydroxide Gel, add 10 mL of dilute sulfuric acid, heat gently to boil while shaking, cooling and filter. Take 5 mL of the filtrate, use this solution as the test solution and perform the test (not more than 5 ppm). Test for Acid-Neutralizing Capacity Weigh precisely about 0.2 g of Dried Aluminum Hydroxide Gel in a flask equipped with a stopper and perform the test: The volume of 0.1 mol/L of hydrochloric acid VS consumed is not less than 250 mL per 1.0 g of Aluminum Hydroxide Gel. Assay Transfer an accurately measured quantity of Dried Aluminum Hydroxide Gel, equivalent to about 22 g of Aluminum Hydroxide Gel, to a beaker, add 15 mL of hydrochloric acid and heat gently until solution is clear. Cool, transfer to a volumetric flask, dilute with water to make 500 mL and mix. Pipet 20 mL of this solution into a beaker and add 30 mL of 0.05 mol/L disodium ethylenediaminetetraacetate VS and 20 mL of acetic acid ammonium acetate buffer TS with continuous stirring, then heat the solution near the boiling point for 5 minutes. Cool and add 55 mL of ethanol and 2 mL of dithizone TS. Titrate the solution with 0.05 mol/L of zinc sulfate VS until the color changes from pale dark green to pale red. Perform a blank determination, substituting 20 mL of water for the sample and make any necessary correction. Each mL of 0.05 mol/L of disodium ethylenediaminetetraacetate VS = 2.5490 mg of Al2O3 Packaging and Storage Preserve in tight containers.

Dried Aluminum Hydroxide Gel

Dried Aluminum Hydroxide gel contains not less than 50.0% of aluminum oxide (Al2O3: 101.96). Description Dried Aluminum Hydroxide Gel is a white and amorphous powder. Dried Aluminum Hydroxide gel is odorless and has no taste. Dried Aluminum Hydroxide Gel is practically insoluble in water, in ether or in ethanol Most of Dried Aluminum Hydroxide Gel is soluble in dilute hydrochloric acid or in sodium hydroxide TS. Identification Dissolve 0.2 g of Dried Aluminum Hydroxide Gel in 20 mL of dilute hydrochloric acid by warming and centrifuge. The supernatant responds to Qualitative test for aluminum salt. Purity (1) Acidity of solutionAdd 1.0 g of Dried Aluminum Hydroxide Gel in 25 mL of water and centrifuge: the supernatant is neutral. (2) ChlorideAdd 1.0 g of Dried Aluminum Hydroxide Gel to 30 mL of dilute hydrochloric acid, shake and heat gently to boiling. Cool, dilute with water to make 100 mL and centrifuge. Add 6 mL of dilute hydronitric acid to 5 mL of the supernatant and dilute with water to make 50 mL. Use this solution as the test solution. Prepare the control solution with 0.4 mL of 0.01 mol/L of hydrochloric acid (not more than 0.284%). (3) SulfateAdd 1.0 g of Dried Aluminum Hydroxide Gel to 15 mL of dilute hydrochloric acid. shake and

Dried Aluminum Hydroxide Gel Fine Granules

Dried Aluminum Hydroxide Gel Fine Granules contain not less than 47.0% of aluminum oxide (Al2O3: 101.96).

KP 9 47 and perform the test with this solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.021%). (3) SulfateTo the residue obtained in (6), add 3 mL of dilute hydrochloric acid, heat on a water-bath for 10 minutes, dilute with 50 mL of water and filter. To 2.0 mL of the filtrate, add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test with this solution as the test solution. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.480%). (4) Heavy metalsTo 1.5 g of Natural Aluminum Silicate, add 50 mL of water and 5 mL of hydrochloric acid, boil gently for 20 minutes while shaking, then cool, centrifuge, remove the supernatant liquid, wash the residue with two 10 mL volumes of water, centrifuging each time, combine these washings with the filtrate and add strong ammonia water drop-wise until a precipitate just produces. Add dilute hydrochloric acid drop-wise with vigorous shaking and redissolve the precipitate. Heat the mixture with 0.45 g of hydroxylamine hydrochloride, cool and add 0.45 g of sodium acetate, 6 mL of dilute acetic acid and water to make 150 mL. Perform the test with 50 mL of this solution as the test solution. Prepare the control solution with 2.0 mL of standard lead solution, 0.15 g of hydroxylamine hydrochloride, 0.15 g of sodium acetate, 2 mL of dilute acetic acid and water to make 50 mL (not more than 40 ppm). (5) ArsenicTo 1.0 g of Natural Aluminum Silicate, add 5 mL of dilute hydrochloric acid, heat gently to boil while shaking well, cool rapidly and centrifuge. Mix the residue with 5 mL of dilute hydrochloric acid with shaking, centrifuge, then add 10 mL of water to the residue and repeat the extraction in the same manner. Concentrate the combined extracts in a water-bath to 5 mL, use this solution as the test solution and perform the test (not more than 2 ppm). (6) Soluble saltsEvaporate 50 mL of the supernatant liquid obtained in (1) on a water-bath to dryness and ignite the residue at 700 for 2 hours: the weight of the ignited residue is not more than 40 mg. (7) Fluoride(i) Apparatus: Use a hard glass apparatus as illustrated in the figure. Ground-glass joints may be used.

Method of Preparation Prepare as directed under Granules, with Dried Aluminum Hydroxide Gel. Identification Dissolve 0.2 g of Dried Aluminum Hydroxide Gel in 20 mL of dilute hydrochloric acid by warming and centrifuge. The supernatant responds to Qualitative Test for aluminum salt. Particle Size Distribution Test It meets the requirement. Test for Acid-Neutralizing Capacity Proceed as directed in the Acid-neutralizing Capacity under Dried Aluminum Hydroxide Gel: the volume of 0.1 mol/L of hydrochloric acid VS consumed is not less than 235 mL per 1.0 g of Aluminum Hydroxide Gel. Assay Proceed as directed in the Assay under Dried Aluminum Hydroxide Gel. Each mL of 0.05 mol/L of disodium ethylenediaminetetraacetate VS = 2.5490 mg of Al2O3 Packaging and Storage Preserve in tight containers

Natural Aluminum Silicate

Description Natural Aluminum Silicate is a white or slightly colored powder, is odorless and tasteless. Natural Aluminum Silicate is practically insoluble in water, in ethanol or in ether. Natural Aluminum Silicate, 1 g, dissolves when heated in 20 mL of sodium hydroxide solution (1 in 5), with some decomposition, leaving a large amount of insoluble substance. Identification (1) To 0.5 g of Natural Aluminum Silicate, add 3 mL of diluted sulfuric acid (1 in 3), heat until white fumes evolve, cool, add 20 mL of water and filter. Render the filtrate slightly acidic with ammonia TS: the solution responds to the Qualitative Tests for aluminum salt. (2) Prepare a bead by fusing dibasic sodium ammonium phosphate on a platinum loop. Place the bead in contact with Natural Aluminum Silicate and fuse again: an infusible material appears in the bead, producing, upon cooling, an opaque bead with a web-like structure. Purity (1) Acidity or alkalinityShake 5.0 g of Natural Aluminum Silicate with 100 mL of water and centrifuge: the supernatant liquid so obtained is neutral. (2) ChlorideTo 5.0 g of Natural Aluminum Silicate, add 100 mL of water, boil gently for 15 minutes while shaking, then cool, add water to restore the original volume and centrifuge. To 10 mL of the supernatant liquid, add 6 mL of dilute nitric acid, dilute to 50 mL with water

48 Monographs, Part I The content of fluoride (F) is not more than 0.01%. Loss on Drying Not more than 20.0% (1 g, 105 C, 3 hours). Adsorptive Power To 0.10 g of Natural Aluminum Silicate, add 20 mL of a solution of methylene blue (3 in 2000), shake for 15 minutes, allow to stand for 5 hours at 37 2 C and centrifuge. Dilute 1.0 mL of the supernatant liquid with water to make 200 mL. Place 50 mL of the solution in a Nessler tube and observe horizontally or vertically against a white background: the color of the solution is not more intense than that of the following control solution. Control solutionDilute 1.0 mL of a solution of methylene blue (3 in 2000) with water to make 400 mL and use 50 mL of this solution.
A: Distilling flask of about 300-mL capacity B: Steam generator of about 1000-mL capacity, add a boiling stone to prevent bumping C: Condenser D: Receiver (200-mL volumetric flask) E: Steam-introducing tube having an inside diameter of about 8 mm F, G: Rubber tube with a pinch-coke H: Thermometer

Packaging and Storage Preserve in well-closed containers.

Synthetic Aluminum Silicate

Description Synthetic Aluminum Silicate is a white powder, is odorless and tasteless. Synthetic Aluminum Silicate is practically insoluble in water, in ethanol or in ether. Synthetic Aluminum Silicate, 1 g, dissolves when heated in 20 mL of a solution of sodium hydroxide (1 in 5), leaving a small amount of insoluble substance. Identification Proceed as directed in the Identification under Natural Aluminum Silicate. Purity (1) Acidity or alkalinityShake 1.0 g of Synthetic Aluminum Silicate with 20 mL of water and centrifuge: the supernatant liquid so obtained is neutral. (2) ChlorideProceed as directed in the Purity under Natural Aluminum Silicate. (3) SulfateTo 2.0 mL of the supernatant liquid obtained in (2), add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test with this solution as the test solution. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.480%). (4) Heavy metalsWeigh about 3.0 g of Synthetic Aluminum Silicate and proceed as directed in the Purity (4) under Natural Aluminum Silicate. Prepare the control solution with 3.0 mL of standard lead solution (not more than 30 ppm). (5) ArsenicTo 1.0 g of Synthetic Aluminum Silicate, add 10 mL of dilute hydrochloric acid, proceed as directed in the Purity (5) under Natural Aluminum Silicate (not more than 2 ppm). Loss on Drying Not more than 20.0% (1 g, 105 C, 3

(ii) Procedure: Transfer 5.0 g of Natural Aluminum Silicate to the distilling flask A with the aid of 20 mL of water, add about 1 g of glass fiber and 50 mL of diluted purified sulfuric acid (1 in 2) and connect A to the distillation apparatus, previously washed with steam through the steam introducing tube E. Connect the condenser C with the receiver D containing 10 mL of 0.01 mol/L sodium hydroxide VS and 10 mL of water so that the lower end of C is immersed in the solution. Heat A gradually until the temperature of the solution in A reaches 130 C, then open the rubber tube F, close the rubber tube G and boil water in the steam generator B vigorously. Simultaneously, heat A and maintain the temperature of the solution in A between 135 C and 145 C Adjust the distilling rate to about 10 mL per minute. Collect about 170 mL of the distillate, then stop the distillation, wash C with a small quantity of water, combine the washings with the distillate, add water to make exactly 200 mL and use this solution as the test solution. Perform the test with the test solution as directed in the procedure of determination for fluoride under the Oxygen Flask Combustion Method. No corrective solution is used in this procedure. Amount (mg) of fluoride (F: 19.00) in the test solution = amount (mg) of fluoride in 5 mL of the standard solution
AT 200 AS V

KP 9 49 hours). Test for Acid-Neutralizing Capacity Weigh accurately about 1g of Synthetic Aluminum Silicate, transfer to a glass-stoppered flask, add 200 mL of 0.1 mol/L hydrochloric acid VS, exactly measured, stopper the flask, and shake at 37 2 C for 1 hour. Filter, pipet 50 mL of the filtrate, and titrate by stirring well the excess hydrochloric acid with 0.1mol/L sodium hydroxide VS until the pH of the solution changes to 3.5. The volume of 0.1 mol/L hydrochloric acid VS consumed is not less than 50.0 mL per g of Synthetic Aluminum Silicate. Packaging and Storage Preserve in well-closed containers.

50 Monographs, Part I solution as directed under the Gas Chromatography according to the following conditions. Determine each peak area of these solutions by the automatic integration method: the area of each peak other than that of major peak from the test solution is not larger than 1/3 of the area of major peak from the standard solution and the total area of all peaks is not larger than the area of major peak from the standard solution. Operating conditions Detector: A hydrogen flame-ionization detector. Column: A glass column, about 3 mm in inside diameter and about 2 m in length, packed with siliceous earth for gas chromatography (150 - 180 m in particle diameter) coated with a mixture of branched hydrocarbon of petroleum hexamethyltetracosane group for gas chromatography and potassium hydroxide at the ratios of 2% and 1%, respectively. Column temperature: Inject at a constant temperature of about 125 C, maintain the temperature for 5 minutes, raise at the rate of 5 C per minute to 150 C and maintain at a constant temperature of about 150 C for 15 minutes. Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of amantadine is about 11 minutes. System suitability System performance: Dissolve 0.15 g of naphthalene in 5 mL of the test solution and add chloroform to make 100 mL. When the procedure is run with 2 L of this solution under the above operating conditions, naphthalene and amantadine are eluted in this order with the resolution between these peaks being not less than 2.5. Detection sensitivity: Adjust the detection sensitivity so that the peak height of amantadine obtained from 2 L of the standard solution composes about 10% of the full scale. Time span of measurement: About twice as long as the retention time of amantadine after the solvent peak. Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.2 g of Amantadine Hydrochloride, previously dried, dissolve in 2 mL of formic acid, add exactly 15 mL of 0.1 mol/L perchloric acid VS and heat on a water-bath for 30 minutes. After cooling, add glacial acetic acid to make 70 mL and titrate the excess perchloric acid with 0.1 mol/L sodium acetate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 18.771 mg of C10H17NHCl Packaging and Storage Preserve in well-closed con-

Amantadine Hydrochloride
NH2 HCl

C10H17NHCl: 187.71 Amantadine Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of amantadine hydrochloride (C10H17NHCl). Description Amantadine Hydrochloride is a white crystalline powder, is odorless and has a bitter taste. Amantadine Hydrochloride is very soluble in formic acid, freely soluble in water, in methanol or in ethanol and practically insoluble in ether. Identification (1) To 0.1 g of Amantadine Hydrochloride, add 1 mL of pyridine and 0.1 mL of acetic anhydride, dissolve by boiling for 1 minute, add 10 mL of dilute hydrochloric acid and cool in ice-water. Filter the crystals separated, wash with water and dry at 105 C for 1 hour: the residue melts between 147 C and 151 C. (2) Determine the infrared spectra of Amantadine Hydrochloride and amantadine hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Amantadine Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride. pH Dissolve 1.0 g of Amantadine Hydrochloride in 5 mL of water: the pH of this solution is between 4.0 and 6.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Amantadine Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Amantadine Hydrochloride according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Amantadine Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.50 g of Amantadine Hydrochloride in 10 mL of water, add 10 mL of sodium hydroxide TS and 10 mL of chloroform and shake. Filter the chloroform layer through absorbent cotton with 3 g of anhydrous sodium sulfate on a funnel and use the filtrate as the test solution. Pipet exactly 1 mL of the test solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with 2 L each of the test solution and the standard

KP 9 51 tainers. 10 cm and air-dry the plate. Allow the plate to stand in the saturated iodine vapor: any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution.
2 Cl

Ambenonium Chloride
Cl CH2CH3 H2C N CH2CH2NH O C O C NHCH2CH2 CH2CH3 N CH2 Cl

Loss on Drying Not more than 11.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.2% (1 g).

CH2CH3

CH2CH3

C28H42C14N4O2: 608.47 Ambenonium Chloride contains not less than 98.5% and not more than 101.0% of ambenonium chloride (C28H42C14N4O2), calculated on the dried basis. Description Ambenonium Chloride is a white powder, is odorless and has a bitter taste. Ambenonium Chloride is freely soluble in water, in methanol or in glacial acetic acid, soluble in ethanol, and slightly soluble in acetic anhydride. Ambenonium Chloride is hygroscopic. Melting pointAbout 205 C (with decomposition). Identification (1) Determine absorption spectra of the solutions of Ambenonium Chloride and Ambenonium Chloride RS, in methanol (1 in 5000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Ambenonium Chloride and Ambenonium Chloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Ambenonium Chloride (1 in 100) responds to the Qualitative Tests for chloride. Purity (1) Clarity and color of solutionDissolve 1.0 g of Ambenonium Chloride in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Ambenonium Chloride according to Method 4 and perform the test. Use a solution of magnesium nitrate in ethanol (1 in 5). Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 0.10 g of Ambenonium Chloride in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add methanol to make exactly 20 mL. Pipet exactly 1 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of n-butanol, formic acid and water (12 : 6 : 5) to a distance of about
o

Assay Weigh accurately about 0.3 g of Ambenonium Chloride and dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3). Titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 30.424 mg of C28H42Cl4N4O2 Packaging and Storage Preserve in tight containers.

Amidotrizoic Acid
CO2H I O CH3 C HN I NH I O C CH3

C11H9I3N2O4: 613.91 Amidotrizoic Acid contains not less than 98.0% and not more than 101.0% of amidotrizoic acid (C11H9I3N2O4), calculated on the dried basis. Description Amidotrizoic Acid is a white, crystalline powder and is odorless. Amidotrizoic Acid is slightly soluble in ethanol, very slightly soluble in water and practically insoluble in ether. Amidotrizoic Acid dissolves in sodium hydroxide TS. Identification (1) Heat 0.1 g of Amidotrizoic Acid over flame: a purple gas is evolved. (2) Determine the infrared spectra of Amidotrizoic Acid and amidotrizoic acid RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Clarity and color of solution Dissolve 1.0 g of Amidotrizoic Acid in 10 mL of 0.2 mol/L sodium hydroxide TS: the solution is clear and colorless. (2) Primary aromatic aminesDissolve 0.20 g of Amidotrizoic Acid in 5 mL of water and 1 mL of sodium hydroxides TS, add 4 mL of a solution of sodium nitrite (1 in 100) and 10 mL of 1 mol/L hydrochloric acid TS,

52 Monographs, Part I shake and allow to stand for 2 minutes. Add 5 mL of ammonium sulfamate TS, shake well, allow to stand for 1 minute and add 0.4 mL of a solution of -naphthol in ethanol (1 in 10), 15 mL of sodium hydroxide TS and water to make exactly 50 mL. Determine the absorbance of this solution at 485 nm using a solution, prepared in the same manner, as the blank: the absorbance is not more than 0.15. (3) Soluble halidesDissolve 2.5 g of Amidotrizoic Acid in 20 mL of water and 2.5 mL of ammonia TS, add 20 mL of dilute nitric acid and water to make 100 mL, allow to stand for 15 minutes with occasional shaking and filter. Discard the first 10 mL of the filtrate, transfer the subsequent 25 mL of the filtrate to a Nessler tube and add ethanol to make 50 mL. Proceed as directed under the Chloride Limit Test using this solution as the test solution. Prepare the control solution as follows: to 0.10 mL of 0.01 mol/L hydrochloric acid VS, add 6 mL of dilute nitric acid and water to make 25 mL, then ethanol to make 50 mL. (4) IodineDissolve 0.20 g of Amidotrizoic Acid in 2.0 mL of sodium hydroxide TS, add 2.5 mL of 0.5 mol/L sulfuric acid TS, allow to stand for 10 minutes with occasional shaking, add 5 mL of chloroform, shake well and allow to stand: the solution is colorless in the chloroform layer. (5) Heavy metalsProceed with 2.0 g of Amidotrizoic Acid according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (6) ArsenicPrepare the test solution with 0.6 g of Amidotrizoic Acid according to Method 3 and perform the test (not more than 3.3 ppm). Loss on Drying Not more than 7.0% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Transfer about 0.5 g of Amidotrizoic Acid, accurately weighed, to a saponification flask, dissolve in 40 mL of sodium hydroxide TS, add 1 g of zinc powder, connect to a reflux condenser, boil for 30 minutes, cool and filter. Wash the flask and the filter paper with 50 mL of water and combine the washings and the filtrate. Add 5 mL of glacial acetic acid to this solution and titrate with 0.1 mol/L silver nitrate VS until the color of the precipitate changes from yellow to green (indicator: 1 mL of tetrabromophenolphthalein ethyl ester TS). Each mL of 0.1 mol/L silver nitrate VS = 20.464 mg of C11H9I3N2O4 Packaging and Storage tight containers. Preserve in light-resistant,

Amikacin
OH HO H2N OH O HO OH O NH2 HO N H HO H O NH2 O NH2 HO O

C22H43N5O13: 585.60 Amikacin contains not less than 900 g (potency) of amikacin (C22H43N5O13) per mg, calculated on the anhydrous basis. Description Amikacin is a white to light grayish white wool-like powder. Identification Weigh about 60 mg each of Amikacin and Amikacin Sulfate RS, dissolve each in water to make the solutions so that each mL contains 6 mg, and use these solutions as the test solution and the standard solution, respectively. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of methanol, concentrated ammonia, and chloroform (60:30:25) in a 230 x 230 x 90 mm developing chamber for 5 hours and 30 minutes, in which 200 x 30 mm Whatman filter paper or an identical filter paper strip is hung in the front of the chamber, the plate is slanted for the silica gel layer to face toward the front side of the chamber, and the cover with holes is placed on the top. The holes are sealed with scotch tape except the holes above the filter paper. Air-dry the plate, and spray evenly the mixture of 100 mL of 1 % ninhydrinbutanol solution and 1 mL of pyridine, and heat at 110 C for 10 minutes: the spots obtained from the test solution and the standard solution is the same in the Rf value. Crystalliity Test It meets the requirement. Specific Optical Rotation [ ]D : +97 ~ +105(0.5 g calculated on the anhydrous basis, water, 25 mL, 100 mm). pH The pH of a solution obtained by dissolving 0.1 g of Amikacin in 10 mL of water is between 9.5 and 11.5. Water Not more than 8.5 % (0.1 g, volumetric titration, direct titration). Residue on Ignition Not more than 1.0 % (1 g). Assay The Cylinder-plate method (1) Agar media
25

KP 9 53 for seed and base layer- Use the medium in I 2 1) (1) 2 under Microbial Assay for Antibiotics. (2) Test organism- Bacillus subtilis ATCC 6633. (3) Weigh accurately about 15 mg (potency) of Amikacin, and dissolve in sterile distilled water to make the solution so that each mL contains 400 g (potency), and use the solution as the test stock solution. Take exactly a suitable amount of the test stock solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration test solution and the low concentration test solution, respectively. Separately, weigh accurately about 15 mg (potency) of Amikacin Sulfate RS, dissolve in 0.05 mol/L phosphate buffer solution, pH 6.0 to make the solution so that each mL contains 400 g (potency), and use the solution as the standard stock solution. Keep the standard stock solution between 5 C and 15 C, and use within 30 days. Take exactly a suitable amount of the standard stock solution in use, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in tight containers. purple color develops. (2) To 20 mg of Amikacin Sulfate add 2 mL of 0.06 mol/L phosphate buffer solution, pH 5.6, and add 1 mL of ninhydrin TS and boil: the blue-purple color develops. (3) To 10 mg of Amikacin Sulfate add 1 mL of water, add 1 mL of 1 mol/L of sodium hydroxide TS, and mix the solution and add 2 mL of cobalt nitrate TS: the purple color develops. (4) To 20 mg of Amikacin Sulfate add 2 mL of water, and add 1 mL of barium chloride TS: the solution turns to turbid. (5) Weigh about 50 mg each of Amikacin Sulfate and Amikacin Sulfate RS, dissolve each in water to make the solutions so that each mL contains 10 mg, and use these solutions as the test solution and the standard solution, respectively. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the mixture of methanol, concentrated ammonia, water, and chloroform (4:2:1:1), and air-dry the plate. Spray evenly 0.2 % ninhydrin unsaturated butanol solution: the spots obtained from the test solution and the standard solution is the same in the Rf value. Specific Optical Rotation water, 25 mL, 100 mm).

[ ]20 D : +69 ~ +79 (0.25 g,

Amikacin Sulfate
OH HO H2N OH O HO OH O NH2 O OH HO NH O NH2 HO O

pH The pH of a solution obtained by dissolving 0.1 g of Amikacin Sulfate in 10 mL of water is between 6.0 and 7.5. Purity Heavy metals Proceed with 1.0 g of Amikacin Sulfate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Sterility Test It meets the requirement, when Amikacin Sulfate is used in a sterile preparation. Bacterial Endotoxins Less than 0.50 EU per mg of amikacin, when Amikacin Sulfate is used in a sterile preparation. Loss on Drying Not more than 5.0 % (0.1 g, in vacuum, 60 C, 3 hours). Assay The Cylinder-plate method (1) Agar media 1 for seed and base layer- Use the medium in I 2 1) (1) under Microbial Assay for Antibiotics. (2) Test organism- Bacillus subtilis ATCC 6633. (3) Weigh accurately about 15 mg (potency) of Amikacin Sulfate, and dissolve in sterile distilled water to make the solution so that each mL contains 400 g (potency), pipet exactly a suitable amount of this solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high

xH2SO4

H H2N

C22H43N5O13xH2SO4 Amikacin Sulfate is the sulfate of a derivative of kanamycin. Amikacin Sulfate contains not less than 635 g (potency) of amikacin (C22H43N5O13: 585.60) per mg, calculated on the dried basis. Description Amikacin Sulfate is a white to yellowish white powder, and is almost odorless and tasteless. Amikacin Sulfate is very soluble in water, and practically insoluble in ethanol or in ether. Identification (1) To 50 mg of Amikacin Sulfate add 3 mL of water, and add 4 mL of anthrone TS: the blue-

54 Monographs, Part I concentration test solution and low concentration test solution, respectively. Separately, weigh accurately about 15 mg (potency) of Amikacin Sulfate RS, dissolve in 0.05 mol/L phosphate buffer solution, pH 6.0 to make the solution so that each mL contains 400 g (potency), and use the solution as the standard stock solution. Keep the standard stock solution between 5 C and 15 C, and use within 30 days. Take exactly a suitable amount of the standard stock solution in use, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in tight containers. and use this solution as the test solution. Separately, weigh accurately about 0.12 g of Aminocaproic Acid RS, previously dried 105 C for 30 minutes, dissolve in water to make exactly 10 mL, then pipet exactly 5 mL of this solution, add exactly 2 mL of internal standard solution and dilute with water to make 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Aminocaproic Acid to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of Aminocaproic Acid (C6H13NO2) = amount (mg) of Aminocaproic Acid RS
QT QS
o

Aminocaproic Acid
H 2NCH 2CH 2CH 2CH 2CH 2CO 2H

Internal standard solutionDissolve 25 mg of methionine in 20 mL of water. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: Dissolve 0.55 g of sodium 1heptanesulphonate to make 1000 mL and use this solution as solution A. Dissolve 10 g of monobasic potassium phosphate in 300 mL of solution A, add 250 mL of methanol, then add 300 mL of solution A and mix. Adjust pH to 2.2 with phosphoric acid, and add solution A to make 1000 mL. Flow rate: 0.7 mL/minute. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, Aminocaproic acid and methionine are eluted in this order with the resolution between their peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area is not more than 2.0%. Packaging and Storage Preserve in tight containers.

C6H13NO2: 131.17 Aminocaproic Acid contains not less than 98.5% and not more than 101.5% of aminocaproic acid (C6H13NO2), calculated on the anhydrous basis. Description Aminocaproic Acid is a fine white crystalline powder and is odorless. It is freely soluble in water, acid, or alkali, slightly soluble in methanol or in ethanol, and practically insoluble in chloroform or in ether. A solution of Aminocaproic Acid is neutral. Melting pointAbout 205 C. Identification Determine the infrared spectra of Aminocaproic Acid and Aminocaproic Acid RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity Heavy metalsProceed with 1.0 g of Aminocaproic Acid according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Water Not more than 0.5% (1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.12 g of Aminocaproic Acid and add water to make exactly 10 mL. Pipet exactly 5 mL of this solution, add exactly 2 mL of the internal standard solution and dilute with water to make 100 mL

KP 9 55 methylrozaniline chloride]. Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid in dioxane VS = 13.12 mg of C6H13NO2 Packaging and Storage Preserve in tight containers.

Aminocaproic Acid Tablets

Aminocaproic Acid Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of aminocaproic acid (C6H13NO2: 131.17). Method of Preparation Prepare as directed under Tablets, with Aminocaproic Acid. Identification Powder two Aminocaproic Acid Tablets, add 10 mL of water, shake well, with 100 mL of acetone and filter. Mix the filtrates with shaking and allow to stand for 15 minutes to be crystallized. Filter the crystals through a glass filter (G4) and wash with 25 mL of acetone and dry at 105 C for 30 minutes and cool. With the residue, proceed as directed in the Identification under Aminocaproic Acid. Dissolution Test Perform the test with 1 tablet of Aminocaproic Acid Tablets at 100 revolutions per minute according to Method 1 under the Dissolution Test, using 900 mL of water as dissolution solution. Take the dissolved solution after 45 minutes from the start of the test and use this solution as the test solution. Separately, weigh accurately sufficient quantity of Aminocaproic Acid RS, previously dried at 105 C for 30 minutes, dissolve in water, so that each mL contains about 500 g of aminocaproic acid and use this solution as the standard solution. To 1 mL each of the test solution and the standard solution add 20.0 mL of borate buffer solution, pH 9.5, and 3.0 mL of sodium -naphthoquinone-4-sulfonate TS, prepared before using, (1 in 500), shake, and allow to stand in a water-bath at 65 5 C for 45 minutes. After cooling, add water to make exactly 50 mL and mix. Determine the absorbances of these solutions at 460 nm as directed under Ultraviolet-visible Spectrophotometry, using the blank solution as the blank. The dissolution rate of Aminocaproic Acid Tablets in 45 minutes is not less than 75%. Borate buffer solution, pH 9.5Dissolve 6.185 g of boric acid and 7.930 g of potassium chloride in water, add 60 mL of 0.1 mol/L of sodium hydroxide VS and add water to make 2 L. Add 0.1 mol/L Sodium hydroxide, if necessary, to adjust to a pH of 9.5 0.1. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Aminocaproic Acid Tablets. Weigh accurately a portion of the powder, equivalent to about 0.5 g of aminocaproic acid (C6H13NO2), add 100 mL of glacial acetic acid, dissolve by heating, cool and titrate with 0.1 mol/L perchloric acid in dioxane VS until a blue color develops [indicator: 10 drops of chlorobenzene TS (1 in 500) of
o

Aminophylline Hydrate
O H3C N H N H2NCH2CH2NH2 O N CH3 N 2
X H 2O

C14H16N8O4 C2H8N2xH2O Aminophylline Hydrate contains not less than 84.0% and not more than 86.0% of theophylline (C7H8N4O2: 180.16) and not less than 14.0% and not more than 15.0% of ethylenediamine (C2H8N2: 60.10), calculated on the anhydrous basis. Description Aminophylline Hydrate is a white to pale yellow granule or powder, is odorless or has slightly ammonia-like odor and has a bitter taste. Aminophylline Hydrate is soluble in water, slightly soluble in methanol, and practically insoluble in ethanol or in ether. To 1 g of Amimophylline Hydrate, add 5 mL of water and shake: it dissolves almost completely. Separation of crystals begins in 2 to 3 minutes and these crystals dissolve on the addition of a small amount of ethylenediamine Aminophylline Hydrate is gradually colored by light and gradually loses ethylenediamine in air. Identification (1) Dissolve 0.75 g of Aminophylline Hydrate in 30 mL of water and use this solution as the test solution. When 1 mL of dilute hydrochloric acid is added to 20 mL of the test solution, a precipitate is gradually formed. Filter the precipitate, recrystallize from water and dry at 105 C for 1 hour: the crystals so obtained melt between 271 C and 275 C. (2) Dissolve 0.1 g of the crystals obtained in (1) in 50 mL of water and to 2 mL of this solution, add tannic acid TS drop-wise: a white precipitate is produced and this precipitate dissolves upon drop-wise addition of tannic acid TS. (3) To 10 mg of the crystals obtained in (1), add 10 drops of hydrogen peroxide TS and 1 drop of hydrochloric acid and evaporate on a water-bath to dryness: the residue shows a yellow-red color. Invert the dish containing the residue over a vessel containing 2 to 3 drops of ammonia TS: the color of the residue changes to red-

56 Monographs, Part I purple, which is destroyed on the addition of 2 to 3 drops of sodium hydroxide TS. (4) Dissolve 10 mg of the crystals obtained in (1) in 5 mL of water, add 3 mL of ammonia-ammonium chloride buffer solution, pH 8.0 and 1 mL of cupric sulfatepyridine TS and mix. Add 5 mL of chloroform to the mixture and shake: the chloroform layer develops a green color. (5) To 5 mL of the test solution obtained in (1), add 2 drops of cupric sulfate TS: a purple color develops. Add 1 mL of cupric sulfate TS: the color changes to blue and green precipitates are formed on staining. pH Dissolve 1.0 g of Aminophylline Hydrate in 25 mL of water: the pH of this solution is between 8.0 and 9.5. Purify (1) Clarity and color of solution Dissolve 1.0 g of Aminophylline Hydrate in 10 mL of hot water: the solution is clear and colorless to pale yellow. (2) Heavy metalsProceed with 1.0 g of Aminophylline Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Water Not more than 7.9% (0.3 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay (1) TheophyllineWeigh accurately about 0.25 g of Aminophylline Hydrate and dissolve in 50 mL of water and 8 mL of ammonia TS by gentle warming on a water-bath. Add exactly 20 mL of 0.1 mol/L silver nitrate VS, warm on a water-bath for 15 minutes, allow to stand between 5 C and 10 C for 20 minutes, collect the precipitate by suction and wash with three 10 mL portions of water. Combine the filtrate and washings and add dilute nitric acid to make neutral. Add 3 mL of dilute nitric acid and titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ammonium ferric sulfate). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L silver nitrate VS = 18.016 mg of C7H8N4O2 (2) EthylenediamineWeigh accurately about 0.5 g of Aminophylline Hydrate, dissolve in 30 mL of water and titrate with 0.1 mol/L hydrochloric acid VS (indicator: 3 drops of bromphenol blue TS). Each mL of 0.1 mol/L hydrochloric acid VS = 3.0049 mg of C2H8N2 Packaging and Storage tight containers. Preserve in light-resistant,

Aminophylline Injection
Aminophylline Injection is an aqueous solution for injection. Aminophylline Injection contains not less than 75.0% and not more than 86.0% of theophylline (C7H8N4O2: 180.16) and not less than 13.0% and not more than 20.0% of ethylenediamine (C2H8N2: 60.10) of the labeled amount of aminophylline. The concentration of Aminophylline Injection is expressed as the quantity of aminophylline hydrate (C16H24N10 O42H2O: 456.46). Method of Preparation Prepare as directed under Injections, with Aminophylline hydrate. Aminophylline Injection may be prepared with Theophylline and its equivalent Ethylenediamine, instead of Aminophylline hydrate. Aminophylline Injection may contain not more than 60 mg of ethylenediamine as a stabilizer for each g of Aminophylline hydrate. Description Aminophylline Injection is a clear and colorless liquid. Aminophylline Injection has a slightly bitter taste. Aminophylline Injection gradually changes in color by light. pHBetween 8.0 and 10.0. Identification To a volume of Aminophylline Injection, equivalent to 0.75 g of Aminophylline hydrate according to the labeled amount, add water to make 30 mL. Proceed with this solution as directed in the Identification under Aminophylline hydrate. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement. It

Determination of Volume of Injection in Containers It meets the requirement. Assay (1) TheophyllineTo an accurately measured volume of Aminophylline Injection, equivalent to about 0.2 g of theophylline (C7H8N4O2) (about 0.25 g of Aminophylline Hydrate), add 15 mL of water, 8 mL of ammonia TS and 20 mL of silver nitrate TS and warm in a water-bath for 15 minutes. Cool to between 5 C and 10 C for 20 minutes, filter the precipitate through a glass filter (G4) and wash with three 10 mL volumes of water. Dissolve the precipitate in 5 mL of nitric acid and wash the filter with three 10 mL volumes of water. Combine the nitric acid solution and washings and titrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ammonium ferric sulfate TS).

KP 9 57 Each mL of 0.1 mol/L ammonium thiocyanate VS = 18.016 mg of C7H8N4O2 (2) EthylenediamineTo an accurately measured volume of Aminophylline Injection equivalent to about 30 mg of ethylenediamine (C2H8N2)(about 0.2 g of Aminophylline hydrate), add water to make 30 mL and titrate with 0.1 mol/L hydrochloric acid VS (indicator: 2 to 3 drops of bromphenol blue TS) Each mL of 0.1 mol/L hydrochloric acid VS = 3.0049 mg of C2H8N2 Packaging and Storage hermetic containers. Preserve in light-resistant, tion solution. Take the dissolved solution after 45 minutes from the start of the test, filter, make any necessary dilution and use this solution as the test solution. Separately, weigh accurately about sufficient quantity of Aminophylline RS, previously dried at 105 C for 4 hours, make the same concentration as the test solution and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution at 269 nm as directed under Ultraviolet-visible Spectrophotometry. The dissolution rate of Aminophylline Tablets in 45 minutes is not less than 75%. Uniformity of Dosage Units It meets the requirement. Assay (1) TheophyllineWeigh accurately and powder not less than 20 Aminophylline Tablets. Weigh accurately a portion of the powder, equivalent to about 2 g of aminophylline hydrate (C16H24N10O42H2O), add 50 mL of water and 15 mL of ammonia TS, warm at 50 C and allow to stand for 30 minutes with occasional shaking. Cool to the room temperature and add water to make exactly 200 mL. Pipet exactly 50 mL of this solution, centrifuge, pipet exact amount of the supernatant, equivalent to 0.2 g of theophylline, into a conical flask and add water to make about 40 mL. Then add 8 mL of ammonia TS, add 20 mL of 0.1 mol/L silver nitrate VS, warm in waterbath for 15 minutes, then cool at between 5 C and 10 C for 20 minutes, filter the precipitate through a glass filter (G4) and wash with three 10 mL volumes of water. Collect the filtrate and washings, add nitric acid to make acidic, then add 3 mL of nitric acid. After cooling, titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ammonium ferric sulfate TS). Each mL of 0.1 mol/L silver nitrate VS = 18.016 mg of C7H8N4O2 (2) EthylenediamineWeigh accurately and powder not less than 20 Aminophylline Tablets. Transfer a portion, weighed accurately, of the powder, equivalent to about 0.35 g of aminophylline hydrate (C16H24N10O4 2H2O) into a conical flask, add 200 mL of water, digest at 50 C with frequent shaking for 30 minutes. After cooling, filter this solution to another conical flask, wash with water until the last washing is neutral to litmus and titrate with 0.1 mol/L hydrochloric acid VS (indicator: 2 to 3 drops of methyl orange TS). Each mL of 0.1 mol/L hydrochloric acid VS = 3.0049 mg of C2H8N2 Packaging and Storage Preserve in tight containers.

Aminophylline Tablets
Aminophylline Tablets contain not less than 75.0% and not more than 86.0% of the labeled amount of theophylline (C7H8N4O2: 180.16) and not less than 12.0% and not more than 14.0% of the labeled amount of ethylenediamine (C2H8N2: 60.10). The amount of Aminophylline Tablets is expressed as the quantity of aminophylline hydrate (C16H24N10O42H2O: 456.46). Method of Preparation Prepare as directed under Tablets, with Aminophylline Hydrate. Identification (1) Weigh a portion of powdered Aminophylline Tablets, equivalent to 0.5 g of Aminophylline Hydrate according to labeled amount, add 25 mL of water, shake well, then filter. This filtrate changes moistened red litmus paper to blue. Mix the filtrate with 1 mL of 3 mol/L of hydrochloric acid, then obtain the crystals of Theophylline. Cool, if necessary, and precipitate. Filter the precipitate (the filtrate is used for identification (2)), wash with small volume of cold water and dry at 105 C for 1 hour. Proceed with this crystal as directed in the Identification (3) under Aminophylline Hydrate. And recrystalize this crystal with water, dry at 105 C for 1 hour, and the melting point of this crystal is not less than 270 C and not more than 274 C. (2) To the filtrate from (1), add 0.5 mL of benzenesulfonyl chloride and 5 mL of 1 mol/L sodium hydroxide TS to make alkaline, shake for 10 minutes, and then add 5 mL of 3 mol/L hydrochloric acid to make acidic, then obtain the crystals of ethylenediamine disulfonamide, wash, recrystalize, dry at for 105 C for 1 hour: the melting point of this crystals is not less than 164 C and not more than 171 C. Dissolution Test This test applies to naked tablets. Perform the test with 1 tablet of Aminophylline Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water, as the dissolu-

58 Monographs, Part I (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 31.386 mg of C20H23NHCl Packaging and Storage tight containers. Preserve in light-resistant,

Amitriptyline Hydrochloride
HCl CH3 CHCH2CH2N CH3

C20H23NHCl: 313.86 Amitriptyline Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of amitriptyline hydrochloride C20H23NHCl). Description Amitriptyline Hydrochloride is a colorless crystal or a white to pale yellowish crystalline powder. Amitriptyline Hydrochloride has a bitter taste and a numbing effect. Amitriptyline Hydrochloride is freely soluble in water, in ethanol or in glacial acetic acid, soluble in acetic anhydride, and practically insoluble in ether. pHDissolve 1.0 g of Amitriptyline Hydrochloride in 20 mL of water: the pH of this solution is between 4.0 and 5.0. Identification (1) Dissolve 5 mg of Amitriptyline Hydrochloride in 3 mL of sulfuric acid: a red color is observed. Add 5 drops of potassium bichromate TS to this solution: it turns dark brown. (2) Acidify 1 mL of a solution of Amitriptyline Hydrochloride (1 in 500) with 0.5 mL of dilute nitric acid and add 1 drop of silver nitrate TS: a white precipitate is produced. (3) Determine absorption spectra of solutions of Amitriptyline Hydrochloride and amitriptyline hydrochloride RS, in water (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Melting Point Between 195 C and 198 C.

Amitriptyline Hydrochloride Injection

Amitriptyline Hydrochloride Injection is a sterile solution of Amitriptyline Hydrochloride in water for injection. Amitriptyline Hydrochloride Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of amitriptyline hydrochloride (C20H23NHCl: 313.86). Method of Preparation Prepare as directed under Injections, with Amitriptyline Hydrochloride. Identification (1) Pipet 1 mL of Amitriptyline Hydrochloride Injection into a separator containing 10 mL of water and 1 mL of 1 mol/L sodium hydroxide TS, mix and extract with two 10 mL volumes of dichloromethane. Evaporate the extracts on a steam-bath just to dryness. Dissolve the residue in methanol, add 1 mL of 1.2 mol/L hydrochloric acid, then add methanol to make 100 mL. Dilute 10 mL of this solution with methanol to make 100 mL and use this solution as the test solution. Separately, prepare the standard solution with Amitriptyline Hydrochloride RS as directed for the preparation of the test solution. Determine the absorption spectra of the test solution and the standard solution, respectively, as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit maximum at the same wavelength. (2) The retention time of the major peak in the chromatogram of the test preparation corresponds to that in the chromatogram of the standard preparation, as obtained in the Assay. pH Between 4.0 and 6.0. Sterility Test It meets the requirement. Pyrogen Test Amitriptyline Hydrochloride Injection, diluted with Isotonic Sodium Chloride Injection to a concentration of 2.5 mg of Amitriptyline Hydrochloride per mL according to the labeled amount, meets the requirement, the test dose being 1 mL per kg of rabbit. Foreign Insoluble Matter Test It meets the requirement.

Purity (1) Clarity and color of solutionDissolve 1.0 g of Amitriptyline Hydrochloride in 20 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Amitriptyline Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Amitriptyline Hydrochloride, previously dried and dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid

KP 9 59

Insoluble Particulate Matter Test for Injections meets the requirement.

It

Amitriptyline Hydrochloride Tablets

Amitriptyline Hydrochloride Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of amitriptyline hydrochloride (C20H23NHCl: 313.86). Method of Preparation Prepare as directed under Tablets, with Amitriptyline Hydrochloride. Identification (1) Weigh a portion of powdered Amitriptyline Hydrochloride Tablets, equivalent to 0.1 g of Amitriptyline Hydrochloride according to the labeled amount. Add 10 mL of chloroform, shake thoroughly and filter. Evaporate the filtrate on a water-bath to about 2 mL, add ether until turbidity is produced and allow to stand. Filter the crystals through a glass filter (G4) and proceed as directed under the Identification (1) and (2) under Amitriptyline Hydrochloride. (2) Determine the absorption spectrum of a solution of the crystals obtained in (1) (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 238 nm and 240 nm and a minimum between 228 nm and 230 nm. Dissolution Test Perform the test with 1 tablet of Amitriptyline Hydrochloride Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of diluted phosphate buffer solution, pH 6.8 (1 in 2) as a dissolution solution. Take 20 mL or more of the dissolved solution 60 minutes after starting the test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet the subsequent V mL of the filtrate, add diluted phosphate buffer solution, pH 6.8 (1 in 2), to make exactly V mL, so that each mL contains about 11 g of amitriptyline hydrochloride (C20H23N.HCl) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately about 55 mg of Amitriptyline Hydrochloride RS, previously dried at 105 C for 2 hours and dissolve in diluted phosphate buffer solution, pH 6.8 (1 in 2) to make exactly 250 mL. Pipet exactly 5 mL of this solution, add diluted phosphate buffer solution, pH 6.8 (1 in 2) to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 239 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Amitriptyline Hydrochloride Tablets in 60 minutes is not less than 70%. Dissolution rate (%) with respect to the labeled amount of amitriptyline hydrochloride (C20H23NHCl) =
WS AT V ' 1 18 AS V C

Determination of Volume of Injection in Containers It meets the requirement. Assay Take accurately equivalent to about 50 mg of Amitriptyline Hydrochloride, add water to make 250 mL and use this solution as the test solution. Separately, dissolve an accurately weighed portion of Amitriptyline Hydrochloride RS in water, previously dried at 60 C, 0.67 kPa and dilute quantitatively with water to obtain a solution having a known concentration of about 0.2 mg per mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the peak area of amitriptyline, AT and AS , for the test solution and the standard solution, respectively. Amount (mg/mL) of Amitriptyline Hydrochloride (C20H23NHCl) = 250 C AT
V AS

C: Concentration of the standard solution (mg/mL), V: Volume of Amitriptyline Hydrochloride Injection taken (mL). Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Temperature: A room temperature. Mobile phase: Dissolve 11.04 g of monobasic sodium phosphate in 900 mL of water, adjust with phosphoric acid to a pH of 2.5 0.5, dilute with water to make 1000 mL and prepare a mixture of this solution and acetonitrile (58 : 42). Flow rate: 2 mL/minute System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the symmetry factor is not more than 2.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area is not more than 2.0. Packaging and Storage hermetic containers. Preserve in light-resistant

60 Monographs, Part I

WS : Amount of Amitriptyline Hydrochloride RS (mg)

C: Labeled amount of amitriptyline hydrochloride (C20H23NHCl) in 1 tablet (mg). Uniformity of Dosage Units It meets the requirement.
O

Amlodipine Besylate
H3C H N O O O H Cl O NH2 CH3 SO3H

Assay Weigh accurately and powder not less than 20 Amitriptyline Hydrochloride Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of amitriptyline hydrochloride (C20H23NHCl) and add mobile phase to make exactly 250 and filter. Discard the first 20 mL volume of the filtrate, and use next filtrate as the test solution. Separately, weigh accurately about 50 mg of Amitriptyline Hydrochloride RS, previously dried at 60 C, 0.67 kPa until the constant mass is attained and dissolve in mobile phase to make exactly 250 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution an the standard solution as diretected under the Liquid Chromatography according to the following conditions and calculate peak areas of amitriptyline, AT and AS , from the test solution and the standard solution, respectively,. Amount (mg) of amitriptyline hydrochloride (C20H23NHCl) = amount (mg) of Amitriptyline Hydrochloride RS
AT AS

H3C

and enantiomer C20H25N2O5ClC6H6O3S : 567.05 Amlodipine Besylate contains not less than 97.0% and not more than 102.0% of amlodipine besylate (C20H25N2O5ClC6H6O3S), calculated on the anhydrous basis. Description Amlodipine Besylate is a white powder. Amlodipine Besylate is freely soluble in methanol, sparingly soluble in ethanol, and slightly soluble in water or 2-propanol. Identification (1) Dissolve 5.0 mg each of Amlodipine Besylate and Amlodipine Besylate RS, respectively, in 1 vol% solution of 0.1 mol/L hydrochloric acid TS in methanol to make 100 mL. Determine the absorption spectra of the solutions respectively, as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Amlodipine Besylate and Amlodipine Besylate RS, previously dried, as directed in the paste method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) The principal spot in the chromatogram obtained with the test solution (2) of i) Related substances is the same in color and Rf value as the principal spot with the standard solution (2. Specific Optical Rotation [ ]20 D : Between -0.10 and +0.10 (0.25 g, methanol, 25 mL, 100 mm) Purity Related substances) Dissolve 0.140 g of the Amlodipine Besylate in methanol to make exactly 2 mL and use this solution as the test solution (1). To 1.0 mL of this solution add methanol to make exactly 10 mL and use this solution as the test solution (2). Separately, dissolve 70.0 mg of Amlodipine Besylate RS in 1.0 mL of methanol and use this solution as the standard solution (1). To 1.0 mL of the standard solution (1) add methanol to make 10 mL and use this solution as the standard solution (2). To 3.0 mL of the standard solution (2) add methanol to make 100 mL and use this solution as the standard solution (3). To 1.0 mL of the standard

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column 4 mm in inside diameter and 30 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (between 5 and 10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of Liquid A and acetonitrile (58 : 42) Liquid A: Dissolve 11.04 g of sodium dihydrogen phosphate in 900 mL of water, adjust to pH 2.5 0.5 with phosphoric acid, and add water to make 1000 mL. Flow rate: 2 mL/minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the symmetry factor of the peak of amitriptyline is not more than 2.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of amitriptyline is not more than 2.0%. Packaging and Storage Preserve in tight containers.

KP 9 61 solution (2) add methanol to make 100 mL and use this solution as the standard solution (4). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test solutions and the standard solutions on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with upper layer of a mixture of methyl isobutyl ketone, water and glacial acetic acid (50 : 25 : 25) to a distance of about 15 cm and dry the plate at 80 C for 15 minutes. Examine under ultraviolet light (main wavelength: 254 nm and 366 nm): any spot, other than the principal spot, in the chromatogram obtained with the test solution (1) is not more intense than that with the standard solution (3) (not more than 0.3%) and more intense spot than the one in the chromatogram from the standard solution (4) is not more than 2 spots (not more than 0.1%). This test is not valid unless the chromatogram from the standard solution (1) shows 2 clearly separated minor spots with Rf values of about 0.18 and 0.22. ) Dissolve 50.0 mg of Amlodipine Besylate in mobile phase to make exactly 50 mL and use this solution as the test solution (1). To 5.0 mL of the test solution (1) add mobile phase to make exactly 100 mL and use this solution as the test solution (2). Separately, dissolve 50.0 mg of Amlodipine Besylate RS in mobile phase to make exactly 50 mL, pipet 5.0 mL of this solution, add mobile phase to make exactly 100 mL and use this solution as the standard solution (1). To 3.0 mL of the test solution (1) add mobile phase to make exactly 100 mL, pipet 5.0 mL of this solution, add mobile phase to make exactly 50 mL and use this solution as the standard solution (2). Dissolve 5 mg of Amlodipine Besylate in 5 mL of strong hydrogen peroxide, heat at 70 C for 45 minutes and use this solution as the standard solution (3). Perform the test with exactly 10 L each of the test (1) and the standard solutions (2) and (3) as directed under Liquid Chromatography according to the following conditions: Amlodipine besylate related substance I {3-Ethyl-5-methyl-2[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl pyridine-3,5-dicarboxylate} obtained with the test solution is not more than the area of the principal peak in the chromatogram from the standard solution (2) (0.3%). Total area of all the other related substances is not more than the area of the principal peak in the chromatogram from the standard solution (2) (0.3%). Disregard any peak due to benzene sulfonate (relative retention : about 0.2) and 0.1 times the area of the principal peak with the standard solution (2). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 237 nm). Column: A stainless steel column about 3.9 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: Mix 15 volumes of acetonitrile, 35 volumes of methanol and 50 volumes of a solution prepared as follows: dissolve 7.0 mL of triethylamine in 1000 mL of water and adjust to pH 3.0 0.1 with phosphoric acid. Flow rate: 1.0 mL/minute Time span of measurement: About 3 times as long as the retention time of amlodipine beginning after the solvent peak. System suitability System performance: When the procedure is run with 10 L of the standard solution (3) under the above operating conditions, minimum resolution is not less than 4.5 between the peaks corresponding to amlodipine and related substance I; relative retention time of related substance I with reference to amlodipine (retention time = about 7 minutes) is about 0.5. For the calculation of content, multiply the peak area of the related substance I by 2. Water Not more than 0.5% (3.0 g, volumetric titration, direct titration) Residue on Ignition Not more than 0.2% (1 g). Assay Perform the test with the test solution (2) and the standard solution (1) of Related substances ) as directed above conditions in Related substances ) and calculate the percentage content of amlodipine besylate from the areas of the peaks, AT and AS , respectively. Amounts(mg) of amlodipine besylate (C20H25N2O5ClC6H6O3S) = amounts (mg) of Amlodipine Besylate RS Packaging and Storage tight containers
AT AS

Preserve in light-resistant,

Ammonia Water
Ammonia Water contains not less than 9.5 w/v% and not more than 10.5 w/v% of ammonia (NH3: 17.03) Description Ammonia Water is a clear, colorless liquid and has a very pungent, characteristic odor. Ammonia Water is alkaline. 20 : Between 0.95 and 0.96. Specific Gravity d 20 Identification (1) Hold a glass rod moistened with hydrochloric acid near the surface of Ammonia Water: dense white fumes are produced. (2) Hold moistened red litmus paper near the surface of Ammonia Water: it turns blue. Purity (1) Residue on evaporationEvaporate 10.0 mL of Ammonia Water to dryness and dry the residue at
o 105 C for 1 hour: the weight of the residue is not more

62 Monographs, Part I than 2.0 mg. (2) Heavy metalsEvaporate 5.0 mL of Ammonia Water to dryness on a water-bath, add 1 mL of dilute hydrochloric acid to the residue and evaporate to dryness. Dissolve the residue in 2 mL of dilute acetic acid, add water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 2.5 mL of standard lead solution, 2 mL of dilute acetic acid and water to make 50 mL (not more than 5 ppm). (3) Potassium permanganate-reducing substances To 10.0 mL of Ammonia Water, add 40 mL of dilute sulfuric acid while cooling and add 0.10 mL of 0.02 mol/L potassium permanganate VS: the red color of the potassium permanganate does not disappear within 10 minutes. Assay Measure exactly 5 mL of Ammonia Water, add 25 mL of water and titrate with 0.5 mol/L sulfuric acid VS (indicator: 2 drops of methyl red TS). Each mL of 0.5 mol/L sulfuric acid VS = 17.031 mg of NH3 Packaging and Storage Preserve in tight containers and store at not exceeding 30 C. tion: a red-purple precipitate is produced in the aqueous layer. Shake the mixture: a red-purple color is produced in the chloroform layer. (3) To 0.4 g of Amobarbital, add 0.1 g of anhydrous sodium carbonate and 4 mL of water, shake and add a solution of 0.3 g of p-nitrobenzyl chloride in 7 mL of ethanol. Heat the mixture in a water-bath for 30 minutes under a reflux condenser and allow to stand for 1 hour. Filter the crystals produced, wash with 7 mL of sodium hydroxide TS and a small portion of water, recrystalize from ethanol and dry at 105 C for 30 minutes: the crystals so obtained melt between 168 C and 173 C or between 150 C and 154 C. Melting Point Between 157 C and 160 C.

Amobarbital
O CH3CH2 NH (CH 3)2CHCH 2CH2 O H N O

C11H18N2O3: 226.27 Amobarbital, when dried, contains not less than 99.0% and not more than 101.0% of amobarbital (C11H18N2O3). Description Amobarbital is a white crystal or crystalline powder, is odorless and has a slightly bitter taste. Amobarbital is freely soluble in ethanol, in acetone or in ether, sparingly soluble in chloroform and practically insoluble in water. Amobarbital dissolves in sodium hydroxide TS or sodium carbonate TS. pHThe pH of a saturated solution of Amobarbital is between 5.0 and 5.6. Identification (1) Boil 0.2 g of Amobarbital with 10 mL of sodium hydroxide TS: the gas evolved changes moistened red litmus paper to blue. (2) Dissolve 50 mg of Amobarbital in 2 to 3 drops of ammonia-ammonium chloride buffer solution, pH 10.7 and 5 mL of diluted pyridine (1 in 10). Add 5 mL of chloroform and 0.3 mL of cupric sulfate TS to the solu-

Purity (1) Clarity and color of solution Dissolve 0.5 g of Amobarbital in 5 mL of sodium hydroxide TS: the solution is clear and colorless. (2) ChlorideDissolve 0.30 g of Amobarbital in 20 mL of acetone and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: take 0.30 mL of 0.01 mol/L hydrochloric acid VS, 20 mL of acetone and 6 mL of dilute nitric acid and add water to make 50 mL (not more than 0.035%). (3) SulfateDissolve 0.40 g of Amobarbital in 20 mL of acetone and add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: take 0.40 mL of 0.005 mol/L sulfuric acid VS, 20 mL of acetone and 1 mL of dilute hydrochloric acid and add water to make 50 mL (not more than 0.048%). (4) Heavy metalsProceed with 1.0 g of Amobarbital according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) Readily carbonizable substancesPerform the test with 0.5 g of Amobarbital. The solution has no more color than Color Matching Fluid A. Loss on Drying Not more than 1.0% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g).

Assay Weigh accurately about 0.5 g of Amobarbital, previously dried, and dissolve in 5 mL of ethanol and 50 mL of chloroform. Titrate with 0.1 mol/L potassium hydroxide-ethanol VS until the color of the solution changes from yellow through pale blue to purple (indicator: 1 mL of alizarin yellow GG-thymolphthalein TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L potassium hydroxide-ethanol VS = 22.627 mg of Cl1H18N2O3 Packaging and Storage Preserve in well-closed con-

KP 9 63 tainers. acid, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.018%). (3) SulfateDissolve 2.0 g of Amobarbital Sodium for Injection in 49 mL of water, add 1 mL of glacial acetic acid, shake and filter. Discard the first 10 mL of the filtrate and to the subsequent 25 mL of the filtrate, add 2.5 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.40 mL of 0.005 mol/L sulfuric acid VS, add 0.5 mL of glacial acetic acid,1 mL of dilute hydrochloric acid and water to make 50 mL (not more than 0.019%). (4) Heavy metalsDissolve 2.0 g of Amobarbital Sodium for Injection in 45 mL of water, add 5 mL of dilute hydrochloric acid, shake vigorously and warm in a water-bath for 2 minutes with occasional shaking. Cool, add 30 mL of water, shake and filter. Discard the first 10 mL of the filtrate, add 1 drop of phenolphthalein TS to the subsequent 40 mL of the filtrate, add ammonia TS until a slight red color develops and add 2.5 mL of dilute acetic acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 2.5 mL of dilute hydrochloric acid, add 1 drop of phenolphthalein, add ammonia TS until a pale red color develops and add 2.5 mL of dilute acetic acid, 2.0 mL of standard lead solution and water to make 50 mL (not more than 20 ppm). (5) Neutral or basic substancesDissolve about 1 g of Amobarbital Sodium for Injection, accurately weighed, in 10 mL of water and 5 mL of sodium hydroxide TS, then add 40 mL of chloroform and shake well. Separate the chloroform layer, wash with two 5mL volumes of water and filter. Evaporate the filtrate on a water-bath to dryness and dry the residue at 105 C for 1 hour: the weight of the residue is not more than 0.30%. (6) Readily carbonizable substancesPerform the test with 0.5 g of Amobarbital Sodium for Injection: the solution has no more color than Color Matching Fluid A. Loss on Drying Not more than 1.0% (1 g, 105 C, 4 hours). Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injection meets the requirement. It

Amobarbital Sodium for Injection

O CH3CH2 N (CH3)2CHCH2CH2 O H N ONa

C11H17N2NaO3: 248.25 Amobarbital Sodium for Injection is a preparation for injection which is dissolved before use. When dried, Amobarbital Sodium for Injection contains not less than 98.5% and not more than 101.0% of amobarbital sodium (C11H17N2NaO3) and not less than 92.5% and not more than 107.5% of the labeled amount of amobarbital sodium (C11H17N2NaO3). Method of Preparation Prepare as directed under Injections with Amobarbital Sodium. Description Amobarbital Sodium for Injection is a white crystal or a crystalline powder, is odorless and has a bitter taste. Amobarbital Sodium for Injection is freely soluble in water or in ethanol, and practically insoluble in ether or in chloroform. pH The pH of a solution of Amobarbital Sodium for Injection (1 in 10) is between 10.0 and 11.0. Identification (1) Dissolve 1.5 g of Amobarbital Sodium for Injection in 20 mL of water and add 10 mL of dilute hydrochloric acid with stirring: a white precipitate is produced. Collect the precipitate, wash with four 10mL volumes of water and dry at 105 C for 3 hours: it melts between 157 C and 160 C. With this precipitate, proceed as directed in the Identification under Amobarbital. (2) Ignite 0.5 g of Amobarbital Sodium for Injection, cool and dissolve the residue in 10 mL of water: the solution responds to the Qualitative Tests (1) for sodium salt. Purity (1) Clarity and color of solutionDissolve 1.0 g of Amobarbital Sodium for Injection in 10 mL of freshly boiled and cooled water: the solution is clear and colorless. (2) ChlorideDissolve 1.0 g of Amobarbital Sodium for Injection in 49 mL of water, add 1 mL of glacial acetic acid, shake and filter. Discard the first 10 mL of the filtrate and to the subsequent 30 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.30 mL of 0.01 mol/L hydrochloric acid VS, add 0.5 mL of glacial acetic

Uniformity of Dosage Units It meets the requirement of the Content Uniformity Test. Assay Weigh accurately the contents of not less than 10 samples of Amobarbital Sodium for Injection. Weigh accurately about 0.5 g of the contents, previously dried, transfer to a separator, dissolve in 20 mL of water, add 5 mL of ethanol and 10 mL of dilute hydrochloric acid and extract with 50 mL of chloroform, then with three 25 mL

64 Monographs, Part I volumes of chloroform. Combine the chloroform extracts, wash with two 5 mL volumes of water and extract the washings with two 10 mL volumes of chloroform. Filter the combined chloroform extracts into a conical flask and wash the filter paper with three 5 mL volumes of chloroform. Combine the filtrate and the washings and add 10 mL of ethanol. Titrate with 0.1 mol/L potassium hydroxide-ethanol VS until the color of the solution changes from yellow through pale blue to purple (indicator: 2 mL of alizarin yellow GG-thymolphthalein TS). Perform a blank determination with a mixture of 160 mL of chloroform and 30 mL of ethanol and make any necessary correction. Each mL of 0.1 mol/L potassium hydroxide-ethanol VS = 24.825 mg of C11H17N2NaO3 Packaging and Storage Preserve in hermetic containers. bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. (5) Weigh about 0.2 g of Amoxicillin Hydrate and Amoxicillin RS, dissolve each in 0.1 mol/L hydrochloric acid TS to make exactly 50 mL, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of methanol, chloroform, water and pyridine (9:8:3:1), air-dry the plate, and spray evenly a ninhydrin solution in methanol (3 in 1000): the spots obtained from the test solution and the standard solution are the same in the Rf value. pH The pH of a solution obtained by dissolving 20 mg of Amoxicillin Hydrate in 10 mL of water is between 3.5 and 6.0. Purity Related substancesWeigh accurately about 3 mg of Amoxicillin Hydrate, dissolve in 2 mL of the mobile phase A, and use this solution as the test solution. Weigh accurately about 3 mg of Amoxicillin Hydrate RS, dissolve in 5 mL of the mobile phase A, pipet exactly 200 L of this solution, and add the mobile phase A to make 2 mL. Pipet 500 L of this solution, and add the mobile phase A to make 2 mL, and use this solution as the standard solution. Perform the test with exactly 50 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area. Each peak area other than amoxicillin from the test solution is not more than the peak area of amoxicillin from the standard solution (not more than 1 %). Also, total peak areas other than amoxicillin from the test solution are not more than the peak area of amoxicillin from the standard solution (not more than 3 %). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) Column temperature: A constant temperature of about 25 C. Mobile phase: Control the gradient by mixing the mobile phase A and B as directed in the following table. Mobile phase A: To 990 mL of 0.05 mol/L potassium dihydrogen phosphate buffer solution, pH 5.0, add 10 mL of acetonitrile. Mobile phase B: To 800 mL of 0.05 mol/L potassium dihydrogen phosphate buffer solution, pH 5.0, add 200 mL of acetonitrile.

Amoxicillin Hydrate
HO O HO O O N H CH3 CH3 S H H 3H2O

N H NH2

C16H19N3O5S3H2O: 419.45 Amoxicillin Hydrate contains not less than 900 g (potency) per mg of amoxicillin (C16H19N3O5S: 365.40), calculated on the anhydrous basis. Description Amoxicillin Hydrate is a white to yellowwhite, crystal or crystalline powder. Amoxicillin Hydrate is slightly soluble in water or methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Spot 0.1 mL of 1 % ninhydrin solution on filter paper, and dry at 105 C for 5 minutes. To this spot 0.1 mL of 0.2 % Amoxicillin Hydrate solution, dry at 105 C for 5 minutes, and cool to a room temperature: the red-purple color develops. (2) Weigh 20 mg of Amoxicillin Hydrate, dissolve in 10 mL of water, and add 1 mL of Fehlings TS: the redpurple color develops immediately. (3) To 2 mL of the aqueous solution of Amoxicillin Hydrate (1 in 5000) add 1 mL of a solution of mercuric (II) sulfate in 2.5 mol/L sulfuric acid solution (3 in 20), heat for 2 minutes in boiling water bath, and add 1 mL of sodium nitrite solution (1 in 1000) and heat for 2 minutes: a red-brown color is developed. (4) Determine the infrared spectra of Amoxicillin Hydrate and Amoxicillin RS, as directed in the potassium

KP 9 65 Packaging and Storage Preserve in tight containers.

Time (min) 0 8 33 48 49 64

Mobile phase A (vol %) 92 92 0 0 92 92

Mobile phase B (vol %) 8 8 100 100 8 8

Amphotericin B
OH HO O HOOC H CH3 O CH3 OH OH NH2 OH OH OH OH O H3C OH O CH3 OH

Flow rate: 1.0 mL per minute. System suitability System repeatability: When the test is repeated 3 times with 50 L of the standard solution under the operating conditions, the relative standard deviation of the peak areas of amoxicillin is not more than 1.0 %. Water Not less than 11.5 and not more than 14.5 % (0.1 g, volumetric titration, direct titration). Assay Weigh accurately about 0.1 g (potency) each of Amoxicillin Hydrate and Amoxicillin RS, dissolve each in water, add 15.0 mL of the internal standard solution and water to make exactly 100 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS , of the peak area of amoxicillin to that of the internal standard. Amount [g (potency)] of amoxicillin (C16H19N3O5S) = Amount [g (potency)] of Amoxicillin RS
QT QS

C47H73NO17: 924.08 Amphotericin B is a polyene macrolide substance having antifungal activity produced by the growth of Streptomyces nodosus. Amphotericin B contains not less than 750 g (potency) of amphotericin B (C47H73NO17) per mg, calculated on the dried basis. Description Amphotericin B is a yellow to orange powder. Amphotericin B is soluble in dimethylsulfoxide, and practically insoluble in water, in ethanol, or in ether. Identification Weigh about 25 mg of Amphotericin B, dissolve in 5 mL of dimethylsulfoxide, and add ethanol anhydrous to make 50 mL. Determine the absorption spectrum of this solution as directed under Ultravioletvisible Spectrophotometry, it exhibits maxima between 360 nm and 364 nm, and between 379 nm and 383 nm, and between 403 nm and 407 nm. pH The pH of a suspension obtained by suspending 0.3 g of Amphotericin B in 10 mL of water is between 3.5 and 6.0. Purity Amphotericin AWeigh accurately about 50 mg of Amphotericin B, dissolve in 10 mL of dimethylsulfoxide, add purified methanol to make exactly 50 mL. Pipet exactly 4 mL of this solution, add purified methanol to make exactly 50 mL, and use this solution as the test solution. Separately, weigh accurately about 20 mg of Nystatin RS, dissolve in 40 mL of dimethylsulfoxide, add purified methanol to make exactly 200 mL. Pipet exactly 4 mL of this solution, add purified methanol to make exactly 50 mL, and use this solution as the nystatin standard solution. Weigh accurately about 50 mg of Amphotericin B RS, dissolve in 10 mL of dimethylsulfoxide, add purified methanol to make exactly 50 mL. Pipet exactly 4 mL of this solution, add purified methanol to make exactly 50 mL, and use this solution as the amphotericin B standard solution. Separately, pipet exactly 10 mL of dimethylsulfoxide, add purified methanol to make exactly 50 mL. Pipet exactly 4 mL of this solution, add purified methanol to make exactly 50 mL, and use this

Internal standard solutionWeigh accurately 0.7 g of sodium benzoate, dissolve in water to make exactly 1000 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography Mobile phase: To 750 mL of water add 6.3 g of ammonium formate, and adjust the pH to 6.0 with formic acid or ammonia TS. To this solution add 30 mL of methanol and water to make exactly 1000 mL. Flow rate: Adjust the flow rate so that the retention time of the internal standard is about 1.5 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the internal standard and amoxicillin are eluted in this order with the resolution between these peaks being not less than 2.0.

66 Monographs, Part I solution as the blank test solution. Determine the absorbances of these solutions at 282 nm and at 304 nm of this solution as directed under Ultraviolet-visible Spectrophotometry (Not more than 5.0 %, w/w). Amount [%] of amphotericin A =
[( B S 2 ) (b S 1 )] 625 amount (g) of Amphoteric in B [( B a)-(b A)]

1% A: E1 cm (282 nm) of nystatin 1% B: E1 cm (282 nm) of amphotericin B 1% a: E1 cm (304 nm) of nystatin

amount of these solutions before use, add dimethylsulfoxide to make solutions so that each mL contains 200 g (potency) and 50 g (potency), dilute each solution in 0.2 mol/L phosphate buffer solution, pH 10.5 to make the solution so that each mL contains 10.0 g (potency) and and 2.5 g (potency), use these solutions as the high concentration test solution and low concentration test solution, and the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in light-resistant, tight containers ( Below 15 C)

b: (304 nm) of amphotericin B S1: Absorbance at 282 nn of the test solution S2: Absorbance at 304 nn of the test solution Sterility Test It meets the requirement, when Amphotericin B is used in a sterile preparation. Pyrogen Test It meets the requirement, when Amphotericin B is used in a sterile preparation,. Weigh 50 mg of Amphotericin B, dissolve in the mixture of 39.2 mg of desoxycholic aicd and 1.6 mL of sodium hydroxide (1 in 200), adjust the pH between 7.2 and 8.0 with hydrochloric acid, add distilled water for injection to make the solution so that each mL contains 1.0 mg (potency), and use the solution as the test solution. Use five rabbits in the test. If a body temperature of each rabbit is not more than 1.11 C, compared to the control body temperature, it meets the requirement. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Loss on Drying Not more than 5.0 % (0.1 g, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 0.5 % (1 g). Assay The Cylinder-plate method (1) Agar medium 1 under Mifor seed layer- Use the medium I 2 1) (4) crobial Assay for Antibiotics. (2) Agar medium for transfer- Use the medium in I 2 1 under Microbial Assay for Antibiotics. 1) (3) (3) Liquid medium for suspension- Use the medium in I 2 3) (1) under Microbial Assay for Antibiotics. (4) Test organism- Saccharomyces cerevisiae ATCC 9763. (5) Cylinder-agar plate- Use Petri dish plates without dispensing the agar medium for base layer but with only dispensing 8.0 mL of the seeded agar medium. (6) Weigh accurately about 20 mg (potency) each of Amphotericin B and Amphotericin B RS, dissolve in dimethylsulfoxide to make the solution so that each mL contains 1 mg (potency), and use these solutions as the test stock solution and the standard stock solution. Keep the test stock solution and standard stock solution below 5 C, and use within 24 hours. Take exactly a suitable

1% E1 cm

Ampicillin Hydrate
HO O O O N H CH3 CH3 S H H 3H2O

N H H2N H

C16H19N3O4S3H2O: 403.45 Ampicillin Hydrate contains not less than 900 g (potency) per mg of ampicillin (C16H19N3O4S: 349.41), calculated on the anhydrous basis. Description Ampicillin. Hydrate is a white to light yellow-white, crystal or crystalline powder. Ampicillin Hydrate is sparingly soluble in water, slightly soluble in methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Spot 0.1 mL of 1 % ninhydrin solution on filter paper, and dry at 105 C for 5 minutes. To this spot additionally 0.1 mL of 0.1 % Ampicillin Hydrate solution, dry at 105 C for 5 minutes, and cool: the purple color develops. (2) Weigh 10 mg of Ampicillin. Hydrate, suspend in 1 mL of water, and add 2 mL of Fehlings TS and 2 mL of water: the red-purple color develops immediately. (3) To 2 mL of the solution of Ampicillin. Hydrate that each mL contains 1 mg, add 0.5 mL of phenol and 5 mL of sodium hypochlorite TS: an odor of benzaldehyde evolves persistently and orage precipitates occurs within 3 to 5 minutes. (4) Determine the infrared spectra of Ampicillin Hydrate and Ampicillin Hydrate RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers.

KP 9 67 pH The pH of a solution obtained by dissolving 0.1 g of Ampicillin Hydrate in 10 mL of water is between 3.5 and 6.0. Sterility Test It meets the requirement, when Ampicillin Hydrate is used in a sterile preparation. Water Not less than 12.0 and not more than 15.0 % (0.1 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg (potency) each of Ampicillin Hydrate and Ampicillin Hydrate RS, dissolve each in 80 mL of water, and add water to make exactly 100 mL. Pipet exactly 5 mL each of these solutions, add water to make exactly 100 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak area of ampicillin, AT and AS . Amount [g (potency)] of ampicillin (C16H19N3O4S) = Amount [g (potency)] of Ampicillin Hydrate RS

AT AS

low-white crystal or crystalline powder. Ampicillin Hydrate is very soluble in water, freely soluble in ethanol, and slightly soluble in ether Identification (1) Perform the tests according to Identification (1), (2) and (3) in Ampicillin Hydrate. (2) Determine the absorption spectrum of Ampicillin Sodium as directed in potassium bromide disk method under Infrared Spectrophotometry, it exhibits absorption at the wave numbers about 3400 cm 1 , 1760 cm 1 , 1660 cm 1 , 1600 cm 1 , 1510 cm 1 , 1450 cm 1 , 1400 cm 1 , 1320 cm 1 , and 1200 cm 1 . (3) Ampicillin Sodium responds to the Qualitative Tests 1) for sodium salt. Specific Optical Rotation [ ] 20 D : +246 ~ +272 (1.0 g calculated on the anhydrous basis, water, 100 mL, 100 mm). pH The pH of a solution obtained by dissolving 0.1 g of Ampicillin Sodium in 10 mL of water is between 8.0 and 10.0. Sterility Test It meets the requirement, when Ampicillin Sodium is used in a sterile preparation. Bacterial Endotoxins Less than 0.15 EU per mg of ampicillin, when Ampicillin Sodium is used in a sterile preparation. Water Not more than 2.0 % (0.2 g, volumetric titration, direct titration). Assay Perform the test according to Assay in Ampicillin Hydrate. Corresponding to the potency specified in the labeling, weigh accurately about 50 mg (potency) of Ampicillin Sodium, dissolve in 80 mL of water, and add water to make exactly 100 mL. Pipet exactly 5 mL of this solution, add water to make exactly 100 mL, and use this solution as the test solution. Packaging and Storage Preserve in tight containers.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 229 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 ~ 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 ~ 10 m in particle diameter) Mobile phase: Dissolve 1.13 g of potassium dihydrogen phosphate in water, add 250 mL of methanol, 1 mL of acetic acid, and add water to make 1000 mL. Packaging and Storage Preserve in tight containers.

Ampicillin Sodium
NaO O O O N H CH3 CH3 S H H

Amyl Nitrite
C5H11NO2: 117.15 Amyl Nitrite is the nitrous acid ester of 3-methyl-1butanol and contains a small quantity of 2-methyl-1butanol and the nitrous acid esters of other homologies. Amyl Nitrite contains not less than 90.0% and not more than 101.0% of amyl nitrite (C5H11NO2). Description Amyl Nitrite is a clear, pale yellowish liquid and has a characteristic, fruity odor. Amyl Nitrite is miscible with ethanol or with ether.

N H H2N H

C16H18N3NaO4S: 371.39 Ampicillin Sodium contains not less than 845 g (potency) per mg of ampicillin (C16H19N3O4S: 349.41), calculated on the anhydrous basis. Description Ampicillin Sodium is a white to light yel-

68 Monographs, Part I Amyl Nitrite is practically insoluble in water. Amyl Nitrite is affected by light and by heat. Amyl Nitrite is volatile at room temperature and flammable even at a low temperature. Boiling pointAbout 97 C. Identification Determine the infrared spectra of Amyl Nitrite and Amyl Nitrite RS as directed in the liquid film method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Gravity
20 : Between 0.871 and 0.880. d 20

Anthralin
OH O OH

Dithranol

C14H10O3: 226.23

Anthralin contains not less than 97.0% and not more than 102.0% of anthralin (C14H10O3), calculated on the dried basis. Description Anthralin is a yellow powder. Anthralin is odorless and tasteless. Anthralin is freely soluble in chloroform, acetone or benzene, sparingly soluble in ethanol, in ether or in glacial acetic acid and practically insoluble in water. Anthralin is dissolves in sodium hydroxide TS Identification (1) Determine the absorption spectra of solutions of Anthralin and Anthralin RS in chloroform (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Anthralin and Anthralin RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit the same intensities of absorption at the same wavenumbers. Melting Point Between 178 C and 181 C (Method 1).

Purity (1) AcidTo 5 mL of Amyl Nitrite, add a mixture of 1.0 mL of 1 mol/L sodium hydroxide VS, 10 mL of water and 1 drop of phenolphthalein TS, shake and allow to stand for 1 minute: the light red color of the water layer does not disappear. (2) WaterAllow 2.0 mL of Amyl Nitrite to stand in ice-water: no turbidity is produced. (3) AldehydeTo 3 mL of a mixture of equal volumes of silver nitrate TS and aldehyde free-ethanol, add ammonia TS dropwise until the precipitate first formed is redissolved. Add 1.0 mL of Amyl Nitrite and warm between 60 C and 70 C for 1 minute: a brown to black color is not produced. (4) Residue on evaporationEvaporate 10.0 mL of Amyl Nitrite in a water-bath in a draft, carefully protecting from flame and dry the residue at 105 C for 1 hour: the weight of the residue is not more than 1.0 mg. Assay Weigh accurately a volumetric flask containing 10 mL of ethanol, add about 0.5 g of Amyl Nitrite and weigh accurately again. Add exactly 25 mL of 0.1 mol/L silver nitrate VS, then add 15 mL of potassium chlorate solution (1 in 20) and 10 mL of dilute nitric acid, stopper the flask immediately and shake vigorously for 5 minutes. Dilute with water to make exactly 100 mL, shake and filter through dry filter paper. Discard the first 20 mL of the filtrate, measure exactly 50 mL of the subsequent filtrate and titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ammonium ferric sulfate TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L silver nitrate VS = 35.144 mg of C5H11NO2 Packaging and Storage Preserve in light-resistant, hermetic containers not exceeding 10 mL capacity. Store in a cold place with avoidance of fire.
o o

Purity (1) Acid or baseSuspend Anthralin in water and filter: the filtrate is neutral using Litmus paper. (2) ChlorideSuspend approximately 1.0 g of Anthralin in 15 mL of water and filter. Collect 5 mL of the filtrate, make acidic with nitric acid and add 2 to 3 drops of silver nitrate TS: the turbidity of this solution is not more than that of 5 mL of the filtrate. (3) SulfateCollect 5 mL of filtrate from (2), add 3 drops of 3 mol/L hydrochloric acid and 5 drops of barium chloride TS: the turbidity of this solution is not more than that of 5 mL of the filtrate. Loss on Drying Not more than 0.5% (1 g, silica gel, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.25 g of Anthralin, dissolve in dichloromethane to make exactly 100 mL, pipet exactly 10 mL of this solution and add dichloromethane to make exactly 100 mL. Pipet exactly 5 mL of this solution, add 5.0 mL of the internal standard solution and the mobile phase to make exactly 25 mL and use this solution as the test solution. Separately weigh accurately a portion of Anthralin RS, dissolve in dichloromethane to

KP 9 69 make 250 g/mL. Pipet exactly 5 mL of this solution, add 5.0 mL of the internal standard solution and the mobile phase to make exactly 25 mL and use this solution as the standard solution. Perform the test with 10 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Anthralin to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of Anthralin (C14H10O3) = C
QT QS

(C14H10O3: 226.23) when labeled amount of Anthralin in Anthralin Ointment is not less than 0.1%. Anthralin Ointment contains not less than 90.0% and not more than 130.0% of the labeled amount of anthralin (C14H10O3: 226.23) when the labeled amount of Anthralin in Anthralin Ointment is not more than 0.1%. Method of Preparation Prepare as directed under the Ointments with Anthralin. Identification Retention time of major peak of the test solution from Assay is the same as that of the standard solution. Assay Weigh accurately about 5 g of Anthralin Ointment in 100 mL of beaker and disperse with 20 mL of dichloromethane and 10 mL of glacial acetic acid. Filter the content with dichloromethane using Whatman filter paper No. 4 to 100 mL volumetric flask. Wash the precipitate with dichloromethane to make 100 mL. Weigh accurately about 0.5 mg of anthralin (C14H10O3), add 2.0 mL of the internal standard solution and the mobile phase to make exactly 25 mL and use this solution as the test solution. Separately, weigh accurately a portion of Anthralin RS and dissolve in dichloromethane to make 0.25 mg/mL. Pipet exactly 2 mL of this solution, add 2.0 mL of the internal standard solution and the mobile phase to make exactly 25 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under Assay of Anthralin. Amount(mg) of Anthralin (C14H10O3) = 200
C QT V QS

C: Concentration of the standard solution (g/mL). Internal standard solutionWeigh a sufficient amount of o-nitroanilin, dissolve in a small volume of dichloromethane and add n-hexane to make 500 g/mL. Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 354 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and 25 cm in length, packed with porous silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of n-hexane, dichloromethane and glacial acetic acid (82 : 12 : 6). Flow rate: 2 mL/minute. System suitability System performance: Dissolve 10 mg of Anthralin RS and 20 mg of danthrone in and dilute with dichloromethane to make 100 mL. To 5 mL of this solution add 5 mL of n-hexane and mobile phase to make 25 mL. When the procedure is run with 10 L of this solution under the above operating conditions, anthralin and danthrone are eluted in this order with the resolution between these peaks being not less than 1.3. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area to that of the internal standard.is not more than 2.0%. Packaging and Storage tight containers. Preserve in light-resistant,

C: Concentration of the standard solution (mg/mL). V: Volume of the test solution collected (mL). Packaging and Storage well-closed containers. Preserve in light-resistant,

L-Arginine
HN

Hydrochloride
H C NH2 COOH HCl

CNHCH2CH2CH2 H2N

Anthralin Ointment
Dithranol Ointment Anthralin Ointment contains not less than 90.0% and not more than 115.0% of the labeled amount of anthralin

L-Arginine L-Arginine

Hydrochloride

C6H14N4O2HCl: 210.66

Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of L-arginine hydrochloride (C6H14N4O2 HCl).
L-Arginine

Description

Hydrochloride is a white crys-

70 Monographs, Part I tal or crystalline powder, is odorless and has a slight, characteristic taste. L-Arginine Hydrochloride is freely soluble in water or in formic acid, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Determine the infrared spectra of LArginine Hydrochloride and L-Arginine Hydrochlorode RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) A solution of L-Arginine Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride. pH Dissolve 1.0 g of L-Arginine Hydrochloride in 10 mL of water: the pH of this solution is between 4.7 and 6.2. Specific Optical Rotation [ ]20 D : Between +21.5 and +23.5 (after drying, 2 g, 6 mol/L hydrochloric acid TS, 25 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 1.0 g of L-Arginine Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 0.6 g of LArginine Hydrochloride and prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.028%). (3) AmmoniumPerform the test with 0.25 g of LArginine Hydrochloride, using the distillation under reduced pressure. Prepare the control solution with 5.0 mL of standard ammonium solution (not more than 0.02%). (4) Heavy metalsProceed with 1.0 g of L-Arginine Hydrochloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) ArsenicPrepare the test solution with 1.0 g of L-Arginine Hydrochloride according to Method 1 and perform the test (not more than 2 ppm). (6) Related substancesDissolve 0.20 g of LArginine Hydrochloride in 10 mL of water and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add water to make exactly 10 mL. Pipet exactly 1 mL of this solution and add water to make exactly 25 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of dehydrated ethanol, water, n-butanol and strong ammonia water (2 : 1 : 1 : 1) to a distance of about 10 cm and dry the plate at 100 C for 30 minutes. Spray evenly a solution of ninhydrin in acetone (1 in 50) on the plate and heat at 80 C for 5 minutes: any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution.

Loss on Drying Not more than 0.2% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.1 g of L-Arginine Hydrochloride, previously dried, dissolve in 2 mL of formic acid, add exactly 15 mL of 0.1 mol/L perchloric acid VS and heat in a water-bath for 30 minutes. After cooling, add 45 mL of glacial acetic acid and titrate the excess perchloric acid with 0.1 mol/L sodium acetate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 10.533 mg of C6H14N4O2HCl Packaging and Storage Preserve in tight containers.

Arginine Hydrochloride Injection

L-Arginine

Hydrochloride Injection

Arginine Hydrochloride Injection is an aqueous solution for injection. Arginine Hydrochloride Injection contains not less than 9.5% and not more than 10.5% of arginine hydrochloride (C6H14N4O2HCl: 210.66). Method of Preparation Arginine Hydrochloride 100 g Water for injection sufficient quantity To make 1000 mL Prepare as directed under Injections, with the above ingredients. No preservative is added. Description Arginine Hydrochloride Injection is a clear, colorless liquid. Identification (1) To 5 mL of a solution of Arginine Hydrochloride Injection (1 in 100), add 1 mL of ninhydrin TS and heat for 3 minutes: a blue-purple color is observed. (2) To 5 mL of a solution of Arginine Hydrochloride Injection (1 in 10), add 2 mL of sodium hydroxide TS and 1 to 2 drops of a solution of -naphthol in ethanol (1 in 1000), allow to stand for 5 minutes and add 1 to 2 drops of sodium hypochlorite TS: a red-orange color is observed. pH Between 5.0 and 6.0.

KP 9 71 Sterility Test It meets the requirement. Pyrogen It meets the requirement when the test is performed with Arginine Hydrochloride Injection stored in a container in a volume exceeding 10 mL. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement. It drochloride and Arotinolol Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) The solution of Arotinolol Hydrochloride (1 in 200) responds to Qualitative Tests (2) for Chloride. Purity (1) Heavy metalsProceed with 1.0 g of Arotinolol Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesDissolve 50 mg of Arotinolol Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 100 mL. Pipet exactly 1 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 40 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, methanol, acetone and strong ammonia water (30 : 10 : 10 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Loss on Drying Not more than 0.2 g (1 g, in vacuum, 105 C, 4 hours).
HCl

Determination of Volume of Injection in Containers It meets the requirement. Assay Pipet exactly 20 mL of Arginine Hydrochloride Injection, add 7.5 mol/L hydrochloric acid TS to make exactly 100 mL and determine the optical rotation, D, as directed under the Optical Rotation Determination at 20 1 C in a 100 mm cell. Amount (mg) of arginine hydrochloride (C6H14N4O2HCl) = D 4444 Packaging and Storage Preserve in hermetic containers.

Arotinolol Hydrochloride
OH N (CH 3)3CNHCH 2CCH 2S H S S C NH2 O

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 1.5 g of Arotinolol Hydrochloride, previously dried and dissolve in dimethylsulfoxide to make exactly 25 mL. Pipet exactly 5 mL of this solution, add 100 mL of water and 5 mL of sodium hydroxide TS and extract with three 50 mL volumes of dichloromethane. Filter each dichloromethane extract through a pledget of absorbent cotton with anhydrous sodium sulfate on it. Evaporate combined filtrate to dryness in vacuum. Dissolve the residue in 70 mL of glacial acetic acid and titrate with 0.05 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L perchloric acid VS = 20.400 mg of C15H21N3O2S3HCl Packaging and Storage tight containers. Preserve in light-resistant,

and enantiomer C15H21N3O2S3HCl: 408.00

Arotinolol Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of arotinolol hydrochloride (C15H21N3O2S3HCl). Description Arotinolol Hydrochloride is a white to pale yellow crystalline powder. Arotinolol Hydrochloride is freely soluble in dimethylsulfoxide, slightly soluble in methanol or in water, very slightly soluble in dehydrated ethanol, and practically insoluble in ether. A solution of Arotinolol Hydrochloride in methanol (1 in 125) shows no optical rotation. Identification (1) Determine absorption spectra of solutions of Arotinolol Hydrochloride and arotinolol hydrochloride RS, in methanol (1 in 75000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Arotinolol Hy-

72 Monographs, Part I

Ascorbic Acid
H HO C CH2OH O O H OH OH

Each mL of 0.05 mol/L iodine VS = 8.806 mg of C6H8O6 Packaging and Storage tight containers. Preserve in light-resistant,

Ascorbic Acid Injection

C6H8O6: 176.12 Vitamin C Injection Ascorbic Acid Injection is an aqueous solution for injection. Ascorbic Acid Injection contains not less than 95.0% and not more than 115.0% of the labeled amount of ascorbic acid (C6H8O6: 176.12). Method of Preparation Prepare as directed under Injections, with the sodium salt of ascorbic acid. Description Ascorbic Acid Injection is a clear, colorless liquid. Identification (1) Measure a volume of Ascorbic Acid Injection, equivalent to 0.5 g of Ascorbic Acid according to the labeled amount, and add water to make 25 mL. Proceed with 5 mL each of the solution as directed in the Identification (1) under Ascorbic Acid. (2) Measure a volume of Ascorbic Acid Injection, equivalent to 5 mg of Ascorbic Acid according to the labeled amount. Add a solution of metaphosphoric acid (1 in 50) to make 5 mL and proceed with this solution as directed in the Identification (2) under Ascorbic Acid. (3) Ascorbic Acid Injection responds to the Qualitative Tests (1) for sodium salt. pH Between 5.6 and 7.4.

Vitamin C

Ascorbic Acid, when dried, contains not less than 99.0% and not more than 101.0% of ascorbic acid (C6H8O6). Description Ascorbic Acid is a white crystal or a white crystalline powder, is odorless and has an acid taste. Ascorbic Acid is freely soluble in water, sparingly soluble in ethanol, and practically insoluble in ether. Melting pointAbout 190 C (with decomposition) Identification (1) To 5 mL each of solution of Ascorbic Acid (1 in 50), add 1 drop of potassium permanganate TS or 1 to 2 drops of 2,6-dichlorophenol-indophenol sodium TS: the color of the solution is discharged immediately in each case . (2) Dissolve 0.1 g of Ascorbic Acid in 100 mL of a solution of metaphosphoric acid (1 in 50). To 5 mL of this solution, add iodine TS until the color of the solution becomes pale yellow. Then add 1 drop of a solution of cupric sulfate (1 in 1000) and 1 drop of pyrrole and
o warm the mixture at 50 C for 5 minutes: a blue color develops.

Specific Optical Rotation [ ]20 D : Between +20.5 and +21.5(2.5 g, water, 25 mL, 100 mm). pH Dissolve 1.0 g of Ascorbic Acid in 20 mL of water: the pH of this solution is between 2.2 and 2.5. Purity (1) Clarity and color of solutionDissolve 1.0 of Ascorbic Acid in 20 mL of water: the solution is clear and colorless. (2) Heavy metalsPerform the test with 1.0 g of Ascorbic Acid according to Method 1. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 0.2% (1 g, silica gel, 24 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Ascorbic Acid, previously dried, dissolve in 50 mL of a solution of meta-phosphoric acid (1 in 50) and titrate with 0.05 mol/L iodine VS (indicator: 1 mL of starch TS).

Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement. It

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of Ascorbic Acid Injection, equivalent to about 0.1 g of ascorbic acid (C6H8O6), previously diluted with metaphosphoric acidacetic acid TS, if necessary and add metaphosphoric acid-acetic acid TS to make exactly 200 mL. Pipet exactly 2 mL of this solution and proceed with this solution as directed in the Assay under Ascorbic Acid Powder.

KP 9 73 Each mL of 2,6-dichlorophenol-indophenol sodium TS for titration = A mg of C6H8O6 Packaging and Storage Preserve under nitrogen atmosphere, in hermetic containers. dichlorophenol-indophenol sodium TS for titration. 2,6-Dichlorophenol-indophenol sodium TS for titration. PreparationDissolve 42 mg of sodium bicarbonate in 50 mL of water, add 50 mg of 2,6dichlorophenol-indophenol sodium and water to make 200 mL and filter. Prepare before use. StandardizationWeigh accurately about 50 mg of Ascorbic Acid RS, previously dried in a desiccator (silica gel) for 24 hours, and dissolve in metaphosphoric acidacetic acid TS to make exactly 100 mL. Pipet exactly 2 mL of this solution, shake with 8 mL of metaphosphoric acid-acetic acid TS and 2 mL of hydrogen peroxide TS and titrate with 2,6-dichlorophenol-indophenol sodium until a light red color persists for 5 seconds. Perform a blank determination and make any necessary correction. Calculate the quantity (A mg) of ascorbic acid (C6H8O6) equivalent to 1 mL of this test solution. Pakaging and Storage Preserve in tight containers.

Ascorbic Acid Powder

Vitamin C Powder Ascorbic Acid Powder contains not less than 95.0% and not more than 120.0% of the labeled amount of ascorbic acid (C6H8O6: 176.12). Method of Preparation Prepare as directed under Powders, with Ascorbic Acid. Identification (1) Weigh a portion of Ascorbic Acid Powder, equivalent to 0.5 g of Ascorbic Acid according to the labeled amount, add 30 mL of water, shake for 1 minute and filter. Proceed with 5 mL each of the filtrate as directed in the Identification (1) under Ascorbic Acid. (2) Weigh a portion of Ascorbic Acid Powder, equivalent to about 10 mg of Ascorbic Acid according to the labeled amount, add 10 mL of a solution of metaphosphoric acid (1 in 50), shake for 1 minute and filter. Proceed with 5 mL of the filtrate as directed in the Identification (2) under Ascorbic Acid. Purity RancidityAscorbic Acid Powder is free from any unpleasant or rancid odor and taste. Particle Size Distribution Test for Preparations It meets the requirement. Uniformity of Dosage Units (divided) It meets the requirement. Assay Weigh accurately a portion of Ascorbic Acid Powder, equivalent to about 0.1 g of ascorbic acid (C6H8O6) according to the labeled amount, extract with several successive portions of metaphosphoric acidacetic acid TS, combine the extracts and filter. Wash the residue with metaphosphoric acid-acetic acid TS. Combine the filtrate and washings and add metaphosphoric acid-acetic acid to make exactly 200 mL. Pipet exactly 2 mL of this solution and shake with 8 mL of metaphosphoric acid-acetic acid TS and 2 mL of hydrogen peroxide TS. Titrate with 2,6-dichlorophenol-indophenol sodium TS for titration until a light red color persists for 5 seconds. Perform a blank determination and make any necessary correction. Each mL of 2,6-dichlorophenol-indophenol sodium TS for titration = A mg of C6H8O6 A is decided by the following standardization of 2,6-

Ascorbic Acid Tablets

Vitamin C Tablets Ascorbic Acid Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of ascorbic acid (C6H8O6: 176.12). Method of Preparation Prepare as directed under Tablets, with Ascorbic Acid. Identification (1) Weigh a portion of powdered Ascorbic Acid Tablets, equivalent to 0.5 g of Ascorbic Acid according to labeled amount, add 30 mL of water, shake for 1 minute and filter. Proceed with 5 mL of the filtrate as directed in the Identification (1) under Ascorbic Acid. (2) Pipet 2 mL of the filtrate, add sodium hydroxide TS to make neutral, then add 2 drops of uranyl acetate TS: a red brown color develops. And then add 2 mL of sodium hydroxide TS: a yellow color develops. (3) Weigh a portion of powdered Ascorbic Acid Tablets, equivalent to 10 mg of Ascorbic Acid according to labeled amount, add 10 mL of a solution of metaphosphoric acid (1 in 50), shake for 1 minute and filter. Proceed with 5 mL of the filtrate as directed in the Identification (2) under Ascorbic Acid. Dissolution Test Perform the test with 1 tablet of Ascorbic Acid Tablets at 50 revolutions per minute according to the Method 2 under the Dissolution Test, using 900 mL of the water as the dissolution solution. After 45 minutes from the start of the test, take not less than 20 mL of the dissolution solution and filter the solution using a membrane filter having the pore size of not more than 0.8 m. Proceed with the filtrate as directed in the Assay.

74 Monographs, Part I The dissolution rate of Ascorbic Acid Tablets in 45 minutes is not less than 75%. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Ascorbic Acid Tablets, equivalent to about 0.1 g of ascorbic acid (C6H8O6) and proceed with this powder as directed in the Assay under Ascorbic Acid Powder. Packaging and Storage tight containers. Preserve in light-resistant, Control solutionDissolve 0.1 g of salicylic acid in water and add 1 mL of glacial acetic acid and water to make 1000 mL. Add 1.0 mL of this solution to a solution prepared by transferring 1 mL of freshly prepared dilute ammonium ferric sulfate TS and 1 mL of ethanol to a Nessler tube and diluting with water to make 50 mL. Allow to stand for 30 seconds. (3) ChlorideBoil 1.8 g of Aspirin in 75 mL of water for 5 minutes, cool, add water to make 75 mL and filter. To 25 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.015%). (4) SulfateTo 25 mL of the filtrate obtained in (3), add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.040%). (5) Heavy metalsDissolve 2.5 g of Aspirin in 30 mL of acetone, add 2 mL of dilute acetic acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 2.5 mL of standard lead solution, 30 mL of acetone, 2 mL of dilute acetic acid and water to make 50 mL (not more than 10 ppm). (6) Readily carbonizable substancesWeigh 0.5 g of Aspirin and perform the test. The solution has no more color than Color Matching Fluid Q. Loss on Drying Not more than 0.5% (3 g, silica gel, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 1.5 g of Aspirin, previously dried, add exactly 50 mL of 0.5 mol/L sodium hydroxide VS and boil gently for 10 minutes using a reflux condenser closed with a carbon dioxide-absorbing tube (soda lime). Cool and titrate excess sodium hydroxide with 0.25 mol/L sulfuric acid VS (indicator: 3 drops of phenolphthalein TS) immediately. Perform a blank determination and make any necessary correction. Each mL of 0.5 mol/L sodium hydroxide VS = 45.04 mg of C9H8O4 Packaging and Storage Preserve in well-closed containers.

Aspirin
CO2H O O C CH3

Acetylsalicylic Acid

C9H8O4: 180.16

Aspirin, when dried, contains not less than 99.5% and not more than 101.0% of aspirin (C9H8O4). Description Aspirin is a white crystal, granule or powder, is odorless and has a slight acid taste. Aspirin is freely soluble in ethanol or in acetone, soluble in ether, and slightly soluble in water. Aspirin dissolves in sodium hydroxide TS and in sodium carbonate TS. In moist air, aspirin gradually hydrolyzes to salicylic acid and acetic acid. Melting pointAbout 136 C (bath fluid is heated at 130 C previously). Identification (1) Boil 0.1 g of Aspirin in 5 mL of water for 5 to 6 minutes, cool. and add 1 to 2 drops of ferric chloride TS: a red-purple color is produced. (2) Boil 0.5 g of Aspirin in 10 mL of sodium carbonate TS for 5 minutes and add 10 mL of dilute sulfuric acid: the odor of acetic acid is perceptible and a white precipitate is produced. Filter the precipitate, add 3 mL of ethanol and 3 mL of sulfuric acid to the filtrate and heat: the odor of ethyl acetate is perceptible. Purity (1) Clarity of solutionDissolve 0.5 g of Aspirin in 10 mL of warm sodium carbonate TS: the solution is clear. (2) Salicylic acidDissolve 2.5 g of Aspirin in ethanol to make 25 mL and add 1.0 mL of this solution to a solution prepared by transferring 1 mL of a freshly prepared dilute ammonium ferric sulfate TS to a Nessler tube and diluting with water to make 50 mL. Allow to stand for 30 seconds: the color of the solution is not more intense than that of the following control solution.

Aspirin Tablets
Acetylsalicylic Acid Tablets Aspirin Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of aspirin (C9H8O4: 180.16).

KP 9 75 Internal standard solutionDissolve about 10 mg of Theophylline RS in mobile phase to make 100 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and between 15 and 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (between 5 and 10 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of 0.085% phosphoric acid and methanol (60 : 40). Flow rate: 1.0 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the internal standard and aspirin are eluted in this order with the resolution between their peaks being not less than 3.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area is not more than 2.0%. Packaging and Storage Preserve in well-closed containers.

Method of Preparation Prepare as directed under Tablets, with Aspirin. Identification (1) Weigh a portion of powdered Aspirin Tablets, equivalent to 0.1 g of Aspirin according to the labeled amount, add 10 mL of water and boil for 5 to 6 minutes. After cooling, filter and add 1 to 2 drops of ferric chloride TS to the filtrate: a red-purple color develops. (2) Weigh a portion of powdered Aspirin Tablets, equivalent to 0.5 g of Aspirin according to the labeled amount, extract with two 10 mL volumes of warm ethanol and filter the combined extracts. Evaporate the filtrate to dryness and boil the residue with 10 mL of sodium carbonate TS for 5 minutes. Proceed as directed in the Identification (2) under Aspirin. Purity Salicylic acidWeigh accurately and powder not less than 20 Aspirin Tablets. Weigh accurately an amount equivalent to about 1.0 g of Aspirin, add mobile phase to make 100 mL and use this solution as the test solution. Separately, weigh accurately about 15 mg of Salicylic Acid RS, dissolve in mobile phase to make 100 mL. Pipet exactly 5 mL of this solution, add mobile phase to make exactly 50 mL and use this solution as the standard solution. Perform the test with test solution and the standard solution as directed under Assay (not more than 0.15%). Disintegration Test It meets the requirement.. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Aspirin Tablets. Weigh accurately an amount equivalent to about 0.25 g of aspirin (C9H8O4), add mobile phase, shake well, add mobile phase to make 200 mL, then filter. Discard the first 10 mL of the filtrate, measure exactly 2 mL of subsequent filtrate, add 4.0 mL of internal standard solution and mobile phase to make 100 mL. Use this solution as the test solution. Separately, weigh accurately about 25 mg of Aspirin RS, previously dried in desiccator (silica gel) for 5 hours, dissolve in mobile phase to make exactly 20 mL. Pipet exactly 2 mL of this solution, add 4.0 mL of internal standard solution and mobile phase to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of aspirin to that of internal standard, for the test solution and the standard solution, respectively. Amount (mg) of aspirin (C9H8O4) = amount (mg) of Aspirin RS
QT 10 QS

Aspirin Aluminum
CO2
-

O O C CH3 Al (OH)
2+

Aluminum Acetyl Salicylate

C18H15NA1O9: 416.30

Aspirin Aluminum contains not less than 83.0% and not more than 90.0% of aspirin (C9H8O4: 180.16) and not less than 6.0% and not more than 7.0% of aluminum (Al: 26.98), calculated on the anhydrous basis. Description Aspirin Aluminum is a white, crystalline powder. Aspirin Aluminum is odorless or has a slightly acetic odor. Aspirin Aluminum is practically insoluble in water, in methanol, in ethanol or in ether. Aspirin Aluminum dissolves, with decomposition, in sodium hydroxide TS or sodium carbonate TS. Identification (1) Dissolve 0.1 g of Aspirin Aluminum in 10 mL of sodium hydroxide TS by heating, if necessary. Neutralize 2 mL of this solution with hydrochloric

76 Monographs, Part I acid and add 1 to 2 drops of ferric chloride TS: a redpurple color develops. (2) Determine the absorption spectrum of the test solution obtained in the Assay (1) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 277 nm and 279 nm. (3) Place 2 g of Aspirin Aluminum in a platinum crucible and ignite until charred. To the residue, add 1 g of anhydrous sodium carbonate and ignite for 20 minutes. After cooling, to the residue, add 15 mL of dilute hydrochloric acid, shake and filter: the filtrate responds to the Qualitative Tests for aluminum salt. Purity (1) SalicylateUsing AT2 and AS2 obtained in the Assay (1), calculate the amount of salicylate [as salicylic acid (C7H6O3: 138.12)] by the following equation: salicylate content is not more than 7.5%, calculated on the anhydrous basis. Amount (mg) of salicylic acid (C7H6O3)
A = amount (mg) of Salicylic Acid RS T 2 1 AS 2 4

with six 20 mL volumes of chloroform. Combine all chloroform extracts and add chloroform to make exactly 200 mL. Measure exactly 10 mL of this solution, add chloroform to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 90 mg of Salicylic Acid RS, previously dried in a desiccator (silica gel) for 3 hours, and dissolve in chloroform to make exactly 200 mL. Measure exactly 5 mL of this solution, add chloroform to make exactly 200 mL and use this solution as the standard solution (1). Then weigh accurately about 90 mg of Aspirin RS, previously dried in a desiccator (silica gel) for 5 hours and dissolve in chloroform to make exactly 200 mL. Measure exactly 10 mL of this solution, add chloroform to make exactly 100 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Ultravioletvisible Spectrophotometry. Determine the absorbances, AT1 and AS1 , of the test solution and the standard solution (1) at 278 nm and absorbances, AT2 and AS2 , of the test solution and the standard solution (2), respectively, at 308 nm. Then determine the absorbance, AS3 of the standard solution (2) at 278 nm. Amount (mg) of aspirin (C9H8O4)

(2) Heavy metalsPlace 2.0 g of Aspirin Aluminum in a porcelain crucible, cover the crucible loosely and ignite at a low temperature until charred. After cooling, add 2 mL of nitric acid and 1 mL of sulfuric acid to the content of the crucible, heat gently the crucible until white fumes are evolved and continue the heating until white fumes are no longer evolved, then ignite between 500 C and 600 C until the carbon is incinerated. When the incineration is not completed, add 2 mL of nitric acid and 1 mL of sulfuric acid and heat gently in the same manner, then ignite between 500 C and 600 C to incinerate completely. After cooling, add 2 mL of hydrochloric acid and proceed as directed in Method 2 and perform the test. Prepare the control solution by using the same quantities of the same reagents as directed for the preparation of the test solution and add 2.0 mL of standard lead solution and water to make 50 mL (not more than 10 ppm). (3) ArsenicDissolve 1.0 g of Aspirin Aluminum in 15 mL of sodium hydroxide TS. To this solution, add 1 drop of phenolphthalein TS and with stirring, add dropwise hydrochloric acid until the red color of the solution disappears. Then add 2 mL of hydrochloric acid, cool with occasional shaking for 10 minutes and filter with a glass filter (G3). Wash the residue with two 5 mL volumes of 1 mol/L hydrochloric acid TS and combine the filtrate and the washings. Use this solution as the test solution and perform the test (not more than 2 ppm). Water Not more than 4.0% (0.15 g, volumetric titration, direct titration).

AT 1
= amount (mg) of Aspirin RS

AT 2 AS1 AS 2 AS 3

(2) AluminumWeigh accurately about 0.4 g of Aspirin Aluminum and dissolve in 10 mL of sodium hydroxide TS. Add drop-wise 1 mol/L hydrochloric acid TS until the pH of the solution to about 1, add 20 mL of acetic acid-ammonium acetate buffer solution, pH 3.0 and 0.5 mL of Cu-PAN TS and heat. While boiling, titrate with 0.05 mol/L disodium ethylenediaminetetraacetate VS until the color of the solution changes from red to yellow and persists for 1 minute. Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L disodium ethylenediaminetetraahetate VS = 1.3491 mg of Al Packaging and Storage Preserve in well-closed containers.

Atenolol
OH O O H N CH3

Assay (1) AspirinWeigh accurately about 0.1 g of Aspirin Aluminum, add 40 mL of sodium fluoride TS and shake for 5 minutes. Allow the solution to stand for 10 minutes with frequent shaking. Extract the solution

CH3 H2N

and enantiomer

KP 9 77 C14H22N2O3: 266.34 Atenolol, when dried, contains not less than 99.0% and not more than 101.0% of atenolol (C14H22N2O3). Description Atenolol is a white to pale yellow crystalline powder. Atenolol is freely soluble in methanol or in glacial acetic acid, soluble in dehydrated ethanol, and slightly soluble in water. A solution of Atenolol in methanol (1 in 25) shows no optical rotation. Identification (1) Determine the absorption spectra of a solution of Atenolol and Atenolol RS, in methanol (5 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths.. (2) Determine the infrared spectra of Atenolol and Atenolol RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 152 C and 156 C. Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.1 g each of Atenolol and Atenolol RS, previously dried, dissolve separately in the mobile phase to make exactly 100 mL, respectively. Pipet exactly 5 mL each of these solutions, add the mobile phase to make exactly 50 mL, pipet exactly 5 mL each of these solutions again, add the mobile phase to make exactly 50 mL, and use these solutions as the test solution and the standard solution respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate each peak area of Atenolol, AT and AS , for the test solution and the standard solution, respectively. Amount (mg) of atenolol (C14H22N2O3) = Amount (mg) of Atenolol RS
AT AS

tetrabutylammonium hydrogensulfate in 1000 mL of this solution. Flow rate: Adjust the flow rate so that the retention time of atenolol is about 8 minutes. System suitability Test for required detectability: To exactly 10 mL of the standard solution add mobile phase to make exactly 50 mL. Confirm that the peak area of atenolol obtained with 10 L of this solution is equivalent to 14 to 26% of that obtained with 10 L of the standard solution. System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of atenolol are not less than 5000 and not more than 1.5, respectively. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of atenolol is not more than 1.0%. Time span of measurement: About 4 times as long as the retention time of atenolol. Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hour).

Purity (1) Heavy metalsProceed with 2.0 g of Atenolol according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of Standard lead solution (not more than 20 ppm). (2) Related substances Dissolve 50 mg of Atenolol in the mobile phase to make 25 mL, and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add the mobile phase to make 200 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: any area peak other than atenolol obtained with the test solution is not larger than 1/2 times the peak area of atenolol from the standard solution, and the total area of all peaks other than atenolol from the test solution is not larger than the peak area of atenolol with the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 226 nm). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecysilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 3.4 g of potassium dihydrogen phosphate in 1000 mL of water, and adjust to pH 3.0 with phosphoric acid. To 40 volume of this solution add 9 volume of methanol and 1 volume of tetrahydrofuran. Dissolve 1.0 g of sodium 1-octanesulfonate and 0.4 g of

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 226 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Mobile phase: Dissolve 1.1 g of sodium 1heptanesulfonate and 0.71 g of anhydrous sodium dihydrogen phosphate in 700 mL of water, add 2 mL of dibutylamine, and adjust the pH to 3.0 with 0.8 mol/L phosphoric acid TS. Add 300 mL of methanol to this solution and mix well.

78 Monographs, Part I Flow rate: 1.7 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the theoretical plates are not less than 5000 and the symmetry factor is not more than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area is not more than 2.0%. Packaging and Storage Preserve in tight containers. not less than 80%. Dissolution rate (%) with respect to the labeled amount of Atenolol (C14H22N2O3) = 900 C D AT
AS

C: Concentration of Atenolol in the standard solution (mg/mL). D: Dilution times of the test solution. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Atenolol Tablets. Weigh accurately a portion of the powder, equivalent to about 0.25 g of atenolol (C14H22N2O3), add the mobile phase, and sonicate for 15 minutes. Add the mobile phase to make exactly 1000 mL, centrifuge, pipet exactly 2 mL of the supernatant, add the mobile phase to make 50 mL, and use this solution as the test solution. Separately, weigh exactly about 25 mg of Atenolol RS, add the mobile phase to make 100 mL, pipet exactly 2 mL of this solution, add the mobile phase to make 50 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed in the Assay under Atenolol. Amount (mg) of atenolol (C14H22N2O3) = amount (mg) of Atenolol RS AT 10
AS

Atenolol Tablets
Atenolol tablets contains not less than 90.0% and not more than 110.0% of the labeled amount of atenolol (C14H22N2O3: 266.34). Method of Preparation Prepare as directed under Tablets, with Atenolol. Identification (1) To a portion of powdered Atenolol Tablets, equivalent to 0.1 g of Atenolol according to labeled amount, add 15 mL of methanol, mix well, warm at 50 C, shake for 5 minutes. Filter, evaporate the filtrate on a water-bath to dryness, add 0.1 mol/L hydrochloric acid to this residue, shake on warming, and filter. To this filtrate, add a sufficient amount of 1 mol/L sodium hydroxide TS to make alkaline, extract with 10 mL of chloroform, remove water from the chloroform layer using anhydrous sodium sulfate, and filter. Evaporate the filtrate to dryness on a water-bath, dry this residue at 105 C for 1 hour, and use this residue as the test sample. Determine the infrared spectra of the test sample and Atenolol RS, powdered finely according to the same method, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The retention time of main peak of the test solution for Assay and the standard solution is the same. Dissolution Test Perform the test with 1 tablet of Atenolol Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water as the dissolution solution. Take 20 mL or more of the dissolve solution 30 minutes after starting the test, and filter through a membrane filter with pore size of not more than 0.8 m. Add phosphoric acid solution (1 in 1000) to this filtrate so that each mL contains 10 g of Atenolol, and use this solution as the test solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, prepared according to the Assay, respectively, as directed in the Assay. The dissolution rate of Atenolol Tablets in 30 minutes is

Packaging and Storage Preserve in tight containers.

Atracurium Besylate
O O S H3CO H3CO N H3CO H3CO
+

OOCH3 OCH3

. 2

O O

O O

N CH3 OCH3 OCH3

CH3

C53H72N2O122C6H5O3S2 : 1243.48 Atracurium Besylate contains not less than 96.0% and not more than 102.0% of atracurium besylate (C53H72N2O122C6H5O3S2), calculated on the anhydrous basis. Description Atracurium Besylate is a white to yellowish-white solid. Atracurium Besylate is very soluble in dichloromethane, in acetonitrile, or in ethanol and soluble in water. Atracurium Besylate is hygroscopic. Identification (1) Determine the infrared spectra of

KP 9 79 Atracurium Besylate and Atracurium Besylate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The 3 principal isomeric peaks from test solution are same in retention time as those from standard solution in Assary. Purity (1) Heavy metalsProceed with 1.0 g of Atracurium Besylate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Methyl benzenesulfonateWeigh exactly 0.10 g of Atracurium Besylate, dissolve mobile phase A to make exactly 10 mL and use this solution as the test solution. Separately, weigh exactly 20.0 mg of Atracurium Besylate RS, add acetonitrile to make exactly 100 mL, pipet 1.0 mL of this solution and add solution A to make exactly 200 mL and use this solution as the standard solution. Perform the test with 100 L of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions and measure the responses for the methyl benzenesulfonate peaks: the peak area from the test solution is not greater than that from the standard solution (0.01%). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 217 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with base-deactivated octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: The chromatograph is programmed as follows with mobile phase A and mobile phase B. Mobile phase A and mobile phase BPrepare as directed in the Assay.
Time (minutes) 0 0~5 5 ~ 15 15 ~ 25 25 ~ 30 30 ~ 38 38 ~ 45 Mobile phase A (vol%) 80 80 80 75 75 75 55 55 0 0 Mobile phase B (vol%) 20 20 20 25 25 25 45 45 100 100 Elution Equilibration Isocratic Linear gradient Isocratic Linear gradient Linear gradient isocratic

12.0. System repeatability: When the test is repeated 2 times with the standard solution under the above operating conditions, the responses for duplicate injections do not differ from each other by more than 12%. (3) TolueneWeigh accurately about 0.20 g of Atracurium Besylate, dissolve in acetonitrile to make exactly 10 mL and use this solution as the test solution. Separately, weigh accurately 0.1 g of Atracurium Besylate RS, dissolve in acetonitrile to make exactly 100 mL, pipet 5.0 mL of this solution, add acetonitrile to make exactly 50 mL and use this solution as the standard solution. Perform the test with 1 L of the test solution and the standard solution as directed under Gas Chromatography according to the following conditions and measure the responses for the major peaks: the area of toluene peak from the test solution is not greater than that from the standard solution (not more than 0.5%). Operating conditions Detector: A hydrogen flame-ionization detector. Column: A fused-silica analytical column 0.53 mm in inside diameter and 30 m in length, coated the inner surface with 5% phenyl-95% methyl polysiloxane for Gas Chromatography 5 m in thickness. A fused-silica guard column 0.53 mm in inside diameter and 5 m in length, coated the inner surface with deactivated with phenylmethyl siloxane. Column temperature: Program as follows; Initially, the column temperature is maintained at 35 C for 5 minutes, then increased at a rate of 8 C per minute to 175 C, followed by an increase at a rate of 35 C per minutes to 260 C, and maintained at 260 C for at least 16 minutes. Injection port temperature: A constant temperature of about 70 C. Detector temperature: A constant temperature of about 260 C. Carrier gas: Helium Flow rate: 35 mL/second System suitability System performance: Weigh accurately each of dichloromethane, dioxane, trichloroethylene, and chloroform and dissolve in acetonitrile to make the solution that contains 12.0, 7.6, 1.6, and 1.2 g per mL, respectively. When the procedure is run with 1 L of this solution under the above operating conditions, the resolution between two peaks is not less than 1.0. System repeatability: Weigh accurately each of dichloromethane, dioxane, trichloroethylene, and chloroform and dissolve in acetonitrile to make the solution that contains 12.0, 7.6, 1.6, and 1.2 g per mL, respectively. When the procedure is run with 1 L of this solution under the above operating conditions, the relative standard deviation of each peak area is not more than 15%. (4) Related substancesUse the test solution of the

Flow rate: 1.0 mL/minute System suitability System performance: Pipet 1 mL of the test solution and 5 mL of 0.02% methyl benzenesulfonate in acetonitrile and add mobile phase to make 100 mL. When the procedure is run with this solution under the above operating condition, the resolution between the transtrans isomer and methyl benzenesulfonate is not less than

80 Monographs, Part I Assay as the test solution. To 1.0 mL of the standard solution of the Assay add mobile phase A of the Assay to make 100 mL and use this solution as the standard solution. Perform the test with 20 L of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions and measure all the peak areas, except the three main isomeric peaks. Calculate the percentage of each related substances in the portion of Atracurium Besylate taken by the formula: indiridual impurity is not more than 1.0% and total impurities is mot more than 3.5%.
A Amount (%) of each impurity = 10000 C i W

Mobile phase: Program as follows with the mobile phase A and the mobile phase B in isocratic or in linear gradient. Mobile phase A: The mixture of buffer solution, acetonitrile and methanol (75 : 20 : 5) Mobile phase B: The mixture of buffer solution, methanol and acetonitrile (50 : 30 : 20)
Time (minutes) 0 0~5 5 ~ 15 15 ~25 25 ~30 Mobile phase A (%) 80 80 80 40 40 40 0 Mobile phase B (%) 20 20 20 60 60 60 100 Elution Equilibration Isocratic Linear gradient Isocratic Linear gradient

AS

Ai :the peak area for each related substances obtained from the test solution AS :the peak area for the cis-cis isomer obtained from the standard solution W:the weight of Atracurium Besylate taken to prepare the test solution (mg) C:the concentration of the cis-cis isomer in the standard solution (mg/mL)
System suitability When the test is repeated more than 2 times with 20 L of the standard solution under the operating conditions of the Assay, the difference of the peak area of ciscis isomer is not more than 10%. Water Not more than 5.0% (1 g, volumetric titration, direct titration) Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.1 g each of Atracurium Besylate and Atracurium Besylate RS, dissolve in mobile phase A to make exactly 100 mL and use the solutions as the test solution and the standard solution, respectively. Perform the test with exactly 20 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine total area, AT and AS , of the three isomeric peaks, respectively. Amount (mg) of atracurium besylate (C53H72N2O122C6H5O3S2) = amount (mg) of Atracurium Besylate RS
AT AS

Flow rate: 1.0 mL/minute System suitability System performance: When the procedure is run with the standard solution under the above operating condition, the relative retention times are about 0.8, 0.9, and 1.0 for the trans-trans isomer, the cis-trans isomer, and the cis-cis isomer, respectively. The resolution between the trans-trans isomer and the cis-trans isomer and between the cis-trans isomer and the cis-cis isomer is not less than 1.1. System repeatability: When the test is repeated 5 times with the standard solution under the above operating conditions, the relative standard deviation of the peak area of each isomer is not more than 2.0%. Buffer solutionWeigh about 10.2 g of monobasic potassium phosphate, and dissolve in about 950 mL of water, while stirring, adjust with phosphoric acid to a pH of 3.1, add water to make 1000 mL. Packaging and Storage Preserve in light-resistant, tight containers. Store at a temperature of 2 C to 8 C.

Atropine Sulfate Hydrate

H O O NCH3 H CH2OH 2 C CH H2SO4 H2O

[(C17H23NO3)2.H2SO4H2O: 694. 83] Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with base-deactivated octadecylsilanized silica gel for Liquid Chromatography (5 to 10 m in particle diameter). Atropine Sulfate Hydrate, when dried, contains not less than 98.0% and not more than 101.0% of atropine sulfate [(C17H23NO3)2H2SO4: 676.82] Description Atropine Sulfate Hydrate is a colorless crystals or a white, crystalline powder and is odorless.

KP 9 81 Atropine Sulfate Hydrate is very soluble in water or in glacial acetic acid, freely soluble in ethanol and practically insoluble in ether. Melting pointBetween 188 C and 194 C (with decomposition). Introduce a capillary tube charged with dried sample into a bath previously heated to 180 C and continue to heat at the rate of rise of about 3 C per minute. Atropine Sulfate Hydrate is affected by light. Identification (1) To 1 mg of Atropine Sulfate Hydrate, add 3 drops of fuming nitric acid and evaporate the mixture in a water-bath to dryness. Dissolve the residue in 1 mL dimethylformamide and add 5 to 6 drops of tetraethylammonium hydroxide TS: a red-purple color develops. (2) To 2 mL of a solution of Atropine Sulfate Hydrate (1 in 50), add 4 to 5 drops of hydrogen tetrachloroaurate TS: a lusterless, yellowish white precipitate is formed. (3) To 5 mL of a solution of Atropine Sulfate Hydrate (1 in 25), add 2 mL of ammonia TS and allow to stand for 2 to 3 minutes. Collect the precipitate, wash with water and dry in a desiccator (in vacuum, silica gel) for 4 hours: it melts between 115 C and 118 C. (4) A solution of Atropine Sulfate Hydrate (1 in 20) responds to the Qualitative Tests for sulfate. Purity (1) Clarity and color of solutionDissolve 0.5 g of Atropine Sulfate Hydrate in 10 mL of water: the solution is clear and colorless. (2) AcidDissolve 1.0 g of Atropine Sulfate Hydrate in 20 mL of water and add 0.30 mL of 0.02 mol/L sodium hydroxide VS and 1 drop of methyl red-methylene blue TS: a green color develops. (3) Related SubstancesDissolve 0.25 g of Atropine Sulfate Hydrate in 1 mL of diluted hydrochloric acid (1 in 10), add water to make 15 mL and use this solution as the test solution. (i) To 5 mL of the test solution, add 2 to 3 drops of chloroplatinic acid TS: no precipitate is formed. (ii) To 5 mL of the test solution, add 2 mL of ammonia TS and shake vigorously: the turbidity of the solution is not greater than that of the following control solution. Control solutionTo 0.30 mL of 0.01 mol/L hydrochloric acid VS, add 6 mL of dilute nitric acid and water to make 50 mL. To this solution, add 1 mL of silver nitrate TS and allow 7 mL of the mixture to stand for 5 minutes. (4) HyoscyamineWeigh accurately about 1 g of Atropine Sulfate Hydrate, previously dried and dissolve in water to make exactly 10 mL: the specific optical rotation [ ]20 D of this solution in a 100 mm cell is between 0.60 and +0.10. (5) Readily carbonizable substancesTake 0.20 g of Atropine Sulfate Hydrate and perform the test: the solution has no more color than Color Matching Fluid A. Loss on Drying Not more than 4.0% (0.5 g, in vacuum, P2O5, 110 C, 4 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Dissolve about 0.25 g of Atropine Sulfate Hydrate, previously dried and accurately weighed, in 30 mL of glacial acetic acid. If necessary, dissolve by warming and cool. Titrate with 0.05 mol/L perchloric acid VS until the color of the solution changes from purple through blue to blue-green (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L perchloric acid VS = 33.841 mg of (C17H23NO3)2H2SO4 Packaging and Storage tight containers. Preserve in light-resistant,

Atropine Sulfate Injection

Atropine Sulfate Injection is an aqueous solution for injection. Atropine Sulfate Injection contains not less than 93.0% and not more than 107.0% of the labeled amount of atropine sulfate hydrate [(C17H23NO3)2H2SO4H2O: 694.83]. Method of Preparation Prepare as directed under Injections, with Atropine Sulfate Hydrate. Description Atropine Sulfate Injection is a clear, colorless liquid. pH Between 4.0 and 6.0. Identification (1) Evaporate a volume of Atropine Sulfate Injection, equivalent to 1 mg of Atropine Sulfate Hydrate according to the labeled amount, in a water-bath to dryness. Proceed with the residue as directed in the Identification (1) under Atropine Sulfate Hydrate. (2) Evaporate an exactly measured volume of Atropine Sulfate Injection, equivalent to 5 mg of Atropine Sulfate Hydrate according to the labeled amount, on a water bath to dryness. After cooling, dissolve the residue in 1 mL of ethanol, and use this solution as the test solution. If insoluble substance remains, crush it, allow to stand, and use the supernatant liquid as the test solution. Separately, dissolve 10 mg of Atropine Sulfate Hydrate RS in 2 mL of ethanol, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of acetone, water and strong ammonia water (90 : 7 : 3) to a distance of about 10 cm, and dry the plate at 80 C for 10 minutes. After cooling, spray evenly Dragendorffs TS for spraying on the plate: the

82 Monographs, Part I spots obtained from the test solution and the standard solution show an orange color and the same Rf value. (3) Atropine Sulfate Injection responds to the Qualitative Tests for sulfate. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 75 EU/mg of Atropine Sulfate Hydrate Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement. It Mobile phase: To 0.4 g of sodium lauryl sulfate add 500 mL of diluted phosphoric acid (1 in 1000) to dissolve, and adjust the pH to 3.0 with sodium hydroxide TS. To 240 mL of this solution add 70 mL of tetrahydrofuran, and mix. Flow rate: Adjust the flow rate so that the retention time of atropine is about 16 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the internal standard and atropine are eluted in this order with the resolutions between these peaks being not less than 3. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviations of the ratio of the peak area of atropine to that of the internal standard is not more than 1.5%. Packaging and Storage hermetic containers. Preserve in light-resistant,

Determination of Volume of Injection in Containers It meets the requirement. Assay To an exactly measured volume of Atropine Sulfate Injection, equivalent to about 5 mg of atropine sulfate hydrate [(C17H23NO3)2H2SO4H2O], add exactly 3 mL of the internal standard solution and water to make 50 mL and use this solution as the test solution. Separately, weigh accurately about 25 mg of Atropine Sulfate Hydrate RS (determine previously its loss on drying in the same manner as directed under Atropine Sulfate Hydrate), and add water to make exactly 50 mL. Pipet exactly 10 mL of this solution, add 3 mL of internal standard solution and water to make 50 mL, and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine the ratios, QT and

Atropine Sulfate Tablets

Atropine Sulfate Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of atropine sulfate hydrate [(C17H23NO3)2H2SO4 H2O: 694.83]. Method of Preparation Prepare as directed under Tablets, with Atropine Sulfate hydrate. Identification (1) Weigh a portion of finely powdered Atropine Sulfate Tablets, equivalent to about 1 mg of Atropine Sulfate Hydrate according to the labeled amount, add 1 drop of strong ammonium hydroxide and 2 mL of chloroform, mix, separate the chloroform layer and evaporate the chloroform on a water-bath. Proceed with the residue as directed in the Identification (1) under Atropine Sulfate hydrate. (2) Weigh a portion of finely powdered Atropine Sulfate Tablets, equivalent to about 5 mg of Atropine Sulfate Hydrate according to the labeled amount, shake with 5 mL of methanol, filter through glass filter (G4) and use this filtrate as the test solution. Separately, weigh accurately about 50 mg of Atropine Sulfate Hydrate RS, add ethanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 50 L of the test solution and 10 L of the standard solution on a plate of silica gel for thin-layer chromatography. Develop the chromatogram with a mixture of chloroform and diethyl amine (9 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly chloroplatinic acid-potassium iodide TS on the plate: the spots obtained from the test solution and the standard solution show a purple color and the same

QS , of the peak area of atropine to that of the internal standard for the test solution and the standard solution.
Amount (mg) of atropine sulfate hydrate [(C17H23NO3)2 H2SO4H2O] = WS
QT 1 1 .0266 QS 5

WS : Amount of Atropine Sulfate RS, calculated based

on the dried basis (mg) Internal standard solutionA solution of etilefrine hydrochloride (1 in 1000). Operating conditions Detector: A ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C.

KP 9 83 Carrier gas: Nitrogen. Flow rate: 25 mL/minute. System suitability System performance: When the procedure is run with 1 L of the standard solution under the above operating conditions with the resolution. being not less than 4.0 and with the symmetry factor being not more than 2.0. System repeatability: When the test is repeated 6 times with 1 L of the standard solution under the above operating conditions,the relative standard deviation of the ratio of the peak area is not more than 2.0%. pH 9.0 Phosphate buffer solutionDissolve 34.8 g of dibasic potassium phosphate in 900 mL of water and adjust to pH 9.0 with 3 mol/L hydrochloric acid or 1 mol/L sodium hydroxide solution. Packaging and Storage tight containers. Preserve in light-resistant,

Rf value.
(3) Atropine Sulfate Tablets respond to the Qualitative Tests for sulfate. Disintegration Test It meets the requirement, provided that the time limit of the test is 15 minutes. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Atropine Sulfate Tablets. Weigh accurately a portion of the powder, equivalent to about 1 mg of atropine sulfate hydrate [(C17H23NO3)2H2SO4H2O], transfer to a separator in 5 mL of a pH 9.0 buffer solution and add 2.0 mL of the internal standard solution. Adjust with 1 mol/L sodium hydroxide to a pH of 9.0 and extract with two 10 mL volumes of dichloromethane. Filter the dichloromethane layer through 1 g of anhydrous sodium sulfate on a pledget of absorbent cotton, evaporate the filtrate with the aid of nitrogen current to dryness, dissolve the residue in 2.0 mL of dichloromethane and use this solution as the test solution. Separately, weigh accurately about 10 mg of Atropine Sulfate Hydrate RS, previously dried at 120 C for 4 hours and dissolve in water to make exactly 100 mL. Prepare this solution before use. Transfer exact 10 mL of this solution to a separator, add 2.0 mL of the internal standard solution and 5.0 mL of a pH 9.0 buffer solution and adjust to a pH of 9.0 with 1 mol/L sodium hydroxide. Extract with two 10 mL volumes of dichloromethane, filter the extracts through 1 g of anhydrous sodium sulfate on a pledget of absorbent cotton, evaporate the filtrate with the aid of nitrogen current to dryness, dissolve the residue in 2.0 mL of dichloromethane and use this solution as the standard solution. Perform the test with 1 L each of the test and the standard solution as directed under the Gas Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of atropine sulfate to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of atropine sulfate hydrate [(C17H23NO3)2 H2SO4H2O] = Amount (mg) of Atropine Sulfate Hydrate RS
1 QT 1.0266 10 QS

Azathioprine
N NO2 N H3C N S H N

C9H7N7O2S: 277.26 Azathioprine, when dried, contains not less than 98.5% and not more than 101.0% of azathioprine (C9H7N7O2S). Description Azathioprine is a pale yellow crystal or crystalline powder and is odorless. Azathioprine is sparingly soluble in dimethylformamide or in pyridine, very slightly soluble in water or in dehydrated ethanol, and practically insoluble in chloroform or in ether. Azathioprine dissolves in sodium hydroxide TS or in ammonia TS. Azathioprine is gradually colored by light. Melting pointAbout 240 C (with decomposition). Identification (1) Dissolve 10 mg of Azathioprine in 50 mL of water by warming. To 5 mL of this solution, add 1 mL of dilute hydrochloric acid and 10 mg of zinc powder and allow to stand for 5 minutes: a yellow color is produced. Filter this solution: the filtrate responds to the Qualitative Tests for primary aromatic amines and a red color is produced. (2) Dissolve 10 mg of Azathioprine in 50 mL of water by warming. To 1 mL of this solution, add 0.5 mL of phosphotungstic acid TS and 0.5 mL of dilute hydroch-

Internal standard solutionAn aqueous solution of Homatropine Hydrobromide (5 in 100000). Prepare this solution before use. Operating conditions Detector: A hydrogen flame-ionization detector. Column: A glass column, about 2 mm in inside diameter and about 1.8 m in length, having 50% phenyland 50% methyl-polysiloxane coated at the ratio of 3% on siliceous earth. Column temperature: About 225 C.

84 Monographs, Part I loric acid: a white precipitate is formed. (3) Prepare the test solution by proceeding with 30 mg of Azathioprine according to the Oxygen Flask Combustion Method, using 20 mL of water as the absorbing liquid: the test solution responds to the Qualitative Tests (1) for sulfate. (4) Dissolve 10 mg of Azathioprine in 100 mL of 2 mol/L hydrochloric acid TS. To 5 mL of this solution, add water to make 50 mL and determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotomtry: it exhibits a maximum between 278 nm and 282 nm. Purity (1) Clarity and color of solutionDissolve 0.5 g of Azathioprine in 50 mL of dimethylformamide: the solution is clear and shows a pale yellow color. (2) Acid or alkaliAdd 100 mL of water to 2.0 g of Azathioprine, shake well for 15 minutes, centrifuge for 5 minutes at 10000 revolutions per minute and filter. Discard the first 20 mL of the filtrate, add 2 drops of methyl red TS to 40 mL of the subsequent filtrate and use this solution as the test solution. (i) Add 0.10 mL of 0.02 mol/L hydrochloric acid VS to 20 mL of the test solution: a red color develops. (ii) Add 0.10 mL of 0.02 mol/L sodium hydroxide VS to 20 mL of the test solution: a yellow color develops. (3) SulfateTo 25 mL of the filtrate obtained in (2), add 1 mL of dilute hydrochloric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.038%). (4) Heavy metalsProceed with 2.0 g of Azathioprine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) ArsenicPrepare the test solution with 1.0 g of Azathioprine, according to Method 3 and perform the test (not more than 2 ppm). (6) Related substancesDissolve 10 mg of Azathioprine in 80 mL of the mobile phase by warming, cool, add the mobile phase to make 100 mL and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add water to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of both solutions by the automatic integration method: the total area of all peaks other than that of Azathioprine from the test solution is not larger than 1/2 of the peak area of Azathioprine from the standard solution. Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 296 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Adjust to a pH of 2.5 by adding diluted phosphoric acid (3 in 2000) to diluted 0.05 mol/L monobasic potassium phosphate TS (1 in 2). To 800 mL of this solution, add 200 mL of methanol. Flow rate: Adjust the flow rate so that the retention time of Azathioprine is about 8 minutes. System suitability System performance: Dissolve 10 mg of Azathioprine in 80 mL of water by warming, cool and add water to make 100 mL. To 2 mL of this solution add 2 mL of a solution, separately prepared by dissolving 60 mg of benzoic acid in 3 mL of methanol and diluting with water to make 10 mL, and add the mobile phase to make 25 mL. When the procedure is run with 20 L of this solution under the above operating conditions, azathioprine and benzoic acid are eluted in this order with the resolution between their peaks being not less than 9. System repeatability: When the test is repeated 6 times with 20 L of this solution under the above operating conditions, the relative standard of the peak areas of azathioprine is not more than 2.0%. Detection sensitivity: To exactly 5 mL of the standard solution add water to make exactly 50 mL. Adjust the detection sensitivity so that the peak area of azathioprine obtained from 20 L of this solution is between 8 and 12% of that of azathioprine obtained from 20L of the standard solution. Time span of measurement: About three times as long as the retention time of Azathioprine after the solvent peak. Loss on Drying Not more than 0.5% (1 g, 105 C, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Azathioprine, previously dried, add 80 mL of dimethylformamide and warm to dissolve. After cooling, titrate with 0.1 mol/L tetramethylammonium hydroxide VS until the color of the solution changes from yellow through yellow-green to blue-green (indicator: 1 mL of thymol bluedimethylformamide TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 27.726 mg of C9H7N7O2S Packaging and Storage well-closed containers. Preserve in light-resistant,

KP 9 85 dissolve in 20 mL of dimethylsulfoxide for spectrophotometry and add 0.1 mol/L hydrochloric acid TS to make exactly 500 mL. Measure exactly 3 mL of this solution, add 0.1 mol/L hydrochloric acid TS to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution at 280 nm, respectively as directed under the Ultraviolet-visible Spectrophotometry. Amount (mg) of azathioprine (C9H7N7O2S) = amount (mg) of Azathioprine RS Packaging and Storage tight containers.
AT AS

Azathioprine Tablets
Azathioprine Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of azathioprine (C9H7N7O2S: 277.26). Method of Preparation Prepare as directed under Tablets, with Azathioprine. Identification (1) Weigh a quantity of powdered Azathioprine Tablets, equivalent to 10 mg of Azathioprine according to the labeled amount. Add 50 mL of water, shake well while warming and filter. Proceed with 5 mL of the filtrate as directed in the Identification (1) under Azathioprine. (2) Proceed with 1 mL of the filtrate obtained in (1) as directed in the Identification (2) under Azathioprine. (3) Determine the absorption spectrum of the test solution in the Assay as directed under the Ultravioletvisible Spectrophotometry: it exhibits a maximum between 278 nm and 282 nm. (4) Weigh a portion of powdered Azathioprine Tablets, equivalent to 0.1 g of Azathioprine to the labeled amount. Add 10 mL of a solution of strong ammonia water in methanol (1 in 10), shake well, filter and use the filtrate as the test solution. Separately, dissolve 0.1 g of Azathioprine RS in 10 mL of a solution of strong ammonia water in methanol (1 in 10) and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, a solution of strong ammonia water in methanol (1 in 10), n-butyl formate and 1,2-dichloroethane (15 : 10 : 5 : 2) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots from the test solution and the standard solution show the same Rf value. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Azathioprine Tablets. Weigh accurately a portion of the powder, equivalent to about 0.1 g of azathioprine (C9H7N7O2S), add 20 mL of dimethylsulfoxide for Spectrophotometry, shake well, add 0.1 mol/L hydrochloric acid TS to make exactly 500 mL and filter. Discard the first 20 mL of the filtrate, measure exactly 3 mL of the subsequent filtrate, add 0.1 mol/L hydrochloric acid TS to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 0.1 g of Azathioprine RS, previously dried at 105 C for 5 hours,

Preserve in light-resistant,

Azelastine Hydrochloride
O H N N NCH3

HCl

Cl

and enantioner C22H24ClN3OHCl : 418.36 Azelastine Hydrochloride contains not less than 98.5% and not more than 101.0% of azelastine hydrochloride (C22H24ClN3OHCl), calculated on the dried basis. Description Azelastine Hydrochloride is a white crystalline powder. Azelastine Hydrochloride is soluble in anhydrous ethanol, slightly soluble in water, and practically insoluble in ether, in n-hexane, or in toluene. Identification (1) Determine the infrared spectra of Azelastine Hydrochloride and Azelastine Hydrochloride RS, previously dried, as directed under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) A solution of Azelastine Hydrochloride in water (1 in 50) responds to the Qualitative Tests for chlorides. Specific Optical Rotation [ ]20 D : Between -0.05 and +0.50 (previously dried, 1.0 g, dichloromethane, 20 mL, 100 mm)

86 Monographs, Part I and dry the plate for 1 hour at 115 C. After cooling examine under ultraviolet light (main wavelength: 254 nm) and spray potassium bithmus iodide TS and then 5 w/v% solution of sodium nitrous : Two spots of related substances obtained with the test solution are not darker than each spot from the standard solutions (2) and (3) (0.1%); those spots and spots other than principal spot are not darker than spots from the standard solution (1) (0.1%). Disregard the spot remaining on the origin of the plate. Loss on Drying Not more than 1.0% (1g, 105 C, constant mass). Assay Weigh accurately about 0.3 g of Azelastine Hydrochloride, dissolve in 5 ml of anhydrous formic acid, add 30 ml of acetic anhydride and titrate quickly with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 41.84 mg of C22H24ClN3OHCl Packaging and Storage tight containers. Preserve in light-resistant,

Purity (1) Acidity or alkalinityTo 10 ml of solution of Azelastine Hydrochloride in water (1 in 100) add 0.2 ml of bromthymol blue solution. Not more than 0.1 mL of 0.1 mol/L hydrochloric acid VS or 0.1 mol/L sodium hydroxide VS is required to change the color of the solution. (2) 1-Methyl-4-(2-benzoylhydrazino)azepane Weigh accurately 0.1 g of Azelastine Hydrochloride and dissolve in 12 mL of methanol and add water to make exactly 20 mL and use this solution as the test solution. Separately, weigh accurately 5.0 mg of 1-Methyl-4-(2benzoylhydrazino)azepane Hydrochloride RS and dissolve in 60% methanol to exactly 100 mL. Pipet 5.0 mL of this solution and dilute in 60% methanol to make exactly 50 mL and use this solution as the standard solution. Perform the test with 10 L of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of the peak of 1-methyl-4-(2-benzoylhydrazino) azepane from the test solution is not larger than that of principal peak from the standard solution. (not more than 0.1%) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (10 m in particle diameter). Mobile phase: A 0.02 mol/L solution of sodium octanesulfonate in the mixture of methanol, water and anhydrous acetic acid (60 : 40 : 1). Flow rate: 1.5 mL/minute. (3) Related substancesWeigh accurately 0.5 g of Azelastine Hydrochloride and dissolve in the mixture of methanol and dichloromethane (1 : 1) to make exactly 10 mL and use this solution as the test solution. Pipet exactly 0.1 mL of the test solution and add the mixture of methanol and dichloromethane (1 : 1) to make exactly 100 mL and use this solution as standard solution (1). Separately, weigh accurately 5 mg of 4-chlorobenzylprazi none, dissolve in the mixture of methanol and dichloromethane (1 : 1) to make exactly 100 mL and use this solution as the standard solution (2). Also weigh accurately 5 mg of 1-Methylazepan-4-one Hydrochloride RS, dissolve in the mixture of methanol and dichloromethane (1 : 1) to make exactly 100 mL and use this solution as the standard solution (3). Perform the test with these solutions as directed under Thin Layer Chromatography according to the following conditions. Spot 10 L each of the test solution, the standard solutions (1), (2), and (3) on a plate coated with silica gel with fluorescent indicator for thin layer chromatography. Develop the plate with the mixture of toluene, ethanol and 10 mol/L ammonia water (60 : 40 : 1) to a distance of about 10 cm

Azithromycin Hydrate
H3C H3C HO H3C OH N CH3 OH CH3 HO O O H3C N CH3

H3C O CH3

CH3

O CH3

CH3 O CH3 O CH3 OH

2H2O

C38H72N2O122H2O: 785.02 Azithromycin Hydrate contains not less than 950 g (potency) and not more than 1030 g (potency) per mg of azithromycin (C38H72N2O12: 748.98), calculated on the anhydrous basis. Description Azithromycin Hydrate is a white to grayish white crystalline powder. Azithromycin Hydrate is freely soluble in acetonitrile, in dimethylformamide, in methanol, in ethanol, in acetone, in isopropanol, in ethylacetate, or in chloroform. Identification (1) Determine the infrared spectra of Azithromycin Hydrate and Azithromycin Hydrate RS, as

KP 9 87 directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. (2) Determine the retention times according to the procedure of Assay: the retention time of the principal peak in the chromatogram obtained with test solution is the same as that of the principal peak in the chromatogram obtained with the standard solution. Specific Optical Rotation [ ] 20 D : -45 ~ -49(0.2 g calculated on the anhydrous basis, dehydrated ethanol, 10 mL, 100 mm). pH The pH of a solution obtained by dissolving 0.2 g of Azithromycin Hydrate in 10 mL of 50 % methanol solution is between 9.0 and 11.0. Purity (1) Heavy metalsProceed with 2.0 g of Azithromycin Hydrate to Method 2 and perform the test. Prepare the control solution with 5.0 mL of standard lead solution (not more than 25 ppm). (2) Total related substancesPerform the test according to the procedure of Assay. Prepare the test solution and as follows. Weigh accurately about 33 mg of Azithromycin Hydrate, dissolve in 5 mL of acetonitrile, add the mixture of 0.02 mol/L potassium dihydrogen phosphate solution and acetonitrile (71:29), pH 8.0 adjusted with 10 mol/L potassium hydroxide to make 100 mL, and use this solution as the test solution. Perform the test with 50 L of the test solution as directed under the Liquid Chromatography according to the following operating conditions, and obtain the peak areas of azithromycin and each related substance (not more than 5.0 %). Amount [%] of total related substances
total peak areas other than the principal peak = 100 total peak areas

for thin-layer chromatography, develop the plate with the mixture of ethylacetate, hexane and diethylamine (15:5:2) as the developing solvent mixture, and heat the plate at 100 C for 10 minutes. Spray evenly the color developing solution on the plate and heat the plate at 100 C. The color developing solution mixture is prepared by dissolving 3 g of vaniline in 100 mL of ethanol and adding 1.5 mL of sulfuric acid. The spots other than the principal spot obtained from the test solution are not more intense than the principal spot from the standard solution. Water Not less than 4.0 % and not more than 5.0 % (0.2 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.3 % (1 g). Assay Weigh accurately about 33 mg (potency) each of Azithromycin Hydrate and Azithromycin Hydrate RS, dissolve each in acetonitrile to make exactly 200 mL, pipet exactly 2 mL each of these solutions, add the internal standard solution to make exactly 100 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 50 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS, of the peak area of azithromycin to that of the internal standard. Amount [g (potency)] of azithromycin (C38H72N2O12) = Amount [g (potency)] of Azithromycin Hydrate RS

QT QS

Operating conditions Mobile phase: Adjust the pH of the mixture of 0.02 mol/L of potassium dihydrogen phosphate solution and acetonitrile (75:25) to 11.0 with 10 mol/L potassium hydroxide TS. Flow rate: 1.2 mL per minute. Time span of measurement: About 80 minutes. (3) Erythromycin A iminoetherWeigh accurately an appropriate amount of Azithromycin Hydrate, dissolve in the mixture of methanol and chloroform (1:1) to make the solution so that each mL contains 25 mg, and use this solution as the test solution. Separately, weigh accurately 2.5 mg (potency) of Erythromycin A iminoether RS, dissolve in the mixture of methanol and chloroform (1:1), to make the solution so that each mL contains 0.125 mg, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 50 L of each solution on a plate of silica gel with fluorescent indicator

Internal standard solutionWeigh accurately 1.5 mg of diphenhydramine hydrochloride, dissolve in the mixture of 0.02 mol/L potassium dihydrogen phosphate solution and acetonitrile (71:29), pH 8.0 adjusted with 10 mol/L potassium hydroxide to make 1000mL Operating conditions Detector: An electrochemical detector Column: A stainless steel column, about 4.6 mm in inside diameter and about 150 mm in length, packed with alumina based hydrocarbon for liquid chromatography Mobile phase: Adjust pH of the mixture of 0.02 mol/L potassium dihydrogen phosphate solution and acetonitrile (71:29) to pH 11.0 with 10 mol/L potassium hydroxide. Flow rate: 1.5 mL per minute Packaging and Storage Preserve in tight containers.

88 Monographs, Part I rately, weigh accurately an amount of Ampicillin RS, equivalent to 20 mg (potency), and dissolve in water to make exactly 100 mL. Pipet 5 mL of this solution, add 10 mL of 0.05 mol/L phosphate buffer solution, pH 7.0 and water to make exactly 25 mL, and use this solution as the standard solution. To exactly 10 mL each of the test solution and the standard solution add exactly 2 mL of sodium hydroxide TS, allow to stand for exactly 15 minutes, add exactly 2 mL of 1 mol/L hydrochloric acid TS, exactly 10 mL of 0.3 mol/L potassium hydrogen phthalate buffer solution, pH 4.6, and exactly 10 mL of 0.005 mol/L iodine VS, allow to stand for exactly 20 minutes without exposure to light. Titrate each of these solutions with 0.01 mol/L sodium thiosulfate VS until the color of the solution turns to colorless. Separately, to exactly 10 mL each of the test solution and the standard solution add exactly 10 mL of 0.3 mol/L potassium hydrogen phthalate buffer solution, pH 4.6 and exactly 10 mL of 0.005 mol/L iodine VS, and perform a blank determination with the same manner. Determine the consumed amounts (mL) of 0.005 mol/L iodine VS, VT and VS, of the test solution and the standard solution: the amount of ampicillin is not more than 1.0 %. Amount (%) of free ampicillin (C16H19N3O4S) = VT
VS

Bacampicillin Hydrochloride
O O CH3 H N CH3 CH3 S H H HCl O O O CH3 O O

N H H2N H

C21H27N3O7SHCl: 501.98 Bacampicillin Hydrochloride contains not less than ampicillin (C16H19N3O4S: 349.41) 626 g (potency) per mg of Bacampicillin Hydrochloride, calculated on the anhydrous basis. Description Bacampicillin Hydrochloride is white to pale yellow crystalline powder, and has a characteristic odor and bitter taste. Bacampicillin Hydrochloride is freely soluble in methanol or in ehtanol, soluble in water, and practically insoluble in ether. Identification (1) Determine the infrared spectra of Bacampicillin Hydrochloride and Bacampicillin Hydrochloride RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Weigh an appropriate amount each of Bacampicillin Hydrochloride and Bacampicillin Hydrochloride RS, dissolve in ethanol and make solutions so that each mL contains 2 mg, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the mixture of methylene chloride, chloroform and ethanol (10:1:1) to a distance of about 10 cm, and air-dry the plate. Spray evenly 0.3 % ninhydrin-ethanol on the plate: the spots obtained from the test solution and the standard solution is same in the Rf value. pH The pH of a solution obtained by dissolving 0.2 g of Bacampicillin Hydrochloride in 10 mL of water is between 3.0 and 5.0. Purity Free ampicillin Weigh accurately about 0.1 g of Bacampicillin Hydrochloride, transfer into a 100 mL separator, add exactly 15 mL of ice-cold 0.05 mol/L phosphate buffer solution, pH 7.0, then add 25 mL of icecold chlorform, shake, and abandon the chloroform layer. Repeat the procedure twice with two 25 mL portions of ice-cold chloroform. Centrifuge the water layer, filter the supernatant, and use the filtrate as the test solution. Sepa-

Amount(mg) of AmpicillinRS taken 100 Amount(mg) of 20 Bacampicil lin Hydrochlor ide taken
Water Not more than 1.0 % (1.0 g, volumetric titration, direct titration). Assay Weigh accurately about 40 mg (potency) each of Bacampicillin Hydrochloride and Bacampicillin Hydrochloride RS, dissolve each in water to make exactly 100 mL, and use these solutions as the test solution and the standard solution. Perform the test with exactly 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT and AS , of bacampicillin of these solutions. Amount [g (potency)] of ampicillin (C16H19N3O4S) = Amount [g (potency) of ampicillin] of Bacampicillin Hydrochloride RS
AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 150 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of

KP 9 89 luble in acetone, in chloroform or in ether. Identification To 5 mg of Bacitracin add 5 mL of pdimethylaminobenzaldehyde TS, shake and mix for one or two minutes, then add one drop of a solution of sodium nitrite (1 in 100): a green to dark green color deveolps. pH The pH of a solution obtained by dissolving 1 g of Bacitracin in 10 mL of water is between 5.5 and 7.5. Purity Heavy metalsProceed with 1.0 g of Bacitracin according to Method 1 and perform the test. Prepare the control solution with 3.0 mL of standard lead solution (not more than 30 ppm). Sterility Test It meets the requirement, when Bacitracin is used in a sterile preparation. Pyrogen Test It meets the requirement, when Bacitracin is used in a sterile preparation. Weigh appropriate amount of Bacitracin, dissolve in water, make the solution so that each mL contains 300 Units (potency), and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Loss on Drying Not more than 5.0 % (0.1 g, in vacuum, 60 o C , 3 hours).
L-Leu D-Phe L-His D-Glu D-Orn L-Asn Y

about 25 o C Mobile phase: To 500 mL of diluted 2 mol/L sodium dihydrogen phosphate TS (1 in 100), add diluted 0.05 mol/L disodium hydrogen phosphate TS (2 in 5) to adjust the pH to 6.8. To 500 mL of this solution add 500 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of bacampicillin is about 6.5 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of bacampicillin are not less than 10000 and not more than 2, respectively. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the operating conditions, the relative standard deviation of the peak areas of bacampicillin is not more than 2.0 %. Packaging and Storage Preserve in tight containers.

Bacitracin
H H2N H N S O CH3 R

Residue on Ignition Not more than 3.0 % (1 g).

X

L-Lys

D-Asp

Bacitracin A Bacitracin B1 Bacitracin B2 Bacitracin B3

X L-Lle L-Lle L-Val L-Lle

Y L-Lle L-Lle L-Lle L-Val

R CH3 H CH3 CH3

Asp: aspartic acid Asn: asparagines Glu: glutamic acid His: histidine Ile: isoleucine

Leu: leucine Lys: lysine Orn: ornithine Phe: phenyalanine

Bacitracin is a mixture of peptide substances having antibacterial activity including bacitracin A as the main component produced by the growth of Bacillus subtilis or Bacillus licheniformis. Bacitracin contains not less than 40 Units (potency) per mg of bacitracin A (C66H103N17O16S: 1422.69), calculated on the dried basis. Description Bacitracin is white to light brown powder. Bacitracin is freely soluble in water, soluble in ethanol, in methanol or in glacial acetic acid, and practically inso-

Assay The Cylinder-plate method (1) Culture medium Agar media for seed and base layer- Use the me1 under Microbial Assay for Antibiodium in I 2 1) (6) tics. (2) Test organism- Micrococcus luteus ATCC 10240. (3) Weigh accurately about 5 mg (potency) of Bacitracin, and dissolve and dilute in 1 % phosphate buffer solution, pH 6.0 to make the solution so that each mL contains 2.0 and 0.5 units (potency) and use these solutions as the high concentration test solution and low concentration test solution. Separately, weigh accurately about 5 mg (potency) of Bacitracin RS, and dissolve in 1 % phosphate buffer solution, pH 6.0 to make the solution so that each mL contains 20 units (potency), and use this solution as the standard stock solution. Keep the stock standard solution at not exceeding 10 C and use within 2 days. Take exactly a suitable amount of the stock standard solution before use, add 1 % phosphate buffer solution, pH 6.0 to make solutions so that each mL contains 2.0 and 0.5 units (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in tight containers.

90 Monographs, Part I dine in butanol and add butanol to make 100 mL. (2) The diluted hydrochloric acid solution of Bacitracin Zinc (1 in 10) responds to the Qualitative Test for zinc salt. pH The pH of a saturated solution of Bacitracin Zinc is between 6.0 and 7.5.
L-Leu D-Phe L-His D-Glu D-Orn L-Asn Y L-Lys

Bacitracin Zinc
H H2N H N S O CH3 R

Loss on Drying Not more than 5.0 % (0.1 g, in vacuum, 60 C, 3 hours). Assay 1) bacitracin The Standard curve method (1) Culture medium Agar media for seed and base 1 under Microbial layer- Use the medium in I 2 1) (6) Assay for Antibiotics. . (2) Test organism- Use Micrococcus luteus ATCC 10240 as test organism. (3) Weigh accurately an appropriate amount of Bacitracin Zinc, and dissolve in 0.01 mol/L hydrochloric acid TS to make the solution so that each mL contains 100 units (potency), pipet exactly an appropriate amount of this solution and dilute in 1 % phosphate buffer solution, pH 6.0 to make the solution so that each mL conains 1.00 units (potency), and use this solution as the test solution. Separately, weigh accurately an appropriate amount of Bacitracin RS, dissolve in 0.01 mol/L hydrochloric acid TS to make the solution so that each mL contains 100 units (potency), and use this solution as the standard stock solution. Pipet exactly an appropriate amount of this standard stock solution and dilute in 1 % phosphate buffer solution, pH 6.0 to make the solutions so that each mL contains 0.64, 0.80, 1.00, 1.25, and 1.56 units (potency) and use these solutions as the standard solutions. Also use 1.0 unit (potency) per mL as the mid-diluted solution. Perform the test with these solutions according to the Standard curve method (II 4) as directed under Microbial Assay for Antibiotics. 2) Zinc Weigh accurately about 0.2 g (potency) of Bacitracin Zinc, dissolve in 0.01 mol/L hydrochloric acid TS, and make the solution 100 mL. Pipet exactly 2 mL of this solution and add 0.001 mol/L hydrochloric acid TS to make 200 mL and use this solution as the test solution. Separately, weigh accurately about 3.11 g of zinc oxide, add 80 mL of 1 mol/L hydrochloric acid TS, heat to dissolve, allow to cool down to room temperature, and add water to make exactly 250 mL. Pipet exactly an appropriate amount of this solution, dilute 0.001 mol/L hydrochloric acid TS to make the solutions contain 0.5, 1.5 and 2.5 g of zinc per mL, and use these solutions as the standard solutions. Perform the test with the test solution and the standard solutions as directed under the Atomic Absorption Spectrophotometry. The operating wavelength is 213.8 nm. Determine absorbances and calculate the amount of zinc according to the calibration curve method. The blank test solution is 0.001 mol/L hydrochloric acid TS. Amount [%] of zinc = C 100000
W (100 m )

D-Asp

Bacitracin A Bacitracin B1 Bacitracin B2 Bacitracin B3

X L-Lle L-Lle L-Val L-Lle

Y L-Lle L-Lle L-Lle L-Val

R CH3 H CH3 CH3

Asp: aspartic acid Asn: asparagine Glu: glutamic acid His: histidine Ile: isoleucine

Leu: leucine Lys: lysine Orn: ornithine Phe: phenyalanine

Bacitracin Zinc contains not less than 40 Units (potency) per mg of bacitracin A (C66H103N17O16S: 1422.69), calculated on the dried basis. Bacitracin Zinc also contains not less than 2.0 % and not more than 10 % of zinc (Zn: 65.41), calculated on the dried basis Description Bacitracin Zinc is white to light yellow powder, is odorless or a tint of odor, and has a bitter taste. Bacitracin Zinc is slightly soluble in water or in ethanol, very slightly soluble in ether, and practically insoluble in chloroform. Bacitracin Zinc is hygroscopic. Identification (1) Weigh appropriate amount of Bacitracin Zinc, dissolve in 1 % ethylenediamine tetraaceticacid dihydrogendisodium, make the solution so that each mL contains 6 mg, and use this solution as the test solution. Separately, weigh appropriate amount of Bacitracin RS, dissolve in 1 % ethylenediamine tetraaceticacid dihydrogendisodium, make the solution so that each mL contains 6 mg, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of n-butanol, glacial acetic acid, water, pyridine, and ethanol (60:15:10:6:5), and air-dry the plate. Then spray evenly ninhydrin-acetic acid TS on the plate: the spots obtained from the test solution and the standard solution exhibit the same Rf value. The nihydrin acetic acid TS is pepared by dissolving 1 g of ninhydrin and 1 mL of pyri-

KP 9 91 (4) Related substancesDissolve 50 mg of Baclofen in 50 mL of the mobile phase and use this solution as the test solution. Pipet 1.0 mL and 1.5 mL of the test solution, to each add the mobile phase to make exactly 100 mL and use these solutions as the standard solutions (1) and (2), respectively. Perform the test with 25 L each of the test solution and the standard solutions (1) and (2) as directed under the Liquid Chromatography according to the following conditions. Determine each peak height of these solutions: each height of the peaks other than the peak of baclofen from the test solution is not larger than the peak height of baclofen from the standard solution (1) and the total height of these peaks is not larger than the peak height of baclofen from the standard solution (2). Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 268 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of methanol and diluted glacial acetic acid (1 in 900) (3 : 2). Flow rate: Adjust the flow rate so that the retention time of baclofen is about 4 minutes. Time span of measurement: About 3 times as long as the retention time of baclofen after the solvent peak. System suitability Test for required detection: Adjust the sensitivity so that the peak height of baclofen obtained from 25 L of the standard solution (1) is between 5 mm and 10 mm. System performance: Dissolve 0.40 g of Baclofen and 5 mg of methyl parahydroxybenzoate in 200 mL of the mobile phase. To 10 mL of this solution, add the mobile phase to make 100 mL. When the procedure is run with 25 L of this solution under the above operating conditions, baclofen and methyl parahydroxybenzoate are eluted in this order with the resolution between their peaks being not less than 5. System repeatability: When the test is repeated 6 times with 25 L of the standard solution (1) under the above operating conditions, the relative standard deviation of the peak heights of baclofen is not more than 3.0%. Water Not more than 1.0% (1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.3% (1 g). Assay Weigh accurately about 0.5 g of Baclofen, dissolve in 80 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue to greenish blue (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

C: Amount [g /mL] of zinc in the test solution W: Amount [mg] of Bacitracin Zinc taken m: loss on drying (%) Packaging and Storage Preserve in tight containers.

Baclofen
H HOH2CHC NH2

Cl

and enantiomer C10H12ClNO2: 213.66

Baclofen contains not less than 98.5% and not more than 101.0% of baclofen (C10H12ClNO2), calculated on the anhydrous basis. Description Baclofen is a white to pale yellowish white, crystalline powder. Baclofen is freely soluble in glacial acetic acid, slightly soluble in water, very slightly soluble in methanol or in ethanol and practically insoluble in ether. Baclofen dissolves in dilute hydrochloric acid. Identification (1) To 5 mL of a solution of Baclofen (1 in 1000), add 1 mL of ninhydrin TS and heat in a waterbath for 3 minutes: a blue-purple color develops. (2) Determine the absorption spectra of solutions of Baclofen and Baclofen RS in 0.1 mol/L hydrochloric acid TS (1 in 2000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Perform the test with Baclofen as directed under the Flame Coloration Test (2): a green color appears. Purity (1) ChlorideDissolve 0.5 g of Baclofen in 50 mL of glacial acetic acid and add water to make 100 mL. To 100 mL of this solution, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.30 mL of 0.01 mol/L hydrochloric acid VS, add 5 mL of glacial acetic acid, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.21%). (2) Heavy metalsProceed with 2.0 g of Baclofen according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Baclofen according to Method 3 and perform the test (not more than 2 ppm).

92 Monographs, Part I mL so that each mL contains about 10 g of baclofen (Cl0Hl2ClNO2) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately about 10 mg of Baclofen RS (separately determined the water content), dissolve in water to make exactly 100 mL, then pipet 10 mL of this solution, add water to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and

Each mL of 0.1 mol/L perchloric acid VS = 21.366 mg of Cl0Hl2ClNO2 Packaging and Storage Preserve in well-closed containers.

Baclofen Tablets
Baclofen Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of baclofen (Cl0Hl2ClNO2: 213.66). Method of Preparation Prepare as directed under Tablets, with Baclofen. Identification (1) To a portion of powdered Baclofen Tablets, equivalent to 10 mg of Baclofen according to the labeled amount, add 10 mL of water, shake well and filter. To 5 mL of the filtrate, add 1 mL of ninhydrin TS and proceed as directed in the Identification (1) under Baclofen. (2) To a portion of powdered Baclofen Tablets, equivalent to 25 mg of Baclofen according to the labeled amount, add 50 mL of 0.1 mol/L hydrochloric acid TS, shake for 15 minutes and filter. Determine the absorption spectrum of the filtrate as directed under the Ultravioletvisible Spectrophotometry: it exhibits maxima between 257 nm and 261 nm, between 264 nm and 268 nm and between 272 nm and 276 nm. (3) To a portion of powdered Baclofen Tablets, equivalent to 10 mg of Baclofen according to the labeled amount, add 2 mL of a mixture of methanol and glacial acetic acid (4 : 1), shake well, centrifuge and use the supernatant liquid as the test solution. Separately, dissolve 10 mg of Baclofen RS in 2 mL of a mixture of methanol and glacial acetic acid (4 : 1) and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (4 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spot from the test solution and that from the standard solution show the same Rf value. Dissolution Test Perform the test with 1 tablet of Baclofen Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 500 mL of water as the dissolution solution. Take 20 mL or more of the dissolve solution 45 minutes after starting the test and filter. Discard the first 10 mL of the filtrate, pipet V mL of the subsequent, filtrate, add water to make exactly V'

AS , of the test solution and the standard solution at 220

nm, respectively, as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate (%) of Baclofen Tablets in 45 minutes is not less than 70%. Dissolution rate (%) with respect to the labeled amount of baclofen (Cl0Hl2ClNO2) = W S
AT V ' 1 50 AS V C

WS : Amount (mg) of Baclofen RS.

C: Labeled amount (mg) of baclofen (Cl0Hl2ClNO2) in 1 tablet. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Baclofen Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of baclofen (Cl0Hl2ClNO2), add 130 mL of 0.1 mol/L hydrochloric acid TS, shake for 10 minutes, add 0.1 mol/L hydrochloric acid TS to make exactly 200 mL and centrifuge. Pipet 10 mL of the supernatant liquid, add 2 drops of phenolphthalein TS, neutralize with dilute sodium hydroxide TS, add water to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 0.25 g of Baclofen RS (separately determined the water content) and dissolve in 0.1 mol/L hydrochloric acid TS to make exactly 100 mL. Pipet 10 mL of this solution and add 0.1 mol/L hydrochloric acid TS to make exactly 100 mL. Pipet 10 mL of this solution, add 2 drops of phenolphthalein TS, neutralize with dilute sodium hydroxide TS, add water to make exactly 50 mL and use this solution as the standard solution. Pipet 2 mL each of the test solution and the standard solution, to each add 4 mL of ninhydrin-stannous chloride TS, shake, heat in a water-bath for 20 minutes and shake at once vigorously for 2 minutes. After cooling, to each solution, add a mixture of water and n-propanol (1 : 1) to make exactly 25 mL. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 570 nm as directed under the Ultraviolet-visible Spectrophotometry, using a blank prepared with 2 mL of water in the same manner. Amount (mg) of baclofen (Cl0Hl2ClNO2) = amount (mg) of Baclofen RS,

KP 9 93 (2) Related substancesDissolve 5.0 mg of Bambuterol Hydrochloride in the mobile phase, dilute to 10.0 mL with the mobile phase, and use this solution as the test solution. Separately, dissolve 1.0 mg of Formoterol Packaging and Storage Preserve in well-closed conFumarate Dihydrate RS in the mobile phase, dilute to 10 tainers. mL with the mobile phase. Mix 0.8 mL of this solution with 0.4 mL of the test solution, dilute to 100 mL with the mobile phase, and use this solution as the standard Bambuterol Hydrochloride solution (1). Dilute 1.0 mL of the test solution with the mobile phase to 50 mL. Dilute 2.0 ml of the solution to CH3 20 mL with the mobile phase, and use this solution as the H OH H standard solution (2). Use the mobile phase as the blank N CH3 N O H 3C solution. Perform the test with 20 L each of the blank H 3C CH3 solution, the test solution, and the standard solutions as O directed under the Liquid Chromatography according to HCl CH3 the following conditions. The area of any peak, other N O than from the principal peak, from the test solution is not H 3C greater than the area of the principal peak from the stanO dard solution (2) (not more than 0.2%); the sum of the and enantiomer areas of all the peaks, other than from the principal peak, is not greater than 3 times the area of the principal peak C18H29N3O5,HCl : 403.90 from the standard solution (2) (not more than 0.6%). Disregard any peak obtained with the blank solution and any Bambuterol Hydrochloride contains not less than 98.5% peak with an area less than 0.25 times the area of the and not more than 101.5% of bambuterol hydrochloride principal peak obtained with the standard solution (2). (C18H29N3O5,HCl), calculated on the anhydrous basis. Operating conditions Description Bambuterol Hydrochloride is a white, Detector: An ultraviolet absorption photometer (wacrystalline powder. velength: 214 nm) Bambuterol Hydrochloride is freely soluble in water, and Column: A stainless steel column about 4.6 mm insoluble in ethanol. side diameter and 15 cm in length, packed with baseBambuterol Hydrochloride shows polymorphism. deactivated octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Identification (1) Determine the infrared absorption Mobile phase: Dissolve 1.3 g of sodium octanesulspectra of Bambuterol Hydrochloride and Bambuterol phonate in 430 mL of a mixture of methanol and acetoniHydrochloride RS as directed in the potassium bromide trile (75:25). Mix this solution with 570 mL of phosphate disk method under the Infrared Spectrophotometry: both buffer solution. spectra exhibit similar intensities of absorption at the Flow rate: 1.5 mL/min. same wavenumbers. If any difference appears between System suitability the spectra, dissolve Bambuterol Hydrochloride and System performance: Adjust the sensitivity of the Bambuterol Hydrochloride RS in a mixture of acetone system so that the height of the principal peak from the and water (6 : 1), cool in ice to precipitate, then dry both standard solution (2) is about 50% of the full scale of the precipitates in vacuum at 50 C to constant weight, and recorder. When the procedure is run with 20 L of the repeat the test with precipitates. standard solution (1), the retention times for formoterol (2) The solution of Bambuterol Hydrochloride in waand bambuterol are about 7 and 9 minutes, respectively, ter (1 in 50) responds to the Qualitative Tests for chlowith the resolution between the peaks of bambuterol and ride (2nd method). formoterol being not less than 5.0. Time span of measurement: 1.5 times the retention Specific Optical Rotation [ ]20 time of bambuterol. D : Between 0.10 and +0.01 (0.4 g calculated on the anhydrous basis, water, Phosphate buffer solutionDissolve 6.90 g of so20 mL, 100 mm). dium dihydrogen phosphate monohydrate in water to make 1000 mL, and adjust the pH to 3.0 with 5% phosPurity (1) Acidity or alkalinity Dissolve 4.0 g of phoric acid. Bambuterol Hydrochloride in water to make exactly 20 mL. To 10 mL of the solution, add 0.2 mL of methyl red Water Not more than 0.5% (0.5 g, volumetric titration, TS and 0.2 mL of 0.01 mol/L hydrochloric acid. The sodirect titration). lution is red. When 0.4 mL of 0.01 mol/L sodium hydroxide is added, the solution becomes yellow. Residue on Ignition Not more than 0.1% (1 g). calculated on the anhydrous basis
AT AS

94 Monographs, Part I Purity (1) Clarity and color of solution Dissolve 1.0 g of Bamethan Sulfate in 20 mL of water: the solution is clear and has no more color than the following control solution. Control solutionTo 1.5 mL of Color Matching Fluid O, add diluted hydrochloric acid (1 in 40) to make 200 mL. (2) ChloridePerform the test with 3.5 g of Bamethan Sulfate. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.002%). (3) Heavy metalsProceed with 2.0 g of Bamethan Sulfate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Bamethan Sulfate according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.10 g of Bamethan Sulfate in 2 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 2 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform and methanol (7 : 2) in a developing vessel saturated with ammonia vapor to a distance of about 12 cm and air-dry the plate. Spray evenly Dragendorffs TS for spraying on the plate, air-dry for 15 minutes, spray Dragendorffs TS for spraying again, then, after 1 minute, spray evenly a solution of sodium nitrite (1 in 20) and immediately put a glass plate on the plate. Examine the plate after 30 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.75 g of Bamethan Sulfate, previously dried, dissolve in 80 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 51.67 mg of (C12H19NO2)2H2SO4 Packaging and Storage Preserve in tight containers.

Assay Dissolve 0.320 g of Bambuterol Hydrochloride, accurately weighed, in 50 mL of ethanol, add 5 mL of 0.01 mol/L hydrochloric acid, and titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Read the volume added between the two points of inflexion. Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L sodium hydroxide VS = 40.39 mg of C18H30ClN3O5 Packaging and Storage Preserve in well-closed containers.

Bamethan Sulfate
OH HO CCH 2NHCH 2CH2CH 2CH3 H 2 H2SO 4

and enantiomer (C12H19NO2)2H2SO4: 516.65 Bamethan Sulfate, when dried, contains not less than 99.0% and not more than 101.0% of bamethan sulfate[(C12H19NO2)2 H2SO4]. Description Bamethan Sulfate is a white crystal or crystalline powder, is odorless and has a bitter taste. Bamethan Sulfate is freely soluble in water or in glacial acetic acid, soluble in methanol, slightly soluble in ethanol and practically insoluble in ether.. Melting point About 169 C (with decomposition) Identification (1) To 1 mL of a solution of Bamethan Sulfate (1 in 1000), add 5 mL of a solution of pnitrobenzene-diazonium ftuoroborate (1 in 2000) and 10 mL of boric acid-potassium chloride-sodium hydroxide buffer solution, pH 9.2: an orange-red color is observed. (2) Determine the absorption spectra of the solutions of Bamethan Sulfate and Bamethan Sulfate RS in 0.01 mol/L hydrochloric acid TS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Bamethan Sulfate and Bamethan Sulfate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) A solution of Bamethan Sulfate (1 in 100) responds to the Qualitative Tests for sulfate pH Dissolve 1.0 g of Bamethan Sulfate in 10 mL of water: The pH of this solution is between 4.0 and 5.5.

KP 9 95 test solution. Prepare the control solution as follows: take 0.30 mL of 0.01 mol/L hydrochloric acid VS, 20 mL of acetone and 6 mL of dilute nitric acid and add water to make 50 mL (not more than 0.035%). (3) SulfateDissolve 0.40 g of Barbital in 20 mL of acetone and add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: take 0.40 mL of 0.005 mol/L sulfuric acid VS, 20 mL of acetone and 1 mL of dilute hydrochloric acid and add water to make 50 mL (not more than 0.048%). (4) Heavy metalsProceed with 1.0 g of Barbital according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) Readily carbonizable substancesPerform the test with 0.5 g of Barbital. The solution has not more color than Color Matching Fluid A. Loss on Drying Not more than 1.0% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.4 g of Barbital, previously dried and dissolve in 5 mL of ethanol and 50 mL of chloroform. Titrate with 0.1 mol/L potassium hydroxideethanol VS until the color of the solution changes from yellow through pale blue to purple (indicator: 1 mL of alizarin yellow GG-thymolphthalein TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L potassium hydroxide-ethanol VS = 18.419 mg of C8H12N2O3 Packaging and Storage Preserve in well-closed containers.

Barbital
O CH3CH2 CH3CH2 O H N O

NH

C8H12N2O3: 184.19 Barbital, when dried, contains not less than 99.0% and not more than 101.0% of barbital (C8H12N2O3). Description Barbital is a colorless or white crystal or crystalline powder, is odorless and has a slightly bitter taste. Barbital is freely soluble in acetone or in pyridine, soluble in ethanol, sparingly soluble in ether and slightly soluble in water or in chloroform. Barbital dissolve in sodium hydroxide TS and in ammonia TS. pHIts saturated solution is between 5.0 and 6.0. Identification (1) Boil 0.2 g of Barbital with 10 mL of sodium hydroxide TS: the gas evolved changes moistened red litmus paper to blue. (2) Dissolve 50 mg of Barbital in 5 mL of diluted pyridine (1 in 10), add 0.3 mL of cupric sulfate TS, shake and allow to stand for 5 minutes: a red-purple precipitate is formed. Shake the mixture with 5 mL of chloroform: a red-purple color develops in the chloroform layer. Separately, dissolve 50 mg of Barbital in 2 to 3 drops of ammonia-ammonium chloride buffer solution, pH 10.7 and 5 mL of diluted pyridine (1 in 10). Add 5 mL of chloroform and 0.3 mL of cupric sulfate TS to the solution: a red-purple precipitate is produced in the aqueous layer. The red-purple precipitate is not dissolve in the chloroform by shaking. (3) To 0.4 g of Barbital, add 0.1 g of anhydrous sodium carbonate and 4 mL of water, shake and add a solution of 0.3 g of p-nitrobenzyl chloride in 7 mL of ethanol. Heat the mixture in a water-bath under a reflux condenser for 30 minutes and allow to stand for 1 hour. Collect the separated crystals, wash with 7 mL of sodium hydroxide TS and a small amount of water, recrystalize from a mixture of ethanol and chloroform (1 : 1) and dry at 105 C for 30 minutes: the crystals melt between 192 C and 196 C. Melting Point Between 189 C and 192 C

Barium Sulfate
BaSO4: 233.39 Description Barium Sulfate is a white powder, is odorless and tasteless. Barium Sulfate is practically insoluble in water, in ethanol or in ether. Barium Sulfate does not dissolve in hydrochloric acid, in nitric acid or in sodium hydroxide TS. Identification (1) Mix 0.5 g of Barium Sulfate with 2 g each of anhydrous sodium carbonate and anhydrous potassium carbonate in a crucible, heat the mixture until fusion is complete, treat the cooled mass with hot water and filter. The filtrate, acidified with hydrochloric acid, responds to the Qualitative Tests for sulfate. (2) Wash the hot water-insoluble residue obtained in (1) with water, dissolve in 2 mL of acetic acid and filter,

Purity (1) Clarity and color of solution Dissolve 0.5 g of Barbital in 5 mL of sodium hydroxide TS: the solution is clear and colorless. (2) ChlorideDissolve 0.30 g of Barbital in 20 mL of acetone and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the

96 Monographs, Part I if necessary: the solution responds to the Qualitative Tests for barium salt. Purity (1) Acidity or alkalinityAgitate 1.0 g of Barium Sulfate with 20 mL of water for 5 minutes: the solution is neutral. (2) PhosphateBoil 1.0 g of Barium Sulfate with 3 mL of nitric acid and 5 mL of water for 5 minutes, cool and add water to restore the original volume. Filter through a filter paper, previously washed with dilute nitric acid, to the filtrate, add an equal volume of ammonium molybdate TS and allow to stand between 50 C and 60 C for 1 hour: no yellow precipitate is produced. (3) SulfidePlace 10 g of Barium Sulfate in a 250 mL Erlenmeyer flask, add 10 mL of dilute hydrochloric acid and water to make 100 mL and boil for 10 minutes: the gas evolved does not darken moistened lead acetate paper. (4) Heavy metalsBoil 5.0 g of Barium Sulfate with 2.5 mL of glacial acetic acid and 50 mL of water for 10 minutes, cool, add 0.5 mL of ammonia TS and water to make 100 mL and filter. Perform the test with 50 mL of this filtrate. Prepare the control solution with 2.5 mL of standard lead solution, 1.25 mL of glacial acetic acid, 0.25 mL of ammonia TS and water to make 50 mL (not more than 10 ppm). (5) ArsenicPrepare the test solution with 2.0 g of Barium Sulfate according to Method 1 and perform the test (not more than 1 ppm). (6) Hydrochloric acid-soluble substances and soluble barium saltsCool the solution obtained in (3), add water to make 100 mL and filter. Evaporate 50 mL of the filtrate on a water-bath to dryness, add 2 drops of hydrochloric acid and 10 mL of warm water, filter through filter paper for assay and wash with 10 mL of warm water. Evaporate the combined filtrate and washings on a water-bath to dryness and dry the residue at 105 C for 1 hour: the residue weighs not more than 15 mg. Shake the residue, if any, with 10 mL of water and filter. To the filtrate, add 0.5 mL of dilute sulfuric acid and allow to stand for 30 minutes: no turbidity is produced. Packaging and Storage Preserve in well-closed containers.

Beclomethasone Dipropionate, when dried, contains not less than 97.0% and not more than 103.0% of beclomethasone dipropionate (C28H37ClO7). Description Beclomethasone Dipropionate is a white to pale yellow powder and is odorless. Beclomethasone Dipropionate is freely soluble in chloroform, soluble in methanol, sparingly soluble in ethanol or in dioxane, slightly soluble in ether and practically insoluble in water. Melting point About 208 C (with decomposition). Identification (1) Dissolve 2 mg of Beclomethasone Dipropionate in 2 mL of sulfuric acid: initially a yellowish color develops and gradually changes through orange to dark red-brown. To this solution, add carefully 10 mL of water: the color changes to bluish green and a flocculent precipitate is formed. (2) Dissolve 10 mg of Beclomethasone Dipropionate in 1 mL of methanol, add 1 mL of Fehlings TS and heat: a red to red-brown precipitate is formed. (3) Prepare the test solution with 20 mg of Beclomethasone Dipropionate as directed under the Oxygen Flask Combustion Method, using a mixture of 1 mL of sodium hydroxide TS and 20 mL of water as an absorbing liquid: the test solution responds to the Qualitative Tests for chloride. (4) Determine the infrared spectra of Beclomethasone Dipropionate and Beclomethasone Dipropionate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectro-photometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears between the spectra, dissolve Beclomethasone Dipropionate and Beclomethasone Dipropionate RS in ethanol, then evaporate the ethanol to dryness and repeat the test with the residues. Specific Optical Rotation [ ]20 D : Between +88 and +94 (after drying, 0.1 g, dioxane, 10 mL, 100 mm). Purity (1) Heavy metalsProceed with 0.5 g of Beclomethasone Dipropionate according to Method 2 and perform the test. Prepare the control solution with 1.5 mL of standard lead solution (not more than 30 ppm) (2) Related substancesDissolve 20 mg of Beclomethasone Dipropionate in 5 mL of a mixture of chloroform and methanol (9 : 1) and use this solution as the test solution. Pipet 1 mL of the test solution, add a mixture of chloroform and methanol (9 : 1) to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of 1,2-dichloroethane, methanol and water (475 : 25 : 1) to a distance of about 15

Beclomethasone Dipropionate
O C H HO H3C H H3C CH2O O O H CH3 Cl O H C O C CH2CH3

CH2CH3

C28H37ClO7 : 521.04

KP 9 97 cm and air-dry the plate. Spray evenly alkaline blue tetrazolium TS on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.5 g). Asssy Weigh accurately, about 20 mg each of Beclomethasone Dipropionate and Beclomethasone Dipropionate RS, previously dried and dissolve each in methanol to make exactly 50 mL. Pipet 10 mL each of the test solution and the standard solution, add exactly 10 mL of the internal standard solution and methanol to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Beclomethasone Dipropionate to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of C28H37ClO7 = amount (mg) of Beclomethasone Dipropionate RS
QT QS

Packaging and Storage Preserve in tight containers.

Benserazide Hydrochloride
HO H HOCH2C NH2 O C NHNHCH2 OH HCl OH

and enantiomer C10H15N3O5HCl: 293.70 Benserazide Hydrochloride contains not less than 98.0% and not more than 101.0% of benserazide hydrochloride (C10H15N3O5HCl), calculated on the anhydrous basis. Description Benserazide Hydrochloride is a white to grayish white, crystalline powder. Benserazide Hydrochloride is freely soluble in water or in formic acid, soluble in methanol, very slightly soluble in ethanol and practically insoluble in ether. Benserazide Hydrochloride is hygroscopic. Benserazide Hydrochloride is gradually affected by light. A solution of Benserazide Hydrochloride (1 in 100) shows no optical rotation. pHDissolve 1.0 g of a solution of Benserazide Hydrochloride in 100 mL of water: the pH of this solution is between 4.0 and 5.0. Identification (1) Determine the absorption spectra of the solutions of Benserazide Hydrochloride and Benserazide Hydrochloride RS in 0.1 mol/L hydrochloric acid TS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Benserazide Hydrochloride and Benserazide Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry,: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) To 10 mL of a solution of Benserazide Hydrochloride (1 in 30), add silver nitrate TS: a white precipitate is produced. To a portion of this precipitate, add dilute nitric acid: the precipitate does not dissolve. Purity (1) Clarity and color of solutionDissolve 0.5 g of Benserazide Hydrochloride in 10 mL of water and perform the test with this solution as directed under the Ultraviolet-visible Spectrophotometry: the absorbance of this solution at 430 nm is not more than 0.10. (2) Heavy metalsProceed with 1.0 g of Benserazide Hydrochloride in according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesPerform the test without exposure to daylight, using light-resistant vessels. Dissolve 0.25 g of Benserazide Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet 1 mL and 3 mL of the test solution, add methanol to

Internal standard solutionA solution of testosterone propionate in methanol (1 in 4000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 20 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of acetonitrile and water (3 : 2). Flow rate: Adjust the flow rate so that the retention time of Beclomethasone Dipropionate is about 6 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, beclomethasone dipropionate and the internal standard are eluted in this order with the resolution between their peaks being not less than 8. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of beclomethasone dipropionate to that of the internal standard is not more than 1.0 %.

98 Monographs, Part I make exactly 200 mL and use these solutions as the standard solutions (1) and (2), respectively. Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 2 L each of the test solution and the standard solutions (1) and (2) on a plate of cellulose for thin-layer chromato-graphy. Develop the plate with a solution of formic acid in sodium chloride TS (1 in 1000) to a distance of about 10 cm and air-dry the plate. Spray evenly sodium carbonate TS, air-dry and then spray evenly Folins TS on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution (2) and the numbers of the spots which are more intense than the spot from the standard solution (1) are not more than 2. Water Not more than 2.5% (0.5 g, volumetric titration, direct titration). Use a solution of salicylic acid in methanol for Karl Fischer Method (3 in 20) instead of methanol for Karl Fischer Method. Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.3 g of Benserazide Hydrochloride, dissolve in 5 mL of formic acid, add 50 mL of glacial acetic acid and titrate immediately with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 29.370 mg of C10H15N3O5HCl Packaging and Storage tight containers. Preserve in light-resistant, trate and heat for 5 minutes in a water-bath. After cooling, add 10 mL of water and 0.5 g of zinc powder, heat for 5 minutes, cool and filter: the filtrate responds to the Qualitative Tests for primary aromatic amines. The color of the solution is red. (2) To 2 mL of a solution of Benzalkonium Chloride (1 in 1000), add a mixture of 0.2 mL of a solution of bromophenol blue (1 in 2000) and 0.5 mL of sodium hydroxide TS: a blue color is observed. Add 4 mL of chloroform to this solution and shake vigorously: the blue color shifts to the chloroform layer. Collect the chloroform layer and add dropwise, with stirring, a solution of sodium lauryl sulfate (1 in 1000): the chloroform layer turns colorless. (3) Determine the absorption spectra of the solutions of Benzalkonium Chloride and Benzalkonium Chloride RS in 0.1 mol/L hydrochloric acid VS (1 in 2000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensitiesof absorption at the same wavenumbers. (4) To 1 mL of a solution of Benzalkonium Chloride (1 in 100), add 2 mL of ethanol, 0.5 mL of dilute nitric acid and 1 mL of silver nitrate TS: a white precipitate is produced. This precipitate does not dissolve on the addition of dilute nitric acid, but dissolves on the addition of ammonia TS. Purity (1) Clarity and color of solutionDissolve 1.0 g of Benzalkonium Chloride in 10 mL of water: the solution is clear and colorless, or pale yellow. (2) Petroleum ether-soluble substancesTo 3.0 g of Benzalkonium Chloride, add water to make 50 mL, then add 50 mL of dehydrated ethanol and 5 mL of 0.5 mol/L sodium hydroxide TS and extract with three 50 mL volumes of petroleum ether. Combine the petroleum ether extracts and wash with three 50 mL volumes of dilute ethanol. After shaking well with 10 g of anhydrous sodium sulfate, filter through a dry filter paper and wash the filter paper with two 10 mL volumes of petroleum ether. Evaporate the petroleum ether on a water-bath by heating and dry the residue at 105 C for 1 hour: the residue is not more than 1.0%. Water Not more than 15.0% (0.2 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately 0.15 g of Benzalkonium Chloride and dissolve in 75 mL of water. Adjust the pH between 2.6 and 3.4 by adding drop-wise diluted dilute hydrochloric acid (1 in 2), add 1 drop of methylorange TS and titrate with 0.02 mol/L sodium tetraphenylboron VS until the color of the solution becomes red. Each mL of 0.02 mol/L sodium tetraphenylboron VS = 7.080 mg of C22H40ClN Packaging and Storage Preserve in tight containers

Benzalkonium Chloride
Benzalkonium Chloride is represented by the formula [C6H5CH2N(CH3)2R]Cl, in which R extends from C8H17 to C18H37, with C12H25 and C14H29 comprising the major portion. Benzalkonium Chloride contains not less than 95.0% and not more than 105.0% of benzalkonium chloride (C22H40ClN: 354.01) calculated on the anhydrous basis. Description Benzalkonium Chloride is a white to yellowish white powder, colorless to pale yellow, gelatinous pieces or jelly-like fluid or mass. Benzalkonium Chloride has a characteristic odor. Benzalkonium Chloride is very soluble in water or in ethanol and practically insoluble in ether. A solution of Benzalkonium Chloride foams strongly when shaken. Identification (1) Dissolve 0.2 g of Benzalkonium Chloride in 1 mL of sulfuric acid, add 0.1 g of sodium ni-

KP 9 99 Benzalkonium Chloride Concentrated Solution 50, add water to make 50 mL, then add 50 mL of dehydrated ethanol and 5 mL of 0.5 mol/L sodium hydroxide TS and extract with three 50 mL volumes of petroleum ether. Combine the petroleum ether extracts and wash with three 50 mL volumes of dilute ethanol. After shaking well with 10 g of anhydrous sodium sulfate, filter through a dry filter paper and wash the filter paper with two 10 mL volumes of petroleum ether. Evaporate the petroleum ether on a water-bath by heating and dry the residue at 105 C for 1 hour: the residue is not more than 1.0%. Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately 0.30 g of Benzalkonium Chloride Concentrated Solution 50 and dissolve in 75 mL of water. Adjust the pH between 2.6 and 3.4 by adding drop-wise diluted dilute hydrochloric acid (1 in 2), add 1 drop of methylorange TS and titrate with 0.02 mol/L sodium tetraphenylboron VS until the color of the solution becomes red. Each mL of 0.02 mol/L sodium tetraphenylboron VS = 7.080 mg of C22H40ClN Packaging and Storage Preserve in tight containers.

Benzalkonium Chloride Concentrated Solution 50

Benzalkonium Chloride Concentrated Solution 50 is an aqueous solution, presented as [C6H5CH2N(CH3)2R] Cl, where R ranges from C8H17 to C18H37 and mainly consisting of C12H25 and C14H29. Benzalkonium Chloride Concentrated Solution 50 contains not less than 50.0% and not more than 55.0% of benzalkonium chloride (C22H40ClN: 354.01). Description Benzalkonium Chloride Concentrated Solution 50 is a clear, colorless to pale yellow liquid or jelly-like fluid and has a characteristic odor. Benzalkonium Chloride Concentrated Solution 50 is very soluble in water or in ethanol and practically insoluble in ether. A solution prepared by adding water to Benzalkonium Chloride Concentrated Solution 50 vigorously foams when shaken. Identification (1) Dissolve 0.4 g of Benzalkonium Chloride Concentrated Solution 50 in 1 mL of sulfuric acid, add 0.1 g of sodium nitrate, and heat for 5 minutes on a water bath. After cooling, add 10 mL of water and 0.5 g of zinc powder, heat for 5 minutes, cool, and filter: the filtrate responds to the Qualitative Tests for primary aromatic amines. The color of the solution is red. (2) To 2 mL of a solution of Benzalkonium Chloride Concentrated Solution 50 (1 in 500), add a mixture of 0.2 mL of a solution of bromophenol blue (1 in 2000) and 0.5 mL of sodium hydroxide TS: a blue color develops. Add 4 mL of chloroform to this solution, and shake vigorously: the blue cool shifts to the chloroform layer. Collect the chloroform layer, and add dropwise, with stirring, a solution of sodium lauryl sulfate (1 in 1000): the chloroform layer turns colorless. (3) Determine the absorption spectra of the solutions of Benzalkonium Chloride Concentrated Solution 50 and Benzalkonium Chloride Concentrated Solution 50 RS in 0.1 mol/L hydrochloric acid VS (1 in 1000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) To 1 mL of a solution of Benzalkonium Chloride Concentrated Solution 50 (1 in 50), add 2 mL of ethanol, 0.5 mL of dilute nitric acid and 1 mL of silver nitrate TS; a white precipitate is produced. This precipitate does not dissolve on the addition of dilute nitric acid, but dissolves on the addition of ammonia TS. Purity (1) Clarity and color of solution Dissolve 2.0 g of Benzalkonium Chloride Concentrated Solution 50 in 10 mL of water: the solution is clear and colorless to pale yellow. (2) Petroleum ether-soluble substancesTo 6.0 g of

Benzalkonium Chloride Solution

Benzalkonium Chloride Solution is an aqueous solution containing not more than 50.0 w/v% of Benzalkonium Chloride. Benzalkonium Chloride Solution contains not less than 93.0% and not more than 107.0% of the labeled amount of benzalkonium chloride (C22H40ClN: 354.01). Method of Preparation Dissolve Benzalkonium Chloride in Water or Purified Water. Benzalkonium Chloride Solution is also prepared by diluting Benzalkonium Chloride Concentrated Solution 50 with Water or Purified Water. Description Benzalkonium Chloride Solution is a clear, colorless to pale yellow liquid, and has a characteristic odor. Benzalkonium Chloride Solution foams strongly on shaking. Identification (1) Evaporate a volume of Benzalkonium Chloride Solution, equivalent to 0.2 g of Benzalkonium Chloride according to the labeled amount, on a water-bath to dryness and proceed with the residue as directed in the Identification (1) under Benzalkonium Chloride. (2) To a volume of Benzalkonium Chloride Solution, equivalent to 10 mg of Benozalkonium Chloride accord-

100 Monographs, Part I ing to the labeled amount, add water to make 10 mL. Proceed with 2 mL of this solution as directed in the Identification (2) under Benzalkonium Chloride. (3) To a volume of Benzalkonium Chloride Solution, equivalent to 1 g of Benozalkonium Chloride according to the labeled amount, add water or concentrate on a water-bath, if necessary, to make 10 mL. To 1 mL of this solution, add 0.1 mol/L hydrochloric acid VS to make 200 mL and proceed as directed in the Identification (3) under Benzalkonium Chloride. (4) To a volume of Benzalkonium Chloride Solution, equivalent to 0.1 g of Benozalkonium Chloride according to the labeled amount, add water or concentrate on a water-bath, if necessary, to make 10 mL. Proceed with 1 mL of this solution as directed in the Identification (4) under Benzalkonium Chloride. Assay Pipet a volume of Benzalkonium Chloride Solution, equivalent to about 0.15 g of benzalkonium chloride (C22H40ClN), dilute with water to make 75 mL, if necessary and proceed as directed in the Assay under Benzalkonium Chloride. Each mL of 0.02 mol/L sodium tetraphenylboron VS = 7.080 mg of C22H40ClN Packaging and Storage Preserve in tight containers. water. Identification (1) Dissolve 0.1 g of Benzathine Penicillin G Hydrate in 1 mL of 1 mol/L sodium hydroxide, shake for 2 minutes, add 2 mL of ether, shake for one minute, stand, take ether layer and dry. To resulting residues add 2 mL of glacial acetic acid and dissolve. To this solution add 1mL of potassium dichromate TS: a yellow precipitate develops. (2) Dissolve 0.1 g of Benzathine Penicillin G Hydrate in 2 mL of 1 mol/L sodium hydroxide, shake for 2 minutes, extract with 3 mL of ether twice, and take ether layers and dry. To resulting residues add 1 mL of 50 % ethanol and 5 mL of picric acid TS, heat for 5 minutes at 90 C and cool slowly. To the produced precipitate add 50 % ethanol containing small amount of picric acid, recrystallize, and determine melting point: metlting point is about 214 C. (3) Determine the absorption spectra of the solutions of Benzathine Penicillin G Hydrate and Benzathine Penicillin G Hydrate RS in methanol (1 in 200), as directed under Ultraviolet-visible Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Benzathine Penicillin G Hydrate and Benzathine Penicillin G Hydrate RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Benzathine Penicillin G Weigh accurately about 50 mg of Benzathine Penicillin G Hydrate, dissolve in methanol and make exactly 100 mL. Determine absorbance of this solution at 263 nm with methanol as control solu1% tion, and obtain E1 cm .
2

Benzathine Penicillin G Hydrate

O HO O N H H H O N S H CH3 CH3

.
N H H N

Amount [%] of Benzathine Penicillin G

4H2O
1% = E1 cm

100 7

(C16H18N2O4S)2 C16H20N24H2O: 981.19 Benzathine Penicillin G Hydrate is the N, Ndibenzylethylenediamine salt of a penicillin compound having antibacterial activity produced by the growth of Penicillium species. Benzathine Penicillin G Hydrate contains not less than 1050 Units (potency) per mg of penicillin G sodium (C16H17N2NaO4S: 356.37), and benzathine penicillin G is not less than 85.0 %, calculated on the dried basis. Description Benzathine Penicillin G Hydrate is a white crystalline powder. Benzathine Penicillin G Hydrate is sparingly soluble in dimethylformamide or in formamide, slightly soluble in methanol or in ethanol, and very slightly soluble in

pH The pH of a saturated solution of Benzathine Penicillin G Hydrate is between 5.0 and 7.5. Sterility Test It meets the requirement, when Benzathine Penicillin G Hydrate is used in a sterile preparation. Pyrogen Test It meets the requirement, when Benzathine Penicillin G Hydrate is used in a sterile preparation. Weigh appropriate amount of Benzathine Penicillin G Hydrate, dissolve in saline injection solution, make a suspension so that each mL contains 2000 Units (potency), and use the suspension as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not more than 8.0% (0.1 g, volumetric titration, direct titration).

KP 9 101 Description Benzbromarone is a white to pale yellow powder, is odorless and tasteless. Benzbromarone is very soluble in dimethylformamide, freely soluble in acetone or in chloroform, soluble in ether, sparingly soluble in ethanol and practically insoluble in water. Identification (1) Determine the absorption spectra of the solutions of Benzbromarone and Benzbromarone RS in 0.01 mol/L sodium hydroxide TS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Benzbromarone and Benzbromarone RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 149 C and 153 C

Assay Weigh accurately an appropriate amount of Benzathine Penicillin G Hydrate, and dissolve in 1 mol/L sodium hydroxide TS to make the solution containing 2000 units (potency) per mL, and use this solution as the test solution. Pipet exactly 2 mL of the test solution, transfer into a separator, allow to stand for 15 minutes, add exactly 2.0 mL of diluted hydrochloric acid (1 in 10) and 10 mL of 0.01 mol/L iodine VS and allow to stand for 15 minutes, if necessary, add about 5 mL of carbon tetrachloride and shake. Titrate this solution with 0.01 mol/L sodium thiosulfate VS until the color of the solution changes to colorless. When carbon tetrachloride is added, titrate until the color of the layer of carbon tetrachloride changes to colorless. If necessary, use 0.2 mL to 0.5 mL of starch solution TS as indicator solution. Separately, weigh accurately an appropriate amount of Penicillin G Sodium RS, dissolve in 1 % phosphate buffer, pH 6.0 and make the solution contain 2000 units (potency) per mL, and use this solution as the standard solution. Pipet exactly 2 mL of the standard solution, transfer into a separator, add 2.0 mL of 1 mol/L sodium hydroxide TS, allow to stand for 15 minutes, and titrate as the above. Separately, weigh accurately an appropriate amount of Benzathine Penicillin G Hydrate, suspend in 1 % phosphate buffer, pH 6.0 and make the suspension contain 2000 units (potency) per mL. Pipet exactly 2 mL each of this suspension and the standard solution, transfer each into separators, add exactly 10 mL of 0.01 mol/L iodine VS, if necessary, add about 5 mL of carbon tetrachloride and shake. Titrate this solution with 0.01 mol/L sodium thiosulfate VS, perform a blank determination and make any necessary correction. Determine the consumed amounts (mL) of 0.01 mol/L iodine VS, VT and VS , of the test solution and the standard solution. Amount (g) of penicillin G sodium (C16H17N2NaO4S) = amount (g) of Penicillin G Sodium RS
VT VS

Packaging and Storage Preserve in tight containers.

Benzbromarone
O CH2CH3 Br

C O Br

OH

C17H12Br2O3: 424.08 Benzbromarone, when dried, contains not less than 98.5% and not more than 101.0% of benzbromarone (C17H12Br2O3).

Purity (1) SulfateDissolve 1.0 g of Benzbromarone in 40 mL of acetone and add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.40 mL of 0.005 mol/L sulfuric acid VS add 40 mL of acetone, 1 mL of dilute hydrochloric acid and water to make 50 mL (not more than 0.019%). (2) Soluble halidesDissolve 0.5 g of Benzbromarone in 40 mL of acetone and add 6 mL of dilute nitric acid and water to make 50 mL. Proceed with this solution as directed under the Chloride Limit Test. Prepare the control solution as follows: to 0.25 mL of 0.01 mol/L hydrochloric acid VS, add 40 mL of acetone, 6 mL of dilute nitric acid and water to make 50 mL. (3) Heavy metalsProceed with 2.0 g of Benzbromarone according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) IronPrepare the test solution with 1.0 g of Benzbromarone according to Method 3 and perform the test according to Method A. Prepare the control solution with 2.0 mL of standard iron solution (not more than 20 ppm). (5) Related substancesDissolve 0.10 g of Benzbromarone in 10 mL of chloroform and use this solution as the test solution. Pipet 1 mL of the test solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, acetone and glacial acetic acid (100 : 2 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.

102 Monographs, Part I of Benzethonium Chloride and Benzethonium Chloride RS in 0.1 mol/L hydrochloric acid VS (1 in 5000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) To 1 mL of a solution of Benzethonium Chloride (1 in 100), add 2 mL of ethanol, 0.5 mL of dilute nitric acid and 1 mL of silver nitrate TS: a white precipitate is produced. This precipitate does not dissolve on addition of dilute nitric acid, but dissolves on addition of ammonia TS. Melting Point Between 158 C and 164 C (after drying). Purity AmmoniumDissolve 0.1 g of Benzethonium Chloride in 5 mL of water, add 3 mL of sodium hydroxide and boil this solution. The wet red litmus paper does not change to blue in color by gas produced from the solution. Loss on Drying Not more than 5.0% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g).
Cl

Loss on Drying Not more than 0.5% (1 g, in vacuum at a pressure not exceeding 0.67 kPa, P2O5, 50 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.6 g of Benzbromarone, previously dried, dissolve in 30 mL of dimethylformamide and titrate with 0.1 mol/L tetramethylammonium hydroxide VS (indicator: 5 drops of thymol bluedimethylformamide TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 42.41 mg of Cl7Hl2Br2O3 Packaging and Storage tight containers. Preserve in light-resistant,

Benzethonium Chloride
CH3 H3C C CH3 CH2 CH3 C CH3 CH3 O(CH2)2O(CH2)2NCH2 CH3

C27H42ClNO2: 448.08 Benzethonium Chloride, when dried, contains not less than 97.0% and not more than 101.0% of benzethonium chloride(C27H42ClNO2). Description Benzethonium Chloride is a colorless or white crystal and is odorless. Benzethonium Chloride is very soluble in ethanol, freely soluble in water and practically insoluble in ether. A solution of Benzethonium Chloride foams strongly when shaken. Identification (1) Dissolve 0.2 g of Benzethonium Chloride in 1 mL of sulfuric acid, add 0.1 g sodium nitrate and heat for 5 minutes in a water-bath. After cooling, add 10 mL of water and 0.5 g of zinc powder, heat for 5 minutes, cool and filter: the filtrate responds to the Qualitative Tests for primary aromatic amines, with a red color observed. (2) To 2 mL of a solution of Benzethonium Chloride (1 in 1000), add a mixture of 0.2 mL of a solution of bromophenol blue (1 in 2000) and 0.5 mL of sodium hydroxide TS: a blue color is observed. Add 4 mL of chloroform to this solution and shake vigorously: the blue color shifts to the chloroform layer. Collect the chloroform layer and add dropwise a solution of sodium lauryl sulfate (1 in 1000) with stirring: the chloroform layer turns colorless. (3) Determine the absorption spectra of the solutions

Assay Weigh accurately 0.2 g of Benzethonium Chloride, previously dried, dissolve in 75 mL of diluted dilute hydrochloric acid (1 in 2) dropwise to adjust the pH 2.6 to 3.4, then add 1 drop of methyl orange TS and titrate with 0.02 mol/L sodium tetraphenylboron VS until the solution becomes red. Each mL of 0.02 mol/L sodium tetraphenylboron VS = 8.962 mg of C27H42ClNO2 Packaging and Storage tight containers. Preserve in light-resistant,

Benzethonium Chloride Solution

Benzethonium Chloride Solution contains not less than 93.0% and not more than 107.0% of the labeled amount of benzethonium chloride (C27H42ClNO2: 448.08). Method of Preparation Dissolve Benzethonium Chloride in Water or Purified Water. Description Benzethonium Chloride Solution is a clear, colorless liquid and is odorless. Benzethonium Chloride Solution foams strongly when shaken. Identification (1) Evaporate a volume of Benzethonium Chloride Solution, equivalent to 0.2 g of Benzethonium Chloride according to the labeled amount, on a water-bath to dryness and proceed with the residue as di-

KP 9 103 rected in the Identification (1) under Benzethonium Chloride. (2) To a volume of Benzethonium Chloride Solution, equivalent to 10 mg of Benzethonium Chloride according to the labeled amount, add water to make 10 mL and proceed with 2 mL of this solution as directed in the Identification (2) under Benzethonium Chloride. (3) To a volume of Benzethonium Chloride Solution, equivalent to 1 g of Benzethonium Chloride according to the labeled amount, add water or concentrate on a waterbath, if necessary, to make 10 mL. To 1 mL of this solution, add 0.1 mol/L hydrochloric acid VS to make 500 mL, and determine the absorption spectrum as directed under the Ultraviolet-visible Spectrophotometry : it exhibits maxima abetween 262 nm and 264 nm, between 268 nm and 270 nm, and between 274 nm and 276 nm. (4) To a volume of Benzethonium Chloride Solution, equivalent to 0.1 g of Benzethonium Chloride according to the labeled amount, add water or concentrate on a water-bath, if necessary, to make 10 mL and proceed with 1 mL of this solution as directed in the Identification (4) under Benzethonium Chloride. Purity (1) NitriteAdd 1.0 mL of Benzethonium Chloride Solution to a mixture of 1 mL of a solution of glycine (1 in 10) and 0.5 mL of dilute acetic acid: no gas is evolved. (2) Oxidizing substancesTo 5 mL of Benzethonium Chloride Solution, add 0.5 mL of potassium iodide TS and 2 to 3 drops of dilute hydrochloric acid: no yellow color is observed. Assay Pipet a volume of Benzethonium Chloride Solution, equivalent to about 0.2 g of benzethonium chloride (C27H42ClNO2), dilute with water to make 75 mL, if necessary, and proceed as directed in the Assay under Benzethonium Chloride. Each mL of 0.02 mol/L sodium tetraphenylboron VS = 8.962 mg of C27H42ClNO2 Packaging and Storage tight containers. Preserve in light-resistant, Benzoic Acid is freely soluble in ethanol, in acetone, or in ether, soluble in hot water and slightly soluble in water. Identification Dissolve 1 g of Benzoic Acid in 8 mL of sodium hydroxide TS and add water to make 100 mL: this solution responds to the Qualitative Tests (2) for benzoate. Melting Point Between 121 C and 124 C.

Purity (1) Heavy metalsDissolve 1.0 g of Benzoic Acid in 25 mL of acetone, add 2 mL of dilute acetic acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution as follows: to 2.0 mL of standard lead solution, add 2 mL of dilute acetic acid, 25 mL of acetone and water to make 50 mL (not more than 20 ppm). (2) Chlorinated compoundsTake 0.5 g of Benzoic Acid and 0.7 g of calcium carbonate in a crucible, mix with a small amount of water and dry. Ignite it at about 600 C, dissolve in 20 mL of dilute nitric acid and filter. Wash the residue with 15 mL of water, combine the filtrate and the washing, add water to make 50 mL and add 0.5 mL of silver nitrate TS: this solution has no more turbidity than the following control solution. Control solutionDissolve 0.7 g of calcium carbonate in 20 mL of dilute nitric acid and filter. Wash the residue with 15 mL of water, combine the filtrate and the washings, add 1.2 mL of 0.01 mol/L hydrochloric acid VS and water to make 50 mL and add 0.5 mL of silver nitrate TS. (3) Potassium permanganate-reducing substancesAdd 0.02 mol/L potassium permanganate VS drop-wise to a boiling mixture of 100 mL of water and 1.5 mL of sulfuric acid, until a red color persists for 30 seconds. Dissolve 1.0 g of Benzoic Acid in this boiling solution and add 0.50 mL of 0.02 mol/L potassium permanganate VS: a red color persists for at least 15 seconds. (4) Phthalic acidTo 0.10 g of Benzoic Acid, add 1 mL of water and 1 mL of resorcinsulfuric acid TS and heat the mixture in an oil-bath heated at a temperature between 120 C and 125 C . After evaporating the water, heat the residue for 90 minutes, cool and dissolve in 5 mL of water. To 1 mL of the solution, add 10 mL of a solution of sodium hydroxide (43 in 500), shake, then examine under light at a wavelength between 470 nm and 490 nm: the green fluorescence of the solution is not more intense than of the following control solution. Control solutionDissolve 61 mg of potassium biphthalate in water to make exactly 1000 mL. Measure exactly 1 mL of the solution, add 1 mL of resorcinsulfuric acid TS and proceed as directed above. (5) Readily carbonizable substances Perform the
o o

Benzoic Acid
CO 2H

C7H6O2: 122.12 Benzoic Acid, when dried, contains not less than 99.5% and not more than 101.0% of benzoic acid (C7H6O2). Description Benzoic Acid is a white crystal or crystalline powder, is odorless, or has a faint, benzaldehyde-like odor.

104 Monographs, Part I test using 0.5 g of Benzoic Acid. The solution has no more color than Color Matching Fluid Q. Loss on Drying Not more than 0.5% (1 g, silica gel, 3 hours). Residue on Ignition Not more than 0.05% (1 g). tween 4.0 and 5.5. Purity (1) Clarity and color of solutionWhen 1 g of Benzydamine Hydrochloride is dissolved in 10 mL of water, the resultant solution is clear. (2) Heavy metals Proceed with 1.0 g of Benzydamine Hydrochloride according to Method 1 and perform the thest. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Primary aminesDissolve 50 mg of Benzydamine Hydrochloride in 10 mL of ethanol, and add 0.1 mL of hydrochloric acid and 2 mL of the solution of 4dimethylaminobenzaldehyde in ethanol (1 in 20). The yellow color obtained is not more intense than that obtained by treating 10 mL of the solution of 2aminobenzoic acid in ethanol (0.5 g/mL) in the same manner. (4) Related substancesDissolve 25 mg of Benzydamine Hydrochloride in a mixture of methanol and water (1 : 1) to make exactly 10 mL, and use this solution as the test solution. Dissolve 5 mg of Benzydamine Hydrochloride Related Substance I RS (3dimethylaminopropyl 2-benzylaminobenzoate hydrochloride) and 12.5 mg of Benzydamine Hydrochloride Related Substance II RS (3-(1,5-dibenzyl-1H-indazol-3yl)oxypropyldimethylamine hydrochloride) in a mixture of methanol and water (1 : 1) to make exactly 100 mL. Dilute 1.0 mL of this solution with a mixture of methanol and water (1 : 1) to 10 mL, and use this solution as the standard solution (1). Dissolve 2.5 mg of Benzydamine Hydrochloride Related Substance III RS (1-benzyl-1Hindazol-3-ol) in a mixture of methanol and water (1 : 1) to make exactly 100 mL. Pipet 1 mL of this solution, dilute with a mixture of methanol and water (1 : 1) to 10 mL, and use this solution as the standard solution (2). Dilute 0.1 mL of the test solution with a mixture of methanol and water (1 : 1) to 100 mL, and use the solution as the standard solution (3). The standard solution (4) contains equal volumes of the test solution, the standard solution (1) and the standard solution (2). Perform the test with 20 L each of the test solution and the standard solutions as directed under the Liquid Chromatography according to the following conditions. Determine the area of each peak by the automatic integration method. The areas of any peak corresponding to the related substance I or the related substance II are not greater than the area of the corresponding peak from the standard solution (1) (not more than 0.2% of the related substance I; not more than 0.5% of the related substance II); the area of any peak corresponding to the related substance III is not greater than the area of the peak from the standard solution (2) (0.1%); the area of any other secondary peak is not greater than the area of the peak from the standard solution (3) (0.1%); and the sum of the areas of any such peaks is not greater than 1%. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 320 nm).

Assay Weigh accurately about 0.5 g of Benzoic Acid, previously dried, dissolve in 25 mL of neutralized ethanol and 25 mL of water and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Each mL of 0.1 mol/L sodium hydroxide VS = 12.212 mg of C7H6O2 Packaging and Storage Preserve in well-closed containers.

Benzydamine Hydrochloride

HCl
N N

N(CH3)2

C19H23N3OHCl : 345.87 Benzydamine Hydrochloride contains not less than 99.0% and not more than 101.0% of benzydamine hydrochloride (C19H23N3OHCl), calculated on the dried basis. Description Benzydamine Hydrochloride is a white crystalline powder. Benzydamine Hydrochloride is very soluble in water, freely soluble in ethanol or in chloroform, and practically insoluble in ether. Identification (1) Determine the infrared absorption spectra of Benzydamine Hydrochloride and Benzydamine Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The solution of Benzydamine Hydrochloride in water (1 in 50) responds to the Qualitative Tests for chloride. pH The pH of a solution obtained by dissolving 1 g of Benzydamine Hydrochloride in 10 mL of water is be-

KP 9 105 Column: A stainless steel column about 4.6 mm inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A a constant temperature of about 25 o C . Mobile phase: Variable mixtures of mobile A and mobile phase B, and program the chromatograph as follows. Mobile phase AAn aqueous solution containing 0.01 mol/L potassium dihydrogen phosphate and 0.005 mol/L sodium octyl sulphate, adjusted to pH 3.0 0.1 with phosphoric acid. Mobile phase BMethanol. Time (min) 0 0 ~ 20 20 ~ 22 22 ~ 30 Mobile Mobile phase A (%) phase B (%) 50 50 50 30 50 70 30 50 70 50 50 50 Elution equilibrium linear gradient linear gradient isocratic

Berberine Chloride Hydrate

O O

ClN CH3O CH3O

XH2O

Berberine Hydrochloride Berberine Chloride

C20H18ClNO4xH2O

Berberine Chloride Hydrate contains not less than 95.0% and not more than 102.0% of berberine chloride (C20H18ClNO4: 371.81) calculated on the anhydrous basis. Description Berberine Chloride Hydrate is a yellow crystal or crystalline powder, is odorless or has a faint, characteristic odor and has a very bitter taste. Berberine Chloride Hydrate is sparingly soluble in methanol, slightly soluble in ethanol, and very slightly soluble in water. Identification (1) Determine the absorption spectra of the solutions of Berberine Chloride Hydrate and Berberine Chloride Hydrate RS ( 1 in 100000 ) as directed under the Ultraviolet-visible Spetrophotometry: both spectra exhibit similar intensities at the same wavelengths. (2) Determine the infrared spectra of Berberine Chloride Hydrate and Berberine Chloride Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 0.1 g of Berberine Chloride Hydrate in 20 mL of water by warming, add 0.5 mL of nitric acid, cool and filter after allowing to stand for 10 minutes. To 3 mL of the filtrate, add 1 mL of silver nitrate TS and collect the produced precipitate: the precipitate does not dissolve in dilute nitric acid, but it dissolves in an excess amount of ammonia TS. Purity (1) AcidShake thoroughly 0.10 g of Berberine Chloride Hydrate with 30 mL of water and filter. To the filtrate, add 2 drops of phenolphthalein TS and 0.10 mL of 0.1 mol/L sodium hydroxide VS: the yellow color changes to an orange to red color. (2) SulfateShake 1.0 g of Berberine Chloride Hydrate with 48 mL of water and 2 mL of dilute hydrochloric acid for 1 minute and filter. Discard the first 5 mL of the filtrate, take the subsequent 25 mL of the filtrate, add water to make 50 mL and perform the test with this solution as the test solution. Prepare the control solution

Flow rate: 1.5 mL/min. System suitability System performance: When the procedure is run with 20 L of the standard solution (4) under the above operating conditions, the retention time of benzydamine is about 10 minutes, with the resolution between the peak of benzydamine and any adjacent peak being not less than 2.5. Loss on Drying Not more than 0.5% (1 g, 0.67 kPa, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve 0.3 g of Benzydamine Hydrochloride, accurately weighed, in 100 mL of glacial acetic acid, and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 34.59 mg of C19H23N3OHCl Packaging and Storage Preserve in well-closed containers.

106 Monographs, Part I with 0.50 mL of 0.005 mol/L sulfuric acid VS, 1 mL of dilute hydrochloric acid, 5 to 10 drops of bromophenol blue TS and water to make 50 mL (not more than 0.048%). (3) Heavy metalsProceed with 1.0 g of Berberine Chloride Hydrate according to Method 2 and perform the test. Prepare the control solution with 3.0 mL of standard lead solution (not more than 30 ppm). (4) Related substancesDissolve 10 mg of Berberine Chloride Hydrate in 100 mL of mobile phase and use this solution as the test solution. Pipet 4 mL of the test solution, add the mobile phase to make exactly 100 mL, use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of both solutions by the automatic integration method: total of the peak areas of peaks other than berberine of the test solution is not larger than the peak area of berberine of the standard solution. Operating conditions Detector, column, column temperature, mobile phase, flow rate and selection of column: Proceed as directed in the operating conditions in the Assay. Time span of measurement: About 2 times as long as the retention time of berberine, after the solvent peak. Detection sensitivity: Adjust the detection sensitivity so that the peak height of berberine obtained from 10 L of the standard solution is about 10 % of the full scale. Water 8.0% to 12.0% (0.1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 10 mg of Berberine Chloride Hydrate and dissolve in mobile phase to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Berberine Chloride Hydrate RS (separately determined water content) and dissolve in the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak areas, AT and AS of berberine in each solution. Amount (mg) of berberine chloride (C20H18ClNO4) = amount (mg) of Berberine Chloride Hydrate RS, calculated on the anhydrous basis
AT AS

tadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C . Mobile phase: Dissolve 3.4 g of monobasic potassium phosphate and 1.7 g of sodium lauryl sufate in 1000 mL of a mixture of water and acetonitrile (1 : 1). Flow rate: Adjust the flow rate so that the retention time of berberine is about 10 minutes. Selection of column: Dissolve each 1 mg of berberine chloride and palmatin chloride in the mobile phase to make 10 mL. Proceed with 10 L of this solution under the above operating conditions and calculate the resolution. Use a column giving elution of palmatin and berberine in this order with the resolution between these peaks being not less than 1.5 %. Packaging and Storage tight containers. Preserve in light-resistant,
o

Berberine Tannate
Berberine Tannate is a compound of berberine and tannic acid. Berberine Tannate contains not less than 27.0% and not more than 33.0% of berberine (C20H19NO5: 353.37), calculated on the anhydrous basis. Description Berberine Tannate is a yellow to pale yellow-brown powder, is odorless or has a faint, characteristic odor and is tasteless. Berberine Tannate is practically insoluble in water, in methanol, in ethanol or in ether. Identification (1) To 0.1 g of Berberine Tannate, add 10 mL of ethanol and heat in a water-bath for 3 minutes with shaking. Cool, filter and to 5 mL of the filtrate, add 1 drop of ferric chloride TS: a blue-green color is produced and on allowing to stand, a bluish black precipitate is formed. (2) Dissolve 10 mg of Berberine Tannate and Berberine Tannate RS in 10 mL of methanol and 0.4 mL of 1 mol/L hydrochloric acid TS and add water to make 200 mL. To each 8 mL of these solutions add water to make 25 mL. Determine the absorption spectra of the solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Berberine Tannate and Berberine Tannate RS, previously dried, as directly in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Acid To 0.10 g of Berberine Tannate add 30 mL of water and filter after shaking well. To the filtrate add 2 drops of phenolphthalein TS and 0.10 mL of

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 345nm). Column: A stainless steel column about 4 mm in inside diameter and about 25 cm in length, packed with oc-

KP 9 107 0.1 mol/L sodium hydroxide VS: the color of the solution changes from yellow to orange to red. (2) Chloride Shake 1.0 g of Berberine Tannate with 38 mL of water and 12 mL of dilute nitric acid for 5 minutes and filter. Discard the first 5 mL of the filtrate, to 25 mL of the subsequent filtrate, add water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.50 mL of 0.01 mol/L hydrochloric acid VS by adding 6 mL of dilute nitric acid, 10 to 15 drops of bromophenol blue TS and water to make 50 mL (not more than 0.035%). (3) SulfateShake 1.0 g of Berberine Tannate with 48 mL of water and 2 mL of dilute hydrochloric acid for 1 minute and filter. Discard the first 5 mL of the filtrate, take the subsequent 25 mL of the filtrate, add water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS, 1 mL of dilute hydrochloric acid, 5 to 10 drops of bromphenol blue TS and water to make 50 mL (not more than 0.048%). (4) Heavy metalsProceed with 1.0 g of Berberine Tannate according to Method 2 and perform the test. Prepare the control solution with 3.0 mL of standard lead solution (not more than 30 ppm). (5) Related substancesDissolve 10 mg of Berberine Tannate in 100 mL of the mobile phase and use this solution as the test solution. Pipet 4 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of both solutions by the automatic integration method: the total of the peak areas other than berberine of the test solution is not larger than the peak area of berberine of the standard solution. Operating conditions. Detector, column, column temperature, mobile phase, flow rate and selection of column: Proceed as directed in the operating conditions in the Assay. Test for required detectability: To exactly 2 mL of the standard solution, add the mobile phase to make exactly 20 mL. Confirm that the peak area of berberine obtained with 10 L of this solution is equivalent to 7 to 13% of that with 10 L of the standard solution Detection sensitivity: Adjust the detection sensitivity so that the peak height of berberine obtained from 10 L of the standard solution is about 10 % of the full scale. Time span of measurement: About 2 times as long as the retention time of berberine, after the solvent peak. Water Not more than 6.0% (0.7 g, volumetric titration, direct titration). Residue on Ignition Not more than 1.0% (1 g). Assay Weigh accurately 30 mg of Berberine Tannate and dissolve in mobile phase to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Berberine Chloride RS (separately determined water content) and dissolve in the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak areas, AT and AS of berberine in each solution. Amount (mg) of berberine (C20H19NO5) = amount (mg) of Berberine Chloride RS, calculated on the anhydrous basis
AT 0.9504 AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 345 nm). Column: A stainless steel column about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 3.4 g of monobasic potassium phosphate and 1.7 g of sodium lauryl sufate in 1000 mL of a mixture of water and acetonitrile (1 : 1). Flow rate: Adjust the flow rate so that the retention time of berberine is about 10 minutes. Selection of column: Dissolve each 1 mg of Berberine Chloride and palmatin chloride in the mobile phase to make 10 mL. Proceed with 10 L of this solution under the above operating conditions and calculate the resolution. Use a column giving elution of palmatin and berberine in this order with the resolution between these peaks being not less than 1.5%. Packaging and Storage tight containers. Preserve in light-resistant,

Betahistine Mesilate
N CH2CH2NHCH3 2 CH3SO3H

C8H12N22CH4O3S: 328.41 Betahistine Mesilate, when dried, contains not less than 98.0% and not more than 101.0% of betahistine mesilate(C8H12N22CH4O3S). Description Betahistine Mesilate is a white crystal or crystalline powder. Betahistine Mesilate is very soluble in water, freely soluble in methanol or in glacial acetic acid, sparingly so-

108 Monographs, Part I luble in dehydrated ethanol, very slightly soluble in acetic anhydride and practically insoluble in ether. Betahistine Mesilate dissolves in dilute hydrochloric acid. Betahistine Mesilate is hygroscopic. Identification (1) Determine the absorption spectra of the solutions of Betahistine Mesilate and Betahistine Mesilate RS in 0.1 mol/L hydrochloric acid (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Betahistine Mesilate and Betahistine Mesilate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) To 30 mg of Betahistine Mesilate, add 0.1 g of sodium nitrate and 0.1 g of anhydrous sodium carbonate, mix well and ignite gradually. After cooling, dissolve the residue in 2 mL of dilute hydrochloric acid and 10 mL of water, filter, if necessary and to the filtrate, add 1 mL of barium chloride TS: a white precipitate is produced. Melting Point Between 110 C and 114 C (after drying). Purity (1) Heavy metalsProceed with 1.0 g of Betahistine Mesilate according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 50 mg of Betahistine Mesilate in 10 mL of a mixture of water and acetonitrile (63:37), and use this solution as the test solution. Pipet 1 mL of the test solution, add the mixture of water and acetonitrile (63:37) to make exactly 100 mL, and use this solutions as the standard solution. Perform the test with the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of the peak other than betahistine with the test solution is not larger than 1/10 times the peak area of betahistine with the standard solution, and the total area of the peaks other than the peak area betahistine with the test solution is not larger than 1/2 times the peak area of betahistine with the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 261 nm) Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 35 C. Mobile phase: To 5 mL of diethyl amine and 20 mL of acetic anhydride, add water to make 1000 mL. Dissolve 2.3 g of sodium lauryl sulfate in 630 mL of this solution, and add 370 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of betahistine is about 5 minutes. System suitability Test for required detectability: To exactly 5 mL of the standard solution, add the mixture of water and acetonitrile (63:37) to make exactly 50 mL. Confirm that the peak area of betahistine obtained with 20 L of this solution is equivalent to 7 to 13% of that with 20 L of the standard solution. System performance: Dissolve 10 mg of betahistine mesilate and 10 mg of 2-vinylpyridine in 50 mL of the mixture of water and acetonitrile (63:37). To 2 mL of this solution, add the mixture of water and acetonitrile (63:37) to make 50 mL. When the procedure is run with 20 L of this solution under the above operating conditions, 2-vinylpyridine and betahistine are eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of betahistine is not more than 1.0%. Time span of measurement: About twice as long as the retention time of betohistine after the solvent peak. Loss on Drying Not more than 1.0% (1 g, in vacuum, P2O5, 70 C, 24 hours). Residue on Ignition Not more than 0.1% (1 g).

Assay Weigh accurately 0.2 g of Betahistine Mesilate, previously dried, dissolve in 1 mL of glacial acetic acid, add 50 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 16.420 mg of C8H12N22CH4O3S Packaging and Storage Preserve in tight containers.

Betamethasone
O H HO H3C H H3C C CH2OH OH H CH3 F O H

C22H29FO5: 392.46 Betamethasone, when dried, contains not less than 96.0% and not more than 103.0% of betamethasone (C22H29FO5).

KP 9 109 Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 4 hours). Residue on Ignition Not more than 0.5% (0.1 g, platinum crucible). Assay Dissolve about 20 mg each of Betamethasone and Betamethasone RS, previously dried and accurately weighed, in methanol to make exactly 50 mL. Pipet 5 mL each of these solutions, add exactly 5 mL each of the internal standard solution, then add methanol to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of betamethasone to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of betamethasone (C22H29FO5) = amount (mg) of Betamethasone RS QT
QS

Description Betamethasone is a white to pale yellowish white, crystalline powder and is odorless. Betamethasone is sparingly soluble in methanol, in ethanol, in acetone or in dioxane, very slightly soluble in ether or in chloroform and practically insoluble in water. Melting point About 240 C (with decomposition). Identification (1) Proceed 10 mg of Betamethasone as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as an absorbing liquid and prepare the test solution: the test solution so obtained responds to the Qualitative Tests for fluoride. (2) Dissolve 1.0 mg each of Betamethasone and Betamethasone RS in 10 mL each of ethanol. To 2.0 mL each of these solutions, add 10 mL each of phenylhydrazine hydrochloride TS, mix by shaking, heat in a waterbath at 60 C for 20 minutes. After cooling, determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry using a solution, prepared with 2.0 mL of ethanol in the same manner, as the blank: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Betamethasone and Betamethasone RS, previously dried, according to the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, dissolve each of Betamethasone and Betamethasone RS in acetone, evaporate to dryness and repeat the test on the residues. Specific Optical Rotation [ ]20 D : Between +118 and +126 (after drying, 0.1 g, methanol, 20 mL, 100 mm). Purity (1) Heavy metalsProceed with 0.5 g of Betamethasone according to Method 2 and perform the test. Prepare the control solution with 1.5 mL of standard lead solution (not more than 30 ppm). (2) Related substancesDissolve 10 mg of Betamethasone in 5 mL of a mixture of chloroform and methanol (9 : 1) and use this solution as the test solution. Pipet 1 mL of the test solution, add a mixture of chloroform and methanol (9 : 1) to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of dichloromethane, ether, methanol and water (385 : 75 : 40 : 6) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.

Internal standard solutionA solution of butyl parahydroxybenzoate in methanol (1 in 1750). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column, about 4 mm in inside diameter and 15 to 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of water and acetonitrile (3 : 2). Flow rate: Adjust the flow rate so that the retention time of betamethasone is about 4 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, betamethasone and the internal standard are eluted in this order with the resolution between these peaks being not less than 10. System repeatability: When the test is repeated 6 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of betamethasone to that of the internal standard is not more than 1.0%. Packaging and Storage tight containers. Preserve in light-resistant,

110 Monographs, Part I

Betamethasone Tablets
Betamethasone Tablets contain not less than 90.0% and not more than 107.0% of the labeled amount of betamethasone (C22H29FO5: 392.46). Method of Preparation Prepare as directed under Tablets, with Betamethasone. Identification Pulverize Betamethasone Tablets. To a portion of the powder, equivalent to 2 mg of Betamethasone according to the labeled amount, add 20 mL of methanol, shake for 5 minutes, and filter. Evaporate the filtrate on a water bath to dryness, dissolve the residue after cooling in 2 mL of methanol, filter if necessary, and use the solution as the test solution. Separately, dissolve 2 mg of Betamethasone RS in 2 mL of methanol, and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on the plate of silica gel with a fluorescent indicator for thin-layer chromatography, develop with a mixture of 1-butanol, water and acetic anhydride (3:1:1) to a distance of about 10 cm, and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the principal spots from the test solution and the standard solution show the same Rf value. Dissolution Test Perform the test with 1 tablet of Betamethasone Tablets at 50 revolutions per minute according to the paddle method under the Dissolution Test, using the mixture of 900 mL of water as the dissolution medium. Withdraw 20 mL or more of the dissolution medium 30 minutes after starting the test, and filter through a membrane filter with a pore size not exceeding 0.45 m. Discard the first 10 mL of the filtrate, pipet the subsequent V mL of the filtrate, add water to make exactly V mL so that each mL contains about 0.56 g of betamethasone (C22H29FO5) according to the labeled amount, and use this solution as the test solution. Separately, weigh accurately about 28 mg of Betamethasone RS, previously dried in a desiccator (in vacuum, P2O5) for 4 hours, dissolve in methanol to make exactly 100 mL. Pipet 5 mL of this solution, and use this solution as the standard solution. Perform the test with 100 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine the peak areas, AT and AS , of betamethasone for the test solution and the standard solution. The dissolution rate in 30 minutes is not less than 85%. Dissolution rate (%) with respect to the labeled amount of betamethasone (C22H29FO5)

=WS

AT V ' 1 9 AS V C 5

WS : Amount (mg) of Betamethasone RS

C: Labeled amount (C22H29FO5) in 1 tablet (mg) of betamethasone

Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 241 nm). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of methanol and water (3:2). Flow rate: Adjust the flow rate so that the retention time of betamethasone is about 7 minutes. System suitability System performance: When the procedure is run with 100 L of the standard solution under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of betamethasone are not less than 3000 and not more than 2.0, respectively. System repeatability: When the test is repeated 6 times with 100 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of betamethasone is not more than 1.0%. Uniformity of Dosage Units It meets the requirement, when the content uniformity test is performed according to the following method. To 1 tablet of Betamethasone Tablets, add V mL of water so that each mL contains about 50 g of betamethasone (C22H29FO5) according to the labeled amount, add exactly an amount of the internal standard solution equivalent to 2 mL per 50 g of betamethasone, shake vigorously for 10 minutes, centrifuge, and use the supernatant liquid as the test solution. Separately, weigh accurately about 20 mg of Betamethasone RS, previously dried in a desiccator (in vacuum, P2O5) for 4 hours, and dissolve in acetonitrile to make exactly 200 mL. Pipet 5 mL of this solution, add exactly 20 mL of the internal standard solution, and use this solution as the standard solution. Perform the test with 50 g each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of betamethasone to that of the internal standard for the test solution and the standard solution. Amount (mg) of betamethasone (C22H29FO5) = Amount (mg) of Betamethasone RS
QT V Q S 400

KP 9 111 Internal standard solutionA solution of butyl parahydroxybenzoate in acetonitrile (1 in 40000) Operating conditionsProceed as directed in the assay. System suitability System performance: When the procedure is run with 50 L of the standard solution under the above operating conditions, betamethasone and the internal standard are eluted in this order with the resolution between these peaks being not less than 10. System repeatability: When the test is run 6 times with 50 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of betamethasone to that of the internal standard is not more than 1.0%. Assay Weigh accurately not less than 20 Betamethasone Tablets and powder. Weigh accurately a portion of powder, equivalent to about 5 mg of betamethasone(C22H29FO5), add 25 mL of water, then add exactly 50 mL of the internal standard solution, and shake vigorously for 10 minutes. Filter through a membrane filter with pore size of not more than 0.5 m, discard the first 5 mL of the filtrate, and use the subsequent filtrate as the test solution. Separately, weigh accurately about 20 mg of Betamethasone, previously dried in a desiccator (in vacuum, P2O5) for 4 hours, and dissolve in acetonitrile to make exactly 20 mL of the internal standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of betamethasone to that of the internal standard for the test solution and the standard solution. Amount (mg) of betamethasone (C22H29FO5) = Amount (mg) of Betamethasone RS
QT 1 QS 4

with 20 L of the standard solution under the above operating conditions, Betamethasone and internal standard is eluted in this order with the resolution between these peaks being not less than 10. System repeatability: When the test is repeated 6 times with the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of betamethasone to that of the internal standard is not more than 2.0%. Packaging and Storage Preserve in well-closed containers.

Betamethasone Dipropionate
O H HO H3C H H F O H H3C C CH2O O O C CH3 O C CH2CH3 CH2CH3

C28H37FO7: 504.59 Betamethasone Dipropionate, when dried, contains not less than 97.0% and not more than 103.0% of betamethasone dipropionate (C28H37FO7) and not less than 3.4% and not more than 4.1% of fluorine (F: 19.00).. Description Betamethasone Dipropionate is a white to pale yellowish white, crystalline powder and is odorless. Betamethasone Dipropionate is freely soluble in acetone, in dioxane or in chloroform, soluble in methanol, sparingly soluble in ethanol, slightly soluble in ether and practically insoluble in water or in hexane. Betamethasone Dipropionate is gradually affected by light. Identification (1) To 1 mL of a solution of Betamethasone Dipropionate in methanol (1 in 10000), add 4 mL of isoniazid TS and heat in a water-bath for 2 minutes: a yellow color is observed. (2) Proceed with 10 mg of Betamethasone Dipropionate as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid: the test solution so obtained responds to the Qualitative Tests for fluoride. (3) Determine the absorption spectra of the solutions of Betamethasone Dipropionate and Betamethasone Dipropionate RS in methanol (3 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Betamethasone Dipropionate and Betamethasone Dipropionate RS, pre-

Internal standard solutionA solution of butyl parahydroxybenzoate in acetonitrile (1 in 10000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column 4 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of water and acetonitrile (3:2). Flow rate: Adjust the flow rate so that the retention time of betamethasone is about 4 minutes. System suitability System performance: When the procedure is run

112 Monographs, Part I viously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between +63 and +70 (after drying, 50 mg, dioxane, 10 mL, 100 mm). Melting Point Between 176 C and 180 C. Purity (1) FluorideTo 0.10 g of Betamethasone Dipropionate, add 10.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20), shake for 10 minutes and filter through a membrane filter (0.4 m pore size). Place 5.0 mL of the filtrate in a volumetric flask and add 10 mL of a mixture of alizalin complexone TS, acetic acidpotassium acetate buffer solution, pH 4.3 and cerous nitrate TS (1 : 1 : 1), add water to make 20 mL, allow to stand for 1 hour and use this solution as the test solution. Separately, place 1.0 mL of standard fluorine solution in a volumetric flask, add 5.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20), then 10 mL of a mixture of alizarin complexone TS, acetic acid-potassium acetate buffer solution, pH 4.3 and cerous nitrate TS (1 : 1 : 1), proceed in the same manner as the preparation of the test solution and use this solution as the standard solution. Place 5.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20) in a volumetric flask, proceed in the same manner as the preparation of the test solution and use this solution as the blank. Determine the absorbances of the test solution and the standard solution at 600 nm as directed under the Ultraviolet-visible Spectrophotometry: the absorbance of the test solution is not greater than that of the standard solution (not more than 0.012%). (2) Heavy metalsProceed with 1.0 g of Betamethasone Dipropionate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesPerform the test without exposure to daylight using light-resistant vessels. Dissolve 10 mg of Betamethasone Dipropionate in 10 mL of chloroform and use this solution as the test solution. Pipet 3 mL of the test solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test as directed under the Thin-layer Chromatography with the test solution and the standard solution. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform and acetone (7 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 1.0% (0.5 g, 105 C, 3 hours).

Residue on Ignition Not more than 0.2% (0.5 g, platinum crucible). Assay (1) Betamethasone DipropionateWeigh accurately 15 mg of Betamethasone Dipropionate, previously dried and dissolve in methanol to make exactly 100 mL. Pipet 5 mL of this solution and dilute with methanol to make exactly 50 mL. Determine the absorbance A of this solution at the wavelength of a maximum absorption at about 239 nm as directed under the Ultraviolet-visible Spectrophotometry. Amount (mg) of betamethasone dipropionate A (C28H37FO7) = 312 10000 (2) FluorineWeigh accurately 10 mg of Betamethasone Dipropionate, previously dried and proceed as directed in the determination for fluorine under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid. Packaging and Storage tight containers. Preserve in light-resistant,

Betamethasone Sodium Phosphate

O H HO H3C H H3C C CH2OPO3Na2 OH H CH3 F O H

C22H28FNa2O8P: 516.41 Betamethasone Sodium Phosphate contains not less than 97.0% and not more than 103.0% of betamethasone sodium phosphate (C22H28FNa2O8P), calculated on the anhydrous basis. Description Betamethasone Sodium Phosphate is a white to pale yellowish white, crystalline powder or mass and is odorless. Betamethasone Sodium Phosphate is freely soluble in water, sparingly soluble in methanol, slightly soluble in ethanol and practically insoluble in ether. Betamethasone Sodium Phosphate is hygroscopic. Melting point About 213 C (with decomposition). Identification (1) Dissolve 2 mg of Betamethasone Sodium Phosphate in 2 mL of sulfuric acid: a brown col-

KP 9 113 or develops and gradually changes to blackish brown. (2) Prepare the test solution with 10 mg of Betamethasone Sodium Phosphate as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as an absorbing liquid: the test solution responds to the Qualitative Tests (2) for fluoride. (3) Take 40 mg of Betamethasone Sodium Phosphate in a platinum crucible and carbonize by heating. After cooling, add 5 drops of nitric acid and incinerate by heating. To the residue, add 10 mL of diluted nitric acid (1 in 50) and boil for several minutes. After cooling, neutralize the solution with ammonia TS, filter, if necessary and use this solution as the test solution. The test solution responds to the Qualitative Tests for sodium salt and for phosphate. (4) Determine the infrared spectra of Betamethasone Sodium Phosphate and Betamethasone Sodium Phosphate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between +99 and +105 (0.1 g, calculated on the anhydrous basis, water, 10 mL, 100 mm). pH Dissolve 0.10 g of Betamethasone Sodium Phosphate in 20 mL of water: the pH of this solution is between 7.5 and 9.0. Purify (1) Clarity and color of solutionDissolve 0.25 g of Betamethasone Sodium Phosphate in 10 mL of water: the solution is clear and colorless. (2) Free phosphoric acidWeigh accurately about 20 mg of Betamethasone Sodium Phosphate, dissolve in 20 mL of water and use this solution as the test solution. Separately, pipet 4 mL of phosphoric acid standard solution, add 20 mL of water and use this solution as the standard solution,. To each of the test solution and the standard solution, add exactly 7 mL of dilute sulfuric acid, exactly 2 mL of ammonium molybdate-sulfuric acid TS and exactly 2 mL of p-methylaminophenol sulfate TS, shake well and allow to stand at 20 1 oC for 15 minutes. To each, add water to make exactly 50 mL and allow to stand at 20 1 oC for 15 minutes. Perform the test with the test solution and the standard solutions as directed under the Ultraviolet-visible Spectrophotometry, using a solution prepared with 20 mL of water in the same manner as the blank. Determine the absorbances, AT and

W: Amount (mg) of Betamethasone Sodium Phosphate, calculated on the anhydrous basis. (3) BetamethasoneDissolve 20 mg of Betamethasone Sodium Phosphate in exactly 2 mL of methanol and use this solution as the test solution. Separately, dissolve 20 mg of Betamethasone RS in exactly 10 mL of methanol. Pipet 1 mL of this solution, add methanol to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a freshly prepared mixture of n-butanol, water and acetic anhydride (3 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spot from the test solution corresponding to the spot from the standard solution is not more intense than the spot from the standard solution. Water Not more than 10.0% (0.2 g, volumetric titration, back titration). Assay Weigh accurately about 20 mg each of Betamethasone Sodium Phosphate and Betamethasone Sodium Phosphate RS (determined its water content before using in the same manner as Betamethasone Sodium Phosphate) and dissolve each in methanol to make exactly 20 mL. Pipet 5 mL each of these solutions, add exactly 5 mL of the internal standard solution, then add methanol to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of betamethasone phosphate to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of betamethasone sodium phosphate (C22H28FNa2O8P) = amount (mg) of dried Betamethasone Sodium Phosphate RS QT
QS

Internal Standard solutionA solution of butyl parahydroxybenzoate in methanol (1 in 5000). Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter). Column temperature: A constant temperature of

AS , of the test solution and the standard solution, respectively, at 730 nm: the amount of free phosphoric acid is not more than 0.5%. Amount (%) of free phosphoric acid (H3PO4) =
AT 1 10 . 32 AS W

114 Monographs, Part I about 25 C . Mobile phase: Dissolve 1.6 g of tetra-nbutylammonium bromide, 3.2 g of dibasic sodium phosphate and 6.9 g of monobasic potassium phosphate in 1000 mL of water and add 1500 mL of methanol. Flow rate: Adjust the flow rate so that the retention time of betamethasone phosphate is about 5 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, betamethasone phosphate and the internal standard are eluted in this order with the resolution between these peaks being not less than 10. System repeatability: When the procedure is run 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deivation o the ratios of the peak area of betamethasone phosphate to that of the internal standard is not more than 1.0%. Packaging and Storage Preserve in tight containers.

Specific Optical Rotation [ ]20 D : Between +77 and +83 (after drying, 0.10 g, methanol, 20 mL, 100 mm) Purity Related substancesPerform this procedure without exposure to light. Dissolve 20 mg of Betamethasone Valerate in 5 mL of a mixture of chloroform and methanol (9 : 1) and use this solution as the test solution. Pipet 1 mL of the test solution, add a mixture of chloroform and methanol (9 : 1) to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform and methanol (9 : 1) to a distance of about 12 cm and air-dry the plate. Spray evenly alkaline blue tetrazolium TS on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.2% (0.5 g, platinum crucible).

Betamethasone Valerate
O H HO CH3 H H3C CH2OH O O C H CH3 F O H CH2CH2CH2CH3

C27H37FO6: 476.58 Betamethasone Valerate, when dried, contains not less than 97.0% and not more than 103.0% of betamethasone valerate (C27H37FO6). Description Betamethasone Valerate is a white crystalline powder and is odorless. Betamethasone Valerate is freely soluble in chloroform, soluble in ethanol, sparingly soluble in methanol, slightly soluble in ether and practically insoluble in water. Melting point About 190 C (with decomposition). Identification (1) Proceed with 10 mg of Betamethasone Valerate as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid and prepare the test solution: the test solution responds to the Qualitative Tests for fluoride. (2) Determine the infrared spectra of Betamethasone Valerate and Betamethasone Valerate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.

Assay Dissolve about 10 mg each of Betamethasone Valerate and Betamethasone Valerate RS, previously dried and accurately weighed, in methanol to make exactly 100 mL. Pipet 10 mL each of these solutions, add 10 mL each of the internal standard solution and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operation conditions and calculate the ratios, QT and

QS , of the peak area of Betamethasone Valerate to that

of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of Betamethasone Valerate (C27H37FO6) = amount (mg) of Betamethasone Valerate RS
QT QS

Internal standard solutionA solution of isoamyl benzoate in methanol (1 in 1000). Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 20 to 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter). Column temperature: A constant temperature of

KP 9 115 about 25 C. Mobile phase: A mixture of methanol and water (7 : 3). Flow rate: Adjust the flow rate so that the retention time of Betamethasone Valerate is about 10 minutes. Selection of column: Proceed with 10 L of the standard solution under the above operating conditions and calculate the resolution. Use a column giving elution of Betamethasone Valerate and the internal standard in this order with the resolution between their peaks being not less than 5. Packaging and Storage Preserve in tight containers. 10 mL, and use this solution as the test solution. Perform the test with 20 L of the test solution as directed under the Liquid Chromatography according to the following conditions, and determine the area for the peaks. Calculate the percentage of each impurity according to the peak area percentage method. The sum of all impurities found is not more than 1.0%. Amount (%) of each related substance = 100
Ai AS

Ai is the peak area of each individual peak, other than the main betaxolol peak AS is the sum of all peak areas

Betaxolol Hydrochloride
O CH3

O OH

N H

CH3

H Cl

C18H29NO3HCl : 343.89) Betaxolol Hydrochloride contains not less than 98.5% and not more than 101.5% of betaxolol hydrochloride (C18H29 NO3HCl), calculated on the dried basis. Description Betaxolol Hydrochloride is a white, crystalline powder. Betaxolol Hydrochloride is freely soluble in water, in methanol, or in chloroform. Identification (1) Determine the infrared absorption spectra of Betaxolol Hydrochloride and Betaxolol Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) When the solution of Betaxolol Hydrochloride (1 in 100) is mixed with 1 drop of dilute nitric acid, it responds to the Qualitative Tests for chloride. Melting Point Between 113 C and 117 C.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 273 nm). Column: A stainless steel column 4.6 mm inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: a mixture of buffer solution and acetonitrile (85:15) Flow rate: 1.5 mL/min System suitability System performance: Dissolve 20 mg of Betaxolol Hydrochloride RS and 10 mg of alprenolol hydrochloride in 10 mL, and use this solution as the resolution solution. When the procedure is run with 20 L of the resolution solution according to the above operating conditions, the relative retention times are about 0.9 for alprenolol and 1.0 for betaxolol, the resolution between the two peaks is not less than 1.0, and the symmetry factors for the two peaks is not more than 2.0. System repeatability: When the test is repeated 5 times with 20 L of the resolution solution according to the above conditions, the relative standard deviation of the peak area of betaxolol is not more than 2.0%. Buffer solution Prepare a solution of 0.025 mol/L monobasic potassium phosphate containing 0.1 % of tetrabutylammonium bromide. Adjust with 0.025 mol/L phosphoric acid to a pH of 3.0. Loss on Drying Not more than 1.0% (1 g, 65 C, 2 hours) Residue on Ignition Not more than 0.1% (1 g) Assay Dissolve about 300 mg of Betaxolol Hydrochloride, accurately weighed, in 50 mL of glacial acetic acid, add 7 mL of mercuric acetate TS, and titrate with 0.1 N perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 34.39 mg of

pH The pH of a solution obtained by dissolving 1 g of Betaxolol Hydrochloride in 50 mL of water is between 4.5 and 6.5. Purity (1) Heavy metalsProceed with 1.0 g of Betaxolol Hydrochloride, according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 20.0 mg of Betaxolol Hydrochloride in the mobile phase to make exactly

116 Monographs, Part I C18H29NO3HCl. Packaging and Storage Preserve in tight containers. L each of the test solution and the standard solution on a plate of cellulose for thin-layer chromatography. Develop the plate with a mixture of a solution of ammonium chloride acetate (1 in 100), acetone, 1-butanol and formic acid (20:20:20:1) to a distance of about 10 cm, and dry the plate at 105 C for 15 minutes. Spray evenly hydrogen hexachloroplatinate (IV)-potassium iodide TS on the plate, and allow to stand for 30 minutes: the spot other than the principal spot from the test solution is not more than intense than the spot from the standard solution. Loss on Drying Not more than 1.0% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.4 g of Bethanechol Chloride, previously dried, dissolve in 2 mL of glacial acetic acid, add 40 mL of acetic anhydride, and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 19.668 mg of C7H17ClN2O2 Packaging and Storage Preserve in tight containers.
o

Bethanechol Chloride
H CH3CCH2N(CH3)3 Cl OCONH 2

and enantiomer C7H17ClN2O2: 196.68

Bethanechol Chloride, when dried, contains not less than 98.0% and not more than 101.0% of bethanechol chloride (C7H17ClN2O2). Description Bethanechol Chloride is a colorless or white crystal or a white, crystalline powder. Bethanechol Chloride has a slight amine-like odor, and has a slight, saline taste. Bethanechol Chloride is very soluble in water, freely soluble in glacial acetic acid, and sparingly soluble in dehydrated ethanol. Bethanechol Chloride is hygroscopic. A solution of Bethanechol Chloride (1 in 10) shows no optical rotation. Identification (1) To 2 mL of a solution of Bethanechol Chloride (1 in 40), add 0.1 mL of a solution of cobaltous chloride (1 in 100), then add 0.1 mL of potassium ferrocyanide TS: a green color is observed, and almost entirely fades within 10 minutes. (2) To 1 mL of a solution of Bethanechol Chloride (1 in 100), add 0.1 mL of iodine TS: a brown precipitate is produced, and the solution shows a greenish brown color. (3) Determine the infrared spectra of Bethanechol Chloride and Bethanechol Chloride RS as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) A solution of Bethanechol Chloride (1 in 100) responds to the Qualitative Tests for chloride. Melting Point About 217 C or about 221 C (after drying) Purity (1) Heavy metalsProceed with 1.0 g of Bethanechol Chloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 1.0 g of Bethanechol Chloride in 2.5 mL of water, and use this solution as the test solution. Pipet 1 mL of the test solution, add water to make exactly 100 mL, and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 1

Bezafibrate
O O N H Cl H3C CO2H CH3

C19H20ClNO4 : 361.82 Bezafibrate contains not less than 98.5% and not more than 101.0% of bezafibrate (C19H20ClNO4), calculated on the dried basis. Description Bezafibrate is a white, crystalline powder. Bezafibrate is freely soluble in dimethylformamide, soluble in methanol, slightly soluble in dehydrated ethanol, and practically insoluble in water. Identification (1) Determine the absorption spectra of the solutions of Bezafibrate and Bezafibrate RS in methanol (1 in 100000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared absorption spectra of Bezafibrate and Bezafibrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorp-

KP 9 117 tion at the same wavenumbers. (3) Perform the test with Bezafibrate as directed under the Flame Coloration Test (2): it shows a green color. Melting Point Between 181 C and 186 C. nol and diluted 0.5 mol/L ammonium acetate TS (1 in 50) (7 : 3) to make exactly 50 mL. Confirm that the peak area of bezafibrate obtained with 5 L of this solution is equivalent to 7 to 13% of that with 5 L of the standard solution. System performance: Dissolve 20 mg of Bezafibrate and 10 mg of 4-chlorobenzoate in 70 mL of methanol, and add diluted 0.5 mol/L ammonium acetate TS (1 in 50) to make exactly 100 mL. When the procedure is run with 5 L of this solution under the above operating conditions, 4-chlorobenzoate and bezafibrate are eluted in this order with the resolution between these peaks being not less than 3. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of bezafibrate is not more than 2.0%. Time span of measurement: About 2.5 times as long as the retention time of bezafibrate beginning after the solvent peak. Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Accurately weigh about 0.7 g of Bezafibrate, previously dried, dissolve in 50 mL of dehydrated ethanol, and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Perform a blank determination in the same manner and make any necessary correction. Each mL of 0.1 mol/L sodium hydroxide VS = 36.18 mg of C19H20ClNO4 Packaging and Storage Preserve in tight containers.

Purity (1) ChloridesDissolve 3.0 g of Bezafibrate in 15 mL of dimethylformamide, dilute with water to 60 mL, shake well, allow to stand for at least 12 hours, and filter. To 40 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL, and use this solution as the test solution. Separately, mix 0.70 mL of 0.01 mol/L hydrochloric acid, 10 mL of dimethylformamide, and 6 mL of dilute nitric acid. Dilute this solution with water to 50 mL, and use this solution as the control solution (not more than 0.012%). (2) Heavy metalsProceed with 2.0 g of Bezafibrate, according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve 0.1 g of Bezafibrate in 35 mL of methanol, add diluted 0.5 mol/L ammonium acetate TS (1 in 50) to make 50 mL, and use this solution as the test solution. Pipet 1.0 mL of the test solution, add 70 mL of methanol and diluted 0.5 mol/L ammonium acetate TS (1 in 50) to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the areas of the peaks having the relative retention times of about 0.65 and 1.86 with respect to bezafibrate obtained from the test solution are not larger than 0.5 times the peak area of bezafibrate from the standard solution; the area of the peak other than those and other than bezafibrate from the test solution is not larger than 0.2 times the peak area of bezafibrate from the standard solution; and the sum of the total area of the peaks other than the peak of bezafibrate from the test solution is not larger than 0.75 times the peak area of bezafibrate from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column about 4.6 mm inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of methanol and diluted dehydrated acetic acid (1 in 100) (9:4). Flow rate: Adjust the flow rate so that the retention time of bezafibrate is about 6 minutes. System suitability Test for required detectability: Measure exactly 5 mL of the standard solution, and add a mixture of metha-

Bifonazole
H C N

and enantiomer C22H18N2: 310.39 Bifonazole, when dried, contains not less than 98.5% and not more than 101.0% of bifonazole (C22Hl8N2). Description Bifonazole is a white to pale yellow powder, is odorless and tasteless. Bifonazole is freely soluble in dichloromethane, soluble in methanol, sparingly soluble in ethanol, slightly soluble in ether and practically insoluble in water.

118 Monographs, Part I A solution of Bifonazole in methanol (1 in 100) does not show optical rotation. Identification (1) Determine the absorption spectra of the solutions of Bifonazole and Bifonazole RS in methanol (1 in 100000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Bifonazole and Bifonazole RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 147 C and 151 C Purity (1) ChlorideTo 2.0 g of Bifonazole add 40 mL of water, warm for 5 minutes, cool and filter. To 10 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL, Perform the test using this solution as the test solution. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.021%). (2) SulfateTo 10 mL of the filtrate obtained in (1), add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (0.048%). (3) Heavy metalsProceed with 2.0 g of Bifonazole according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Related substancesPerform this test without exposure to daylight, using light-resistant vessels. Dissolve 0.10 g of Bifonazole in 10 mL of methanol and use this solution as the test solution. Pipet 3 mL of the test solution and add methanol to make exactly 100 mL. Pipet 25 mL and 5 mL of this solution, add methanol to make exactly 50 mL each and use these solutions as the standard solutions (1) and (2), respectively. Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate and strong ammonia water (49 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spot with Rf value of about 0.20 from the test solution is not more intense than the spot from the standard solution (1). And the spots other than the spot mentioned above and the principal spot from the test solution are not more intense than the spot from the standard solution (2). Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.15 g of Bifonazole, previously dried and dissolve in dichloromethane to make exactly 50 mL. Pipet 5 mL of this solution in a glassstoppered Erlenmeyer flask, add 10 mL of water, 5 mL of dilute sulfuric acid and 25 mL of dichloromethane and add 2 to 3 drops of a solution of methyl yellow in dichloromethane (1 in 500) as indicator and titrate, while shaking vigorously, with 0.01 mol/L sodium lauryl sulfate VS by a buret with 0.02-mL minimum graduation. The end point is reached when the color of the dichloromethane layer changes from yellow to orange-red after dropwise addition of 0.01 mol/L sodium lauryl sulfate VS, strong shaking and standing for a while. Each mL of 0.01 mol/L sodium lauryl sulfate VS = 3.1039 mg of C22Hl8N2 Packaging and Storage tight containers. Preserve in light-resistant,

Biperidene Hydrochloride

CH2CH2C

OH

HCl

and enantiomer C21H29NOHCl: 347.92 Biperiden Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of biperiden hydrochloride (C21H29NOHCl). Description Biperiden Hydrochloride is a white to brownish and yellowish white, crystalline powder. Biperiden Hydrochloride is freely soluble in formic acid, slightly soluble in methanol or in ethanol and practically insoluble in ether..
o Melting pointAbout 270 C (with decomposition).

Identification (1) Dissolve 20 mg of Biperiden Hydrochloride in 5 mL of phosphoric acid: a green color is observed. (2) Dissolve 10 mg of Biperiden Hydro-chloride in 5 mL of water by heating, cool and add 5 to 6 drops of bromine TS: a yellow precipitate is produced. (3) Determine the absorption spectra of the solutions of Biperiden Hydrochloride and Biperiden Hydrochloride RS (1 in 2000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar

KP 9 119 intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Biperiden Hydrochloride and Biperiden Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (5) Dissolve 20 mg of Biperiden Hydrochloride in 10 mL of water by heating and cool: the solution responds to the Qualitative Tests for chloride. Purity (1) Acid or alkaliTo 1.0 g of Biperiden Hydrochloride, add 50 mL of water, shake vigorously, filter and to 20 mL of the filtrate, add 1 drop of methyl red TS: no red to yellow color is observed. (2) Heavy metalsProceed with 1.0 g of Biperiden Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Biperiden Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.10 g of Biperiden Hydrochloride in 20 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 50 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform, methanol and strong ammonia water (80 : 15 : 2) to a distance of about 15 cm and air-dry the plate. Spray evenly Dragendorffs TS on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.4 g of Biperiden Hydrochloride, previously dried and dissolve in 5 mL of formic acid, add 60 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometic titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 34.792 mg of C21H29NOHCl Packaging and Storage well-closed containers. Preserve in light-resistant,

Bisacodyl
N O H3C C O CH O O C CH3

C22H19NO4: 361.39 Bisacodyl, when dried, contains not less than 98.5% and not more than 101.0% of bisacodyl (C22H19NO4). Description Bisacodyl is a white, crystalline powder. Bisacodyl is freely soluble in glacial acetic acid, soluble in acetone, slightly soluble in ethanol or in ether and practically insoluble in water. Bisacodyl dissolves in dilute hydrochloric acid. Identification (1) Determine the absorption spectra of the solutions of Bisacodyl and Bisacodyl RS in ethanol (3 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Bisacodyl and Bisacodyl RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 132 C and 136 C

Purity (1) ChlorideDissolve 1.0 g of Bisacodyl in 30 mL of acetone and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.35 mL of 0.01 mol/L hydrochloric acid VS, add 30 mL of acetone, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.012%). (2) SulfateDissolve 1.0 g of Bisacodyl in 2 mL of dilute hydrochloric acid and add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.35 mL of 0.005 mol/L sulfuric acid VS, add 2 mL of dilute hydrochloric acid and water to make 50 mL (not more than 0.017%). (3) Heavy metalsProceed with 2.0 g of Bisacodyl according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Related substancesDissolve 0.20 g of Bisacodyl in 10 mL of acetone and use this solution as the test solution. Pipet 1 mL of the test solution, add acetone to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with flu-

120 Monographs, Part I orescent indicator for thin-layer chromatography, Develop the plate with a mixture of methyl ethyl ketone, chloroform and xylene (1 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.5 g of Bisacodyl, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from orange-yellow to green (indicator: 0.5 mL of -naphtholbenzeine TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 36.139 mg of C22H19NO4. Packaging and Storage Preserve in well-closed containers.

solution show the same Rf value. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 Bisacodyl Suppositories, make fine fragments carefully and mix uniformly. Weigh accurately a portion of the fragments, equivalent to about 10 mg of bisacodyl (C22H19NO4), add 40 mL of tetrahydrofuran, warm to 40 C, dissolve by shaking, cool and add tetrahydrofuran to make exactly 50 mL. Pipet 5 mL of this solution, add exactly 5 mL of the internal standard solution and add the mobile phase to make 100 mL. Cool this solution in ice for 30 minutes, centrifuge, filter the supernatant liquid through a membrane filter with pore size of 0.5 m, discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately about 10 mg of Bisacodyl RS, previously dried at 105 C for 2 hours and dissolve in tetrahydrofuran to make exactly 50 mL. Pipet 5 mL of this solution, proceed in the same manner as the test solution and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of bisacodyl to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of bisacodyl (C22H19NO4) = amount (mg) of Bisacodyl RS
QT QS

Bisacodyl Suppositories
Bisacodyl Suppositories contain not less than 90.0% and not more than 110.0% of the labeled amount of bisacodyl (C22H19NO4: 361.39). Method of Preparation Prepare as directed under Suppositories, with Bisacodyl. Identification (1) To a portion of Bisacodyl Suppositories, equivalent to 6 mg of Bisacodyl according to the labeled amount, add 20 mL of ethanol, warm in a waterbath for 10 minutes, shake vigorously for 10 minutes and allow to stand in ice-water for 1 hour. Collect the supernatant after centrifugation, filter the supernatant and to 2 mL of the filtrate, add ethanol to make 20 mL. Determine the absorption spectrum of the solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 261 nm and 265 nm. (2) Use the filtrate obtained in (1) as the test solution, Separately, dissolve 6 mg of Bisacodyl RS in 20 mL of ethanol and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of methyl ethyl ketone, chloroform and xylene (1 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spot from the test solution and the standard

Internal standard solutionA solution of ethyl parahydroxybenzoate in acetonitrile (3 in 100000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of 0.01 mol/L citric acid TS, acetonitrile and methanol (2 : 1 : 1). Flow rate: Adjust the flow rate so that the retention time of bisacodyl is about 8 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the internal standard and bisacodyl are eluted in this order with the resolution between their peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 20 L each of the standard solution under the above operating conditions, the relative standard devia-

KP 9 121 tion of the ratios of the peak area of bisacodyl to that of the internal standard is not more than 1.0%. Packaging and Storage Preserve in tight containers. standard solution, respectively. Amount (mg) of bisacodyl (C22H19NO4) Q = amount (mg) of Bisacodyl RS T QS Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 265 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography the particle size is 5 to 10 m. Column temperature: A room temperature. Mobile phase: 55 mL of 0.074 mol/L sodium acetate solution, adjust pH 7.4 with 2.5 vol% acetic acid, add 45 mL of acetonitrile. Flow rate: 2 mL/minute. System suitability System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area is not more than 2.0%. Packaging and Storage Preserve in well-closed containers (under 30 C).

Bisacodyl Tablets
Bisacodyl Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of bisacodyl (C22H19NO4: 361.39). Bisacodyl Tablets are enteric coated. Method of Preparation Prepare as directed under Tablets, with Bisacodyl. Identification To a quantity of powdered Bisacodyl Tablets, equivalent to about 0.3 g of Bisacodyl according to the labeled amount, add 100 mL of acetone, shake, heat in a water-bath to boiling, filter and evaporate to about 20 mL. Add 200 mL of water, evaporate acetone in a water-bath, under the nitrogen gas. Cool, filter with glass filtrator (G4) after 30 minutes and discard the filtrate. Dissolve the residue with 50 mL of acetone, evaporate to about 15 mL, add about 75 mL of water, heat in a water-bath for 15 minutes and cool. Crystallize by scratching the wall of beaker, filter, dry at 105 C for about 15 minutes and proceed the test with the residue as directed under Identification (2) of Bisacodyl. Disintegration Test When the procedure is run as directed for the enteric coated tablets, the tablets do not disintegrated after 60 minutes in the first fluid, but then disintegrated within 45 minutes in the second fluid. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 Bisacodyl Tablets and powder. Weigh accurately a portion of the powder, equivalent to about 25 mg of bisacodyl (C22H19NO4), add 10 mL of water, shake thoroughly for 15 minutes and shake vigorously for 15 minutes, add 30 mL of acetonitrile, shake for 15 minutes and shake vigorously for 15 minutes, add acetonitrile to make 50 mL, mix and filter. Discard the first 10 mL of the filtrate and exactly take the subsequent 5 mL of the filtrate. Add exactly 5 mL of the internal standard. Use the this solution as the test solution. Separately, Bisacodyl RS, dried at 105 C for 2 hours, weigh accurately 25 mg, and add acetonitrile to make exactly 50 mL. Take exactly 5 mL of this solution, and treat as directed to prepare the test solution. Use this solution as the standard solution. Perform the test with each 20 L of the test solution and the standard solution, as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and Q S of the peak area of Biscodyl to that of the internal standard for the test solution and

Bismuth Subgallate
Dermatol Bismuth Subgallate, when dried, contains not less than 47.0% and not more than 51.0% of bismuth (Bi: 208.98). Description Bismuth Subgallate is a yellow powder, is odorless and tasteless. Bismuth Subgallate is practically insoluble in water, in ethanol or in ether. Bismuth Subgallate dissolves in dilute hydrochloric acid, in dilute nitric acid or in dilute sulfuric acid on warming. Bismuth Subgallate dissolves in sodium hydroxide TS, forming a clear, yellow solution, which turns red immediately. Bismuth Subgallate is affected by light. Identification (1) Ignite 0.5 g of Bismuth Subgallate: it chars at first and leaves finally a yellow residue. The residue responds to the Qualitative Tests for bismuth salt. (2) To 0.5 g of Bismuth Subgallate, add 25 mL of water and 20 mL of hydrogen sulfide TS and shake well. Filter off the blackish brown precipitate and add 1 drop of ferric chloride TS to the filtrate: a blue-black color is produced. Purity (1) Clarity of solution Dissolve 1.0 g of Bismuth Subgallate in 40 mL of diluted sodium hydrox-

122 Monographs, Part I ide TS (1 in 8): the solution is clear. (2) SulfateIgnite 3.0 g of Bismuth Subgallate in a porcelain crucible and cautiously dissolve the residue in 2.5 mL of nitric acid by warming. Pour the solution into 100 mL of water, shake and filter. Evaporate 50 mL of the filtrate in a water-bath to 15 mL. Add water to make 20 mL, filter again and use the filtrate as the test solution. To 5 mL of the test solution, add 2 to 3 drops of barium nitrate TS: no turbidity is produced. (3) NitrateTo 0.5 g of Bismuth Subgallate, add 5 mL of dilute sulfuric acid and 25 mL of ferrous sulfate TS, shake well and filter. Superimpose carefully 5 mL of the filtrate on sulfuric acid: no red-brown color develops at the zone of contact. (4) AmmoniumDissolve 1.0 g of Bismuth Subgallate in 5 mL of sodium hydroxide TS and heat: the gas evolved does not change moistened red litmus paper to blue. (5) CopperTo 5 mL of the test solution obtained in (2), add 1 mL of ammonia TS and filter: no blue color develops in the filtrate. (6) LeadIgnite 1.0 g of Bismuth Subgallate at about 500 C in a porcelain crucible, dissolve the residue in a smallest possible amount of nitric acid added drop-wise, evaporate over a low flame to dryness and cool. Add 5 mL of a solution of potassium hydroxide (1 in 6) to the residue, boil carefully for 2 minutes, cool and centrifuge. Take the supernatant liquid in a test tube, add 10 drops of potassium chromate TS and acidify the solution by adding glacial acctic acid drop-wise: neither turbidity nor a yellow precipitate is produced. (7) SilverTo 5 mL of the test solution obtained in (2), add 0.5 mL of nitric acid and 2 to 3 drops of dilute hydrochloric acid: no turbidity is produced. (8) Alkaline earth metals and alkali metalsBoil 1.0 g of Bismuth Subgallate with 40 mL of diluted acetic acid (1 in 2) for 2 minutes, cool, add water to make 40 mL and filter. To 20 mL of the filtrate, add 2 mL of dilute hydrochloric acid, boil, immediately pass hydrogen sulfide thoroughly through the solution, filter the precipitate produced and wash with water. Combine the filtrate and the washings, add 5 drops of sulfuric acid and evaporate to dryness. Ignite as directed under the Residue on Ignition: the weight of the residue does not more than 5.0 mg. (9) ArsenicMix well 0.20 g of Bismuth Subgallate with 0.20 g of calcium hydroxide and ignite the mixture. Dissolve the residue in 5 mL of dilute hydrochloric acid, use this solution as the test solution and perform the test (not more than 10 ppm). (10) Gallic acidTo 1.0 g of Bismuth Subgallate, add 20 mL of ethanol, shake for 1 minute and filter. Evaporate the filtrate in a water-bath to dryness: the weight of the residue does not more than 5.0 mg. Loss on Drying Not more than 6.0% (1 g, 105 C, 3 hours). Assay Weigh accurately 0.5 g of Bismuth Subgallate,
o

previously dried, ignite at about 500 C for 30 minutes and cool. Dissolve the residue in 5 mL of diluted nitric acid (2 in 5) by warming and add water to make exactly 100 mL. Measure exactly 30 mL of this solution, add 200 mL of water and titrate with 0.02 mol/L disodium ethylenediaminetetraacetate VS until the color of the solution changes from red-purple to yellow (indicator: 2 to 3 drops of xylenol orange TS). Each mL of 0.02 mol/L disodium ethylenediaminetetraacetate VS = 4.180 mg of Bi Packaging and Storage well-closed containers. preserve in light-resistant,

Bismuth Subnitrate
Bismuth Subnitrate, when dried, contains not less than 71.5% and not more than 74.5% of bismuth (Bi: 208.98). Description Bismuth Subnitrate is a white powder. Bismuth Subnitrate is practically insoluble in water, in ethanol or in ether. Bismuth Subnitrate readily dissolves in hydrochloric acid or nitric acid without effervescence. Bismuth Subnitrate is slightly hygroscopic and changes moistened blue litmus paper to red. Identification Bismuth Subnitrate responds to the Qualitative Tests for bismuth salt and for nitrate. Purity (1) ChlorideDissolve 0.7 g of Bismuth Subnitrate in 2 mL of water and 2 mL of nitric acid and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: evaporate 2 mL of nitric acid on a water-bath to dryness, add 0.70 mL of 0.01 mol/L hydrochloric acid VS, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.035%). (2) SulfateDissolve 3.0 g of Bismuth Subnitrate in 3.0 mL of warmed nitric acid, pour this solution into 100 mL of water and shake. Concentrate the filtrate in a water-bath to 30 mL, filter and use this filtrate as the test solution. To 5 mL of the test solution, add 2 to 3 drops of barium nitrate TS: no turbidity is produced. (3) AmmoniumBoil 0.10 g of Bismuth Subnitrate with 5 mL of sodium hydroxide TS: the gas evolved does not change moistened red litmus paper to blue. (4) CopperTo 5 mL of the test solution obtained in (2), add 2 mL of ammonia TS and filter: no blue color develops. (5) LeadTo 1.0 g of Bismuth Subnitrate, add 5 mL of a solution of sodium hydroxide (1 in 6), boil carefully for 2 minutes, cool and centrifuge. Transfer the supernatant liquid to a test tube, add 10 drops of potassium chromate TS and add drop-wise acetic acid to render the

KP 9 123 solution acid: no turbidity or yellow precipitate is produced. (6) SilverTo 5 mL of the test solution obtained in (2), add 0.5 mL of nitric acid and 2 to 3 drops of dilute hydrochloric acid: no turbidity is produced. (7) Alkaline earth metals and alkali metals Boil 2.0 g of Bismuth Subnitrate with 40 mL of diluted acetic acid (1 in 2) for 2 minutes, cool, add water to make 40 mL and filter. To 20 mL of the filtrate, add 2 mL of dilute hydrochloric acid, boil, immediately pass hydrogen sulfide thoroughly through the solution, filter and wash the residue with water. Combine the filtrate and the washings, add 5 drops of sulfuric acid, evaporate to dryness and ignite as directed under the Residue on Ignition: the residue does not exceed 5.0 mg. (8) ArsenicTo 0.20 g of Bismuth Subnitrate, add 2 mL of sulfuric acid, heat until white fumes evolve, dilute cautiously with water to make 5 mL, use this solution as the test solution and perform the test (not more than 10 ppm). Loss on Drying Not more than 3.0% (2 g, 105 C, 2 hours). Assay Weigh accurately 0.4 g of Bismuth Subnitrate, previously dried, dissolve in 5 mL of diluted nitric acid (2 in 5) by warming and add water to make exactly 100 mL. Pipet 25 mL of the solution, add 200 mL of water and titrate with 0.02 mol/L disodium ethylenediaminetetraacetate VS until the color of the solution changes from red-purple to yellow (indicator: 5 drops of xylenol orange TS). Each mL of 0.02 mol/L disodium ethylenediaminetetraacetate VS = 4.180 mg of Bi Packaging and Storage Preserve in well-closed containers. ethyl acetate. Identification (1) Determine the infrared absorption spectra of Bisoprolol Fumarate and Bisoprolol Fumarate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The retention time of the principal peak from the test solution corresponds to that from the standard solution, as obtained in the Assay. Specific Optical Rotation [ ]20 D : Between 2 and +0.2 (0.2 g, methanol 20 mL, 100 mm) Purity (1) Heavy metalsProceed with 1.0 g of Bisoprolol Fumarate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Fumaric acidDissolve about 0.5 g of Bisoprolol Fumarate, accurately weighed, in 70 mL of dehydrated alcohol. Add 8.0 mL of 0.1 mol/L tetrabutylammonium hydroxide VS, stir for 2 minutes, and titrate with 0.1 mol/L tetrabutylammonium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction. Not less than 14.8% and not more than 15.4% of fumaric acid is found, calculated on the anhydrous basis. Each mL of 0.1 mol/L tetrabutylammonium hydroxide VS = 5.804 mg of fumaric acid (C4H4O4) (3) Related substances Prepare the test solution as directed in the Assay. Perform the test with 10 L of the test solution as directed under the Liquid Chromatography according to the procedure in the Assay, and measure peak areas. When the percentage of total related substances in the portion of Bisoprolol Fumarate is calculated, not more than 0.5% of total related substances is found. Amount (%) of total related substances = 100
H N COOH CH3

Bisoprolol Fumarate
OH O CH3 O H3C O 2 CH3

Ai AS

.
HOOC

(C18H31NO4)2C4H4O4 : 766.96 Bisoprolol Fumarate contains not less than 97.5% and not more than 102.0% of bisoprolol fumarate (C18H31NO4)2C4H4O4, calculated on the anhydrous basis. Description Bisoprolol Fumarate is a white crystalline powder. Bisoprolol Fumarate is very soluble in water or in methanol, freely soluble in ethanol, in glacial acetic acid or in chloroform, and sparingly soluble in acetone or in

Ai is the sum of all the peak areas, excluding the peak areas of fumaric acid and bisoprolol. AS is the sum of all the peak areas in the chromatograpm.

Water Not more than 0.5%. (1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve 50 mg of Bisoprolol Fumarate, accurately weighed, in a mixture of acetonitrile and water (35:65) to make exactly 50 mL, and use this solution as

124 Monographs, Part I the test solution. Separately, weigh accurately about 50 mg of Bisoprolol Fumarate RS, dissolve in 35% acetonitrile solution to make exactly 50 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine the peak areas, AT and AS , of Bisoprolol Fumarate. Amount (mg) of bisoprolol fumarate [(C18H31NO4)2C4H4O4] = 50 C AT
AS
OH OH O OH O OH O O N H

Bleomycin Sulfate
O NH2 H N H N N H H H2N CH3 HN H O H N N H O HO O H HO CH3 H CH3 O CH3 H N O H N H S H NH2 NH2 N O N O R

x H2SO4
OH O NH2 OH

C : the concentration (mg/mL) of Bisoprolol Fumarate RS in the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 273 nm). Column: A stainless steel column about 4.6 mm inside diameter and 12.5 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 10 m in particle diameter). Mobile phase: To 1000 mL of 35% acetonitrile solution, add 5 mL of heptafluorobutyric acid, 5 mL of diethylamine, and 2.5 mL of formic acid, mix, and filter. Flow rate: 1.0 mL/min. System suitability System performance: Dissolve 25.0 mg of propranolol hydrochloride and 50.0 mg of Bisoprolol Fumarate in 35% acetonitrile solution to make 50 mL. When the procedure is run with 10 L of this solution as directed in the Liquid chromatography under the above operating conditions, the resolution between the peaks of bisoprolol and propranolol is not less than 7.0. When the procedure is run with 10 L of the standard solution, the symmetry factor is not more than 2.0. System repeatability: When the test is repeated 5 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of bisoprolol is not more than 2.0%. Packaging and Storage resistant containers. Preserve in tight, light-

Bleomycin acid: R = OH Bleomycin A1: R = NHCH2CH2CH2SOCH3 Bleomycin dimethyl: A2 R = NHCH2CH2CH2SCH3 CH3 Bleomycin A2: R = NHCH2CH2CH2S+ XCH3 Bleomycin A2'-a: R = NHCH2CH2CH2CH2NH2 Bleomycin A2'-b: R = NHCH2CH2CH2NH2 Bleomycin A5: R = NH(CH2)3NH(CH2)4NH2 Bleomycin B1: R = NH2 Bleomycin B2: R = NH(CH2)4NHC(NH)NH2 Bleomycin B4: R= NH(CH2)4NHC(NH)NH(CH2)4NHC(NH)NH2 Bleomycin Sulfate is the sulfate of a mixture of substances having antitumor activity produced by the growth of Streptomyces verticillus. Bleomycin Sulfate contains not less than 1400 g (potency) and not more than 2000 g (potency) per mg of bleomycin A2 (C55H84ClN17O21S3: 1451.01), calculated on the dried basis. Description Bleomycin Sulfate is a white to yellowish white powder. Bleomycin Sulfate is freely soluble in water, slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Weigh 10 mg of Bleomycin Sulfate, dissolve in 2 mL of water, and add barium chloride TS to this solution; a white precipitate develops. This precipitate does not dissolve in diluted nitric acid, but dissolve in excess ammonia TS. (2) Weigh 4 mg of Bleomycin Sulfate, add 5 L of copper sulfate TS and water to make 100 mL. Determine the absorption spectrum of this solution as directed under Ultraviolet-visible Spectrophotometry, it exhibits maxima between 240 nm and 243 nm, and between 288 nm and 293 nm, and minimum between 267 nm and 270 nm. pH The pH of a solution obtained by dissolving 50 mg of Bleomycin Sulfate in 10 mL of water is between 4.5 and 6.0.

KP 9 125 Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter) Column temperature: A constant temperature of about 40 C Mobile phase: Control the gradient by mixing the mobile phase A and B as directed in the following table. Mobile phase A : A mixture of the mobile phase stock solution and methanol (9:1) Mobile phase B : A mixture of the mobile phase stock solution and methanol (3:2) Time ( min) 0 ~ 60 60 ~ 75 Mobile phase A (vol %) 100 0 0 Mobile phase B (vol %) 0 100 100

Purity Content of copper Weigh accurately about 15 mg of Bleomycin Sulfate, dissolve in diluted hydrochloric acid (1 in 100), make exactly 10 mL, and use this solution as the test solution. Pipet exactly 10 mL each of the test solution and the copper standard solution, add 10 mL of zinc dibenzyldithiocarbamate TS, shake vigorously for one minute, allow to stand to separate the layers, take the lower layer, if necessary, add 1 g of anhydrous sodium sulfate, shake to dehydrate and filter. Separately, make a blank solution with the same manner except the addition of copper standard solution. Determine the absorbance of the test solution, the standard solution and the blank solution at 435 nm, AT , AS and AO , as directed under Ultraviolet-visible Spectrophotometry, and calculate the content of copper according to the following equation (not more than 0.02 %). Amount [%] of copper =
1.5 ( AT - AO ) Amount (mg) of the sample taken ( AS - AO )

Sterility Test It meets the requirement, when Bleomycin Sulfate is used in a sterile preparation. Pyrogen Test It meets the requirement, when Bleomycin Sulfate is used in a sterile preparation. Weigh appropriate amount of Bleomycin Sulfate, dissolve in water, make the solution so that each mL contains 200 g, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Histamine It meets the requirement, when Bleomycin Sulfate is used in a sterile preparation. Weigh appropriate amount of Bleomycin Sulfate, dissolve in water, make the solution so that each mL contains 300 g, and use the solution as the test solution. Loss on Drying Not more than 3.0 % (50 mg, in vacuum, 60 C, 3 hours). Content Ratio of Bleomycin Weigh accurately 10 mg (potency) of Bleomycin Sulfate, dissolve in 20 mL of water, and use this solution as the test solution. Perform the test with 20 L of the test solution as directed under the Liquid Chromatography according to the following operating conditions, and determine each peak area. Based on the % of peak area, bleomycin A2 (the first principal peak) is between 55 % and 70 %, bleomycin B2 (the second principal peak) is between 25 % and 32 %, the total peak area of bleomycin A2 and bleomycin B2 is not less than 85 %, the peak area of dimethylbleomycin A2 ( a peak having the relative retention time of 1.5 ~ 2.5 against bleomycin A2) is not more than 5.5 %, and the total area of the rest peaks is not more than 9.5 %. Operating conditions

Flow rate: About1.2 mL per min System suitability System performance: When the procedure is run with 20 L of the test solution under the above operating conditions, bleomycin A2 and bleomycin B2 are eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 20 L of the test solution under the above operating conditions, the relative standard deviation of the each peak area is not more than 2.0 %. Mobile phase stock solution: Dissolve 0.96 g of sodium 1-pentanesulfonate and 1.86 g of disodium dihydrogen ethylenediamine tetraacetate dehydrate in 1000 mL of water and 5 mL of glacial acetic acid, and adjust the pH to 4.3 with ammonia TS. Time span of measurement: Twenty minutes after elution of the peak of demethylbleomycin A2 beginning after the solvent peak. Assay The Cylinder-plate method (1) Agar media for seed and base layerPeptone 10.0 g Glycerine 10.0 g Meat extract 10.0 g Agar 15.0 20.0 g Sodium chloride 1.5 g Water 1000 mL Mix all the ingredients, and sterilize. Adjust the pH of the solution so that it will be 6.9 to 7.1 after sterilization. (2) Liquid media for suspending the test organism Peptone 10.0 g Sodium chloride 3.0 g Meat extract 10.0 g Glycerine 10.0 g Water 1000 mL Mix all the ingredients, and sterilize. Adjust the pH of the solution so that it will be 6.9 to 7.1 after sterilization. (3) Test organism - Mycobacterium smegmatis ATCC 607. (4) Preparation of seeded agar layer Cultivate the

126 Monographs, Part I test organism, on the slant of the agar medium for transferring the test organism at 27 C for 40 to 48 hours, then inoculate the test organism thus obtained in 100 mL of the liquid media for suspending the test organism, cultivate with shaking at between 25 C and 27C for 5 days, and use this as the suspension of test organism. Store the suspension of test organism at a temperature not exceeding 5 C, and use within 14 days. Add 0.5 mL of the suspension of test organism in 100 mL of the agar medium for seed previously kept at 48 C, mix thoroughly, and use as the seeded agar layer. (5) Agar plate Proceed as directed in I 5 under the Microbial Assay for Antibiotics. Use 5.0 mL of agar medium for base layer and 8.0 mL of the agar medium for seed. (6) Weigh accurately about 5 mg (potency) of Bleomycin Sulfate, dissolve in 0.1 mol/L phosphate buffer solution, pH 6.8 to make the solutions so that each mL contains 30.0 g (potency) and 15.0 g (potency), and use these solutions as the high concentration test solution and the low concentration test solution. Separately, weigh accurately an amount of Bleomycin Hydrochloride RS, previously dried, equivalent to about 5 mg (potency) and dissolve in 0.1 mol/L phosphate buffer solution, pH 6.8 to make the solution so that each mL contains 150.0 g (potency) and use the solution as the standard stock solution. Keep the standard solution at not exceeding 5 C and use within 30 days. Take exactly a suitable amount of the standard stock solution, add 0.1 mol/L phosphate buffer solution, pH 6.8 to make solutions so that each mL contains 30.0 g (potency) and 15.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in tight containers (2 ~ 8 C). C14H10BrN3O: 316.15 Bromazepam, when dried, contains not less than 99.0% and not more than 101.0% of bromazepam (C14H10BrN3O). Description Bromazepam is a white to pale yellowish white crystal or crystalline powder and is odorless. Bromazepam is freely soluble in dimethylformamide or in glacial acetic acid, sparingly soluble in chloroform, slightly soluble in methanol or in dehydrated ethanol, very slightly soluble in ether and practically insoluble in water. Bromazepam dissolves in dilute hydrochloric acid. Melting point About 245 C (with decomposition). Identification (1) Determine the absorption spectra of the solutions of Bromazepam and Bromazepam RS in dehydrated ethanol (1 in 200000) under the Ultravioletsponds to the Qualitative Tests for borate. Purity (1) Clarity and color of solution Dissolve 1.0 g of Boric acid in 25 mL of water or in 10 mL of hot ethanol: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Boric Acid according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 0.40 g of Boric Acid according to Method 1 and perform the test (not more than 5 ppm). Loss on Drying Not more than 0.5% (2 g, silica gel, 5 hours). Assay Weigh accurately 1.5 g of Boric Acid, previously dried, add 15 g of D-Sorbitol and 50 mL of water, ant dissolve by warming. After cooling, titrate with 1 mol/L sodium hydroxide VS (indicator: 2 drops of phenolphthalein TS). Each mL of 1 mol/L sodium hydroxide VS = 61.83 mg of H3BO3 Packaging and Storage Preserve in well-closed containers.

Bromazepam
H N O

Br

Boric Acid
H3BO3: 61.83 Boric Acid, when dried, contains not less than 99.5% and not more than 101.0% of boric acid (H3BO3). Description Boric Acid is a colorless or white crystal or crystalline powder, odorless and has a slight, characteristic taste. Boric Acid is freely soluble in warm water, in hot ethanol or in glycerin, soluble in water or in ethanol and practically insoluble in ether. pH A solution of Boric Acid (1 in 20) is between 3.5 and 4.1. Identification A solution of Boric acid (1 in 20) re-

KP 9 127 visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the absorption spectra of Bromazepam and Bromazepam RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelnumbers. Purity (1) Heavy metalsProceed with 1.0 g of Bromazepam in a platinum crucible according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Related substancesDissolve 50 mg of Bromazepam in 5 mL of a mixture of acetonitrile and methanol (3 : 2) and use this solution as the test solution. Pipet 1 mL of the test solution and add a mixture of acetonitrile and methanol (3 : 2) to make exactly 50 mL. Pipet 5 mL of this solution, add a mixture of acetonitrile and methanol (3 : 2) to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, dehydrated ethanol and strong ammonia water (38 : 1 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution and the spot of the starting point are not more than 2 and not more intense than the spot from the standard solution. Loss on Drying Not more than 0.20% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.4 g of Bromazepam, previously dried, dissolve in 80 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 31.615 mg of C14H10BrN3O Packaging and Storage Preserve in well-closed containers.

C14H20Br2N2HCl: 412.59 Bromhexine Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of bromhexine (C14H20Br2N2HCl). Description Bromhexine Hydrochloride is a white crystal or crystalline powder, is odorless and tasteless. Bromhexine Hydrochloride is freely soluble in formic acid, sparingly soluble in methanol and slightly soluble in water or ethanol and practically insoluble in ether. pH The pH of saturated solution of Bromhexine Hydrochloride is between 3.0 and 5.0. Melting point About 239 C (with decomposition). Identification (1) Dissolve 3 mg each of Bromhexine Hydrochloride and Bromhexine Hydrochloride RS in 0.01 mol/L hydrochloric acid TS to make 100 mL. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Bromhexine Hydrochloride and Bromhexine Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Add 20mL of water to 1 g of Brohexine Hydrochloride. After thorough shaking, add 3 mL of sodium hydroxide TS and extract with four 20 mL portions of ether. Neutralize the water layer with dilute nitric acid: the solution responds to the Qualitatve Test (2) for chloride. Purity (1) Heavy metalsProceed with 2.0 g of Bromhexine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesPerform this test without exposure to daylight, using light-resistant vessels. Dissolve 50 mg of Bromhexine Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution and add the mobile phase to make exactly 20 mL. Pipet 1 mL of this solution, add the mobile phase to make exactly 25 mL and this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine each peak area by the automatic integration method: each peak area other than bromhexine from the test solution is not larger than the peak area of bromhexine from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 245 nm) Column: A stainless steel column about 5 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5

Bromhexine Hydrochloride
Br CH3 CH2 N HCl

Br

NH2

128 Monographs, Part I m in particle diameter). Column temperature: A constant temperature of about 40 C Mobile phase: Dissolve 1.0 g of potassium hydrogen phosphate in 900 mL of water, adjust the pH to 7.0 with 0.5 mol/L sodium hydroxide TS and add water to make 1000 mL. To 200 mL of this solution 800 mL of acetonitrile. Flow rate: Adjust flow rate so that the retention time of bromhexine is about 6 minutes. System suitability System performance: To 50 mg of bamethane sulfate add 0.5 mL of the test solution and add the mobile phase to make 10 mL. When the procedure is run with 5 L of this solution under the above operating conditions, bamethane and bromhexine are eluted in this order with the resolution between these peaks being not less than 7. Detection sensitivity: Adjust the detection sensitivity so that the peak height of bromhexine from 5 L of the standard solution is between 5 mm and 15 mm. Time span of measurement: About 2 times as long as the retention time of bromhexine after the solvent peak. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.5 g of Bromhexine Hydrochloride, previously dried, dissolve in 2 mL of formic acid, add 60 mL of acetic anhydride and warm in a 50 C water-bath and cool. Titrate with 0.1 mol/L perchloric acid until the color of the solution changes from purple through blue-green to yellow-green (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 41.26 mg of C14H20Br2N2HCl Packaging and Storage well-closed containers. Preserve in light-resistant,

Bromocriptine Mesilate contains not less than 98.0% and not more than 101.0% of bromocriptine mesilate (C32H40BrN5O5CH4O3S), calculated on the dried basis. Description Bromocriptine Mesilate is a white to pale yellowish white or pale brownish white, crystalline powder and is odorless or has a faint characteristic odor. Bromocriptine Mesilate is very soluble in glacial acetic acid, freely soluble in methanol, sparingly soluble in ethanol, very slightly soluble in acetic anhydride, in dichloromethane or in chloroform and practically insoluble in water or in ether. Bromocriptine Mesilate is gradually affected by light. Identification (1) Dissolve 2 mg of Bromocriptine Mesilate in 1 mL of methanol, add 2 mL of pdimethylaminobenzaldehyde-ferric chloride TS and shake: a purplish blue color is observed. (2) Determine the absorption spectra of the solutions of Bromocriptine Mesilate and Bromocriptine Mesilate RS in methanol (3 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorptioni at the same wavelengths. (3) Determine the infrared spectra of Bromocriptine Mesilate and Bromocriptine Mesilate RS, previously dried, as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) Perform the test with Bromocriptine Mesilate as directed under the Flame Coloration Test (2): a green color is observed. Specific Optical Rotation [ ]20 D : Between +95 and +105 [0.1 g previously dried, a mixture of methanol and dichloromethane (1 : 1), 10 mL, 100 mm]. Purity (1) Clarity and color of solutionDissolve 0.10 g of Bromocriptine Mesilate in 10 mL of methanol: the solution is clear and has no more color than the following control solution. Control solutionTo 2.5 mL of cobaltous chloride stock CS, 6.0 mL of ferric chloride stock CS and 1.0 mL of cupric sulfate stock CS, add diluted hydrochloric acid (1 in 40) to make exactly 100 mL. (2) Heavy metalsProceed with 1.0 g of Bromocriptine Mesilate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesPerform this test without exposure to daylight, using light-resistant vessels. Dissolve 0.10 g of Bromocriptine Mesilate in 10 mL of a mixture of methanol and chloroform (1 : 1) and use this solution as the test solution. Pipet 1 mL of the test solution, add a mixture of methanol and chloroform (1 : 1) to make exactly 200 mL and use this solution as the standard solution (1). Pipet 10 mL of the standard solution (1), add a mixture of methanol and chloroform (1 : 1) to make exactly 20 mL and use this solution as the standard

Bromocriptine Mesilate
O (CH3)2CH H C H N O N H N N H O CH3 H O CH2CH(CH 3)2 CH3SO 3H OH

HN Br

C32H40BrN5O5CH4O3S: 750.70

KP 9 129 solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solutions (1) and (2), as a band with 1 cm in width, on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate immediately with a mixture of dichloromethane, dioxane, ethanol and strong ammonia water (1800 : 150 : 50 : 1) to a distance of about 10 cm and dry the plate under reduced pressure for 30 minutes. Spray evenly Dragendorffs TS on the plate, then spray evenly hydrogen peroxide TS, cover the plate with a glass plate and examine: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution (1) and the spot other than the principal spot, which is more intense than the spot from the standard solution (2), is not more than one. Loss on Drying Not more than 3.0% (1 g, not exceeding 0.67 kPa, 80 C, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.6 g of Bromocriptine Mesilate, dissolve in 80 mL of a mixture of glacial acetic acid and acetic anhydride (7 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 75.07 mg of C32H40BrN5O5CH4O3S Packaging and Storage Preserve in light-resistant, tight containers. Store at a temperature below 18 C. Bromovalerylurea dissolve in sodium hydroxide TS. Identification (1) Boil 0.2 g of Bromovalerylurea with 5 mL of a solution of sodium hydroxide (1 in 10): the gas evolved changes moistened red litmus paper to blue. Boil this solution with an excess of dilute sulfuric acid: the odor of valeric acid is perceptible, (2) To 0.1 g of Bromovalerylurea, add 0.5 g of anhydrous sodium carbonate and decompose thoroughly by gentle heating. Dissolve the residue in 5 mL of hot water, cool, acidify with acetic acid and filter: the filtrate responds to the Qualitative Tests (2) for bromide. Melting Point Between 151 C and 155 C.

Purity (1) Acidity or alkalinityTo 1.5 g of Bromovalerylurea, add 30 mL of water, shake for 5 minutes and filter: the filtrate is neutral. (2) ChloridePerform the test with a 10 mL of the filtrate obtained in (1). Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.028%). (3) SulfatePerform the test with 10 mL of the filtrate obtained in (1). Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.038%). (4) Heavy metalsProceed with 2.0 g of Bromovalerylurea according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) ArsenicDissolve 0.5 g of Bromovalerylurea in 5 mL of sodium hydroxide TS, use this solution as the test solution and perform the test (not more than 4 ppm). (6) Readily carbonizable substancesPerform the test with 0.5 g of Bromovalerylurea: the solution has no more color than Color Matching Fluid A. Loss on Drying Not more than 0.5% (1 g, 80 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.4 g of Bromovalerylurea, previously dried, in an Erlenmeyer flask, add 40 mL of sodium hydroxide TS and boil gently for 20 minutes under a reflux condenser. Cool, wash the lower part of the reflux condenser and the mouth of the flask with 30 mL of water and combine the washings with the solution in the Erlenmeyer flask. Add 5 mL of nitric acid and exactly 30 mL of 0.1 mol/L silver nitrate VS and titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ferric ammonium sulfate TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L silver nitrate VS = 22.307 mg of C6H11BrN2O2 Packaging and Storage Preserve in well-closed con-

Bromvalerylurea
Br (CH3)2CHC H O C NH O C NH2

and enantiomer C6H11BrN2O2: 223.07 Bromovalerylurea, when dried, contains not less than 98.0% and not more than 101.0% of bromovalerylurea (C6H11BrN2O2). Description Bromovalerylurea is a white crystal or crystalline powder, is odorless and has a slightly bitter taste. Bromovalerylurea is soluble in ethanol, sparingly soluble in ether and very slightly soluble in water. Bromovalerylurea dissolves in sulfuric acid, nitric acid or hydrochloric acid and precipitates are produced on the addition of water.

130 Monographs, Part I tainers. Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Bucumolol Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.10 g of Bucumolol Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution and add methanol to make exactly 50 mL. Pipet 5 mL of this solution, add methanol to make exactly 25 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methanol and ammoniaammonium chloride buffer solution, pH 10.7 (30 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.4 g of Bucumolol Hydrochloride, previously dried, add 45 mL of glacial acetic acid, dissolve by warming at 60 C and cool. Add 105 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometic titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 34.183 mg of C17H23NO4.HCl Packaging and Storage Preserve in well-closed containers.

Bucumolol Hydrochloride
OH OCH2CCH2NHCH(CH3)3 H O O HCl

CH3

C17H23NO4HCl: 341.83 Bucumolol Hydrochloride, when dried, contains not less than 99.0% of bucumolol hydrochloride (C17H23NO4HCl). Description Bucumolol Hydrochloride is a white crystal or crystalline powder. Bucumolol Hydrochloride is freely soluble in water, sparingly soluble in methanol or in ethanol, slightly soluble in glacial acetic acid and practically insoluble in ether. Melting point About 228 C (with decomposition) Identification (1) Dissolve 10 mg of Bucumolol Hydrochloride in 10 mL of diluted ethanol (1 in 2) and observe under ultraviolet light (main wavelength: 365 nm): the solution has a yellow-green fluorescence. Render this solution alkaline by adding sodium hydroxide TS: the fluorescence disappears. Acidify the solution by adding dilute hydrochloric acid: the fluorescence reappears. (2) Dissolve 0.1 g of Bucumolol Hydrochloride in 5 mL of water and add 5 drops of Reinecke salt TS: a light red precipitate is formed. (3) Determine the absorption spectra of the solutions of Bucumolol Hydrochloride and Bucumolol Hydrochloride RS (1 in 60000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Bucumolol Hydrochloride and Bucumulol Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (5) A solution of Bucumolol Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride.
1% Absorbance E1 cm (296 nm): Between 330 and 360 (after drying, 40 mg, water, 2500 mL).

Bufetolol Hydrochloride
OH OCH2CHCH2NHC(CH3)3 HCl

OCH2

Purity (1) Clarity and color of solutionDissolve 1.0 g of Bucumolol Hydrochloride in 20 mL of water: the solution is clear and colorless to pale yellow. (2) Heavy metalsProceed with 1.0 g of Bucumolol

C18H29NO4HCl: 359.89 Bufetolol Hydrochloride, when dried, contains not less than 98.5% of bufetolol hydrochloride (C18H29NO4HCl).

KP 9 131 Residue on Ignition Not more than 0.1% (1 g). Description Bufetolol Hydrochloride is a white crystal or crystalline powder. Bufetolol Hydrochloride is freely soluble in water or in methanol, soluble in ethanol or in glacial acetic acid and practically insoluble in ether. A solution of Bufetolol Hydrochloride (1 in 10) is optically inactive. Identification (1) To 5 mL of a solution of Bufetolol Hydrochloride (1 in 100), add 5 drops of Reinecke salt TS: a pale red precipitate is produced. (2) Determine the absorption spectra of the solutions of Bufetolol Hydrochloride and Bufetolol Hydrochloride RS (1 in 20000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Bufetolol Hydrochloride and Bufetolol Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) A solution of Bufetolol Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride. Melting Point Between 153 C and 157 C. Purity (1) Clarity and color of solutionDissolve 1.0 g of Bufetolol Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 0.5 g of Bufetolol Hydrochloride. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.038%). (3) Heavy metalsProceed with 2.0 g of Bufetolol Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Related substancesDissolve 0.20 g of Bufetolol Hydrochloride in 5 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, acetone, ethanol and strong ammonia water (40 : 20 : 5 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Assay Weigh accurately 0.4 g of Bufetolol Hydrochloride, previously dried, dissolve in 10 mL of glacial acetic acid, add 50 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometic titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 35.989 mg of C18H29NO4HCl Packaging and Storage Preserve in tight containers.

Bufexamac
O CH3CH2CH2CH2O CH2 C NHOH

Cl2H17NO3: 223.27 Bufexamac, when dried, contains not less than 98.0% and not more than 101.0% of bufexamac (Cl2H17NO3). Description Bufexamac is a white to pale yellowish white crystal or crystalline powder, has a faint, characteristic odor and is tasteless. Bufexamac is freely soluble in dimethylformamide, sparingly soluble in methanol or in ethanol and practically insoluble in water or ether. Melting point About 162 C (with decomposition). Identification (1) To 5 mL of a solution of Bufexamac in methanol (1 in 5000), add 1 drop of ferric chloridemethanol TS and shake: a dark red color develops. (2) Determine the absorption spectra of the solutions of Bufexamac and Bufexamac RS in ethanol (1 in 100000) as directed tinder the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Bufexamac and Bufexamac RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Clarity and color of solution Reconstitute 0.20 g of Bufexamac in 20 mL of ethanol: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Bufexamac according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Bufexamac according to Method 3 and perform the test (not more than 2 ppm).

132 Monographs, Part I (4) Related substancesDissolve 0.20 g of Bufexamac in 10 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Use a plate of silica gel with fluorescent indicator for thin-layer chromato-graphy, moisten the surface of the plate evenly by spraying with 0.1 mol/L disodium ethylene-diaminetetraacetate TS and dry at 110 C for about 30 minutes. Spot 15 L each of the test solution and the standard solution on the plate. Develop the plate with a mixture of chloroform, cyclohexane, methanol and glacial acetic acid (6 : 4 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Bufexamac, previously dried, dissolve in 40 mL of dimethylformamide and titrate with 0.1 mol/L tetramethylammonium hydroxide-methanol VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L tetramethylammonium hydroxide-methanol VS = 22.327 mg of Cl2H17NO3 Packaging and Storage Preserve in tight containers. matography. Use a plate of silica gel for thin-layer chromatography, moisten the surface of the plate evenly by spraying with 0.1 mol/L disodium ethylenediaminetetraacetate TS and dry the plate at 110 C for about 30 minutes. Spot 5 L each of the test solution and the standard solution on the plate. Develop the plate with a mixture of pentane, ethyl acetate and glacial acetic acid (7 : 4 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly ferric chloride TS on the plate: the spot from the sample solution and the standard solution show a red-brown color and the same Rf value. Assay Weigh accurately a quantity of Bufexamac Cream, equivalent to about 50 mg of bufexamac (Cl2H17NO3), dissolve in 40 mL of methanol and add methanol to make exactly 50 mL. Pipet 10 mL of this solution, add exactly 5 mL of the internal standard solution and the mobile phase to make 100 mL, filter and use the filtrate as the test solution. Separately, weigh accurately 50 mg of Bufexamac RS, previously dried at 105 C for 4 hours and dissolve in methanol to make exactly 50 mL. Pipet 10 mL of this solution, add exactly 5 mL of the internal standard solution, add the mobile phase to make 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Bufexamac to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of bufexamac (Cl2H17NO3) = amount (mg) of Bufexamac RS
QT QS

Internal standard solutionA solution of diphenylimidazole in methanol (1 in 5000).

Bufexamac Cream
Bufexamac Cream contains not less 90.0% and not more than 110.0% of the labeled amount of bufexamac (Cl2H17NO3: 223.27). Method of Preparation Creams, with Bufexamac. Prepared as directed under

Description Bufexamac Cream is white. pH Between 4.0 and 6.0 Identification To a quantity of Bufexamac Cream, equivalent to 50 mg of Bufexamac according to the labeled amount, add 10 mL of tetrahydrofuran, shake well, centrifuge and use the supernatant liquid as the test solution. Separately, dissolve 50 mg of bufexamac for Assay in 10 mL of methanol and use this solution as the standard solution. Perform the test with the test solution and standard solution as directed under the Thin-layer Chro-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 275 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 2.5 g of sodium 1octanesulfonate and 0.6 g of disodium ethylenediaminetetraacetate in 850 mL of water and add 400 mL of methanol, 400 mL of acetonitrile and 8 mL of glacial acetic acid. Flow rate: Adjust the flow rate so that the retention time of Bufexamac is about 6 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above op-

KP 9 133 erating conditions, bufexamac and the internal standard are eluted in this order with the resolution between these peaks being not less than 8 System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of bufexamac to that of the internal standard is not more than 1.0%. Packaging and Storage Preserve in tight containers. tion. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Bufexamac to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of bufexamac (Cl2H17NO3) = amount (mg) of Bufexamac RS
QT QS

Bufexamac Ointment
Bufexamac Ointment contains not less than 90.0% and not more than 110.0% of the labeled amount of bufexamac (Cl2H17NO3: 223.27). Method of Preparation Prepare as directed under Ointments, with Bufexamac. Identification To a quantity of Bufexamac Ointment, equivalent to 50 mg of Bufexamac according to the labeled amount, add 5 mL of tetrahydrofuran, shake well, add 5 mL of dehydrated ethanol, shake, centrifuge and use the supernatant liquid as the test solution. Separately, dissolve 50 mg of Bufexamac RS 10 mL of methanol and use this solution as the standard solution. Perform the test with the test solution and standard solution as directed under the Thin-layer Chromatography. Use a plate of silica gel for thin-layer chromatography, moisten the surface of the plate evenly by spraying with 0.1 mol/L disodium ethylenediaminetetraacetate TS and dry the plate at 110 C for about 30 minutes. Spot 5 L each of the test solution and the standard solution on the plate. Develop the plate with a mixture of pentane, ethyl acetate and glacial acetic acid (7 : 4 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly ferric chloride TS on the plate: the spot from the test solution and the standard solution show a red-brown color and the same Rf value. Assay Weigh accurately a quantity of Bufexamac Ointment, equivalent to about 50 mg of bufexamac (Cl2H17NO3), add 40 mL of tetrahydrofuran, warm to 40 C. dissolve by shaking, cool and add tetrahydrofuran to make exactly 50 mL. Pipet 10 mL of this solution, add exactly 5 mL of the internal standard solution and the mobile phase to make 100 mL and filter, if necessary, through a membrane filter of 0.45m porosity. Discard the first 20 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately 50 mg of Bufexamac RS, previously dried at 105 C for 4 hours and dissolve in tetrahydrofuran to make exactly 50 mL. Pipet 10 mL of this solution, add exactly 5 mL of the internal standard solution and the mobile phase to make 100 mL and use this solution as the standard solu-

Internal standard solutionA solution of diphenylimidazole in methanol (1 in 5000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 275 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 2.5 g of sodium 1octanesulfonate and 0.6 g of disodium ethylenediaminetetraacetate in 850 mL of water and add 400 mL of methanol, 400 mL of acetonitrile and 8 mL of glacial acetic acid. Flow rate: Adjust the flow rate so that the retention time of Bufexamac is about 6 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, bufexamac and the internal standard are eluted in this order with the resolution between these peaks being not less than 8. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of bufexamac to that of the internal standard is not more than 1.0%. Packaging and Storage Preserve in tight containers.

Buflomedil Hydrochloride
OCH3 O N

HCl
H3 CO OCH3

C17H25NO4HCl : 343.85 Buflomedil Hydrochloride contains not less than 98.5% and not more than 101.5% of buflomedil hydrochloride

134 Monographs, Part I (C17H25NO4HCl), calculated on the dried basis. Description Buflomedil Hydrochloride is a white, crystalline powder. Buflomedil Hydrochloride is freely soluble in water, soluble in ethanol, and very slightly soluble in acetone. Melting pointAbout 195 C (with decomposition). Identification (1) Dissolve 25.0 mg each of Buflomedil Hydrochloride and Buflomedil Hydrochloride RS in ethanol to make exactly 50 mL. Dilute 2.0 mL of the solutions with ethanol to 20 mL. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared absorption spectra of Buflomedil Hydrochloride and Buflomedil Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 40 mg each of Buflomedil Hydrochloride and Buflomedil Hydrochloride RS in 2 mL of methanol, and use these solutions the test solution and the standard solution, respectively. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography, develop with a mixture of toluene, isopropanol, and triethyamine (50 : 50 : 5) to a distance of about 15 cm, and air-dry the plate. Examine in ultraviolet light at 254 nm, and compare the principal spot from the test solution with that from the standard solution. Their Rf values are the same. (4) The solution of Buflomedil Hydrochloride in water (1 in 200) responds to the Qualitative Tests for chloride. pH The pH of a solution obtained by dissolving 2.5 g of Buflomedil Hydrochloride in 50 mL of water is between 5.0 and 6.5. Purity (1) Clarity and color of solutionWhen 2.5 g of Buflomedil Hydrochloride is dissolved in 50 mL water, the resultant solution is colorless and clear. (2) Heavy metals Proceed with 2.0 g of Buflomedil Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve 0.10 g of Buflomedil Hydrochloride in the mobile phase to make exactly 10 mL, and use this solution as the test solution. Dilute 0.5 mL of the test solution to 100 mL with the mobile phase. Dilute 5 mL of this solution to 10 mL with the mobile phase, and use this solution as the standard solution (1). Separately, dissolve 2 mg of Buflomedil Hydrochloride Related Substance I RS [4-(pyrrolidin-1-yl)1-(4-hydroxy-2,6-dimethoxyphenyl)butan-1-one] in the mobile phase, add 0.5 mL of the test solution and dilute to 100 mL with the mobile phase, and use this solution as the standard solution (2). Perform the test with 10 L each of the test solution and the standard solutions as directed under the Liquid Chromatography according to the following conditions. Determine the area of each peak by the automatic integration method. The peak area of any related substance from the test solution is not more than the principal peak area from with the standard solution (1) (0.25%); and the sum of the peak areas of all related substances is not more than twice the principal peak area from the standard solution (1) (0.5%). Disregard any peak areas that are not more than 0.2 times the principal peak area from the standard solution (1) (0.05%). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column about 4.6 mm inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: At a constant temperature of about 40 C. Mobile phase: A mixture of phosphate buffer solution at pH 2.5 and acetonitrile (55:45). Flow rate: 1 mL/min. System suitability System performance: When the procedure is run with 10 L of the standard solution (2) under the above operating conditions, the resolution between the peak of buflomedil and the related substance I is not less than 5.0. Time span of measurement: 2 times as long as the retention time of buflomedil. Phosphate buffer solutionDissolve 9.25 g of potassium dihydrogen phosphate in water to make 1000 mL and adjust the solution pH to 2.5 with phosphoric acid. Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve 0.3 g of Buflomedil Hydrochloride, accurately weighed, in 15 mLof glacial acetic acid, add 35 mL of acetic anhydride, and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 34.385 mg of C17H25NO4HCl Packaging and Storage Preserve in tight containers.

KP 9 135 num crucible and heat until the reaction is complete. After cooling, to the residue, add 14 mL of dilute sulfuric acid and 6 mL of water, boil for 5 minutes, filter, wash the residue, with 10 mL of water, combine the filtrate and the washing and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.021%). (3) Heavy metalsProceed with 2.0 g of Bumetanide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Bumetanide according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesPerform this test without exposure to daylight, using light-resistant vessels. Dissolve 0.10 g of Bumetanide in 10 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution and add methanol to make exactly 100 mL. Pipet 2 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, glacial acetic acid, cyclohexane and methanol (32 : 4 : 4 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Bumetanide, previously dried, dissolve in 50 mL of ethanol and titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry), Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L sodium hydroxide VS = 36.442 mg of C17H20N2O5S Packaging and Storage tight containers. Preserve in light-resistant,

Bumetanide
CO2H

O H2N S O O NHCH2CH2CH2CH3

C17H20N2O5S: 364.42 Bumetanide, when dried, contains not less than 98.5% and not more than 101.0% of bumetanide(C17H20N2O5S). Description Bumetanide is a white crystal or crystalline powder. Bumetanide is freely soluble in pyridine, soluble in methanol and in ethanol, slightly soluble in ether and practically insoluble in water. Bumetanide dissolves in sodium hydroxide TS. Bumetanide is gradually colored by light. Identification (1) Dissolve 10 mg of Bumetanide in 1 mL of pyridine, add 2 drops of cupric sulfate TS, shake, add 3 mL of water and 5 mL of chloroform, shake and allow to stand: a pale blue color develops in the chloroform layer. (2) Dissolve 40 mg each of Bumetanide and Bumetanide RS in 100 mL of phosphate buffer solution, pH 7.0. To 10 mL of these solutions, add water to make 100 mL and determine their absorption spectra as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Bumetanide and Bumetanide RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 232 C and 237 C

Purity (1) Clarity and color of solution Dissolve 50 mg of Bumetanide in 2 mL of a solution of potassium hydroxide (1 in 30) and 8 mL of water: the solution is clear and has no more color than the following control solution. Control solutionsPipet 0.5 mL each of cobaltous chloride stock CS, ferric chloride stock CS and cupric sulfate stock CS, mix and add diluted hydrochloric acid (1 in 40) to make exactly 100 mL. (2) Chloride Mix well 0.5 g of Bumetanide with 0.7 g of potassium nitrate and 1.2 g of anhydrous sodium carbonate, transfer, in small portions, to a red-hot plati-

136 Monographs, Part I side diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: Dissolve 1.44 g of sodium lauryl sulfate in a suitable amount of water, add 10 mL of glacial acetic acid, 500 mL of acetonitrile and water to make 1000 mL. Flow rate: Adjust the flow rate so that the retention time of bunazosin is about 5 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution and a solution of Procaine Hydrochloride in the mobile phase (1 in 20000)-(1:1) under the above operating conditions, procaine and bunazosin are eluted in this order with the resolution between their peaks being not less than 3.0. Detection sensitivity: Adjust the detection sensitivity so that the peak height of bunazosin obtained from 20 L of the standard solution is 20 to 60% of the full-scale. Time span of measurement: About 6 times of the retention time of bunazosin. Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.3 g of Bunazosin Hydrochloride, previously dried, dissolve in 6 mL of formic acid, add exactly 15 mL of 0.1 mol/L perchloric acid and heat for 20 minutes in a water-bath. After cooling, add 20 mL of glacial acetic acid and titrate the excess perchloric acid with 0.1 mol/L sodium acetate VS (potentiometic titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 40.99 mg of C19H27N5O3HCl Packaging and Storage well-closed containers. Preserve in light-resistant,

Bunazosin Hydrochloride
O CH3O N N N C CH2CH2CH3 HCl N CH3O NH2

C19H27N5O3.HCl: 409.91 Bunazosin Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of bunazosin hydrochloride (C19H27N5O3HCl). Description Bunazosin Hydrochloride is a white crystalline powder. Bunazosin Hydrochloride is very soluble in formic acid, slightly soluble in methanol, very slightly soluble in dehydrated ethanol and practically insoluble in ether.
o Melting point About 273 C (with decomposition).

Identification (1) Dissolve 0.1 g of Bunazosin Hydrochloride in 10 mL of 0.2 mol/L hydrochloric acid TS and boil for 3 minutes over a flame: butylic acid like odor is perceptible. (2) Determine the infrared spectra of Bunazosin Hydrochloride and Bunazosin Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Bunazosin Hydrochloride (1 in 100) responds to the Qualitative Tests for chloride. Purity (1) Heavy metalsProceed with 1.0 g of Bunazosin Hydrochloride according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 50 mg of Bunazosin Hydrochloride in 50 mL of the mobile phase and use this solution as the test solution. To exactly 1 mL of the test solution, add the mobile phase to make exactly 200 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography, according to the following conditions. Determine each peak area of both solutions by the automatic integration method: the total area of the peaks other than the peak of bunazosin from the test solution is not larger than the peak area of bunazosin from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in in-

Bupranolol Hydrochloride
H 3C OH OCH 2CCH2NHNC(CH 3)3 H Cl HCl

C14H22ClNO2.HCl: 308.24 Bupranolol Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of bupranolol hydrochloride (C14H22ClNO2HCl).

KP 9 137 solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of methanol, strong ammonia water and water (16 : 4 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.18 g of Bupranolol Hydrochloride, previously dried, dissolve in 60 mL of a mixture of acetic anhydride and glacial acetic acid (2 : 1) by warming, cool and titrate with 0.1 mol/L perchloric acid VS (potentiometic titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 30.824 mg of C14H22ClNO2.HCl Packaging and Storage Preserve in well-closed containers.

Description Bupranolol Hydrochloride is a white crystalline powder. Bupranolol Hydrochloride is sparingly soluble in methanol, in ethanol or in glacial acetic acid, very slightly soluble in acetic anhydride and practically insoluble in ether. pH Dissolve 1.0 g of Bupranolol Hydrochloride in 1000 mL water: the pH of this solution is between 5.2 and 6.2. . Identification (1) Take 10 mg of Bupranolol Hydrochloride in a test tube, mix with 25 mg of potassium iodide and 25 mg of oxalic acid, cover the mouth of the test tube with a filter paper, moistened with a solution of 2,6dibromoquinone chloride in ethanol (1 in 100) and heat gently for several minutes. Expose the filter paper to ammonia gas: the filter paper acquires a blue color. (2) Determine the absorption spectra of the solutions of Bupranolol Hydrochloride and Bupranolol Hydrochloride RS in 0.1 mol/L hydrochloric acid TS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Bupranolol Hydrochloride and Bupranolol Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) A solution of Bupranolol Hydrochloride (1 in 200) responds to the Qualitative Tests for chloride.
1% Absorbance E1 cm (275 nm): Between 57 and 60 (after drying, 50 mg, 0.1 mol/L hydrochloric acid TS, 500 mL).

Melting Point Between 223 C and 226 C. Purity (1) Clarity and color of solutionDissolve 0.10 g of Bupranolol Hydrochloride in 15 mL of water: the solution is clear and colorless. (2) AcidDissolve 0.10 g of Bupranolol Hydrochloride in 15 mL of freshly boiled and cooled water and add 1 drop of methyl red TS: a light red color is observed. To this solution, add 50 L of 0.01 mol/L sodium hydroxide VS: the color changes to yellow. (3) SulfatePerform the test with 0.10 g of Bupranolol Hydrochloride. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.168%). (4) Heavy metalsProceed with 1.0 g of Bupranolol Hydrochloride according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) ArsenicPrepare the test solution with 1.0 g Bupranolol Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (6) Related substancesDissolve 0.30 g of Bupranolol Hydrochloride in 10 mL of methanol and use this

Buspirone Hydrochloride
O N

N N O

HCl

C21H31N5O2HCl : 421.96 Buspirone Hydrochloride contains not less than 97.5% and not more than 102.5% of buspirone hydrochloride (C21H31N5O2HCl), calculated on the anhydrous basis. Description Buspirone Hydrochloride is a white, crystalline powder. Buspirone Hydrochloride is very soluble in water, freely soluble in methanol or in dichlormethane, sparingly soluble in ethanol or in acetonitrile, very slightly soluble in ethyl acetate, and practically insoluble in hexane.

138 Monographs, Part I Identification (1) Determine the infrared absorption spectra of Buspirone Hydrochloride and Buspirone Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) When the procedure in the Assay is performed, the relative retention time of the principal peak from the test solution corresponds to that from the standard solution. Purity (1) Heavy metalsProceed with 1.0 g of Buspirone Hydrochloride, according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (20 ppm). (2) ChlorideDissolve about 0.4 g, accurately weighed, in 20 mL of water. Add 3 mL of nitric acid and 20 mL of 0.1 mol/L silver nitrate VS, gently boil the mixture for about 5 minutes, and filter. Rinse the flask with about 80 mL of water divided into small portions, and filter each portion. To all combined filtrates, add 2 mL of 8% ferric ammonium sulfate, and stir rapidly while titrating from a buret with 0.1 mol/L ammonium thiocyanate VS to a faint red-brown endpoint (8.0 8.8%). Each mL of 0.1 mol/L silver nitrate VS = 3.545 mg of chloride Water Not more than 0.5% (1 g, volumetric titration, direct titration).
H3C

Column: A stainless steel column 3.0 mm inside diameter and 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of phosphate buffer solution and acetonitrile (60:40). Flow rate: 2 mL/min. System suitability System performance: When the procedure is run with 25 L of the standard solution according to the above operating conditions, the resolution between the peaks of buspirone hydrochloride and the internal standard is not less than 4.0. System repeatability: When the test is repeated 5 times with 25 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of buspirone hydrochloride to that of the internal standard is not more than 2.0. Buffer solutionDissolve 1.36 g of monobasic potassium phosphate in water to make 1000 mL, and adjust the solution with 10% sodium hydroxide to a pH of 7.5. Packaging and Storage resistant containers. Preserve in tight, light-

Busulfan
O S O OCH2CH2CH2CH2O O S O CH3

Residue on Ignition Not more than 0.5% (1 g). Assay Dissolve about 50 mg each of Buspirone Hydrochloride and Buspirone Hydrochloride RS, accurately weighed, in 1 mol/L to make 25 mL, dilute with water to make 100 mL. To 10 mL of these solutions, add 10 mL of the internal standard, and dilute with water to make 50 mL. Use these solutions as the test solution and the standard solution, respectively. Perform the test with 25 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS , of the peak area of Buspirone Hydrochloride. Amount (mg) of buspirone hydrochloride (C21H31N5O2HCl) = amount (mg) of Buspirone Hydrochloride RS
QT QS

C6H14O6S2: 246.30 Busulfan contains not less than 98.5% and not more than 101.0% of busulfan (C6H14O6S2), calculated on the dried basis. Description Busulfan is a white, crystalline powder and is odorless. Busulfan is slightly soluble in ether, very slightly soluble in ethanol and practically insoluble in water. Identification (1) To 0.1 g of Busulfan, add 10 mL of water and 5 mL of sodium hydroxide TS, dissolve by heating and use this solution as the test solution. (i) To 7 mL of the test solution, add 1 drop of potassium permanganate TS: the red-purple color of potassium permanganate TS changes from blue-purple trough blue to green. (ii) Acidify 7 mL of the test solution with dilute sulfuric acid and add 1 drop of potassium permanganate TS: the color of potassium permanganate TS remains. (2) Determine the infrared spectra of Busulfan and Busulfan RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.

Internal standard solutionDissolve 0.25 g of propyl parahydroxybenzoate in methanol to make 100 mL. Dilute 25 mL of this solution with water to 500 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm).

KP 9 139 soluble matter to settle and finally decanting the supernatant liquid through a sintered-glass filter (G4). Evaporate the collection of extracts to about 10 mL, add phenolphthalein TS and neutralize with 0.1 mol/L sodium hydroxide. Evaporate to dryness, add about 30 mL of water and proceed as directed under the Assay of Busulfan with reflux condenser (indicator : 3 drops of phenolphthalein TS). Each mL of 0.1 mol/L sodium hydroxide VS = 12.315 mg of C6H14O6S2 Packaging and Storage well-closed containers. Preserve in light-resistant,

Melting Point

Between 115 C and 118 C

Purity (1) SulfateTo 1.0 g of Busulfan, add 40 mL of water and dissolve by heating. Cool in ice for 15 minutes and filter. Wash the residue with 5 mL of water, combine the washings with the filtrate and add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.019%). (2) Heavy metalsProceed with 1.0 g of Busulfan according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 2.0% (1 g, in vacuum, P2O5, 60 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Busulfan, add 40 mL of water and boil gently under a reflux condenser for 30 minutes. Cool and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Each mL of 0.1 mol/L sodium hydroxide VS = 12.315 mg of C6H14O6S2 Packaging and Storage well-closed containers. Preserve in light-resistant,

Caffeine
O H3C N N O N CH3 CH3 N

Anhydrous Caffeine

C8H10N4O2: 194.19

Caffeine, when dried, contains not less than 98.5% and not more than 101.0% of caffeine (C8H10N4O2). Description Caffeine is a white crystal or powder, is odorless and has a bitter taste. Caffeine is freely soluble in chloroform, sparingly soluble in water, in acetic anhydride or in glacial acetic acid and slightly soluble in ethanol or in ether. pH A solution of Caffeine (1 in 100) is between 5.5 and 6.5 Identification (1) To 2 mL of a solution of Caffeine (1 in 500), add tannic acid TS drop-wise: a white precipitate, which dissolves upon the drop-wise addition of tannic acid TS, is produced. (2) To 10 mg of Caffeine, add 10 drops of hydrogen peroxide TS and 1 drop of hydrochloric acid and evaporate on a water-bath to dryness: the residue acquires a yellow-red color. Invert the residue over a vessel containing 2 to 3 drops of ammonia TS: the color turns a redpurple and disappears upon the addition of 2 to 3 drops of sodium hydroxide TS. (3) Dissolve 10 mg of Caffeine in water to make 50 mL. To 5 mL of this solution, add 3 mL of diluted acetic acid (3 in 100) and 5 mL of pyridine (1 in 10), mix, add 2 mL of diluted sodium hypochlorite TS (1 in 5) and allow to stand for 1 minute. Add 2 mL of sodium thiosulfate TS and 5 mL of sodium hydroxide TS to the solution: a yellow color develops. Melting Point Between 235 C and 238 C.

Busulfan Tablets
Busulfan Tablets contains not less than 93.0% and not more than 107.0% of the labeled amount of busulfan (C6H14O6S2: 246.30). Method of Preparation Prepare as directed under Tablets, with Busulfan. Identification Powder Busulfan Tablets and extract the powder several times with acetone. Collect these extracts and evaporate to dryness in a water-bath, with the aid of a current of air. The residue responds to Identification test (1) and (2) of Busulfan, and melts at about 115 C. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh and finely powder not less than 40 tablets (caution: guard against accidental inhalation of fine powder). Weigh accurately a portion of powder, equivalent to about 80 mg of busulfan (C6H14O6S2) and transfer to a beaker. Extract with four 20 mL volumes of acetone, each time stirring the mixture well, then allowing the in-

140 Monographs, Part I Purity Test (1) ChlorideDissolve 2.0 g of Caffeine in 80 mL of hot water, cool rapidly to 20 C, add water to make 100 mL and use this solution as the test stock solution. To 40 mL of the test stock solution, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.011%). (2) SulfateTo 40 mL of the test stock solution obtained in (1), add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (3) Heavy metalsProceed with 2.0 g of Caffeine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Related substancesDissolve 0.10 g of Caffeine in 10 mL of chloroform and use this solution as the test solution. Pipet 1 mL of the test solution and add chloroform to make exactly 100 mL. Pipet 1 mL of this solution, add chloroform to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform and ethanol (9 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. (5) Readily carbonizable SubstancesPerform the test using 0.5 g of Caffeine: the solution has no more color than Color Matching Fluid D. Loss on Drying Not more than 0.5% (1 g, 80 C, 4 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately 0.4 g of Caffeine, previously dried, dissolve in 70 mL of a mixture of acetic anhydride and glacial acetic acid (6 : 1) and titrate with 0.1 mol/L perchloric acid VS until the solution changes from purple through green to yellow (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 19.419 mg of C8Hl0N4O2 Packaging and Storage Preserve in tight containers.

Caffeine Hydrate
O H 3C N H2O N O N CH3 CH3 N

C8Hl0N4O2H2O: 212.21 Caffeine Hydrate, previously dried, contains not less than 98.5% and not more than 101.0% of caffeine (C8Hl0N4O2: 194.19). Description Caffeine Hydrate is a white, soft crystal or powder, is odorless and has a slightly bitter taste. Caffeine Hydrate is freely soluble in chloroform, sparingly soluble in water, in glacial acetic acid or in acetic anhydride, slightly soluble in ethanol and very slightly soluble in ether. pH A solution of Caffeine Hydrate (1 in 100) is between 5.5 and 6.5. Caffeine Hydrate effloresces in dry air. Identification (1) To 2 mL of a solution of Caffeine Hydrate (1 in 500), add tannic acid TS drop-wise: a white precipitate, which dissolves upon the drop-wise addition of tannic acid TS, is produced. (2) To 10 mg of Caffeine Hydrate, add 10 drops of hydrogen peroxide TS and 1 drop of hydrochloric acid and evaporate to dryness on a water-bath: the residue acquires a yellow-red color. Invert the residue over a vessel containing 2 to 3 drops of ammonia TS: the color turns red-purple and disappears upon the addition of 2 to 3 drops of sodium hydroxide TS. (3) Dissolve 10 mg of Caffeine Hydrate in water to make 50 mL. To 5 mL of this solution, add 3 mL of diluted acetic acid (3 in 100) and 5 mL of a solution of pyridine (1 in 10), mix, add 2 mL of diluted sodium hypochlorite TS (1 in 5) and allow to stand for 1 minute. Add 2 mL of sodium thiosulfate TS and 5 mL of sodium hydroxide TS to the solution: a yellow color develops. Melting Point ing). Between 235 C and 238 C (after dry-

Purity (1) ChlorideDissolve 2.0 g of Caffeine Hydrate in 80 mL of hot water, cool rapidly to 20 C, add water to make 100 mL and use this solution as the test stock solution. To 40 mL of the test stock solution, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.011%). (2) SulfateTo 40 mL of the test stock solution obtained in (1), add 1 mL of dilute hydrochloric acid and

KP 9 141 water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (3) Heavy metalsProceed with 2.0 g of Caffeine Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Related substancesDissolve 0.10 g of Caffeine Hydrate in 10 mL of chloroform and use this solution as the test solution. Pipet 1 mL of the test solution and add chloroform to make exactly 100 mL. Pipet 1 mL of this solution, add chloroform to make exactly 10 mL and use this solution as the standard solution. Perform the test as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thinlayer chromatography. Develop the plate with a mixture of chloroform and ethanol (9 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. (5) Readily carbonizable substancePerform the test using 0.5 g of Caffeine Hydrate: the solution has no more color than Color Matching Fluid D. Loss on Drying Between 0.5 and 8.5% (1 g, 80 C, 4 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately about 0.4 g of Caffeine Hydrate, previously dried, dissolve in 70 mL of a mixture of acetic anhydride and glacial acetic acid (6 : 1) and titrate with 0.1 mol/L perchloric acid VS until the solution changes from purple through green to yellow (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 19.419 mg of C8Hl0N4O2 Packaging and Storage Preserve in tight containers. ringly soluble in ethanol and practically insoluble in ether. Identification (1) Dissolve 1 g of Caffeine and Sodium Benzoate in 10 mL of water in a separator, add 1 drop of phenolphthalein TS and add carefully 0.01 mol/L sodium hydroxide VS dropwise until a pale red color develops. Extract with three 20 mL volume of chloroform by thorough shaking and separate the chloroform layer from the water layer [use the water layer for test (2).]. Filter the combined chloroform extracts, evaporate the filtrate to dryness in a water-bath and proceed the following tests with the residue. (i) To 2 mL of a solution of the residue (1 in 500), add tannic acid TS drop-wise: a white precipitate, which dissolves upon the drop-wise addition of tannic acid TS, is produced. (ii) To 10 mg of the residue, add 10 drops of hydrogen peroxide TS and 1 drop of hydrochloric acid, evaporate to dryness on a water-bath: the residue acquires a yellow-red color. Invert the residue over a vessel containing 2 to 3 drops of ammonia TS: the color turns red-purple and disappears upon the addition of 2 to 3 drops of sodium hydroxide TS. (iii) Dissolve 10 mg of the residue in water to make 50 mL. To 5 mL of this solution, add 3 mL of diluted acetic acid (3 in 100) and 5 mL of a solution of pyridine (1 in 10), mix, add 2 mL of diluted sodium hypochlorite TS (1 in 5) and allow to stand for 1 minute. Add 2 mL of sodium thiosulfate TS and 5 mL of sodium hydroxide TS to the solution: a yellow color develops. (2) To 5 mL of the water layer obtained in (1), add 5 mL of water: the solution responds to the Qualitative Tests (2) for benzoate. (3) Heat Caffeine and Sodium Benzoate: white fumes are evolved. Ignite furthermore and to the residue, add hydrochloric acid: bubbles are produced and the solution responds to the Qualitative Tests (1) for sodium salt. Purity (1) Clarity and color of solution Dissolve 1.0 g of Caffeine and Sodium Benzoate in 5 mL of water: the solution is clear and colorless. (2) AlkaliDissolve 1.0 g of Caffeine and Sodium Benzoate in 20 mL of water and add 1 or 2 drops of phenolphthalein TS: no red color develops. (3) ChlorideDissolve 0.5 g of Caffeine and Sodium Benzoate in 10 mL of water and add 30 mL of ethanol, 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.70 mL of 0.01 mol/L hydrochloric acid VS, 30 mL of ethanol and water to make 50 mL (not more than 0.050%). (4) Chlorinated compoundsDissolve 1.0 g of Caffeine and Sodium Benzoate in 40 mL of water, add 10 mL of dilute sulfuric acid and extract with two 2 mL volumes of ether. Allow the combined ether extracts to evaporate at room temperature to dryness. Place this residue and 0.7 g of calcium carbonate in a crucible, mix with a small amount of water and dry. Ignite at about 600 C, dissolve the residue in 20 mL of dilute nitric acid and filter. Wash the residue with 15 mL of water, combine the

Caffeine and Sodium Benzoate

Caffeine and Sodium Benzoate, when dried, contains not less than 48.0% and not more than 50.0% of caffeine (C8H10N4O2: 194.19) and not less than 50.0% and not more than 52.0% of sodium benzoate (C7H5NaO2: 144.10). Description Caffeine and Sodium Benzoate is a white powder, is odorless and has a slightly bitter taste. Caffeine and Sodium Benzoate is freely soluble in water, soluble in glacial acetic acid or in acetic anhydride, spa-

142 Monographs, Part I filtrate and the washings and add water to make 50 mL. To this solution, add 0.5 mL of silver nitrate TS: the solution shows no more turbidity than the following control solution to which 0.5 mL of silver nitrate TS has been added. Control solutionDissolve 0.7 g of calcium carbonate in 20 mL of dilute nitric acid and filter. Wash the residue with 15 mL of water, combine the filtrate and the washings and add 1.2 mL of 0.01 mol/L hydrochloric acid VS and water to make 50 mL. (5) Heavy metalsDissolve 2.0 g of Caffeine and Sodium Benzoate in 47 mL of water, add slowly, with vigorous stirring, 3 mL of dilute hydrochloric acid and filter. Discard the first 5 mL of the filtrate, neutralize the subsequent 25 mL of the filtrate with ammonia TS and add 2 mL of dilute acetic acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 2.0 mL of standard lead solution by adding 2 mL of dilute acetic acid and water to make 50 mL (not more than 20 ppm). (6) ArsenicPrepare the test solution with 1.0 g of Caffeine and Sodium Benzoate according to Method 1 and perform the test (not more than 2 ppm). (7) Phthalic acidTo 0.10 g of Caffeine and Sodium Benzoate, add 1 mL of water and 1 mL of resorcinsulfuric acid TS and heat the mixture in an oil-bath heated at a temperature between 120 C and 125 C to evaporate the water, then heat the residue for further 90 minutes, cool and dissolve in 5 mL of water. To 1 mL of the solution add 10 mL of a solution of sodium hydroxide (43 in 500), shake, then examine under light at a wavelength between 470 nm and 490 nm: the green fluorescence of the solution is not more intense than that of the following control solution. Control solutionDissolve 61 mg of potassium biphthalate in water to make exactly 1000 mL. Pipet exactly 1 mL of the solution, add 1 mL of resorcin-sulfuric acid TS and proceed as directed above. (8) Readily carbonizable substances Proceed with 0.5 g of Caffeine and Sodium Benzoate and perform the test: the solution has no more color than Color Matching Fluid A. Loss on Drying Not more than 3.0% (2 g, 80 C, 4 hours). Assay (1) Sodium benzoateWeigh accurately 0.2 g of Caffeine and Sodium Benzoate, previously dried, dissolve by warming in 50 mL of a mixture of acetic anhydride and glacial acetic acid for nonaqueous titration (6 : 1), cool and titrate with 0.1 mol/L perchloric aciddioxane VS to the first equivalence point (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid-dioxane VS = 14.411 mg of C7H5NaO2 (2) CaffeineContinue the titration in (1) with 0.1 mol/L perchloric acid-dioxane VS from the first equivalence point to the second equivalence point (potentiometric titration, Endpoint Detection Method in Titrimetry). Each mL of 0.1 mol/L perchloric acid-dioxane VS = 19.419 mg of C8H10N4O2 Packaging and Storage Preserve in well-closed containers.

Calamine
Calamine is zinc oxide with a small proportion of ferric oxide. Calamine contains, after ignition, not less than 98.0 and not more than 100.5% of zinc oxide (ZnO: 81.37). Description Calamine is a pale pinkish fine powder, is odorless and tasteless. Calamine is insoluble in water. Calamine dissolves in hydrochloric acid. Identification (1) Dissolve 1 g of Calamine in 10 mL of 3 mol/L hydrochloric acid and filter: the filtrate responds to the Qualitative Tests for zinc. (2) Treat 1 g of Calamine with 10 mL of 3 mol/L hydrochloric acid, heat to boiling and filter: the filtrate produces reddish color upon the addition of ammonium thiocyanate TS. Purity (1) Acid-insoluble substancesDissolve 2.0 g of Calamine in 50 mL of 3 mol/L hydrochloric acid. Filter of an insoluble residue remains. Wash with water, dry at 105 C for 1 hour, cool and weigh: the weight of the residue does not exceed 40 mg (not more than 2.0%). (2) Alkaline substancesAdd 1.0 g of Calamine to 20 mL of water in a water-bath for 15 minutes. Filter and add 2 drops of phenolphthalein TS and 0.20 mL of 0.05 mol/L sulfuric acid: the color of the solution is colorless. (3) LeadAdd 15 mL of water to 1 g of Calamine, stir, then add 3 mL of glacial acetic acid. Warm on a steam-bath until dissolved. Filter the mixture and add 5 drops of potassium chromate TS: no turbidity is produced. (4) ArsenicDissolve 0.25 g of Calamine in 35 mL of water and use this solution as the test solution and perform the test (not more than 8 ppm). (5) CalciumDissolve 1 g of Calamine in 25 mL of 3 mol/L hydrochloric acid for 30 minutes and add 6 mol/L ammonium hydroxide to the filtrate until the precipitate first formed is dissolved, then add 5 mL more of

KP 9 143 6 mol/L ammonium hydroxide. To 10 mL of this solution, add 2 mL of ammonium oxalate TS: not more than a slight turbidity is produced. (6) Calcium or magnesiumTo another 10 mL portion of the solution prepared for the test for Calcium, add 2 mL of dibasic sodium phosphate TS: not more than a slight turbidity is produced. Loss on Ignition Not more than 2.0% (2 g). Assay Dissolve about 1.5 g of freshly ignited Calamine, accurately weighed, with 50 mL of 0.5 mol/L sulfuric acid VS. Filter the mixture and wash the residue on the filter with hot water until last washing is neutral. To the combined filtrate and washings, add 2.5 g of the ammonium chloride, cool and titrate with 1 mol/L sodium hydroxide VS (indicator: 3 drops of methyl orange TS). Perform a blank determination and make any necessary correction. Each mL of 0.5 mol/L sulfuric acid = 40.69 mg of ZnO Packaging and Storage Preserve in well-closed containers. metry: both spectra exhibit similar intensities of absorption at the same wave numbers. (2) Retention time of the main peak obtained from test solution and standard solution in the Assay is the same. Purity Related substancesPerform the test as directed under the Liquid Chromatography according to the conditions in Assay. Determine the peak area of each solution according to the automatic integration. Calculate the peak areas of related substances, that are eluted within twice the retention time of Calcitriol obtained from the test solution. Peak area of any individual related substance is not more than 5.0% of principal peak area and the sum of the related substances is not more than 1.0%. Disregard any peak with an area less than 0.1 times that of the peak in the chromatogram obtained from standard solution (2). Assay Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air. Weigh accurately 1 mg of Calcitriol, and dissolve in the mobile phase to make exactly 10 mL, use this solution as the test solution. Weigh accurately 1 mg of Calcitriol RS, and dissolve in the mobile phase to make exactly 10 mL, and use this solution as the standard solution (1). Add the mobile phase to 1.0 mL of this solution to make exactly 100 mL, use this solution as the standard solution (2). Keep 2 mL of standard solution (1) at 80 for 30 minutes, use this solution as the standard solution (3). Perform the test with 50 L each of test solution and standard solution (1) as directed under the Liquid Chromatography according to the following conditions, and calculate the peak areas of Calcitriol of each solution, AT and AS , respectively. Amount (mg) of calcitriol (C27H44O3) = 10 C
AT AS

Calcitriol
H H3C CH3 H HO CH3 CH3

CH2

HO H H

C : Concentration (mg/mL) of Calcitriol obtained from standard solution (1). Operating conditions Detector: Ultraviolet-visible absorption Photometer (wavelength: 230 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for the liquid chromatography. Column temperature: A constant teperature of about 40 C. Mobile phase: A mixture of acetonitrile and trishydroxymethylaminomethane bufffer solution (550 : 450). Flow rate: 1.0 mL/minute. System suitability System performance: When the procedure is run with 50 L of the standard solution (3) under the above operating conditions: the relative retention time of precalcitriol peak to calcitriol peak is about 0.9, the resolu-

OH

C27H44O3 : 416.64 Calcitriol contains not less than 97.0% and not more than 103.0% of calcitriol (C27H44O3). Description Calcitriol is a white crystals. Calcitriol is freely soluble in ethanol, soluble in fatty oils, practically insoluble in water. Calcitriol is sensitive to air, heat and light. A reversible isomerisation to pre-calcitriol takes place in solution, depending on temperature and time. Identification (1) Determine the infrared spectra of Calcitriol and Calcitriol RS as directed in the potassium bromide disk method under the Infrared Spectrophoto-

144 Monographs, Part I tion between the peaks due to Calcitriol and pre-calcitriol is not less than 3.5. Perform the test with 50 L of the standard solution (1) according to the above operating conditions, the number of theoretical plates is not than 10000. System repeatability: When the test is repeated 6 times with 50 L of the standard solution (1) under the above operating conditions: the relative standard deviation of peak area of calcitriol is not more than 1%. Trishydroxymethylaminomethane buffer solution Dissolve 1.0 g of trishydroxymethylaminomethane in 1000 mL of water. Adjust the pH between 7.0 and 7.5. Packaging and Storage Preserve in a light-resistant, tight containers, store under nitrogen at between 2 and 8 C. The contents of an opened container are to be used immediately. (5) Iron, aluminum or phosphateDissolve, in a Nessler tube, 1.0 g of Calcium Chloride Hydrate in 20 mL of water and 1 drop of dilute hydrochloric acid, boil, then cool, add 3 drops of ammonia TS and heat the solution to boil: no turbidity or precipitate is produced. (6) BariumDissolve 0.5 g of Calcium Chloride Hydrate in 5 mL of water, add 2 drops of dilute hydrochloric acid and 2 mL of potassium sulfate TS and allow to stand for 10 minutes: no turbidity is produced. (7) ArsenicPrepare the test solution with 1.0 g of Calcium Chloride Hydrate according to Method 1 and perform the test (not more than 2 ppm). Assay Weigh accurately about 0.4 g of Calcium Chloride Hydrate and dissolve in water to make exactly 200 mL. Measure exactly 20 mL of this solution, add 40 mL of water, 2 mL of 8 mol/L potassium hydroxide TS and 0.1 g of NN indicator and titrate with 0.02 mol/L of disodium ethylenediaminetetraacetate VS until the color of the solution changes from red-purple to blue. Each mL of 0.02 mol/L disodium ethylenediaminetetraacetate VS = 2.9402 mg of CaCl22H2O Packaging and Storage Preserve in tight containers.

Calcium Chloride Hydrate

CaCl22H2O: 147.02 Calcium Chloride Hydrate contains not less than 96.7% and not more than 103.3% of calcium chloride hydrate (CaCl22H2O). Description Calcium Chloride Hydrate is a white granule or mass and is odorless. Calcium Chloride Hydrate is very soluble in water, soluble in ethanol, and practically insoluble in ether. Calcium Chloride Hydrate is deliquescent. Identification A solution of Calcium Chloride Hydrate (1 in 10) responds to the Qualitative Test for calcium salt and for chloride. pH Dissolve 1.0 g of Calcium Chloride Hydrate in 20 mL of freshly boiled and cooled water: the pH of this solution is between 4.5 and 9.2. Purity (1) Clarity and color of solutionA solution of 1.0 g of Calcium Chloride Hydrate in 20 mL of water is clear and colorless. (2) SulfateTake 1.0 g of Calcium Chloride Hydrate and perform the test. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (3) HypochloriteDissolve 0.5 g of Calcium Chloride Hydrate in 5 mL of water, add 2 to 3 drops of dilute hydrochloric acid and 2 to 3 drops of zinc iodide-starch TS: no blue color is observed immediately. (4) Heavy metalsProceed with 2.0 g of Calcium Chloride Hydrate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm).

Calcium Chloride Injection

Calcium Chloride Injection is an aqueous solution for injection. Calcium Chloride Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of calcium chloride (CaCl2: 110.98). The concentration of Calcium Chloride Injection is expressed as the quantity of calcium chloride (CaCl2: 110.98). Do not perform the Pyrogen Test with Calcium Chloride Injection. Method of Preparation Prepare as directed under Injections, with Calcium Chloride Hydrate. Description Calcium Chloride Injection is a clear colorless liquid. Identification Calcium Chloride Injection responds to the Qualitative Test for calcium salt and for chloride. pH Between 4.5 and 7.5. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement. It

KP 9 145 Water Weigh accurately about 0.2 g of Calcium Folinate in a dried titration flask and dissolve in 25 mL of glacial acetic acid. Add 10.0 mL of standard watermethanol solution, titrate with Karl Fischer TS to the end point and perform the test: it is not more than 17.0%. Perform a blank determination and make any necessary correction. Assay Weigh accurately about 20 mg of Calcium Folinate, dissolve in the mobile phase to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 17.5 mg of Calcium Folinate RS, calculated on the anhydrous basis, dissolve in the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine the peak areas, AT and AS, of folinate for the test solution and the standard solution, respectively. Amount (mg) of calcium folinate (C20H21CaN7O7) = amount (mg) of Calcium Folinate RS, AT calculated on the anhydrous basis A S Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: To 860 mL of water, add 100 mL of acetonitrile and 15 mL of tetrabutylammonium hydroxide-methanol TS, adjust the pH to 7.5 with 2 mol/L monobasic sodium phosphate TS and add water to make 1000 mL. Flow rate: Adjust the flow rate so that the retention time of folinate is about 10 minutes. System suitability System performance: Dissolve 17.5 mg of folic acid in 100 mL of the mobile phase and to 5 mL of this solution add 20 mL of the standard solution. When the procedure is run with 20 L of this solution under the above operating conditions, folinate and folic acid are eluted in this order with the resolution of their peaks being not less than 3.6. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of each peak area of folinate is not more than 2.0%. Packaging and Storage tight containers. Preserve in light-resistant,

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of Calcium Chloride Injection, equivalent to about 0.4 g of calcium chloride (CaCl2) and proceed as directed in the Assay under Calcium Chloride Hydrate. Each mL of 0.02 mol/L disodium ethylenediaminetetraacetate VS = 2.2197 mg of CaCl2 Packaging and Storage Preserve in hermetic containers.

Calcium Folinate
H2N H N H N H N N O CHO CH2NH CONH C CO2
-

H CH2CH2CO2-

Ca2+

Calcium Leucovorin

C20H21CaN7O7: 511.50

Calcium Folinate contains not less than 95.0% and not more than 102.0% of calcium folinate (C20H21CaN7O7), calculated on the anhydrous basis. Description Calcium Folinate is a pale yellow to yellow powder, is odorless and tasteless. Calcium Folinate is very soluble in water, freely soluble in glacial acetic acid and practically insoluble in ethanol or in ether. Calcium Folinate is gradually affected by light. Identification (1) Determine the absorption spectra of solutions (1 in 100000) of Calcium Folinate and Calcium Folinate RS as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Calcium Folinate and Calcium Folinate RS, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Calcium Folinate (1 in 100) responds to the Qualitative Tests (2), (3) and (4) for calcium salt. Purity (1) Clarity and color of solution Dissolve 1.0 g of Calcium Folinate in 10 mL of water: the solution is clear and yellow. (2) Heavy metalsProceed with 0.40 g of Calcium Folinate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 50 ppm).

146 Monographs, Part I drate and perform the test. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.048%). (4) Heavy metalsDissolve 1.0 g of Calcium Gluconate Hydrate in 30 mL of water and 2 mL of dilute acetic acid by warming, cool and add water to make 50 mL. Perform the test with this solution as the test solution. Prepare the control solution with 2.0 mL of standard lead solution, 2 mL of dilute acetic acid and water to make 50 mL (not more than 20 ppm). (5) ArsenicDissolve 0.6 g of Calcium Gluconate Hydrate in 5 mL of water by warming, add 5 mL of dilute sulfuric acid and 1 mL of bromine TS and concentrate in a water-bath to 5 mL use this solution as the test solution and perform the test (not more than 3.3 ppm). (6) Sucrose and reducing sugarsTo 0.5 g of Calcium Gluconate Hydrate, add 10 mL of water and 2 mL of dilute hydrochloric acid and boil the solution for 2 minutes. After cooling, add 5 mL of sodium carbonate TS, allow to stand for 5 minutes, add water to make 20 mL and filter. To 5 mL of the filtrate, add 2 mL of Fehlings TS and boil for 1 minute: no orange-yellow to red precipitate is produced immediately. Loss on Drying Not more than 1.0% (1 g, 80 C, 2 hours). Assay Weigh accurately 0.4 g of Calcium Gluconate Hydrate, previously dried, dissolve in 100 mL of water, add 2 mL of 8 mol/L potassium hydroxide TS and 0.1 g of NN indicator and titrate immediately with 0.05 mol/L disodium ethylenediaminetetraacetate VS until the color of the solution changes from red-purple to blue. Each mL of 0.05 mol/L disodium ethylenediaminetetraacetate VS = 22.420 mg of C12H22CaO14H2O Packaging and Storage Preserve in well-closed containers.

Calcium Gluconate Hydrate

OH HOH2C C H OH C H H C OH OH C H CO2 Ca2+ H2O

Calcium Gluconate

C12H22CaO14H2O: 448.39

Calcium Gluconate Hydrate, when dried, contains not less than 99.0% and not more than 104.0% of calcium gluconate hydrate (C12H22CaO14H2O). Description Calcium Gluconate Hydrate is a white, crystalline powder or granule. Calcium Gluconate Hydrate is freely soluble in hot water, soluble in water and practically insoluble in dehydrated ethanol. Identification (1) To 10 mg each of Calcium Gluconate Hydrate and Calcium Gluconate Hydrate RS, add 1 mL of water each, dissolve by warming, and use these solutions as the test solution and standard solution, respectively. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethanol, water, ammonia solution and ethyl acetate (5:3:1:1) to a distance of about 10 cm, air-dry the plate, and heat the plate at 110 for 20 minutes. After cooling, spray evenly a mixture containing molybdate sulfate TS and cerium sulfate TS on the plate, air-dry, and heat at 110 C for 10 minutes: the spots with the test solution and the standard solution are the same in the Rf value and color tone. (2) A solution of Calcium Gluconate Hydrate (1 in 40) responds to the Qualitative Tests for calcium salt. Specific Optical Rotation nD : +6 ~ +11 (after drying, 0.5g, water, 25 mL, 100 mm). pH Dissolve 1.0 g of Calcium Gluconate Hydrate in 20 mL of water by warming: the pH of the solution is between 6.0 and 8.0. Purity (1) Clarity of solutionDissolve 1.0 g of Calcium Gluconate Hydrate in 50 mL of water by warming: the solution is clear. (2) ChlorideTake 0.40 g of Calcium Gluconate Hydrate and perform the test. Prepare the control solution with 0.80 mL of 0.010 mol/L hydrochloric acid VS (not more than 0.071%). (3) SulfateTake 1.0 g of Calcium Gluconate Hy20

Calcium Gluconate Injection

Calcium Gluconate Injection is an aqueous solution for injection. Calcium Gluconate Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of total calcium (Ca: 40.08). An adequate amount of calcium saccharate or other suitable calcium salts may be added as stabilizers. Method of Preparation Prepare as directed under Injections, with Calcium Gluconate Hydrate. Indicate the amount of total calcium if any stabilizers are added. Description Calcium Gluconate Injection is a clear and colorless liquid. Identification (1) Pipet about 5.0 mL of Calcium Glu-

KP 9 147 conate Injection. Add 0.7 mL of glacial acetic acid and 1 mL of freshly distilled phenylhydrazine and heat the solution in a water-bath for 30 minutes. Cool and perform the test according to Identification (1) under Calcium Gluconate Hydrate. (2) An aqueous solution of Calcium Gluconate Injection (1 in 5) responds to the Qualitative Tests for calcium. pH Between 6.0 and 8.2. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 0.17 EU per g of Calcium Gluconate Hydrate. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement. It nary temperature and yields the anhydride at 120 C. Identification A solution of Calcium Lactate Hydrate (1 in 20) responds to the Qualitative Tests for calcium salt and for lactate. Purity (1) Clarity of solutionDissolve 1.0 g of Calcium Lactate Hydrate in 20 mL of water by warming: the solution is clear. (2) Acid or alkaliTo the solution obtained in (1), add 2 drops of phenolphthalein TS: no red color is produced. Then add 0.50 mL of 0.1 mol/L sodium hydroxide VS: a red color develops. (3) Heavy metalsDissolve 1.0 g of Calcium Lactate Hydrate in 30 mL of water and 5 mL of dilute acetic acid by warming, cool, add water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 2.0 mL of standard lead solution and 2 mL of dilute acetic acid and dilute with water to make 50 mL (not more than 20 ppm). (4) Magnesium or alkali metalsDissolve 1.0 g of Calcium Lactate Hydrate in 40 mL of water, add 0.5 g of ammonium chloride, boil, then add 20 mL of ammonium oxalate TS. Heat the mixture in a water-bath for 1 hour, cool, dilute with water to make 100 mL and filter, To 50 mL of the filtrate, add 0.5 mL of sulfuric acid, evaporate to dryness and ignite between 450 C and 550 C to a constant mass: the weight of the residue is not more than 5 mg. (5) ArsenicDissolve 0.5 g of Calcium Lactate Hydrate in 2 mL of water and 3 mL of hydrochloric acid, use this solution as the test solution. and perform the test (not more than 4 ppm). (6) Volatile fatty acidWarm 1.0 g of Calcium Lactate Hydrate with 2 mL of sulfuric acid: an odor of acetic acid or butyric acid is not perceptible. Loss on Drying Between 25.0 and 30.0% (1 g, 80 C, 1 hour at first, then 120 C, 4 hours).

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure accurately a volume of Calcium Gluconate Injection equivalent to about 0.4 g of Calcium Gluconate, add 100 mL of water, 2 mL of 8 mol/L potassium hydroxide TS and 0.1 mg of NN indicator. Immediately after the addition, titrate with 0.05 mol/L disodium ethylenediamine-tetraacetate VS until the solution becomes blue. Each mL of 0.05 mol/L disodium ethlendiaminetetraacetate VS = 2.0040 mg of Ca Packaging and Storage Preserve in hermetic containers.

Calcium Lactate Hydrate

OH CH3CCO2 H
-

Ca
2

2+

5H2O

C6H10CaO6.5H2O: 308.29 Calcium Lactate Hydrate, when dried, contains not less than 97.0% and not more than 101.0% of calcium lactate (C6H10CaO6: 218.22). Description Calcium Lactate Hydrate is a white powder or granule, is odorless and has a slightly acid taste. 1 g Of Calcium Lactate Hydrate dissolves gradually in 20 mL of water, is slightly soluble in ethanol and practically insoluble in ether. Calcium Lactate Hydrate is partly efflorescent at ordi-

Assay Weigh accurately 0.5 g of Calcium Lactate Hydrate, previously dried, add water, dissolve by heating in a water-bath, cool and add water to make exactly 100 mL. Pipet 20 mL of this solution, add 80 mL of water and 1.5 mL of 8 mol/L potassium hydroxide TS and allow to stand for 3 to 5 minutes. Add 0.1 g of NN indicator and titrate immediately with 0.02 mol/L disodium ethylenediaminetetraacetate VS until the color of the solution changes from red to blue. Each mL of 0.02 mol/L disodium ethylenediaminetetraacetate VS = 4.3644 mg of C6H10CaO6. Packaging and Storage Preserve in tight containers.

148 Monographs, Part I tion as the test solution and perform the test (not more than 5 ppm). (4) m-AminophenolTo 0.10 g of Calcium paminosalicylate Hydrate, add 5 mL of 0.1 mol/L disodium ethylenediaminetetraacetate TS, previously cooled in ice-water and dissolve by shaking vigorously. Add immediately 3 mL of ammonia-ammonium chloride buffer solution, pH 11.0, previously cooled in ice-water and shake. Add 2 mL of 4-amino-N,N-diethylaniline sulfate TS, shake, add 10.0 mL of cyclohexane and 4 mL of diluted potassium ferricyanide TS (1 in 10) and shake immediately for 20 seconds. Centrifuge this solution, wash the separated cyclohexane layer with two 5 mL volumes of diluted ammonia TS (1 in 14), add 1 g of anhydrous sodium sulfate, shake and allow to stand for 5 minutes: the clear cyclohexane layer has no more color than the following control solution. Control solutionDissolve 50 mg of maminophenol in water and dilute with water to make exactly 500 mL. Measure exactly 20 mL of this solution and add water to make exactly 100 mL. Take 5.0 mL of this solution, add 3 mL of ammonia-ammonium chloride buffer solution, pH 11.0, previously cooled in ice-water and treat this solution in the same manner as the test solution. Water Between 23.3% and 26.3% (0.1 g, volumetric titration, direct ditration) Assay Weigh accurately about 0.2 g of Calcium paminosalicylate Hydrate, dissolve in 60 mL of water and 0.75 mL of dilute hydrochloric acid by warming on a water-bath. After cooling, add water to make exactly 100 mL and use this solution as the test solution. Measure exactly 30 mL of the test solution, transfer to an iodine flask and add exactly 25 mL of 0.05 mol/L bromine VS and 20 mL of a solution of potassium bromide (1 in 4). Add immediately 14 mL of a mixture of glacial acetic acid and hydrochloric acid (5 : 2), stopper the flask immediately and allow to stand for 10 minutes with occasional shaking. Add cautiously 6 mL of potassium iodide TS and shake gently. After 5 minutes, titrate the produced iodine with 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of starch TS). Perform a blank determination and make any necessary connection. Each mL of 0.05 mol/L bromine VS = 3.187 mg of C7H5CaNO3 Packaging and Storage tight containers. Preserve in light-resistant,

Calcium p-aminosalicylate Hydrate

COOO2 Ca2+ 7H2O

NH2

Pas-calcium

C7H5CaNO33H2O: 251.23

Calcium p-aminosalicylate Hydrate contains not less than 97.0% and not more than 103.0% of calcium paminosalicylic acid (C7H5CaNO3: 191.20), calculated on the anhydrous basis. Description Calcium p-aminosalicylate Hydrate is a white to slightly colored powder, is odorless and has a slightly bitter taste. Calcium p-aminosalicylate Hydrate is very slightly soluble in water and practically insoluble in ethanol, in acetone or in chloroform. A saturated solution of Calcium p-aminosalicylate Hydrate is alkaline. Identification (1) To 50 mg of Calcium paminosalicylate Hydrate, add 100 mL of water, shake well and filter. To 10 mL of the filtrate, add 1 mL of 1 mol/L hydrochloric acid TS, shake and add 1 drop of ferric chloride TS: a red-purple color develops. (2) Determine the infrared spectra of Calcium paminosalicylate Hydrate and calcium p-aminosalicylate Hydrate RS, as directed in the potassium bromide disk method under the Infrared Spectro-photometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) To 3 g of Calcium p-aminosalicylate Hydrate, add 15 mL of ammonium chloride TS and 15 mL of water, heat on a water-bath for 10 minutes: the solution responds to the Qualitative Tests 1), 2) and 3) for calcium salt. Purity (1) ChlorideDissolve 1.0 g of Calcium paminosalicylate Hydrate in 15 mL of dilute nitric acid and add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.70 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.025%). (2) Heavy metalsProceed with 1.0 g of Calcium paminosalicylate Hydrate according to method 3 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicDissolve 0.40 g of Calcium paminosalicylate Hydrate in 20 mL of 0.1 mol/L hydrochloric acid TS by warming on a water-bath, use this solu-

KP 9 149

Calcium p-aminosalicylate Granules

Pas-calcium Granules Calcium p-aminosalicylate Granules contain not less than 95.0% and not more than 105.0% of the labeled amount of calcium p-aminosalicylate hydrate (C7H5CaNO33H2O: 254.25). Method of Preparation Prepare as directed under Granules, with Calcium p-aminosalicylate Hydrate. Identification Powder Calcium p-aminosalicylate Granules, weigh a portion of the powder, equivalent to 50 mg of calcium p-aminosalicylate Hydrate according to the labeled amount, add 100 mL of water, mix well and filter. To 10 mL of the filtrate, add 1 mL of 1 mol/L hydrochloric acid TS, shake and add 1 drop of ferric chloride TS: a red-purple color develops. Particle Size Distribution Test for Preparations It meets the requirement. Disintegration Test It meets the requirement. Uniformity of Dosage Unit (divided) quirement. It meets the re-

Calcium Pantothenate, when dried, contains not less than 5.7% and not more than 6.0% of nitrogen (N: 14.01) and not less than 8.2% and not more than 8.6% of calcium (Ca: 40.08). Description Calcium Pantothenate is a white powder, is odorless and has bitter taste. Calcium Pantothenate is freely soluble in water, very slightly soluble in ethanol and practically insoluble in ether. Calcium Pantothenate is hygroscopic. pHThe pH of a solution of Calcium Pantothenate (1 in 20) is between 7.0 and 9.0. Identification (1) Dissolve 50 mg of Calcium Pantothenate in 5 mL of sodium hydroxide TS and filter. Add 1 drop of copper(II) sulfate TS to the filtrate: a deep blue color develops. (2) Add 5 mL of sodium hydroxide TS to 50 mg of Clacium Pantothenate, and boil for 1 minute. After cooling, add diluted hydrochloric acid (1 in 10) to adjust the solution to a pH between 3 and 4, and add 2 dops of iron(III) chloride TS: a yellow color develops. (3) A solution of Calcium Panthothenate (1 in 10) responds to the Qualitative Tests for calcium salt. Specific Optical Rotation [ ]20 D : Between +25.0 and -28.5 (after drying, 1 g, water, 20 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 1.0 g of Calcium Pantothenate in 20 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Calcium Pantothenate according to method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) AlkaloidsDissolve 50 mg of Calcium Pantothenate in 5 mL of water, add 0.5 mL of hexaammonium heptamolybdate TS and 0.5 mL of a solution of phosphoric acid (1 in 10): no white turbidity is produced. Loss on Drying Not more than 5.0% (1 g, 105 C, 4 hours). Assay (1) NitrogenProceed with about 50 mg of Calcium Pantothenate, previously dried and accurately weighed, as directed under Nitrogen Determination. (2) CalciumWeigh accurately about 0.4 g of Calcium Pantothenate, previously dried, and dissolve in 30 mL of water by warming. After cooling, add exactly 25 mL of 0.05 mol/L disodium ethylenediamine tetraacetate VS, then 10 mL of ammonia-ammonium chloride buffer solution, pH 10.7, and tittrate the excess disodium ethylenediamine tetraacetate with 0.05 mol/L magnesium chloride VS until the color of the solution changes from blue-purple to red-purple (indicator: 40 mg of eriochrome black T-sodium chloride indicator). Perform a

Assay Powder Calcium p-aminosalicylate Granules, weigh accurately a portion of the powder, equivalent to about 0.4 g of calcium p-aminosalicylate hydrate (C7H5CaNO33H2O: 251.23), add 120 mL of water and 1.5 mL of dilute hydrochloric acid and dissolve by heating on a water-bath. After cooling, add water to make exactly 200 mL and filter. Pipet 30 mL of the filtrate, transfer to an iodine flask and proceed as directed in the Assay under Calcium p-aminosalicylate Hydrate. Each mL of 0.05 mol/L bromine VS = 4.238 mg of C7H5CaNO33H2O Packaging and Storage tight containers. Preserve in light-resistant,

Calcium Pantothenate
CH3 OH HO CH2 C C O C NHCH 2CH2CO2
2+

Ca 2

CH3 H

C18H32CaN2O10 : 476.53

150 Monographs, Part I blank determination and make any necessary correction. Each mL of 0.05 mol/L disodium ethylenediamine tetraacetate VS = 2.0039 mg Ca Packaging and Storage Preserve in tight containers. conditions. Determine the peak heights, HT and HS, of styrene for the test solution and the standard solution: HT is not larger than HS. Operating conditions Detector: A hydrogen flame-ionization detector. Column: A stainless steel column, about 3 mm in inside diameter and about 2 m in length, having polyethylene glycol 20M coated at the ratio of 15% on siliceous earth for gas chromatography (149 to 177 m in particle diameter). Column temperature: A constant temperature of about 90 C. Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of styrene is about 9 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, the number of theoretical plates of the peak of styrene is not less than 800 and the symmetry factor of the peak is between 0.8 and 1.2. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of each peak height of styreme is not more than 5.0%. (5) SodiumPipet 20 mL of the 50 mL solution obtained in Assay (1), add 0.02 mol/L hydrochloric acid to make exactly 500 mL and use this solution as the test solution. Separately, weigh accurately 0.2542 g of sodium chloride, previously dried at 130 C for 2 hours and dissolve in 0.02 mol/L hydrochloric acid to make exactly 1000 mL. Pipet a suitable volume of this solution and dilute with 0.02 mol/L hydrochloric acid to make a solution containing 1 to 3 g of sodium (Na: 22.99) per mL and use these solutions as the standard solutions. Perform the test with the test solution and the standard solutions as directed under the Atomic Absorption Spectrophotometry according to the following conditions and determine the amount of sodium in the test solution using the calibration curve obtained from the standard solutions: the amount of sodium is not more than 1%. Gas used: Combustible gas: Acetylene. Supporting gas: Air. Lamp: A sodium hollow-cathode lamp. Wavelength: 589.0 nm. Loss on Drying Not more than 10.0% (1 g, in vacuum, 80 C, 5 hours). Microparticles (1) Apparatus: Use an apparatus as shown in the figure. Actual volume to the mark of 20 cm at which the sedimentation tube is inserted: 550 mL. Single suction volume: 10 mL. (2) Procedure: Weigh accurately about 5.5 g of Calcium Polystyrene Sulfonate, previously dried, add 300

Calcium Polystyrene Sulfonate

Calcium Polystyrene Sulfonate is a cation exchange resin prepared as the calcium form of the sulfonated styrene divinylbenzene copolymer. Calcium Polystyrene Sulfonate, when dried, contains not less than 7.0% and not more than 9.0% of calcium (Ca: 40.08). Each g of Calcium Polystyrene Sulfonate, when dried, exchanges with 53 to 71 mg of potassium (K: 39.10). Description Calcium Polystyrene Sulfonate is a pale yellowish white to pale yellow powder, is odorless and tasteless. Calcium Polystyrene Sulfonate is practically insoluble in water, in ethanol or in ether. Identification (1) Take Calcium Polystyrene Sulfonate and Calcium Polystyrene Sulfonate RS, previously dried and determine the infrared spectra as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Mix 0.5 g of Calcium Polystyrene Sulfonate with 10 mL of dilute hydrochloric acid, filter and neutralize the filtrate with ammonia TS: the solution responds to the Qualitative Tests for calcium salt. Purity (1) AmmoniumPlace 1.0 g of Calcium Polystyrene Sulfonate in a flask, add 5 mL of sodium hydroxide TS, cover the flask with a watch glass having a moistened strip of red litmus paper on the underside and boil for 15 minutes: the gas evolved does not change the red litmus paper to blue (not more than 5 ppm). (2) Heavy metalsProceed with 2.0 g of Calcium Polystyrene Sulfonate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Calcium Polystyrene Sulfonate according to Method 3 and perform the test (not more than 2 ppm). (4) StyreneTo 10.0 g of Calcium Polystyrene Sulfonate, add 10 mL of acetone, shake for 30 minutes, centrifuge and use the supernatant as the test solution. Separately, dissolve 10 mg of styrene in acetone to make exactly 100 mL. Pipet 1 mL of this solution, dilute with acetone to make exactly 100 mL and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following

KP 9 151 mL of water of 25 C and mix for 5 minutes. Transfer this turbid solution to the sedimentation tube, J, keeping a temperature at 25 C, add water of 25 C to 2 mm below the mark, F of 20 cm of the sedimentation tube, J and then insert the pipet. Open the two-way stopcock, C, exhaust air, add exactly water from the vent-hole, D to the mark, F of 20 cm and close the two-way stopcock, C. Shake the apparatus well vertically and horizontally, disperse Calcium Polystyrene Sulfonate in water and then open the two-way stopcock and allow to stand at 25 1 C for 5 hours and 15 minutes. Then, draw exactly the meniscus of the turbid solution in sedimentation tube, J, up to the mark of pipet bulb, A, by suction, open the twoway stopcock, C, to the outlet of pipet, H and transfer exactly measured 20 mL of the turbid solution to a weighing bottle. Repeat the procedure and combine exactly measured 20 mL of the turbid solution. Evaporate 20 mL of this turbid solution on a water-bath to dryness, dry to constant mass at 105 C and weigh the residue as WS (g). Pipet 20 mL of used water and weigh the residue in the same manner as WB (g). Calculate the difference mi (g) of the weight between WS and WB and calculate the amount of microparticles (S) by the following equation: the amount of microparticles is not more than 0.1%. |WS - WB| (g) V (mL) S (%) = 20 (mL) amount of sample (g) 100 V: Actual volume (mL) to the mark of 20 cm at which the suction part of pipet is inserted. fer the mixture and wash out completely with the aid of a small volume of 3 mol/L hydrochloric acid TS, to a column, 12 mm in inside diameter and 70 mm in length, packed with a pledged of fine glass wool in the bottom, placing a volumetric flask as a receiver under the column. Then collect about 45 mL of eluate, adding 3 mol/L hydrochloric acid TS to the column and add water to make exactly 50 mL. Pipet 20 mL of this solution, adjust with ammonia water to a pH of exactly 10. Titrate immediately with 0.05 mol/L disodium ethylenediaminetetraacetate VS until the red-purple color of the solution disappears and a blue color is observed (indicator: 40 mg eriochrome black T-sodium chloride indicator). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L disodium ethylenediaminetetraacetate VS = 2.0039 mg of Ca (2) Potassium exchange capacityPipet 50 mL of standard potassium stock solution into a glass-stoppered flask containing about 1.0 g of dried Calcium Polystyrene Sulfonate, accurately weighed, stir for 120 minutes, filter and discard the first 20 mL of the filtrate. Pipet 5 mL of the subsequent filtrate and add 0.02 mol/L hydrochloric acid TS to make exactly 100 mL. Pipet 10 mL of this solution, add 0.02 mol/L hydrochloric acid TS to make exactly 1000 mL and use this solution as the test solution. Separately, measure exactly a suitable volume of standard potassium stock solution, dilute with water to make solutions containing 0.5 to 2.5 g of potassium (K: 39.10) per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Atomic Absorption Spectrophotometry according to the following conditions and determine the amount, Y (mg), of potassium in 1000 mL of the test solution, using the calibration curve obtained from the standard solution. The exchange capacity for potassium per g of dried Calcium Polystyrene Sulfonate is 53 to 71 mg, calculated by the following equation. Exchange capacity (mg) for potassium (K) per g of dried X - 100Y Calcium Polystyrene Sulfonate = W X: Amount (mg) of potassium in 50 mL of standard potassium stock solution before exchange, W: Amount (g) of dried Calcium Polystyrene Sulfonate taken. Gas used: Combustible gas- Acetylene. Supporting gas: Air. Lamp: A potassium hollow-cathode lamp. Wavelength: 766.5 nm. Andreagen pipet Assay (1) CalciumWeigh accurately about 1.0 g of Calcium Polystyrene Sulfonate, previously dried, and disperse in 5 mL of 3 mol/L hydrochloric acid TS. TransPackaging and Storage Preserve in tight containers.

A: Mark of pipet bulb, B: Pipet bulb for suction, C: Two-way stopcock, D: Vent-hole, E: Suction part of pipet, F: Mark of 20 cm, G: Base line of 0 cm, H: Outlet of pipet, I: Capillary tube of pipet, J: Sedimentation tube.

152 Monographs, Part I d-Camphor RS, add exactly 5 mL each of the internal standard solution, dissolve in dehydrated methanol to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 2 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of d-Camphor to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of d-camphor (C10Hl6O) = amount (mg) of d-Camphor RS QT
QS

d-Camphor
H3C O CH3 CH3

C10Hl6O: 152.23 d-Camphor contains not less than 96.0% and not more than 101.0 % of d-camphor (C10Hl6O). Description d-Camphor is a colorless or white, translucent crystal, crystalline powder or mass. d-Camphor has a characteristic, agreeable odor and a slightly bitter taste, followed by a pleasant, cooling sensation. Camphor is freely soluble in ethanol, in ether or in carbon disulfide and slightly soluble in water. d-Camphor slowly volatilizes at room temperature. Identification Dissolve 0.1 g of d-Camphor in 2 mL of methanol, add 1 mL of 2,4-dinitrophenylhydrazine TS and heat for 5 minutes in a water-bath: an orange-red precipitate is formed. Specific Optical Rotation [ ]20 D : Between + 41.0 and + 43.0 (5g, ethanol, 50 mL, 100 mm). Melting Point Between 177 C and 182 C

Internal standard solutionA solution of methyl salicilate in dehydrated ethanol (1 in 25). Operating conditions Detector: A hydrogen flame-ionization detector. Column: A column, about 3 mm in inside diameter and 3 m in length, packed with 10% of polyethylene glycol 20M for gas chromatography supported on 180 to 250 m mesh silanized siliceous earth for gas chromatography. Column temperature: A constant temperature of about 160 C. Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of d-Camphor is about 6 minutes. System suitability System performance: When the procedure is run with 2 L of the standard solution under the above operating conditions, d-Camphor and the internal standard are eluted in this order, with a resolution between these peaks being not less than 7. System repeatability: When the test is repeated 6 times with 2 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of d-Camphor to that of the internal standard is not more than 1.0 %. Packaging and Storage Preserve in tight containers.

Purity (1) WaterShake 1.0 g of d-Camphor with 10 mL of carbon disulfide: the solution is clear. (2) Chlorinated compoundsMix 0.20 g of finely powdered d-Camphor with 0.4 g of sodium peroxide in a dried, porcelain crucible. Heat the crucible gently by the open flame until the incineration is complete. Dissolve the residue in 20 mL of warm water, acidify with 12 mL of dilute nitric acid and filter the solution into a Nessler tube. Wash the test tube and the filter with three 5 mL volumes of hot water, adding the washings to the filtrate. After cooling, add water to make 50 mL, then add 1 mL of silver nitrate TS, mix well and allow to stand for 5 minutes: the turbidity of the solution does not exceed that of the following control solution. Control solutionPrepare in the same manner as described above, using 0.20 mL of 0.01 mol/L hydrochloric acid VS. (3) Non-volatile residueHeat 2.0 g of d-camphor in a water-bath until sublimation is completed, then dry the residue at 105 C for 3 hours: the weight of the residue is not more than 1.0 mg. Assay Weigh accurately 0.1 g each of d-Camphor and

dl-Camphor
H3C O CH3 CH3

and enantiomer C10Hl6O: 152.23 dl-Camphor contains not less than 96.0% and not more

KP 9 153 than 101.0% of dl-camphor (C10Hl6O). Description dl-Camphor is a colorless or white, translucent crystal, crystalline powder or mass. dl-Camphor has a characteristic, agreeable odor and has a slightly bitter taste followed by a pleasant, cooling sensation. dl-Camphor is freely soluble in ethanol, in ether or in carbon bisulfide and slightly soluble in water. dl-Camphor slowly volatilizes at room temperature. Identification Dissolve 0.1 g of dl-Camphor in 2 mL of methanol, add 1 mL of 2,4-dinitrophenyl-hydradine TS and heat for 5 minutes in a water-bath: an orange-red precipitate is formed. Specific Optical Rotation [ ]20 D : Between - 1.5 and + l.5 (5 g, ethanol, 50 mL, 100 mm). Melting Point Between 175 C and 180 C

= amount (mg) of dl-Camphor RS

QT QS

Internal standard solutionA solution of methyl salicilate in dehydrated ethanol (1 in 25). Operating conditions Detector: A hydrogen flame-ionization detector. Column: A column, about 3 mm in inside diameter and about 3 m in length, packed with 10% of polyethylene glycol 20M for gas chromatography supported on 180 to 250 m mesh silanized siliceous earth for gas chromatography. Column temperature: A constant tem-perature of about 160 C. Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of dl-Camphor is about 6 minutes. System suitability System performance: When the procedure is run with 2 L of the standard solution under the above operating conditions, dl-Camphor and the internal standard are eluted in this order, with a resolution between these peaks being not less than 7. System repeatability: When the test is repeated 6 times with 2 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of dl-Camphor to that of the internal standard is not more than 1.0 %. Packaging and Storage Preserve in tight containers.

Purity (1) WaterShake 1.0 g of dl-Camphor with 10 mL of carbon bisulfide: the solution is clear. (2) Chlorinated compoundsMix 0.20 g of finely powdered dl-Camphor with 0.4 g of sodium peroxide in a dried, porcelain crucible. Heat the crucible gently by the open flame until the incineration is complete. Dissolve the residue in 20 mL of warm water, acidify with 12 mL of dilute nitric acid and filter the solution into a Nessler tube. Wash the test tube and the filter with three 5 mL volumes of hot water, adding the washings to the filtrate. After cooling, add water to make 50 mL, then add 1 mL of silver nitrate TS, mix well and allow to stand for 5 minutes: the turbidity of the solution does not exceed that of the following control solution. Control solutionPrepare in the same manner as described above, using 0.20 mL of 0.01 mol/L hydrochloric acid VS. (3) Non-volatile residueHeat 2.0 g of dl-Camphor in a water-bath until sublimation is completed, then dry the residue at 105 C for 3 hours: the weight of the residue is not more than 1.0 mg. Assay Weigh accurately 0.1 g each of dl-Camphor and dl-Camphor RS, add exactly 5 mL each of the internal standard solution, dissolve in dehydrated methanol to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 2 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of dl-camphor to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of dl-camphor (C10Hl6O)

Captopril
H HS CH3 O H O

OH

C9H15NO3S : 217.29 Captopril contains not less than 97.5% and not more than 102.0% of captopril (C9H15NO3S), calculated on the dried basis. Description Captopril is white crystal or crystalline powder. Captopril is very soluble in methanol, freely soluble in ethanol and soluble in water. Melting pointsAbout 106 C Identification Determine the infrared spectra of Captopril and Captopril RS, previously dried, as directed in the potassium bromide disk method under the Infrared

154 Monographs, Part I Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation -134 mm)
o

ing not less than 3.0. Loss on Drying Not more than 1.0% (1 g, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.3 g of Captopril, dissolve in 100 mL of water, add 20 mL of dilute sulfuric acid and 1 g of potassium iodide and shake. Titrate with 1/60 mol/L potassium iodate VS (indicator: 2 mL of starch TS). Perform a blank determination and make any necessary correction. Each mL of 1/60 mol/L potassium iodate = 21.729 mg of C9H15NO3S Packaging and Storage Preserve in tight containers.

[ ]20 D :

Between -125

and

(dried, 0.1 g, dehydrated ethanol, 10 mL, 100

Purity (1) Heavy metalsProceed with 1 g of Captopril according to the Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) ArsenicPrepare the test solution with 1 g of Captopril according to Method 1 and perform the test. (not more than 2 ppm). (3) Related substancesDissolve 50.0 mg of Captopril in methanol to make exactly 25 mL and use this solution as the test solution(use immediately after preparation). Separately, weigh accurately captopril disulfide RS, dissolve in methanol to make 10 g per mL and use this solution as the standard solution. Perform the test with 20 L each of these solutions as directed under the Liquid Chromatography according to the following conditions and calculate the peak area, AT and AS , of captopril disulfide of these solutions: the amount of captoprile disulfide is not more than 1.0% and the peak response of each impurity is not more than 40% of the main peak in the chromatogram of the standard solution(not more than 0.2%) and the sum of the impurity peak responses is not more than the main peak response in the chromatogram of the standard solution(not more than 0.5%). Amount (%) of captopril disulfide =
C S AT 100 C u AS

Captopril Tablets
Captopril Tablets contain not less than 90.0% and not more than 110.0% of labeled amount of captopril (C9H15NO3S: 217.29). Method of Preparation Prepare as directed under Tablets, with Captopril. Identification weigh accurately and Powder a portion of Captopril Tablets, equivalent to 0.1 g of Captopril, put in the flask, add 25 mL of methanol, mix, centrifuge and use the supernatant as the test solution. Separately weigh a suitable amount of Captopril RS, dissolve in a suitable amount of methanol so as to contain 4 mg of Captopril per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under Thin Layer Chromatograph. Spot 50 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plates with a mixture of toluene, glacial acetic acid and methanol (75 : 25 : 1) to a distance of about 15 cm and air-dry the plate. Spray with a freshly prepared mixture of ammonium hydroxide and a solution of 0.04 % 5,5-dithio-bis-(2-nitrobenzoic acid) (1 : 6). The Rf value of the principal spot of the test solution is the same as that of standard solution. Related Substances CaptoprilndisulfideUse the test solution under the Assay. Separately, weigh accurately a suitable portion of Captopril Disulfide RS and dissolve in a suitable volume of mobile phase so as to contain 50 g of captopril disufide per mL. Use this solution as the standard solution. Avoid the test solution and the standard solution with contact of air and use them within 8 hours of preparation. Perform the test with 20 L each of the test solution and the standard solution as directed un-

CS: the concentration of captopril disulfide RS in the standard solution (g/mL) Cu: the concentration of captopril in the test solution (g/mL) Operating conditions Detector: An ultraviolet absorption photometer ( wavelength: 220 nm ). Column: A stainless steel column 3.9 mm in inside diameter and 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particles diameter ). Mobile phase: A mixture of tetrahydrofuran methanol solution (9 in 100) and phosphoric acid solution (1 in 2000) (33 : 67). Selection of column: Dissolve Captopril RS, Captopril Disulfide RS and 3-Acetylthio-2-Methylpropanoic Acid RS in methanol to make 10 g per mL, respectively. Proceed with 20 L of this solution under the above operating conditions. Use a column giving elution of captopril, 3-acetylthio-2-methylpropanoic acid and captopril disulfide in this order with the resolution between peaks of captopril and 3-acetylthio-2-methylpropanoic acid be-

KP 9 155 der the the Liquid Chromatography according to the following operating conditions. Determine peak areas, AT and AS , of captopril disulfide of the test solution and the standard solution, respectively: the amount of captopril disulfide is not more than 3.0%.
A Amount (%) of captopril disulfide = 2500 C T W AS

C: Concentration of the standard solution (mg/mL), W: Amount of sample (mg). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column:A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilylated silica gel (contains 15% of CH group) for liquid chromatography (3 to 10 m in particle diameter). Mobile phase: A mixture of methanol, water and phosphoric acid (450 : 550 : 0.5). Flow rate: 1.0 mL/minute. System suitability System performance: When the procedure is run with 20 L of the standard solution, as directed under the above operating conditions, Captopril and captopril disulfide are eluted in this order with the resolution between these peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with with 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation between the peak areas of captopril is not more than 2.0%. Dissolution Test Perform the test with 1 tablet of Captopril Tablets at 50 revolutions per minute according to Method 1 under Dissolution Test, using 900 mL of 0.01 mol/L hydrochloric acid. as a dissolution solution. Take 20 mL or more of the dissolved solution 20 minutes after starting the test, filter and use the filtrate as the test solution. Separately, weigh accurately a suitable amount of Captopril RS, dissolved in a suitable amount of water so as to contain same amount of Captopril per mL as the test solution and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution at 212 nm as directed under the Ultraviolet-visible Spectrophotometry : The dissolution rate (%) with respect to the labeled amount of Captopril Tablets in 20 minutes is not less than 80%. Uniformity of Dosage Units It meets the requirement. Assay Transfer not less than 20 Captopril Tablets to a suitable volumetric flask, add mobile phase to fill the flask to about half of its capacity and sonicate for 15 minutes. Dilute with mobile phase to volume, shake by mechanical means for 15 minutes and filter. Dilute quantita-

tively and stepwise if necessary, with mobile phase to obtain a solution having a concentration of about 1 mg of Captopril per mL. Separately, dissolve suitable quantities of Captopril RS and Captopril Disulfide RS in mobile phase to obtain a solution having known concentrations of about 1 mg per mL and 50 g per mL, respectively. Use this solution as the standard solution. Avoid the test solution and the standard solution with contact of air and use them within 8 hours of prepration. Proceed with 20 L each of the test solution and the standard solution according to the Liquid Chromatography under the following operating conditions. Determine peak areas, AT and

AS , of Captopril of the test solution and the standard solution, respectively. Amount(mg) of Captopril (C9H15NO3S) in 1 mL of test solution = C
AT AS

C: Concentration (mg/mL) of Captopril in the standard solution, Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm length, packed with octadecylsilylated silica gel (contains 15% of CH group) for liquid chromatography (5 to 10 m in particle diameter). Mobile phase: A mixture solution of methanol, water and phosphoric acid (450 : 550 : 0.5). Flow rate: 1.0 mL/minute. System suitability System performance: When the procedure is run with 20 L of the stndard solution, as directed under the above operating conditions, Captopril and captopril disulfide are eluted in this order with the resolution between these peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the peak areas of Captopril is not more than 2.0%. Packaging and Storage Preserve in well-closed containers.

Carbamazepine

N C O NH2

156 Monographs, Part I each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of toluene and methanol (19 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly potassium bichromate-sulfuric acid TS on the plate: the spots other than the principal spot from the test solution is not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve 50 mg of Carbamazepine, previously dried and accurately weighed, in ethanol to make exactly 250 mL. Dilute 5 mL of this solution with ethanol to make exactly 100 mL. Perform the test as directed under the Ultraviolet-visible Spectrophotometry and determine the absorbance, A, of this solution at the wavelength of a maximum absorption at about 285 nm. Amount (mg) of carbamazepine (C15H12N2O) A = 490 50000 Packaging and Storage Preserve in tight containers.

C15Hl2N2O: 236.27 Carbamazepine, when dried, contains not less than 97.0% and not more than 103.0% of carbamazepine (C15Hl2N2O) Description Carbamazepine is a white to slightly yellowish white powder, is odorless and tasteless at first and leaves a slightly bitter aftertaste. Carbamazepine is freely soluble in chloroform, sparingly soluble in ethanol or in acetone and very slightly soluble in water or in ether. Identification (1) To 0.1 g of Carbamazepine, add 2 mL of nitric acid and heat in a water-bath for 3 minutes: an orange-red color is produced. (2) To 0.1 g of Carbamazepine, add 2 mL of sulfuric acid and heat in a water-bath for 3 minutes: a yellow color is produced with a green fluorescence. (3) Examine Carbamazepine under ultraviolet light: the solution shows an intense blue fluorescence. (4) Determine the absorption spectra of solutions of Carbamazepin and Carbamazepin RS in ethanol (1 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Melting Point Between 189 C and 193 C

Purity (1) Clarity and color of solutionDissolve 1.0 g of Carbamazepine in 10 mL of chloroform: the solution is clear and colorless to pale yellow. (2) AcidTo 2.0 g of Carbamazepine, add exactly 40 mL of water, stir well for 15 minutes and filter through a glass filter. To 10 mL of this filtrate, add 1 drop of phenolphthalein TS and 0.50 mL of 0.01 mol/L sodium hydroxide VS: a red color is produced. (3) AlkaliTo 10 mL of the filtrate obtained in (2), add 1 drop of methyl red TS and 0.50 mL of 0.01 mol/L hydrochloric acid VS: a red color is produced. (4) ChlorideDissolve 0.25 g of Carbamazepine in 30 mL of acetone, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.20 mL of 0.01 mol/L hydrochloric acid VS, add 30 mL of acetone, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.028%). (5) Heavy metalsProceed with 2.0 g of Carbamazepine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (6) Related substancesDissolve 0.25 g of Carbamazepine in 10 mL of chloroform and use this solution as the test solution. Separately, dissolve 5.0 mg of iminodibenzyl in chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L

Carbazochrome Sodium Sulfonate hydrate

CH3 O N SO3Na H H2NCONHN 3H2O

and enantiomer Carbazochrome Sodium Sulfonate C10Hl1N4NaO5S 3H20 : 376.32 Carbazochrome Sodium Sulfonate Hydrate contains not less than 98.0% and not more than 102.0% of carbazochrome sodium sulfonate (C10Hl1N4NaO5S: 322.28), calculated on the anhydrous basis. Description Carbazochrome Sodium Sulfonate is a orange-yellow, crystal or crystalline powder. Carbazochrome Sodium Sulfate is sparingly soluble in water, very slightly soluble in ethanol and practically insoluble in ether. A solution of Carbazochrome Sodium Sulfonate(1 in 100) shows no optical rotation. Melting point About 210 C (with decomposition). Identification (1) Determine the absorption spectra of solutions of Carbazochrome Sodium Sulfonate and Car-

KP 9 157 bazochrome Sodium Sulfonate RS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Carbazochrome Sodium Sulfonate and Carbazochrome Sodium Sulfonate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Carbazochrome Sodium Sulfonate (1 in 100) responds to the Qualitative Tests (1) for sodium salt. pH Dissolve 0.8 g of Carbazochrome Sodium Sulfonate in 50 mL of water by warming and cool: the pH of this solution is between 5.0 and 6.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Carbazochrome Sodium Sulfonate in 50 mL of water by wanting and allow to cool: the solution is clear. Perform the test with this solution as directed under the Ultraviolet-visible Spectrophotometry: the absorbance at 590 nm is not more than 0.070. (2) Heavy metalsProceed with 1.0 g of Carbazochrome Sodium Sulfonate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 50 mg of Carbazochrome Sodium Sulfonate in 100 mL of water and use this solution as the test solution. Pipet 2 mL of the test solution, add water to make exactly 200 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution and the standard solution by the automatic integration method: the total area of the peaks other than the peak of carbazochrome sulfonate from the test solution is not larger than the peak area of carbazochrome sulfonate from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 360 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 1.2 g of monobasic ammonium phosphate in 1000 mL of water and filter through a membrane filter, if necessary. To 925 mL of this solution, add 75 mL of ethanol, shake and adjust with phosphoric acid to a pH of 3. Flow rate: Adjust the flow rate so that the retention time of carbazochrome sulfonate is between 6 and 8 minutes. System suitability Test for required detectability: Take exactly 2 mLof the standard solution, add the mobile phase to make exactly 20 mL. The peak area of Carbazochrome Sulfonate from 10 mL of this solution is 7% to 13% of the peak area of Carbazochrome Sulfonate from the standard solution. System performance: Dissolve 10 mg each of Carbazochrome Sodium Sulfonate and carbazochrome in 100 mL of water by warming. When the procedure is run with 10 L of this solution under the above operating conditions, Carbazochrome sulfonate and Carbazochrome are eluted in this order with the resolution between their peaks being not less than 3. Detection sensitivity: Adjust the detection sensitivity so that the peak height of carbazochrome sulfonate obtained from 10 L of the standard solution composes about 5% of the full scale. Time span of measurement: About 3 times as long as the retention time of carbazochrome sulfonate after the solvent peak. Water Between 13.0% and 16.0% (0.3 g, volumetric titration, direct titration). Assay Weigh accurately about 0.25 g of Carbazochrome Sodium Sulfonate, dissolve in 50 mL of water, apply to a chromatographic column, 10 mm in diameter, previously prepared with 20 mL of strongly acidic ion exchange resin for column chromatography (type H) and allow to flow at a rate of 4 mL per minute. Wash the column with 150 mL of water, combine the washing and the former eluent solution and titrate with 0.05 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L sodium hydroxide VS = 16.114 mg of C10H11N4NaO5S Packaging and Storage Preserve in well-closed containers.

L-Carbocysteine
H HO 2CCH2SCH2 C NH 2 CO2H

Carbocysteine
L-Carbocysteine,

C5H9NO4S: 179.19

when dried, contains not less than 98.5% and not more than 101.0% of L-carbocysteine (C5H9NO4S). Description
L-Carbocysteine

is a white crystalline

158 Monographs, Part I powder, is odorless and has a slightly acid taste. L-Carbocysteine is very slightly soluble in water and practically insoluble in ethanol, in glacial acetic acid or in ether. L-Carbocysteine dissolves in dilute hydrochloric acid or in sodium hydroxide TS. Melting pointAbout 186 C (with decomposition) Identification (1) To 0.2 g of L-Carbocysteine, add 1 mL of lead acetate TS and 3 mL of water, shake, add 0.2 g of sodium hydroxide and heat over a flame for 1 minute: a dark brown to black precipitate is formed. (2) Determine the infrared absorption spectra of LCarbocysteine and Carbocysteine RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between -33.5 C and -36.5 C. Weigh accurately 5 g of L-Carbocysteine, previously dried, dissolve in 20 mL of water and a suitable volume of a solution of sodium hydroxide (13 in 100) and adjust the pH with 1 mol/L hydrochloric acid TS or 0.1 mol/L hydrochloric acid TS to 6.0 and add water to make exactly 50 mL. Determine the optical rotation of this solution in a 100 mm cell. Purity (1) Clarity and color of solutionDissolve 1.0 g of L-Carbocysteine in 10 mL of a solution of sodium hydroxide TS: the solution is clear and colorless. (2) ChlorideDissolve 0.20 g of L-Carbocysteine in 10 mL of water and 20 mL of nitric acid and add water to make 50 mL. Perform the test using this solution as the test solution. Control solutionTo 0.40 mL of 0.01 mol/L hydrochloric acid VS, add 20 mL of nitric acid and water to make 50 mL (not more than 0.071%). (3) AmmoniumPerform the test with 0.25 g of LCarbocysteine using the distillation under reduced pressure. Prepare the control solution with 5.0 mL of standard ammonium solution (not more than 0.02%). (4) Heavy metalsProceed with 1.0 g of LCarbocysteine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) ArsenicPrepare the test solution with 1.0 g of L-Carbocysteine according to Method 3 and perform the test (not more than 2 ppm). (6) Related substancesDissolve 0.30 g of LCarbocysteine in 10 mL of 0.2 mol/L sodium hydroxide TS and use this solution as the test solution. Pipet 2 mL of the test solution and add 0.2 mol/L sodium hydroxide TS to make exactly 100 mL. Pipet 1 mL of this solution and add 0.2 mol/L sodium hydroxide TS to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution, in 15 mm length along the starting line on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (3 : 1 : 1) to a distance of about 10 cm, air-dry the plate and then dry at 100 C for 30 minutes. Spray evenly a solution of ninhydrin in acetone (1 in 50) on the plate and heat at 80 C for 5 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.3% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.25 g of L-Carbocysteine, previously dried, dissolve in exactly 20 mL of 0.1 mol/L perchloric acid VS and 50 mL of glacial acetic acid and titrate the excess perchloric acid with 0.1 mol/L sodium acetate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 17.919 mg of C5H9NO4S Packaging and Storagte Preserve in tight containers.

Carbon Dioxide
CO2: 44.01 Carbon Dioxide contains not less than 99.5% and not more than 101.0 vol% of carbon dioxide (CO2). Description Carbon Dioxide is a colorless gas at room temperature and under atmospheric pressure and is odorless. 1 mL volume of Carbon Dioxide dissolve in 1 mL of water and the solution is slightly acidic. 1000 mL of Carbon Dioxide at 0 C and under a pressure of 101.3 kPa weighs about 1.978 g. Identification (1) Put a flaming wood splinter into Carbon Dioxide: the flame is extinguished immediately. (2) Pass Carbon Dioxide into calcium hydroxide TS: a white precipitate is produced. Collect the precipitate and add acetic acid: it dissolves with effervescence. Purity Maintain containers of Carbon Dioxide between 18 C and 22 C for not less than 6 hours prior to the test and correct the volume of Carbon Dioxide to 20 C and under a pressure of 101.3 kPa. (1) AcidPlace 50 mL of freshly boiled and cooled

KP 9 159 water in a Nessler tube and pass 1000 mL of Carbon Dioxide for 15 minutes through an introducing tube about 1 mm in diameter extending to 2 mm from the bottom of the Nessler tube, then add 0.10 mL of methyl orange TS: the solution has no more color than the following control solution. Control solutionTo 50 mL of freshly boiled and cooled water in a Nessler tube, add 0.10 mL of methyl orange TS and 1.0 mL of 0.01 mol/L hydrochloric acid VS. (2) Hydrogen phosphide, hydrogen sulfide or reducing organic substancesPlace 25 mL of silver nitrateammonia TS and 3 mL of ammonia TS in each two Nessler tubes A and B and designate the solution in each tube as solutions A and B, respectively. Pass 1000 mL of Carbon Dioxide into solution A in the same manner as directed in (1): the turbidity and color of this solution are the same as that of solution B. (3) Carbon monoxideIntroduce 5.0 mL of Carbon dioxide into a gas-cylinder or a syringe for gas chromatography from a metal cylinder holding gas under pressure and fitted with a pressure reducing valve, through a directly connected polyvinyl tube. Perform the test with this according to the Gas Chromatography under the following conditions: no peak is observed at the same retention time as that of the carbon monoxide. Operating conditions Detector: A thermal-conductivity detector. Column: A column about 3 mm in inside diameter and about 3 m in length, packed with 300 to 500 m zeolite for gas chromatography (0.5 nm in poreous size). Column temperature: A constant temperature of about 50 C. Carrier gas: Hydrogen or helium. Flow rate: Adjust the flow rate so that the retention time of carbon monoxide is about 20 minutes. System suitability System performance: To 0.1 mL each of carbon monoxide and air in a gas mixer add carrier gas to make 100 mL and mix well. When the procedure is run with 5.0 mL of the mixed gas under the above operating conditions, oxygen, nitrogen and carbon monoxide are eluted in this order with a well-resolving of their peaks. Detection sensitivity: Adjust the sensitivity so that the peak height of carbon monoxide obtained from 5.0 mL of the mixed gas used in the system performance is about 10 cm. (4) Oxygen and nitrogenIntroduce 1.0 mL of Carbon Dioxide into a gas-measuring tube or syringe for gas chromatography from metal cylinder under pressure with a pressure-reducing valve through a directly connected polyvinyl chloride tube. Perform the test as directed under Gas Chromatography according to the following conditions and determine the peak area, AT, of air. Separately, introduce 0.50 mL of nitrogen into the gas mixer, draw carrier gas into the mixer to make exactly 100 mL, allow to mix thoroughly and use this mixture as the standard gas mixture. Perform the test with 1.0 mL of the mixture in the same manner as directed in the case of Carbon Dioxide and determine the peak area, AS, of nitrogen: AT is smaller than AS and no other peak appears. Operating conditions Detector: A thermal-conductivity detector. Column: A column, about 3 mm in inside diameter and about 3 m in length, packed with silica gel for gas chromatography (300 to 500 m in particle diameter). Column temperature: A constant temperature of about 50 C. Carrier gas: Hydrogen or helium. Flow rate: Adjust the flow rate so that the retention time of nitrogen is about 2 minutes. System suitability System performance: Collect 0.5 mL of nitrogen in a gas mixer, add Carbon Dioxide to make 100 mL, mix well and proceed with 1.0 mL of the mixture under the above operating conditions. Use a column giving wellresolved peaks of nitrogen and Carbon Dioxide in this order. Detection sensitivity: Adjust the detection sensitivity so that the peak height of nitrogen obtained from 1.0 mL of the standard gas mixture composes about 50% of the full scale. Assay For the withdrawing of Carbon Dioxide, proceed as directed in the Purity. Place 125 mL of a solution of potassium hydroxide (1 in 2) in a gas pipet of suitable capacity. Measure exactly about 100 mL of Carbon Dioxide in a 100 mL gas buret filled with water. Force the entire volume of gas into the gas pipet and shake for 5 minutes. Draw some of the unabsored gas into the gas buret, measure the volume, force the residual back upon the surface of the liquid in the gas pipet and repeat this procedure until a constant volume of the residual reading is obtained. Determine the volume, V (mL) of the residual gas and correct its volume, V to 20 C and under a pressure of 101.3 kPa. Volume (mL) of carbon dioxide (CO2) = calculated volume (mL) of the sample calculated volume, V (mL) Packaging and Storage Preserve in pressure metal cylinders not exceeding 40 C.

160 Monographs, Part I tive filter paper. Wash the beaker with warm water, filter the washing solution, wipe the beaker and stirring rod with a small piece of quantitative filter paper, place all the filter papers in the porcelain crucible, cover the crucible heat slowly to carbonize, then ignite at 800 C for 1 hour. Weigh the mass after cooling in the desicator with silica gel, determine the amount of the residue to the dehydrated carboplatin is between 52.0% and 53.0%. (4) 1,1-cyclobutanedicarboxylic acidWeigh accurately about 50 mg of Carboplatin, dissolve in mobile phase to make exactly 50 mL, and use this solution as the test solution. Separately, weigh accurately about 50 mg of 1,1-cyclobutanedicarboxylic acid, dissolve in mobile phase to make exactly 100 mL, add the mobile phase to 2.0 mL of this solution to make 200 mL, and use this solution as the standard solution. Perform the test with 100 L of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and measure the peak areas of 1,1-cyclobutanedicarboxylic acid obtained from the test solution and the standard solution, AT and AS , respectively, by the automatic integration method, calculate the amount of 1,1-cyclobutanedicarboxylic acid: not more than 0.5%. Amount (%) of 1,1-cyclobutanedicarboxylic acid =5
C AT W AS

Carboplatin
O O Pt O O NH3 NH3

C6H12N2O4Pt : 371.25 Carboplatin contains not less than 98.0% and not more than 102.0% of carboplatin (C6H12N2O4Pt), calculated on the anhydrous basis. Description Carboplatin is a white crystalline powder. Carboplatin is slightly soluble in water, and practically insoluble in acetone or in ethanol. Melting pointabout 200 (decomposition) Identification Determine the infrared spectra of Carboplatin and Carboplatin RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. pH pH of a solution of Carboplatin (1 in 100) is between 5.0 and 7.0. Purity (1) Clarity and color of solutionDissolve 0.1 g of carboplatin in water to make exactly 10 mL and determine the transmittance of this solution using water as a blank as directed under the Ultraviolet-visible Spectrophotometry at the 440 nm: not less than 97%. (2) Insoluble substancesTransfer about 1 g of carboplatin, accurately weighed, to a beaker. Add 100 mL of water, and dissolve by stirring for 30 minutes. Filter through a tared glass filter. Rinse the beaker with water, and filter the rinsings. Dry the glass filter at 130 10 C to constant mass: the amount of residue is not more than 0.5%. (3) PlatinumTransfer about 0.25 g of Carboplatin, accurately weighed, to a 600-mL beaker. Add 400 mL of water, and slowly dissolve by heating almost to the boiling point, stirring frequently with a glass rod. Dissolve it complately, boil for about 10 minutes, cool for 1 minute without stirring, filter with quantitative filter paper. Transfer the filtrate to a 600-mL beaker, wash the quantitative filter paper with the warm water, combine the washings and the filterate. Evaporate this solution until to about 300 mL. Place a glass stirring rod in the beaker, and heat the solution to boiling, add slowly about 10 mL of hydrazine hydrate to the center of beaker by dropwise addition. Add 2 drops of 10 mol/L sodium hydroxide TS, heat for about 10 minutes to coagulate the precipitate for ease of filtration, cool, filter with quantita-

C : Concentration (g/mL) of 1,1-cyclobutane dicarboxylic acid in the standard solution. W : Weight (mg) of Carboplatin taken to prepare the test solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength : 220 nm) Column: A stainless steel column, about 4.0 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography. Mobile phase: Dissolve 8.5 g of tetrabutylammonium hydrogen sulfate in 80 mL of water. Add 3.4 mL of phosphoric acid, and adjust with 10 mol/L sodium hydroxide to a pH of 7.55. Add 20 mL of this solution to a mixture of 880 mL of water and 100 mL of acetonitrile, mix. Flow rate: 2 mL/minute System suitability System performance: Mix 1.0 mL of the standard solution and 1.0 mL of the standard solution in the Assay, and use this solution as the system suitability solution. When the procedure is run with 100 L of this solution under the above operating conditions, the relative retention times of carboplatin peak and 1,1-cyclobutane dicarboxylic acid peak are 0.65 and 1.0, respectively, with the resolution between carboplatin and 1,1-cyclobutane dicarboxylic acid peaks being not less than 2.5, and the column efficiency, determined from the 1,1-cyclobutane

KP 9 161 dicarboxylic acid peak, is not less than 1500 theoretical plates. System repeatability: when the test is repeated 6 times with 20 L of system suitability solution under the above operating conditions, the relative standard deviation of peak area obtained from 1,1-cyclobutane dicarboxylic acid is not more than 10 %. (5) Related substancesDissolve 50 mg of Carboplatin in water, and use this solution as the test solution. Separately, dissolve 25 mg of Carboplatin RS in water to make exactly 100 mL, add water to 1.0 mL of this solution to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. The total peak areas other than carboplatin obtained from the test solution and 1,1cyclobutanedicarboxylic acid by automatic integration method: is not more than 2 times peak area of carboplatin obtained from standard solution (0.5 %), and each peak area is not more than the peak area of carboplatin obtained from the standard solution (0.25 %). Operating conditions Detector, column, Mobile phase, System suitability correspond to the the operating conditions in the Assay. Water Not more than 0.5 % (1 g, dehydraed form amide, volumetric titration, dirct titration). Assay Weigh accurately about 50 mg each of Carboplatin and Carboplatin RS, dissolve in water to make exactly 50 mL, and use these solutions as the test solution and the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, measure the peak areas of Carboplatin and Carboplatin RS, AT and AS , by automatic integration method. Amount (mg) of carboplatin (C6H12N2O4Pt) = amount (mg) of Carboplatin RS
AT AS

less than 3.0, the numbers of theoretical plates is not less than 2500, and the symmetry factor of Carboplatin peak is not more than 2.5. System repeatability: When the test is repeated 5 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of peak area of carboplatin is not more than 1.2%. Packing and Storage Preserve in light-resistant, tight containers.

Carisoprodol
CH3 O O

H3C

N H

O H3C

NH2 CH3

C12H24N2O4 : 260.33 Carisoprodol contains not less than 98.0% and not more than 102.0% of carisoprodol (C12H24N2O4), calculated on the dried basis. Description Carisoprodol is a white, crystalline powder, has slightly specific odor and a bitter taste. Carisoprodol is freely soluble in acetone, in ethanol or in chloroform and very slightly soluble in water. Identification (1) Determine the infrared spectra of Carisoprodol and Carisoprodol RS as directed in the potassium bromide disk method under Infrared Spectrophotometry: both spectra exhibit slmilar intensities of absorption at the same wavenumbers. (2) Weigh 0.1 g each of Carisoprodol and Carisoprodol RS, dissolve in 1.0 mL each of chloroform, and use these solutions as the test solution and the standard solution. Perform the test with these solutions according to meprobamate under the Purity: the main spots from the test solution and the standard solution have the same Rf value. Melting Point Between 91 C and 94 C. Purity (1) Heavy metalsProceed with 2.0 g of Carisoprodol according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Meprobamate Weigh 0.1 g of Carisoprodol and dissolve in 10 mL of chloroform, use this solution as the test solution. Separately, weigh 10 mg of Meprobamate RS, dissolve in 10 mL of chloroform, and use this solution as the standard solution. Spot 10 L and 5 L of the test solution and the standard solution respectively on the plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform and acetone

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm) Column: A stainless steel column, about 4.0 mm in inside diameter and about 30 cm in length, packed with aminopropylsilanized silica gel for liquid chromatography (between 3 and 10 m in particle diameter). Mobile phase : A mixture of acetonitrile and water (87 : 13) Flow rate: 2 mL/minute System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the ratio of mass distribution is not

162 Monographs, Part I (4 : 1) to a distance of about 15 cm and air-dry the plate. Spray evenly, repeatedly by turns, with antimony trichloride TS and 3% solution of furfural in chloroform until the black spot is developed, heat the plate at 110 for 15 minutes,the spot of meprobamate from test solution is not more than intense than the spot from standard solution (not more than 0.5%). Loss on Drying Not more than 0.5% (1 g, vaccum, 60 C, 3 hours). Assay Weigh acculately about 0.4 g of Carisoprodol, add 10 mL of pyridine and 1 drop of phenolphthalein TS, and titrate with 0.1 mol/L sodium methoxide until pink color is developed. Add 25.0 mL of 0.1 mol/L sodium methoxide to this solution, heat for 30 minutes with reflux condenser and cool. Add 40 mL of ethanol to this solution, and titrate with 0.1 mol/L hydrochloric acid VS(Indicator: 7 drops of phenolphthalein TS). Perform the blank, and make any necessary correction. 1 mL of 0.1 mol/L Sodium Methoxide = 26.033 mg of C12H24N2O4. Packaging and Storage Preserve in tight containers. traviolet-visible Spectrophotometry: both spectra exhibits similar intensities of absorption at the same wavelength. (3) Determine the infrared spectra of Carmofur and Carmofur RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. Purity (1) Heavy metalsProceed with 2.0 g of Carmofur according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesDissolve 0.2 g of Carmofur in 10 mL of a mixture of methanol and glacial acetic acid (99 : 1) and use this solution as the test solution. Pipet 1 mL of the test solution, add a mixture of methanol and glacial acetic acid (99 : 1) to make exactly 500 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 15 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of toluene and acetone (5 : 3) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. After exposure of the plate to bromine vapor for 30 seconds, spray evenly a solution of fluorescein in ethanol (1 in 2500): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, in vacuum, 50 C, 3 hours). C11Hl6FN3O3: 257.26 Carmofur, when dried, contains not less than 98.0% and not more than 101.0% of carmofur (C11Hl6FN3O3). Description Carmofur is a white crystalline powder. Carmofur is very soluble in dimethylformamide, freely soluble in glacial acetic acid, soluble in ether, sparingly soluble in methanol or in dehydrated ethanol and practically insoluble in water. Melting point About 111 C (with decomposition). Identification (1) Proceed with 5 mg of Carmofur as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid and prepare the test solution: the test solution responds to the Qualitative Tests (2) for fluoride. (2) Determine the absorption spectra of solutions of Carmofur and Carmofur RS in a mixture of methanol and phosphoric acid - acetic acid - boric acid buffer solution, (pH 2.0), (9 : 1) (1 in 100000) as directed under the UlResidue on Ignition Not more than 0.1% (1 g).

Carmofur
O C N NHCH2(CH2)4CH3 O

NH F O

Assay Weigh accurately about 0.5 g of Carmofur, previously dried, dissolve in 20 mL of dimethylformamide and titrate with 0.1 mol/L tetramethylammonium hydroxide-methanol VS until the color of the solution changes from yellow through blue-green to blue (indicator: 3 drops of thymol blue-dimethylformamide TS). Each mL of 0.1 mol/L tetramethylammonium hydroxidemethanol VS = 25.726 mg of C11Hl6FN3O3 Packaging and Storage Preserve in tight containers

KP 9 163 and add methanol to make exactly 100 mL. Pipet 1 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard soution on a plate of silica gel with a fluorescent indicator for thinlayer chromatography. Develop the plate with a mixture of chloroform, methanol and strong ammonia water (50 : 20 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.5 g of Carteolol Hydrochloride, previously dried, add 30 mL of glacial acetic acid, dissolve by heating in a water-bath and cool. After adding 70 mL of acetic anhydride, titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry ). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 32.883 mg of C16H24N2O3HCl Packaging and Storage Preserve in well-closed containers.

Carteolol Hydrochloride
H N O HCl H OCH2CCH2NH OH CH3 C CH3 CH3

and enantiomer C16H24N2O3HCl: 328.83 Carteolol Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of carteolol hydrochloride (C16H24N2O3HCl). Description Carteolol Hydrochloride is a white crystal or crystalline powder. Carteolol Hydrochloride is soluble in water, sparingly soluble in methanol, very slightly soluble in ethanol or in glacial acetic acid and practically insoluble in ether. The solution of Carteolol Hydrochloride (1 in 20) shows no optical rotation. pHDissolve 1.0 g of Carteolol Hydrochloride in 100 mL of water: the pH of this solution is between 5.0 and 6.0. Melting pointAbout 277 C (with decomposition). Identification (1) Dissolve 0.1 g of Carteolol Hydrochloride in 5 mL of water and add 5 drops of Reinecke salt TS: a pale red precipitate is produced. (2) Determine the absorption spectra of solutions of Carteolol Hydrochloride and Carteolol Hydrochloride RS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both sprectra exhibit similar intensities of absorption at the same wavenumbers. (3) Determine the infrared spectra of Carteolol Hydrochloride and Carteolol Hydrochloride RS as directed in the potassium cholride disk method under the Infrared Spectrophotometry: both spectra exhibit similar in tensities of absorption at the same wavenumbers. (4) A solution of Carteolol Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride. Purity (1) Clarity and color of solutionDissolve 1.0 g of Carteolol Hydrochloride in 30 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Carteolol Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Carteolol Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.20 g of Carteolol Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet 2 mL of the test solution

Carvedilol
H O OH H N O OCH3

HN

and enantiomer C24H26N2O4 : 406.47 Carvedilol, when dried, contains not less than 99.0% and not more than 101.0% of carvedilol (C24H26N2O4). Description Carvedilol is a white crystalline powder. Carvedilol is slightly soluble in ethanol and practically insoluble in water. Carvedilol shows polymorphism. Identification Determine the infrared spectra of Carvedilol and Carvedilol RS, previously dried, as directed

164 Monographs, Part I in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears between the spectra, dissolve Carvedilol and Carvedilol RS in 2-propanol respectively, evaporate to dryness and repeat the test on the residues. Purity (1) Heavy metalsProceed with 2.0 g of Carvedilol according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Related substances Dissolve 25.0 mg of Carvedilol, accurately weighed, in mobile phase to exactly 10 mL and use this solution as the test solution. Pipet 1.0 mL of the test solution, add mobile phase to make exactly 100 mL, pipet 1.0 mL of this solution, add mobile phase to make exactly 10 mL and use this solution as the standard solution (1). Separately, dissolve 5.0 mg of carvedilol related substance I{(2RS-1-benzyl[2-(2methoxyphenoxy)ethyl]amino-3-(9H-carbazol-4-yloxy) propan-2-ol)} in 5 mL of the test solution and add mobile phase to make exactly 100 mL and use this solution as the standard solution (2). To 1.0 mL of standard solution (2) add mobile phase to make exactly 100 mL, pipet 2.0 mL of this solution, add mobile phase to make exactly 10 mL and use this solution as the standard solution (3). Perform the test with exactly 20 L each of the test solution and the standard solutions (1) and (2) as directed under Liquid Chromatography according to the following conditions. Multiply by 2 as correction factor in the calculation of content of related substance II. The area of the related substance II obtained in the test solution is not more than twice that of the principal peak in the chromatogram obtained with standard solution (1) (0.2%), the area of the related substance I is not more than twice that of the corresponding peak in the chromatogram obtained with the standard solution (3) (0.02%), the area of any other impurity is not more than the area of the principal peak in the chromatogram from the standard solution (1) (0.1%), the total area of peaks obtained with the test solution is not more than 5 times that of the principal peak in the chromatogram from the standard solution (1) (0.5%). Disregard the peaks that is less than the area of the principal peak in the chromatogram from the standard solution (3) (0.01%). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 12.5 cm in length, packed with octylsilyl silica gel for Liquid Chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 55 C. Mobile phase: To 1.77 g of potassium dihydrogen phosphate add water to make 650 ml, adjust to pH 2.0 with phosphoric acid and add 350 ml of acetonitrile. Flow rate: 1.5 mL/minute. Time span of measurement: About 8 times as long as the retention time of carvedilol beginning after the solvent peak. Retention time with reference to the peak of carvedilol (about 4 minutes): 0.6, 3.5, and 6.7 for related substance II, I, and III, respectively. System suitability System performance: When the procedure is run with 20 L of the standard solution (2) under the above operating condition, the resolution between the peaks of carvedilol and related substance I is not less than 17. Loss on Drying Not more than 0.5 % (1 g, 105 C, constant mass). Residue on Ignition Not more than 0.1 % (1 g). Assay Weigh accurately about 0.35 g of Carvedilol, previous dried, dissolve in 60 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 40.649 mg of C24H26N2O4 Packaging and Storage well-closed containers. Preserve in light-resistant,

Cefaclor Hydrate
HO O H2N N H H N O H S H O Cl

H2O

C15H14ClN3O4SH2O: 385.82 Cefaclor Hydrate contains not less than 860 g (potency) and not more than 1050 g (potency) per mg of (C15H14ClN3O4S: 367.81), calculated on the anhydrous basis. Description Cefaclor Hydrate is a white to light yellow-white, crystal or crystalline powder, odorless or has a little of characteristic odor, and has a bit of bitter taste. Cefaclor Hydrate is soluble in water, sparingly soluble in methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Weigh 10 mg of Cefaclor Hydrate, dissolve in 2 mL of water, add 3 mL of hydroxylamine hydrochloride TS, allow to stand for 3 minutes, add 1 mL

KP 9 165 of acidic ferric ammonium sulfate TS and shake: a redpurple color develops. (2) Weigh an appropriate amount of Cefaclor Hydrate and Cefaclor RS, dissolve each in water to make the solutions contain 4 mg per mL, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of acetonitrile, ethylactate, water and formic acid (30:10:10:1), spray evenly ninhydrin TS: the spots obtained from the test solution and the standard solution are the same in the Rf value. (3) Weigh 2 mg of Cefaclor Hydrate, dissolve in water and make to 100 mL. Determine the absorption spectrum of this solution as directed under Ultraviolet-visible Spectrophotometry, it exhibits maximum between 263 nm and 267 nm. (4) Determine the absorption spectra of the solutions of Cefaclor Hydrate and Cefaclor RS as directed in the paste method under Infrared Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wavenumvers. (5) Perform the test with Cefaclor Hydrate as directed under Flame Coloration Test: a green color appears. Purity Related substancesWeigh accurately about 50 mg of Cefaclor Hydrate, dissolve in sodium dihydrogen phosphate TS, pH 2.5 to make exactly 10 mL, and use this solution as the test solution. Pipet exactly 2 mL of the test solution, add sodium dihydrogen phosphate TS, pH 2.5 to make exactly 100 mL, and use this solution as the standard solution. Perform the test with exactly 20 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area. If necessary, proceed with 20 L of sodium dihydrogen phosphate TS, pH 2.5 in the same manner as the above to compensate the base line to make correction. The peak areas other than cefaclor from the test solution are not more than 1/2 of the peak area of cefaclor from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A constant temperature of about 25 C. Mobile phase: Control the gradient by mixing the mobile phase A and B as directed in the following table. Mobile phase A: Dissolve 7.8 g pg sodium dihydrogen phosphate in 1000 mL of water, and adjust the pH to 4.0 with phosphoric acid. Mobile phase B: To 550 mL of the mobile phase A add 450 mL of acetonitrile for liquid chromatography. Time (min) 0 ~ 30 30 ~ 45 45 ~ 55 Mobile phase A (vol %) 95 75 75 0 0 Mobile phase B (vol %) 5 25 25 100 100

Flow rate: 1.0 mL per minute. System suitability Test for required detectability: Measure exactly 1 mL of the standard solution, and add sodium dihydrogen phosphate TS, pH 2.5 to make exactly 20 mL. Confirm that the peak area of cefaclor obtained from 20 L of this solution is equivalent to 4 to 6 % of that from 20 L of the standard solution. System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of cefaclor are not less than 40000 and 0.8 to 1.3, respectively. System repeatability: When the test is repeated 3 times with 20 L of the standard solution under the operating conditions, the relative standard deviation of the peak areas and the retention times of cefaclor are not more than 2.0 %, respectively. Time span of measurement: About 2.5 times as long as the retention time of cefaclor beginning after the solvent peak. Specific Optical Rotation [ ]20 D : +105 ~ +120(0.1 g calculated on the anhydrous basis, water, 25 mL, 100 mm). Crystalliity Test It meets the requirement.

pH The pH of a solution obtained by dissolving 0.25 g of Cefaclor Hydrate in 10 mL of water is between 3.0 and 4.5. Water Not less than 3.0 and not more than 8.0% (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg (potency) each of Cefaclor Hydrate and Cefaclor Hydrate RS, dissolve each in a suitable amount of the mobile phase to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and obtain the peak areas of cefaclor, AT and AS. Amount [g (potency)] of cefaclor (C15H14ClN3O4S) = Amount [g (potency)] of Cefaclor RS
AT AS

166 Monographs, Part I Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 265 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Mobile phase: Weigh 1.0 g of sodium 1pentanesulfonate, dissolve in 750 mL of water, add 10 mL of triethylamine, mix well, and adjust the pH to 2.5 with phosphoric acid. To this solution add 200 mL of methanol. Flow rate: Adjust the flow rate so that the retention time of cefaclor is about 4 minutes. Packaging and Storage Preserve in tight containers.

Cefadroxil Hydrate
HO O O N H NH2 H N H H S CH3

O HO

H2O

C16H17N3O5SH2O: 381.40 Cefadroxil Hydrate contains not less than 900 g (potency) and not more than 1050 g (potency) per mg of cefadroxil (C16H17N3O5S: 363.39), calculated on the anhydrous basis Description Cefadroxil Hydrate is a white to light yellow-white powder, has a bit of characteristic odor, but is tasteless. Cefadroxil Hydrate is sparingly soluble in water, and practically insoluble in ethanol or in ether. Identification (1) Determine the absorption spectra of the solutions of Cefadroxil Hydrate and Cefadroxil Hydrate RS in ethanol (1 in 50000), as directed under Ultraviolet-visible Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Cefadroxil Hydrate and Cefadroxil Hydrate RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. Purity Related substancesWeigh accurately about 50 mg of Cefadroxil Hydrate, dissolve in a suitable amount of the mobile phase A, add the mobile phase to make exactly 50 mL, and use this solution as the test solution. Weigh accurately about 10 mg of D--4Hydroxyphenylglycine RS, dissolve in a suitable amount of the mobile phase A, add the mobile phase to make exactly 10 mL, and use this solution as the related substance I solution. Weigh accurately about 10 mg of 7aminodesacetoxycephalosporanic acid RS, dissolve in a suitable amount of phosphate buffer solution, pH 7.0, add the buffer to make exactly 10 mL, and use this solution as the related substance II solution. Pipet exactly 1 mL each of the related substance I solution and related substance II solution, add the mobile phase A to make 100.0 mL, and use this solution as the related substance III solution. Weigh accurately about 10 mg each of dimethylformamide and dimethylacetamide, dissolve in a suitable amount of the mobile phase A, add the mobile phase to make exactly 10 mL, pipet exactly 1 mL of this solution, add the mobile phase A to make exactly 100.0 mL, and use this solution as the related substance IV solution. Pipet exactly one mL of the related substance III

Cefaclor Capsules
Cefaclor Capsules contain not less than 90.0 % and not more than 120 % of the labeled amount of cefaclor (C15H14ClN3O4S: 367.81). Mehtod of Preparation Prepare as directed under Capsules, with Cefaclor Hydrate. Identification Proceed as directed in the Identification (1) and (2) under Cefaclor Hydrate. Disintegration Test It meets the requirement . Uiformity of Dosage Units It meets the requirement. Water Not more than 8.0 % (0.1 g, volumetric titration, direct titration). Assay Weigh accurately the contents of not less than twenty Cefaclor Capsules, weigh accurately about 50 mg (potency) of the contents, according to the labeled potency, dissolve, make exactly 50 mL, and use this solution as the test solution. Perform the test as directed in the Assay under Cefaclor Hydrate. Amount [g (potency)] of cefaclor (C15H14ClN3O4S) = Amount [g (potency)] of Cefaclor RS
AT AS

Packaging and Storage Preserve in tight containers.

KP 9 167 solution, add the mobile phase A to make exactly 25 mL, and use this solution as the related substance V solution. Perform the test with 20 L of each of the test solution, the related substance III solution, the related substance IV solution and the related substance V solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the amount of each of related substances corresponding to the peak area (Related substance I: not more than 1 %, other related substances: not more than 3.0 %, total related substances: not more than 3.0 %). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 100 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Mobile phase: Control the gradient by mixing the mobile phase A and B as directed in the following table. Mobile phase A: Phosphate buffer solution, pH 5.0. Mobile phase B: Methanol Time (min) 0~1 1 ~ 20 20 ~ 23 23 ~ 30 Mobile phase A (vol %) 98 98 70 70 98 98 Mobile phase B (vol %) 2 2 30 30 2 2 solutions as the test solution and the standard solution. Perform the test with each of the test solution and the standard solution as directed under Ultraviolet-visible Spectrophotometry, and determine absorptions at 264 nm, AT and AS . Amount [g (potency)] of cefadroxil (C16H17N3O5S) = Amount [g (potency)] of Cefadroxil RS Packaging and Storage tight containers.
AT AS

Preserve in light-resistant,

Cefalexin Hydrate
HO O CH3 N H H N O H S H O H2N

H2 O

C16H17N3O4SH2O: 365.40 Cefalexin Hydrate contains not less than 900 g (potency) per mg of cefalexin (C16H17N3O4S: 347.39), calculated on the anhydrous basis. Description Cefalexin Hydrate is a white to light yellowish white crystals or crystalline powder. Cefalexin Hydrate is sparingly soluble in water, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) To 5 mg of Cefalexin Hydrate add 1 mL of hydroxylamine hydrochloride TS, allow to dissolve and stand for 3 minutes, add 1 mL of acidic ferric ammonium sulfate TS and mix: the red-brown to brown color develops. (2) To 20 mg of Cefalexin Hydrate add two to three drops of 80 % sulfuric acid containing 1 % nitric acid: the yellow color develops. (3) To 20 mg of Cefalexin Hydrate add five drops of 1 % glacial acetic acid, two drops of 1 % cupric sulfate solution and one drop of 2 mol/L sodium hydroxide, and mix: the yellowish green color develops. (4) Weigh 3 mg of Cefalexin Hydrate, dissolve in water and add water to make 100 mL. Determine the absorption spectrum of the solution as directed under Ultraviolet-visible Spectrophotometry, it exhibits maximum between 261 nm and 263 nm. (5) Determine the infrared spectra of Cefalexin Hydrate and Cefalexin RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers.

Flow rate: 1.5 mL per minutes. Relative retention with reference to cefadroxil (retention time is about 6 minutes): dimethylformamide is about 0.4, and dimethylacetamide is about 0.75. System performance: When the procedure is run with the related substance III solution under the above operating conditions, the resolution between the peak of related substance I and that of related substance II is not less than 5.0. Specific Optical Rotation [ ]20 D : +164 ~ +182 (0.6 g, calculated on the anhydrous basis, water, 100 mL, 100 mm). pH The pH of a solution obtained by dissolving 0.5 g of Cefadroxil Hydrate in 10 mL of water is between 4.0 and 6.0. Water Not less than 4.2 % and not more than 6.0% (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg of Cefadroxil Hydrate and Cefadroxil Hydrate RS, dissolve each in a suitable amount of the water, then add water to make exactly 250 mL. Pipet exactly 10 mL each of these solutions, add water to make exactly 100 mL, and use these

168 Monographs, Part I

+144 ~ +158 Specific Optical Rotation [ ]20 D : (0.125 g calculated on the anhydrous basis, water, 25 mL, 200 mm). pH The pH of a solution obtained by dissolving 0.5 g of Cefalexin Hydrate in 10 mL of water is between 3.0 and 5.5. Water Not less than 4.0 % and not more than 8.0% (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg (potency) each of Cefalexin Hydrate and Cefalexin Hydrate RS, dissolve each in water to make exactly 50 mL. Pipet exactly 5 mL of each solution, add exactly 5 mL of the internal standard solution and water to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and calculate the ratios, QT and QS , of the peak area of cefalexin to that of the internal standard. Amount [g (potency)] of cefalexin (C16H17N3O4S) = Amount [g (potency)] of Cefalexin Hydrate RS
Q T QS

C16H15N2NaO6S2: 418.42 Cefalotin Sodium contains not less than 850 g (potency) per mg of cefalotin (C16H16N2O6S2: 396.44), calculated on the anhydrous basis. Description Cefalotin Sodium is a white to light yellowish white crystals or crystalline powder. Cefalotin Sodium is freely soluble in water, slightly soluble in methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) To 20 mg of Cefalotin Sodium add two to three drops of 80 % sulfuric acid containing 1 % nitric acid: the yellowish green color develops, and the color turns to red-brown shortly. (2) Determine the absorption spectra of the 0.0025 % aqueous solutions of Cefalotin Sodium and Cefalotin Sodium RS from 220 nm to 310 nm as directed under Ultraviolet-visible Spectrophotometry, both spectra exhibit maximum and minimum at the same wavelengths. (3) Place 20 mg of Cefalotin Sodium in platinum dish, ignite, add 5 mL of water and 0.5 mL of hydrochloric acid, dissolve, and filter. The filtrate responds to the Qualitative Tests 1) for sodium salt. Specific Optical Rotation water, 100 mL, 100 mm).
[ ]20 D : +124 ~ +134 (5.0 g,

Internal standard solutionWeigh accurately about 0.1 g of Cephaloglycin RS, dissolve in the mixture of water and methanol (9:1) to make 100 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 ~ 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 ~ 10 m in particle diameter) Mobile phase: Adjust the pH of 0.05 mol/L potassium dihydrogenphosphate solution to 3.0 with phosphoric acid. To 800 mL of this solution add 200 mL of methanol and mix. Packaging and Storage Preserve in tight containers.

pH The pH of a solution obtained by dissolving 2.5 g of Cefalexin Hydrate in 10 mL of water is between 5.0 and 7.5. Sterility Test It meets the requirement, when Cefalotin Sodium is used in a sterile preparation. Pyrogen Test It meets the requirement, when Cefalotin Sodium is used in a sterile preparation,. Weigh an appropriate amount of Cefalotin Sodium, dissolve in water, make the solution containing 50 mg (potency) per mL, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not more than 1.5% (0.5 g, volumetric titration, direct titration). Assay Weigh accurately about 20 mg (potency) each of Cefalotin Sodium and Cefalotin Sodium RS, dissolve each in the mobile phase to make exactly 20 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT and AS , of cefalotin of these solutions.

Cefalotin Sodium
NaO O N S H N O H S H O CH3 O O

KP 9 169 and very slightly soluble in ethylacetate, in chloroform, in ether or in benzene. Identification Dissolve Cefamandole Nafate in water to make the solution contain 1 mg per mL, and use this solution as the test solution. Separately, dissolve Cefamandole Nafate RS in water to make the solution contain 1 mg per mL, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot the test solution and the standard solution on a plate of silica gel for thinlayer chromatography. Develop the plate with the developing solvent which is a mixture of n-butanol, glacial acetic acid and water (4:1:1), and air-dry the plate. Spray evenly the mixture of starch TS, glacial acetic acid and 0.1 mol/L iodine solution (50:3:1): the white spots on purple backgrounds, obtained from the test solution and the standard solution are the same in the Rf value. pH The pH of a solution obtained by dissolving 1 g of Cefamandole Nafate in 10 mL of water is between 3.5 and 7.0. Sterility Test It meets the requirement, when Cefamandole Nafate is used in a sterile preparation. Bacterial Endotoxins Less than 0.15 EU per mg of Cefamandole Nafate, when it is used in a sterile preparation. Water Not more than 2.0% (0.5 g, volumetric titration, direct titration). Assay The Standard curve method (1) Culture medium Agar media for seed and base layer- Use the me1 or 2 under Microbial Assay for dium in I 2 1) (3) Antibiotics. (2) Test organism- Staphylococcus aureus ATCC 6538P. (3) Weigh accurately an appropriate amount of Cefamandole Nafate, dissolve in 0.1 mol/L phosphate buffer solution, pH 8.0 to make the solution so that each mL contains 1 mg (potency), hydrolyze at 37 C for 60 minutes in water bath, and pipet exactly an appropriate amount of this solution and dilute in 1 % phosphate buffer solution, pH 6.0 to make solution so that each mL contains 2.00 g (potency) and use this solution as the test solution. Separately, weigh accurately an appropriate amount of Cefamandole Nafate RS, dissolve in 0.1 mol/L phosphate buffer solution, pH 8.0 to make the solution so that each mL contains 1 mg (potency), and allow to stand at 37 C in water bath for 60 minutes. Keep the standard stock solution at not exceeding 5 C and use within one day. Pipet exactly an appropriate amount of this solution and dilute in 1 % phosphate buffer solution, pH 6.0 to make solutions so that each mL contains 1.28, 1.60, 2.00, 2.50, and 3.12 g (potency), and use these solutions as the standard solutions. Also use the solution containing

Amount [g (potency)] of cefalotin (C16H16N2O6S2) = Amount [g (potency)] of Cefalotin Sodium RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 17 g of sodium acetate trihydrate in 790 mL of water, and add 0.6 mL of glacial acetic acid. If necessary, adjust the pH to 5.9 with 0.1 mol/L sodium hydroxide TS or glacial acetic acid. To this solution add 150 mL of acetonitrile and 70 mL of ethanol. System suitability System performance: Heat the standard solution in a water bath of 90 o C for 10 minutes, and cool. Measure exactly 2.5 mL of this solution, and add the mobile phase to make exactly 100 mL. When the procedure is run with 10 L of this solution under the above operation conditions, the resolution between peak of cefalotin and the peak, having the relative retention time of 0.5 with respect to cefalotin is not less than 9, and the symmetry factor of the peak of cefalotin is not more than 1.8. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of cefalotin is not more than 1.0 %. Packaging and Storage Preserve in tight containers.

Cefamandole Nafate
NaO H O H O H N O H H S O H3C N O N S N N N

C19H17N6NaO6S2: 512.50 Cefamandole Nafate contains not less than 810 g (potency) and not more than 1000 g (potency) per mg of cefamandole (C18H18N6O5S2: 462.51), calculated on the anhydrous basis Description Cefamandole Nafate is a white powder. Cefamandole Nafate is soluble in water or in ethanol,

170 Monographs, Part I 2.00 g (potency) per mL as the standard mid-diluted solution. Perform the test with these solutions according to the Standard curve method (II 4) as directed under Microbial Assay for Antibiotics. Packaging and Storage tight containers. Preserve in light-resistant, phosphate buffer solution, pH 6.0 to make exactly 100 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and calculate the ratios, QT and QS , of the peak area of cefapirin to that of the internal standard. Amount [g (potency)] of cefapirin (C17H17N3O6S2) = Amount [g (potency)] of Cefapirin Sodium RS
NaO O N H N S O H H S N O CH3 O O

Cefapirin Sodium

QT QS

Internal standard solutionA solution of vanillin (1 in 1000) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A constant temperature of about 40 C. Mobile phase: Weigh 7.0 g of sodium dihydrogenphosphate dihydrate dissolve in 800 mL of water, adjust the pH to 2.5 ~2.7 with diluted phosphoric acid (1 in 15), and add water to make 1000 mL. To 930 mL of this solution add 70 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of cefapirin is about 7 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, cefapirin and the internal standard are eluted in this order with the resolution between these peaks being not less than 4.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of cefapirin to that of the internal standard is not more than 1.0 %. Packaging and Storage Preserve in tight containers.

C17H16N3NaO6S2: 445.45 Cefapirin Sodium contains not less than 855 g (potency) and not more than 1000g (potency) per mg of cefapirin (C17H17N3O6S2: 423.46), calculated on the anhydrous basis. Description Cefapirin Sodium is a white to yellowish white powder. Identification (1) Determine the infrared spectra of Cefapirin Sodium and Cefapirin Sodium RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Cefapirin Sodium responds to the Qualitative Tests 1) for sodium salt. pH The pH of a solution obtained by dissolving 0.1 g of Cefapirin Sodium in 10 mL of water is between 6.5 and 8.5. Sterility Test It meets the requirement, when Cefapirin Sodium is used in a sterile preparation. Pyrogen Test It meets the requirement, when Cefapirin Sodium is used in a sterile preparation,. Weigh appropriate amount of Cefapirin Sodium, dissolve in water, make the solution containing 0.1 g (potency) per mL, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not more than 2.0% (0.5 g, volumetric titration, direct titration).

Cefatrizine Propylene Glycol

HO O N H NH2 H N H H S N NH S N O

Assay Weigh accurately about 0.1 g (potency) each of Cefapirin Sodium and Cefapirin Sodium RS, dissolve each in 1 % phosphate buffer solution, pH 6.0 to make exactly 100 mL. Pipet exactly 5 mL of each solution, add exactly 5 mL of the internal standard solution and 1 %

O HO H H3C

.
OH OH

KP 9 171 Amount [g (potency)] of cefatrzine (C18H18N6O5S2) = Amount [g (potency)] of Cefatrizine Propylene Glycol RS
AT AS

C18H18N6O5S2 (C3H8O2)n Cefatrizine Propylene Glycol contains not less than 780 g (potency) per mg of cefatrizine (C18H18N6O5S2: 462.50), calculated on the anhydrous basis. Description Cefatrizine Propylene Glycol is a white to yellowish white powder, odorless, and has a bit of bitter taste. Cefatrizine Propylene Glycol is sparingly soluble in water, and practically insoluble in methanol, in ethanol or in ether. Identification (1) To 5 mg of Cefatrizine Propylene Glycol add 1 mL of hydroxylamine hydrochloride TS, allow to dissolve and stand, add 1 mL of acidic ferric ammonium sulfate TS and mix: the red-brown to brown color develops. (2) Dissolve 5 mg of Cefatrizine Propylene Glycol in 5 mL of water, add 1 mL of ammonium chlorideammonia TS, mix, and add 1 mL of 4aminoantipyrin TS and 1 mL of potassium ferric hexacyanate TS: the red color develops. (3) To 10 mg of Cefatrizine Propylene Glycol add 1 mL of water, allow to dissolve, add 1 mL of potassium periodate TS, allow to stand for 5 minutes, add 1 mL of sulfurous acid water, shake, and add 1 mL of fuchsinsulfurous acid TS and allow to stand for 30 minutes: the red-purple color develops. pH The pH of a solution obtained by dissolving 0.1 g of Cefatrizine Propylene Glycol in 10 mL of water is between 3.5 and 6.0. Absorbance 2000 mL).
1% E1 cm (270 nm): 190 ~ 220 (40 mg, water,

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 270 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Mobile phase: Dissolve 2.04 g of potassium dihydrogenphosphate in water to make 1500 mL, and add 500 mL of methanol. Flow rate: Adjust the flow rate so that the retention time of cefatrizine is about 6 minutes. System suitability System performance: Weigh accurately about 5 mg (potency) of Cefadroxil and about 10 mg of Cefatrizine Propylene Glycol, dissolve in 50 mL of water. When the procedure is run with 10 L of the solution under the above operating conditions, cefadroxil and cefatrizine are eluted in this order with the resolution between these peaks being not less than 4. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak areas of cefatrizine are not more than 1.0 %. Packaging and Storage Preserve in tight containers.

Cefazolin Sodium
NaO O S CH3 O N H N N H O N H S S N N N N

Purity Heavy metalsProceed with 1.0 g of Cefatrizine Propylene Glycol according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Water Not more than 2.0% (0.5 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg (potency) each of Cefatrizine Propylene Glycol and Cefatrizine Propylene Glycol RS, dissolve each in water to make exactly 25 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and calculate the peak areas, AT and AS , of cefatrizine of these solutions.

C14H13N8NaO4S3: 476.49 Cefazolin Sodium contains not less than 860 g (potency) per mg of (C14H14N8O4S3: 454.51), calculated on the anhydrous basis. Description Cefazolin Sodium is a white to light yellow-white, crystal or crystalline powder, odorless and has a bit of bitter taste and salty taste. Cefazolin Sodium is freely soluble in water, slightly soluble in methanol, and practically insoluble in ethanol or in ether. Identification (1) Weigh 10 mg of Cefazolin Sodium,

172 Monographs, Part I dissolve in 2 mL of water, add 3 mL of hydroxylamine hydrochloride TS, allow to stand for 5 minutes, add 1 mL of acidic ferric ammonium sulfate TS and shake: a redbrown color develops. (2) Weigh 50 mg of Cefazolin Sodium, add 50 mL of 0.1 mol/L hydrochloric acid TS, heat for 10 minutes in water bath, and cool. Pipet 20 mL of this solution in a separator, add 20 mL of chloroform, shake well, filter through fat-free cotton and anhydrous sodium sulfate. To 10 mL of the filtrate add 1 mL of cobalt acetate TS: a light blue color develops. (3) Weigh 10 mg of Cefazolin Sodium, dissolve in water and make to 500 mL. Determine the absorption spectrum of this solution as directed under Ultravioletvisible Spectrophotometry, it exhibits maxima between 270 nm and 274 nm. (4) Cefazolin Sodium responds to the Qualitative Tests 1) for sodium salt. Specific Optical Rotation water, 25 mL, 100 mm).
[ ] 20 D :

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 ~ 200 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) Mobile phase: Dissolve 2.27 g of disodium hydrogen phosphate and 0.47 g of citric acid in water to make 935 mL, and add 65 mL acetonitrile. Flow rate: Adjust the flow rate so that the retention time of cefazolin is about 8 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, cefazolin and p-acetoanisidide are eluted in this order with the resolution between these peaks being not less than 4. Packaging and Storage tight containers. Preserve in light-resistant,

-18 ~ -25(2.5 g,

pH The pH of a solution obtained by dissolving 1 g of Cefazolin Sodium in 10 mL of water is between 4.5 and 6.5. Absorbance 100 mL).
1% E1 cm

Cefixime Hydrate
O HO O OH O N H N S N H2N O H S H O N CH2

(272 nm): 264 ~ 292 (1.6 mg, water,

Sterility Test It meets the requirement when tested when Cefazolin Sodium is used in a sterile preparation. Bacterial Endotoxins Less than 0.10 EU per mg of cefazolin, when Cefazolin Sodium is used in a sterile preparation. Water Not more than 3.0% (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 0.1 g (potency) each of Cefazolin Sodium and Cefazolin RS, dissolve each in a suitable amount of the internal standard solution to make exactly 100 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS, of the peak area of cefazolin to that of the internal standard. Amount [g (potency)] of cefazolin (C14H14N8O4S3) = Amount [g (potency)] of cefazolin RS
QT QS

3 H2 O

C16H15N5O7S23H2O: 507.50 Cefixime Hydrate contains not less than 900 g (potency) per mg of cefixime (C16H15N5O7S2: 453.45), calculated on the anhydrous basis. Description Cefixime Hydrate is a white to light yellowish white crystalline powder, odorless or has a little bit of a characteristic odor. Cefixime Hydrate is freely soluble in methanol or in dimethylsulfoxide, sparingly soluble in acetone, slightly soluble in ethanol, and practically insoluble in water, in ethylacetate, in ether or in hexane. Identification (1) Weigh 10 mg of Cefixime Hydrate, dissolve in 0.5 mL of sodium hydrogen carbonate solution (21 in 2500) and 1.5 mL of water, add 3 mL of hydroxylamine hydrochloride TS, allow to stand for 5 minutes, and 1 mL of acidic ferric ammonium sulfate TS and mix with shaking: the red-brown color develops. (2) Weigh 1 mg of Cefixime Hydrate, dissolve in one drop of sodium hydrogen carbonate solution (21 in 2500) and 4 mL of water, add 1 mL of dilute hydrochloric acid, cool down in ice bath and add 1 mL of fresh prepared sodium nitrite solution (1 in 100), mix with shaking, al-

Internal standard solutionWeigh 0.55 g of pacetoanisidide, and dissolve in 1000 mL of 0.1 mol/L phosphate buffer solution, pH 7.0.

KP 9 173 low to stand for 2 minutes in ice water, add 1 mL of ammonium sulfamate TS in ice bath with shaking, allow to stand for one minute, and add 1 mL of N-1naphtylethylenediamine dihydrochloride solution (1 in 1000) and mix with shaking; the red-purple color develops. (3) Determine the absorption spectrum of a solution of Cefixime Hydrate in 0.1 mol/L phosphate buffer solution, pH 7.0 (1 in 62500) as directed under Ultravioletvisible Spectrophotometry, it exhibits maximum between 286 nm and 290 nm, and minimum between 248 nm and 256 nm. Specific Optical Rotation [ ] 20 D : -73 ~ -88(0.45 g calculated on the anhydrous basis, 50 mL, 100 mm). pH The pH of a saturated solution of Cefixime Hydrate in water is between 2.4 and 4.1. Absorbance E1 cm (288 nm): 500 ~ 550 (70 mg calculated on the anhydrous basis, 0.1 mol/L phosphate buffer solution, pH 7.0, 5 L). Water Not more than 12.0 % (0.1 g, volumetric titration, direct titration). Assay Weigh accurately about 0.11 mg (potency) each of Cefixime Hydrate and Cefixime RS, dissolve each in 0.1 mol/L phosphate buffer solution, pH 7.0 to make exactly 100 mL, pipet exactly 10 mL each of these solutions, add 10 mL of the internal standard solution and 0.1 mol/L phosphate buffer solution, pH 7.0 to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS, of the peak area of cefixime to that of the internal standard. Amount [g (potency)] of cefixime (C16H15N5O7S2) = Amount [g (potency)] of Cefixime RS
QT QS
HO

this solution add 110 mL of acetonitrile, degas, and use the solution. Flow rate: Adjust the flow rate so that the retention time of cefixime is about 9 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, cefixime and 2-naphthalenesulfonic acid are eluted in this order with the resolution between these peaks being not less than 4.0. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of cefixime to that of the internal standard is not more than 2.0 %. Packaging and Storage Preserve in tight containers.

1%

Cefoperazone Sodium
CH3 N N HN O O O H H N S O H H O N S N CH3 NaO O N N N

C25H26N9NaO8S2: 667.65 Cefoperazone Sodium contains not less than 870 g (potency) and not more than 1015g (potency) per mg of cefoperazone (C25H27N9O8S: 645.67), calculated on the anhydrous basis. Description Cefoperazone Sodium is a white to yellowish white crystalline powder, odorless, and has a little of bitter taste. Cefoperazone Sodium is freely soluble in water, sparingly soluble in methanol, and practically insoluble in ethanol or in ether. Identification (1) Determine the infrared spectra of Cefoperazone Sodium and Cefoperazone RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Weigh an appropriate amount each of Cefoperazone Sodium and Cefoperazone RS, dissolve in the mixture of acetone and water (9:1) to make the solutions so that each mL contains 1.0 mg, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of methylethylketone, glacial acetic acid, and water

Internal standard solutionWeigh 0.37 g of 2naphthalenesulfonic acid, dissolve in 100 mL of 0.1 mol/L phosphate buffer solution, pH 7.0. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 250 ~ 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter) Mobile phase: To 25 mL of tetrabutylammonium hydroxide TS add water to make 1000 mL, adjust the pH to 6.5 with diluted phosphoric acid (1 in 10). To 290 mL of

174 Monographs, Part I (18:3:3). Then allow the plate under ultra-violet light (main wavelength: 254 nm); the spots obtained from the test solution are the same with the corresponding spots from the standard solution in the Rf value, respectively. (3) Determine the retention times according to the procedure of Assay: the retention time of the principal peak in the chromatogram obtained with test solution is the same as that of the principal peak in the chromatogram obtained with the standard solution (4) Cefoperazone Sodium responds to the Qualitative Tests 1) for sodium salt. Crystalliity Test It meets the requirement. velength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) Mobile phase: To 60.1 g of acetic acid and 0.101 g of triethylamine add water to make exactly 1000 mL. To 1.20 mL of this solution add 2.80 mL of dilute acetic acid and 120 mL of acetonitrile and water to make exactly 1000 mL. Flow rate: 1 mL per minute System suitability System performance: When the procedure is run with 25 L of the standard solution under the above operation conditions, the internal standard and cefoperazone are eluted in this order with the resolution between these peaks being not less than 1.5. System repeatability: When the test is repeated 6 times with 25 L of the standard solution under the operating conditions, the relative standard deviation of the ratios of the peak area of cefoperazone to that of the internal standard is not more than 1.0 %. Packaging and Storage Preserve in tight containers.

pH The pH of a solution obtained by dissolving 2.5 g of Cefoperazone Sodium in 10 mL of water is between 4.5 and 6.5. Sterility Test It meets the requirement, when Cefoperazone Sodium is used in a sterile preparation. Bacterial Endotoxins Less than 0.20 EU per mg (potency) of cefoperazone, when Cefoperazone Sodium is used in a sterile preparation. Water Crystalline powder: not more than 5.0 % (0.2 g, volumetric titration, direct titration). Freeze-dried powder: not more than 2.0 % (0.5 g, volumetric titration, direct titration). Assay Weigh accurately about 0.1 g (potency) of Cefoperazone Sodium, dissolve in the mobile phase to make exactly 100 mL, pipet exactly 10 mL of this solution, add 10.0 mL of the internal standard solution and the mobile phase to make exactly 50 mL, and use this solution as the test solution. Separately, weigh accurately about 0.1 g (potency) of Cefoperazone RS, dissolve in 5 mL of 0.1 mol/L phosphate buffer solution, pH 7.0, and add the mobile phase to make exactly 100 mL. Pipet exactly 10 mL of this solution, add 10.0 mL of the internal standard solution and the mobile phase to make exactly 50 mL, and use this solution as the standard solution. Perform the test with 25 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS, of the peak area of cefoperazone to that of the internal standard. Amount [g (potency)] of cefoperazone (C25H27N9O8S) = Amount [g (potency)] of Cefoperazone RS
QT QS

Cefotaxime Sodium
NaO CH3 O S H2N N O N H N H S H O N O CH3 O O

C16H16N5NaO7S2: 477.45 Cefotaxime Sodium contains not less than 914 g (potency) per mg of cefotaxime (C16H17N5O7S2: 455.47), calculated on the dried basis. Description Cefotaxime Sodium is a white to light yellowish white crystalline powder and odorless, or has a bit of characteristic odor. Cefotaxime Sodium is freely soluble in water, sparingly soluble in methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) To 2 mg of Cefotaxime Sodium add 0.05 mL of water and 2 mL of sulfuric acidformalde hyde TS, mix with shaking: the yellow color develops. When this solution is heated in a boiling water bath for one minute, the color turns to brown. (2) Determine the infrared spectra of Cefotaxime Sodium and Cefotaxime Sodium RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Weigh about 20 mg each of Cefotaxime Sodium

Internal standard solutionWeigh accurately about 50 mg of acetanilide, add the mobile phase to make exactly 100 mL. Operating conditions Detector: An ultraviolet absorption photometer (wa-

KP 9 175 and Cefotaxime Sodium RS, dissolve in 5 mL of the mixture of methanol and 0.067 mol/L phosphate buffer solution, pH 8.0, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of 2 mol/L ammonium acetate solution, pH 6.2 adjusted with acetic acid, and acetone (85:15). Then allow the plate under ultraviolet light (main wavelength: 254 nm): the spots obtained from the test solution are the same with the corresponding spots from the standard solution in the Rf value, respectively. (4) Place 50 mg of Cefotaxime Sodium in platinum dish, ignite, add 10 mL of water and 1 mL of hydrochloric acid, dissolve, and filter. The filtrate responds to the Qualitative Tests 1) for sodium salt. Specific Optical Rotation [ ] 20 D : +58 ~ +64 (0.1 g after being dried, water, 10 mL). pH The pH of a solution obtained by dissolving 1 g of Cefotaxime Sodium in 10 mL of water is between 4.5 and 6.5. Absorbance (235 nm): 360 ~ 390 (20 mg calculated on the dried basis, water, 1000 mL). Purity (1) Related substancesPerform the test according to the Assay of Cefotaxime Sodium. Weigh accurately about 25 mg of Cefotaxime Sodium, dissolve in the mobile phase to make exactly 25 mL, pipet exactly 1 mL of this solution and add the mobile phase to make 100 mL, and use this solution as the test solution. Time span of measurement is about 3 times as long as the retention time of cefotaxime beginning after the solvent peak (Each peak area other than cefotaxime is not more than 1.0 % and the total of these peak areas is not more than 3.0 %). (2) DimethylanilineWeigh accurately about 1.0 g of Cefotaxime Sodium, add 5 mL of 1 mol/L sodium hydroxide TS and 1 mL of the internal standard solution, if necessary, centrifuge, use the supernatant as the test solution. Separately, weigh accurately about 50 mg of dimethylaniline, add 2 mL of hydrochloric acid and water to make 50 mL. Pipet exactly 1.0 mL, add 5.0 mL of 1 mol/L sodium hydroxide TS and 1.0 mL of the internal standard solution, if necessary, centrifuge, use the supernatant as the standard solution. Perform the test with the test solution and the standard solution as directed under the Gas Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of dimethylaniline to that of the internal standard for the test solution and the standard solution, respectively. (not more than 20 ppm)
1% E1 cm

Amount (ppm) of dimethylaniline = iline taken amount (mg) of dimethylan the content (%) of dimethylan iline 1 QT amount of CefotaximeSodium taken 50 QS 100000 Internal standard solutionDissolve 50.0 mg of naphthalene in cyclohexane and make 50 mL. Pipet 5.0 mL of this solution, add cyclohexane to make 100 mL, and use this solution as the internal standard solution. Operating conditions Detector: A hydrogen flame ionization detector. Column: A column, about 2 mm in inside diameter and 2 m in length, packed with diatomaceous earth for gas chromatography impregnated with 3 % m/m of a mixuture of polyphenylmethylsiloxane. Column temperature: 120 o C Injector and detector temperature: 150 o C Carrier gas: Nitrogen gas Flow rate: 30 mL/minute.

Sterility Test It meets the requirement, when Cefotaxime Sodium is used in a sterile preparation.
Bacterial Endotoxins Less than 0.05 EU per mg (potency) of cefotaxime, when Cefotaxime Sodium is used in a sterile preparation. Loss on Drying Not more than 3.0 % (1 g, 105 C, 3 hours). Assay Weigh accurately about 25 mg (potency) each of Cefotaxime Sodium and Cefotaxime Sodium RS, dissolve each in the mobile phase to make exactly 25 mL, pipet exactly 2 mL each of these solutions, add the mobile phase to make exactly 200 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT and AS , of cefotaxime of these solutions.

Amount [g (potency)] of cefotaxime (C16H17N5O7S2) = Amount [g (potency)] of Cefotaxime Sodium RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 235 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 ~ 200 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter)

176 Monographs, Part I

Mobile phase: A mixture of phosphate buffer, pH 7.0 and methanol (100:18). Flow rate: 1.0 mL per minute System suitability System performance: Add 1.0 mL of diluted hydrochloric acid to 4.0 mL of the test solution. Heat the solution at 40 C for 2 hours. Add 5.0 mL of 0.05 mol/L phosphate buffer solution, pH 6.6 and 1.0mL of diluted sodium hydroxide TS and 1 mL of the standard solution. Use this solution as the reference solution. When the procedure is run with the reference solution under the above operation conditions, the resolution between the two peaks is not less than 3.5.
Packaging and Storage Preserve in tight containers.

with the developing solvent which is a mixture of chloroform, acetone, and formic acid (10:9:1) to a distance of about 10 cm, and air-dry the plate. Spray evenly pdimethylaminocinnamaldehyde TS on the plate and heat the plate at about 80 C for 5 minutes. The red-purple spots obtained from the test solution and the standard solutions are the same in the Rf value. (4) Cefoxitin Sodium responds to the Qualitative Tests 1) for sodium salt.
Specific Optical Rotation [ ] 20 +200 ~ +218 D : (0.25 g calculated on the anhydrous basis, methanol, 25 mL, 100 mm). pH The pH of a solution obtained by dissolving 1 g of Cefoxitin Sodium in 10 mL of water is between 4.2 and 7.0. Absorbance E1 cm (262 nm): 190 ~ 210(2.0 mg calculated on the anhydrous basis, 0.05 mol/L phosphate buffer solution, pH 7.0, 100 mL). Purity Cefoxitin lactone Weigh accurately about 0.1 g of Cefoxitin Sodium, dissolve in methanol to make exactly 10 mL, and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 100 mL, and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of chloroform, acetone, and formic acid (10:9:1) to a distance of about 10 cm, and air-dry the plate. Spray evenly p-dimethylaminocinnam aldehyde TS on the plate and heat the plate at about 80 C for 5 minutes. The spots other than the principal spot obtained from the test solution are not more intense than those obtained from the standard solution (Not more than 1.0 %). Sterility Test It meets the requirement, when Cefoxitin Sodium is used in a sterile preparation. Bacterial Endotoxins Less than 0.10 EU per mg of cefoxitin, when Cefoxitin Sodium is used in a sterile preparation. Water Not more than 2.0 % (0.5 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg (potency) of Cefoxitin Sodium, dissolve in the mobile phase to make exactly 100 mL, and use this solution as the test solution. Separately, weigh accurately about 10 mg (potency) of Cefoxitin RS, dissolve in water to make 20 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard so1%

Cefoxitin Sodium
NaO O N S H N O O S H CH3 O NH2 O O

C16H17N3NaO7S2: 449.44 Cefoxitin Sodium contains not less than 860 g (potency) per mg of cefoxitin (C16H17N3O7S2: 427.46), calculated on the anhydrous basis.
Description Cefoxitin Sodium is a white to light yellowish white particle or powder, and has a little bit of a characteristic odor. Cefoxitin Sodium is very soluble in water, soluble in methanol, slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Weigh 50 mg of Cefoxitin Sodium, dissolve in 2 mL of water, add 2 mL of hydroxylamine hydrochloride TS and 2 mL of 1 mol/L sodium hydroxide TS, allow to stand for 5 minutes, add 3 mL of 1 mol/L hydrochloric acid TS and 1 mL of acidic ferric ammonium sulfate TS and mix with shaking; the redbrown color develops. (2) Weigh 25 mg of Cefoxitin Sodium, dissolve in 0.05 mol/L phosphate buffer solution, pH 7.0 and make 1000 mL. Determine the absorption spectrum of this solution as directed under Ultraviolet-visible Spectrophotometry, it exhibits maxima between 234 nm and 238 nm, and between 260 nm and 264 nm. (3) Weigh 20 mg each of Cefoxitin Sodium and Cefoxitin RS, dissolve in methanol to make 10 mL, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate

KP 9 177

lution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT and AS, of cefoxitin of these solutions. Amount [g (potency)] of cefoxitin (C16H17N3O7S2) = Amount [g (potency)] of Cefoxitin RS
AT AS

and Cefradine Hydrate RS in methanol, evaporate to dryness, and repeat the test on the residues.
Crystalliity Test It meets the requirement. pH The pH of a solution obtained by dissolving 0.1 g of Cefradine Hydrate in 10 mL of water is between 3.5 and 6.0. Purity (1) Heavy metals Proceed with 2.0 g of Cefradine Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Arsenic Prepare the test solution with 2.0 g of Cefradine Hydrate according to Method 4 and perform the test (not more than 1 ppm). (3) CefalexinWeigh accurately about 0.1 g of Cefradine Hydrate, dissolve in the mobile phase, make exactly 50 mL, and use this solution as the test solution. Separately, weigh accurately about 25 mg of Cefalexin RS, dissolve in the mobile phase, make exactly 250 mL, and use this solution as the standard solution. Perform the test with exactly 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine the peak areas of cefalexin. The peak area of cefalexin obtained from the test solution is not greater than that from the standard solution (Not more than 5.0 %). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 150 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 6.8 g of potassium dihydrogen phosphate in 800 mL of water and adjust the pH to 3.0 with dilute phosphoric acid (1 in 10). To 700 mL of this solution add 100 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of cefalexin is about 10 minutes. System suitability System performance: When the procedure is run with 5 L of the test solution under the above operation conditions, cefalexin and cefradine are eluted in this order with the resolution between these peaks being not less than 4.0. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the operating conditions, the relative standard deviation of peak area of cefalexin is not more than 2.0 %. Sterility Test It meets the requirement, when Cefradine Hydrate is used in a sterile preparation.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A room temperature. Mobile phase: A mixture of water, methanol and glacial acetic acid (50 : 50 : 1) Flow rate: Adjust the flow rate so that the retention time of cefoxitin is about 6 minutes. Packaging and Storage Preserve in tight containers.

Cefradine Hydrate
HO O CH3 N H H N O H S H O H2N

2H2O

C16H19N3O4S2H2O: 385.44 Cefradine Hydrate contains not less than 900 g (potency) and not more than 1050 g (potency) per mg of cefradine (C16H19N3O4S: 349.41), calculated on the anhydrous basis.
Description Cefradine Hydrate is a white to light yellowish white crystalline powder, is odorless or has a little bit of a characteristic odor and bitter taste. Cefradine Hydrate is sparingly soluble in water, slightly soluble in methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Place 50 mg of Cefradine Hydrate in a stopered test tube, add 2 mL of water and 1 mL of hydrochloric acid, boil for 10 minutes in water bath and cool: the brown-red color develops. (2) Determine the infrared spectra of Cefradine Hydrate and Cefradine RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears between the spectra, dissolve each of Cefradine Hydrate

178 Monographs, Part I Bacterial Endotoxins Less than 0.20 EU per mg of cefradine, when Cefradine Hydrate is used in a sterile preparation. Water Not more than 6.0 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 80 mg (potency) each of Cefradine Hydrate and Cefradine RS, dissolve each in water to make exactly 100 mL, and use these solutions as the test stock solution and the standard stock solution. Pipet exactly 10 mL each of the test stock solution and the standard stock solution, add the internal standard solution to make exactly 20 mL, and use these solutions as the test solution and the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS, of the peak area of Cefradine to that of the internal standard.

Ceftazidime Hydrate
O HO O N H N S N H2N O H S H CH3 CH3 O N O

+
N

5H2O

C22H22N6O7S25H2O: 636.65 Ceftazidime Hydrate contains not less than 950 g (potency) per mg of ceftazidime (C22H22N6O7S2: 546.58), calculated on the dried basis.
Description Ceftazidime Hydrate is a white to light yellowish white powder. Ceftazidime Hydrate is freely soluble in dimethylsulfoxide or in acetic acid, slightly soluble in water or in methanol, and very slightly soluble in dimethylformamide. Ceftazidime Hydrate is soluble in 2 mol/L hydrochloric acid, and in 2 mol/L sodium hydroxide. Identification Determine the retention times according to the procedure of Assay: the retention time of the principal peak in the chromatogram obtained with test solution is the same as that of the principal peak in the chromatogram obtained with the standard solution. Crystalliity Test quirement.

Amount [g (potency)] of cefradine (C16H19N3O4S) = Amount [g (potency)] of Cefradine RS

QT QS

Internal standard solutionWeigh about 0.2 g (potency) of Amoxicillin RS, dissolve in water to make 100 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) Mobile phase: The mixture of 0.0052 mol/L sodium acetate, methanol and glacial acetic acid (800:200:0.12) Flow rate: 1.0 mL per minute. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operation conditions, amoxicillin and cefradine are eluted in this order. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the operating conditions, the relative standard deviation of the ratios of the peak area of cefradine to that of the internal standard is not more than 2.0 %. Packaging and Storage tight containers.

Ceftazidime Hydrate meets the re-

pH The pH of a solution obtained by dissolving 50 mg of Ceftazidime Hydrate in 10 mL of water is between 3.0 and 4.0. Purity High molecular polymerWeigh accurately about 0.4 g of Ceftazidime Hydrate, dissolve in the mobile phase, make exactly 100 mL, and use this solution as the test solution. Use the test solution immediately after its preparation. Separately, weigh accurately about 4 mg of Ceftazidime High Moleuclar polymer RS, dissolve in the mobile phase, make exactly 100 mL, pipet exactly 5 mL of this solution, add the mobile phase to make exactly 50 mL, and use this solution as the standard solution. Use the standard solution within one hour after its preparation. Perform the test with exactly 100 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine the peak heights of the high molecular polymer, HT and HS (Not more than 0.05 %).

Preserve in light-resistant,

Amount (%) of the high molecular polymer

KP 9 179

Amount (mg/mL) of the high molecular H T polymer in the standard solution 0.1 = H S Amount (mg/mL) of Ceftazidime Hydrate

Ceftriaxone Sodium Hydrate

O NaO CH3 O N H N O N H2N H S H ONa O N N N S N CH3

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 235 nm) Column: A stainless steel column, about 9 mm in inside diameter and about 500 mm in length, packed with hydrophilic gel for gel permeation liquid chromatography Column temperature: 20 ~ 25 C. Mobile phase: 0.1 mol/L potassium monohydrogen phosphate solution Flow rate: 1.0 mL per minute Sterility Test It meets the requirement, when Ceftazidime Hydrate is used in a sterile preparation. Bacterial Endotoxins Less than 0.10 EU per mg of ceftazidime, when Ceftazidime Hydrate is used in a sterile preparation. Loss on Drying Not less than 13.0 % and not more than 15.0 % (0.1 g, in vacuum, 60 C, 3 hours). Assay Weigh accurately about 0.1 g (potency) each of Ceftazidime Hydrate and Ceftazidime RS, dissolve each in phosphate buffer solution, pH 7.0, to make exactly 100 mL. Pipet exactly 10 mL each of these solutions, add water to make exactly 100 mL, and use these solutions as the test solution and the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak area of ceftazidime, AT and AS. Prepare the test solution and the standard solution under light resistant conditions.

3 1/2 H2O

C18H16N8Na2O7S33H2O: 661.60 Ceftriaxone Sodium Hydrate contains not less than 871 g (potency) per mg of ceftriaxone (C18H18N8O7S3: 554.58), calculated on the anhydrous basis.
Description Ceftriaxone Sodium Hydrate is a white to light yellowish white crystalline powder. Ceftriaxone Sodium Hydrate is freely soluble in water or in propyleneglycol, sparingly soluble in methanol, slightly soluble in ethanol, and practically insoluble in chloroform. Identification (1) Determine the infrared spectra of Ceftriaxone Sodium Hydrate and Ceftriaxone Sodium RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Ceftriaxone Sodium Hydrate responds to the Qualitative Tests 1) for sodium salt. pH The pH of a solution obtained by dissolving 1.2 g of Ceftriaxone Sodium Hydrate in 10 mL of water is between 6.0 and 8.0. Sterility Test It meets the requirement, when Ceftriaxone Sodium Hydrate is used in a sterile preparation. Bacterial Endotoxins Less than 0.20 EU per mg of ceftriaxone, when Ceftriaxone Sodium Hydrate is used in a sterile preparation. Water Not less than 8.0 % and not more than 11.0 % (0.1 g, volumetric titration, direct titration). Assay Weigh accurately about 4.5 mg (potency) each of Ceftriaxone Sodium Hydrate and Ceftriaxone Sodium RS, dissolve each in the mobile phase to make exactly 100 mL, and use these solutions as the test solution and the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak area of ceftazidime, AT and AS.

Amount [g (potency)] of ceftazidime (C22H22N6O7S2) = Amount [g (potency)] of Ceftazidime RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column packed with octadecylsilanized silica gel for liquid chromatography (3 ~ 10 m in particle diameter) Mobile phase: To 40 mL of acetonitrile and 200 mL of phosphate buffer solution, pH 7.0 add water to make 200 mL. Flow rate: 2.0 mL per minute. Packaging and Storage Preserve in light-resistant, tight containers (Nitrogen-filled, 0 ~ 5 C).

180 Monographs, Part I Water Not more than 1.5 % (1.0 g, volumetric titration, direct titration). Isomer Ratio Perform the test according to Assay, and calculate the content ratio of isomer A by using the following equation (Not less than 0.48 and not more than 0.55).

Amount [g (potency)] of ceftriaxone (C18H18N8O7S3) = Amount [g (potency)] of Ceftriaxone Sodium RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 3.2 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A room temperature Mobile phase: Weigh 2g of tetra-n-heptylammonium bromide and 2g of tetra-n-decylammonium bromide, dissolve in 620 mL of acetonitrile, and add 320 mL of water, 55 mL of phosphate buffer solution, pH 7.0, and 5.0 mL of citric acid buffer solution, pH 5.0. Flow rate: 1.0 mL per minute. Packaging and Storage Preserve in tight containers.

Amount of isomer A in Cefuroxime Axetil =

The peak area of isomer A the peak area of isomer A + the peak area of isomer B

Cefuroxime Axetil
H3C O CH3 O O N H N S O H H O O CH3 O O O N O NH2

Assay Weigh accurately about 30 mg (potency) of Cefuroxime Axetil and Cefuroxime Axetil RS, dissolve each in methanol and add methanol to make exactly 25 mL. Pipet exactly 10 mL of each solution, add exactly 5.0 mL of the interal standard solution and 3.8 mL of methanol, mix, add 0.2 mol/L ammonium dihydrogen phosphate solution to make exactly 50 mL, and use these solutions as the test solution and the standard solution. Use these solutions within 8 hours. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, HT and HS, of the peak height of cefuroxime axetil to that of the internal standard.

Amount [g (potency)] of cefuroxime (C16H16N4O8S) = Amount [g (potency)] of Cefuroxime Axetil RS

HT HS

C20H22N4O10S: 510.47 Cefuroxime Axetil contains not less than 745 g (potency) and not more than 875 g (potency) per mg of cefuroxime (C16H16N4O8S: 424.39), calculated on the anhydrous basis.
Description Cefuroxime Axetil is a white to dark yellow powder. Cefuroxime Axetil is soluble in dimethylsulfoxide, in dimethylformamide, in 1,4-dioxane, in chloroform, in methanol, in acetone, in glacial acetic acid or in ethylacetate, and slightly soluble in ethanol, in ether or in toluene. Cefuroxime Axetil dissolves in 2 mol/L sodium hydroxide TS. Identification Determine the infrared spectra of Cefuroxime Axetil and Cefuroxime Axetil RS, as directed in the paste method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Crystalliity Test It meets the requirement.

HT = (the peak height of isomer A + the peak height of isomer B, in the test solution)/(the peak height of the internal standard) HS = (the peak height of isomer A + the peak height of isomer B, in the standard solution)/(the peak height of the internal standard) Internal standard solutionWeigh accurately about 54 mg of acetanilide, add methanol to make 10 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 278 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with methylsilanized silica gel for liquid chromatography (3 ~ 10 m in particle diameter) Mobile phase: To 620 mL of 0.2 mol/L ammonium dihydrogen phosphate add methanol to make exactly 1000 mL. Flow rate: 1.5 mL per minute System suitability System performance: When the procedure is run with the standard solution under the above operation conditions, the internal standard, cefuroxime axetil isomer B and cefuroxime axetil isomer A are eluted in this

KP 9 181

order with the resolution between isomer A and isomer B being not less than 1.5. Theoretical plates of the peak of isomer A is not less than 3000 and the symmetry factor of the peak of isomer A is not more than 1.5. System repeatability: When the test is repeated 5 times with 5 L of the standard solution under the operating conditions, the relative standard deviation of every peak is not more than 2.0 %. Relative retention time: The relative retention time of isomer B to the internal standard is about 2 and that of isomer A is about 2.25.
Packaging and Storage Preserve in tight containers.

Sterility Test It meets the requirement, when Cefuroxime Sodium is used in a sterile preparation. Pyrogen Test It meets the requirement, when Cefuroxime Sodium is used in a sterile preparation,. Weigh an appropriate amount of Cefuroxime Sodium, dissolve in water, make the solution so that each mL contains 50 mg (potency), and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not more than 3.5 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 0.15 g (potency) each of Cefuroxime Sodium and Cefuroxime Sodium RS, dissolve in water to make exactly 100 mL. Pipet exactly 2 mL of each of these solutions, add water to make exactly 200 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with each of the test solution and the standard solution as directed under Ultraviolet-visible Spectrophotometry, and determine the absorbance at absorption maximum around 273 nm, AT and AS, of these solutions.

Cefuroxime Sodium
NaO CH3 O O N H N S O H H O N O NH2 O O

C16H15N4NaO8S: 446.37 Cefuroxime Sodium contains not less than 856 g (potency) per mg of cefuroxime (C16H16N4O8S: 424.39), calculated on the anhydrous basis.
Description Cefuroxime Sodium is a white to light yellowish white crystal or crystalline powder, is odorless, or has a little bit of a characteristic odor. Cefuroxime Sodium is freely soluble in water, sparingly soluble in methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Determine the absorption spectra of Cefuroxime Sodium and Cefuroxime Sodium RS as directed in the paste method under Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumvers. (2) Determine the absorption spectra of the 0.0015 % aqueous solutions each of Cefuroxime Sodium and Cefuroxime Sodium RS from 190 nm to 450 nm as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit maximum and minimum at the same wavelengths. (3) Cefuroxime Sodium responds to the Qualitative Tests 1) for sodium salt. Specific Optical Rotation [ ] 20 D : +59 ~ +266(0.5 g calculated on the anhydrous basis, water, 100 mL, 100 mm). pH The pH of a solution obtained by dissolving 1 g of Cefuroxime Sodium in 10 mL of water is between 6.0 and 8.5.

Amount [g (potency)] of cefuroxime (C16H16N4O8S) = Amount [g (potency)] of Cefuroxime Sodium RS

AT AS

Packaging and Storage Preserve in tight containers.

Cetirizine Dihydrochloride
Cl N N H O COOH

2HCl

and enantiomer C21H25ClN2O32HCl : 461.81 Cetirizine Dihydrochloride contains not less than 99.0% and not more than 100.5% of cetirizine dihydrochloride (C21H25ClN2O32HCl), calculated on an anhydrous basis.
Description Cetirizine Dihydrochloride is a white powder. Cetirizine Dihydrochloride is freely soluble in water, and practically insoluble in acetone or in methylene chloride. Identification (1) Dissolve 20.0 mg of Cetirizine Di-

182 Monographs, Part I

hydrochloride in 0.1 mol/L hydrochloric acid to make 100 mL. Dilute 10.0 mL of this solution with 0.1 mol/L hydrochloric acid to 100 mL. Determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry. Absorption maximum occurs at 231 nm, and the specific absorbance at this wavelength ranges from 359 to 381. (2) Determine the infrared absorption spectra of Cetirizine Dihydrochloride and Cetirizine Dihydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 10 mg of Cetirizine Dihydrochloride in water to make 5 mL, and use this solution as the test solution. Separately, dissolve 10 mg of Cetirizine Dihydrochloride RS in water to make 5 mL, and use this solution as the standard solution (1). Separately, dissolve 10 mg of chlorphenamine maleate RS in water to make 5 mL. To 1 mL of the solution, add 1 mL of the standard solution (1). Use this solution as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for thin-layer chromatography, develop with a mixture of methylene chloride, methanol, and ammonia (90 : 10 : 1) to a distance of about 15 cm, and dry the plate in cool air. Examine in ultraviolet light at 254 nm, and compare the principal spot from the test solution with that from the standard solution (1). Their Rf values are the same. The test is valid when 2 clearly separated spots are observed with the standard solution (2). (4) The solution of Cetirizine Dihydrochloride in water (1 in 100) responds to the Qualitative Tests for chloride (2nd method).
pH The pH of a solution obtained by dissolving 1.0 g of Cetirizine Dihydrochloride in 20 mL of water is between 1.2 and 1.8. Purity (1) Clarity and color of solutionWhen 2.0 g of Cetirizine Dihydrochloride is dissolved in 20 mL of water, the resultant solution is clear. (2) Heavy metals Proceed the test with 2.0 g of Cetirizine Dihydrochloride according to Method 1. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve 20.0 mg of Cetirizine Dihydrochloride in the mobile phase to make 100 mL, and use this solution as the test solution. Separately, dissolve 5.0 mg of Cetirizine Dihydrochloride RS and 5.0 mg of cetirizine related substance I RS {(RS)-1-[(4chlorophenyl)phenylmethyl] piperazine} in the mobile phase to make 25 mL. Dilute 1.0 mL of the solution to 100 mL with the mobile phase, and use this solution as the standard solution (1). Dilute 2.0 mL of the test solution to 50 mL with the mobile phase. Dilute 5.0 mL of this solution to 100 mL with the mobile phase, and use

this solution as the standard solution (2). Perform the test with 20 L each of the test solution, the standard solutions (1), and (2) as directed under the Liquid Chromatography according to the following conditions. Determine the area of each peak by the automatic integration method. The peak area of any related substance, excluding the principal peak area corresponding to cetirizine, from the test solution is not more that the area of the principal peak from the standard solution (2) (0.2%); and the sum of the peak areas of all related substances from the test solution is not more than 1.5 times the area of the principal peak from the standard solution (2) (0.3%). Disregard any peak areas that are not more than 0.1 times the principal peak area from the standard solution (2).
Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column about 4.6 mm inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of acetonitrile, water, and dilute sulfuric acid (93 : 6.6 : 0.4). Flow rate: 1.0 mL/min. System suitability System performance: Adjust the sensitivity of the system so that the peak heights of cetirizine and the related substance I are about 50% of the full scale of the recorder. When the procedure is run with 20 L of the standard solution (1) under the above operation conditions, the resolution between the peaks of cetirizine and the related substance I is not less 3, and the symmetry factor is not more than 2.0. Loss on Drying Not more than 0.5% (1 g, 105 C, constant weight). Residue on Ignitio Not more than 0.2% (1 g). Assay Dissolve 0.100 g of Cetirizine Dihydrochloride in 70 mL of a mixture of water and acetone (30 : 70), titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L sodium hydroxide VS = 15.394 mg of C21H25ClN2O32HCl

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Cetraxate Hydrochloride

KP 9 183 Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column, about 6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Adjust the pH of a mixture of water, methanol and 0.5 mol/L ammonium acetate TS (15 : 10 : 4) to 6.0 with acetic acid. Flow rate: Adjust the flow rate so that the retention time of cetraxate is about 10 minutes. System suitability System performance: Dissolve 20 mg of Cetraxate Hydrochloride and 10 mg of phenol in 100 mL of water. To 2 mL of this solution, add water to make 20 mL. When the procedure is run with 10 L of this solution under the above operating conditions, cetraxate and phenol are eluted in this order with the resolution between these peaks being not less than 5. Detection sensitivity: Adjust the detection sensitivity so that the peak height of cetraxate obtained from 10 L of the standard solution is not less than 20 mm.

O H2NH2C H C H O CH2CH2CO 2H HCl

C17H23NO4HCl: 341.83 Cetraxate Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of cetraxate hydrochloride (C17H23NO4HCl).
Description Cetraxate Hydrochloride is a white crystal or crystalline powder. Cetraxate Hydrochloride is soluble in methanol, sparingly soluble in water or in ethanol and practically insoluble in ether. Melting point About 236 C (with decomposition). Identification (1) Determine the absorption spectra of the solutions of Cetraxate Hydrochloride and Cetraxate Hydrochloride RS in methanol (1 in 2500) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Dissolve 0.5 g of Cetraxate Hydrochloride in 5 mL of a mixture of water and 2-propanol (1 : 1) by warming, cool to below 25 oC. Filter, dry the formed crystals in vacuum for 4 hours, and further dry at 105 oC for 1 hour. Determine the infrared spectra of the dried matter and Cetraxate Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Cetraxate Hydrochloride (1 in 100) responds to the Qualitative Tests (2) for chloride. Purity (1) Heavy metalsProceed with 2.0 g of Cetraxate Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Cetraxate Hydrochloride according to Method 3 and perform the test with a solution of magnesium nitrate in ethanol (1 in 5) (not more than 2 ppm). (3) Cis isomerDissolve 0.10 g of Cetraxate Hydrochloride in 10 mL of water and use this solution as the test solution. To exactly 5 mL of the test solution, add water to make exactly 100 mL. To exactly 2 mL of this solution, add water to make exactly 50 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution and the standard solution by the automatic integration method: the area of the peak which has a retention time 1.3 to 1.6 times that of cetraxate from the test solution is not larger than the peak area of cetraxate from the standard solution.

(4) 3-(p-Hydroxyphenyl)propionic acidTo about 0.10 g of Cetraxate Hydrochloride, add exactly 2 mL of the internal standard solution and methanol to make 10 mL and use this solution as the test solution. Separately, dissolve 25 mg of 3-(p-hydroxyphenyl)propionic acid in methanol to make exactly 100 mL. To exactly 2 mL of this solution, add exactly 2 mL of the internal standard solution and methanol to make 10 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratio, QT and QS , of the peak area of 3-(p-hydroxyphenyl) propionic acid to that of the internal standard: QT is not larger than QS .
Internal standard solutionA solution of Caffeine in methanol (1 in 4000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column, about 6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C Mobile phase: Adjust the pH of a mixture of water, methanol and 0.5 mol/L ammonium acetate TS (15 : 5 : 2) to 5.5 with acetic acid.

184 Monographs, Part I

Flow rate: Adjust the flow rate so that the retention time of 3-(p-hydroxyphenyl)propionic acid is about 7 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, 3-(p-hydroxy phenyl) propionic acid and the internal standard are eluted in this order with the resolution between these peaks being not less than 5.0. Detection sensitivity: Adjust the detection sensitivity so that the peak height of 3-(p-hydroxyphenyl) propionic acid obtained from 10 L of the standard solution is not less than 30 mm. (5) Related substancesDissolve 0.10 g of Cetraxate Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform, methanol and glacial acetic acid (20 : 4 : 3) to a distance of about 10 cm and airdry the plate. Spray evenly ninhydrin TS on the plate and heat at 90 C for 10 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.5 g of Cetraxate Hydrochloride, previously dried, dissolve in 100 mL of water and adjust the pH of this solution to between 7.0 and 7.5 with dilute sodium hydroxide TS. To this solution, add 10 mL of formalin, stir for about 5 minutes and titrate with 0.1 mol/L sodium hydroxide VS for over about 20 minutes (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Chloral Hydrate contains not less than 99.5% and not more than 101.0% of chloral hydrate (C2H3Cl3O2).
Description Chloral Hydrate is a colorless crystal, has a pungent odor and an acrid, slightly bitter taste. Chloral Hydrate is very soluble in water and freely soluble in ethanol or in ether. Chloral Hydrate slowly volatilizes in air. Identification (1) Dissolve 0.2 g of Chloral Hydrate in 2 mL of water and add 2 mL of sodium hydroxide TS: the turbidity is produced and it separates into two clear layers by warming. (2) Heat 0.2 g of Chloral Hydrate with 3 drops of aniline and 3 drops of sodium hydroxide TS: the disagreeable odor of phenylisocyanide (poisonous) is perceptible. Purity (1) Clarity and color of solutionDissolve 1.0 g of Chloral Hydrate in 2 mL of water: the solution is clear and colorless. (2) AcidDissolve 0.20 g of Chloral Hydrate in 2 mL of water and add 1 drop of methyl orange TS: a yellow color is observed. (3) ChloridePerform the test with 1.0 g of Chloral Hydrate. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.011%). (4) Chloral alcoholateWarm 1.0 g of Chloral Hydrate with 10 mL of sodium hydroxide TS, filter the upper layer, add iodine TS to the filtrate until a yellow color is observed and allow the solution to stand for 1 hour: no yellow precipitate is produced. (5) BenzeneWarm the solution obtained in (1) with 3 mL of water: no odor of benzene is perceptible. Residue on Ignition Not more than 0.10% (1 g). Assay Weigh accurately about 4 g of Chloral Hydrate in a glass-stoppered flask, add 10 mL of water and 40 mL of 1 mol/L sodium hydroxide VS and allow the mixture to stand for exactly 2 minutes. Titrate the excess sodium hydroxide immediately with 0.5 mol/L sulfuric acid VS (indicator: 2 drops of phenolphthalein TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L sodium hydroxide VS = 34.183 mg of C17H23NO4HCl

Packaging and Storage Preserve in tight containers.

Each mL of 1 mol/L sodium hydroxide VS = 165.40 mg of C2H3Cl3O2.

Packaging and Storage Preserve in tight containers.

Chloral Hydrate
OH Cl3C C OH H

Chlorambucil
Cl N HO O

C2H3Cl3O2: 165.40

Cl

KP 9 185

C14H19Cl2NO2: 304.21 Chlorambucil contains not less than 98.0% and not more than 101.0% of chlorambucil (C14H19Cl2NO2), calculated on the anhydrous basis.
Description Chlorambucil is a milk-white granuleshaped powder. Chlorambucil is practically insoluble in water and soluble in acetone. Chlorambucil dissolves in dilute sodium hydroxide TS. Identification (1) Dissolve 50 mg of Chlorambucil in 5 mL of acetone and dilute with water to make 10 mL. Add 1 drop of 1 mol/L sulfuric acid and, then, add 4 drops of silver nitrate TS: no opalescence is observed immediately. Warm this solution on a steam-bath: opalescence develops. (2) Determine the infrared absorption spectra of solutions of Chlorambucil and Chlorambucil RS in carbon disulfide (1 to 125) as directed under the Infrared Spectrophotometry, using solid cell with 1 mm of thickness: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point

Disintegration Test Place 1 tablet in each of six tubes of baskets. If the tablet has a soluble external coating, immerse the basket in water at room temperature for 5 minutes. Operate the apparatus, using the first solution maintained at 37 2 C as the immersion fluid. After 30 minutes of operation in the first solution, lift the basket from the fluid, and observe the tablets. If the tablets have not disintegrated completely, substitute the second solution maintained at 37 2 C as the immersion fluid, and continue the test for total period time, including previous exposure to water and the first solution, equal to 45 minutes. If one or two tablets do not disintegrate, proceed the test with additional 12 tablets and determine: tablets which do not disintegrate are not more than 2 out of total 18 tablets. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Chlorambucil Tablets. Weigh accurately a portion of the powder, equivalent to about 2 mg of chlorambucil (C14H19Cl2NO2), add 50 mL of ethanol, shake cautiously, add 5.0 mL of 0.1 mol/L hydrochloric acid and 2.0 mL of internal standard solution, shake by mechanical means for 5 minutes and dilute with ethanol to make exactly 100 mL. Filter and use this filtrate as the test solution. Separately, weigh accurately about 20 mg of Chlorambucil RS, previously dried in a desiccators (silica gel) for 24 hours and dissolve in ethanol to make exactly 20 mL. To 2.0 mL of this solution, add 50 mL of ethanol, 5.0 mL of 0.1 mol/L hydrochloric acid and 2.0 mL of internal standard solution with cautious shaking and dilute with ethanol to make exactly 100 mL. Use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine the relative peak areas compared to internal standard, QT and QS , of Chlorambucil of the test solution and the standard solution, respectively.

Between 65 C and 69 C.

Water Not more than 0.5% (1 g, volumetric titration, direct titration ). Assay Weigh accurately 0.2 g of Chlorambucil, dissolve in 10 mL of acetone and dilute with 10 mL of water. Titrate with 0.1 mol/L sodium hydroxide (indicator: 2 drops of phenolphthalein TS).

Each mL of 0.1 mol/L sodium hydroxide = 30.421 mg of C14H19Cl2NO2

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Chlorambucil Tablets
Chlorambucil Tablets contain not less than 85.0% and not more than 110.0% of the labeled amount of the chlorambucil (C14H19Cl2NO2: 304.22).
Preparation Prepare as directed under Tablets, with Chlorambucil. Identification Weigh a portion of powered Chlorambucil Tablets, equivalent to 16 mg of Chlorambucil, add 20 mL of carbon disulfide and shake to mix. Filter and evaporate the filtrate to dryness. Dissolve the residue in 2 mL of carbon disulfide. Proceed the test with the solution as directed in the Identification (1) under Chlorambucil.

Amounts (mg) of chlorambucil (C14H19Cl2NO2) = amount (mg) of Chlorambucil RS

QT 1 Q S 10

Internal standard solutionAn ethanol solution (1 in 2500) of propyl p-oxybenzoate. Operating Conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 2 mm in inside diameter and about 25 to 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: Mix 500 mL of ethanol and 1.0 mL of

186 Monographs, Part I

glacial acetic acid and add water to make 1000 mL. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the resolution between peaks of Chlorambucil and internal substance is not less than 2.0. System repeatability: When the test is repeated 6 times with the standard solution, the relative standard deviation of the peak area of Chlorambucil is not more than 2.0%.
Packaging and Storage Preserve in well-closed containers (light-resistant, well-closed containers in case of uncoated tablets).

in water is between 4.5 and 7.5.

Absorbance 1000 mL).
1% E1 cm (278 nm): 289 ~ 307 (20 mg, water,

Purity (1) Heavy metalsProceed with 2.0 g of Chloramphenicol according to Method 2 and perform the test. Prepare the control solution with 5.0 mL of standard lead solution (not more than 25 ppm). (2) Free chlorineTo 5 mL of 0.1 % aqueous solution of Chloramphenicol, add 2 to 3 drops of silver nitrate TS: no precipitate appears. Sterility Test It meets the requirement, when Chloramphenicol is used in a sterile preparation. Pyrogen Test It meets the requirement, when Chloramphenicol is used in a sterile preparation,. Weigh an appropriate amount of Chloramphenicol, dissolve in saline injection solution to make the solution so that each mL contains 5.0 mg, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Loss on Drying Not more than 0.5 % (1.0 g, 100 C). Residue on Ignition Not more than 0.1 % (1 g). Assay Weigh accurately about 50 mg (potency) each of Chloramphenicol and Chloramphenicol RS, dissolve in the mobile phase to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas of chloramphenicol, AT , AS.

Chloramphenicol
OH O Cl N H Cl H OH H NO2

C11H12Cl2N2O5: 323.13 Chloramphenicol contains not less than 900 g (potency) of per mg of chloramphenicol (C11H12Cl2N2O5), calculated on the dried basis.
Description Chloramphenicol is a whtie to yellowish white crystals or crystalline powder, is odorless, and has a bitter taste. Chloramphenicol is freely soluble in methanol or in ethanol, slightly soluble in water or in ether. Identification (1) Weigh 10 mg of Chloramphenicol, dissolve in 1 mL of 50 % ethanol, add 3 mL of calcium chloride solution (1 in 100) and 50 mg of zinc powder, and heat for 10 minutes in water bath. Decant the supernatant into a test tube, add 0.1 g of glacial acetic acid and 2 drops of benzoyl chloride, mix with shaking for 1 minute, add 10 drops of ferric chloride TS (if the solution is not clear, add dilute hydrochloric acid): a purple to red-purple color develops. If the test is run as above except the addition of zinc powder, the solution is colorless. (2) To 50 mg of Chloramphenicol, add 3 mL of sodium hydroxide ethanol TS, and heat for 15 minutes in water bath and cool down: The solution responds to the Qualitative Tests for chloride. Specific Optical Rotation [ ] 20 D : +18.5 ~ +21.5 (1.25 g, ethanol anhydrous, 25 mL (dissolve with heating), 200 mm). Melting Point pH

Amount [g (potency)] of chloramphenicol (C11H12Cl2N2O5) = Amount [g (potency)] of Chloramphenicol RS

AT AS

Between 149 C and 153 C .

The pH of a saturated solution of Chloramphenicol

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Mobile phase: A mixture of water, methanol and acetic acid (550:450:1) Flow rate: 1.5 mL per minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the theoretical plate number is not less than 1800, and the symmetry factor is not more than

KP 9 187

2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the principal peak area is not more than 1.0 %.
Packaging and Storage Preserve in tight containers .

Amount [g (potency)] of chloramphenicol (C11H12Cl2N2O5) = Amount [g (potency)] Chloramphenicol RS

AT AS

Packaging and Storage Preserve in tight containers .

Chloramphenicol Palmitate
O O H O CI O2N H OH N H CI

Chloramphenicol Sodium Succinate

O O Cl Cl N H H O R1 R3 H NO2

C27H42Cl2N2O6: 561.54 Chloramphenicol Palmitate contains not less than 555 g (potency) and not more than 595 g (potency) of per mg of chloramphenicol (C11H12Cl2N2O5: 323.13).
Description Chloramphenicol Palmitate is a whtie to grayish white crystalline powder or powder, and has not a taste. Chloramphenicol Palmitate is freely soluble in acetone or in chloroform, soluble in methanol or in ethanol, and practically insoluble in water. Identification (1) Determine the absorption spectrum of 0.0025 w/v % solution of Chloramphenicol Palmitate in ethanol anhydrous as directed under Ultravioletvisible Spectrophotometry, it exhibits maximum between 268 nm and 274 nm. (2) Weigh 50 mg of Chloramphenicol Palmitate, add 2 mL of potassium hydroxide ethanol TS, heat for 15 minutes in water bath, and cool down: the solution responds to the Qualitative Tests for chloride. Specific Optical Rotation [ ] 20 D : +21 ~ +25 (1.25 g, ethanol anhydrous, 25 mL (dissolve with heating), 200 mm). Melting Point

1 Isomer: R1 = CO-CH2-CH2-CO2Na, R3 = H 3 Isomer: R1 = H, R3 = CO-CH2-CH2-CO2Na C15H15Cl2N2NaO8: 445.18 Chloramphenicol Sodium Succinate contains not less than 650 g (potency) of per mg of chloramphenicol (C11H12Cl2N2O5: 323.13).
Description Chloramphenicol Sodium Succinate is a whtie to yellow-brown crystalline powder or powder. Chloramphenicol Sodium Succinate is freely soluble in water or in methanol. Identification (1) To 50 mg of Chloramphenicol Sodium Succinate, add 5 mL of pyridine, 5 mL of 1 mol/L sodium hydroxide TS, mix with shaking, and heat in water bath for several minutes: The pyridine layer turns to deep red color. (2) To 0.1 g mg of Chloramphenicol Sodium Succinate, add 0.2 g of resorcin and 0.2 mL of sulfuric acid, and heat until the color turns to deep red. To this solution, add water slowly: an orage color with green fluorescence develops. (3) Determine the absorption spectrum of 0.002 w/v % aqueous solution of Chloramphenicol Sodium Succinate as directed under Ultraviolet-visible Spectrophotometry, it exhibits maximum between 271 nm and 281 nm. (4) Weigh 50 mg of Chloramphenicol Sodium Succinate, add 2 mL of potassium hydroxide ethanol TS, heat for 15 minutes in water bath, and cool down: the solution responds to the Qualitative Tests for chloride. (5) Chloramphenicol Sodium Succinate responds to the Qualitative Tests 1) for sodium salt. Specific Optical Rotation water, 25 mL, 200 mm).
[ ] 20 D :

Between 87 C and 95 C .

Assay Weigh accurately about 90 mg of Chloramphenicol Palmitate and about 50 mg (potency) of Chloramphenicol RS, dissolve each in ethanol anhydrous to make exactly 100 mL, pipet exactly 5 mL each of these solutions, add ethanol anhydrous to make exactly 250 mL, and use these solutions as the test solution and the standard solution, respectively. Determine the absorbances, AT and AS, at 271 nm of the test solution and the standard solution as directed under Ultraviolet-visible Spectrophotometry.

+5 ~ +8 (1.25 g,

188 Monographs, Part I pH The pH of a solution obtained by dissolving 2.5 g of Chloramphenicol Sodium Succinate in 10 mL of water is between 6.4 and 7.0. Sterility Test It meets the requirement, when Chloramphenicol Sodium Succinate is used in a sterile preparation. Pyrogen Test It meets the requirement, when Chloramphenicol Sodium Succinate is used in a sterile preparation. Weigh an appropriate amount of Chloramphenicol Sodium Succinate, dissolve in Isotonic Sodium Chloride Injection to make a solution so that each mL contains 5.0 mg, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not more than 5.0 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately an amount of Chloramphenicol Sodium Succinate and Chloramphenicol Sodium Succinate RS, dissolve each in water to make solutions so that each mL contains 30 g (potency), and use these solutions as the test solution and the standard solution, respectively. Determine the absorbances, AT and AS, at 276 nm of the test solution and the standard solution as directed under Ultraviolet-visible Spectrophotometry.

sparingly soluble in ethanol, very lightly soluble in ether and practically insoluble in water. Chlorodiazepoxide dissolves in dilute hydrochloric acid. Chlorodiazepoxide is gradually affected by light. Melting point About 240 C (with decomposition).
Identification (1) Determine the absorption spectra of solutions of Chlordiazepoxide and Chlordiazepoxide RS in 0.1 mol/L hydrochloric acid TS (1 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Chlordiazepoxide and Chlordiazepoxide RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. (3) Proceed with Chlordiazepoxide as directed under the Flame Coloration Test (2) and perform the test: a green color develops. Purity (1) Heavy metalsProceed with 1.0 g of Chlordiazepoxide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancePerform the test without exposure to daylight, using light-resistant vessels. Dissolve 0.20 g of Chlordiazepoxide in exactly 10 mL of a mixture of methanol and ammonia TS (97 : 3) and use this solution as the test solution. Pipet 1 mL of the test solution, add a mixture of methanol and ammonia TS (97 : 3) to make exactly 200 mL and use this solution as the standard solution (1). Separately, dissolve 10 mg of 2amino-5-chlorobenzophenone RS in methanol to make exactly 200 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thinlayer Chromatography. Spot 25 L of the test solution and 5 L each of the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate and dehydrated ethanol (19 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution (1). Spray evenly a solution of sodium nitrite in 1 mol/L hydrochloric acid TS (1 in 100) on the plate, allow to stand for 1 minute and spray evenly N-(1-naphthyl)N'-diethyl-ethylenediamine oxalateacetone TS on the plate: the spots from the test solution are not more intense than the spots from the standard solution (2). Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, 60 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.6 g of Chlordiazepox-

Amount [g (potency)] of chloramphenicol (C11H12Cl2N2O5) = Amount [g (potency)] Chloramphenicol Sodium Succinate RS

AT AS

Packaging and Storage Preserve in tight containers .

Chlordiazepoxide
NHCH3 N

Cl

C16H14ClN3O: 299.76 Chlordiazepoxide, when dried, contains not less than 98.5% and not more than 101.0% of chlordiazepoxide (Cl6H14ClN3O).
Description Chlordiazepoxide is a white to pale yellow crystal or crystalline powder. Chlordiazepoxide is freely soluble in glacial acetic acid,

KP 9 189

ide, previously dried and dissolve in 50 mL of glacial acetic acid. Titrate with 0.1 mol/L perchloric acid VS until the color of the supernatant liquid changes from purple through blue purple to blue (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 29.976 mg of Cl6H14ClN3O
Packaging and Storage tight containers.

standard solutions (1) and (2) as directed under the Thinlayer Chromatography. Spot 25 L of the test solution and 10 L each of the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Proceed as directed in the Purity (2) under Chlordiazepoxide.
Particle Size Distribution Test It meets the requirement. Uniformity of Dosage Units(divided) It meets the requirement. Assay Perform this test without exposure to daylight, using light-resistant vessels. Weigh accurately a quantity of Chlordiazepoxide Powder, equivalent to about 0.1 g of chlordiazepoxide (Cl6H14ClN3O), transfer to a glassstoppered flask, add exactly 90 mL of methanol, stopper, shake vigorously for 15 minutes and centrifuge. Pipet 10 mL of the supernatant liquid, add exactly 5 mL of the internal standard solution, add methanol to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 0.1 g of Chlordiazepoxide RS, previously dried in a desiccator (in vacuum, P2O5,

Preserve in light-resistant,

Chlordiazepoxide Powder
Chlordiazepoxide Powder contains not less than 93.0% and not more than 107.0% of the labeled amount of chlordiazepoxide (Cl6H14ClN3O: 299.76).
Method of Preparation Prepare as directed under Powders, with Chlordiazepoxide. Identification (1) Weigh a portion of Chlordiazepoxide Powder, equivalent to 10 mg of Chlordiazepoxide according to the labeled amount, add 100 mL of 0.1 mol/L hydrochloric acid TS, shake and filter. To 5 mL of the filtrate, add 0.1 mol/L hydrochloric acid TS to make 100 mL, determine the absorption spectrum as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 244 nm and 248 nm and between 306 nm and 310 nm and a minimum between 288 nm and 292 nm. (2) Weigh a portion of Chlordiazepoxide Powder, equivalent to 20 mg of Chlordiazepoxide according to the labeled amount, add 10 mL of methanol, shake for 5 minutes, then filter by suction through a glass filter, evaporate the filtrate with the aid of a current of air to dryness and dry the residue in vacuum at 60 C for 1 hour. Determine the infrared absorption spectrum of the residue as directed in the potassium bromide disk method under the Infrared Spectrophotometry: it exhibits absorption at the wave numbers of about 1625 cm-1, 1465 cm-1, 1265 cm-1, 850 cm-1 and 765 cm-1. Purity Related substancesPerform this test without exposure to daylight, using light-resistant vessels, To a portion of Chlordiazepoxide Powder, equivalent to 50 mg of Chlordiazepoxide according to the labeled amount, add exactly 5 mL of a mixture of methanol and ammonia TS (97 : 3), shake, centrifuge and use the supernatant liquid as the test solution. Separately, dissolve 50 mg of Chlordiazepoxide RS in a mixture of methanol and ammonia TS (97 : 3) to make exactly 50 mL and use this solution as the standard solution (1). Dissolve 5.0 mg of 2amino-5-chlorobenzophenone RS in methanol to make exactly 200 mL and use this solution as the standard solution (2). Perform the test with the test solution and the

60 C ) for 4 hours, dissolve in methanol, add exactly 5 mL of the internal standard solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and calculate the ratios, QT and

QS , of the peak area of Chlordiazepoxide to that of the internal standard for the test solution and the standard solution, respectably.
Amount (mg) of chlordiazepoxide (Cl6H14ClN3O) = amount (mg) of Chlordiazepoxide RS
QT QS

Internal standard solutionA solution of isobutyl salicylate in methanol (1 in 20), Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 25 to 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of methanol and 0.02 mol/L monobasic ammonium phosphate TS (7 : 3). Flow rate: Adjust the flow rate so that the retention time of Chlordiazepoxide is about 5 minutes. System suirability System performance: When the procedure is run

190 Monographs, Part I

with 10 L of the standard solution under the above operating conditions, Chlordiazepoxide and internal standard are eluted in this order with the resolution between these peaks neing not less than 9. System repeatability: When the test is repeated 6 times, the relative standard deviation of the ratios of the peak area of Chlordiazepoxide to that of internal standard.
Packaging and Storage tight containers.

of the test solution and 10 L each of the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Proceed as directed in the Purity (2) under Chlordiazepoxide.
Dissolution Test Perform the test with 1 tablet of Chlordiazepoxide Tablets at 100 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of the Dissolution medium solution 2 as the dissolution solution. Take 30 mL or more of the dissolved solution after 60 minutes from the start of the test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipette the subsequent V mL, add the Dissolution medium solution 2 to make exactly V' mL so that each mL contains about 3.7 g of chlordiazepoxide (Cl6H14ClN3O) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately about 12 mg of Chlordiazepoxide RS, previously dried in a desiccator for 4 hours (in vacuum, P2O5, 60 C) and dissolve in the Dissolution medium solution 2 to make exactly 200 mL. Pipette 3 mL of this solution, add the Dissolution medium solution 2 to make exactly 50 mL and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectably, at 260 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Chlordiazepoxide Tablets in 60 minutes is not less than 70%.

Preserve in light-resistance,

Chlordiazepoxide Tablets
Chlordiazepoxide Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of chlordiazepoxide (Cl6H14ClN3O: 299.76).
Method of Preparation Prepare as directed under Tablets, with Chlordiazepoxide. Identification (1) Weigh a portion of powdered Chlordiazepoxide Tablets, equivalent to 10 mg of Chlordiazepoxide according to the labeled amount, add 100 mL of 0.1 mol/L hydrochloric acid TS, shake and filter. To 5 mL of the filtrate, add 0.1 mol/L hydrochloric acid TS to make 100 mL. Determine the absorption spectrum of this solution as directed under the Ultraviolet-visible absorption spectrophotometry: it exhibit the maximum absorption at the wavelength between 244 and 248 nm and the minimum absorption at the wavelength 288 and 292 nm. (2) Weigh a portion of powdered Chlordiazepoxide Tablets, equivalent to 10 mg of Chlordiazepoxide according to the labeled amount, add 10 mL of ether, shake vigorously and centrifuge. Evaporate 5 mL of the supernatant liquid by warming on a water-bath to dryness. Determine the infrared absorption spectrum of the residue as directed in the potassium bromide disk method under the Infrared Spectrophotometry: it exhibits absorption at the wave numbers of about 1625 cm-1, 1465 cm-1, 1265 cm-1, 850 cm-1 and 765 cm-1. Purity Related substancesPerform the test without exposure to daylight, using light-resistant vessels, To a portion of powdered Chlordiazepoxide Tablets, equivalent to 50 mg of Chlordiazepoxide according to the labeled amount, add exactly 5 mL of a mixture of methanol and ammonia TS (97 : 3), shake, centrifuge and use the supernatant liquid as the test solution. Separately, dissolve 50 mg of Chlordiazepoxide RS in a mixture of methanol and ammonia TS (97 : 3) to make exactly 50 mL and use this solution as the standard solution (1). Dissolve 5.0 mg of 2-amino-5-chlorobenzophenone RS in methanol to make exactly 200 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 25 L

Dissolution rate (%) with respect to the labeled of chlordiazepoxide (Cl6H14ClN3O) = W S AT V ' 1 27
AS V C

WS : Amount (mg) of Chlordiazepoxide RS.

C: Labeled amount (mg) of chlordiazepoxide (Cl6H14ClN3O) in 1 tablet. Uniformity of Dosage Units It meets the requirement. Assay Perform the test without exposure to daylight, using light-resistant vessels. Weigh accurately a portion of powdered Chlordiazepoxide Tablets, equivalent to about 0.1 g of chlordiazepoxide (Cl6H14ClN3O), transfer to a glass-stoppered flask, add 10 mL of water and shake well to disintegrate. Add 60 mL of methanol, shake well, add methanol to make exactly 100 mL and centrifuge. Pipet 10 mL of the supernatant liquid, add exactly 5 mL of the internal standard solution, add methanol to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Chloroiazepoxide RS, previously dried in a desiccator (in vacuum, P2O5, 60 C) for 4 hours, dissolve in 1 mL of water and methanol, add exactly 5 mL of the internal standard solution, add methanol to make exactly 100 mL and use this solution as the standard solution. and proceed as directed in the assay under Chlordiazepoxide Powder.

KP 9 191

Amount (mg) of chlordiazepoxide (Cl6H14ClN3O) = amount (mg) of Chlordiazepoxide RS

QT 10 QS

Internal standard solutionA solution of isobutyl salicylate in methanol (1 in 20). Packaging and Storage tight containers.

Preserve in light-resistant,

Chlordiazepoxide Hydrochloride
NHCH3 N

Cl

HCl

C16H14ClN3OHCl: 336.22 Chlordiazepoxide Hydrochloride contains not less than 98.0% and not more than 102.0% of chlordiazepoxide hydrochloride (C16H14ClN3OHCl), calculated on the dried basis.
Description Chlordiazepoxide Hydrochloride is a white crystalline powder and is odorless. Chlordiazepoxide Hydrochloride is soluble in water or in ethanol and sparingly soluble in hexane. Chlordiazepoxide Hydrochloride is affected by light. Identification (1) To about 20 mg of Chlordiazepoxide Hydrochloride, add 5 mL of hydrochloric acid and 10 mL of water and heat to boiling to hydrolyze. To the cooled solution, add 2 mL of sodium nitrite solution (1 in 1000), 1 mL of ammonium sulfamate solution (1 in 200) and 1 mL of N-1-naphthylethylenediamine dihydrochloride solution (1 in 1000): a reddish violet color is observed. (2) The relative retention time of the major peak in the chromatogram of the test solution corresponds to that of the Standard solution obtained as directed in the Assay. (3) Determine the infrared spectra of Chlordiazepoxide Hydrochloride and Chlordiazepoxide Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 212 C and 218 C (with decomposition). Purity

Chlordiazepoxide Hydrochloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesTransfer about 50 mg of Chlordiazepoxide Hydrochloride to a volumetric flask and dissolve in 2.5 mL of acetone by shaking. Allow to stand until precipitate and use the supernatant as the test solution. Separately, weigh a portion of 7-Chloro-1,3dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one-4-oxide RS, dissolve in acetone to make the concentration of 100 g per mL and use this solution as the standard solution (1). Weigh a portion of 2-Amino-5-chlorobenzophenone RS, dissolve in acetone to make the solution containing 10 g per mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thinlayer Chromatography. Spot 50 L of the test solution and 10 L each of the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate, previously not equilibrated with the developer, with ethyl acetate until the solvent front has moved about three-fourths of the length of the plate, air-dry the plate. Spray evenly 1 mol/L sulfuric acid in the plate, dry the plate at 105 C for 15 minutes and then spray sodium nitrite (1 in 1000), ammonium sulfamate (1 in 200) and N-(1-naphthyl)ethylenediamine hydrochloric acid (1 in 1000) sequentially. The spots, equivalent to those from the standard solutions, from the test solution are not more intense than the spots from the standard solutions (1) and (2).
Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, 60 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Perform the test under the conditions protected from light. Transfer about 0.1 g of Chlordiazepoxide Hydrochloride, accurately weighed, to a volumetric flask, dissolve in mobile phase by sonicating for minutes, dilute with mobile phase to make 50 mL and mix. Transfer 10 mL of the resulting solution, dilute with mobile phase to make 100 mL, mix and use this solution as the test solution. Separately, weigh a portion of Chlordiazepoxide Hydrochloride RS and accurately dissolve in mobile phase to make the concentration of 1 mg per mL. Pipet 10 mL of this solution, add mobile phase to make exactly 100 mL and use this solution as a standard solution. Perform the test with 5 L each of the test solution and the standard solution and calculate the peak areas, AT and

AS , the test solution and the standard solution respectively.

Amount (mg) of Chlordiazepoxide Hydrochloride (C16H14ClN3OHCl) = 0.5 C
AT AS

(1) Heavy metalsProceed with 1.0 g of

192 Monographs, Part I C: Concentration of Chlordiazepoxide Hydrochloride RS solution (g/mL). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of water and methanol (60 : 40). Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, the theoretical plates are not less than 3600, and the symmetry factor is no more than 2.0 System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of chlordiazepoxide hydrochloride is not more than 2.0 %. Packaging and Storage tight containers.

(1 in 20000).
Dissolution Test Perform the test with 1 capsule of Chlordiazepoxide Hydrochloride Capsules at 100 revolutions per minute according to Method 1 under the Dissolution Test, using 900 mL of water as a dissolution solution. Take the dissolved solution after 30 minutes from the beginning of the test and filter through a membrane filter with pore size of not more than 0.8 m. Pipet V mL of the filtrate accurately, add water to make the concentration of about 6 g per mL with the volumes of V mL and use this solution as the test solution. Separately, weigh accurately 30 mg of Chlordiazepoxide Hydrochloride RS and dissolve in water to make exactly 100 mL. Pipet 2 mL of this solution, add water to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 245 nm, using water as the blank, as directed under the Ultraviolet-visible Spectrophotometry. Remove the contents of 12 Chlordiazepoxide Hydrochloride Capsules as completely as possible with the aid of a current of air, determine the absorbance at the same dilution and in the same manner as for the Capsules and make any necessary modifications. The dissolution rate of Chlordiazepoxide Hydrochloride Capsules in 30 minutes should be not less than 85%.

Preserve in light-resistant,

Chlordiazepoxide Hydrochloride Capsules

Chlordiazepoxide Hydrochloride Capsules contain not less than 90.0% and not more than 110.0% of the labeled amount of chlordiazepoxide hydrochloride (C16H14ClN3OHCl: 336.22).
Method of Preparation Prepare as directed under Capsules, with Chlordiazepoxide Hydrochloride. Identification (1) The solution used for measurement of absorbance in the Assay exhibits maxima at 245 2 nm and 311 2 nm and the ratio, A245 / A311 , is between 2.90 and 3.45. (2) A portion of the contents of Chlordiazepoxide Hydrochloride Capsules responds to Identification (1) under Chlordiazepoxide Hydrochloride. Purity Related substancesAccurately weigh a portion of Chlordiazepoxide Hydrochloride Capsules, equivalent to about 25 mg of chlordiazepoxide hydrochloride (C16H14ClN3OHCl) and perform the test as directed in Purity (2) under Chlordiazepoxide Hydrochloride. Use 15 L of 7-chloro-1,3-dihydro-5-phenyl-2H1,4-benzodiazepin-2-one-4-oxide in acetone (1 in 1000) and 10 L of 2-amino-5-chlorobenzophenone in acetone

Dissolution rate (%) of the labeled quantity of chlordiazepoxide hydrochloride capsules (C16H14ClN3OHCl) = WS
AT V ' 90 1 AS V C 5

WS: Amount of Chlordiazepoxide Hydrochloride RS (mg), C: Labeled quantity of chlordiazepoxide hydrochloride (C16H14ClN3OHCl) (mg).
Uniformity of Dosage Units It meets the requirement when the content uniformity test is performed according to following method. Perform this procedure without exposure of daylight using light-resistant vessels. Transfer the contents of 1 capsule to a 200 mL volumetric flask, dissolve in and dilute with water to volume, and filter, discarding the first 20 mL of the filterate. Dilute a portion of the filtrate quantitatively and step-wise with 0.1N hydrochloric acid to obtain a solution having a concentration of about 6 g of Chlordiazepoxide Hydrochloride per mL. Dissolve a suitable quantity of Chlordiszeposide Hydrocholride RS, accurately weighed, in 0.1 N hydrochloric acid to obtain a standard solution having a known concentration of about 6 g per mL. Concomitantly determine the absorbances AT and AS of the test solution and the standard solution at the wavelength of maximum absorbance of about 245nm, with a suitable spectrophotometer, using 0.1 mol/L hydrochloric acid as the blank.

KP 9 193
ration for injection which is reconstituted before use and contains not less than 93.0% and not more than 107.0% of the labeled amount of chlordiazepoxide hydrochloride (C16H14ClN3OHCl: 336.22).
Method of Preparation Prepare as directed under Injections, with Chlordiazepoxide Hydrochloride. Description Chlordiazepoxide Hydrochloride for Injection is a white crystalline powder and is odorless. Chlordiazepoxide Hydrochloride for Injection is soluble in water or in ethanol and sparingly soluble in hexane. Chlordiazepoxide Hydrochloride for Injection is affected by light. Identification Perform the test, as directed under Chlordiazepoxide Hydrochloride. pH The pH of the solution containing about 1.0 g of Chlordiazepoxide Hydrochloride for Injection in 100 mL of water is between 2.5 and 3.5. Purity Heavy metals and related substances Perform the tests according to Methods (1) and (2), as directed under Chlordiazepoxide Hydrochloride. Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, 60 C, 4 hours). Sterility Test It meets the requirement. Bacterial Endotoxins Less than 3.57 EU per 1 mg of Chordiazepoxide Hydrochloride. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Calculate the quantity, in mg, of C16H14ClN3OHCl in the

A Capsule by the formula = T C T

AS

T : Labeled amount (mg) of Chlordiazepoxide Hydrochloride in the 1 Capsule. D : Concentration (g/mL) of Chlordiazepoxide Hydrochloride in the test solution, based on the labeled amount per Capsule and the extent of dilution C : Concentration (g/mL) of Chlordiazepoxide Hydrochloride RS in the standard solution
Assay Perform this procedure without exposure to daylight, using light-resistant vessels. Weigh accurately the contents of not less than about 20 Chlordiazepoxide Hydrochloride Capsules and determine the average weight per capsule. Mix the combined contents and transfer an accurately weighed portion of the powder, equivalent to about 60 mg of chlordiazepoxide hydrochloride (C16H14ClN3OHCl), to a volumetric flask. Add methanol to make 100 mL, mix and filter and discard the first 15 mL of the filtrate. Pipet 5 mL of the clear filtrate into a volumetric flask and add sulfuric acid in ethanol (1 in 360) to make 100 mL. Pipet 10 mL of this solution into a volumetric flask and dilute with sulfuric acid in ethanol (1 in 360) to make 50 mL and use this solution as the test solution. Dissolve an accurately a portion of Chlordiazepoxide Hydrochloride in methanol to obtain a solution having a known concentration of about 60 mg per mL. Dilute this solution quantitatively and step-wise with sulfuric acid in ethanol (1 in 360) to obtain a standard solution having a known concentration of about 6 g per mL. Concomitantly determine the absorbances of the test solution and the standard solution at the wavelength of maximum absorbance at about 245 nm, as directed under the Ultraviolet-visible Spectrophotometry, using sulfuric acid in ethanol (1 in 360) as the blank. Calculate the absorbances, AT and AS , for the test solution and the standard solution, respectively.

It

Uniformity of Dosage Units It meets the requirement. Assay Proceed as directed under Chlordiazepoxide Hydrochloride. Packaging and Storage hermetic containers.

Amount (mg) of chlordiazepoxide hydrochloride

A (C16H14ClN3OHCl) = 10 C T AS

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C: Concentration of Chlordiazepoxide Hydrochloride RS solution (g/mL).

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Chlorhexidine Gluconate Solution

Chlorhexidine Gluconate Solution is a solution of digluconate of chlorhexidine. Chlorhexidine Gluconate Solution contains not less than 19.0 w/v% and not more than 21.0 w/v% of chlorhexidine gluconate (C22H30Cl2N102C6H12O7: 897.76).

Chlordiazepoxide Hydrochloride for Injection

Chlordiazepoxide Hydrochloride for Injection is a prepa-

194 Monographs, Part I

to make exactly 100 mL. Pipet 5 mL of the solution, add 20 mL of water and 5 mL of 1 mol/L hydrochloric acid TS and proceed as directed for the preparation of the test solution.
Residue on Ignition Not more than 0.1% (2 g, after evaporation). Assay Pipet 2 mL of Chlorhexidine Gluconate Solution, evaporate to dryness on a water-bath, dissolve the residue in 60 mL of glacial acetic acid for nonaqueous titration and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Description Chlorhexidine Gluconate Solution is a clear, colorless or pale yellow liquid, is odorless and has a bitter taste. Chlorhexidine Gluconate Solution is miscible with glacial acetic acid or with water. One mL of Chlorhexidine Gluconate Solution is miscible with not more than 5 mL of dehydrated ethanol or not more than 3 mL of acetone. A white turbidity is produced by further addition of dehydrated ethanol or acetone. Chlorhexidine Gluconate Solution is gradually colored by light. 20 Specific gravity d 20 : Between 1.06 and 1.07. Identification (1) To 50 L of Chlorhexidine Gluconate Solution, add 5 mL of methanol, 1 mL of bromine TS and 1 mL of 8 mol/L sodium hydroxide TS: a deep red color is observed. (2) To 0.5 mL of Chlorhexidine Gluconate Solution, add 10 mL of water and 0.5 mL of cupric sulfate TS: a white precipitate is produced. Heat to boiling: the precipitate changes to pale purple. (3) To 10 mL of Chlorhexidine Gluconate Solution, add 5 mL of water, cool on ice and add 5 mL of sodium hydroxide TS dropwise with stirring: a white precipitate is produced. Collect the precipitate by filtration, wash with water, recrystalize from diluted ethanol (7 in 10) and dry at 105 C for 30 minutes: the crystals thus obtained melt between 130 C and 134 C. (4) Neutralize the filtrate obtained in (3) with 5 mol/L hydrochloric acid TS. To 5 mL of this solution, add 0.65 mL of glacial acetic acid and 1 mL of freshly distilled phenylhydrazine and heat in a water-bath for 30 minutes. After cooling, scratch the inner wall of the vessel with a glass rod to induce crystallization. Collect the crystals, dissolve in 10 mL of hot water, add a small amount of activated charcoal and filter. Cool the filtrate, scratch the inner side of the vessel, collect the formed crystals and dry: the crystals thus obtained melt at about 195 C (with decomposition). pH Add 5.0 mL of Chlorhexidine Gluconate Solution to water to make 100 mL: the pH of this solution is between 5.5 and 7.0. Purity p-ChlroanilineTo 2.0 mL of Chlorhexidine Gluconate Solution, add water to make exactly 100 mL. Pipet 5 mL of the solution and add 20 mL of water and 5 mL of 1 mol/L hydrochloric acid TS. Add 0.3 mL of sodium nitrite TS, shake and allow to stand for 2 minutes. Add 4 mL of ammonium sulfamate TS and then allow to stand for 1 minute. Add 5 mL of N-(1-naphthyl)-Ndiethylethylenediamine oxalate-acetone TS, allow to stand for 10 minutes, add 1 mL of ethanol and then add water to make 50 mL: the color of the solution is not more intense than the following control solution.

Each mL of 0.1 mol/L perchloric acid VS = 22.444 mg of C22H30Cl2N102C6H12O7

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Chlorhexidine Hydrochloride
NH Cl NH C NH NH C NH(CH2)6NH NH C NH NH C NH Cl 2HCl

C22H30Cl2N102HCl: 578.37 Chlorhexidine Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of chlorhexidine hydrochloride (C22H30Cl2N102HCl).
Description Chlorhexidine Hydrochloride is a white, crystalline powder, is odorless and has a bitter taste. Chlorhexidine Hydrochloride is soluble in formic acid, slightly soluble in methanol or in warm methanol and practically insoluble in water, in ethanol or in ether. Chlorhexidine Hydrochloride is gradually affected by light. Identification (1) Dissolve 10 mg of Chlorhexidine Hydrochloride in 5 mL of methanol by warming and add 1 mL of bromine TS and 1 mL of 8 mol/L sodium hydroxide TS: a deep red color is observed. (2) Dissolve 0.3 g of Chlorhexidine Hydrochloride in 10 mL of 6 mol/L hydrochloric acid TS, cool in ice and add 10 mL of 8 mol/L sodium hydroxide TS drop-wise with stirring: a white precipitate is produced. Collect the precipitate, wash with water, recrystallize from diluted ethanol (7 in 10) and dry at 105 C for 30 minutes: the crystals so obtained melt between 130 C and 134 C. (3) Dissolve 0.1 g of Chlorhexidine Hydrochloride in 50 mL of dilute nitric acid: the solution responds to the Qualitative Tests for chloride. Purity

Control solutionDissolve 20 mg of p-chloroaniline in 10 mL of 1 mol/L hydrochloric acid TS and add water

(1) Heavy metalsProceed with 2.0 g of

KP 9 195
Chlorhexidine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicTo 1.0 g of Chlorhexidine Hydrochloride in a crucible, add 10 mL of a solution of magnesium nitrate in ethanol (1 in 10), fire the ethanol to burn and heat gradually to incinerate. If a carbonized substance remains, moisten with a small volume of nitric acid and ignite to incinerate. Cool, add 10 mL of dilute hydrochloric acid to the residue, dissolve by warming in a waterbath, use this solution as the test solution and perform the test (not more than 2 ppm). (3) p-ChloroanilineDissolve 0.10 g of Chlorhexidine Hydrochloride in 2 mL of formic acid and add 15 mL of 1 mol/L hydrochloric acid TS and 20 mL of water immediately. Add 0.3 mL of sodium nitrite TS, shake and allow to stand for 2 minutes. Add 4 mL of ammonium sulfamate TS and then allow to stand for 1 minute. Add 5 mL of N-(1-naphthyl)-N-diethylethylene diamineoxalate-acetone TS, allow to stand for 10 minutes, add 1 mL of ethanol and then add water to make 50 mL: the color of the solution is not more intense than the following control solution.

Chlormadinone Acetate
CH3 O H3C O H3C H CO OCCH3

H O H Cl

C23H29C1O4: 404.93 Chlormadinone Acetate, when dried, contains not less than 98.0% and not more than 101.0% of chlormadinone acetate (C23H29C1O4).
Description Chlormadinone Acetate is a white to pale yellow crystal or crystalline powder and is odorless. Chlormadinone Acetate is freely soluble in chloroform, soluble in acetonitrile, slightly soluble in ethanol or in ether and practically insoluble in water. Identification (1) Dissolve 2 mg of Chlormadinone Acetate in 1 mL of ethanol and add 1 mL of mdinitrobenzene TS and 1 mL of a solution of potassium hydroxide (1 in 5): a red-purple color develops. (2) To 50 mg of Chlormadinone Acetate, add 2 mL of potassium hydroxide-ethanol TS and boil in a water-bath for 5 minutes. After cooling, add 2 mL of diluted sulfuric acid (2 in 7) and boil gently for 1 minute: the odor of ethylacetate is perceptible. (3) Determine the infrared spectra of Chlormadinone Acetate and Chlormadinone Acetate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) Perform the test with Chlormadinone Acetate as directed under the Flame Coloration Test (2): a green color appears. Specific Optical Rotation [ ]20 D : Between -10.0 C and -14.0 C (after drying, 0.2 g, acetonitrile, 10 mL, 100 mm). Melting Point between 211 C and 215 C. Purity (1) Heavy metalsProceed with 1.0 g of Chlormadinone Acetate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Chlormadinone Acetate according to Method 3 and perform the test (not more than 2 ppm).

Control solutionDissolve 20 mg of p-chloroaniline in 10 mL of 1 mol/L hydrochloric acid TS and add water to make exactly 100 mL. Pipet 5 mL of the solution and add water to make exactly 100 mL. To 2.0 mL of this solution, add 2 mL of formic acid, 15 mL of 1 mol/L hydrochloric acid TS and 20 mL of water and proceed in the same manner.
Loss on Drying Not more than 2.0% (1 g, 130 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.2 g of Chlorhexidine Hydrochloride, previously dried, dissolve in 2.0 mL of formic acid, add 60 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 14.459 mg of C22H30Cl2N102HCl

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196 Monographs, Part I

(3) Related substancesDissolve 20 mg of Chlormadinone Acetate in 10 mL of acetonitrile and use this solution as the test solution. Pipet 1 mL of the test solution, add acetonitrile to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of these solutions by the automatic integration method: the total area of peaks other than the peak of Chlormadinone Acetate from the test solution is not larger than the peak area of Chlormadinone Acetate from the standard solution. to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Ultraviolet-visible Spectrophotometry and determine the absorbance, AT and AS , at 285 nm, for the test solution and the standard solution, respectively. Amount (mg) of chlormadinone acetate (C23H29ClO4) = amount (mg) of Chlormadinone Acetate RS
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AT AS

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Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 236 nm). Column: A stainless steel column, about 6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of acetonitrile and water (13 : 7). Flow rate: Adjust the flow rate so that the retention time of Chlormadinone Acetate is about 10 minutes. System suitability Test for required detection: Pipet exactly 5 mL of the standard solution, add acetonitrile to make exactly 50 mL. The peak area of Chlormadinone Acetate obtained from 10 L of this solution is between 7 and 13% of the peak area of Chlormadinone Acetate obtained from 10 L of the standard solution. System performance: Dissolve 8 mg of Chlormadinone Acetate and 2 mg of butyl parahydroxybenzoate in 100 mL of acetonitrile. When the procedure is run with 10 L of this solution under the above operating conditions, butyl parahydroxybenzoate and chlormadinone acetate are eluted in this order, with the resolution between their peaks being not less than 8.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution according to the above conditions: the relative standard deviation of the peak areas of chlormadinone acetate is not more than 1.0%. Time span of measurement: About 1.5 times as long as the retention time of chlormadinone acetate after the solvent peak.
Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 4 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately about 20 mg each of Chlormadinone Acetate and Chlormadinone Acetate RS, previously dried, and dissolve in ethanol to make exactly 100 mL. Pipet 5 mL each of these solutions, add ethanol

Chlorothiazide
O NH2SO2 S NH O

Cl

C7H6ClN3O4S2: 295.72 Chlorothiazide contains not less than 98.0% and not more than 102.0% of chlorothiazide (C7H6ClN3O4S2) calculated on the dried basis.
Description Chlorothiazide is a white crystalline powder and is odorless. Chlorothiazide is very slightly soluble in water, freely soluble in dimethyl formamide or in dimethyl sulfoxide, slightly soluble in methanol or in pyridine, and practically insoluble in ether, in benzene or in chloroform. Melting point About 340 C (with decomposition) Identification (1) Determine the absorption spectra of solutions of Chlorothiazide and Chlorothiazide RS in sodium hydroxide solution (1 in 250) (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit the similar intensities of absorption at the same wavelength and the difference of absorption is not less than 3.0% when calculated on the dried basis at the maximum absorption wavelength of about 292 nm. (2) Determine the infrared absorption spectra of Chlorothiazide and Chlorothiazide RS as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) ChlorideDissolve 1.0 g of Chlorothiazide in a mixture of 10 mL of water and 10 mL of sodium hydroxide (1 in 10) solution. Cool in an ice-bath and add 20 mL of water and 5 mL of nitric acid: a flocculent, white precipitate is formed. Titrate with 0.05 mol/L of silver nitrate (potentiometric titration, Endpoint Detection Me-

KP 9 197
thod in Titrimetry), using a silver-silver chloride electrode system: not more than 0.28 mL is required (not more than 0.05%). (2) Heavy metalsProceed with 1.0 g of Chlorothiazide according to Method 2 and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (3) SeleniumUsing a solution of 0.20 g of Chlorothiazide in 25 mL of dilute nitric acid (1 in 30) in 1000 mL of oxygen flask as the absorbing liquid, proceed as directed under Oxygen Flask Combustion Method. After combustion, wash inside of the A, B and C with 10 mL of water, transfer the solution of the oxygen flask to the 150 mL of beaker using 20 mL of water, heat slowly until boiling and then boil for 10 minutes, cool to the room temperature and use this solution as the test solution. Separately, take 6 mL exactly of the selenium standard solution in 25 mL of dilute nitric acid (1 to 30) and 25 mL of water and use this solution as the standard solution. After adjusting pH to 2.0 0.2 by adding ammonia solution (1 to 2) to each of the test solution and the standard solutions, dilute each solution with exactly 60 mL of water, transfer to the each separatory funnel using 10.0 mL of water and wash each separatory funnel with 10.0 mL of water. After dissolving 0.2 g of hydroxylamine in this solution followed by addition of 5.0 mL of 2,3-diaminonaphtalene TS with stirring and closing with stopper and stand for 100 minutes at room temperature. After adding 5.0 mL of cyclohexane, shaking vigorously for 2 minutes, standing, separating two layers, discard aqueous layer and take cyclohexane layer, after water of the cyclohexane extract is removed on centrifuge. Perform the test as directed under the Ultraviolet-visible Spectrophotometry using the solution obtained according to the same method by adding 25 mL of water to 25 mL of dilute nitric acid as the reference solution. Absorbance of the test solution when measured at near 380 nm at absorbance maximum wavelength is not larger than that obtained from the standard solution (not more than 0.003%). (4) 4-Amino-6-chloro-1,3-benzenedisulfonamide Weigh accurately a portion of 4-amino-6-chloro-1,3benzenedisulfonamide RS dissolve in mobile phase, to make each mL of mobile phase containing 1.5 g of 4amino-6-chloro-1,3-benzene-disulfonamide and use this solution as the standard solution. Perform the test with 20 L each of the test solution obtained from the Assay and the standard solution as directed under the Liquid Chromatography according to the operating conditions under the Assay: the amount of 4-amino-6-chloro-1,3benzene disulfonamide is not more than 1.0% when main peak areas, AT and AS of 4-amino-6-chloro-1,3benzenedisulfonamide of the test solution and the standard solution, respectively, are measured. Amount (mg) of 4-amino-6-chloro-1,3A benzenedisulfonamide = 0 .2 C T AS

C: Concentration (g/mL) of 4-Amino-6-chloro-1,3benzenedisulfonamide RS in standard solution.

Loss on Drying Not more than 1.0% (1 g, 105 C, 1 hour). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 30 mg of Chlorothiazide in 200 mL of volumetric flask, put a volume of acetonitrile, not more than 10% of the total volume, add the mobile phase to make exactly 200 mL and use this solution as the test solution. Separately, weigh a portion of Chlorothiazide RS and 4-Amino-6-chloro-1,3-benzenedisul fonamide RS accurately, add the mobile phase to make each mL contains 0.15 mg of Chlorothiazide and 1.5 g of 4-amino-6-chloro-1,3-benzenedisulfonamide and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine the peak areas, AT and AS, of the test solution and the standard solution, respectably.

Amount (mg) of Chlorothiazide (C7H6ClN3O4S2) = 200 C

AT AS

C: Concentration (mg/mL) of Chlorothiazide RS in the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadesyl silanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of 0.1 mol/L sodium hydrogen phosphate and acetonitrile (9 : 1) whose pH is adjusted to 3.0 0.1 by adding phosphoric acid. Flow rate: 1.2 mL/min. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, 4-amino-6-chloro-1,3-benzenedisul fonamide and chlorothiazide are eluted in this order with the resolution of these peaks being not less than 3.5. System repeatability: When the test is repeated 6 times with 20 L of the standard solution: the relative standard deviation of the peak areas of chlorothiazide is not more than 1.5%.
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198 Monographs, Part I

lution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine the peak areas, AT and AS , of the test solution and the standard solution, respectively. Amount (mg) of chlorothiazide (C7H6ClN3O4S2) = Amount (mg) of Chlorothiazide RS
AT 10 AS

Chlorothiazide Tablets
Chlorothiazide Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of chlorothiazide (C7H6ClN3O4S2: 295.72).
Method of Preparation Prepare as directed under Tablets, with Chlorothiazide. Identification (1) Powder 1 tablet of Chlorthiazide Tablets and fuse with a pellet of sodium hydroxide: the ammonia fumes produced turn moistened red litmus paper blue and the residue responds to the Qualitative Test for sulfite. (2) The retention time of the main peak obtained from the test solution as directed in the Assay corresponds to that from the standard solution. Dissolution Test Perform the test with 1 tablet of Chlorothiazide Tablets at 75 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of 0.05 mol/L phosphate buffer solution, pH 8.0, as a dissolution solution prepared from 50 mL of 0.2 mol/L potassium dihydrogen phosphate buffer solution, 42.3 mL of 0.2 mol/L sodium hydroxide and 200 mL of water. Take a volume of dissolved solution after 60 minutes from the start of the test, filter, dilute with a volume of dissolution solution, if necessary and use this solution as the test solution. Separately, weigh accurately a portion of Chlorothiazide RS, previously dried at 105 C for 1 hour, dissolve in the dissolution solution to make same concentration as the test solution and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 294 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Chlorothiazide Tablets in 60 minutes is not less than 75%. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Chlorothiazide Tablets. Weigh accurately a quantity of powder, equivalent to about 0.25 g of chlorothiazide (C7H6ClN3O4S2) in a volumetric flask, add 50 mL of 0.05 mol/L sodium dihydrogen phosphate and sonicate for about 2 minutes. Add 100 mL of acetonitrile and sonicate about for 3 minutes, dilute with water to make exactly 500 mL, mix, filter and use this solution as the test solution. Seperately, weigh accurately 25 mg of Chlorothiazide RS, previously dried at 105 C for 1 hour, add 5 mL of 0.05 mol/L sodium dihydrogen phosphate, followed by 20 mL of acetonitrile, sonicate with occasional shaking often about for 3 minutes. Add water to make exactly 50 mL, mix, filter and use this solution as the standard solution. Perform the test with 15 L each of the test so-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, packed with octadesylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of 0.08 mol/L sodium dihydrogen phosphate and methanol (95 : 5) whose pH is adjusted to 2.9 0.1 by adding phosphoric acid. Flow rate: 2 mL/minute. System suitability System performance: When the procedure is run with 15 L of the standard solution under the above operating conditions, the symmetry factor is not more than 2.0. System repeatability: When the test is repeated 6 times with 15 L of the standard solution: the relative standard deviation of the peak areas of chlorothiazide is not more than 2.0%.
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Chlorphenesin Carbamate
OH Cl OCH2CHCH2O O C NH2

Cl0H12ClNO4: 245.66 Chlorphenesin Carbamate, when dried, contains not less than 98.0% and not more than 102.0% of chlorphenesin carbamate(Cl0H12ClNO4 ).
Description Chlorphenesin Carbamate is a white crystal or crystalline powder, is odorless and has a slightly bitter taste. Chlorphenesin Carbamate is freely soluble in methanol, in ethanol or in pyridine, soluble in isopropanol, sparingly soluble in ether, slightly soluble in water and practically insoluble in hexane. A solution of Chlorphenesin Carbamate in ethanol (1 in 20) shows no optical rotation.

KP 9 199
Identification (1) Determine the absorption spectra of solutions of Chlorphenesin Carbamate and Chlorphenesin Carbamate RS in ethanol (3 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Chlorphenesin Carbamate and Chlorphenesin Carbamate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Perform the test with Chlorphenesin Carbamate as directed under the Flame Coloration Test (2): a green color appears. Melting Point

Between 88 C and 91 C .

Purity (1) Heavy metalsDissolve 2.0 g of Chlorphenesin Carbamate in 20 mL of ethanol and add 2 mL of dilute acetic acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 2.0 mL of standard lead solution, add 20 mL of ethanol, 2 mL of dilute acetic acid and water to make 50 mL (not more than 10 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Chlorphenesin Carbamate according to Method 3 and perform the test (not more than 2 ppm). (3) Related substances(i) Chlorphenesin-2carbamate: Dissolve 0.10 g of Chlorphenesin Carbamate in 20 mL of a mixture of n-hexane for liquid chromatography and isopropanol(7 : 3) and use this solution as the test solution. Perform the test with 10 L of the test solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak area, Aa , of Chlorphenesin Carbamate and the peak area,

Ab , of chlorphenesin-2-carbamate by the automatic integration method: the ratio, Ab /(Aa + Ab ) , is not larger
than 0.007.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with silica gel for liquid chromatography (5 m in particle diameter). Column temperature: At constant temperature.of about 40 C. Mobile phase: a mixture of n-hexane for liquid chromatography, isopropanol and glacial acetic acid (700 : 300 : 1) Flow rate: Adjust the flow rate so that the retention time of Chlorphenesin Carbamate is about 9 minutes. System suitability Test for required detection: Pipet 1 mL of the test solution, add a mixture of hexane for liquid chromatography and isopropanol (7 : 3) to make 100 mL, and use

this solution as the test solution in the system suitability. The peak area of chlorphenesin carbamate obtained from 10 L of this solution is between 40 and 60% of the peak area of chlorphenesin carbamate obtained from the test solution of the system suitability. System performance: Dissolve 0.1 g of Chlorphenesin Carbamate in methanol to make 50 mL. To 25 mL of this solution, add 25 mL of dilute sodium hydroxide TS and warm at 60 C for 20 minutes. To 20 mL of this solution, add 5 mL of 1 mol/L hydrochloric acid TS, shake well with 20 mL of ethylacetate, allow to stand to separate the ethyl acetate layer and take the ethyl acetate layer. When the procedure is run with 10 L of this solution under the above operating conditions, chlorphenesin, chlorphenesin carbamate and chlorphenesin-2- carbamate are eluted in this order, with the ratios of the retention time of chlorphenesin and chlorphenesin-2carbamate to chlorphenesin carbamate being about 0.7 and about 1.2, respectively and with the resolution between the peaks of chlorphenesin and Chlorphenesin Carbamate being not less than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the test solution in the system suitability under the above operation conditions, the relative standard deviation of the peak area of chlorphenesin carbamate is not more than 2.0%. (ii) Other related substances: Dissolve 0.10 g of Chlorphenesin Carbamate in 10 mL of ethanol and use this solution as the test solution. Pipet 1 mL of the test solution and add ethanol to make exactly 20 mL. Pipet 2 mL of this solution, add ethanol to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 50 L each of the test solution and the standard solution on a plate of silica gel for liquid chromatography. Develop the plate with a mixture of ethyl acetate, methanol and strong ammonia water (17 : 2 : 1) to a distance of about 10 cm and air-dry the plate. Allow to stand in iodine vapor for 20 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.2% (1 g, in vacuum, silica gel, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Chlorphenesin Carbamate, previously dried, dissolve in 20 mL of pyridine, add exactly 50 mL of 0.1 mol/L potassium hydroxide-ethanol VS and warm at 70 C for 40 minutes. After cooling, add 100 mL of ethanol and titrate the excess potassium hdroxide with 0.1 mol/L hydrochloric acid VS until the color of the solution changes from blue through blue-green to yellow (indicator: 1 mL of thymol blue TS). Perform a blank determination and make any necessary correction.

200 Monographs, Part I

Each mL of 0.1 mol/L potassium hydroxide-ethanol VS = 24.566 mg of Cl0H12ClNO4
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1% Absorbance E1 cm (265 nm): Between 210 and 220 (after drying, 5 mg, 0.25 mol/L sulfuric acid TS, 250 mL).

Melting Point Between 130 C and 135 C.

Chlorpheniramine Maleate
N CH2CH2N(CH3)2 C H Cl C H CO2H H C CO2H

and enantiomer C16H19ClN2.C4H4O4: 390.86 Chlorpheniramine Maleate, when dried, contains not less than 98.0% and not more than 101.0% of dlchlorpheniramine maleate (C16H19ClN2C4H4O4).
Description Chlorpheniramine Maleate is a white, fine crystal, is odorless and has a bitter taste. Chlorpheniramine Maleate is very soluble in glacial acetic acid, freely soluble in water or in methanol, and sparingly soluble in ethanol. A solution of Chlorpheniramine Maleate shows no optical rotation. Identification (1) Determine the absorption spectra of solutions of Chlorpheniramine Maleate and Chlorpheniramine Maleate RS in 0.1 mol/L hydrochloric acid TS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Chlorpheniramine Maleate and Chlorpheniramine Maleate RS, previously dried, as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.. (3) Dissolve 0.1 g of Chlorpheniramine Maleate in 5 mL of methanol, and use this solution as the test solution. Separately, dissolve 56 mg of maleinic acid in 10 mL of methanol, and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot each 5 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ether, methanol, glacial acetic acid and water (70 : 20: 7 : 3) to a distance of about 12 cm and air-dry the plates. And examine the plate under ultraviolet light (main wavelength: 254 nm): a spot among two of the spots obtained from the test solution shows the similar intense and same Rf value with the spot obtained from standard solution. pH Dissolve about 1.0 g of Chlorpheniramine Maleate in 100 mL of freshly boiled and cooled water: the pH of this solution is between 4.0 and 5.5.

Purity (1) Clarity and color of solution Dissolve 1.0 g of Chlorpheniramine Maleate in 50 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with1.0 g of Chlorpheniramine Maleate according to Method 4, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm).,. (3) Related substancesDissolve 0.10 g of Chlorpheniramine Maleate in 100 mL of the mobile phase, and use this solution as the test solution. Pipet exactly 3 mL of the test solution and add the mobile phase to make exactly 100 mL. Pipet exactly 2 mL of this solution, add the mobile phase again to make exactly 20 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions; and determine each peak area of both solutions according to the automatic integration method: the peak area other than the peak of Maleic acid and Chlorpheniramine obtained from the test solution is not larger than 2/3 times the peak area of Chlorpheniramine obtained from the standard solution. The total area of the peaks other than the peaks of maleic acid and chlorpheniramine obtained from the test solution is not larger than the peak area of chlorpheniramine obtained from the standard solution.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 225 nm). Column: A stainless steel column 3.9 mm in inside diameter and 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 8.57 g of ammonium dihydrogen phosphate and 1 mL of phosphoric acid in water to make 1000 mL. To 800 mL of this solution add 200 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of chlorpheniramine is about 11 minutes. System suitability Test for required detectability: To exactly 2.5 mL of the standard solution add the mobile phase to make exactly 25 mL. Confirm that the peak area of chlorpheniramine obtained with 20 L of this solution is equivalent to 7 to 13% of that with 20 L of the standard solution. System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the number o theoretical plates and

KP 9 201
the symmetry factor of the peak of chlorpheniramine are not less than 40000 and not more than 2.0, respectively. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of chlorpheniramine is not more than 4.0%. Time span of measurement: About 4 times as long as the retention time of chlorpheniramine after the solvent peak.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve about 0.4 g of Chlorpheniramine Maleate, previously dried and accurately weighed, in 20 mL of glacial acetic acid. Titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue-green to green (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

(2) Determine the infrared spectra of dChlorpheniramine Maleate and d-Chlorpheniramine Maleate RS, previously dried, as directed under Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. (3) Dissolve 0.1 g of d-Chlorpheniramine Maleate in 5 mL of methanol, and use this solution as the test solution. Separately, dissolve 56 mg of maleic acid in 10 mL of methanol, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 5 L each of the test solution and standard solution on a plate of silica gel for thin-layer chromatography. Deveop the plate with a mixture of ether, methanol, glacial acetic acid and water (70 : 20 : 7 : 3) to a distance of about 12 cm, and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm), a spot among two of the spots obtained from the test solution shows the same intense to the spot with the stasndard solution, and its R value is about 0.4.
Specific Optical Rotation [ ]20 D : Between +39.5 and +43.0 (after drying, 0.5 g, dimethylformamide, 10 mL, 100 mm). Melting Point Between 111 C and 115 C. pH Dissolve 1.0 g of d-Chlorpheniramine Maleate in 100 mL of freshly boiled and cooled water: the pH of this solution is between 4.0 and 5.0.
1% Absorbance E1 cm (265 nm): Between 210 and 220 (after drying, 5 mg, 0.25 mol/L sulfuric acid TS, 250 mL).

Each mL of 0.1 mol/L perchloric acid VS = 19.543 mg of C16H19ClN2C4H4O4

Packaging and Storage tight containers.

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d-Chlorpheniramine Maleate
N CH2CH2N(CH3)2 C H Cl C H CO2H H C CO2H

C16H19ClN2C4H4O4: 390.86 d-Chlorpheniramine Maleate, when dried, contains not less than 99.0% and not more than 101.0% of dchlorpheniramine maleate (C16H19ClN2C4H4O4).
Description d-Chlorpheniramine Maleate is a white, crystal powder, is odorless and has a bitter taste. d-Chlorpheniramine Maleate is very soluble in methanol or in glacial acetic acid, and freely soluble in imethylformamide or in ethanol. d-Chlorpheniramine Maleate is dissolves in dilute hydrochloric acid. Identification (1) Determine the absorption spectra of solutions of d-Chlorpheniramine Maleate and dChlorpheniramine Maleate RS in hydrochloric acid TS (3 in 100000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths

Purity (1) Clarity and color of solutionDissolve 1.0 g of d-Chlorpheniramine Maleate in 50 mL of water: the solution is colorless and clear. (2) Heavy metalsProceed 1.0 g of dChlorpheniramine Maleate according to Method 4, and perform the test.Prepare the control solution with 2.0 mL of standard lead solution (not mre than 20 ppm). (3) Related substancesDissolve 0.10 g of dChlorpheniramine Maleate in 100 mL of the mobile phase, and use this solution as the test solution. Pipet 3 mL of the test solution, and add the mobile phase to make exactly 100 mL. Pipet 2 mL of this solution, add the mobile phase to make exactly 20 mL, and use this solution as the standard solution. Perform the test with exactly 20 L each of the test solution and standard solution as directed under Liquid Chromatography according to the following conditions; and determine each peak area by the automatic integration method: the peak area other than maleic acid and chlorpheniramine is not larger than 2/3 times thepeak area of chlorpheniramine obtained from the standard solution, and the total area of the peaks other than maleic acid and chlorpheniramine is not larger than the peak area of chlorpheniramine with the standard solution.

202 Monographs, Part I

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 225 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 L in particle diameter). Column temperature: A constant temperature of about 25. Mobile phase: Dissolve 8.57 g of ammonium dihydrogen phosphate and 1 mL of phosphoric acid in water to make 1000 mL. To 800 mL of this solution add 200 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of chlorpheniramine is about 11 minutes. System suitability Test for required detectability: To exactly 2.5 mL of the standard solution add the mobile phase to make exactly 25 mL. Confirm that the peak area of chlorpheniramine obtained from 20 L of this solution is equivalent to 7 to 13% of that with 20 L of the standard solution. System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of chlorpheniramine are not less than 4000 and not more than 2.0, respectively. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of chlorpheniramine is not more than 4.0%. Time span of measurement: About 4 times as long as the retention time of chlorpheniramine after the solvent peak.
Loss on Drying Not more than 0.5% (1 g, 65 C, 4 hours). Residue on Ignition

Chlorpheniramine Maleate Injection

Chlorpheniramine Maleate Injection is an aqueous solution for injection. Chlorpheniramine Maleate Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of dl-chlorpheniramine maleate (C16H19ClN2 C4H4O4: 390.86).
Method of Preparation Prepare as directed under Injections, with Chlorpheniramine Maleate. Description Chlorpheniramine Maleate Injec-tion is a clear, colorless liquid. pHBetween 4.5 and 7.0. Identification Take a volume of Chlorpheniramine Maleate Injection, equivalent to 25 mg of Chlorpheniramine Maleate according to the labeled amount, add 5 mL of dilute sodium hydroxide TS, and extract with 20 mL of hexane. Wash the hexane layer with 10 mL of water, shake with 0.5 g of anhydrous sodium sulfate for several minutes, and filter. Evaporate the filtrate in a water bath at 50 under a reduced pressure, anddetermine the infrared absorption spectrum of the residue as directed in the liquid film method under Infrared Chromatography: it exhibits absorption at the wavenumbers of about 2940 cm-1, 2810 cm-1, 2770 cm-1, 1589 cm-1, 1491 cm-1, 1470 cm-1, 1434 cm-1, 1091 cm-1 and 1015-1 cm . Sterility Test It meets the requirement. Bacterial Endotoxins Less than 8.8 EU per each mg of Chlorpheniramine Maleate. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter for Injections the requirement.

Not more than 0.1% (1 g).

Assay Weigh accurately 0.3 g of d-Chlorpheniramine Maleate, previously dried, and dissolve in 20 mL of glacial acetic acid. Titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue-green to green (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

It meets

Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer an exactly measured volume of Chlorpheniramine Maleate Injection, equivalent to about 3 mg of chlorpheniramine maleate (C16H19ClN2.C4H4O4), to a separator, add 20 mL of water and 2 mL of sodium hydroxide TS and extract with two 50 mL volumes of ether. Combine the ether extracts, wash with 20 mL of water, and then extract with 20-mL, 20-mL and 5-mL portions of 0.25 mol/L sulfuric acid TS successively. Combine all acid extracts, and add 0.25 mol/L sulfuric acid TS to make exactly 200 mL. Pipet exactly 20 mL of this solution , transfer to a 100-mL separator, add 2 mL of sodium

Each mL of 0.1 mol/L perchloric acid VS = 19.543 mg of C16H19ClN2C4H4O4

Packaging and Storage tight containers.

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KP 9 203
hydroxide TS, and extract with two 50-mL portions of ether. Proceed in the same manner as for the preparation of the test solution, and use the solution as the standard solution. Determine the absorbances AT and AS of the test solution and standard solution at a wavelength of the maximum absorbance at about 265 nm as directed under Ultraviolet-visible Spectrophotometry. Amount (mg) of chlorpheniramine maleate (C16H19ClN2C4H4O4) = amount (mg) of Chlorpheniramine Maleate RS

AT 1 AS 10

nutes, then add the internal standard solution to make exactly 100 mL, and use this solution as the test solution. Separately, weigh accurately about 20 mg of Chlorpheniramine Maleate RS, previously dried at 105 for 3 hours, and add the internal standard to make exactly 100 mL. Pipet 20 mL of this solution, add the internal standard solution to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 30 L each of thetest solution and standard solution as directed under Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of chlorpheniramine to that of the internal standard. Amount (mg) of chlorpheniramine maleate (C16H19ClN2C4H4O4) = amount (mg) of chlorpheniramine maleate RS

QT 1 QS 5

Packaging and Storage hermetic containers.

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Chlorpheniramine Maleate Powder

Chlorpheniramine Maleate Powder contains not less than 93.0% and not more than 107.0% of the labeled amount of dl-chlorpheniramine maleate (C16H19ClN2C4H4O4: 390.86).
Method of Preparation Prepare as directed under Powders, with Chlorpheniramine Maleate. Identification Weigh a portion of Chlorpheniramine Maleate Powder, equivalent to 50 mg of Chlorpheniramine Maleate according to the labeled amount, shake with 40 mL of 0.1 mol/L hydrochloric acid TS, and filter. Transfer the filtrate to a separator, and wash 40 mL of hexane. Add 10 mL of sodium hydroxide TS, and extract with 20 mL of hexane. Wash the hexane layer with 5 mL of water. Centrifuge, if necessary, shake the hexane extract with 0.5 g of anhydrous sodium sulfate for several minutes, and filter. Evaporate the filtrate in a water bath under reduced pressure, and determine the spectrum of the residue as directed in the liquid film method under Infrared Spectrophotometry: it exhibits absorption at the wavenumber of about 2940 cm-1, 2810 cm-1, 2770 cm-1, 1589 cm-1, 1491 cm-1, 1470 cm-1, 1434 cm-1, 1091 cm-1 and 1015 cm-1. Particle Size Distribution Test for Preparations meets the requirement. Uniformity of Dosage Units (divided) quirement.

Internal standard solutionTo 7 mL of a solution of methyl parahydroxybenzoate in methanol (1 in 1000) add water to make 1000 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 265 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 1.0 g of sodium 1-heptane sulfonate in 900 mL of water, add 10 mL of glacial acetic acid and water to make 1000 mL. To 650 mL of this solution add 350 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of chlorpheniramine is about 8 minutes. System suitability System performance: When the procedure is run with 30 L of the standard solution under the above operating conditions, the internal standard and chlorpheniramine are eluted in this order with the resolution between these peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 30 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of chlorpheniramine to that of the internal standard is not more than 1.0%. Range of measurement: About 4 times as long as the retention time of chlorpheniramine after the solvent peak.
Packaging and Storage Preserve in tight containers.

It

It meets the re-

Assay Weigh accurately an amount of Chlorpheniramine Maleate Powder, equivalent to about 4 mg of chlorpheniramine maleate (C16H19ClN2.C4H4O4), add 70 mL of the internal standard solution, shake for 15 mi-

204 Monographs, Part I

= amount (mg) of Chlorpheniramine Maleate RS

Chlorpheniramine Maleate Tablets

Chlorpheniramine Maleate Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of dl-chlorpheniramine maleate (C16H19ClN2C4H4O4: 390.86).
Method of Preparation Prepare as directed under Tablets, with Chlorpheniramine Maleate. Identification Weigh a portion of powdered Chlorpheniramine Maleate Tablets, equivalent to 50 mg of Chlorpheniramine Maleate according to the labeled amount, shake with 40 mL of 0.1 mol/L hydrochloric acid TS and filter. Transfer the filtrate to a separator and wash with 40 mL of hexane. Add 10 mL of sodium hydroxide TS and extract with 20 mL of hexane. Wash the hexane layer with 5 mL of water. Centrifuge, if necessary, shake the hexane extract with 0.5 g of anhydrous sodium sulfate for several minutes and filter. Evapolate the filtrate in a water bath at about 50 under reduced pressure, and determine the spectrum of the residue as directed in the liquid film method under Infrared Spectrophotometry: it exhibits absorption at the wavenumber of about 2940 cm1 , 2810 cm-1 , 2770 cm-1 , 1589 cm-1 , 1491 cm-1, 1470 cm-1 , 1434 cm-1 , 1091 cm-1 and 1015 cm-1 . Disintegration Test It meets the requirement. Uniformity of Dosage Units Perform the test according to the following method: it meets the requirement of the Content uniformity test. To 1 tablet of Chlorpheniramine Maleate Tablets add 10 mL of water, shake to disintegrate the tablet, then add water to make exactly V mL of a solution containing about 80 g of chlorpheniramine maleate (C16H19ClN2 C4H4O4) per mL, and filter through a membrane filter with pore size of not more than 0.5 um. Pipet 5 mL of the filtrate, add exactly 2.5 mL of internal standard solution, add water to make 10 mL, and use this solution as the test solution. Separately, weigh accurately about 20 mg of Chlorpheniramine Maleate RS, previously dried at 105 for 3 hours, and add water to make exactly 100 mL. Pipet 20 mL of this solution, add exactly 25 mL of the internal standard solution, add water to make 100 mL, and use this solution as the standard solution. Perform the test with 30 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the conditions described in the Assay, and determine the ratios, QT and QS , of the peak area of chlorpheniramine to that of the internal standard.

QT V QS 250

Internal standard solutionTo 7 mL of a solution of methyl parahydroxybenzoate (1 in 250) add water to make 1000 mL.
Assay Weigh accurately and Weigh accurately the mass of not less than 20 Chlorpheniramine Maleate Tablets, and powder. Weigh accurately a portion of the powder, equivalent to about 4 mg of chlorpheniramine maleate (C16H19ClN2.C4H4O4), add 70 mL of the internal standard solution, shake for 15 minutes, then add the internal standard solution to make exactly 100 mL, filter through a membrane filter with pore size of not more than 0.5 m, and use this solution as thetest solution. Separately, weigh acculately about 20 mg of Chlorpheniramine Maleate RS, previously dried at 105 for 3 hours, and add the internal standard to make exactly 100 mL. Pipet 20 mL of this solution, add the internal standard solution to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 30 L each of the test solution and standard solution as directed under Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of chlorpheniramine to that of this internal standard.

Amount (mg) of chlorpheniramine maleate (C16H19ClN2.C4H4O4) = amount (mg) of Chlorpheniramine Maleate RS

QT 1 QS 5

Internal standard solutionTo 7 mL of a solution of methyl parahydroxybenzoate in methanol (1 in 1000) add water to make 1000 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 265 nm). Column: Astainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 1.0 g of sodium 1-heptane sulfonate in 900 mL of water, add 10 mL of glacial acetic acid and water to make 1000 mL. To 650 mL of this solution add 350 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of chlorpheniramine is about 8 minutes. System suitability System performance: When the procedure is run with 30 L of the standard solution under the above operating conditions, the internal standard and chlorpheni-

Amount (mg) of chlorpheniramine maleate (C16H19ClN2C4H4O4)

KP 9 205
ramine are eluted in this order with the resolution between these peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 30 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of chlorpheniramine to that of the internal standard is not more than 1.0%. Range of measurement: About 4 times as long as the retention time of chlorpheniramine after the solvent peak.
Packaging and Storage Preserve in tight containers..

within 10 minutes: the pH of this solution is between 4.0 and 5.0.

Purity (1) Clarity and color of solution A solution of 1.0 g of Chlorpromazine Hydrochloride in 20 mL of water, when observed within 10 minutes, is clear and colorless to pale yellow. (2) Heavy metalsProceed with 1.0 g of Chlorpromazine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours).

Chlorpromazine Hydrochloride
CH3 CH2CH2CH2N N CH3 Cl HCl

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.7 g of Chlorpromazine Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

C17H19ClN2SHCl: 355.33 Chlorpromazine Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of chlorpromazine hydrochloride (C17H19ClN2SHCl).
Description Chlorpromazine Hydrochloride is a white to pale yellow, crystalline powder, is odorless and has a faint, characteristic odor. Chlorpromazine Hydrochloride is very soluble in water, freely soluble in ethanol or in glacial acetic acid, sparingly soluble in acetic anhydride and practically insoluble in ether. Chlorpromazine Hydrochloride is gradually colored by light. Identification (1) To 5 mL of a solution of Chlorpromazine Hydrochloride (1 in 1000), add 1 drop of ferric chloride TS: a red color is observed. (2) Dissolve 0.1 g of Chlorpromazine Hydrochloride in 20 mL of water and 3 drops of dilute hydrochloric acid, add 10 mL of picric acid TS and allow to stand for 5 hours. Collect the resulting precipitate, wash with water, recrystallize from a small volume of acetone and dry at 105 C for 1 hour: the crystals so obtained melt between 175 C and 179 C. (3) Dissolve 0.5 g of Chlorpromazine Hydrochloride in 5 mL of water, add 2 mL of ammonia TS and heat in a water-bath for 5 minutes. Cool, filter and render the filtrate acidic with dilute nitric acid: the solution responds to the Qualitative Tests (2) for chloride. Melting Point Between 194 C and 198 C. pH Dissolve 1.0 g of Chlorpromazine Hydrochloride in 20 mL of freshly boiled and cooled water and measure

Each mL of 0.1 mol/L perchloric acid VS = 35.533 mg of C17H19ClN2S HCl

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Chlorpromazine Hydrochloride Injection

Chlorpromazine Hydrochloride Injection is an aqueous solution for injection and contains not less than 95.0% and not more than 105.0% of the labeled amount of chlorpromazine hydrochloride (C17H19ClN2SHCl: 355.33).
Method of Preparation Prepare as directed under Injections, with Chlorpromazine Hydrochloride. Description Chlorpromazine Hydrochloride Injection is a clear, colorless or pale yellow liquid. pHBetween 4.0 and 6.5. Identification (1) Proceed with a volume of Chlorpromazine Hydrochloride Injection, equivalent to 5 mg of Chlorpromazine Hydrochloride according to the labeled amount, as directed in the Identification (1) under Chlorpromazine Hydrochloride. (2) Proceed with a volume of Chlorpromazine Hydrochloride Injection, equivalent to 0.1 g of Chlorpromazine Hydrochloride according to the labeled amount, as directed in the Identification (2) under Chlorpromazine

206 Monographs, Part I

Hydrochloride.
Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injection meets the requirement.

It

Determination of Volume of Inhection in Containers It meets the requirement. Assay Transfer an exactly measured volume of Chlorpromazine Hydrochloride Injection, equivalent to about 0.15 g of chlorpromazine hydrochloride (C17H19ClN2S HCl), to a separator, add 30 mL of water and 10 mL of a solution of sodium hydroxide (1 in 5) and extract with two 30 mL volumes and three 20 mL volumes of ether. Wash the combined ether extracts with successive 10 mL volumes of water until the last washing shows no red color upon the addition of phenolphthalein TS. Concentrate the ether extracts on a water-bath to 20 mL, add 5 g of anhydrous sodium sulfate, allow to stand for 20 minutes and filter through a pledget of absorbent cotton. Wash with ether, combine the washings with the filtrate and evaporate the ether on a water-bath. Dissolve the residue in 50 mL of acetone and 5 mL of glacial acetic acid and titrate with 0.05 mol/L perchloric acid VS until the color of the solution changes from red-purple to bluepurple (indicator: 3 drops of bromocresol green methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

Filter the combined ether extracts through anhydrous sodium sulfate. With the aid of a stream of nitrogen, evaporate the ether to about 5 mL. Quantitatively transfer the solution to a centrifuge tube. Evaporate with a stream of nitrogen and mild heat to dryness. Dissolve the residue in 100 mL of methanol to obtain and use this solution as the test solution. Separately, dissolve a suitable amount of Chlorpromazine RS in methanol to have a concentration of about 0.2 mg per mL, and use this solution as the standard solution. Perform the test as with these solutions directed under the Thin-layer chromatography. Spot 15 L of the test solution and standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a freshly prepared mixture of ethyl acetate that has been saturated with ammonium hydroxide, ether and methanol (75 : 25 : 20) to a distance of about 15 cm. Remove the plate from the chamber, air-dry and spray with iodoplatinate reagent. The Rf value of the principal spot from the test solution corresponds to that obtained from the standard solution. (2) Dilute a volume of the Chlorpromazine Hydrochloride Syrup with an equal volume of water: the resulting solution responds to Qualitative Tests for chloride.

Iodoplatinate reagentThis reagent is prepared by dissolving 0.1 g of platinic chloride in 10 mL of 0.1 mol/L hydrochloric acid, adding 25 mL of potassium iodide solution (1 in 25), 0.5 mL of formic acid and diluting with water to make 100 mL.
Purity Chlorpromazine sulfoxide Transfer an accurately measured volume of the Syrup, equivalent to about 20 mg of chlorpromazine hydrochloride, to a 125 mL separator. Add 10 mL of water and 2 mL of sodirm hydroxide solution (1 in 2), and mix. Extract with three 30 mL portions of ether. Filter the combined ether extracts through anhydrous sodium sulfate. With the aid of stream of nitrogen evaporate the ether to about 5 mL. Quantitatively transfer the solution to a 40 mL centrifuge tube. Evaporate with a stream of nitrogen and mild heat to dryness. Dissolve the residue in 1.0 mL of methanol and use this solution as test solution. Transfer 5 mL of a solution in dilute hydrochloric acid (1 in 100) of Chlorpromazine Hydrochloride RS containing 10.6 mg per mL to a 50 mL volumetric flask. Add 2 mL of 30% hydrogen peroxide and heat at 60 C for 10 minutes. Cool, dilute with 1 mol/L sodium bisulfite solution to volume, and mix. Transfer 10.0 mL of this solution to a 60 mL separator, add 2 mL of sodium hydroxide solution (1 in 2), and mix. Extract with three 30 mL portions of ether. Filter the extracts through ether-wetted anhydrous sodium sulfate into a 250 mL conical flask. Cautiously evaporate the extracts to dryness. Dissolve the residue in 10.0 mL of methanol, and filter if necessary, and used this solution as Chlorpromazine Sulfoxide standard solution. Each mL of this solution contains 1 mg of chlorpromazine sulfoxide. Perform the test with these solutions as directed under the Thin-layer chromatography. Spot 15 L each of the

Each mL of 0.05 mol/L perchloric acid VS = 17.767 mg of C17H19ClN2SHCl

Packaging and Storage Preserve in light-resistant, hermetic containers or color containers.

Chlorpromazine Hydrochloride Syrup

Chlorpromazine Hydrochloride Syrup contains, in each 100 mL, not less than 0.19 g and not more than 0.21 g of chlorpromazine hydrochloride (C17H19ClN2SHCl: 355.33).
Method of Preparation Prepare as directed under Syrups, with Chlorpromazine Hydrochloride. Identification (1) Transfer a volume of Chlorpromazine Hydrochloride Syrup, equivalent to about 20 mg of Chlorpromazine Hydrochloride, to a separator. Add 10 mL of water and 2 mL of sodium hydroxide solution (1 in 2) and mix. Extract with three 30 mL volumes of ether.

KP 9 207
test solution and the Chlorpromazine sulfoxide standard solution to a plate prepared of silica gel for the thin-layer chromatography. Develop the plate with a freshly prepared mixture of ethyl acetate that has been saturated with ammonium hydroxide, ether, and methanol (75 : 25 : 20) until the solvent front has moved about threefourths of the length of the plate. Remove the plate from the chamber, air-dry, and spray with Iodoplatinate reagent. The chromatogram from the Test solution may exhibit a secondary spot whose Rf value corresponds to, and whose size and intensity are not greater than, those of the spot from the Clorpromazine sulfoxide standard solution (5.0%).

Chlorpromazine Hydrochloride Tablets

Chlorpromazine Hydrochloride Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of chlorpromazine hydrochloride (C17H19ClN2S HCl: 355.33).
Method of Preparation Prepare as directed under Tablets, with Chlorpromazine Hydrochloride. Identification (1) Shake a portion of powdered Chlorpromazine Hydrochloride Tablets, equivalent to 0.2 g of Chlorpromazine Hydrochloride according to the labeled amount, with 40 mL of 0.1 mol/L hydrochloric acid TS and filter. To 1 mL of the filtrate, add 4 mL of water and 1 drop of ferric chloride TS: a red color is observed. (2) To 20 mL of the filtrate obtained in (1), add 10 mL of picric acid TS drop-wise and proceed as directed in the Identification (2) under Chlorpromazine Hydrochloride. Dissolution Test Perform the test with 1 tablet of Chlorpromazine Hydrochloride Tablets at 75 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of the Dissolution medium solution 2 as a dissolution solution. Take 20 mL or more of the dissolved solution after 30 minutes from the start of the Dissolution Test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet the subsequent V mL, add the Dissolution medium solution 2 to make exactly V mL so that each mL of the filtrate contains about 5.6 g of chlorpromazine hydrochloride (C17H19ClN2SHCl) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately about 90 mg of Chlorpromazine Hydrochloride RS, previously dried at 105 C for 2 hours, dissolve the Dissolution medium solution 2 to make exactly 200 mL. Pipet 5 mL of this solution, add the Dissolution medium solution 2 to make exactly 100 mL, further pipet 5 mL of this solution, add the Dissolution mediun solution 2 to make exactly 20 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 254 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Chlorpromazine Hydrochloride Tablets in 30 minutes should be not less than 75%.

Iodoplatinate reagentThis reagent is prepared by dissolving 0.1 g of platinic chloride in 10 mL of 0.1 mol/L hydrochloric acid, adding 25 mL of potassium iodide solution (1 in 25), 0.5 mL of formic acid and diluting with water to make 100 mL.
Assay Transfer an accurately measured volume of Chlorpromazine Hydrochloride Syrup, equivalent to about 10 mg of chlorpromazine hydrochloride (C17H19ClN2SHCl), to a volumetric flask, add 0.1 mol/L hydrochloric acid to make 50 mL and mix. Transfer 10 mL of this solution to a separator, add about 20 mL of water and strong ammonia water to make alkaline and extract with four 25 mL volumes of ether. Extract the combined ether layers with 25 mL of 0.1 mol/L hydrochloric acid. Transfer the aqueous layers to a volumetric flask, evaporate ether, add 0.1 mol/L hydrochloric acid to make 250 mL, mix and use this solution as the test solution. Separately, weigh accurately a portion of Chlorpromazine Hydrochloride RS, previously dried at 105 C for 2 hours, dissolve in 0.1 mol/L hydrochloric acid TS to make a solution containing 8 g/mL and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solutions, respectively, at 254 nm and 277 nm, using 0.1 mol/L hydrochloric acid TS as the blank, as directed under the Ultravioletvisible Spectrophotometry.

Amount (mg) of chlorpromazine hydrochloride (C17H19ClN2SHCl) in each mL of the syrup = 1.25 C

( A254 -A277 ) T ( A254 -A277 ) S

C: Concentration of Chlorpromazine hydrochloride in the standard solution (g/mL), V: Volume of Chlorpromazine Hydrochloride Syrup taken (mL), ( A254-A277 )T : Absorbance of the test solution,

( A254-A277 )S : Absorbance of the standard solution.

Packaging and Storage well-closed containers.

Dissolution rate (%) with respect to the labeled amount of chlorpromazine hydrochloride (C17H19ClN2SHCl) = W S AT V ' 1 45
AS V C

Preserve in light-resistant,

WS : Amount (mg) of Chlorpromazine Hydrochloride

208 Monographs, Part I

RS. dium dihydrogen phosphate TS (1 in 2) and acetonitrile (27 : 13). Flow rate: Adjust the flow rate so that the retention time of Chlorpromazine hydrochloride is about 6.5 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the internal standard and chlorpromazine are eluted in this order with the resolution between these peaks the resolution being not less than 10. System repeatability : When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of chlorpromazine to that of the internal standard is not more than 1.0%.
Packaging and Storage tight containers.

C: Labeled amount (mg) of chlorpromazine hydrochloride (C17H19ClN2SHCl) in 1 tablet.

Uniformity of Dosage Units It meets the requirement. Assay Perform the procedure without exposure to daylight using the light-resistant vessels. Weigh accurately and powder not less than 20 Chlorpromazine Hydrochloride Tablets. Weigh accurataly a portion of the powder, equivalent to about 50 mg of chlorpromazine hydrochloride (C17H19ClN2SHCl), add 60 mL of a mixture of dilute phosphoric acid (1 in 500) and dehydrated ethanol, treat with sonicator for 5 minutes, add a mixture (1:1) of dilute phosphoric aicd (1 in500) and dehydrated ethanol after mixed with strongly shaking, add a mixture (1:1) of dilute phosphoric aicd (1 in500) and dehydrated ethanol to make exactly 100 mL, filtrate with membrain filter with a pore size not exceeding 0.45 m. Discard the first 3 mL of the filtrate, take the subsequent 2.5 mL, add exactly 5 mL of the internal standard solution, add a mixture (1:1) of dilute with phosphoric aicd (1 in500) and dehydrated ethanol to make exactly 25 mL, and use this solution as the test solution. Separately, weigh accurately about 25 mg of Chlorpromazine Hydrochloride RS, previously dried at 105 C for 2 hours, dilute with a mixture (1:1) of dilute phosphoric aicd (1 in500) and dehydrated ethanol to make exactly 100 mL. To exactly 5 mL of this solution, add exactly 5 mL of the internal standard solution, dilute with a mixture (1:1) of dilute phosphoric aicd (1 in500) to make exactly 25 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. And calculate the ratios, QT and QS , of the peak area of chlorpromazine hydrochloride to that of the internal standard, for the test solution and the standard solution, respectively.

Preserve in light-resistant,

Chlorpropamide
Cl SO 2NHCONHCH 2CH 2CH 3

C10Hl3ClN2O3S: 276.74 Chlorpropamide, when dried, contains not less than 98.0% and not more than 101.0% of chlorpropamide (C10Hl3ClN2O3S).
Description Chlorpropamide is a white crystal or crystalline powder. Chlorpropamide is freely soluble in methanol or in acetone, soluble in ethanol and slightly soluble in ether and practically insoluble in water. Identification (1) Dissolve 80 mg of Chlorpropamide and Chlorpropamide RS in 50 mL of methanol, then to 1 mL each of these solutions, add 0.01 mol/L hydrochloric acid TS to make 200 mL and determine the absorption spectrum of solutions as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Chlorpropamide and Chlorpropamide RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Perform the test with Chlorpropamide as directed under the Flame Coloration Test (2): a green color appears. Melting Point

Amount (mg) of chlorpromazine hydrochloride (C17H19ClN2SHCl) = amount (mg) of Chlorpromazine Hydrochloride RS QT

QS

Internal standard solutionA solution ethylparahydroxybenzoate in a mixture of diluted phosphoric acid (1 in 500) and dehydrated ethanol (1 : 1) (1 in 4500). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 15 to 30 cm in length, packed with cyano groups chemically bonded silica gel for liquid chromatography (5 m to10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of diluted 0.05 mol/L so-

Between 127 C and 131 C.

Purity (1) AcidTo 3.0 g Chlorpropamide, add 150

KP 9 209
mL of water and warm at 70 C for 5 minutes. Allow to stand in ice-water for 1 hour and filter. To 25 mL of the filtrate, add 2 drops of methyl red TS and 0.30 mL of 0.1 mol/L sodium hydroxide VS: a yellow color develops. (2) ChlorideTo 40 mL of the filtrate obtained in (1), add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.011%). (3) SulfateTo 40 mL of the filtrate obtained in (1), add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.021%). (4) Heavy metalsProceed with 2.0 g of Chlorpropamide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) Related substancesDissolve 0.60 g of Chlorpropamide in acetone to make exactly 10 mL and use this solution as the test solution. Pipet 1 mL of the test solution, add acetone to make exactly 300 mL and use this solution as the standard solution (1). Separately, dissolve 60 mg of p-chlorobenzene sulfonamide in acetone to make exactly 300 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution, the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of cyclohexane, isoamyl alcohol, methanol and strong ammonia water (15 : 10 : 5 : 1) to a distance of about 10 cm and air-dry the plate. After drying the plate at 100 C for 1 hour, spray evenly sodium hypochlorite TS on the plate and air-dry for 15 minutes. Then spray evenly potassium iodide-starch TS on the plate: the spot from the test solution equivalent to the spot from the standard solution (2) is not more intense than the spot from the standard solution (2) and the spots other than the spot mentioned above and other than the principal spot from the test solution is not more intense than the spot from the standard solution (1).
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.5 g of Chlorpropamide, previously dried, dissolve in 30 mL of neutralized ethanol and add 20 mL of water. Titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS).

Chlorpropamide Tablets
Chlorpropamide Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of chlorpropamide (C10Hl3ClN2O3S: 276.74).
Method of Preparation Prepare as directed under Tablets, with Chlorpropamide. Identification Take a quantity of powdered Chlorpropamide Tablets, equivalent to 80 mg of Chlorpropamide according to the labeled amount, add 50 mL of methanol, shake and filter. To 1 mL of the filtrate, add 0.01 mol/L hydrochloric acid TS to make 200 mL and determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 231 nm and 235 nm. Dissolution Test Perform the test with 1 tablet of Chlorpropamide Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of diluted phosphate buffer solution, pH 6.8, (1 in 2) as a dissolution solution. Take 20 mL or more of the dissolved solution 45 minutes after starting the test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet the subsequent V mL of the nitrate, add diluted phosphate buffer solution, pH 6.8, (1 in 2) to make exactly V' mL so that each mL contains about 10 g of chlorpropamide (C10Hl3ClN2O3S) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately about 50 mg of Chlorpropamide RS, previously dried at 105 C for 3 hours, dissolve in 10 mL of methanol and add water to make exactly 50 mL. Pipet 1 mL of this solution, add diluted phosphate buffer solution, pH 6.8, (1 in 2) to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at 232 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Chlorpropamide Tablets in 45 minutes should be not less than 70%.

Dissoution rate (%) with respect to the amount of chlorpropamide (C10Hl3ClN2O3S) = WS

AT V ' 1 18 AS V C

Each mL of 0.1 mol/L sodium hydroxide VS = 27.674 mg of C10Hl3ClN2O3S

Packaging and Storage Preserve in well-closed containers.

WS : Amount (mg) of Chlorpropamide RS. C: Labeled amount (mg) of chlorpropamide (C10Hl3ClN2O3S) in 1 tablet.
Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20

210 Monographs, Part I

Chlorpropamide Tablets. Weigh accurately a quantity of the powder, equivalent to about 50 mg of chlorpropamide (C10Hl3ClN2O3S), add 75 mL of the mobile phase, shake for 10 minutes and add the mobile phase to make exactly 100 mL. Centrifuge this solution, pipet 10 mL of the supernatant liquid, add the mobile phase to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Chlorpropamide RS, previously dried at 105 C for 3 hours and dissolve in the mobile phase to make exactly 100 mL. Pipet 10 mL of this solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine the peak areas, AT and AS , of Chlorpropamide of the test solution and the standard solution, respectively. Amount (mg) of chlorpropamide (C10Hl3ClN2O3S) = amount (mg) of Chlorpropamide RS
AT AS

Cl NH

C7H4ClNO2: 169.57 Chlorzoxazone contains not less than 98.0% and not more than 102.0% of chlorzoxazone (C7H4ClNO2), calculated on the dried basis.
Description Chlorzoxazone is white to pale yellow crystals or crystalline powder, is odorless, tasteless and a little irritant. Chlorzoxazone is freely soluble in dimethylformamide, soluble in methanol, in ethanol or in acetone, slightly soluble in ether and practically insoluble in water. Identification (1) Determine the absorption spectra of solutions of Chlorzoxazone and Chlorzoxazone RS in methanol (1 in 50000) as directed under the Ultravioletvisible Spectrophotometry: the maximum and minimum absorption occurs at the same wavelength. (2) Determine the infrared spectra of Chlorzoxazone and Chlorzoxazone RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 189 C and 194 C. Purity (1) Heavy metalsProceed with 1.0 g of Chlorzoxazone according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesWeigh accurately a portion of Chlorzoxazone, dissolve in methanol, so that each mL contains 20 mg of Chlorzoxazone and use this solution as the test solution. Separately, weigh accurately a portion of 2-Amino-4-chlorophenol RS, previously dried at 105 C for 2 hours and dissolve in methanol so that each mL contains 100 g and 50 g of Chlorzoxazone and use these solutions as the standard solutions (1) and (2). Perform the test with the test solution and the standard solutions and (1) and (2) as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of hexane and dioxane (63 : 37) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution (1) (not more than 0.5 %). Also allow to stand in iodine vapor: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution (2) (not more than 0.25 %). Loss on Drying Not more than 0.5 % (1 g, 105 C, 2 hours).

Operating conditions Detector:An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of diluted glacial acetic acid (1 in 100) and acetonitrile (1 : 1). Flow rate: Adjust the flow rate so that the retention time of chlorpropamide is about 5 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the numbers of theoretical plates are not less than 1500 plates, and the symmetry factor of the peak of chlorpropamide are not less than 1500 and not more than 1.5, respectively. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak areas of chlorpropamide is not more than 1.5%.
Packaging and Storage Preserve in well-closed containers.

Chlorzoxazone

KP 9 211
Uniformity of Dosage Units It meets the requirement. Residue on Ignition Not more than 0.15 % (1 g). Assay Weigh accurately 50 mg of Chlorzoxazone and Chlorzoxazone RS, previously dried at 105 C for 2 hours, dissolve in methanol and dilute with methanol to make exactly 100 mL, respectively. Transfer each 4.0 mL of these solutions to a volumetric flask and dilute with methanol to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with each of the test solution and the standard solution as directed under the Ultraviolet-visible Spectrophotometry using methanol as a blank and determine the absorbances at 282 nm, AT and AS , of the test solution and the standard solution, respectively. Assay Weigh accurately and powder not less than 20 Chlorzoxazone Tablets. Weigh accurately a portion of powder, equivalent to about 0.125 g of chlorzoxazone (C7H4ClNO2) in a volumetric flask, add about 70 mL of acetonitrile and shake vigorously for 30 minutes. Dilute with acetonitrile to make exactly 100 mL and filter. Discard the first 10 mL of the filtrates, transfer 5 mL of next filtrate to a volumetric flask containing 10.0 mL of internal standard solution, dilute with diluted glacial acetic acid (1 in 100) to make exactly 50 mL, mix and use this solution as the test solution. Separately, weigh accurately 0.125 g of Chlorzoxazone RS, previously dried at 105 C for 2 hours, add a volume of acetonitrile to make exactly 100 mL and use this solution as the standard stock solution. Transfer 5 mL of this solution to a volumetric flask containing 10.0 mL of internal standard solution, dilute with diluted glacial acetic acid (1 in 100) to make 50 mL, mix and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and calculate the ratios, Q T and QS of the peak area of chlorzoxazone to that of the internal standard, for the test solution and the standard solution, respectively.

Amount (mg) of chlorzoxazone (C7H4ClNO2) = Amount (mg) of Chlorzoxazone RS

AT AS

Packaging and Storage Preserve in tight container.

Chlorzoxazone Tablets
Chlorzoxazone Tablets contains not less than 90.0 % and not more than 110.0 % of the labeled amount of chlorzoxazone (C7H4ClNO2: 169.56).
Method of Preparation Prepare as directed under Tablets, with Chlorzoxazone. Identification (1) Determine the absorption spectra of solutions (2 in 100000) of Chlorzoxazone and Chlorzoxazone RS in methanol as directed under the Ultravioletvisible Spectrophotometry: the maximum and minimum absorption occurs at the same wavelength. (2) The retention time of the main peak obtained as directed in the Assay from the test solution corresponds to the standard solution. Dissolution Test Perform the test with 1 tablet of Chlorzoxazone Tablets at 75 revolutions per minute according to Method 2 under the Dissolution Test, using 1800 mL of phosphate buffer solution, pH 6.8 as a dissolution solution. Take a portion of dissolved solution after 60 minutes of the test, filter, discard the first 10 mL of the filtrates and use next filtrate as the test solution. Separately, weigh accurately a portion of Chlorzoxazone RS, previously dried at 105 C for 2 hours, dissolve in the dissolution solution to make the same concentration as that of the test solution and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under Assay. The dissolution rate of Chlorzoxazone Tablets in 60 minutes is not less than 75%.

Amount (mg) of chlorzoxazone (C7H4ClNO2) = amount (mg) of Chlorzoxazone RS

QT QS

Internal standard solutionDilute 0.125 g of phenacetin with acetonitrile to make 100 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of water, acetonitrile and glacial acetic acid (70 : 30 : 1). Flow rate: 1.5 mL/minute. System suitability System performance: Dissolve 85 mg of pchlorophenol in 10 mL of acetonitrile. Transfer 1 mL of this solution to a volumetric flask containing 4 mL of the stock solution, 10 mL of the internal standard solution and diluted glacial acetic acid (1 in 100) to make 50 mL and mix. Proceed with this solution under the above operating conditions: phenacetin, chlorzoxazone and pchlorphenol are eluted in this order and the resolution between the cloxazone peak and the p-chlorophenol peak is not less than 2.0. System repeatability: When the test is repeated 6 times with 20 l of the standard solution: the relative

212 Monographs, Part I

standard deviation of the peak areas of Chlorzoxazone is not more than 1.5%.
Packaging and Storage Preserve in tight containers. Purity 7-DehydrocholesterolDissolve 10 mg of Cholecalciferol in 2.0 mL of diluted ethanol (9 in 10), add a solution prepared by dissolving 20 mg of digitonin in 2.0 mL of diluted ethanol (9 in 10), and allow the mixture to stand for 18 hours: no precipitate is formed. Assay Dissolve separately about 30 mg each of Cholecalciferol and Cholecalciferol RS, accurately weighed, in isooctane to make exactly 50 mL. Pipet 10 mL each of these solutions, add 3 mL each of the internal standard solution, then add the mobile phase to make 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS , of the peak area of cholecalciferol to that of the internal standard for the test solution and the standard solutions, respectively. Proceed with the operation avoiding contact with air or other oxidizing agents, and using lightresistant containers.

Cholecalciferol
H H3C H 3C H CH 3

CH 3

CH 2

H HO H

Vitamin D3

C27H44O: 384.65

Cholecalciferol contains not less than 97.0% and not more than 103.0% of cholecalciferol (C27H44O).
Description Cholecalciferol is a white crystal and is odorless. Cholecalciferol is freely soluble in ethanol, in chloroform, in ether or in isooctane, and practically insoluble in water. Cholecalciferol is affected by air and by light. Melting pointBetween 84 C and 88 C. Place Cholecalciferol in a capillary, dry for 3 hours in a desicator (vacuum, not exceeding 2.67 kPa), fire seal the capillary tube immediately, put it in a bath fiuid previously heated to a temperature of about 10 C beolw the expected melting point, and heat at a rate of rise of about 3 per minute, and read the melting point. Identification (1) Dissolve 0.5 mg of Cholecalciferol in 5 mL of chloroform, add 0.3 mL of acetic anhydride and 0.1 mL of sulfuric acid, and mix under shaking: a red color is produced, and rapidly changes through purple and blue to green. (2) Determine the infrared absorption spectra of Cholecalciferol and Cholecalciferol RS, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between + 103 and + 112 (50 mg, ethanol, 10 mL, 100 mm). Prepare the solution without delay, using Cholecalciferol from a container opened not longer than 30 minutes previously, and determine the rotation within 30 minutes after the solution has been prepared.
1% Absorbance E1 cm (265 nm): Between 450 and 490 (10 mg, ethanol, 1000 mL).

Amount (mg) of cholecalciferol (C27H44O) = amount (mg) of Cholecalciferol RS QT

QS

Internal standard solutionA solution of dimethyl phthalate in isooctane (1 in 100). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 10 cm to 30 cm in length, packed with silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of hexane and namylalcohol (997 : 3). Flow rate: Adjust the flow rate so that the retention time of Cholecalciferol is about 25 minutes. Selection of column: Dissolve 15 mg of Cholecalciferol RS in 25 mL of isooctane. Transfer this solution to a flask, heat under a reflux condenser in an oil-bath for 2 hours, and cool to room temperature rapidly. Transfer this solution to a quartz test tube, and irradiate under a short-wave lamp (main wavelength: 254 nm) and a longwave lamp (main wavelength: 365 nm) for 3 hours. To this solution, add the mobile phase to make 50 mL. Proceed with 10 L of this solution under the above operating conditions. Use a column with the ratios of the retention time of previtamin D3, trans-vitamin D3, and tachysterol D3 to that of cholecalciferol being about 0.5, about 0.6, and about 1.1, respectively, and with resolution between previtamin D3 and trans-vitamin D3, and that between cholecalciferol and tachysterol D3 being not less than 1.0.

KP 9 213
Packaging and Storage Preserve in light-resistant, hermetic containers under nitrogen atmosphere, and in a cold place.

Chorionic Gonadotrophin
Chorionic Gonadotrophin is dried preparation of gonadstimulating hormone obtained from the urine of pregnant women through the removal of the virus or non-active process. . Chorionic Gonadotrophin contains not less than 2500 chorionic gonadotrophin units per mg. Chorionic Gonadotrophin contains not less than 80.0% and not more than 125.0% of the labeled amount of Chorionic Gonadotrophin.
Description Chorionic Gonadotrophin is a white to pale yellow-brown powder. Chorionic Gonadotrophin is odorless. Chorionic Gonadotropin is freely soluble in water and practically insoluble in ether. Identification Calculate b by the following equation, using Y3 and Y4 obtained in the Assay: b is less than 120.
E I Y3 Y4 E= f b=

120 units per each mL, and use this solution as the test solution. Inject 5.0 mL of the test solution into the peritoneal cavity of each of two or more of, well nourished, healthy guinea pigs weighing about 350 g and observe the conditions of the animals for more than days : all the animals exhibit no abnormalities.
Specific activity When calculate from the results obtained by the Assay and the following test, the specific activity is not less than 3000 human chorionic gonadotrophin Units per mg protein (1) Test solutionTo an exactly amount of Chorionic Gonadotrophin add water to make a solution so that each mL contains about 500 Units of chorionic gonadotrophin according to the labeled amount (2) Standard solutionWeigh accurately about 10mg of bovine serum albumin, and dissolve in water to make exactly 20mL. To a suitable volume of this solution add water to make four solutions containing exactly 300, 200, 100 and 50g of the albumin per mL, respectively. (3) ProcedurePipet 0.5mL each of the test solution and the standard solutions, put them in glass test tubes about 18mm in inside diameter and about 130 mm in length, add exactly 5mL of alkaline copper TS, mix, and allow the tubes to stand in a water bath at 30 for 10 minutes. Then add exactly 0.5mL of diluted Folins TS (1 in 2), mix, and warm in a water bath at 30 for 20 minutes. Determine the absorbances of these solutions at 750nm as directed under Ultraviolet-visible Spectrophotometry using a solution obtained in the same manner with 0.5mL of water as the blank. Plot the absorbances of the standard solutions on the vertical axis and their protein concentrations on the horizontal axis to prepare a calibration curve, and determine the protein content of the test solution from its absorbance by using this curve. Then calculate the amount of the protein in the sample. Assay (1) Test animalsSelect healthy female albino rats weighing about 45 g. (2) Standard solutionDissolve a quantity of Chorionic Gonadotrophin RS in bovine serum albuminisotonic sodium chloride solution to prepare four kinds of solutions, having 7.5, 15, 30 and 60 units per 2.5 mL, respectively. Inject these solutions into four groups consisting of five test animals each and weigh their ovaries, as directed in procedure of (iv). Inject bovine serum albumin-isotonic sodium chloride solution to another group and use this group as the control group. According to the result of this test, designate the concentration of the RS which will increase the weights of the ovaries about 2.5 times the weight of the ovaries of the control group as a low-dose concentration of the standard solution and the concentration 1.5 to 2.0 times the low-dose concentration as a high dose concentration. Dissolve a quantity of chorionic gonadotrophin RS in bovine serum albumin-isotonic sodium chloride solution and prepare a high-dose standard solution, S H and a low-dose stan-

f : Number of test animals per group.

I = log TH TL

Purity (1) Clarity and color solutionDissolve 0.05 g of Chorionic Gonadotrophin in 5 mL of isotonic sodium chloride solution: the solution is clear and colorless or pale yellow. (2) EstrogenInject subcutaneously into each of three female albino rats or albino mice ovaectomized at least two weeks before the test, single dose of 100 units according to the labeled units dissolved in 0.5 mL of isotonic sodium chloride solution. Take vaginal smear twice daily, on the third, fourth and fifth day. Place the smear thinly on a slide glass, dry, stain with Giemsas TS, wash with water and again dry: no estrus figure is shown microscopically. Loss on Drying Less than 5.0% (0.1 g, in vacuum, phosphorus pentoxide, 4 hours). Bacterial Endotoxins Less than 0.03 EU per each unit of Chorionic Gonadotrophin. Abnormal Toxicity Weigh the suitable amount of Chorionic Gonadotrophin, prepare a solution containing

214 Monographs, Part I

dard solution, SL , whose concentrations are equal to those determined by the above test. (3) Test solutionAccording to the labeled units, weigh accurately a suitable quantity of Chorionic Gonadotrophin, dissolve in bovine serum albumin-isotonic sodium chloride solution and prepare a high-dose sample solution, TH and a low-dose test solution, TL , having units equal to the standard solutions in equal volumes, respectively. (4) ProcedureDivide the test animals at random into 4 groups, A, B, C and D, with not less than 10 animals and equal numbers in each group. Inject subcutaneously 0.5 mL of S H , SL , TH , TL in each group for 5 days. On the sixth day, excise the ovaries and remove the adhering water by lightly pressing between filter paper and immediately weigh the ovaries. (5) CalculationDesignate the weight of ovaries by S H , SL , TH , TL as y1 , y2 , y3 and y4 , respectively. Sum up y1 , y2 , y3 and y4 on each set to obtain Y1 , Y2 , Y3 and Y4 . Units per mg of Chorionic Gonadotrophin = antilog M (units per mL of the high dose of the standard solution)
M = I = log IYa Yb

s2 =

y2 f
n

y 2 : The sum of the squares of each

and y4 .
Y = Y12 + Y22 + Y32 + Y42

y1 , y2 , y3

n = 4( f 1)
L = 2 (C 1)(CM 2 + I 2 )

C=

Yb2 Yb2 4 fs 2 t 2

used to calcuate s2.

t 2 = Value shown in the following tablet against n

t 2 = F1
161.45 18.51 10.129 7.709 6.608 5.987 5.591 5.318 5.117 4.965 4.844 4.747

n
1 2 3 4 5 6 7 8 9 10 11 12

n
13 14 15 16 17 18 19 20 21 22 23 24

t 2 = F1
4.667 4.600 4.543 4.494 4.451 4.414 4.381 4.351 4.325 4.301 4.279 4.260

n
25 26 27 28 29 30 40 60 120

t 2 = F1
4.242 4.225 4.210 4.196 4.183 4.171 4.085 4.001 3.920 3.841

b a

SH T = log H SL TL

Ya = Y1 Y2 + Y3 + Y4 Yb = Y1 Y2 + Y3 Y4
a: Mass (mg) of sample, b: Total volume (mL) of the high dose of the test solution prepared by diluting with bovine serum albuminisotonic sodium chloride solution.
F compared by the following equation should be
2

Packaging and Storage Preserve in light-resistant, tight containers in a cold place.

Chorionic Gonadotrophin for Injection

Chorionic Gonadotrophin for Injection is a preparation for injection which is reconstituted before use. Chorionic Gonadotrophin for Injection contains not less than 80.0% and not more than 125.0% of the labeled units of Chorionic Gonadotrophin.
Method of Preparation Prepare as directed under Injections, with Chorionic Gonadotrophin. Description Chorionic Gonadotrophin for Injection is a white to pale yellow-brown powder or masses. Chorionic Gonadotropin is freely soluble in water. Identification Proceed as directed in the Identification

smaller than F1 against n when s is calculated. And compute L (P = 0.95) by the following equation: L should be not more than 0.3. If F exceeds F1 , or if L exceeds 0.3, repeat the test increasing the number of the test animals ro arranging the assay method in a better way until F is smaller than F or L is not more than 0.3.
F = (Y1 Y2 Y3 + Y4 ) 2 4 fs 2

F : Number of test animals per group.

KP 9 215
under Chorionic Gonadotrophin.
Loss on Drying Not more than 5.0% (1.0 g, in vacuum, P2O5, 4 hours). Sterility Test It meets the requirement. Bacterial Endotoxins Less than 0.03 EU per each mL of Chorionic Gonadotrophin. Insoluble Particulate Matter Test quirement.

(0.2 g calculated on the anhydrous basis, 0.067 mol/L phosphate buffer solution at pH 7.0, 50 mL, 100 mm).
Purity (1) Related substance I Dissolve 0.20 g of Cilazapril Hydrate in methanol to make 5 mL, and use this solution as the test solution. Separately, dissolve 2 mg of cilazapril related substance I RS {1,1dimethylethyl(1S,9S)-9-[[(S)-1-(ethoxycarbonyl)-3phenylpropyl]amino]-10-oxooctahydro-6H-pyridazino[1, 2-][1,2]diazepine-1-carboxylate}, accurately weighed, in methanol to make exactly 50 mL, and use this solution as the standard solution (1). Dissolve 5 mg of cilazapril hydrate related substance I RS and 5 mg of Cilazapril Hydrate in methanol to make exactly 10 mL, and use this solution as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution , the standard solution (1), and the standard solution (2) on a plate of silica gel for thin-layer chromatography, develop with a mixture of ethyl acetate, methanol, hexane, glacial acetic acid, and water (60 : 15 : 15 : 5 : 5) to a distance of about 10 cm, and dry the plate in air. Spray the plate first with a freshly prepared mixture of 12 w/v% acetic acid and potassium iodobismuthate solution (10:1) and then with hydrogen peroxide TS. Any spot, corresponding to the related substance I, from the test solution is not more intense than the spot from the standard solution (1) (0.1%). The test is valid when the chromatogram obtained with the standard solution (2) shows 2 clearly separated spots. (2) Other related substances Dissolve 25 mg of Cilazapril Hydrate, accurately weighed, in the mobile to make 50 mL, and use this solution as the test solution. Dilute 1.0 mL of the test solution to 50 mL with the mobile phase. Dilute 5.0 mL of this solution to 20 mL with the mobile phase, and use this solution as the standard solution (1). Separately, dissolve 10 mg of cilazapril hydrate related substance IV RS {(1S,9S)-9-[[(R)-1(ethoxycarbonyl)-3-phenylpropyl]amino]-10-oxoocta hydro-6H-pyridazino-[1,2-][1,2]diazepine-1-carboxylic acid}in the test solution to make exactly 20 mL, and use this solution as the standard solution (2). Perform the test with 20 L each of the test solution and the standard solutions as directed under the Liquid Chromatography according to the following conditions. In the chromatogram obtained with the test solution: the area of any peak corresponding to the related substance IV is not greater than 0.4 times the area of the principal peak from the standard solution (1) (0.2%); the area of any peak corresponding to cilazapril hydrate related substance II {(1S,9S)-9-[[(S)1-carboxy-3-phenylpropyl] amino]-10-oxooctahydro-6Hpyridazino[1,2-][1,2] diazepine-1-carboxylic acid} is not greater than the area of the principal peak from the standard solution (1) (0.5%); the area of any peak corresponding to cilazapril hydrate related substance III {ethyl(1S,9S)-9-[[(S)-1-(ethoxycarbonyl)-3phenylpropyl] amino]-10-oxooctahydro-6Hpyridazino[1,2-][1,2] diazepine-1-carboxylate} is not greater than 0.2 times the area of the principal peak from

It meets the re-

Insoluble Particulate Matter Test for Injections It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Proceed as directed in the Assay under Chorionic Gonadotrophin. The ratio of the assayed units to the labeled units should be calculated by the following equation.

The ratio of the assayed units to the labeled units = antilog M

Packaging and Storage Preserve in light-resistant, hermetic containers in a cold place.

Cilazapril Hydrate
O H N H N N H CO2H

H2O

O H3C O

C22H31N3O5,H2O : 435.51 Cilazapril Hydrate contains not less than 98.5% and not more than 101.5% of cilazapril (C22H31N3O5), calculated on the anhydrous basis.
Description Cilazapril Hydrate is a white, crystalline powder. Cilazapril Hydrate is freely soluble in methanol or in methylene chloride, and slightly soluble in water. Identification (1) Determine the infrared absorption spectra of Cilazapril Hydrate and Cilazapril Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation
20 [ ]365 nm : 383 to 399

216 Monographs, Part I

the standard solution (1) (0.1%); the area of any peak, apart from the principal peak and the peaks corresponding to the related substances II, III and IV, is not greater than 0.2 times the area of the principal peak from the standard solution (1) (0.1%); and the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak from the standard solution (1) (1%). Disregard any peak with an area less than 0.1 times that of the principal peak from the standard solution (1) and any peak due to the related substance I. well-closed containers.

Cimetidine
HN N NCN H3C CH2SCH2CH2NH C NHCH3

C10H16N6S: 252.34 Cimetidine, when dried, contains not less than 99.0% and not more than 101.0% of cimetidine (C10H16N6S).
Description Cimetidine is a white crystalline powder, is odorless and has a bitter taste. Cimetidine is freely soluble in methanol or in glacial acetic acid, sparingly soluble in ethanol, slightly soluble in water and practically insoluble in ether. Cimetidine dissolves in dilute hydrochloric acid. Cimetidine is gradually colored by light. Identification (1) To 0.1 mL of a solution of Cimetidine in ethanol (1 in 100), add 5 mL of citric acid-acetic anhydride TS and heat in a water bath for 15 minutes: a red-purple color develops. (2) Determine the infrared spectra of Cimetidine and Cimetidins RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. pH Dissolve 0.5 g of Cimetidine in 50 mL of freshly boiled and cooled water, shake for 5 minutes and filter: the pH of the filtrate is between 9.0 and 10.5. Melting Point

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 214 nm). Column: A stainless steel column about 4.6 mm inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: Mix 10 mL of triethylamine and 750 mL of water, adjust to pH 2.30 with phosphoric acid, and add 200 mL of tetrahydrofuran. Flow rate: 1 mL/min. System suitability Test for required detectability: Perform the test with 20 L each of the test solution and the standard solution (1) according to the above operating conditions. The retention times relative to cilazapril are about 0.6 for the related substance II, 0.9 for the related substance IV, and 1.6 for the related substance III. System performance: Perform the test with 20 L each of the standard solutions (1) and (2) according to the above operating conditions. Adjust the sensitivity of the system so that the height of the principal peak from the standard solution (1) is at least 50% of the full scale of the recorder. The test is valid when the resolution between the peaks of cilazapril and the related substance IV from the standard solution (2) is at least 2.5. Time span of measurement: 2 times as long as the retention time of cilazapril. When the related substance I with a relative retention time of 4 to 5 is present, the chromatography should be continued until it is eluted.
Water Between 3.5% and 5.0% (0.30 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve 0.30 g of Cilazapril Hydrate, accurately weighed, in 10 mL of ethanol and add 50 mL of water, and titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Between 140 C and 144 C

Each mL of 0.1 mol/L sodium hydroxide VS = 41.75 mg of C22H31N3O5

Packaging and Storage Preserve in light-resistant,

Purity (1) Clarity and color of solutionDissolve 1.0 g of Cimetidine in 10 mL of methanol: the solution is clear and colorless to pale yellow in color. (2) Heavy metalsProceed with 2.0 g of Cimetidine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicDissolve 1.0 g of Cimetidine in 5 mL of dilute hydrochloric acid and perform the test with this solution (not more than 2 ppm). (4) Related substancesDissolve 0.5 g of Cimetidine in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 100 mL and pipet exactly 1 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with these solutions as directed under the Thinlayer Chromatography. Spot 4 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the mixture of ethyl acetate, methanol and strong ammonia

KP 9 217
water (21 : 2 : 2) to a distance of about 15 cm, air-dry the plate and then dry at 80 C for 30 minutes. Allow the plate to stand in iodine vapor for 45 minutes: any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5 % (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.25 % (1 g). Assay Weigh accurately about 0.24 g of Cimetidine, previous dried, dissolve in 75 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 25.234 mg of C10H16N6S

Packaging and Storage well-closed containers.

Assay Weigh accurately and powder not less than 20 Cimetidine Tablets. Weigh accurately a portion of the powder, equivalent to 0.1 g of cimetidine (C10H16N6S), add 50 mL of methanol, shake, mix for 2 minutes, then add 40 mL of water, ultrasonicate, mix for 15 minutes and add water to make exactly 250 mL. Pipet 5 mL of this solution and add mobile phase to make exactly 200 mL and use this solution as the test solution. Separately, weigh accurately 20 mg of Cimetidine RS, dissolve in a mixture of water and methanol (4 : 1) to make exactly 50 mL, pipet 5 mL of this solution and add mobile phase to make exactly 200 mL and use this solution as the standard solution. Perform the test with 50 mL each of the test solution and standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, AT and AS , of the peak area of Cimetidine, for the test solution and the standard solution, respectively.

Preserve in light-resistant,

Amount (mg) of cimetidine (C10H16N6S) = amount(mg) of Cimetidine RS

AT AS

Cimetidine Tablets
Cimetidine Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of cimetidine (C10H16N6S: 252.34).
Method of Preparation Prepare as directed under Tablets, with Cimetidine. Identification The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that of the standard preparation as obtained in the Assay. Dissolution Test Perform the test with 1 tablet of Cimetidine Tablets at 100 revolutions per minute according to Method 1 under Dissolution Test, using 900 mL of 0.01 mol/L hydrochloric acid as a dissolution solution. Take the dissolved solution 15 minutes after start of the test and make any necessary dilute in 0.01 mol/L hydrochloric acid and use this solution as the test solution. Separately, weigh accurately sufficient quantity of Cimetidine RS and dissolve in 0.01 mol/L hydrochloric acid and make the same concentration as the test solution and use this solution as the standard solution. Determine the absorbances of the test solution and standard solution at the maximum absorption wavelength of about 218 nm as directed under the Ultraviolet-visible Spectrophotometry, using 0.01 mol/L hydrochloric acid as the blank. The dissolution rate of Cimetidine Tablets in 15 minutes is not less than 80%. Uniformity of Dosage Units It meets the requirement.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column about 3.9 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Mobile phase: To 200 mL of methanol, add 0.3 mL of phosphoric acid , add water to make 1000 mL and mix. Flow rate: 2.0 mL/minute. System repeatability: When the test is repeated 6 times with 50 L of the standard solution under the above operating conditions, relative standard deviation of the peak areas is not more than 2.0%.
Packaging and Storage Preserve in well-closed containers.

Cimetidine Hydrochloride
HN N NCN H3C CH2SCH2CH2NH C NHCH3

HCl

C10H16N6SHCl : 288.80 Cimetidine Hydrochloride contains not less than 98.0% and not more than 102.0% of cimetidine hydrochloride (C10H16N6SHCl), calculated on the dried basis .

218 Monographs, Part I

Description Cimetidine Hydrochloride is a white crystalline powder. Cimetidine Hydrochloride is freely soluble in water, and sparingly soluble in ethanol. Identification (1) Dissolve about 15 mg, accurately weighed, of Cimetidine Hydrochloride and Cimetidine Hydrochloride RS, respectively, in 0.05 mol/L sulfuric acid TS to exactly 100 mL. Pipet 5.0 mL each of the solutions, add 0.05 mol/L sulfuric acid TS to make exactly 50 mL and use these solutions as the test solution and the standard solution, respectively. Determine the absorption spectra of the test and standard solutions, respectively, as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Cimetidine Hydrochloride and Cimetidine Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Heavy metalsProceed with 1.0 g of Cimetidine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesWeigh accurately about 0.1 g of Cimetidine Hydrochloride, add mobile phase to make exactly 250 mL, and use this solution as the test solution. Pipet exactly 1.0 mL of the test solution, add mobile phase to make exactly 500 mL, and use this solution as the standard solution. Perform the test with exactly 50 L each of the test and the standard solutions as directed under Liquid Chromatography according to the following conditions. Determine each peak area by the automatic integration method and calculate the percentage of each related substance: Each related substance is not greater than 0.2%, and the sum of all related substances is not more than 1.0%.

of phosphoric acid and water to make 1000 mL. Flow rate: 2 mL/minute System suitability System performance: Dissolve 50 mg of Cimetidine Hydrochloride in 100 mL of 1 mol/L hydrochloric acid TS and heat on a steam bath for about 10 minutes and cool. Pipet 1.0 mL of this solution and add mobile phase to make 250 mL. When the procedure is run with 50 L of this solution under the above operating condition, the resolution between the peaks of cimetidine and its amide analog is not less than 4.0. And when the procedure is run with 50 L of the standard solution under the above operating condition, the capacity factor is not less than 3.0 and the number of theoretical plates is not less than 2000. System repeatability: When the test is repeated 6 times with 50 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of cimetidine is not more than 7.0%.
Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.115 g of Cimetidine Hydrochloride, add 50 mL of water to dissolve, add 50 mL of methanol and water to make exactly 250 mL. Pipet 5.0 mL of this solution, add mobile phase to make exactly 200 mL, and use this solution as the test solution. Separately, weigh accurately about 50 mg of Cimetidine Hydrochloride RS, add a mixture of water and methanol (80 : 20) to make exactly 100 mL. Pipet 5.0 mL of this solution, add mobile phase to make exactly 200 mL, and use this solutions the standard solution. Perform the test with exactly 50 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine the peak area of cimetidine, AT and AS , respectively.

Amount (%) of each related substance = 0 . 2

Ai AS

Amount (mg) of Cimetidine Hydrochloride (C10H16N6SHCl) = Cimetidine Hydrochloride RS (mg)

AT 2 .5 AS

Ai : the peak area of each related substance in the

chromatogram obtained from the test solution AS : the peak area of cimetidine in the chromatogram obtained from the standard solution

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: Dissolve 0.94 g of sodium 1hexanesulfonate in 240 mL of methanol, and add 0.3 mL

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column about 3.9 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: To 200 mL of methanol add 0.3 mL of phosphoric acid and water to make 1000 mL. Flow rate: about 2 mL/minute System suitability System performance: When the procedure is run with 50 L of the standard solution under the above op-

KP 9 219
erating condition, the number of theoretical plates is not less than 1000 and capacity factor is not less than 0.6. System repeatability: When the test is repeated 5 times with 50 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of cimetidine is not more than 2.0%.
Packaging and Storage tight containers.

same. This test is valid when the chromatogram from the standard solution (2) shows two clearly separated spots.
Melting Point Between 118 C and 120 C. Purity (1) Clarity and color of solutionDissolve 0.5 g of Cinnarizine in methylene chloride to make 20 mL. The solution is clear and has no more color than the following control solution.

Preserve in light-resistant,

Cinnarizine
H N N H

Control solutionTo a mixture of 24 mL of Ferric Cholride Calorimetric Stock Solution, 10 mL of Cobaltous Chloride Colorimetric Stock Solution and 4 mL of Cupric Sulfate Colorimetric Stock Solution add 62 mL of 1 w/w% hydrochloric acid and mix. To 2.5 mL of this solution add 97.5 mL of 1 w/w% hydrochloric acid and mix.
(2) Acidity or alkalinitySuspend 0.5 g of Cinnarizine in 15 mL of water. Boil for 2 minutes, cool, and filter. Dilute the filtrate to 20 mL with water, and use this solution as the test solution. To 10 mL of the test solution, add two drops of phenolphthalein TS and 0.25 mL of 0.01 mol/L sodium hydroxide. The solution is pink. When two drops of methyl red TS and 0.25 mL of 0.01 mol/L hydrochloric acid are added to 10 mL of the test solution, the solution is red. (3) Heavy metals Dissolve 1.0 g of Cinnarizine in a mixture of acetone and water (85:15). Add dilute hydrochloric acid until dissolution is complete. Dilute to 20 mL with a mixture of acetone and water (85:15), and use this solution as the test solution. To 12 mL of the test solution, add 2 mL of sodium acetate buffer solution (pH 3.5), and mix with 1.2 mL of thioacetamide. Allow to stand still for 2 minutes. Mix 10 mL of diluted lead standard solution with 2 mL of the test solution, proceed in the same manner as above, and use this solution as the standard solution. The color obtained with the test solution is not more intense than that observed with the standard solution (not more than 20 ppm). (4) Related substancesDissolve 25.0 mg of Cinnarizine in methanol to make exactly 100 mL, and use this solution as the test solution. Separately, dissolve 12.5 mg of Cinnarizine RS and 15.0 mg of Flunarizine Dihydrochloride RS in methanol to make 100 mL. Dilute 1.0 mL of this solution to 20 mL with methanol, and use the solution as the standard solution (1). Dilute 1.0 mL of the test solution to 100 mL with methanol. Dilute 5.0 mL of this solution to 20 mL with methanol, and use this solution as the standard solution (2). Perform the test with 10 L each of methanol (as a blank), the test solution and the standard solutions as directed under the Liquid Chromatography according to the following conditions. In the chromatogram of the test solution, the area of any peak, other than the principal peak, is not greater than the area of the principal peak from the standard solution (2) (0.25%); and the sum of the areas of the peaks, other than the principal peak, is not greater than twice the area of the principal peak from the standard solution (2)

C26H28N2 : 368.51 Cinnarizine contains not less than 99.0% and not more than 101.0% of cinnarizine (C26H28N2), calculated on the dried basis.
Description Cinnarizine is a white powder. Cinnarizine is freely soluble in methylene chloride, soluble in acetone, slightly soluble in ethanol or in methanol, and practically insoluble in water. Identification (1) Dissolve 0.2 g of anhydrous citric acid in 10 mL of acetic anhydride in a water-bath at 80 C and maintain the temperature for 10 minutes. Add about 20 mg of Cinnarizine. A purple color develops. (2) Determine the infrared absorption spectra of Cinnarizine and Cinnarizine RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 10 mg of Cinnarizine in methanol to make 20 mL, and use this solution as the test solution. Separately, dissolve 10 mg of Cinnarizine RS in methanol to make 5 mL, and use this solution as the standard solution (1). Dissolve 10 mg of Cinnarizine RS and 10 mg of Flunarizine Dihydrochloride RS in methanol and dilute to 20 mL with methanol. Use the solution as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solutions on a plate of silica gel with fluorescent indicator for thin-layer chromatography, develop with a mixture of acetone, methanol and 1 mol/L sodium chloride solution (50:30:20) to a distance of about 15 cm, and dry the plate in air. Examine in ultraviolet light at 254 nm, and compare the principal spot from the test solution with that from the standard solution (1). Their Rf values are the

220 Monographs, Part I

(0.5%). Disregard any peak due to the blank and any peak with an area less than 0.2 times the area of the principal peak from the standard solution (2).

Ciprofloxacin Hydrochloride Hydrate

HN N N HCl OH F O O H2O

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column about 4.0 mm inside diameter and 10 cm in length, packed with basedeactivated octadecylsilanized silica gel for liquid chromatography (3 m in particle diameter). Mobile phase: Variable mixtures of mobile A and mobile phase B, and program the chromatograph as follows. Mobile phase A: 1 w/v% ammonium acetate solution Mobile phase B: 0.2 v/v% solution of glacial acetic acid in acetonitrile
Time (min) 0 ~ 20 20 ~ 25 Mobile phase A (%) 75 10 10 Mobile Elution phase B (%) 25 90 linear gradient 90 isocratic switch to initial 25 eluent composition 25 restart gradient

C17H18FN3O3HClH2O: 385.82 Ciprofloxacin Hydrochloride Hydrate contains not less than 98.0% and not more than 102.0% of ciprofloxacin hydrochloride (C17H18FN3O3HCl), calculated on the anhydrous basis.
Description Ciprofloxacin Hydrochloride Hydrate is a pale yellow, crystalline powder. Ciprofloxacin Hydrochloride Hydrate is sparingly soluble in water, slightly soluble in acetic acid or methanol, very slightly soluble in dehydrated ethanol, and practically insoluble in acetone, acetonitrile, ethyl acetate, hexane or dichloromethane. Identification (1) Determine the infrared spectra of Ciprofloxacin Hydrochloride Hydrate and Ciprofloxacin Hydrochloride Hydrate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Dissolve 0.1 g of Ciprofloxacin Hydrochloride Hydrate in water to make 10 mL and use this solution as the test solution. Separately, dissolve 0.1 g of Ciprofloxacin Hydrochloride Hydrate RS in 10 mL of water and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test and the standard solution on a plate of silica gel for thin-layer chromatography. Allow the plate to stand in the container with ammonia gas for 15 minutes. Develop the plate with a mixture of dichloromethane, methanol, strong ammonia water and acetonitrile (4 : 4 : 2 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm and 365 nm): the spot from the test solution exhibits similar intensity and same Rf value as the spot from the standard solution. (3) A solution of Ciprofloxacin Hydrochloride Hydrate (1 in 50) responds to the Qualitative Tests for chloride. pH Dissolve 1.0 g of Ciprofloxacin Hydroxide Hydrate in 40 mL of freshly boiled and cooled water: the pH of this solution is between 3.0 and 4.5.

25 ~ 30 75 30 = 0 75

Flow rate: 1.5 mL/min. System suitability System performance: 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. When the procedure is run with the standard solution (1) under the above operating conditions, the retention times are about 11 minutes for cinnarizine and about 11.5 minutes for flunarizine. The test is valid when the resolution of these peaks between cinnarizine and flunarizine is at least 5.0. If necessary, adjust the time programm for the gradient elution.
Loss on Drying Not more than 0.5% (1 g, vacuum, 60 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve 0.15 g of Cinnarizine, accurately weighed, in 50 mL of a mixture of ethyl methyl ketone and glacial acetic acid (7 : 1), and titrate with 0.1 mol/L perchloric acid VS (indicator: 4 drops of naphtholbenzein TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 18.426 mg of C26H28N2.

Packaging and Storage well-closed containers.

Preserve in light-resistant,

KP 9 221
tion containing about 0.5 mg per mL and use this solution as the standard solution. Perform the test with 10 L each of the test and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and determine the peak area of ciprofloxacin hydrochloride, AT and AS , of the test solution and the standard solution, respectively. Amount (mg) of ciprofloxacin hydrochloride (C17H18FN3O3HCl) = 50 C
AT AS

Purity (1) Heavy metalsProceed with 1.0 g of Ciprofloxacin Hydrochloride Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) SulfatePerform the test with 0.375 g of Ciprofloxacin Hydrochloride Hydrate. Prepare the control solution with 0.30 mL of 0.005 mol/L sulfuric acid VS (not more than 0.04%). (3) Fluoroquinolonic acidWeigh a suitable amount of Ciprofloxacin Hydrochloride Hydrate, dissolve in water to make a solution containing 10 mg per mL and use this solution as the test solution. Separately, weigh accurately about 5.0 mg of Fluoroquinolonic Acid RS, add 50 L of ammonia TS and water to make 50 mL. Pipet 2.0 mL of this solution, add water to make 10 mL and use this solution as the standard solution. Perform the test according to Thin-layer Chromatography with the test solution and the standard solution. Spot 5 L each of the test and the standard solution on a plate with silica gel for thin-layer chromatography. Allow the plate to stand in the container with 50 mL of ammonia TS for 15 minutes. Develop the plate with a mixture of dichloromethane, methanol, strong ammonia water and acetonitrile (4 : 4 : 2 : 1) to a distance of about 15 cm and airdry the plate for 15 minutes. Examine under the ultraviolet light (main wavelength: 250 nm): Rf value corresponding to the principal spot from the standard solution, the spots other than the principal spot from the test solution are not larger or not more intense than the spot from the standard solution (not more than 0.2%). (4) Related substancesPerform the test according to the operating conditions in the Assay. Determine each peak area of impurities, Ai , from the test solution and

C: the concentration of Ciprofloxacin Hydrochloride Hydrate RS in the standard solution, calculated on the anhydrous basis (mg/mL). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 278 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column Temperature: A constant temperature of about 30 C. Mobile phase: A mixture of 0.025 mol/L phosphoric acid adjusted to pH 3.0 0.1 with triethylamine and acetonitrile (87 : 13). Flow rate: 1.5 mL/minute. System suitability System performance: Dissolve Ciprofloxacin Ethylenediamine derivative RS in the standard solution of ciprofloxacin to make a solution containing 0.5 mg per mL. Perform the test with 10 L of this solution according to the above operating conditions. Ciprofloxacin ethylenediamine derivative and ciprofloxacin are eluted in this order and the resolution between ciprofloxacin ethylenediamine derivate and ciprofloxacin are not less than 6. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, relative standard derivation of the peak area of ciprofloxacin is no more than 1.5%.
Packaging and Storage resistant containers.

the total area of all peaks, AT and calculate the content of each peak area of impurities according to the following equation. Amount (%) of each of impurities = 100
Ai AS

Each derivative of ciprofloxacin ethylenediamine and related substance is not more than 0.2% and the total of all related substances is not more than 0.5%.
Water Between 4.7% and 6.7% (0.5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 25 mg of Ciprofloxacin Hydrochloride Hydrate, dissolve in mobile phase to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately a suitable amount of Ciprofloxacin Hydrochloride Hydrate RS (determined formerly water contents as directed under water determination assay), dissolve in mobile phase to make a solu-

Preserve in tight, light-

Ciprofloxacin Hydrochloride Tablets

Ciprofloxacin Hydrochloride Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of ciprofloxacin (C17H18FN3O3 : 331.34).
Method of Preparation Prepare as directed under tab-

222 Monographs, Part I

lets, with Ciprofloxacin Hydrochloride Hydrate.
Identification (1) The retention time of the major peak in the chromatography of the test solution corresponds to that of the standard solution obtained as directed in the Assay. (2) Place a number of tablets, equivalent to 1.5 g of ciprofloxacin, in a suitable flask containing 750 mL of water and sonicate for about 20 minutes. Dilute with water to 1000 mL and centrifuge a portion of this suspension and use the supernatant solution obtained as the test solution. Separately, dissolve a suitable quantity of Ciprofloxacin Hydrochloride Hydrate RS in water to obtain a solution containing 1.5 mg per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Allow the plate to stand in the container with ammonia gas for 15 minutes. Develop the plate with a mixture of dichloromethane, methanol, ammonia TS and acetonitrile (4 : 4 : 2 : 1) to a distance of about 15 cm. Remove the plate and allow it to air-dry for about 15 minutes. Examine under ultraviolet light (main wavelength: 254 nm and 366 nm): the principal spot from the test solution exhibits the same Rf value as the principal spot from the standard solution. Dissolution Test Perform the test with 1 tablet of Ciprofloxacin Hydrochloride Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of 0.01 mol/L hydrochloric acid as a dissolution medium. 30 minutes later after starting the test, take the dissolution medium and filter. If necessary, make a test solution by diluting with the dissolution medium. Separately weigh accurately a portion of ciprofloxacin hydrochloride hydrate and dissolve in the dissolution medium to the same concentration with the test solution and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution at 276 nm as directed under the Ultravioletvisible Spectrophotometry, using the dissolution medium as a blank. The dissolution rate of ciprofloxacin hydrochloride in 30 minutes is not less than 80%. Uniformity of Dosage Units It meets the requirement. Assay Transfer 5 tablets in 400 mL of water and sonicate for about 20 minutes. Dilute with water to make exactly 500 mL. Dilute an accurately measured volume of this solution with water to obtain a solution containing the equivalent of the labeled amount of ciprofloxacin (C17H18FN3O3) about 0.3 mg per mL and use this solution as the test solution. Separately dissolve a suitable amount, accurately weighed, of Ciprofloxacin Hydrochloride Hydrate RS (formerly determined water contents as directed under water determination assay) in water to obtain a solution containing about 0.3 mg per mL and use this solution as the standard solution. Perform the

test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the operating conditions of the Assay of Ciprofloxacin Hydrochloride Hydrate and determine the peak areas, AT and AS , of ciprofloxacin for the test solution and the standard solution, respectively. Amount (mg) of ciprofloxacin (C17H18FN3O3) =
C L AT 331 .34 D A S 367 .81

C : Concentration of ciprofloxacin hydrochloride in the standard solution, calculated on the anhydrous basis (mg/mL). L : Labeled amount of ciprofloxacin in each tablet (mg). D : Concentration of Ciprofloxacin Hydrochloride in the test solution (mg/mL).
Packaging and Storage Preserve in tight containers.

Cisplatin
Cl NH3

Pt
Cl NH3

Cl2H6N2Pt : 300.05 Cisplatin, when dried, contains not less than 98.0% and not more than 102.0% of cisplatin (Cl2H6N2Pt).
Description Cisplatin is a yellow crystalline powder. Identification (1) Add 2 to 3 drops of stannous chloride dihydrate solution (1 in 100) to 5 mL of a solution of Cisplatin in water (1 in 2000): brown precipitate forms. (2) Determine the absorption spectra of the solutions of Cisplatin and Cisplatin RS, respectively, in a solution of sodium chloride (9 in 1000) in 0.01 mol/L hydrochloric acid TS (1 in 2000) as directed under Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Cisplatin and Cisplatin RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) TrichloroammineplatinatePerform this procedure without exposure of daylight, using lightresistant ressels. Weigh 50 mg of Cisplatin, and add a solution of sodium chloride (9 in 1000) to make exactly 100 mL and use this solution as the test solution. Separately, weigh 10 mg of Trichloroammineplatinate RS, previously dried at 80 C for 3 hours, add a solution of

KP 9 223
sodium chloride (3 in 1000) to make exactly 200 mL. Pipet 2.0 mL of this solution, add a solution of sodium chloride (9 in 1000) to make exactly 20 mL and use this solution as the standard solution. Perform the test with exactly 40 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the peak area trichloroammineplatinate from the test solution is not larger than the peak area from the standard solution. Detector: An ultraviolet absorption photometer (wavelength: 310 nm). Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with aminopropylsilyl silica gel for Liquid Chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture solution of ethyl acetate, methanol, water and dimethylformamide (25 : 16 : 5 : 5) Flow rate: Adjust the flow rate so that the retention time of cisplatin is about 4 minutes. System suitability System performance: When the procedure is run with 40 L of the standard solution under the above operating condition, the number of theoretical plates and the symmetry factor of the peak of cisplatin are not less than 3000 and not more than 2.0, respectively. System repeatability: When the test is repeated 6 times with 40 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of cisplatin is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 209 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with quartanaryammonium group introduced octadecylsilanized silica gel for Liquid Chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A solution of ammonium sulfate (1 in 800) Flow rate: Adjust the flow rate so that the retention time of trichloroammineplatinate is about 8 minutes. System suitability System performance: When the procedure is run with 40 L of the standard solution under the above operating condition, the number of theoretical plates and the symmetry factor of the peak of trichloroammineplatinate are not less than 1500 and not more than 2.0, respectively. System repeatability: When the test is repeated 6 times with 40 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of trichloroammineplatinate is not more than 3.0%.
Loss on Drying Not more than 0.1% (1 g, 105 C, 4 hours). Assay Perform this procedure without exposure of daylight, using light-resistant ressels. Weigh accurately about 25 mg each of Cisplatin and Cisplatin RS, previously dried, add dimethylformamide to make exactly 25 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with exactly 40 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine peak area of cisplatin, AT and AS , by the automatic integration method, respectively.

Citric Acid Hydrate

CH2CO2H HO C CO2H H2O

CH2CO2H

C6H8O7H2O: 210.14 Citric Acid Hydrate contains not less than 99.5% and not more than 100.5% of anhydrous citric acid (C6H8O7 : 192.12), calculated on the anhydrous basis.
Description Citric Acid Hydrate is a colorless crystal, white granule or crystalline powder, is odorless and has a strong acid taste. Citric Acid Hydrate is very soluble in water, and freely soluble in ethanol Citric Acid Hydrate is efflorescent in dry air. Identification Determine the infrared spectra of Citric Acid Hydrate and Citric Acid Hydrate RS, previously dried at 105 C for 2 hours, as directed in the potassium bromide disk method under Infrared Spectrophotometry: both spectra similar intensities of absorption at the same wave numbers. Purity (1) Clarity and color of solutionDissolve 2.0 g of Citric Acid Hydrate in water to make 10 mL: the solution is clear and has no more color than the following control solutions (1), (2), or (3). Control solution (1): To 1.5 mL of cobalt (II) chloride colorimetric stock solution and 6.0 mL of iron (III) chloride colorimetric stock solution add water to make 1000 mL.

Amount (mg) of cisplation (Cl2H6N2Pt) = amount (mg) of Cisplation RS AT

AS

Operating conditions

224 Monographs, Part I

Control solution (2): To 0.15 mL of cobalt (II) chloride colorimetric stock solution, 7.2 mL of iron (III) chloride colorimetric stock solution and 0.15 mL of copper (II) sulfate colorimetric stock solution add water to make 1000 mL. Control solution (3): To 2.5 mL of cobalt (II) chloride colorimetric stock solution, 6.0 mL of iron (III) chloride colorimetric stock solution and 1.0 mL of copper (II) sulfate colorimetric stock solution add water to make 1000 mL. (2) SulfatesDissolve 2.0 g of Citric Acid Hydrate in water to make 30 mL, and use this solution as the test solution. Separately, dissolve 0.181 g of potassium sulfate in diluted dehydrated ethanol (3 in 10) to make exactly 500 mL. Pipet exactly 5 mL of this solution, and add diluted dehydrated ethanol (3 in 10) to make exactly 100 mL. To 4.5 mL of this solution add 3 mL of a solution of barium chloride dihydrate (1 in 4), shake, and allow to stand for 1 minute. To 2.5 mL of this solution add 15 mL of the test solution and 0.5 mL of acetic acid, and allow to stand for 5 minutes: the solution has no more turbidity than the following control solution. drate, dissolve in 50 mL of water and titrate with 1 mol/L sodium hydroxide VS (indicator: 2 drops of phenolphthalein TS). Each mL of 1 mol/L sodium hydroxide VS = 64.04 mg of C6H8O7
Packaging and Storage Preserve in tight containers.

Anhydrous Citric Acid

CH2CO2H HO C CO2H

CH2CO2H

C6H8O7: 192.12 Anhydrous Citric Acid contains not less than 99.5% and not more than 101.0% of anhydrous citric acid (C6H8O7), calculated on the anhydrous basis.
Description Anhydrous Citric Acid is a colorless crystal, white granule or crystalline powder. Anhydrous Citric Acid is very soluble in water, and freely soluble in ethanol. Identification Proceed as directed in the Identification under Citric Acid Hydrate. Purity Proceed as directed in the Purity under Citric Acid Hydrate. Water Not more than 1.0% (2 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.55 g of Anhydrous Citric Acid, dissolve in 50 mL of water, and titrate with 1 mol/ L sodium hydroxide VS (indicator: 2 drops of phenolphthalein TS).

Control solutionDissolve 0.180 g of potassium sulfate in water to make exactly 500 mL. Pipet exactly 5 mL of this solution and add water to make exactly 100 mL.
(3) Oxalic acidDissolve 0.80 g of Citric Acid Hydrate in 4 mL of water, add 3 mL of hydrochloric acid and 1 g of zinc, and boil for 1 minute. After allowing to stand for 2 minutes, take the supernatant liquid, add 0.25 mL of a solution of phenyl hydrazinium hydrochloride (1 in 100), heat to boil, and then cool quickly. To this solution add the equal volume of hydrochloric acid and 0.25 mL of a solution of potassium hexacyanoferrate (III) (1 in 20), mix, and allow to stand for 30 minutes: the solution has no more color than the following control solution prepared at the same time.

Control solutionTo 4 mL of a solution of oxalic acid dihydrate (1 in 10000) add 3 mL of hydrochloric acid and 1 g of zinc.
(4) Heavy metalsProceed with 2.0 g of Citric Acid Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) Readily carbonizable substancesPerform the test with 0.5 g of Citric Acid Hydrate, provided that the solution is heated at 90 C for 1 hour and then cool quickly: the solution has no more color than Matching Fluid K.
Water Not less than 7.5 % and not more than 9.0 % (0.5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 1.5 g of Citric Acid Hy-

Each mL of 1 mol/L sodium hydroxide VS = 64.04 mg of C6H8O7

Packaging and Storage Preserve in tight containers.

KP 9 225
ter to make 20 mL, and use this solution as the test solution. The pH of the solution is between 8.0 and 10.0.
Purity Heavy metalsProceed with 2.0 g of Clarithromycin according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). Water Not more than 2.0 % (0.5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1 % (2.0 g).
CH3 O CH3 OH

Clarithromycin
O H3C CH3 O OH H3C O H3C O CH3 O CH3 O H3C O O CH3 CH3 CH3 H3C OH N CH3

HO

C38H69NO13: 747.95 Clarithromycin is a derivative of erythromycin. Clarithromycin contains not less than 900 g (potency) per mg of clarithromycin (C38H69NO13), calculated on the anhydrous basis.
Description Clarithromycin is a white crystalline powder, is odorless, and has a bitter taste. Clarithromycin is soluble in acetone or in chloroform, slightly soluble in methanol, in ethanol, or in ether, and practically insoluble in water. Identification (1) To 5 mg of Clarithromycin add 2 mL of sulfuric acid, and mix with shaking: the red-brown color develops. (2) To 3 mg of Clarithromycin, add 2 mL of acetone, and add 2 mL of hydrochloric acid: the orange color develops and the color turns immediately to red to dark purple. (3) Dissolve Clarithromycin in chloroform to make a solution so that each mL contains about 2.5 mg, and use this solution as the test solution. Separately, dissolve Clarithromycin RS in chloroform to make a solution so that each mL contains about 2.5 mg, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 5 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the mixture of chloroform, methanol and strong ammonia water (100 : 5 : 1), air-dry the plate. Spray evenly sulfuric acid on the plate and heat the plate at 105 C for 10 minutes. The spots obtained from the test solution and the standard solution are the same in the Rf value. Specific Optical Rotation [ ] 20 D : -81 ~ -97 (0.25 g calculated on the anhydrous basis, chloroform, 25 mL, 100 mm). pH Weigh 50 mg of Clarithromycin, dissolve in 20 mL of methanol, and take 10 mL of this solution and add wa-

Assay Weigh accurately about 0.1 g (potency) each of Clarithromycin and Clarithromycin RS, and dissolve each in the mobile phase to make exactly 20 mL. Pipet exactly 2 mL each of these solutions, add 2.0 mL of the internal standard solution, add the mobile phase to make exactly 20 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and calculate the ratios, QT and QS, of the peak area of clarithromycin to that of the internal standard.

Amount [g (potency)] of clarithromycin (C38H69NO13) = Amount [g (potency)] of Clarithromycin RS

QT QS

Internal standard solutionA solution of butyl parahydroxybenzoate in the mobile phase (1 in 20000) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A constant temperature of about 50 C Mobile phase: A mixture of 0.06 mol/L potassium dihydrogenphosphate solution and acetonitrile (13:7) Flow rate: Adjust the flow rate so that the retention time of clarithromycin is about 8 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, clarithromycin and the internal standard are eluted in this order with the resolution between these peaks being not less than 3.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of clarithromycin to that of the internal standard is not more than 2.0 %.

226 Monographs, Part I

Packaging and Storage Preserve in tight containers.

Clebopride Malate
O Cl N H H HO2C H2N OCH3 N HO CO2H

the chromatogram obtained with standard solution (2) according to the above operation condition, shows two clearly separated bands. -naphthol solutionDissolve 5g of -naphthol in 40 mL of 2 mol/L sodium hydroxide TS, add water to make 100 mL.
pH Dissolve 1.0 g of Clebopride Malate in water to make 100 mL: pH of this solution is between 3.8 and 4.2. Purity (1) Clarity and color of solutionDissolve 1.0 g of Clebopride Malate in water to make 100 mL: this solution is colorless and clear. (2) ChlorideDissolve 1.0 g of Clebopride Malate in 20 mL of glacial acetic acid, add 6 mL of dilute nitric acid, dilute with water to 50 mL and perform the test. Prepare the control solution, as follows mix 20 mL of glacial acetic acid, 6 mL of nitric acid and 0.3 mL of 0.01 mol/L hydrochloric acid VS, and add water to make 50 mL. (3) SulphatesDissolve 2.0 g of Clebopride Malate in 20 mL of glacial acetic acid, add water to make 50 mL and perform the test. Prepare the control solution, as follows mix 20 mL of glacial acetic acid and 0.42 mL of 0.005 mol/L sulfuric acid VS, and add water to make 50 mL (not more than 0.01%). (4) Heavy metalsProceed with 1.0 g of Clebopride Malate according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) Related substancesDissolve 0.10 g of Clebopride Malate in the mobile phase to make exactly 100 mL and use this solution as the test solution. Add the mobile phase to 1.0 mL of this solution to make exactly 10 mL and use this solution as the standard solution (1). Separately, dissolve 10 mg of Clebopride Malate RS and 10 mg of metoclopramide hydrochloride RS in the mobile phase to make exactly 100 mL, add the mobile phase to 1.0 mL of this solution to make 10 mL and use this solution as the standard solution (2). Perform the test with 20 L each of the test solution and the standard solutions as directed under the Liquid Chromatography according to the following conditions. The areas of the peak other than the principal peak from the test solution and the two peaks eluted within 2 minutes from the test solution are not greater than the area of the principal peak from standard solution (1) (0.1%); the sum of the areas of all peaks, other than the principal peak and two peaks within 2 minutes, is not greater than 3 times the area of the principal peak with standard solution (1) (0.3%). Disregard any peak from an area less than 0.25 times that of the principal peak with standard solution (1).

and enantiomer C20H24ClN3O2C4H6O5 : 507.96 Clebopride Malate, when dried, containes not less than 98.5% and not more than 101.0% of clebopride malate (C20H24ClN3O2C4H6O5).
Description Clebopride Malate is a white, crystalline powder. Clebopride Malate is sparingly soluble in methanol or in water, slightly soluble in ethanol and practically insoluble in dichloromethane. Melting pointAbout 164 C (with decomposition). Identification (1) Dissolve 20 mg of Clebopride Malate in 1 mL of surfuric acid, add 1 mL of -naphthol TS, mix and observe under daylight ; The solution shows a yellow color with blue fluorescence. (2) Dissolve 20 mg each of Clebopride Malate and Clebopride Malate RS in water to make 100 mL, add water to 10 mL each of these solutions to make 100 mL and use these solutions as test solution and standard solution respactively. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Clebopride Malate and Clebopride Malate RS as directed in the potassium bromide disk method under the infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) Dissolve 5 mg of Clebopride Malate in ethanol to make 10 mL and use this solution as the test solution. Separately, dissolve 5 mg of Clebopride Malate RS in ethanol to make 10 mL and use this solution as the standard solution (1). Dissolve 5 mg of Clebopride Malate RS and 5 mg of metoclopramide hydrochloride RS in ethanol to make 10 mL and use this solution as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of test solution and standard solution as bands of 10 mm on a plate of siliga gel with a fluorescent indicator for thin-layer chromatography. Develop the plates with a mixture of toluene, methanol, acetone and concentrated ammonia TS (70 : 14 : 14 : 2) to a distance of about 15 cm and air-dry the plate and examine under ultraviolet light (main wavelength : 254 nm). The main spot from test solution has the same Rf value as the spot from standard solution (1). This test is valid when

Operating conditions Detector : An ultraviolet-visible absorption photometer (wavelength: 215 nm)

KP 9 227
Column : A stainless steel column, about 4.0 mm in inside diameter and about 12 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature : A room temperature. Mobile phase : A mixture of sodium heptane sulfonate solution (pH 2.5) and acetonitrile (80 : 20). Flow rate : 1.0 mL/minute. System suitability Perform the test with the standard solution (2) according to the above conditions, adjust the sensitity of the system so that the heights of the peaks in the chromatogram obtained are at least 30% of the full scale of the recorder. This test is valid when the retention time of the second peak (clebopride) is about 15 minutes and the relative retention time to the first peak is about 0.45. Time span of measurement : About 2 times as long as the retention time of Clebopride.
Description Clemastine Fumarate is a white, crystalline powder and is odorless. Clemastine Fumarate is sparingly soluble in methanol or in glacial acetic acid, slightly soluble in ethanol, very slightly soluble in ether and practically insoluble in water. Identification (1) To 5 mg of Clemastine Fumarate, add 5 mL of sulfuric acid and shake to dissolve: a yellow color is observed. Slowly drop this solution into 10 mL of water: the yellow color immediately disappears. (2) To 10 mg of Clemastine Fumarate, add 1 mL of fuming nitric acid and evaporate on a water-bath to dryness. Then add 2 mL of diluted hydrochloric acid (1 in 2) and 0.2 g of zinc powder, heat for 10 minutes on a waterbath, cool and filter. Add 20 mL of water to the filtrate. The solution responds to the Qualitative Tests for primary aromatic amines. (3) To 5 mL of a solution of Clemastine Fumarate (1 in 50000), add 5 mL of p-dimethyl-aminobenzaldehyde TS and warm for 10 minutes: a red-purple color is observed. (4) Perform the test with Clemastine Fumarate as directed under the Flame Coloration Test (2): a green color is observed. (5) Dissolve 40 mg of Clemastine Fumarate and 10 mg of fumaric acid in 2 mL each of a mixture of ethanol and water (4 : 1) by gentle warming and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of isopropyl ether, formic acid and water (90 : 7 : 3) to a distance of about 10 cm and air-dry the plate. Examine the plate under ultraviolet light (main wavelength: 254 nm): the spot with larger Rf value

Sodium heptane sulfonate solution (pH 2.5) Dissolve 1.0 g of sodium heptane sulfonate in water to make 1000 mL, adjust to pH 2.5 with phosphoric acid.
Loss on Drying not more than 0.5% (1 g, 105 C, constant mass). Residue on Ignition not more than 0.1% (1 g). Assay Weigh accurately about 0.4 g of Clebopride Malate, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

1 mL of 0.1 mol/L Perchloric acid = 50.80 mg of C20H24ClN3O2C4H6O5

Packaging and Storage well-closed containers..

Preserve in light-resistant,

from the test solution has the same Rf value as the spot from the standard solution.
Specific Optical Rotation [ ]20 D : Between +16 and +18 (after drying, 0.1 g, methanol, 10 mL, 100 mm). Melting Point composition).

Clemastine Fumarate
Cl CH3 N H CH3 O H HO2C C C CO2H H

Between 176 C and 180 C (with de-

C21H26ClNOC4H4O4: 459.96 Clemastine Fumarate, when dried, contains not less than 98.5% and not more than 101.0% of clemastine fumarate (C21H26ClNOC4H4O4).

Purity (1) Clarity and color of solution Dissolve 0.5 g of Clemastine Fumarate in 10 mL of methanol by warming: the solution is clear and colorless. (2) Heavy metalsPerform the test with 1.0 g of Clemastine Fumarate according to Method 2. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Clemastine Fumarate according to Method 3 and perform the test (not more than 2 ppm). (4) Related SubstancesDissolve 0.10 g of Clemastine Fumarate in 5 mL of methanol and use this solution

228 Monographs, Part I

as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 250 mL and use this solution as the standard solution (1). Pipet 5 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform, methanol and strong ammonia water (90 : 10 : 1) to a distance of about 15 cm and air-dry the plate. After spraying evenly Dragendorffs TS on the plate, immediately spray evenly hydrogen peroxide TS: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution (1) and not more than 2 spots from the test solution are more intense than the spot from the standard solution (2).
Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.4 g of Clemastine Fumarate, previously dried, dissolved in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Identification (1) Determine the infrared spectra of Clenbuterol Hydrochloride and Clenbuterol Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Dissolve 10 mg of Clenbuterol Hydrochloride in 10 mL of methanol and use this solution as the test solution. Separately, dissolve 10 mg of Clenbuterol Hydrochloride RS in 10 mL of methanol and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the with a plate mixture of toluene, ethanol and ammonia TS (15:10:0.15) a distance of about 10 cm, dry the plate. Splay evenly 1% sodium nitrite solution in 1 mol/L hydrochloric acid, after standing for 10 minutes, dip the plate in 0.4% naphthylethylenediamine dihydrochloride solution in methanol and remove the plate from the solution and air-dry the plate: the spots from the test solution and the standard solution show same color and same Rf -value. (3) The water solution of Clenbuterol Hydrochloride (1 in 100) exhibits the Qualitative Reaction (2) of chlorides. Specific Optical Rotation [ ]20 D : Between -0.10 and +0.10 (0.30 g, water, 10 mL, 100 mm) pH Dissolve about 0.5 g of Clenbuterol Hydrochloride in 10 mL of water: the pH of this solution is between 5.0 and 7.0. Purity (1) Clarity and color of solution Dissolve 0.5 g of Clenbuterol Hydrochloride in 10 mL of water: turbidity of this solution is not more intense than the following control suspension.

Each mL of 0.1 mol/L perchloric acid VS = 46.00 mg of C21H26ClNOC4H4O4

Packaging and Storage Preserve in tight containers.

Clenbuterol Hydrochloride
OH Cl H N CH3

HCl
CH3 H3C H2N Cl

C12H18Cl2N2OHCl : 313.65 Clenbuterol Hydrochloride contains not less than 99.0% and not more than 101.0% of clenbuterol hydrochloride (C12H18Cl2N2OHCl), calculated on the anhydrous basis.
Description Clenbuterol Hydrochloride is a white, crystalline powder. Clenbuterol Hydrochloride is soluble in water or in ethanol and slightly soluble in acetone. Melting pointAbout 173 C (decomposition)

Control suspensionDissolve 1.0 g of hydrazine sufate in water to make exactly 100 mL, allow to stand for between 4 and 6 hours. Add 25.0 mL of this solution to the solution dissolving 2.5 g of hexamine in water, mix well, allow to stand for 24 hours. Preserve in glass containers and use within 2 months. When this solution is used, add water to 15.0 mL of this suspension to make 1000 mL, mix well 90.0 mL of water and 10.0 mL of this suspension, and use this solution as the control suspension.
(2) Related substancesDissolve 0.1 g of Clenbuterol Hydrochloride in the mobile phase to make exactly 50 mL, use this solution as the test solution. Dilute 0.1 mL of the test solution with water to make 100 mL, use this solution as the standard solution. Perform the test with 5L each of test solution and the standard solution, as directed under the Liquid Chromatography according

KP 9 229
to the following conditions, measure the peak area of each solution according to the automatic integration method. The area of any peak other than the principal peak from the test solution is not greater than the area of the principal peak from the standard solution (0.1%) and the sum of the areas of all the peaks other than the principal peak form the test solution is not greater than 2 times the area of the principal peak from the standard solution (0.2%). Disregard any peak with an area less than 0.1 times the area of the principal peak from the standard solution.

Clindamycin Hydrochloride
H3C H N H H N H Cl

CH3 O HO O S

H3C

Operating conditions Detector: An ultraviolet-visible absorption photometer (wavelength: 215 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 12.5 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter)., Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of phosphate buffer solution, methanol and acetonitrile (60 : 20 : 20). Flow rate : 0.5 mL/minute System suitability System performance: Dissolve 10 mg of clenbuterol related substances (1) RS {1-(4-amino-3,5dichlorophenyl)-2-[(1,1-dimethylethyl)amino]ethanol (cienbuterol-ketone)} with 20 mL of mobile phase, add 5 mL of the test solution, and add mobile phase to make 50 mL. When the procedure is run with 5L of this solution as directed under the above operating conditions: the resolution between the related substance peak and clebuterol peak is not less than 2.5. Phosphate buffer solutionDissolve sodium decanesulfonate and 5 g of potassium dihydrogen phosphate in 900 mL of water, adjust to pH 3.0 with dilute phosphoric acid and dilute to 1000 mL with water.
Water Not more than 1.0 % (volumetric titration, direct titration). Residue on Ignition Not more than 0.1 % (1 g). Assay Dissolve accurately about 0.25 g in 50 mL of ethanol and add 5.0 mL of 0.01 mol/L hydrochloric acid. Titrate with 0.1 mol/L sodium hydroxide. (potentiometric titration Endpoint Detection Method in Titrimetry). Read the volume added between 1st equivalent point and 2nd equivalent point of inflexion.

OH

HCl

C18H33ClN2O5SHCl: 461.44 Clindamycin Hydrochloride contains not less than 800 g (potency) of per mg of clindamycin (C18H33ClN2O5S: 424.98), calculated on the anhydrous basis.
Description Clindamycin Hydrochloride is a whtie to grayish white crystalline powder, and is odorless. Clindamycin Hydrochloride is freely soluble in water, in pyridine, or in methanol, slightly soluble in ethanol, and practically insoluble in acetone or in chloroform. Identification (1) Determine the absorption spectra of Clindamycin Hydrochloride and Clindamycin Hydrochloride RS as directed in paste method under Infrared Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wave numbers. If any difference appears between the spectra, dissolve each of Clindamycin Hydrochloride and Clindamycin Hydrochloride RS in an appropriate solvent, evaporate the solvent to dryness, and repeat the test with the resudes. (2) Dissolve 50 mg each of Clindamycin Hydrochloride and Clindamycin Hydrochloride RS in 10 mL of methanol, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methylethylketone, acetone and water (8:4:1) to a distance of about 10 cm, and air-dry the plate. Spray 0.5 % potassium permanganate solution on the plate, and after 10 minutes spray 0.2 % bromophenol blue solution: The blue spots obtained from the test solution are the same with the corresponding spots from the standard solution in the Rf value. (3) Determine the retention times according to the procedure of Assay: the retention time of the principal peak in the chromatogram obtained with test solution is the same as that of the principal peak in the chromatogram obtained with the standard solution. (4) Clilndamycin Hydrochloride responds to the Qualitative Tests for chloride. Specific Optical Rotation

1 mL of 0.1 mol/L sodium hydroxide = 31.365 mg of C12H18Cl2N2OHCl

Packaging and Storage Preserve in well-closed containers.

[ ]25 D : +135 ~ +150(0.5 g,

230 Monographs, Part I

water, 25 mL, 200 mm).
pH The pH of a solution obtained by dissolving 1 g of Clindamycin Hydrochloride in 10 mL of water is between 3.0 and 5.5. Sterility Test It meets the requirement, when Clindamycin Hydrochloride is used in a sterile preparation. Pyrogen Test It meets the requirement, when Clindamycin Hydrochloride is used in a sterile preparation,. Weigh an appropriate amount of Clindamycin Hydrochloride, dissolve in Isotonic Sodium Chloride Injecton to make the solution so that each mL contains 5.0 mg, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not less than 3.0 % and not more than 6.0 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg (potency) each of Clindamycin Hydrochloride and Clilndamycin Hydrochloride RS, dissolve in the mobile phase to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas AT and AS, of clindamycin for the test solution and the standard solution.

than 90.0 % and not more than 120 % of the labeled amount of clindamycin (C18H33ClN2O5S: 424.98).
Mehtod of Preparation Prepare as directed under Capsules, with Clindamycin Hydrochloride. Identification (1) Proceed as directed in the Identification (2) under Clindamycin Hydrochloride. (2) Determine the retention times according to the procedure of Assay: the retention time of the principal peak in the chromatogram obtained with test solution is the same as that of the principal peak in the chromatogram obtained with the standard solution Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Water Not more than 7.0 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately the mass of contents of not less than 20 Clindamycin Hydrochloride Capsules. Weigh accurately about 45 mg (potency) according to the labeled amount, place in a 15 mL stopperd glass centrifuge tube, add 3.0 mL of 1 % sodium carbonate solution and 3.0 mL of chloroform, centrifuge and discard the aqueous layer, add 1 g of sodium sulfate anhydrous and mix with shaking, add 1.0 mL of chloroform, 1.0 mL of the internal standard solution and 0.6 mL of glacial acetic acid, close the stopper, and heat at 100 C for 2.5 hours and cool down, centrifuge, and take the supernatant solution. Use this solution as the test solution. Separetely, weigh accurately about 45 mg (potency) of Clindamycin Hydrochloride RS, prepare the standard solution with the same procedure as the above test solution preparation. Perform the test with 1 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following operating conditions, and determine the ratios, QT and QS, of the peak area of clindamycin to that of the internal standard.

Amount [g (potency)] of clindamycin (C18H33ClN2O5S) = amount [g (potency)] of Clindamycin Hydrochloride RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 205 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Mobile phase: Dissolve 6.8 g of potassium dihydrogen phosphate in water to make 1000 mL, and adjust the pH to 7.5 with 8 mol/L potassium hydroxide solution. To 500 mL of this solution, add 500 mL of acetonitrile. Flow rate: 1.0 mL per minute.
Packaging and Storage Preserve in tight containers.

Amount [g (potency)] of clindamycin (C18H33ClN2O5S) = Amount [g (potency)] of Clindamycin Hydrochloride RS

QT QS

Internal standard solutionWeigh an appropriate amount of cholestane, dissolve in pyridine to make a solution so that each mL contains 3 mg. Operating conditions Detector: A hydrogen flame-ionization detector Column: A glass column, about 3 mm in inside diameter and about 1 m in length, packed with diatomaceous earth washed with acid and alkali for gas chromatography impregnated with 1 % dimethyl polysiloxan rubber.

Clindamycin Hydrochloride Capsules

Clindamycin Hydrochloride Capsules contains not less

KP 9 231
Column temperature: 200 C Detector temperature: 215 C Carrier gas: Helium Flow rate: 120 mL per minute
Packaging and Storage Preserve in tight containers.

mycin Phosphate is used in a sterile preparation. Weigh an appropriate amount of Clindamycin Phosphate, dissolve in Isotonic Sodium Cholride Injection to make the solution so that each mL contains 5.0 mg, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit.
Histamine It meets the requirement, when Clindamycin Phosphate is used in a sterile preparation. Weigh appropriate an amount of Clindamycin Phosphate, dissolve in Isotonic Sodium Chloride Injection, make a solution so that each mL contains 5 mg, and use the solution as the test solution. Water Not more than 6.0 % (0.2 g, volumetric titration, direct titration). Assay Perform the test according to Assay in Clindamycin Hydrochloride. Use Clindamycin Phosphate RS as the standard substance. Packaging and Storage Preserve in tight containers.

Clindamycin Phosphate
CH3

H
N

H N

Cl

CH3

O
H 3C

OH HO O S CH3 O HO P OH O

C18H34ClN2O8PS: 504.97 Clindamycin Phosphate contains not less than 758 g (potency) of per mg of clindamycin (C18H33ClN2O5S: 424.98), calculated on the anhydrous basis.
Description Clindamycin Phosphate is a whtie to pale yellowish white crystalline powder. Clindamycin Phosphate is freely soluble in water, sparingly soluble in methanol, and practically insoluble in ethanol or in ether. Identification (1) Proceed with Clindamycin Phosphate and Clindamycin Phosphate RS as directed in the Identification (1) under Clindamycin Hydrochloride. Dry 0.2 g each of Clindamycin Phosphate and Clindamycin Phosphate RS at 100 C for 2 hours, and keep them at room temperature for 1 hour, and use them for the test. (2) Weigh about 50 mg each of Clindamycin Phosphate and Clindamycin Phosphate RS, dissolve each in 10 mL of water, and use these solutions as the test solution and the standard solution. Perform the test as directed in the Identification (2) under Clindamycin Hydrochloride. (3) An aqueous solution of Clindamycin Phosphate responds to the Qualitative Tests for phosphate salt. (4) Determine the retention times according to the procedure of Assay: the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the standard solution. pH The pH of a solution obtained by dissolving 0.1 g of Clindamycin Phosphate in 10 mL of water is between 3.5 and 4.5. Sterility Test It meets the requirement, when Clindamycin Phosphate is used in a sterile preparation. Pyrogen Test It meets the requirement, when Clinda-

Clinofibrate
CH2CH3 O CCO2H CH3

CH3 O CCO2H CH2CH3

C28H36O6: 468.58 Clinofibrate, when dried, contains not less than 98.5% and not more than 101.0% of clinofibrate (C28H36O6).
Description Clinofibrate is a white to yellowish white powder, is odorless and has no taste. Clinofibrate is freely soluble in methanol, in dehydrated ethanol, in acetone or in ether and practically insoluble in water. A solution of Clinofibrate in methanol (1 in 20) shows no optical rotation. Melting pointAbout 146 C (with decomposition). Identification (1) Determine the absorption spectra of solutions of Clinofibrate and Clinofibrate RS in dehydrated ethanol (1 in 50000) under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Clinofibrate and Clinofibrate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of

232 Monographs, Part I

absorption at the same wavenumbers.
Purity (1) Heavy metalsProceed with 1.0 g of Clinofibrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Clinofibrate according to Method 3 and perform the test (not more than 2 ppm). (3) Related substancesDissolve 0.10 g of Clinofibrate in 10 mL of acetone and use this solution as the test solution. Pipet 1 mL of the test solution and add acetone to make exactly 50 mL. Pipet 5 mL of this solution, add acetone to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 50 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, cyclohexane and glacial acetic acid (12 : 5 : 3) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Isomer ratio To 50 mg of Clinofibrate, add 0.4 mL of thionyl chloride, stopper tightly, heat in a water-bath at 60 C for 5 minutes with occasional shaking and evaporate the excess thionyl chloride at a temperature not exceeding 60 C under reduced pressure. Dissolve the residue in 2 mL of toluene, previously dried with synthetic zeolite for drying, add 2 mL of a solution of D-(+)-methylbenzylamine in toluene, previously dried with synthetic zeolite for drying (3 in 100), mix gently, allow to stand for 10 minutes and evaporate the toluene at a temperature not exceeding 60 C under reduced pressure. Dissolve the residue in 5 mL of chloroform and use this solution as the test solution. Perform the test with 5 L of the test solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area, Aa , Ab and Ac , of three peaks appear in order near the retention time of 40 minutes: a value, Ab /(Aa + Ab + Ac ) 100, is between 40 and 70.

Selection of column: Proceed with 5 L of the test solution under the above operating conditions. Use a column giving a complete separation of the three peaks.
Loss on Drying Not more than 1.0% (1 g, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.45 g of Clinofibrate, previously dried, dissolve in 40 mL of dehydrated ethanol, add 30 mL of water and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L sodium hydroxide VS = 23.429 mg of C28H36O6

Packaging and Storage Preserve in tight containers.

Clobetasol Propionate
O H3C H HO H3C H H CH3 F O H H3C O O Cl

C25H32ClFO5: 466.97 Clobetasol Propionate, when dried, contains not less than 97.0% and not more than 102.0% of clobetasol propionate (C25H32ClFO5).
Description Clobetasol Propionate is a white crystalline powder. Clobetasol Propionate is freely soluble in acetone or in dichloromethane, sparingly soluble in ethanol and practically insoluble in water. Melting point196 C (with decomposition). Identification (1) Determine the infrared spectra of Clobetasol Propionate and Clobetasol Propionate RS, previously dried, respectively, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The retention time of the principal peak in the chromatogram of the Assay preparation corresponds to that of the standard preparation, both relative to the internal standard, as obtained in the Assay.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 20 C. Mobile phase: A mixture of hexane and isopropanol (500 : 3). Flow rate: Adjust the flow rate so that the retention time of the peak appearing first is about 35 minutes.

KP 9 233
lution as directed under Liquid Chromatography according to the operating conditions of the related substances in Purity and calculate the ratios, QT and QS , of the peak area of clobetasol propionate to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of clobetasol propionate (C25H32ClFO5) = amount (mg) of Clobetasol Propionate RS
QT QS

Specific Optical Rotation

[ ]20 D : Between +96

and

+104 o (after drying, 0.1 g, 1,4-dioxane, 10 mL, 100 mm).

Purity Related substancesWeigh 10.0 mg of Clobetasol Propionate, dissolve in the mobile phase to make 100 mL and use this solution as the test solution (1). Pipet 1 mL of the test solution (1), add the mobile phase to make exactly 50 mL and use this solution as the test solution (2). Separately, weigh accurately about 10 mg of Clobetasol Propionate RS and about 5 mg of 9-Fluoro11-hydroxy-16-methyl-3-oxoandrosta-1,4-diene-17(R) -spiro-2'-[4'-chloro-5'-ethylfurane-3-(2'H)-one] RS, dissolve in the mobile phase to make 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solutions (1) and (2) and the standard solution as directed under the Liquid Chromatography: the area of each peak other than the principal peak of the test solution (1) is not more than 0.5 times (1.0%) of the area of the principal peak of the test solution (2) and the total peak areas of all related substances is not more than 1.25 times (2.5%) of the area of the principal peak of the test solution (2). But, omit the peak which peak area is less than 0.025 times (0.05%) of the area of the principal peak among the peaks of the test solution (2).

Internal standard solutionA solution of Beclometasone Dipropionate in mobile phase (1 in 5000).

Packaging and Storage Preserve in well-closed, lightresistant containers.

Clobetasol Propionate Cream

Clobetasol Propionate Cream contains not less than 90.0% and not more than 115.0% of the labeled amount of clobetasol propionate (C25H32ClFO5:466.97).
Method of Preparation Prepare as directed under Creams, with Clobetasol Propionate. Identification (1) Transfer, accurately weighed, a portion of Clobetasol Propionate Cream, equivalent to about 0.75 mg of clobetasol propionate(C25H32ClFO5), to a 25 mL centrifugal tube, add 10 mL of methanol, stopper, heat in the water-bath at 60 C for 4 minutes, then take out from the water-bath and extract while vigorously shaking. Repeat this procedure several times, cool to room temperature, add 3.5 mL of water and centrifuge for about 10 minutes. Transfer 10 mL of a clear supernatant to a separator, add 1 g of sodium chloride and 10 mL of water and mix well. To this solution, add 5 mL of dichloromethane, extract after sonication for 1 minute, take the dichloromethane layer, evaporate to dryness under the current of nitrogen, dissolve the residue in 0.5 mL of dichloromethane and use this solution as the test solution. Proceed as directed in the Identification (1) under Clobetasol Propionate Ointment. (2) The retention time of the principal peak in the chromatogram of the Assay preparation corresponds to that of the standard preparation, both relative to the internal standard, as obtained in the Assay. Assay Weigh accurately a portion of Clobetasol Propionate Cream, equivalent to about 1 mg of clobetasol propionate (C25H32ClFO5), add 10 mL of dehydrated ethanol, warm in a water-bath with frequent shaking until the cream is completely dispersed and cool in the icebath for 30 minutes. Pipet 5 mL of clear solution after centrifuge, add 5.0 mL of the internal standard solution, mix well and use this solution as the test solution. With

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, having octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of 0.05 mol/L monobasic potassium phosphate (adjust to pH 2.5 by adding phosphate), acetonitrile and methanol (425 : 475 : 100). Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution according to the above operating conditions, the resolution between their peaks of Clobetasol Propionate and 9-fluoro-11-hydroxy16-methyl-3-oxoandrosta-1,4-diene-17(R)-spiro-2'-[4'chloro-5'-ethylfurane-3-(2'H) -one] is not less than 3.0.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 10 mg each of Clobetasol Propionate and Clobetasol Propionate RS, previously dried, dissolve each in mobile phase, add exactly 100 mL of the internal standard solution, add mobile phase to make 250 mL and use these solution as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard so-

234 Monographs, Part I

this test solution, proceed as directed in the Assay under Clobetasol Propionate Ointment. Amount (mg) of clobetasol propionate(C25H32ClFO5) = amount (mg) of Clobetasol Propionate RS
QT 1 Q S 10

Internal standard solutionA solution of Beclometasone Dipropionate in 50% ethanol (1 in 5000).

Packaging and Storage Preserve in tight containers. Store at a temperature not exceeding 30 C.

Clobetasol Propionate Ointment

Clobetasol Propionate Ointment contains not less than 90.0% and not more than 115.0% of the labeled amount of clobetasol propionate (C25H32-ClFO5: 466.97).
Method of Preparation Prepare as directed under Ointments, with Clobetasol Propionate. Identification (1) Transfer a portion of Clobetasol Propionate Ointment, equivalent to about 0.5 mg of clobetasol propionate (C25H32-ClFO5) according to the labled amount, to a 25 mL centrifugal tube, add 10 mL of methanol, stopper, heat in the water-bath at 70 C for 4 minutes, then take out from water-bath and extract while vigorously shaking. Repeat this operation several times, cool in the ice-bath for 5 minutes and centrifuge for about 10 minutes. Pipet 5 mL of a clear supernatant, evaporate to dryness under the current of nitrogen, dissolve the residue in 0.5 mL of dichloromethane and use this solution as the test solution. Separately, weigh about 10 mg of Clobetasol Propionate RS, dissolve in dichloromethane to make 20 mL and use this solution as the standard solution. Perform the test with these solutions as directed and the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of dichloromethanes, acetone and absolute ethanol (100 : 10 : 5) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 245 nm): the principal spot from the test solution shows the same Rf value as the principal spot from the standard solution. (2) The retention time of the principal peak in the chromatogram of the test solution in Assay corresponds to that of the standard solution, both relative to the internal standard, as obtained in the Assay. Assay Weigh accurately a portion of Clobetasol Propionate Ointment, equivalent to about 1 mg of clobetasol propionate (C25H32ClFO5), add 10 mL of absolute ethanol, warm in the water-bath with frequent shaking until

the ointment is completely dispersed and cool in the icebath for 30 minutes. Pipet exactly 5 mL of clear solution after centrifuge, add 5 mL of the internal standard solution, mix well and use this solution as the test solution. Separately, weigh accurately about 10 mg of Clobetasol Propionate RS, dissolve in 50% ethanol to make 100 mL, transfer exactly 5 mL of this solution to a volumetric flask, add 5.0 mL of the internal standard solution, mix well and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under Liquid Chromatography according operating to the following conditions and calculate the ratios, QT and QS , of the peak area of Clobetasol Propionate to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of clobetasol propionate(C25H32ClFO5) = amount (mg) of Clobetasol Propionate RS
QT 1 Q S 10

Internal standard solutionA solution of Beclometasone Dipropionate in 50% ethanol (1 in 5000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 10 cm in length, having octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of water and absolute ethanol (55 : 45). Column temperature: 60 C. Flow rate: 2 mL/minute.
Packaging and Storage Preserve in tight containers. Store at a temperature not exceeding 30 C.

Clofibrate
CH3 O Cl O C CH3 C OC2H5

Cl2Hl5ClO3: 242.70 Clofibrate contains not less than 98.0% and not more than 101.0% of clofibrate (Cl2Hl5ClO3), calculated on the anhydrous basis.
Description Clofibrate is a colorless or pale yellow, clear, oily liquid, has a characteristic odor and taste, which is bitter at first and subsequently sweet. Clofibrate is miscible with methanol, with ethanol, with dehydrated ethanol, with ether or with hexane and practi-

KP 9 235
cally insoluble in water. Clofibrate is gradually decomposed by light.
Identification (1) Determine the absorption spectra of solutions of Clofibrate and Clofibrate RS in ethanol (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similsr intensities of absorption at the same wavelengths. Determine the absorption spectra of solutions of Clofibrate and Clofibrate RS in dehydrated ethanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Clofibrate and Clofibrate RS as directed in the liquid film method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Refractive Index Specific Gravity
20 : Between 1.500 and 1.505. nD 20 : Between 1.137 and 1.144. d 20

tion as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of p-chlorophenol to that of the internal standard, for the test solution and the standard solution, respectively: QT is not greater than QS .

Internal standard solutionA solution of pethoxyphenol in the mobile phase (1 in 30000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 275 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with cyanopropylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of hexane, isopropanol and glacial acetic acid (1970 : 30 : 1). Flow rate: Adjust the flow rate so that the retention time of Clofibrate is about 2 minutes. Selection of column: Dissolve 0.1 g of Clofibrate, 6 mg of p-chlorophenol and 6 mg of p-ethoxyphenol in 1000 mL of hexane. Proceed with 20 L of this solution under the above operating conditions and calculate the resolution. Use a column giving elution of Clofibrate, pchlorophenol and p-ethoxyphenol in this order, with the resolution between the peaks of Clofibrate and pchlorophenol being not less than 5.0 and with the resolution between the peaks of p-chlorophenol and pethoxyphenol being not less than 2.0.
Water Not more than 0.2% (5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Clofibrate, add exactly 50 mL of 0.1 mol/L potassium hydroxide-ethanol VS and heat in a water-bath under a reflux condenser with a carbon dioxide absorbing tube (soda lime) for 2 hours with frequent shaking. Cool and titrate immediately the excess potassium hydroxide with 0.1 mol/L hydrochloric acid VS (indicator: 3 drops of phenolphthalein TS). Perform a blank determination and make any necessary connection.

Purity (1) AcidDissolve 2.0 g of Clofibrate in 100 mL of neutralized ethanol and add 1 drop of phenolphthalein TS and 0.20 mL of 0.1 mol/L sodium hydroxide VS: the solution is red in color. (2) Heavy metalsProceed with 2.0 g of Clofibrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicTo 5.0 g of Clofibrate, add 20 mL of nitric acid and 5 mL of sulfuric acid and heat until white fumes are evolved. After cooling, if necessary, add further 5 mL of nitric acid, heat until white fumes are evolved and repeat this procedure until the solution is colorless to pale yellow. After cooling, add 15 mL of saturated ammonium oxalate solution and heat again until white fumes are evolved. Cool, add water to make 25 mL, use 5 mL of this solution as the test solution and perform the test (not more than 2 ppm).

Standard solutionPrepare a solution according to the above procedure without using Clofibrate as the blank. Transfer 5 mL of the solution to a generator bottle, add 2.0 mL of standard arsenic solution and then proceed as directed in the test solution.
(4) p-ChlorophenolTo 1.0 g of Clofibrate, add exactly 1 mL of the internal standard solution, then add the mobile phase to make 5 mL and use this solution as the test solution. Separately, dissolve 10 mg of pchlorophenol in a mixture of hexane and isopropanol (9 : 1) to make exactly 100 mL. Pipet 10 mL of this solution and add a mixture of hexane and isopropanol (9 : 1) to make exactly 50 mL. Pipet 6 mL of this solution, add exactly 4 mL of the internal standard solution, then add the mobile phase to make exactly 20 mL and use this solu-

Each mL of 0.1 mol/L potassium hydroxide-ethanol VS = 24.270 mg of Cl2Hl5ClO3

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236 Monographs, Part I Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 275 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of acetonitrile and diluted phosphoric acid (1 in 1000) (3 : 2). Flow rate: Adjust the flow rate so that the retention time of Clofibrate is about 10 minutes. Selection of column: Dissolve 50 mg of Clofibrate and 0.3 g of ibuprofen in 50 mL of acetonitrile. Proceed with 10 L of this solution under the above operating conditions and calculate the resolution. Use a column giving elution of ibuprofen and clofibrate in this order with the resolution between their peaks being not less than 6.0.
Packaging and Storage well-closed containers.

Clofibrate Capsules
Clofibrate Capsules contain not less than 93.0% and not more than 107.0% of the labeled amount of clofibrate (Cl2Hl5C1O3: 242.70).
Method of Preparation Capsules, with Clofibrate.

Prepare as directed under

Identification Open the capsules and use the contents. Determine the absorption spectrum of a solution of the contents in ethanol (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 278 nm and 282 nm. Determine the absorption spectrum of a solution of the contents in ethanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 224 nm and 228 nm. Purity p-ChlorophenolCut and open not less than 20 Clofibrate Capsules and proceed with 1.0 g of the well mixed contents as directed in the Purity (4) under Clofibrate. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement Assay Weigh accurately not less than 20 Clofibrate Capsules, open the capsules, rinse the inside of the capsules with a small amount of ether after taking out the contents, evaporate the ether by allowing the capsules to stand at room temperature and weigh the capsules accurately. Weigh accurately an amount of the contents, equivalent to about 0.1 g of clofibrate (Cl2Hl5C1O3), dissolve in acetonitrile to make exactly 100 mL. Pipet 5 mL of this solution, add exactly 5 mL of the internal standard solution and use this solution as the test solution. Separately, weigh accurately about 0.1 g of Clofibrate RS, proceed in the same manner as directed for the test solution and use this solution so obtained as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Clofibrate to that of the internal standard, for the test solution and the standard solution, respectively.

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Clomifene Citrate
Cl C C OCH 2CH2N(CH 2CH3)2 HO CH2CO 2H C CO 2H

CH2CO 2H

C26H28ClNOC6H8O7: 598.08 Clomifene Citrate, when dried, contains not less than 98.0% and not more than 101.0% of clomifene citrate (C26H28ClNOC6H8O7).
Description Clomifene Citrate is a white to pale yellowish white powder and is odorless. Clomifene Citrate is freely soluble in methanol or in glacial acetic acid, soluble in ethanol, sparingly soluble in water and practically insoluble in ether. Clomifene Citrate is gradually colored by light. Melting pointAbout 115 C Identification (1) To 2 mL of a solution of Clomifene Citrate in butanol (1 in 200), add 2 mL of Reinecke salt TS: a light red precipitate is produced. (2) Determine the absorption spectra of solutions of Clomifene Citrate and Clomifene Citrate RS in 0.1 mol/L hydrochloric acid TS (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) A solution of Clomifene Citrate in methanol (1 in 200) responds to the Qualitative Tests (1) and (2) for ci-

Amount (mg) of cloabrate (Cl2Hl5C1O3) = amount (mg) of Clofibrate RS, calculated on the anhydrous basis
QT QS

Interanl standard solutionA solution of ibuprofen in the mobile phase (1 in 100).

KP 9 237
trate salt.
Purity (1) Clarity and color of solutionThe solution of 1.0 g of Clomifene Citrate in 30 mL of methanol is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Clomifene Citrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). Loss on Drying Not more than 1.0% (1 g, in vacuum, P2O5, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Isomer Ratio To 0.10 g of Clomifene Citrate, add 10 mL of water and 1 mL of sodium hydroxide TS and extract with three 15 mL volumes of ether. Wash the combined ether extracts with 20 mL of water, add 10 g of anhydrous sodium sulfate to the combined ether extracts, shake for 1 minute, filter and evaporate the ether of the filtrate. Dissolve the residue in 10 mL of chloroform and use this solution as the test solution. Perform the test with 2 L of the test solution as directed under the Gas Chromatography according to the following conditions. Determine the areas of two adjacent peaks, Aa and Ab ,

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Clomifene Citrate Tablets

Clomifene Citrate Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of the clomifene citrate (C26H28ClNOC6H8O7: 598.08).
Method of Preparation Prepare as directed under Tablets, with Clomifene Citrate. Identification Weigh a portion of powdered Clomifene Citrate Tablets, equivalent to 1 g of Clomifene Citrate according to the labeled amount, shake vigorously with 100 mL of chloroform and filter. Concentrate the filtrate in a water-bath, allow to stand at room temperature, collect the crystals formed and wash with a small volume of chloroform. Proceed with the crystals as directed in the Identification under Clomifene Citrate. Disintegration Test It meets the requirement. Uniformity of Dosage Limits It meets the requirement Assay Weigh accurately and powder not less than 20 Clomifene Citrate Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of clomifene citrate (C26H28ClNOC6H8O7), add 50 mL of methanol, shake for 10 minutes and add methanol to make exactly 100 mL. Centrifuge a volume of this solution, pipet 4.0 mL of the supernatant liquid, add methanol to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately 50 mg of Clomifene Citrate RS, previously dried in a desiccator (in vacuum, P2O5) for 3 hours and dissolve in methanol to make 100 mL. Pipet 4.0 mL of this solution and dilute with methanol to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances as directed under the Ultraviolet-visible Spectrophotometry, AT and

having retention times of about 20 minutes, where Aa is the peak area of shorter retention time and Ab is the peak area of longer retention time: Ab /( Aa + Ab ) is between 0.3 and 0.5.

Operating conditions Detector: A hydrogen flame-ionization detector. Column: A column, about 3 mm in inside diameter and about 1 m in length, having methylsilicone polymer coated at the ratio of 1% on siliceous earth for gas chromatography (125 m to 150 m in particle diameter). Column temperature: A constant temperature of about 195 C. Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of the peak for the first elution of clomifene citrate is about 20 minutes. Selection of column: Proceed with 2 L of the test solution under the above operating conditions: use a column giving the resolution between the two peaks being not less than 1.3.
Assay Weigh accurately about 1 g of Clomifene Citrate, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

AS , of the test solution and the standard solution, respectively, at 295 nm. Amount (mg) of clomifene citrate (C26H28ClNOC6H8O7) = amount (mg) of Clomifene Citrate RS
AT AS

Packaging and Storage Preserve in tight containers.

Each mL of 0.1 mol/L perchloric acid VS = 59.81 mg of C26H28ClNOC6H8O7

238 Monographs, Part I

the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Clomipramine Hydrochloride according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.20 g of Clomipramine Hydrochloride in exactly 10 mL of methanol and use this solution as the test solution. Separately, weigh 20 mg of Imipramine Hydrochloride RS, dissolve in methanol to make exactly 100 mL and use this solution as the standard solution (1). Then pipet 1 mL of the test solution and add methanol to make exactly 50 mL. Pipet 5 mL of this solution, add methanol to make exactly 50 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thinlayer Chromatography. Spot 5 L of the test solution and the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, acetone and strong ammonia water (15 : 5 : 1) to a distance of 10 cm and air-dry the plate. Spray evenly potassium dichromate-sulfuric acid TS on the plate: the spots from the test solution corresponding to that from the standard solution (1) is not more intense than the spot from standard solution (1). Each of the spots other than the principal spot and the above spot from the test solution are not more intense than the spot from the standard solution (2).
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.10% (1 g). Assay Weigh accurately about 0.5 g of Clomipramine Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Clomipramine Hydrochloride

N Cl CH3 CH2CH2CH2N CH3 HCl

C19H23ClN2HCl: 351.31 Clomipramine Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of clomipramine hydrochloride (C19H23ClN2HCl).
Description Clomipramine Hydrochloride is a white to pale yellow, crystalline powder and is odorless. Clomipramine Hydrochloride is very soluble in glacial acetic acid, freely soluble in water, in methanol or in chloroform, soluble in ethanol, sparingly soluble in acetic anhydride, slightly soluble in acetone and practically insoluble in ethyl acetate or in ether. Identification (1) Dissolve 3 mg of Clomipramine Hydrochloride in 1 mL of nitric acid: a deep blue color is observed. (2) Determine the absorption spectra of solutions of Clomipramine Hydrochloride and Clomipramine Hydrochloride RS in 0.1 mol/L hydrochloric acid TS (3 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Take 1 g of Clomipramine Hydrochloride in a separator, dissolve in 10 mL of water, add 5 mL of sodium hydroxide TS and extract with two 30 mL volumes of ether [the water layer is used for Identification (4)]. Combine the ether extracts, add 20 mL of water and shake. Take ether layer, dry with a small portion of anhydrous sodium sulfate and filter. Evaporate the combined extracts by warming a water-bath and proceed the test with the residue as directed under the Flame Coloration Test (2): a green color is observed. (4) The solution neutralized by adding dilute nitric acid to the water layer obtained in (3) responds to the Qualitative Tests for chloride. Melting Point Between 192 C and 196 C pH Dissolve 1.0 g of Clomipramine Hydrochloride in 10 mL of water: the pH of this solution is between 3.5 and 5.0. Purity (1) Clarity and color of solution Dissolve 1.0 g of Clomipramine Hydrochloride in 10 mL of water: the solution is clear and colorless to pale yellow. (2) Heavy metalsProceed with 2.0 g of Clomipramine Hydrochloride according to Method 2 and perform

Each mL of 0.1 mol/L perchloric acid VS = 35.131 mg of C19H23ClN2HCl

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Clonazepam

KP 9 239
silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of nitromethane and acetone (10 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.3% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Clonazepam, previously dried, dissolve in 70 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

H N

O2N

N Cl

C15H10ClN3O3: 315.71 Clonazepam, when dried, contains not less than 99.0% and not more than 101.0% of clonazepam (C15H10ClN3O3).
Description Clonazepam is a white to pale yellow, crystal or crystalline powder. Clonazepam is sparingly soluble in acetic anhydride or acetone, slightly soluble in methanol or in ethanol, very slightly soluble in ether and practically insoluble in water. Clonazepam is gradually colored by light. Melting pointAbout 240 C (with decomposition). Identification (1) Determine the absorption spectra of solutions of Clonazepam and Clonazepam RS in methanol (1 in 100000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Clonazepam and Clonazepam RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. (3) Perform the test with Clonazepam as directed under the Flame Coloration Test (2): a green color appears. Purity (1) ChlorideTo 1.0 g of Clonazepam, add 50 mL of water, allow to stand for 1 hour with occasional shaking and filter. Discard the first 20 mL volumes of the filtrate, take the subsequent 20 mL volumes of the filtrate and add 6 mL of dilute nitric acid and water to make 50 mL. Use this solution as the test solution and perform the test. Prepare the control solution as follows: take 0.25ml of 0.01 mol/L hydrochloric acid VS and add 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.022%). (2) Heavy metalsProceed with 1.0 g of Clonazepam according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 0.25 g of Clonazepam in 10.0 mL of acetone and use this solution as the test solution. Pipet 1.0 mL of the test solution, add acetone to make exactly 100 mL, then pipet 1.0 mL of this solution, add acetone to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of

Each mL of 0.1 mol/L perchloric acid VS = 31.571 mg of C15H10ClN3O3

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Clonidine Hydrochloride
Cl H N N

HCl

NH

Cl

C9H9Cl2N3HCl: 266.56 Clonidine Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of clonidine hydrochloride (C9H9Cl2N3HCl).
Description Clonidine Hydrochloride is a white crystal or crystalline powder. Clonidine Hydrochloride is very soluble in methanol, soluble in water or in ethanol, slighlty soluble in glacial acetic acid and practically insoluble in acetic anhydride or ether. Identification (1) To 5 mL of a solution of Clonidine Hydrochloride (1 in 1000), add 6 drops of Dragendorfs TS: an orange precipitate is produced. (2) Determine the absorption spectra of solutions of Clonidine Hydrochloride and Clonidine Hydrochloride RS in 0.01 mol/L hydrochloride VS (3 in 10000), as directed under the Ultraviolet-visible Spectrometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Clonidine Hy-

240 Monographs, Part I

drochloride and Clonidine Hydrochloride RS, previously dried, as directed in potassium chloride disk method under the Infrared Spectorphotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) A solution (1 in 50) of Clonidine Hydrochloride responds to the Qualitative Tests for chloride.
pH Dissolve 1.0 g of Clonidine Hydrochloride in 20 mL of water: the pH of this solution is between 4.0 and 5.5. Purity (1) Clarity and color of solutionThe solution of 1.0 g of Clonidine Hydrochloride in 20 mL of water, is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Clonidine Hydrochloride according to Method 1,.and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicPrepare the test solution with 0.5 g of Clonidine Hydrochloride, according to Method 3 and perform the test (not more than 4 ppm). (4) Related substancesDissolve 0.20 g of Clonidine Hydrochloride in 2.0 mL of methanol and use this solution as the test solution. Pipet 1.0 mL of the test solution and add methanol to make exactly 100 mL. Take each 1.0 mL and 2.0 mL volumes of this solution, add methanol to make exactly 20 mL each and use these solutions as the standard solutions (1) and (2), respectively. Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot each 2 L of the test solution and the standard solutions (1) and (2) on the plate of silica gel. Develop the plate with a mixture of toluene, dioxane, absolute ethanol and strong ammonia water (10 : 8 : 2 : 1) to a distance of 12 cm and air-dry the plate. Dry the plate at 100 C for 1 hour, spray evenly sodium hypochlorite TS and air-dry the plate for 15 minutes. Then, spray evenly potassium iodide-starch TS to the plate. The spots other than the principal and original spots are not more intense than the spot from standard solution (2) and the number of spots, which are more intense than the spot from standard solution (1), other than the principal and original spots is not more than 3. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.4 g of Clonidine Hydrochloride, previously dried, dissolve in 30 mL of glacial acetic and warm to dissolve. Cool, add 70 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

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Cloperastine Hydrochloride

H O N Cl

HCl

and enatiomer C20H24ClNOHCl: 366.33 Cloperastine Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of cloperastine hydrochloride (C20H24ClNOHCl).
Description Cloperastine Hydrochloride is a white crystal or crystalline powder. Cloperastine Hydrochloride is very soluble in water, in methanol, in ethanol or in glacial acetic acid, and sparingly soluble in acetic anhydride. A solution of Cloperastine Hydrochloride (1 in 10) shows no optical rotation. Identification (1) Determine the absorption spectra of solutions of Cloperastine Hydrochloride and Cloperastine Hydrochloride RS in 0.1 mol/L hydrochloric acid TS (1 in 2500) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Cloperastine Hydrochloride and Cloperastine Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Shake 10 mL of a solution of Cloperastine Hydrochloride (1 in 100) with 2 mL of ammonia TS and 20 mL of ether, and filter. Acidify the filtrate with dilute nitric acid: the solution responds to the Qualitative Tests for chloride. Melting Point

Between 148 C and 152 C

Each mL of 0.1 mol/L perchloric acid VS = 26.656 mg of C9H9Cl2N3HCl

Purity (1) Heavy metalsProceed with 1.0 g of Cloperastine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm).

KP 9 241
(3) Related substancesDissolve 40 mg of Cloperastine Hydrochloride in 50 mL of the mobile phase and use this solution as the test solution. Pipet 1.0 mL of the test solution and add the mobile phase to make exactly 200 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography, according to the following conditions, and determine each peak area of both solutions by the automatic integration method: the areas of two peaks corresponding to the relative retention times of about 0.8 and 3.0 to the retention time of cloperastine obtained from the test solution are not larger than the peak area from the standard solution, respectively, and the area of the peak corresponding to the relative retention time of about 2.0 to cloperastine is not larger than 5/3 of the peak area from the standard solution, and the areas of the peaks other than cloperastine and other than the peaks mentioned above are all not larger than 3/5 of the peak area from the standard solution. The total area of these peaks is not larger than 2 times of the peak area of cloperastine from the standard solution . mixture of acetic anhydride and glacial acetic acid (7:3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 36.63 mg of C20H24ClNOHCl
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Clotiazepam
CH3 O C2H5 S N

N Cl

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 222 nm). Column: A stainless steel column, about 5 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of methanol, 0.1 mol/L monobasic potassium phosphate TS and perchloric acid (500:250:1). Flow rate: Adjust the flow rate so that the retention time of cloperastine is about 7 minutes. Selection of column: Dissolve 30 mg of Cloperastine Hydrochloride and 40 mg of benzophenone in 100 mL of the mobile phase. To 2.0 mL of this solution add the mobile phase to make 50 mL. Perform the test with 20 L of this solution under the above operating conditions: Use a column giving elution of cloperastine and benzophenone in this order with the resolution between these peaks being not less than 6. Detection sensitivity: Adjust the detection sensitivity so that the peak height of cloperastine obtained from 20 L of the standard solution is about 30% of the full scale. Time span of measurement: About 4 times as long as the retention time of cloperastine, beginning after the solvent peak.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Cloperastine Hydrochloride, previously dried, dissolve in 70 mL of a

Cl6H15ClN2OS: 318.82 Clotiazepam, when dired, contains not less than 98.5% and not more than 101.0% of clotiazepam (Cl6H15ClN2OS).
Description Clotiazepam is a white to pale yellowish white crystal or crystalline powder, is odorless and has a slightly bitter taste. Clotiazepam is very soluble in chloroform, freely soluble in methanol, in ethanol, in acetone, in glacial acetic acid or in ethyl acetate, soluble in ether and practically insoluble in water. Clotiazepam dissolves in 0.1 mol/L hydrochloric acid TS. Clotiazepam is gradually colored by light. Identification (1) Dissolve 10 mg of Clotiazepam in 3 mL of sulfuric acid: the solution shows a pale yellow fluorescent under ultraviolet light. (2) Determine the absorption spectra of solutions of Clotiazepam and Clotiazepam RS in 0.1 mol/L hydrochloric acid TS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra of absorption exhibit similar intensities at the same wavelengths. (3) Prepare the test solution with 10 mg of Clotiazepam as directed under the Oxygen Flask Combustion Method, using 10 mL of diluted strong hydrogen peroxide water (1 in 5) as the absorbing liquid. Apply a small amount of water to the upper part of the Apparatus A, pull out C carefully, wash C, B and the inner side of A with 15 mL of methanol and use the obtained solution as the test solution. Add 0.5 mL of dilute nitric acid to 15 mL of the test solution: this solution responds to the Qualitative Tests (2) for chloride. The remaining test so-

242 Monographs, Part I

lution responds to the Qualitative Tests (1) for sulfate.
Melting Point

tight containers.

Between 106 C and 109 C.

Purity (1) Clarity and color of solutionDissolve 1.0 g of Clotiazepam in 10 mL of ethanol: the solution is clear and has no more color than the following control solution.

Control solutionTo 5 mL of Color Matching Fluid C, add 0.01 mol/L hydrochloric acid TS to make 10 mL.
(2) ChlorideTo 1.0 g of Clotiazepam, add 50 mL of water, shake for 30 minutes and filter. To 30 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.015%). (3) Heavy metalsProceed with 2.0 g of Clotiazepam according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Clotiazepam, according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.25 g of Clotiazepam in 10.0 mL of acetone and use this solution as the test solution. Pipet 1.0 mL of the test solution, add acetone to make exactly 20 mL, pipet 2.0 mL of this solution, add acetone to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform and acetone (5 : 1) to a distance of about 10 cm and airdry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 80 C, 3 hours). Residue on Ignition

Clotrimazole
N N C

Cl

C22H17ClN2: 344.84 Clotrimazole, when dried, contains not less than 98.0% and not more than 101.0% of clotrimazole (C22H17ClN2).
Description Clotrimazole is a white, crystalline powder, is odorless and tasteless. Clotrimazole is freely soluble in dichloromethane or in glacial acetic acid, soluble in dimethylformamide, in methanol or in ethanol, slightly soluble in ether and practically insoluble in water. Identification (1) Dissolve 0.1 g of Clotrimazole in 10 mL of 5 mol/L hydrochloric acid TS by heating and cool. To this solution, add 3 drops of Reinecke salt TS: a pale red precipitate is produced. (2) Determine the absorption spectra of solutions of Clotrimazole and Clotrimazole RS in methanol (1 in 5000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Clotrimazole and Clotrimazole RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) Perform the test with Clotrimazole as directed under the Flame Coloration Test (2): a green color appears. Melting Point

Not more than 0.1% (1 g).

Assay Weigh accurately about 0.5 g of Clotiazepam, previously dried, dissolve in 80 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Between 142 C and 145 C.

Each mL of 0.1 mol/L perchloric acid VS = 31.882 mg of Cl6H15ClN2OS

Packaging and Storage

Preserve in light-resistant,

Purity (1) Clarity and color of solutionDissolve 0.5 g of Clotrimazole in 10 mL of dichloromethane: the solution is clear and colorless. (2) ChlorideDissolve 1.0 g of Clotrimazole in 40 mL of dimethylformamide, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.60 mL of 0.01 mol/L hydrochloric acid VS, 40 mL of dimethylformamide, 6 mL of dilute nitric acid and

KP 9 243
water to make 50 mL (not more than 0.021%). (3) SulfateDissolve 0.5 g of Clotrimazole in 10 mL of methanol and add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.50 mL of 0.05 mol/L sulfuric acid VS, 10 mL of methanol, 1 mL of dilute hydrochloric acid and water to make 50 mL (not more than 0.048%). (4) Heavy metalsProceed with 2.0 g of Clotrimazole according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) ArsenicPrepare the test solution with 1.0 g of Clotrimazole according to Method 3 and perform the test (not more than 2 ppm). (6) ImidazoleDissolve 0.10 g of Clotrimazole in exactly 10 mL of dichloromethane and use this solution as the test solution. Separately, dissolve 25.0 mg of imidazole RS in dichloromethane to make exactly 50 mL. Pipet 5.0 mL of this solution, add dichloromethane to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methanol and chloroform (3 : 2) to a distance of about 10 cm and air-dry the plate. Spray evenly sodium hypochlorite TS on the plate and air-dry the plate for 15 minutes, then spray evenly potassium iodide-starch TS on the plate: the spot from the test solution, corresponding to that from the standard solution, is not more intense than that from the standard solution. (7) (2-Chlorophenyl)-diphenylmethanolDissolve 0.20 g of Clotrimazole in dichloromethane to make exactly 10 mL and use this solution as the test solution. Separately, dissolve 10.0 mg of (2-chlorophenyl)diphenylmethanol RS in dichloromethane to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate and strong ammonia water (50 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spot from the test solution, corresponding to that from the standard solution, is not more intense than that from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.35 g of Clotrimazole, previously dried and dissolve in 80 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (poten-

tiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 34.484 mg of C22H17ClN2
Packaging and Storage well-closed containers.

Preserve in light-resistant,

Cloxacillin Sodium Hydrate

NaO O O Cl N O CH3 H2O N H H H N O H CH3 CH3 S

C19H17ClN3NaO5SH2O: 475.88 Cloxacillin Sodium Hydrate contains not less than 825 g (potency) of per mg of cloxacillin (C19H17ClN3NaO5S: 435.88), calculated on the anhydrous basis.
Description Cloxacillin Sodium Hydrate is a whtie to yellow-white crystals or crystalline powder. Cloxacillin Sodium Hydrate is freely soluble in water or in methanol, sparingly soluble in ethanol, and slightly soluble in ether. Identification (1) Determine the absorption spectra of Cloxacillin Sodium Hydrate and Cloxacillin Sodium Hydrate RS as directed in potassium bromide disk method under Infrared Spectrophotometry, both spectra exhibit similar intensities of absorption at the wave numbers about 1765 cm 1 , 1658 cm 1 , 1600 cm 1 , 1495 cm 1 , 1410 cm 1 , 1335 cm 1 , and 770 cm 1 . (2) To 2 mg of Cloxacillin Sodium Hydrate in test tube, add 2 mg of chromotrophic acid and 2 mL of sulfuric acid, and heat at 150 o C : the color of solution turns to olive-green after one to 1.5 minutes, after 2 minutes changes to deep red, and the test sample is carbonized to become gradually black. (3) Cloxacillin Sodium Hydrate responds to the Qualitative Tests for sodium salt. pH The pH of a solution obtained by dissolving 10 mg of Cloxacillin Sodium Hydrate in 10 mL of water is between 4.5 and 7.5. Sterility Test It meets the requirement, when Cloxacillin Sodium Hydrate is used in a sterile preparation. Bacterial Endotoxins Less than 0.20 EU per mg of

244 Monographs, Part I

cloxacillin, when Cloxacillin Sodium Hydrate is used in a sterile preparation.
Water Between 3.0 % and 5.0 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg (potency) each of Cloxacillin Sodium Hydrate and Cloxacillin Sodium Hydrate RS, dissolve in water to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT, and AS, of cloxacillin.

hydrated ethanol or in ether, very slightly soluble in ethanol and practically insoluble in water. Cloxazolam dissolves in dilute hydrochloric acid. Cloxazolam is gradually colored by light. Melting pointAbout 200 C (with decomposition).
Identification (1) Dissolve 10 mg of Cloxazolam in 10 mL of dehydrated ethanol by heating and add 1 drop of hydrochloric acid: the solution shows a pale yellow color and a yellow-green fluorescence under ultraviolet light (main wavelength: 365 nm). Add 1 mL of sodium hydroxide TS to this solution: the color and fluorescence of this solution disappear immediately. (2) Dissolve 10 mg of Cloxazolam in 5 mL of dilute hydrochloric acid by heating in a water-bath for 10 minutes. After cooling, 1 mL of this solution responds to the Qualitative Tests for primary aromatic amines. (3) Place 2 g of Cloxazolam in a flask, add 50 mL of ethanol and 25 mL of sodium hydroxide TS and boil under a reflux condenser for 4 hours. After cooling, neutralize with dilute hydrochloric acid and extract with 30 mL of dichloromethane. Dehydrate with 3 g of anhydrous sodium sulfate, filter and evaporate the dichloromethane of the filtrate. Dissolve the residue in 5 mL of methanol by heating in a water-bath and cool immediately in an ice-bath. Collect the crystals and dry the crystals in vacuum at 60 C for 1 hour: it melts between 87 C and 91 C. (4) Determine the absorption spectra of solutions of Cloxazolam and Cloxazolam RS in ethanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities at the same wavelengths. (5) Proceed with Cloxazolam as directed under the Flame Coloration Test (2): a green color appears.
1% Absorbance E1 cm (244 nm): Between 390 and 410 (after drying, 1 mg, ethanol, 100 mL).

Amount [g (potency)] of cloxacillin (C19H17ClN3NaO5S) = Amount [g (potency)] of Cloxacillin Sodium Hydrate RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Mobile phase: Dissolve 2.72 g of potassium dihydrogen phosphate in water to make 1000 mL, and adjust the pH to 5.0 with 8 mol/L potassium hydroxide solution. To 1000 mL of this solution, add 500 mL of acetonitrile. Flow rate: 1.0 mL per minute.
Packaging and Storage Preserve in tight containers.

Cloxazolam
H N O

O2N O Cl

and enantiomer C17H14Cl2N2O2: 349.21

Cloxazolam, when dried, contains not less than 99.0% and not more than 101.0% of cloxazolam (C17H14Cl2N2O2).
Description Cloxazolam is a white crystal or crystalline powder, is odorless and tasteless. Cloxazolam is freely soluble in glacial acetic acid, sparingly soluble in dichloromethane, slightly soluble in de-

Purity (1) ChlorideTo 1.0 g of Cloxazolam, add 50 mL of water, allow to stand for 1 hour with occasional shaking and filter. To 25 mL of this filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.20 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.014%). (2) Heavy metalsProceed with 1.0 g of Cloxazolam according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPlace 1.0 g of Cloxazolam in a Kjeldahl flask, add 5 mL of sulfuric acid and 5 mL of nitric acid and heat gently. Repeat the addition of 2 mL to 3 mL of nitric acid at times and continue heating until a colorless to pale yellow solution is obtained. After cooling, add 15 mL of saturated ammonium oxalate solution and heat the solution until dense white fumes are evolved and evaporate to a volume of 2 mL to 3 mL. After cooling,

KP 9 245
dilute with water to make 10 mL and perform the test with this solution as the test solution (not more than 2 ppm). (4) Related substancesDissolve 50.0 mg of Cloxazolam in 10.0 mL of dichloromethane and use this solution as the test solution. Pipet 1.0 mL of this solution, add dichloromethane to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Immediately after air-drying, develop the plate with a mixture of toluene and acetone (5 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than that from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Cloxazolam, previously dried and dissolve in 50 mL of glacial acetic acid. Titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue to blue-green (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

soluble in ethanol or in glacial acetic acid, slightly soluble in acetic anhydride, and practically insoluble in ether.
Identification (1) Determine the absorption spectra of solutions of Cocaine Hydrochloride and Cocaine Hydrochloride RS in 0.01 mol/L hydrochloric acid TS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Determine the absorption spectra of solutions of Cocaine Hydrochloride and Cocaine Hydrochloride RS in 0.01 mol/L hydrochloric acid TS (1 in 50000) as directed under the Ultravioletvisible spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths (2) Determine the infrared spectra of Cocaine Hydrochloride and Cocaine Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit the similar intensities at the same wavenumbers. (3) A solution of Cocaine Hydrochloride (1 in 50) responds to the Qualitative Tests (2) for chlorides. Specific Optical Rotation [ ]20 D : Between -70 and 73 (after drying, 0.5 g, water, 20 mL, 100 mm). Purity (1) AcidityDissolve 0.5 g of Cocaine Hydrochloride in 10 mL of water, add 1 drop of methyl red TS, and titrate with 0.01 mol/L sodium hydroxide VS: not more than 1.0 mL is required to obtain a yellow color. (2) CinnamylcocaineDissolve 0.10 g of Cocaine Hydrochloride in 5 mL of water, and add 0.3 mL of diluted sulfuric acid (1 in 20) and 0.10 mL of 0.02 mol/L potassium permanganate VS: the violet color does not disappear entirely within 30 minutes. (3) Isoatropy cocaineDissolve 0.10 g of Cocaine Hydrochloride in 30 ml of water in a beaker. Transfer 5 ml of this solution to a test tube, add 1 drop of ammonia TS, and mix. After the precipitate is coagulated, add 10 ml of water, and transfer the mixture to the former beaker, to which 30 ml of water has been added previously. Wash the test tube with 10 ml of water, combine the washing with the mixture in the beaker, add 3 drops of ammonia TS to the combined mixture, and mix gently: a crystalline precipitate is produced. Allow to stand for 1 hour: the supernatant liquid is clear. Loss on Drying Not more than 1.0% ( 1 g, 105 C, 4 hours).

Each mL of 0.1 mol/L perchloric acid VS = 34.921 mg of C17H14Cl2N2O2

Packaging and Storage tight containers.

Preserve in light-resistant,

Cocaine Hydrochloride
O H C OCH 3 H O NCH3 H O C HCl

C17H21NO4HCl: 339.81 Cocaine Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of cocaine hydrochloride (C17H21NO4HCl).
Description Cocaine Hydrochloride is a colorless crystal or white crystalline powder. Cocaine Hydrochloride is very soluble in water, freely

Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately 0.5 g of Cocaine Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3), and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction.

246 Monographs, Part I

Each mL of 0.1 mol/L perchloric acid VS = 33.981 mg of C17H21NO4HCl

Packaging and Storage resistant containers.

Preserve in tight, light-

KP 9 247
C18H21NO3H3PO41/2H2O: 406.37

Cod Liver Oil

Cod Liver Oil is the fatty oils obtained from fresh livers and pyloric appendages of Gadus macrocephalus Tilesius or Theraga chalcogramma Pallas (Gadidae). Cod Liver Oil contains not less than 2000 vitamin A units and not more than 5000 vitamin A units per g.
Description Cod Liver Oil is a yellow to orange oily liquid, has a characteristic, slightly fishy odor and a mild taste. Cod Liver Oil is miscible with chloroform, with ether or with pertroleum ether. Cod Liver Oil is slightly soluble in ethanol. Decomposition of Cod Liver Oil is accelerated by air or by light. Identification Dissolve 0.1 g of Cod Liver Oil in 10 mL of chloroform, and to 1 mL of this solution, add 3 mL of antimony trichloride TS: a blue color is observed immediately, but the color fades rapidly. Specific Gravity
20 : Between 0.918 and 0.928. d 20

Codeine Phosphate Hydrate contains not less than 98.0 and not more than 101.0% of codeine phosphate (C18H21NO3H3PO4: 397.36), calculated on the anhydrous basis.
Description Codeine Phosphate Hydrate is a white yellowish white crystal or crystalline powder. Codeine Phosphate Hydrate is freely soluble in water in glacial acetic acid, slightly soluble in methanol or ethanol and practically insoluble in ether. pHA solution of Codeine Phosphate Hydrate (1 10) is between 3.0 and 5.0. Codeine Phosphate Hydrate is affected by light.

to or in in

Acid Value Not more than 1.7. Iodine Value Between 130 and 170. Saponification Value Between 180 and 192. Unsaponifiable Matter Not more than 3.0% Purity RancidityNo unpleasant odor of rancid oil is perceptible on warming Cod Liver Oil. Assay Perform the test with about 0.5 g of Cod Liver Oil, weighed accurately, as directed in Method 2 under the Vitamin A Determination. Packaging and Storage Preserve in light-resistant, tight containers and almost well-filled or under nitrogen atmosphere.

Identification (1) Determine the absorption spectra of solutions of Codeine Phosphate Hydrate and Codeine Phosphate Hydrate RS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared absorption spectra of Codeine Phosphate Hydrate and Codeine Phosphate Hydrate RS, previously dried at 105 C for 4 hours, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Codeine Phosphate Hydrate (1 in 20) responds to the Qualitative Tests (1) for phosphate. Specific Optical Rotation [ ]20 D : Between -98 and 102 (0.4 g calculated on the anhydrous basis, water, 20 mL, 100 mm). Purity (1) ChloridePerform the test with 0.5 g of Codeine Phosphate Hydrate. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.021%). (2) SulfatePerform the test with 0.20 g of Codeine Phosphate Hydrate. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.240%). (3) Related substances Dissolve 0.20 g of Codeine Phosphate Hydrate in 10.0 mL of a mixture of 0.01 mol/L hydrochloric acid TS and dehydrated ethanol (4 : 1) and use this solution as the test solution. Pipet 1.0 mL of the test solution, add a mixture of 0.01 mol/L hydrochloric acid TS and dehydrated ethanol (4 : 1) to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of dehydrated ethanol, toluene, acetone and strong ammonia water (14 : 14 : 7 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ul-

Codeine Phosphate Hydrate

CH3 H N

H3PO4

1/2 H2O

O CH3O H H OH

Codeine Phosphate

248 Monographs, Part I

traviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Water Between 1.5% and 3.0% (0.5 g, volumetric titration, direct titration). Asssy Dissolve about 0.5 g of Codeine Phosphate Hydrate, accurately weighed, in 70 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue to greenish blue (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

tion as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and calculate the ratios, QT and QS , of the peak area of codeine to that of the internal standard for the test solution and the standard solutions, respectively. Amount (mg) of codeine phosphate hydrate (C18H21NO3H3PO41/2 H2O) = amount (mg) of Codeine Phosphate RS, calculated on the anhydrous basis 1.0227
QT QS

Each mL of 0.1 mol/L perchloric acid VS = 39.736 mg of C18H21NO3H3PO4

Packaging and Storage tight containers.

Internal Standard soultionA solution of etilefline hydrochloride (3 in 10000). Operating conditions Detector, column, column temperature, mobile phase, flow rate and selection of column: Proceed as directed in the operating conditions in the Assay under 10% Codeine Phosphate Powder.
Packaging and Storage Preserve in tight containers.

Preserve in light-resistant,

1% Codeine Phosphate powder

1 % Codeine Phosphate Powder contains not less than 0.90% and not more the 1.10% of codeine phosphate hydrate (C18H21NO3H3PO41/2H2O: 406.37).
Method of Preparation Codeine Phosphate Hydrate 10 g Lactose a sufficient quantity To make 1000 g Prepare as directed under Powders, with the above ingredients. Identification Determine the absorption spectrum of a solution of 1 % Codeine Phosphate Powder (1 in 100) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 283 nm and 287 nm. Particle Size Distribution Test for preparations meets the requirement. Uniformity of Dosage Units (divided) quirement.

10% Codeine Phosphate Powder

10% Codeine Phosphate Powder contains not less than 9.3% and not more than 10.7% of codeine phosphate hydrate (C18H21NO3H3PO41/2H2O: 406.37).
Method of Preparation Codeine Phosphate hydrate 100 g Lactose a sufficient quantity To make 1000 g

Prepare as directed under Powders, with the above ingredients.

Identification Test Determine the absorption spectrum of a solution of 10% Codeine Phosphate Powder (1 in 1000) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 283 nm and 287 nm. Particle Size Distribution Test for preparations meets the requirement. Uniformity of Dosage Units (divided) quirement

It

It meets the re-

Assay Weigh accurately about 5 g of 1 % Codeine Phosphate Powder, dissolve in water to make exactly 100 mL, then pipet exactly 10 mL of this solution, add exactly 10 mL of the internal standard solution and use this solution as the test solution. Previously, weigh accurately about 50 mg of Codeine Phosphate RS, separately determined the water content in the same manner as Codeine Phosphate Hydrate, dissolve in water to make exactly 100 mL, then pipet 10.0 mL of this solution, add exactly 10 mL of the internal standard solution and use this solu-

It

It meets the re-

Assay Weigh accurately about 2.5 g of 10 % Codeine Phosphate Powder, dissolve in water to make exactly 100 mL, then pipet 2.0 mL of this solution, add exactly 10

KP 9 249
mL of the internal standard solution and water to make 20.0 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Codeine Phosphate Hydrate RS, previously determined the water content in the same manner as Codeine Phosphate Hydrate, dissolve in water to make exactly 100 mL, then pipet 10.0 mL of this solution, add exactly 10 mL of the internal standard solution and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and calculate the ratios, QT and QS , of the peak area of codeine to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of codeine phosphate hydrate (C18H21NO3H3PO41/2 H2O) = amount (mg) of Codeine Phosphate Hydrate RS, calculated on the anhydrous basis 1.0227
QT 5 QS

Codeine Phosphate Tablets

Codeine Phosphate Tablets contains not less than 93.0% and not more than 107.0% of labeled amount of codeine phosphate hydrate (C18H21NO3H3PO41/2H2O: 406.37).
Method of Preparation Prepare as directed under Tablets, with Codeine Phosphate Hydrate. Identification To a portion of powdered Codeine Phosphate Tablets, equivalent to 0.1 g of Codeine Phosphate Hydrate according to the labeled amount, add 20 mL of water, shake and filter. To 2 mL of the filtrate, add water to make 100 mL and determine the absorption spectrum as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 283 nm and 287 nm. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Codeine Phosphate Tablets. Weigh accurately a portion of the powder, equivalent to about 0.1 g of codeine phosphate hydrate (C18H21NO3H3PO41/2H2O), add 30 mL of water, shake, add 20 mL of diluted dilute sulfuric acid (1 in 20), treat the mixture with ultrasonic waves for 10 minutes, and add water to make exactly 100 mL. Filter the solution, then pipet 5 mL of the filtrate, add exactly 10 mL of the internal standard solution and water to make 20 mL, and use this solution as the test solution. Separately, weigh accurately about 50 mg of Codeine Phosphate Hydrate RS (previously determine the water in the same manner as Codeine Phosphate Hydrate), dissolve in water to make exactly 100 mL, then pipet 10 mL of this solution, add exactly 10 mL of the internal standard solution, and use this solution as the standard solution. Perform the test with 20 L each of the sample solution and standard solution as directed under Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS , of the peak area of codeine to that of the internal standard.

Internal standard solutionA solution of etilefline hydrochloride (3 in 10000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and 15 cm to 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in 500 mL of diluted phosphoric acid (1 in 1000) and adjust the pH to 3.0 with sodium hydroxide TS. To 240 mL of this solution, add 70 mL of tetrahydrofuran and mix. Flow rate: Adjust the flow rate so that the retention time of codeine is about 10 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution according to the above operating conditions, codeine and the internal standard are eluted in this order with the resolution between these peaks being not less than 4.0. System repeatability: When the test is repeated 5 times with 20 L of the standard solution according to the above operating conditions, the relative standard deviation of the ratios of the peak area of codeine to that of the internal standard substance is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Amount (mg) of codeine phosphate hydrate (C18H21NO3H3PO41/2H2O) = WS QT 2 1.0227

QS

WS : Amount (mg) of Codeine Phosphate Hydrate RS, calculated on the anhydrous basis

Internal standard solution A solution of etilefline hydrochloride (3 in 10000). Operating conditions

250 Monographs, Part I

Detector, column, column temperature, mobile phase, flow rate and selection of column: Proceed as directed in the operating conditions in the Assay under 10% Codeine Phosphate Powder.
Packaging and Storage Preserve in tight containers.

Colchicine
CH3O NHCOCH3 H CH3O CH3O O OCH3

Separately, weigh 0.3 g of chloroform using the 100 mLvolumetric flask containing about 20 mL of dimethylformamide, add dimethylformamide to make 100 mL. Pipet exactly 2 mL of this solution, add dimethylformamide to make exactly 100 mL, and use this solution as the standard solution (1). Separately weigh accurately about 1.8 g of ethyl acetate, using the 100-mL volumetric flask containing about 20 mL of dimethylformamide, add dimethylformamide to make 100 mL. Pipet exactly 2 mL of this solution, add exactly 2 mL of internal standard solution, add dimethylformamide to make 10 mL, and use this solution as the standard solution (2). Perform the test with each 2 L of the test solution, the standard solution (1) and the standard solution (2), as directed under Gas Chromatography according to the following conditions: the peak area of chloroform obtained from the test solution is not more than the that obtained from the standard solution (1). Determine the ratios of peak area, QT and

C22H25NO6: 399.44 Colchicine, when dired, contains not less than 97.0% and not more than 102.0% of colchicine (C22H25NO6), calculated on the anhydrous basis..
Description Colchicine is a yellowish white powder. Colchine is very soluble in methanol, freely soluble in dimethylformamide, in ethanol or in acetic anhydride and sparingly soluble in water. Colchicine is colored by light. Identification (1) Determine the absorption spectra of solutions of Colchicine and Colchicine RS in ethanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities absorption at the same wavelength. (2) Add each 0.5 mL of solutions of Colchicine and Colchicine RS in methanol (1 in 50) to 1 g of potassium bromide for infrared absorption spectrum, grind thoroughly, dry in vacuum at 80 for 1 hour, determine the absorption spectra of these powders as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between -235 and o 250 (0.1 g calculated on the anhydrous basis and corrected by the amount of ethyl acetate, ethanol, 10 mL, 100 mm) Purity (1) Colchiceine Dissolve 0.1 g of Colchicine in 10 mL of water and to 5 mL of this solution, add 2 drops of ferric chloride TS: no definite green color is produced. (2) Ethyl acetate and Chloroform Weigh accurately 0.6 g of Colchicine, dissolve in exactly 2 mL of internal standard solution, and add dimethylformamide to make 10 mL, and use this solution as the test solution.

QS , of ethyl acetate to the peak area of the internal standard obtained from the test solution and the standard solution. Calculate the amount of ethyl acetate according to the following equation: the amount of ethylacetate is not more than 6.0%.
Amount (mg) of Ethyl acetate =
WS QT 2 W T QS

WS : amount (g) of Ethylacetate WT : amount (g) of Colchicine

Internal standard solutionA solution of 1-propanol in dimethylformamide (3 in 200). Operating conditions Detector: A hydrogen flame ionized detector Column: A fused glass column, about 0.53 mm in inside diameter and about 30 m in length, coated inside surface with polyethylene glycol 20 M for gas chromatograph 1.0 m in thickness. Column temperature: 60 C for 7 minutes then up to 100 C at a rate of 40 C per minute if necessary, and hold at 100 C for 10 minutes. Inject port temperature: A constant temperature of about 130 C. Detector temperature: A constant temperature of about 200 C. Carrier gas: Helium Flow rate: Adjust the folw rate so that the retention time of ethylacetate is about 3 minutes. Split ratio : about 1 : 20 System suitability Test for required detactability: Pipet 2 mL of the standard solution (2), and add dimethylformamide to make exactly 25 mL. Pipet 1 mL of this solution, and add dimethylformamide to make exactly 50 mL. Confirm that the peak area of ethylacetate obtained from 2 L of

KP 9 251
this solution is equivalent to 0.11 and 0.21% of that obtained from 2L of the standard solution(2). System performance: To 1 mL of chloroform add dimethylformamide to make 10 mL. To 1 mL of this solution add 2 mL of ethylacetate, and dimethylformamide to make 10 mL. To 2 mL of this solution add 2 mL of the internal standard solution and dimethylformamide to make 10 mL. When the procedure is run with 2 L of this solution according to the above operation conditions, ethylacetate, chloroform and internal standard are eluted in this order, with the resolution between chloroform and internal standard being not less than 2.0. System repeatability: When the test is repeated 3 times with 2 L of the standard solution (2) according to the above operating conditions, the relative standard deviation of the ratio of peak area of ethylacetate to that of the internal is not more than 3.0%. (3) Related substancesDissolve 60 mg of Colchicine in 100 mL of diluted methanol (1 in 2). Pipet exactly 1 mL of this solution, add diluted methanol (1 in 2) to make exactly 100 mL and use this solution as the test solution. Perform the test with 20 L of the test solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution by the automatic integration method and calculate the total amount of the peaks other than colchicines by the area percentage method: not more than 5.0%. peak of the solvent.
Water Not more than 2.0% (0.5 g, volumetric titration method, back titration) Assay Weigh accurately 0.4 g of Colchicine, previously dried, dissolve in 25 mL of acetic anhydride and titrate with 0.05 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.05 mol/L perchloric acid VS = 19.972 mg of C22H25NO6

Packaging and Storage tight containers.

Preserve in light-resistant,

Colchicine Tablets
Colchicine Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of colchicine (C22H25NO6: 399.44).
Preparation Colchicine.

Prepare as directed under Tablets, with

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column,about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (1.0 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Add methanol to 450 mL of 0.05 mol/L potassium dihydrogen phosphate TS to make 1000 mL, Adjust pH to 5.5 with diluted phosphoric acid (7 in 200) . System suitability Test for required detactability: Pipet exactly 1 mL of the test solution, and add diluted methanol (1 in 2) to make exactly 50 mL. Conform that the peak area of colchicine obtained from 20 L of this solution is equivalent to 1.4 to 2.6% of that obtained from colchicine of the test solution. System performance: When the procedure is run with 20 L of the test solution according to the above operating conditions, the number of theoretical plates is 6000, and the symmetry factor is not more than 1.5. System repeatability: When the test is repeated 6 times with 20 L of the test solution according to the above operation conditions, the relative standard deviation of the peak area of colchicine is not more than 2.0%. Time span of measurement: About 2 times as long as the retention time of colchicine beginning after the

Identification Weigh a portion of powdered Colchicine Tablets, equivalent to 20 mg of Colchicine, add 20 mL of water, allow the solids to settle and filter the supernatant liquid into a separatory funnel. Extract the filtrate with 30 mL of chloroform and evaporate the chloroform extract to dryness. Determine the infrared spectra of the residue and Colchicine RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Dissolution Test Perform the test with 1 tablet of Colchicine Tablets at 100 revolutions per minutes according to Method 1 under the Dissolution Test, using 500 mL of water as a dissolution solution. Filter through a membrane filter with pore size of not more than 0.8 m, after 30 minutes from the start of the test, pipet V mL of the filtrate and extract with 15 mL of chloroform three times. Evaporate the combined extracts to dryness, add chloroform to make exactly 10 mL and use this solution as the test solution. Separately, weigh accurately a portion of Colchicine RS, previously dried at 105 C for 3 hours, dissolve in dissolution solution, prepare the same concentration of solution as the test solution and use this solution as the standard solution. Determine the absorbance of the test solution and the standard solution at 350 nm as directed under the Ultraviolet-visible Spectrophotometry using chloroform as a blank. The dissolution rate of Colchicine Tablets in 30 minutes is not less than 75%.

252 Monographs, Part I

Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Colchicine Tablets. Weigh accurately a portion of the powder, equivalent to about 0.6 mg of colchicine (C22H25NO6), add a mixture of methanol and water (1 : 1), shake by mechanical means for 15 minutes, dilute with the same mixture to make exactly 100 mL, filter and use this solution as the test solution (prepared immediately prior to use). Separately, weigh accurately a portion of Colchicine RS, previously dried at 105 C for 3 hours, dissolve in a mixture of methanol and water (1 : 1) and use this solution as the standard solution so that each mL contains 6 g of Colchicine. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine the peak areas, AT and AS , of colchicine of the test solution and the standard solution, respectively.

Cortisone Acetate
O H3C O H3C H C CH2O OH O C CH3

H O

C23H30O6: 402.48 Cortisone Acetate, when dried, contains not less than 97.0% and not more than 102.0% of cortisone acetate (C23H30O6).
Description Cortisone Acetate is a white crystal or crystalline powder and is odorless. Cortisone Acetate is sparingly soluble in methanol, slightly soluble in dehydrated ethanol and practically insoluble in water. Melting pointAbout 240 C (with decomposition). Identification (1) Add 2 mL of sulfuric acid to 2 mg of Cortisone Acetate and allow to stand for a while: a yellowish green color is produced and it gradually changes to yellow-orange. Examine the solution under ultraviolet light: the solution shows a pale green fluorescence. Add carefully 10 mL of water to this solution: the yelloworange color of the solution is discharged and the solution remains clear. (2) `Determine the absorption spectra of solutions of Cortisone Acetate and Cortisone Acetate RS in methanol (1 in 50000) as directed under the Ultraviolet-visible absorption Photometer: both spectra exhibit similar intensities of absorption at the same wavelengths.. (3) Determine the infrared spectra of Cortisone Acetate and Cortisone Acetate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears between the spectra, dissolve Cortisone Acetate and Cortisone Acetate RS in acetone, respectively, evaporate the solutions to dryness and repeat the test on the residues. Specific Optical Rotation [ ]20 D : Between +207 and +2l6 (after drying, 0.1 g, 10 mL of methanol, 100 mm). Purity Related substancesDissolve 25 mg of Cortisone Acetate in 10 mL of a mixture of acetonitrile, water and glacial acetic acid (70 : 30 : 1) and use this solution as the test solution. Pipet 1 mL of the test solution, add a mixture of acetonitrile, water and glacial acetic acid (70 : 30 : 1) to make exactly 100 mL and use this solution as the standard solution. Perform the test with each 15 L

Amounts (mg) of colchicine (C22H25NO6) = 0 .1 C

AT AS

C: Concentration (g/mL) of the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: Add 45 mL of 0.5 mol/L potassium dihydrogen phosphate and water to make 450 mL. Add 530 mL of methanol to this solution, cool to room temperature and dilute with methanol to make 1000 mL. Adjust pH to 5.5 0.05 with 0.5 mol/L phosphoric acid. Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the retention time of colchicine is between 5.5 and 9.5 minutes, and the number of theoretical plates is not less than 4500. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of colchicine is not more than 2.0%.
Packaging and Storage tight containers.

Preserve in light-resistant,

KP 9 253
of the test solution and the standard solution as directed under the Liquid Chromatography according to the operating conditions. And determine each peak area according to the automatic integration method: the peak area other than cortisone acetate obtained from the test solution is not greater than 1/2 times the peak area of cortisone acetate obtained from the standard solution. The sum of peak area other than the peak area of cortisone acetate obtained from the test solution is not greater than 1.5 times peak area of cortisone acetate obtained from the standard solution.

Residue on Ignition Not more than 0.1% (0.5 g). Assay Dissolve about 10 mg each of Cortisone Acetate and Cortisone Acetate RS, previously dried and accurately weighed, in 50 mL of methanol, add exactly 5 mL each of the internal standard solution, then add methanol to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Cortisone Acetate to that of the internal standard, for the test solution and the standard solution, respectively.

Operating conditions Detector: An ultraviolet-visible Photometer (wavelength: 254nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Control the gradient by mixing the mobile phase A and the mobile phase B as directed in the following table Mobile phase A : A mixture of water and acetonitrile (7 : 3) Mobile phase B : B mixture of acetonitrile and water (7 : 3)
Time after injection of sample (min) 0-5 5-25 25-30 Mobile phase A (vol%) 90 9010 10 Mobile phase B (vol %) 10 1090 90

Amount (mg) of cortisone acetate (C23H30O6) = amount (mg) of Cortisone Acetate RS QT

QS

Internal standard solutionA solution of butyl parahydroxybenzoate in methanol (3 in 5000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of water and acetonitrile (13 : 7). Flow rate: Adjust the flow rate so that the retention time of Cortisone Acetate is about 12 minutes. System suitability System performance: 10 mg of Cortisone Acetate in 50 mL of methanol, add 5 mL of a solution of propylparahydroxybenzoate in methanol (3 in 5000) and add methanol to make 100 mL. When the procedure is run with 10 L of the standard solution under the above operating conditions, cortisone acetate and internal standard sabstance are eluted in this order with the resolution between their peaks being not less than 4.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of peak area of cortisone acetate to that of the internal standard is not more than 1.0%
Packaging and Storage tight containers.

Folw rate: About 1 mL per minute System suitability Test for required detectability: To exactly 1 mL of the standard solution add a mixture of acetonitrile, water and glacial acetic acid (70 : 30 : 1) to make exactly 10 mL. Confirm that the peak area of cortisone acetate obtained with 15 L of this solution is equivalent to 8 to 12 % of that with 15 L of the standard solution System performance: When the procedure is run with 15 L of the test solution under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of cortisone acetate are not less than 10,000 and not more than 1.3, respectively. System repeatability: When the test is repeated 3 times with 15L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of cortisone acetate is not more than 5.0 %. Time span of measurement: About 3 times as long as the retention time of cortisone acetate beginning after the solvent peak.
Loss on Drying Not more than 1.0% (0.5 g, 105 C, 3 hours).

Preserve in light-resistant,

254 Monographs, Part I

graphy. Spot 10 L each of the test solution and standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, hexane, methanol and ammonia solution (30 : 15 : 5 : 1) to a distance of about 10 cm, and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot and other than the spot of the starting point from the test solution are not more than intense than the spot from the standard solution.
Loss on Drying Not more than 0.5 % (1.0 g, 60 C, 4 hours).
N

Croconazole Hydrochloride

Cl O

HCl

H2C

Residue on Ignition Not more than 0.1 % (1.0 g).

C18H15ClN2OHCl : 347.24 Croconazole Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of croconazole hydrochloride (C18H15ClN2OHCl).
Description Croconazole Hydrochloride is a white to pale ellowish white crystalls or cryatalline powder. Croconazole Hydrochloride is very soluble in water, freely soluble in glacial acetic acid, in methanol or in acetone, practically insoluble in ether. Identification (1) Determine the absorption spectra of solutions of Croconazole Hydrochloride and Croconazole Hydrochloride RS in methanol (1 in 20000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the absorption spectra of the solutions of Croconazole Hydrochloride and Croconazole Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve about 50 mg of Croconazole Hydrochloride in 10 mL of water, add 2 mL of sodium hydroxide TS and 20 mL of ether, and shake. Wash the separated aqueous layer with two 10 mL portions of ether, and acidify the solution with 2 mL of dilute nitric acid: the solution responds to the qualitative tests for chloride. Melting Point Between 148 C and 153 C Purity (1) Heavy metals - Proceed with 1.0 g of Croconazole Hydrochloride according to the method 4, and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (2) Related substances - Dissolve 50 mg of Croconazole Hydrochloride in 10 mL of methanol, and use this solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 100 mL, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromato-

Assay Weigh accurately about 0.6 g of Croconazole Hydrochloride, previously dried, dissolve in 10 mL of glacial acetic acid, add 40 mL of acetic anhydride, and titrate with 0.1 mol/L perchloric acid VS [indicator: 1 to 2 drops of a solution of malachite green oxalate in glacial acetic acid (1 in 100)] until the color of this solution changes from blue-green through green to yellow-green. Perform a blank determination, and make any necessary correction.

1 mL of 0.1 mol/L perchloric acid = 34.724 mg of C18H15ClN2OHCl

Packing and Storage Preserve in the well-closed, tight containers.

Cyanamide
H2N
Aminonitrile

N
CH2N2: 42.04

Cyanamide contains not less than 97.0% and not more than 101.0% of cyanamide (CH2N2), calculated on the anhydrous basis.
Description Cyanamide is a white crystal or crystalline powder and has a faint, characteristic odor. Cyanamide is very soluble in water, in methanol or in ethanol and freely soluble in ether. Cyanamide is hygroscopic. pHThe pH of a solution of 1.0 g of Cyanamide in 100 mL of water is between 5.0 and 6.5. Melting pointAbout 46 C. Identification (1) To 1 mL of a solution of Cyanamide (1 in 100), add 1 mL of potassium naphthoquinone sulfonate TS and 0.2 mL of sodium hydroxide TS: a deep red color develops. (2) Drop one or two drops each of the solutions of Cyanamide and Cyanimide RS in acetone (1 in 100) onto

KP 9 255
potassium bromide disks prepared as directed in the potassium bromide disk method under Infrared Sectrophotometry separately and air-dry the disks. Determine the infrared spectra of the disks as directed in the film method under Infrared Spectrometry: both spectra exhibit similar intensities of absorption at the same wave numbers.
Purity (1) Clarity and color of solution Dissolve 1.0 g of Cyanamide in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Cyanamide according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). Water Not more than 1.0% (1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 1 g of Cyanamide and dissolve in water to make exactly 250 mL. Pipet exactly 15 mL of this solution, add 2 to 3 drops of dilute nitric acid, 10 mL of ammonia TS and exactly 50 mL of 0.1 mol/L silver nitrate VS and allow to stand for 15 minutes with occasional shaking. Add water to make exactly 100 mL, filter, discard the first 20 mL of the filtrate and pipet the subsequent exactly 50 mL. After neutralizing this solution with dilute nitric acid, add 3 mL of dilute nitric acid and titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ferric ammonium sulfate TS). Perform a blank determination and make any necessary correction.
NH

O H2N H2N C C CH2CH2 H2C CN N H3C H2C H H3 C O C CH2CH2 N H3C H N Co N N H CH3 CH3 CH2 H

O C NH2 O C NH2

O CH3 O H2N C

CH2CH2

CH3 CH3 H CH2CH2 O C NH2

CH3

O H2 C H O P H H O CH2OH H O

N OH

CH3

CH3 O

Vitamin B12

C63H88CoN14O14P: 1355.37

Cyanocobalamin contains not less than 96.0% and not more than 102.0% of cyanocobalamin (C63H88CoN14O14P), calculated on the dried basis.
Description Cyanocobalamin is a dark red crystal or powder. Cyanocobalamin is sparingly soluble in water and slightly soluble in ethanol Cyanocobalamin is hygroscopic. Identification (1) Determine the absorption spectra of the test solution and the standard solution obtained in the Assay, as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Mix 1 mg of Cyanocobalamin with 50 mg of potassium bisulfate and fuse by igniting. Cool, break up the mass with a glass rod, add 3 mL of water and dissolve by boiling. Add 1 drop of phenolphthalein TS, then add drop-wise sodium hydroxide TS until a light red color just develops. Add 0.5 g of sodium acetate, 0.5 mL of dilute acetic acid and 0.5 mL of a solution of disodium 1nitroso-2-naphthol-3,6-disulfonate (1 in 500): a red to orange-red color is immediately produced. Then add 0.5 mL of hydrochloric acid and boil for 1 minute: the red color does not disappear. (3) Transfer 5 mg of Cyanocobalamin to a distilling flask, dissolve in 5 mL of water and add 2.5 mL of hypophosphorous acid. Connect the flask with a short condenser and dip its tip into a test tube containing 1 mL of a solution of sodium hydroxide (1 in 50). Heat gently for 10 minutes, then distill 1 mL into a test tube. To the test tube, add 4 drops of a saturated solution of ferrous ammonium sulfate, shake gently, then add about 30 mg sodium fluoride and heat the contents to boil. Immediately add drop-wise diluted sulfuric acid (1 in 7) until a clear solution results, then add 3 to 5 drops more of diluted sulfuric acid (1 in 7): a blue to blue-green color develops. pH Dissolve 0.10 g of Cyanocobalamin in 20 mL of

Each mL of 0.1 mol/L silver nitrate VS = 2.1020 mg of CH2N2

Packaging and Storage Preserve in a cold place, tight containers.

Cyanocobalamin

256 Monographs, Part I

freshly boiled and cooled water: the pH of this solution is between 4.2 and 7.0.
Purity (1) Clarity and color of solution Dissolve 20 mg of Cyanocobalamin in 10 mL of water: the solution is clear and red in color. (2) PseudocyanocobalaminDissolve 1.0 mg of Cyanocobalamin in 20 mL of water, transfer the solution to a separator, add 5 mL of the mixture of m-cresol and carbon tetrachloride (1 : 1) and shake vigorously for 1 minute. Allow to separate, draw off the lower layer into another separator, add 5 mL of diluted sulfuric acid (1 in 7), shake vigorously and allow to separate completely, If necessary, centrifuge the mixture: the supernatant liquid is colorless or has no more color than the following control solution.

red to red liquid. Cyanocobalamin Injection is gradually colored by light. pHBetween 4.0 and 5.5.
Identification Determine the absorption spectrum of the test solution obtained in the Assay as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 277 nm and 279 nm, between 360 nm and 362 nm, and between 548 nm and 552 nm. Determine the absorbances, A1 and A2 , of this solution at the wavelengths of maximum absorption between 360 nm and 362 nm and between 548 nm and 552 nm, respectively: the ratio, A 2 / A1 is not less than 0.29 and not more than 0.32. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement . Insoluble Particulate Matter Test for Injections meets the requirement

Control solutionDilute 0.6 mL of 0.02 mol/L potassium permanganate VS with water to make 1000 mL.
Loss on Drying Not more than 12% (50 mg, in vacuum at a pressure not exceeding 0.67 kPa, P2O5, 100 C, 4 hours). Assay Weigh accurately about 20 mg each of Cyanocobalamin and Cyanocobalamin RS (previously determine the loss on drying in the same manner as Cyanocobalamin), dissolve in water to make exactly 1000 mL, respectively, and use these solutions as the test solution and the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 361 nm as directed under the Ultraviolet-visible Spectrophotometry.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of Cyanocobalamin Injection, equivalent to about 2 mg of cyanocobalamin (C63H88CoN14O14P), add water to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 20 mg of Cyanocobalamin RS (previously determined the loss on drying in the same manner as Cyanocobalamin), add water to make exactly 1000 mL and use this solution as the standard solution. With these solutions, proceed as directed in the Assay under Cyanocobalamin.

Amount (mg) of cyanocobalamin (C63H88CoN14O14P) = amount (mg) of Cyanocobalamin RS, calculated on the dried basis
Packaging and Storage tight containers.
AT AS

Amount (mg) of cyanocobalamin (C63H88Co N14O14P) = amount (mg) of Cyanocobalamin RS, calculated on the dried basis
Packaging and Storage hermetic containers.
AT 1 A S 10

Preserve in light-resistant,

Preserve in light-resistant,

Cyanocobalamin Injection
Vitamin B12 Injection Cyanocobalamin Injection is an aqueous solution for injection. Cyanocobalamin Injection contains not less than 95.0% and not more than 115.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P: 1355.37)
Method of Preparation Prepare as directed under Injections, with Cyanocobalamin. Description Cyanocobalamin Injection is a clear, pale

Cyclopentolate Hydrochloride
OH O C H COCH2CH2N CH3 CH2 HCl

and enantiomer C17H25NO3HCl: 327.85

KP 9 257
the spot from the standard solution. Cyclopentolate Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of cyclopentolate hydrochloride (C17H25NO3HCl).
Description Cyclopentolate Hydrochloride is a white, crystalline powder and is odorless or has a characteristic odor. Cyclopentolate Hydrochloride is very soluble in water, freely soluble in ethanol, in glacial acetic acid or in chloroform, sparingly soluble in acetic anhydride and practically insoluble in ether. Identification (1) To 1 mL of a solution of Cyclopentolate Hydrochloride (1 in 100), add 1 mL of Reinecke salt TS: a pale red precipitate is produced. (2) Dissolve 0.2 g of Cyclopentolate Hydrochloride in 2 mL of water, add 2 mL of sodium hydroxide TS and boil for 1 minute. After cooling, add 2 drops of nitric acid: a phenylacetic acid-like odor is perceptible. (3) Determine the infrared spectra of Cyclopentolate Hydrochloride and Cyclopentolate Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) A solution of Cyclopentolate Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride. pH Dissolve 0.20 g of Cyclopentolate Hydrochloride in 20 ml of water: the pH of this solution is between 4.5 and 5.5. Melting Point Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.05% (1 g). Assay Weigh accurately about 0.5 g of Cyclopentolate Hydrochloride, previously dried and dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (4 : 1) and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue-green to yellow-green (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 32.79 mg of C17H25NO3HCl

Packaging and Storage Preserve in tight containers.

Cyclophosphamide Hydrate
O P N NH CH2CH2Cl O CH2CH2Cl H2O

C7H15Cl2N2O2PH2O: 279.10 Cyclophosphamide Hydrate contains not less than 97.0% and not more than 101.0% of cyclophosphamide hydrate (C7H15Cl2N2O2PH2O).
Description Cyclophosphamide Hydrate is a white crystalline powder and is odorless. Cyclophosphamide Hydrate is very soluble in glacial acetic acid, freely soluble in ethanol, acetic anhydride or in chloroform and soluble in water or in ether. Melting pointBetween 45 C and 53 C. Identification (1) Dissolve 0.1 g of Cyclophosphamide Hydrate in 10 mL of water and add 5 mL of silver nitrate TS: no precipitate is produced. Then boil this solution: a white precipitate is produced. Collect the precipitate and add dilute nitric acid to a portion of this precipitate: it does not dissolve. Add excess ammonia TS to another portion of the precipitate: it dissolves. (2) Add 1 mL of diluted sulfuric acid (1 in 25) to 20 mg of Cyclophosphamide Hydrate and heat until white fumes are evolved. After cooling, add 5 mL of water and shake. Neutralize with ammonia TS, then acidify with dilute nitric acid: this solution responds to the Qualitative Tests (2) for phosphate. (3) Determine the infrared spectra of Cyclophosphamide Hydrate and Cyclophosphamide Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar

Between 135 C and 138 C.

Purity (1) Clarity and color of solutionDissolve 1.0 g of Cyclopentolate Hydrochloride in 10 ml of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Cyclopentolate Hydrochloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 0.20 g of Cyclopentolate Hydrochloride in 10 ml of chloroform and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add chloroform to make exactly 20 mL. Pipet exactly 1 mL of this solution, add chloroform to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thinlayer chromatography. Develop the plate with a mixture of isopropanol, n-butyl acetate, water and strong ammonia water (100 : 60 : 23 : 17) to a distance of about 10 cm and air-dry the plate. Spray evenly a solution of sulfuric acid in dehydrated ethanol (1 in 10) on the plate and heat at 120 C for 30 minutes. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than

258 Monographs, Part I

intensities of absorption at the same wavenumbers.
Purity (1) Clarity and color of solution Dissolve 0.20 g of Cyclophosphamide Hydrate in 10 mL of water: the solution is clear and colorless. (2) ChloridePerform the test with 0.40 g of Cyclophosphamide Hydrate at a temperature not exceeding 20C. Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.036%). (3) Heavy metalsProceed with 1.0 g of Cyclophosphamide Hydrate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Water Between 5.5% and 7.0% (0.5 g, volumetric titration, direct titration). Assay Weigh accurately about 0.3 g of Cyclophosphamide Hydrate, add 15 mL of hydrogen chloride-ethanol TS and heat in a water-bath under a reflux condenser for 3.5 hours while protecting from moisture. Distill the ethanol under reduced pressure. Dissolve the residue in 40 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric aciddioxane VS (indicator: 2 drops of methylrosaniline chloride TS) until the color of the solution changes from blue through green to yellow. Perform a blank determination and make any necessary correction.

matogram of the test solution under the Assay corresponds to that of the standard solution under the Assay.
pH Weigh accurately a portion of Cyclophosphamide for Injection, equivalent to about 0.2 g of anhydrous Cyclophosphamide and dissolve in 10 mL of water: pH of the solution is between 3.0 and 9.0 (30 minutes after preparation). Sterility Test It meets the requirement. Bacterial Endotoxins Less than 0.20 EU per mg of Cyclophosphamide. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately the contents of not less than 10 Cyclophosphamide for Injections. Weigh accurately a portion of Cyclophosphamide for Injection, equivalent to 0.10 g of anhydrous cyclophosphamide (C7H15Cl2N2O2P) and proceed as directed in the Assay under Cyclophosphamide Tablets. Packaging and Storage Preserve in not exceeding 30 C, hermetic containers. Not exceeding 25 C is recommended.

Each mL of 0.1 mol/L perchloric acid-dioxane VS = 13.955 mg of C7H15Cl2N2O2PH2O

Packaging and Storage Preserve in tight containers. Store at not exceeding 30 C.

Cyclophosphamide Tablets
Cyclophosphamide Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of anhydrous cyclophosphamide (C7H15Cl2 N2O2P: 261.09).
Method of Preparation Prepare as directed under Tablets, with Cyclophosphamide Hydrate. Identification (1) Weigh a quantity of powdered Cyclophosphamide Tablets, equivalent to 50 mg of Cyclophosphamide, extract with 25 mL of chloroform, then filter. To 2 mL of the filtrate, add 0.5 g of potassium bromide, mix, evaporate chloroform, carefully removing the last trace of solvent in a small vacuum flask and use the residue to prepare a potassium bromide disk. Determine the infrared spectra of residue and Cyclophosphamide Hydrate RS (previously dried) as directed in the potassium bromide disk method under the infrared Spectrophotometry and compare the spectra at wavenumbers between 700 cm-1 and 1600 cm-1: both spectra exhibit the same intensities of absorption at the same wavenumbers. (2) The retention time of the major peak in the chromatogram of the test solution under the Assay corresponds to that of the standard solution under the Assay. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement when the content uniformity test is performed according

Cyclophosphamide for Injection

Cyclophosphamide for Injection is a preparation for injection which is dissolved before use and Cyclophosphamide for Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of anhydrous cyclophosphamide (C7H15Cl2 N2O2P: 261.09).
Method of Preparation Prepare as directed under Injections, with Cyclophosphamide Hydrate and Sodium Chloride. Description Cyclophosphamide for Injection is a white powder. Identification (1) Determine the infrared absorption spectra of Cyclophosphamide for Injection and Cyclophosphamide Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both exhibit the similar intensities of absoption at the same wavenumbers. (2) The retention time of the major peak in the chro-

KP 9 259
to the following method. Place 1 Tablet in a volumetric flask of suitable size so that the final concentration is about 500 g/mL. Fill the flask about two-thirds full of water, shake until the Tablet is completely disintegrated, and dilute with water to volume. Filter, discard the first 10 mL of the filtrate, and use the remaining filtrate as the test solution. Separately, dissolve an accurately weighed quantity of Cyclophosphamide Hydrate RS in water, and dilute with water to obtain a solution having a known concentration of about 500 g/mL. Use this solution as the standard solution. Place in separate test tubes 2.0 mL of the test solution, 2.0 mL of water (a blank), and 2.0 mL of the standard solution. Treat each tube as follows. Add 0.7 mL of perchloric acid solution, mix, and heat at 95 C for 10 minutes. Cool, add 1.0 mL of sodium acetate TS, mix, add 1.6 mL of 4-(p-nitrobenzyl)pyridine solution, mix, and heat at 95 C for 10 minutes. Cool, add 8.0 mL of sodium hydroxide solution, and mix. Within 4 minutes, determine the absorbances of the solutions, as directed under the Ultraviolet-visible Spectrophotometry, at the wavelength of maximum absorbance at 560 nm, against the blank. Determine the absorbances of the test solution and the standard solution AT and As, respectively. Amount (mg) of cyclophosphamide (C7H15Cl2N2O2P: 261.09) in the Tablet =
A T C T 500 AS

lophosphamide Hydrate RS QT 4
QS

Internal standard solutionWeigh 0.185 g of poxyethylbenzoate and dissolve in 250 mL of ethanol and add water to make exactly 1000 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 195 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of water and acetonitrile (70 : 30). Flow rate: 1.5 mL/minute. System suitability System performance: When the procedure is run with 25 L of the standard solution under the above operating conditions, cyclophosphamide and internal standard are eluted in this order with a resolution between their peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 25 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of cyclophosphamide to that of internal standard is not more than 2.0%.
Packaging and Storage Preserve in not exceeding 30 C, tight containers.

T: The labeled quanity (mg) of anhydrous cyclophosphamide in the Tablet C: The concentration (g/mL) of cyclophosphamide corrected based on Cyclophosphamide Hydrate RS
Assay Weigh accurately and powder not less than 20 Cyclophosphamide Tablets. Weigh accurately a portion of the powder, equivalent to 0.1 g of anhydrous cyclophosphamide (C7H15Cl2-N2O2P), add 50 mL of water, shake, mix for 30 minutes and add water to make exactly 100 mL. Filter through a membrane filter, discard the first 40 or 50 mL of the filtrate, pipet 25.0 mL of the filtrate, add 5.0 mL of internal standard solution and add water to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately 25 mg of Cyclophosphamide Hydrate RS (previously determine the water content), dissolve in 25 mL of water, add 5.0 mL of internal standard solution and water to make exactly 50 mL and use this solution as the standard solution. Perform the test with 25 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS, of the peak area of cyclophosphamide to that of the internal standard, for the test solution and the standard solution, respectively.

Cyproheptadine Hydrochloride Hydrate

HCl

1 1/2 H2O

N CH 3

C21H21N.HCl.11/2 H2O: 350.88 Cyproheptadine Hydrochloride Hydrate, when dried, contains not less than 98.5% and not more than 101.0% of cyproheptadine hydrochloride (C21H21NHCl: 323.86).
Description Cyproheptadine Hydrochloride Hydrate is a white to pale yellow, crystalline powder, is odorless and has a slightly bitter taste. Cyproheptadine Hydrochloride Hydrate is freely soluble in methanol or in glacial acetic acid, soluble in chloro-

Amount (mg) of cyclophosphamide (C7H15Cl2 N2O2P) = amount (mg) of anhydrous cyclophosphamide in Cyc-

260 Monographs, Part I

form, sparingly soluble in ethanol, slightly soluble in water and practically insoluble in ether.
Identification (1) Dissolve 0.1 g of Cyproheptadine Hydrochloride Hydrate in 10 mL of methanol, apply 1 drop of this solution on filter paper, air-dry and examine under ultraviolet light (main wavelength: 254 nm): the solution shows a pale blue fluorescence. (2) Weigh 0.1 g of Cyproheptadine Hydrochloride Hydrate, transfer to a separatory funnel, dissolve in 5 mL of chloroform, add 4 mL of water and 1 mL of sodium carbonate TS and shake. Transfer the chloroform layer to another separatory funnel and wash with 4 mL of water by shaking well. Filter the chloroform layer through absorbent cotton moistened previously with chloroform and evaporate the filtrate to dryness. Dissolve the residue in 8 mL of dilute ethanol by warming at 65 C. Rub the inner wall of the container with a glass rod while cooling until crystallization begins and allow to stand for 30 minutes. Collect the crystals and dry at 80 C for 2 hours: the crystals melt between 111 C and 115 C. (3) Determine absorption spectra of the solutions of Cyproheptadine Hydrochloride Hydrate and Cyproheptadine Hydrochloride Hydrate RS, in ethanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) A saturated solution of Cyproheptadine Hydrochloride Hydrate responds to the Qualitative Tests (2) for chloride. Purity (1) AcidDissolve 2.0 g of Cyproheptadine Hydrochloride Hydrate in 25 mL of methanol and add 1 drop of methyl red TS and 0.30 mL of 0.1 mol/L sodium hydroxide VS: a yellow color is observed. (2) Heavy metals Proceed with 1.0 g of Cyproheptadine Hydrochloride Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Between 7.0 and 9.0% (1g, in vacuum at the pressure not exceeding 0.67 kPa, 100 C, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Cyproheptadine Hydrochloride Hydrate, previously dried, and dissolve in 20 ml of glacial acetic acid by warming at 50 C. After cooling, add 40 ml of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Cyproterone Acetate
O CH3 H CH3 H H O Cl H H O CH3 O CH3

C24H29ClO4 : 416.94 Cyproterone Acetate contains not less than 97.0% and not more than 103.0% of cyproterone acetate (C24H29ClO4), calculated on a dried basis.
Description Cyproterone Acetate is a white, crystalline powder. Cyproterone Acetate is very soluble in methylene chloride, freely soluble in acetone, soluble in methanol, sparingly soluble in ethanol, and practically insoluble in water Identification (1) To about 1 mg of Cyproterone Acetate, add 2 mLof sulphuric acid and heat on a water-bath for 2 min, and cool. Add the solution cautiously to 4 mL of water and shake. The solution becomes violet. (2) Determine the infrared absorption spectra of Cyproterone Acetate and Cyproterone Acetate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 20 mg of Cyproterone Acetate in methylene chloride to make 10 mL, and use this solution as the test solution. Separately, dissolve 10 mg of Cyproterone Acetate RS in methylene chloride to make 5 mL, and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography, develop with a mixture of cyclohexane and ethyl acetate (50:50) to a distance of about 15 cm, and dry the plate in air. Examine in ultraviolet light at 254 nm, and the principal spot from the test solution and that from the standard solution show the same Rf value. (4) Incinerate about 30 mg of Cyproterone Acetate with 0.3 g of anhydrous sodium carbonate over a naked flame for about 10 minutes. Cool, dissolve the residue in 5 mL of dilute nitric acid, and filter. To 1 mL of the filtrate, add 1 mL of water. The solution responds to the Qualitative Tests (2) for chloride. (5) In a test tube (about 180 mm 18 mm), place about 15 mg of Cyproterone Acetate and 0.15 mL of phosphoric acid. Close the tube with a stopper through

Each ml of 0.1 mol/L perchloric acid VS = 32.386 mg of C21H21NHCl

Packaging and Storage Preserve in well-closed containers.

KP 9 261
which passes a small test tube (about 100 mm 10 mm) containing water to act as a condenser. On the outside of the smaller tube, hang a drop of 5% lanthanum nitrate solution. Place the apparatus in a water-bath for 5 minutes, then take out the smaller tube. Remove the drop and mix it with 0.05 mL of 0.01 mol/L iodine TS on a tile. Add at the edge 0.05 mL of 2 mol/L ammonia. After 1 to 2 minutes, a blue color develops at the junction of the two drops; the color intensifies and persists for a short time.
Specific Optical Rotation [ ]20 D : Between +152 to +157 (0.25 g previously dried, acetone, 20 mL, 100 mm). Purity Related substancesDissolve 10.0 mg of Cyproterone Acetate in acetonitrile to make exactly 10 mL, and use this solution as the test solution. Dilute 1.0 mL of the test solution to 100 mL with acetonitrile, and use this solution as the standard solution (1). Dissolve 5 mg of Medroxyprogesterone acetate RS in acetonitrile to make exactly 50 mL. Dilute 1.0 mL of the solution to 10 mL with the standard solution (1), and use this solution as the standard solution (2). Perform the test with 20 L each of the test solution and the standard solutions as directed under the Liquid Chromatography according to the following conditions. The sum of the areas of all the peaks, other than the principal peak, from the test solution is not greater than 0.5 times the area of the principal peak from the standard solution (1) (0.5%). Disregard any peak with an area less than 0.05 times that of the principal peak from the standard solution (1).

tion to 100 mL with methanol, and use this solution as the test solution. Using methanol as the blank solution, measure the absorbance (A) of the test solution at 282 nm. Amount (mg) of cyproterone acetate (C24H29ClO4) =
A 50000 414

Packaging and Storage well-closed containers.

Preserve in light-resistant,

L-Cystine
H HOOC S H NH2 S COOH NH2

C6H12N2O4S2: 240.30
L-Cystine, when dried, contains not less than 98.5% and not more than 101.0% of L-cystine (C6H12N2O4S2).

Description L-Cystine is a white crystalline powder. L-Cystine is practically insoluble in water or in ethanol. L-Cystine dissolves in dilute sodium hydroxide TS. Identification (1) Determine the infrared spectra of LCystine and L-Cystine RS in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Examine the thin-layer chromatograms obtained in the test for ninhydrin-positive substances under the Purity (2): The principal spot from the test solution (2) is same in color and Rf value the principal spot from the standard solution (1). (3) To 0.1 g of L-Cystine, add carefully 1 mL of strong hydrogen peroxide TS and 0.1 mL of ferric chloride TS and allow to cool. Add 1 mL of dilute hydrochloric acid TS, 5 mL of water and 1 mL of barium chloride TS: a turbidity or a white precipitate develops within 3 minutes. Specific Optical Rotation [ ]20 D : Between -218 and 224 (after drying, 0.5 g, 1 mol/L hydrochloric acid VS, 25 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 1.0 g of L-Cystine in 10 mL of dilute hydrochloric acid TS: The solution is clear and not more intensely colored than Color Matching Solution F. (2) Ninhydrin-positive substancesDissolve 0.1 g

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column about 4.6 mm inside diameter and 12.5 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m in particle diameter). Mobile phase: A mixture of water and acetonitrile (60:40). Flow rate: 1.5 mL/min. System suitability System performance: Adjust the sensitivity of the system so that the height of the principal peak from the standard solution (1) is at least 50% of the full scale of the recorder. When the procedure is run with the standard solution (2), the resolution between the peaks of cyproterone acetate and medroxyprogesterone acetate is not less than 3.0.
Loss on Drying Not more than 0.5% (1 g, 80 C, not exceeding 0.67 kPa, constant weight). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve 50.0 mg of Cyproterone Acetate in methanol to make exactly 50 mL. Dilute 1.0 mL of the solu-

262 Monographs, Part I

of L-Cystine in 10 mL of 1 mol/L hydrochloric acid VS and use this solution as the test solution (1). Pipet 1.0 mL of the test solution, add water to make 50.0 mL and use this solution as the test solution (2). Separately, dissolve 10 mg of L-Cystine RS in 1 mL of 1 mol/L hydrochloric acid VS, add water to make 50 mL and use this solution as the standard solution (1). To 2 mL of the test solution (2) add water to make 20 mL and use this solution as the standard solution (2). Dissolve 10 mg of L-Cystine RS and 10 mg of arginine hydrochloride in 1 mL of 1 mol/L hydrochloric acid VS, add water to make 25 mL and use this solution as the standard solution (3). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution (1), the test solution (2), the standard solution (1), the standard solution (2) and the standard solution (3) on a plate of silica gel for thin-layer chromatography. Develop the plates with an upper layer of a mixture of concentrated ammonia water and 2-propanol (30 : 70) to a distance of about 15 cm and air-dry the plates. Spray evenly with a solution of ninhydrin VS on plate and heat at 100 C to 150 C for 15 minutes: the spots other than principal spot from the test solution (1) are not more intense than the spot from the standard solution (2) and the chromatogram obtained from the standard solution (3) shows two clearly separated spots. (3) ChloridePerform the test with 0.50 g of LCystine. Prepare the control solution with 0.29 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.020%). (4) SulfateDissolve 0.50 g of L-Cystine in 5 mL of dilute hydrochloric acid TS, add water to make 15 mL and use this solution as the test solution. Prepare the control solution with 0.32 mL of 0.005 mol/L sulfuric acid VS, 5 mL of dilute hydrochloric acid TS and add water to make 15 mL (not more than 0.030%). (5) AmmoniumPerform the test with 0.25 g of LCystine. Prepare the control solution with 5.0 mL of standard ammonium solution (not more than 0.020%). (6) Heavy metalsProceed with 2.0 g of L-Cystine according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (7) IronDissolve 1.0 g of L-Cystine in 10 mL of dilute hydrochloric acid TS in a separator, extract with three quantities, each 10 mL of methyl isobutyl ketone. To the methyl isobutyl ketone layer add 10 mL of water and mix with shaking for 3 minutes. Take the water layer, add 30 mL of acetic acid-sodium acetate buffer solution, pH 4.5 and use this solution as the test solution. Perform the test with the test solution according to Method B. Prepare the control solution with 1.0 mL of standard iron solution (not more than 10 ppm).
Loss on Drying Not more than 0.5% (1 g, 105C, constant mass) Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.1 g of L-Cystine, previously dried, transfer to a capped flask and dissolve in 2 mL of dilute sodium hydroxide VS and 10 mL of water. Add 10 mL of potassium bromide solution (2 in 10), 50.0 mL of 1/60 mol/L potassium bromate VS and 15 mL of dilute hydrochloric acid TS. Stopper the flask and cool in iced water. Allow to stand in the dark for 10 minutes and add 1.5 g of potassium iodide. After 1 minute, titrate with 0.1 mol/L sodium thiosulfate (indicator: 2 mL of starch TS). Perform a blank determination and make any necessary correction.

Each mL of 1/60 mol/L solution of potassium bromate VS = 2.4030 mg of C6H12N2O4S2

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Cytarabine
NH2 N

O HOH2C H H OH H O HO

C9H13N3O5: 243.22 Cytarabine, when dried, contains not less than 98.5% and not more than 101.0% of cytarabine (C9H13N3O5).
Description Cytarabine is a white crystals or crystalline powder. Cytarabine is freely soluble in water, soluble in glacial acetic acid, very slightly soluble in ethanol and practically insoluble in ether. Melting pointAbout 214 C (with decomposition) Identification (1) To 1 mL of a solution of Cytarabine (1 in 1000), add 1 drop of bromine TS, allow to stand for 10 minutes and expel the excess bromine under a current of air. To this solution, add 1 mL of a solution of ascorbic acid (1 in 5000) and 1 mL of ninhydrin TS and heat in a water-bath for 30 minutes: a purple color develops. (2) To 1 mL of a solution of Cytarabine (1 in 100), add 1 mL of orcin-ferric chloride TS and heat in a waterbath for 30 minutes: a green color develops.
1% Absorbance E1cm (282 nm): Between 530 and 570 (after drying, 2 mg, 0.1 mol/L hydrochloric acid TS, 200 mL).

Specific Optical Rotation [ ]20 D : Between +154 and +160 (after drying, 0.1 g, water, 10 mL, 100 mm).

KP 9 263
contains not less than 90.0% and not more than 110.0% of the labeled amount of cytarabine (C9H13N3O5: 243.22).
Method of Preparation Prepare as directed under Injections, with cytarabine. Identification The retention time of the major peak in the chromatogram of the test solution under the Assay corresponds to that in the chromatogram of the standard solution under the Assay. pH Between 4.0 and 6.0 (water, 20 mg/mL). Sterility Test It meets the requirement. Bacterial Endotoxins Less than 0.07 EU per mg of Cytarabine. Uniformity of Dosage Units It meets the requirement. Water Not more than 3.0% (1 g, volumetric titration, direct titration). Assay Take 10 Cytarabine for Injection, dissolve in water to make a solution containing 10 mg of cytarabine (C9H13N3O5) per mL. Pipet 5.0 mL of this solution and add water to make exactly 50 mL. Pipet 3.0 mL of this solution, add 5.0 mL of the internal standard solution and mobile phase to make exactly 25 mL and use this solution as the test solution. Separately, weigh accurately 50 mg of Cytarabine RS (previously dried at 60 C, not more than 0.67 kPa, for 3 hours), dissolve in water to make exactly 50 mL. Pipet 3.0 mL of this solution and add 5.0 mL of the internal standard solutio and mobile phase to make exactly 25 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Cytarabine to that of the internal standard for the test solution and the standard solution, respectively.

pH Dissolve 0.20 g of Cytarabine in 20 mL of water: the pH of this solution is between 6.5 and 8.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Cytarabine in 10 mL of water: the solution is clear and colorless. (2) ChloridePerform the test with 1.0 g of Cytarabine. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.009%). (3) Heavy metalsProceed with 1.0 g of Cytarabine according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Cytarabine according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.10 g of Cytarabine in 10 mL of water and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add water to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with n-butanol saturated with water to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Spray evenly acidic potassium permanganate TS on the plate: any spot other than the principal spot does not appear. Loss on Drying Not more than 1.0% (1 g, in vacuum, silica gel, 4 hours). Residue on Ignition Not more than 0.5% (1 g). Assay Weigh accurately about 0.2 g of Cytarabine, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.05 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Methods in Titrimetry). Perform a blank determination and make any necessary correction.

Amount (mg) of cytarabine (C9H13N3O5) = amount (mg) of Cytarabine RS QT 1

QS

Each mL of 0.05 mol/L perchloric acid VS = 12.161 mg of C9H13N3O5

Packaging and Storage Preserve in tight containers.

Internal standard solution0.14% p-toluenic acid in methanol. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, 4 mm to 4.6 mm in inside diameter and 25 cm to 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Mobile phase: Dissolve 0.69 g of sodium dihydro-

Cytarabine for Injection

Cytarabine for Injection is a preparation for injection which is dissolved before use. Cytarabine for Injection

264 Monographs, Part I

phosphate and 1.34 g of sodium monophosphate in 950 mL of water. Add 50 mL of methanol and mix. Flow rate: 1 mL/minute. System suitability System performance: Weigh a sufficient portion of Uracylarabinoside RS and Cytarabine RS, dissolve in water to make a solution containing 10 g and 600 g per 1 mL, respectively, dilute with same volume of internal standard solution, mix with about four times volume of mobile phase. When the procedure is run with 10 L of this solution under the above operating condition, cytarabine, uracylarabinoside and p-toluenic acid are eluted in this order with a resolution between cytarabine and uracylarabinoside peaks being not less than 2.5. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of cytarabine to that of the internal standard is not more than 2.0%.
Packaging and Storage Preserve in hermetic containers.

mL of water and 2 drops of glacial acetic acid, shake well and filter: the filtrate responds to the Qualitative Tests (1) for sodium salt.
Purity (1) AlkaliTo about 0.7 g of Dantrolene Sodium Hydrate, add 10 mL of water, shake well and centrifuge or filter through a membrane filter. To 5 mL of the supernatant or the filtrate, add 45 mL of water, 3 drops of phenophthalein TS and 0.10 mL of 0.1 mol/L hydrochloric acid VS: a red color is not observed. (2) Heavy metalsProceed with 1.0 g of Dantrolene Sodium Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 50.0 mg of Dantrolene Sodium Hydrate in 20 mL of tetrahydrofuran and 2 mL of glacial acetic acid, add dehydrated ethanol to make exactly 100 mL and use this solution as the test solution. Pipet 1.0 mL of the test solution, add dehydrated ethanol to make exactly 50 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area from the test solution and the standard solution by the automatic integration method: the total area of all peaks other than the peak of dantrolene from the test solution is not larger than the peak area of dantrolene from the standard solution.

Dantrolene Sodium Hydrate

O O O 2N NNa H C N N O 3 1/2 H2O

C14H9N4NaO531/2 H2O: 399.29 Dantrolene Sodium contains not less than 98.0% and not more than 101.0% of dantrolene sodium (C14H9-N4 NaO5), calculated on the anhydrous basis.
Description Dantrolene Sodium Hydrate is a yellowish orange to deep orange, crystalline powder. Dantrolene Sodium Hydrate is soluble in propylene glycol, sparingly soluble in methanol, slightly soluble in ethanol, very slightly soluble in water or in glacial acetic acid and practically insoluble in acetone, in tetrahydrofuran or in ether. Identification (1) Determine the absorption spectra of solutions of Dantrolene Sodium Hydrate and Dantrolene Sodium Hydrate RS in methanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Dantrolene Sodium Hydrate and Dantrolene Sodium Hydrate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both exhibit similar intensity of absorption at the same wavenumbers. (3) To 0.1 g of Dantrolene Sodium Hydrate, add 20

Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 300 nm). Column : A stainless steel column, 4 m in inside diameter and about 15 cm in length, packed with silica gel for liquid chromatography (5 m in particle diameter). Column temperature : A constant temperature of about 30 C. Mobile phase: A mixture of hexane, glacial acetic acid and dehydrated ethanol (90 : 10 : 9). Flow rate : Adjust the flow rate so that the retention time of dantrolene is about 8 minutes. System suitability System performance: Dissolve 5 mg of Dantrolene Sodium Hydrate and 0.1 g of theophylline in 20 mL of tetrahydrofuran and 2 mL of glacial acetic acid and add dehydrated ethanol to make 100 mL. To 10 mL of this solution, add dehydrated ethanol to make 100 mL and use this solution as the standard solution. When the procedure is run with 10 L of the standard solution under the above operating conditions, theophylline and dantrolene are eluted in this order with the resolution between their peaks being not less than 6.0. Detection sensitivity: Adjust the detection sensitivity so that the peak height of dantrolene from 10 L of the standard solution is 10% to 40% of the full scale. Time span of measurement: About twice as long as the retention time of Dantrolene after the solvent peak.

KP 9 265
Water Between 14.5% and 17.0% (0.2 g, volumetric titration, direct titration). Assay Weigh accurately 0.7 g of Dantrolene Sodium Hydrate, dissolve in 180 mL of a mixture of propylene glycol and acetone (1 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 33.624 mg of C14H9N4NaO5

Packaging and Storage Preserve in tight containers.

266 Monographs, Part I

in 50.0 mL of methanol and use this solution as the test solution. Pipet 1.0 mL of this solution accurately and add methanol to make exactly 100 mL and use this solution as the standard solution (1). Pipet 10.0 mL of the standard solution (1), add methanol to make exactly 50 mL and use this as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethylacetate, n-heptane, methanol and 13.5 mol/L of ammonia reagent mixture (20 : 20 : 6 : 1) and air-dry the plate. Spray evenly 0.1% 4-dimethylaminocinnam aldehyde solution of hydrochloric acid and ethanol mixture (1 : 99) on the plate: the spot having a strong intensity second to the principal spot from the test solution is not more intense than that from the standard solution (1). The spots other than the above two spots from the test solution are not more intense than that from the standard solution (2).
Loss on Drying Not more than 1.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 50 mg of Dapsone, dissolve in methanol to make exactly 200 mL. Pipet 5 mL of the solution, add methanol to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Dapsone RS, diluted similarly to the test solution with methanol and use this solution as the standard solution. Perform the test with 10 L each of the test and standard solution as directed under the Liquid Chromatography. Determine each peak area, AT and AS , of Dapsone for the test solution and the standard solution.

Dapsone
O S O

H2N

NH2

D.D.S

C12H12N2O2S: 248.30

Dapsone contains not less than 98.0% and not more than 102.0% of dapsone (C12H12N2O2S), calculated on the anhydrous basis.
Description Dapsone is a white or pale yellow crystalline powder. Dapsone is freely soluble in acetone, slightly soluble in ethanol and practically insoluble in water. Dapsone dissolves in dilute inorganic acid, Identification (1) Determine the absorption spectra of solutions of Dapsone and Dapsone RS in methanol (1 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Dry a suitable amount of Dapsone and Dapsone RS and measure the infrared spectra as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibits similar intensities of absorption at the same wavenumbers. Melting Point

Between 175 C and 181 C.

Purity (1) SeleniumAdd 0.5 mL of a mixture containing perchloric acid and sulfuric acid (1 : 1), 2 mL of nitric acid to about 0.10 g of Dapsone and heat in a steam bath. Cool the solution until a brown gas is not evolved and the solution becomes clear with an evanescent yellowish color. Then, add 4 mL of nitric acid and water to make exactly 50 mL. Use this solution as the test solution. Separately, pipet 3 mL of selenium standard solution, add 0.5 mL of a mixture containing perchloric acid and sulfuric acid (1 : 1), 6 mL of nitric acid and water to make exactly 50 mL. Use this solution as the standard solution. Perform the test on the standard and the test solution as directed under the Atomic Absorption Spectrophotometry. Measure the absorbance AT for the test solution and AS for the standard solution when the reading of the display rapidly rises and, then, reaches a plateau. AT is less than AS (not more than 30 ppm). This test is performed using a hydride generator and a heating cell. Atomizing temperature : When an electric furnace is used, the temperature is set at about 1000 C. Carrier gas: Nitrogen or argon Lamp : Selenium hollow-cathode lamp Wavelength : 196.0 nm

Amount (mg) of dapsone (C12H12N2O2S) = Amount (mg) of Dapsone RS

AT AS

(2) Related substancesDissolve 0.50 g of Dapsone

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with porous silica for liquid chromatography (10 m in particle diameter). Column temperature: A room temperature. Mobile phase: Mix 100 mL of isopropanol, 100 mL of ethylacetate, 100 mL of acetonitrile and pentane to make 1000 mL. Allow the mixture to cool to room temperature. System suitability System repeatability: When the test is repeated 6 times with 10 L each of the standard solution under the

KP 9 267
above operating conditions, the relative standard deviation of the peak area of dapsone is not more than 2.0 %.
Packaging and Storage well-closed containers.

Preserve in light-resistant,

Daunorubicin Hydrochloride
O OH O CH3 OH

Pyrogen Test It meets the requirement, when Daunorubicin Hydrochloride is used in a sterile preparation. Weigh appropriate amount of Daunorubicin Hydrochloride, dissolve in water, make the solution so that each mL contains 2.0 mg, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Histamine It meets the requirement, when Daunorubicin Hydrochloride is used in a sterile preparation,. Weigh appropriate amount of Daunorubicin Hydrochloride, dissolve in water, make the solution so that each mL contains 5.0 mg, and use the solution as the test solution.

O H3C

OH

H3C

HCl
O NH2 OH

Loss on Drying Not more than 8.0 % (0.1 g, in vacuum, 60 C, 3 hours). Assay Weigh accurately an amount of Daunorubicin Hydrochloride and Daunorubicin Hydrochloride RS, equivalent to about 25 mg (potency), dissolve each in a suitable amount of the mobile phase, add exactly 4 mL of the internal standard solution and the mobile phase to make 20 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 5 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS , of the peak area of daunorubicin to that of the internal standard..

C27H29NO10HCl: 563.98

Daunorubicin Hydrochloride is the hydrochloride of an anthracycline substance having antitumor activity produced by the growth of Streptomyces peucetius. Daunorubicin Hydrochloride contains not less than 900 g (potency) per mg of daunoarubicin hydrochloride (C27H29NO10HCl), calculated on the dried basis.
Description Daunorubicin Hydrochloride is a red powder. Daunorubicin Hydrochloride is very soluble in water and in methanol, slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Dissolve 1 ~ 2 mg of Daunorubicin Hydrochloride in 5 mL of 1 mol/L sodium hydroxide: a blue-purple color develops. (2) Determine the absorption spectra of the solutions of Daunorubicin Hydrochloride and Daunorubicin Hydrochloride RS in methanol (1 in 100000), as directed under Ultraviolet-visible Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wavelengths. Specific Optical Rotation [ ] 20 D : +250 ~ +275 (15 mg calculated on the dried basis, methanol, 10 mL, 100 mm). Absorbance E1 cm (495 nm): 210 ~ 250 (10 mg calculated on the dried basis, methanol, 500 mL). pH The pH of a solution obtained by dissolving 50 mg of Daunorubicin Hydrochloride in 10 mL of water is between 4.5 and 6.5. Sterility Test It meets the requirement, when Daunorubicin Hydrochloride is used in a sterile preparation,.
1%

Amount [g (potency)] of Daunorubicin hydrochloride (C27H29NO10HCl) = Amount [g (potency)] of Daunorubicin hydrochloride RS

QT QS

Internal standard solutionA solution of 2naphthalenesulfonic acid in the mobile phase (1 in 100) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) Column temperature: A room temperature Mobile phase: Adjust the pH of a mixture of water and acetonitrile (62:38) to 2.2 0.2 with phosphoric acid Flow rate: Adjust the flow rate so that the retention time of daunorubicin is about 9 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, the internal standard and daunorubicin are eluted in this order with the resolution between these peaks being not less than 2.0.

268 Monographs, Part I

System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the operating conditions, the relative standard deviation of the ratios of the peak area of daunorubicin to that of the internal standard is not more than 2.0 %.
Packaging and Storage Preserve in tight containers.

Deferoxamine Mesilate
O H2N(H2C)5N OH C CH2CH2 O C NH(CH 2)5N OH O C CH2CH2 O C NH(CH 2)5N OH O C CH2 CH3SO3H

C25H48N6O8CH4O3S: 656.79 Deferoxamine Mesilate contains not less than 98.0% and not more than 102.0% of deferoxamine mesilate (C25H48N6O8CH4O3S), calculated on the anhydrous basis.
Description Deferoxamine Mesilate is a white to pale yellow white, crystalline powder. Deferoxamine Mesilate is freely soluble in water and practically insoluble in dehydrated ethanol, in isopropanol or in ether. Melting pointAbout 147 C (with decomposition). Identification (1) To 5 mL of a solution of Deferoxamine Mesilate (1 in 500), add 1 drop of ferric chloride TS: a deep red color is observed. (2) To 50 mg of Deferoxamine Mesilate, add 0.2 g of sodium hydroxide, melt by heating over a small flame and heat further for 2 to 3 seconds. To the residue, add 0.5 mL of water, acidify with dilute hydrochloric acid and warm: the gas evolved changes moistened potassium iodate-starch paper to blue. (3) Determine the infrared spectra of Deferoxamine Mesilate and Deferoxamine Mesilate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both exhibit similar intensities of absorption at the same wavenumbers. pH Dissolve 1.0 g of Deferoxamine Mesilate in 10 mL of water: the pH of this solution is between 3.5 and 5.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Deferoxamine Mesilate in 10 mL of water: the solution is clear and colorless to pale yellow. (2) ChloridePerform the test with 1.0 g of Deferoxamine Mesilate. Prepare the control solution with 0.90 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.032%). (3) SulfatePerform the test with 0.6 g of Deferoxamine Mesilate. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.040%).

(4) Heavy metalsProceed with 2.0 g of Deferoxamine Mesilate according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) ArsenicPrepare the test solution with 1.0 g of Deferoxamine Mesilate according to Method 3 and perform the test. Use a solution of magnesium nitrate in ethanol (1 in 10) (not more than 2 ppm). (6) Related substancesDissolve 50.0 mg of Deferoxamine Mesilate in 50.0 mL of the mobile phase and use this solution as the test solution. Pipet 3.0 mL of the test solution, add the mobile phase to make exactly 50 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution and the standard solution by the automatic integration method: the total area of all peaks other than the peak of deferoxamine from the test solution is not larger than the peak area of deferoxamine from the standard solution.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 20 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 1.32 g of dibasic ammonium phosphate, 0.37 g of disodium ethylenediaminetetraacetate and 1.08 g of sodium 1-heptanesulfonate in 950 mL of water. Adjust pH of this solution with phosphoric acid to 2.8 and add 100 mL of isopropanol to 800 mL of this solution. Flow rate: Adjust the flow rate so that the retention time of deferoxamine is about 15 minutes. System suitability System performance: Dissolve 16 mg of Deferoxamine Mesilate and 4 mg of methylparahydroxybenzoate in 50 mL of the mobile phase. When the procedure is run with 20 L of this solution under the above operating conditions, deferoxamine and methyl p-hydroxybenzoate are eluted in this order with the resolution between their peaks being not less than 4.0. Detection sensitivity: Adjust the detection sensitivity so that the peak height of deferoxamine from 20 L of the standard solution is between 5 mm and 20 mm. Time span of measurement: About twice as long as the retention time of deferoxamine after the solvent peak.
Water Not more than 2.0% (0.2 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 60 mg of Deferoxamine

KP 9 269
Mesilate and Deferoxamine Mesilate RS (determine the content of water before using in the same manner as Deferoxamine Mesilate), dissolve each in 20 mL of water, add 10 mL of 0.05 mol/L sulfuric acid TS and add water to make exactly 50 mL. Pipet 5.0 mL each of these solutions, add exactly 5 mL of 0.05 mol/L sulfuric acid TS and exactly 0.2 mL of ferric chloride TS, then add water to make exactly 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Spectrophotometry, using a solution prepared by adding 0.05 mol/L sulfuric acid TS to 0.2 mL of ferric chloride TS to make exactly 50 mL as the blank and determine the absorbances, AT and AS, for the test solution and the standard solution, respectively, at 430 nm. Amount (mg) of deferoxamine mesilate (C25H48N6O8CH4O3S) = amount (mg) of Deferoxamine Mesilate RS, A calculated on the anhydrous basis T AS
Packaging and Storage Preserve in tight containers.

gradually changes to brown.

Specific Optical Rotation [ ]20 D : Between +29 and +32 (after drying, 0.2 g, dioxane, 10 mL, 100 mm). Melting Point

Between 233 C and 242 C.

Dehydrocholic Acid
H O H3C CH3 H CO2H

CH3

H O H

H O

C24H34O5: 402.52 Dehydrocholic Acid, when dried, contains not less than 98.5% and not more than 101.0% of dehydrocholic acid (C24H34O5).
Description Dehydrocholic Acid is a white crystalline powder, is odorless and has a bitter taste. Dehydrocholic Acid is sparingly soluble in dioxane, slightly soluble in ethanol and practically insoluble in water or in ether. Dehydrocholic Acid dissolves in sodium hydroxide TS. Identification (1) Dissolve 5 mg of Dehydrocholic Acid in 1 mL of sulfuric acid and 1 drop of formalin, allow to stand for 5 minutes and add 5 mL of water: the solution shows a yellow color and a blue-green fluorescence. (2) To 20 mg of Dehydrocholic Acid, add 1 mL of ethanol, shake, add 5 drops of m-dinitrobezene TS and 0.5 mL of a solution of sodium hydroxide (1 in 8) and allow to stand: a purple to red-purple color is observed and

Purity (1) OdorTo 2.0 g of Dehydrocholic Acid, add 100 mL of water and boil for 2 minutes: the solution is odorless. (2) Clarity and color of solutionTo 0.10 g of Dehydrocholic Acid, previously powdered, add 30 mL of ethanol and dissolve by shaking for 10 minutes: the solution is clear and colorless. (3) ChlorideTo 2.0 g of Dehydrocholic Acid, add 100 mL of water, shake for 5 minutes and filter. Take 25 mL of the this solution, add 6 mL of dilute nitric acid, heat in a water-bath for 6 minutes, filter after cooling and collect the clear filtrate. Wash the residue with 10 mL of water, combine the washings and the filtrate, dilute with water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.021%). (4) SulfateAdd 1 mL of dilute hydrochloric acid to 25 mL of the test solution obtained in (3), heat in a water-bath for 6 minutes, filter after cooling and collect the clear filtrate. Wash the residue with 10 mL of water, combine the washings and the filtrate, dilute with water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.5 mL of 0.005 mol/L sulfuric acid VS (not more than 0.048%). (5) Heavy metalsProceed with 1.0 g of Dehydrocholic Acid according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (6) BariumTo the solution obtained in (1), add 2 mL of hydrochloric acid and boil for 2 minutes. Cool, filter and wash with water until 100 mL of the filtrate is obtained. To 10 mL of the filtrate, add 1 mL of dilute sulfuric acid: no turbidity is produced. Loss on Drying Not more than 1.0% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.5 g of Dehydrocholic Acid, previously dried, add 40 mL of neutralized ethanol and 20 mL of water and dissolve by warming. Add 2 drops of phenolphthalein TS, titrate with 0.1 mol/L sodium hydroxide VS, add 100 mL of freshly boiled and cooled water as the end point is approached and continue the titration.

Each mL of 0.1 mol/L sodium hydroxide VS = 40.25 mg of C24H34O5

270 Monographs, Part I

Packaging and Storage Preserve in well-closed containers.

Dehydrocholic Acid Injection

Sodium Dehydrocholate Injection

Purified Dehydrocholic Acid

H O H3C CH3 H CO2H

Dehydrocholic Acid Injection is an aqueous solution for injection. Dehydrocholic Acid Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of dehydrocholic acid (C24H34O5: 402.52).
Method of Preparation Dissolve Purified Dehydrocholic Acid in a solution of Sodium Hydroxide and prepare as directed under Injections.

CH3

H O H

H O

C24H34O5: 402.52 Purified Dehydrocholic Acid, when dried, contains not less than 99.0% and not more than 101.0% of dehydrocholic acid (C24H34O5: 402.52).
Description Purified Dehydrocholic Acid is a white crystalline powder, is odorless and has a bitter taste. Purified Dehydrocholic Acid is sparingly soluble in dioxane, slightly soluble in ethanol and practically insoluble in water or in ether. Purified Dehydrocholic Acid dissolves in sodium hydroxide TS. Identification Purified Dehydrocholic Acid responds to the Identification for Dehydrocholic Acid. Specific Optical Rotation [ ]20 D : Between +29 and +32 (after drying, 0.2 g, dioxane, 10 mL, 100 mm). Melting Point Between 237 C and 242 C. Purity Purified Dehydrocholic Acid responds to the Purity for Dehydrocholic Acid. Loss on Drying Not more than 1.0% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Purified Dehydrocholic Acid responds to the Assay for Dehydrocholic Acid.

Description Dehydrocholic Acid Injection is a clear, colorless to pale yellow liquid and has a bitter taste. pHBetween 9 and 11. Identification Transfer a volume of Dehydrocholic Acid Injection, equivalent to 0.1 g of Purified Dehydrocholic Acid according to the labeled amount, to a separator and add 10 mL of water and 1 mL of dilute hydrochloric acid: a white precipitate is produced. Extract the mixture with three 15 mL volumes of chloroform, combine all of the chloroform extracts, evaporate the chloroform on a water-bath and dry the residue at 105 C for 1 hour: the residue so obtained melts between 235 C and 242 C. Purity Heavy metalsEvaporate a volume of Dehydrocholic Acid Injection, equivalent to 1.0 g of Purified Dehydrocholic Acid according to the labeled amount, on a water-bath to dryness. Proceed with the residue according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Sterility Test It meets the requirement. Bacterial Endotoxins Less than 0.30 EU per 1 mg of Dehydrocholic Acid. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Each mL of 0.1 mol/L sodium hydroxide VS = 40.25 mg of C24H34O5

Packaging and Storage Preserve in well-closed containers.

Determination of Volume of Injection in Containers It meets the requirement. Assay Weigh exactly a volume of Dehydrocholic Acid Injection, equivalent to about 0.5 g of dehydrocholic acid (C24H34O5) and add methanol to make exactly 50 mL. Pipet 5.0 mL of this solution and add 8.0 mL of the internal standard solution. Then add methanol to make exactly 50 mL and use this solution as the test solution.

KP 9 271
Separately weigh accurately 50 mg of Dehydrocholic Acid RS, previously dried, add 8.0 mL of the internal standard solution, add methanol to make exactly 50 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of dehydrocholic acid of the test solution and the standard solution to that of the internal standard, respectively. Amount (mg) of dehydrocholic acid (C24H34O5) = amount (mg) of Dehydrocholic Acid RS QT QS

Deslanoside
O O HO H CH3 H CH3 H CH3 H H H CH3 H CH2OH H O H OH H HO H H OH H H O OH H O O OH H O H H O OH H O O H H H OH CH3 H

10

Internal standard solutionA methanol solution of butyl parahydroxybenzoate (3 in 3200). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of 10 mL of acetic acid with 1000 mL of a mixture of water and acetonitrile (55 : 45). Flow rate : Adjust the flow rate so that the retention time of Dehydrocholiric Acid is about 12 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, dehydrocholinic acid and internal standard are eluted in this order with the resolution between their peaks being not less than 5.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak areas of dehydrocholinic acid to that of the ineternal standard is not more than 2.0%.
Packaging and Storage Preserve in light-resistant, hermetic containers. Colored containers may be used.

C47H74O19: 943.08 Deslanoside, when dried, contains not less than 90.0% and not more than 102.0% of deslanoside (C47H74O19).
Description Deslanoside is a colorless or white crystal or white crystalline powder. Deslanoside is freely soluble in anhydrous pyridine, sparingly soluble in methanol, slightly soluble in ethanol and practically insoluble in water or in ether. Deslanoside is hygroscopic. Identification Transfer 1 mg of Deslanoside to a small test tube, about 10 mm in inside diameter, dissolve in 1 mL of a solution of ferric chloride in glacial acetic acid (1 in 10000) and underlay gently with 1 mL of sulfuric acid: at the zone of contact of two liquids, a brown ring is produced and the color of the upper layer near to the contact zone changes gradually to blue through purple and the entire glacial acetic acid layer changes to a bluegreen color through a deep blue color. Specific Optical Rotation [ ]20 D : Between +6.5 and +8.5 (after drying, 0.50 g, anhydrous pyridine, 25 mL, 100 mm). Purity (1) Clarity and color of solution Dissolve 20 mg of Deslanoside in 10 mL of ethanol and 3 mL of water by warming, cool and dilute to 100 mL with water: the solution is clear and colorless. (2) Related substancesDissolve 10.0 mg of Deslanoside in exactly 5 mL of methanol and use this solution as the test solution. Dissolve 1.0 mg of Deslanoside RS in exactly 5 mL of methanol and use this solution as the standard solution. Perform the test with the test solution

272 Monographs, Part I

and the standard solution as directed under the Thin-layer Chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel for thinlayer chromatography. Develop the plate with a mixture of dichloromethane, methanol and water (84 : 15 : 1) to a distance of about 13 cm and air-dry the plate. Spray evenly dilute sulfuric acid on the plate and heat the plate at 110 C for 10 minutes: the spots other than the principal spot from the test solution are not larger than and more intense than the spot from the standard solution.
Loss on Drying Not more than 8.0% (0.5 g, in vacuum, P2O5, 60 C, 4 hours). Residue on Ignition Not more than 0.5% (0.1 g). Assay Dissolve about 12 mg each of Deslanoside and Deslanoside RS, previously dried and accurately weighed, in 20 mL each of methanol, add water to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Pipet 5.0 mL each of the test solution and the standard solution, transfer to light-resistant, volumetric flasks, shake well with 5 mL each of picric acid TS and 0.5 mL each of a solution of sodium hydroxide (1 in 10), diluted methanol (1 in 4) to make exactly 25 mL and allow to stand at a temperature between 18 C and 22 C for 25 minutes. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 485 nm as directed under the Ultraviolet-visible Spectrophotometry, using a solution prepared with 5 mL of diluted methanol (1 in 5) in the same manner as the blank.
o

Identification (1) Place a volume of Deslanoside Injection, equivalent to 2 mg of Deslanoside according to the labeled amount, in a separatory funnel, add sodium chloride in the ratio of 0.2 g to each mL of this solution and extract with three 10 mL volumes of chloroform. Combine the chloroform extracts, mix uniformly, pipet 15 mL of this solution and evaporate the chloroform under reduced pressure. Proceed with the residue as directed in the Identification under Deslanoside. (2) Place a volume of Deslanoside Injection, equivalent to 1 mg of Deslanoside according to the labeled amount, in a separator, add sodium chloride in the ratio of 0.2 g to each mL of this solution, and extract with three 5-mL portions of chloroform. Combine the chloroform extracts, evaporate the chloroform under reduced pressure, dissolve the residue in 5 mL of methanol, and use this solution as the test solution. Separately, dissolve 1 mg of Deslanoside RS in 5 mL of methanol, and use this solution as the standard solution. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 20 L each of these solutions on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of dichloromethane, methanol and water (84 : 15 : 1) to a distance of about 13 cm and air-dry the plate. Spray evenly dilute sulfuric acid upon the plate, and heat the plate at 110 C for 10 minutes: the spots from the test solution and the standard solution show a black color and have the same Rf value. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Amount (mg) of deslanoside (C47H74O19) A = amount (mg) of Deslanoside RS T AS

Packaging and Storage Preserve in tight containers.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of Deslanoside Injection, equivalent to about 3 mg of deslanoside (C47H74O19). Add 5 mL of methanol and water to make exactly 25 mL. Use this solution as the test solution and proceed as directed in the Assay under Deslanoside.

Deslanoside Injection
Deslanoside Injection is an aqueous solution for injection. Deslanoside Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of deslanoside (C47H74O19: 943.08).
Method of Preparation Dissolve Deslanoside in 10 vol% of ethanol and prepare as directed under Injections. Deslanoside Injection may contain Glycerin. Deslanoside Injection may be prepared with a suitable amount of Ethanol and Water for Injection. Description Deslanoside Injection is a clear and colorless liquid. pHBetween 5.0 and 7.0.

Amount (mg) of deslanoside (C47H74O19) 1 A = amount (mg) of Deslanoside RS T 4 AS

Packaging and Storage hermetic containers.

Preserve in light-resistant,

KP 9 273
Loss on Drying Not more than 1.0% (1 g, 105 C, constant mass). Residue on Ignition Not more than 0.2% (1 g).
OH H CH3 H CH3 F O H

Desoximetasone
O H HO CH3 H

C22H29FO4 : 376.46

Desoximetasone contains not less than 97.0% and not more than 103.0% of desoximetasone (C22H29FO4), calculated on the dried basis.
Description Desoximetasone is a white, crystalline powder. Desoximetasone is very soluble in ethanol, in acetone or in chloroform and practically insoluble in water. Identification (1) Determine the infrared spectra of Desoximetasone and Desoximetasone RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Dissolve 0.1 g of Desoximetasone in a mixture of chloroform and ethanol (3:1) to make 10 mL and use this solution as the test solution. Separately, dissolve 0.1 g of Desoximetasone RS in a mixture of chloroform and ethanol (3:1) to make 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform and ethyl acetate (1:1) to a distance of about 10 cm, and air-dry the plate. Expose the plate with ultraviolet light (main wavelength 254 nm) and spray evenly the plate with ptoluenesulfonic acid in ethanol (1 in 5): the Rf value of the principal spot obtained from the test solution corresponds to that from the standard solution. Melting Point Between 206 C and 218 C, but the range between beginning and end of melting does not exceed 4 C. Specific Optical Rotation [ ]25 D : Between +107 and -112 (0.1 g, after drying, chloroform, 20 mL, 100 mm). Purity Heavy metalsProceed with 1.0 g of Desoximetasone according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm).

Assay Weigh accurately about 40 mg of Desoximetasone, add methanol to make exactly 100 mL, pipet 10.0 mL of this solution, add a mixture of methanol and acetonitrile (1:1) to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 20 mg of Desoximetasone RS, add methanol to make exactly 50 mL, pipet 10.0 mL of this solution, add a mixture of methanol and acetonitrile (1:1) to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and determine the height of the desoximetasone peak in the test solution, HT , and in the standard

solution, H S . Amount (mg) of desoximetasone (C22H29FO4) = amount (mg) of Desoximetasone RS H T

HS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 15 cm in length, packed with octylsilanized silica gel for liquid chromatography (3-10 m in particle diameter). Mobile phase: A mixture of methanol, water and glacial acetic acid (65:35:1). Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the symmetry factor for desoximetasone peak is not more than 2.0. System repeatability: When the test is repeated 5 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the heights of desoximetasone peak is not more than 2.0%.
Packaging and Storage Preserve in well-closed containers.

274 Monographs, Part I

the test solution. Pipet 2 mL of the test solution, add a solution, prepared by dissolving 1.32 g of ammonium formate in water to make 1000 mL and adjusted to pH 3.6 with formic acid, to make 100 mL, and use this solution as the test solution. To exactly 1 mL of the test solution, add the mobile phase to make exactly 100 mL, and use this solution as the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area by the automatic integration method: the peak area other than dexamethasone is not larger than the peak area of dexamethasone obtained from the standard solution, and the total area of the peaks other than the peak of dexamethasone from the sample solution is not larger than 2 times the peak area of dexamethasone from the standard solution

Dexamethasone
O H HO H3C H H F O H H3C CH2OH OH CH3

C22H29FO5: 392.46 Dexamethasone, when dried, contains not less than 97.0% and not more than 102.0% of dexamethasone (C22H29FO5).
Description Dexamethasone is a white to pale yellow crystal or crystalline powder and is odorless. Dexamethasone is sparingly soluble in methanol, in ethanol or in acetone and practically insoluble in water. Melting pointAbout 245 C (with decomposition). Identification (1) Proceed with 10 mg of Dexamethasone as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid: the solution responds to the Qualitative Tests for fluoride. (2) Dissolve separately 1.0 mg of Dexamethasone and Dexamethansone RS in 10 mL of ethanol, pipet 2.0 mL each of these solutions, add 10 mL each of phenylhydrazine hydrochloride TS, shake and warm on a water-

bath at 60 for 20 minutes. Cool and determine the absorption spectra with these solutions as directed under the Ultraviolet-visible Spectrophotometry using a solution, prepared with 2.0 mL of ethanol in the same manner, as the blank: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Dexamethasone and Dexamethasone RS, previously dried, according to the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, dissolve Dexamethasone and Dexamethasone RS in acetone, respectively, evaporate to dryness and repeat the test on the residues.
Specific Optical Rotation [ ]20 D : Between +86 and +94 (after drying, 0.1 g, methanol, 10 mL, 100 mm). Purity (1) Heavy metalsProceed with 1.0 g of Dexamethasone according to Method 2 and perform the test. Prepare the control solution with 3.0 mL of standard lead solution (not more than 30 ppm). (2) Related SubstancesDissolve 0.10 g of Dexamethasone in 10 mL of acetone and use this solution as

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with phenylsilanized silica gel for liquid chromatography (5 m in particle diameter ). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 1.32 g of ammonium formate in 1000 mL of water, and adjust the pH to 3.6 with formic acid. To 670 mL of this solution, add 330 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retention time of dexamethasone is about 13 minutes. System suitability Test for required detectability: To exactly 1 mL of the standard solution add the mobile phase to make exactly 10 mL. Confirm that the peak area of dexamethasone obtained with 10 L of this solution is equivalent to 8 to 12% of that with 10 L of the standard solution. System performance: When the procedure is run with 10L of the standard solution under the above operating conditions, the number of theoretical plates and the symmetry factor of the peak of dexamethasone are not less than 5000 and not more than 1.5, respectively. System repeatability: When the test is repeated 6 times with 10 mL of the standard solution under the above operating conditions, the relative standard deviation of the peak area of dexamethasone is not more than 1.0%. Time span of measurement: About 4 times as long as the retention time of dexamethasone beginning after the solvent peak.
Loss on Drying Not more than 0.5% (0.2 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.2 g, platinum crucible).

KP 9 275
Assay Dissolve about 10 mg each of Dexamethasone and Dexamethasone RS, previously dried and accurately weighed, in 70 mL each of diluted methanol (1 in 2), add exactly 5 mL each of the internal standard solution, then add diluted methanol (1 in 2) to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT

Method of Preparation Prepare as directed under Tablets, with Dexamethasone. Identification Evaporate 10 mL of the methanol extract of Dexamethsone Tablets, obtained as directed under preparation of the test solution in the Assay on a water-bath to dryness, dissolve the residue in 1 mL of chloroform and use this solution as the test solution. Separately, weigh a quantity of Dexamethasone RS and dissolve in chloroform to contain 500 g per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L of the test solution and 20 L of the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methylene chloride and methanol (45 : 4) to a distance of about 15 cm and airdry the plate. Examine under ultraviolet light (main wavelength: 254nm): the Rf values of the principal spot obtained from the test solution and the standard solution are the same. Dissolution Test Perform the test with 1 tablet of Dexamethasone Tablets at 100 revolutions per minute according to Method 1 under the Dissolution Test, using 500 mL of diluted hydrochloric acid (1 in 100). After 45 minutes of the start of the test, extract a filtered aliquot of dissolution solution, equivalent to about 200 g of Dexamethasone, with three 15 mL volumes of chloroform. Evaporate the combined chloroform extract in a water-bath to dryness, cool, dissolve the residue in 20 mL of ethanol and use this solution as the test solution. Separately weigh a quantity of Dexamethasone RS, previously dried at 105 C for 3 hours, dissolve in methanol to contain 10 g per mL and use this solution as the standard solution. Transfer 20 mL each of the test solution and the standard solution to an Erlenmeyer flask, add 2.0 mL of a solution, dissolve 50 mg of blue tetrazolium in 10 mL of methanol. Use 20 mL ethanol for blank test. Add 2 mL of a mixture of tetra-methylammonium hydroxide and ethanol (1 : 9) to each of these flasks and mix. Immediately determine the absorbance, AT and

and QS , of the peak area of dexamethasone of the test solution and the standard solution to that of the internal standard, respectively. Amount (mg) of dexamethasone (C22H29FO5) = amount (mg) of Dexamethasone RS QT
QS

Internal standard solutionA solution of propyl parahydroxybenzoate in diluted methanol (1 in 2) (1 in 1000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of water and acetonitrile (2 : 1). Flow rate: Adjust the flow rate so that the retention time of dexamethasone is about 6 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, dexamethasone and the internal standard are eluted in this order with the resolution between these peaks being not less than 6. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of dexamethasone to that of the internal standard is not more than 1.0%.
Packaging and Storage tight containers.

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AS , of the test solution and the standard solution, respectively, at the wavelength of maximum absorption around 525 nm as directed under the Ultraviolet-visible Spectrophotometry in contrast with blank solution. Not less than 70% of the labeled amount of C22H29FO5 is dissolved in 45 minutes.
Amount (mg) of dexamethasone (C22H29FO5)
A = 10 C 1 T V

Dexamethasone Tablets
Dexamethasone Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of dexamethasone (C22H29FO5: 392.47).

AS

C: Concentration of standard solution (g/mL), V: Volume of the aliquot extracted with chloroform (mL).

276 Monographs, Part I

System suitability System performance : Dissolve 2 mg of methyl poxybenzoate and 4 mg of propyl p-oxybenzoate in 100 mL of a mixture of water and methanol (1:1). When the test is performed with 10 mL of the solution as directed under the above operating conditions, methyl poxybenzoate and propyl p-oxybenzoate are eluted in this order with the resolution between their peaks being not less than 5.0. System repeatability: When the test is repeated 6 times with 10 mL of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of dexamethason to the peak area of the internal standard is not more than 3.0%.
Packaging and Storage Preserve in well-closed containers.

Uniformity of Dosage Units It meets the requirement when the content uniformity test is performed according to the following procedure. Place 1 tablet of Dexamethasone Tablets in a separatory funnel with 15 mL of water and swirl to dissolve the tablet completely. Extract with four 10 mL volumes of chloroform, filter each volume through chloroform-washed cotton into a volumetric flask, add chloroform to make 50 mL and mix. Pipet a volume of this solution, equivalent to about 200 g of Dexamethasone into a glass-stoppered Erlenmeyer flask, evaporate the chloroform on a water-bath to dryness, cool and dissolve the residue in 20.0 mL of alcohol. Separately pipet 20 mL of standard solution in Dissolution test into a stoppered Erlenmeyer flask. Calculate the amount of Dexamethasone in the same manner of Dissolution Test.

Amount (mg) of dexamethasone (C22H29FO5)

A = C T V AS

Dexamethasone Disodium Phosphate

O H HO H3C H H F O H H3C OH CH3 O ONa O P ONa

C: Concentration of standard solution (g/mL), V: Volume of the solution, equivalent to 200 g of Dexamethasone (mL).
Assay Weigh and finely powder not less than 20 Dexamethasone Tablets. Weigh accurately a portion of the powder, equivalent to about 10 mg of Dexamethasone, transfer to a 100 mL volumetric flask and add 30 mL of diluted methanol (1 in 2). After shaking for 30 minutes, add 5 mL exactly of the internal standard solution, diluted methanol (1 in 2) to make 100 mL and mix. Filter and use the filtrate as the test solution. Separately, weigh accurately about 10 mg of Dexamethasone RS, transfer to a 100 mL volumetric flask and add 30 mL of diluted methanol (1 in 2). Add 5 mL of the internal standard solution, diluted methanol (1 in 2) to make 100 mL, mix and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine the peak area ratios, QT and QS ,, of Dexamethasone for the test solution and the standard solution to the internal standard, respectively.

C22H28FNa2O8P: 516.41 Dexamethasone Disodium Phosphate contains not less than 97.0% and not more than 102.0% of dexamethasone disodium phosphate (C22H28FNa2O8P), calculated on the anhydrous and ethanol-free basis.
Description Dexamethasone Disodium Phosphate is a white to pale yellowish crystalline powder, is odorless or has slightly ethanol-like smell. Dexamethasone Disodium Phosphate is freely soluble in water, slightly soluble in ethanol, very slightly soluble in dioxane and practically insoluble in ether. Dexamethasone Disodium Phosphate is hygroscopic. Identification (1) Transfer 20 mg of Dexamethasone Disodium Phosphate to a centrifugal tube, add 5 mL of alkaline phosphatase TS, vigorously shake and allow to stand for 30 minutes. Add 5 mL of ethyl acetate, vigorously shake, centrifuge and use the ethyl acetate layer (upper layer) as the test solution. Separately, transfer 15 mg of Dexamethasone RS to a volumetric flask, add ethyl acetate to dissolve, add ethylacetate to make 5 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on

Amount (mg) of dexamethasone (C22H29FO5) = Amount (mg) of Dexamethasone RS

QT QS

Internal standard solutionA solution of propyl poxybenzoate in a mixture of water and methanol (1:1) (1 in 1000) Operating conditions Use the operating conditions as directed under the Assay of Dexamethasone.

KP 9 277
a plate of silica gel for thin-layer chromatography. Develop the plate in a mixture of chloroform, methanol and water (180 : 15 : 1) to a distance of about 15 cm and airdry the plate. Observe under ultra-violet light (main wavelength: 254 nm). The Rf value of the principal spot obtained from the test solution corresponds to the that obtained from the standard solution. (2) The residue from the ignition of Dexamethasone Disodium Phosphate responds to the Qualitative Tests for sodium and for phosphate.
pH Between 7.5 and 10.5 in a solution (1 in 100). Specific Optical Rotation [ ]20 D : Between +74 and +82 (calculated on the anhydrous and ethanol-free basic, 0.1 g, water, 10 mL, 100 mm). Purity (1) Ethanol Transfer about 0.5 g of Dexamethasone Disodium Phosphate, accurately weighed, to a volumetric flask, add exactly 5 mL of the internal standard solution to dissolve, then add the water to make exactly 10 mL. After shaking, use this solution as the test solution. Separately, pipet diluted ethanol (1 in 50), determine the specific gravity at 25 C, calculate the amount of ethanol and use this solution as the standard stock solution. Pipet 4.0 mL of this solution to a volumetric flask, add exactly 5 mL of the internal standard solution and add water to make exactly 10 mL, shake and use this solution as the standard solution. Perform the test with 2 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following conditions and determine the peak area ratios, QT and QS , of ethanol for the test solution and the standard solution, respectively, to that of the internal standard.

System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, the resolution between the peaks of ethanol and internal standard is not less than 2.0 and factor symmetry for ethanol is not more than 1.5. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of peak height of ethanol to that of the internal standard is not more than 4.0%. The amount of ethanol in Dexamethasone Disodium Phosphate is not more than 8.0%. (2) Phosphate ion Dissolve about 50 mg of Dexamethasone Disodium Phosphate, accurately weighed, in a mixture of 10 mL of water and 5 mL of 1 mol/L sulfuric acid TS in a volumetric flask, by warming, if necessary. Add 1 mL of each of phosphate reagent A and phosphate reagent B, dilute with water to make exactly 25 mL, mix, allow to stand at room temperature for 30 minutes and use this solution as the test solution. Separately, using 5.0 mL of phosphate standard solution instead of 50 mg of Dexamethasone Disodium Phosphate, proceed in the same manner as the preparation of the test solution and use this solution as the standard solution. Determine absorbances of the test solution and the standard solution at 730 nm as directed under the Ultraviolet-visible Spectrophotometry, using water as the blank. The absorbance of the test solution is not more than that of the standard solution (not more than 1.0% of phosphate).

Amount (%) of C2H5OH = 4 S QT

W

QS

Phosphate standard solutionDissolve 143.3 mg of dried monobasic potassium phosphate, KH2PO4, in water to make 1000 mL (0.10 mg PO4/mL). Phosphate reagent ADissolve 5 g of ammonium molybdate in 0.5 mol/L sulfuric acid TS to make 100 mL. Phosphate reagent BDissolve 350 mg of pmethylaminophenol sulfate in 50 mL of water, add 20 g of sodium bisulfite, mix to dissolve and dilute with water to make 100 mL.
(3) Free Dexamethasone Separately inject 20 L of the standard solution and the test solution under the Assay, respectively. Determine the peak areas, AT and

S: Amount of ethanol in the original standard solution (%), W: Amount of Dexamethasone Disodium Phosphate in the test solution (%). Internal standard solutionA mixture of isopropanol and water (1 in 100). Operating conditions Detector: A hydrogen flame ionization detector. Column: A glass column, about 4 mm in inside diameter and about 1.8 m in length, packed with copolymer of 4-vinyl pyridine and styrenedivinyl benzene (80 100 mesh in particle diameter). Column temperature: A constant temperature of about 120 C. Carrier gas: Nitrogen. System suitability

AS of Dexamethasone of the test solution and the standard solution, respectively. (not more than 1.0%). Amount (g) of dexamethasone (C22H29FO5) A = 1000 C T AS

C: Concentration of the Dexamethasone RS in the standard solution (g/mL).

Water Not more than 16.0% (sum of the % of water content and ethanol content, 0.4 g, direct titration).

278 Monographs, Part I

Assay Take accurately about 50 mg of Dexamethasone Disodium Phosphate, dissolve in mobile phase to make exactly 100 mL. Pipet 5.0 mL of this solution and add the mobile phase to make exactly 50 mL and use this solution as the test solution. Separately, dissolve a portion of Dexamethasone Phospahte RS, previously dried at 40 C and 0.67 kPa, in mobile phase to make the concentration of 0.5 mg of Dexamethasone Phospahte per mL and use this solution as the standard solution (1). Weigh accurately a portion of Dexamethasone RS, previously dried at 105 C for 3 hours, dissolve in a mixture of methanol and water (1 : 1) to make the concentration of 50 g of Dexamethasone RS per mL and use this solution as the standard solution (2). Pipet 10.0 mL of the standard solution (1) and 1.0 mL of the standard solution (2), add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak area, AT

Dexamethasone Disodium Phosphate Injection

Dexamethasone Disodium Phosphate Injection is an aqueous solution for Injection. Dexamethasone Disodium Phosphate Injection contains not less than 90.0% and not more than 115.0% of the labeled amount of dexamethasone phosphate (C22H30FO8P: 472.45).
Method of Preparation Prepare as directed under Injections with the Dexamethasone Disodium Phosphate. Identification Pipet a volume of Dexamethasone Disodium Phosphate Injection, equivalent to about 10 mg of dexamethasone phosphate, into a volumetric flask, add water to make 100 mL and mix. Pipet 5 mL of this solution into a separatory funnel and wash with two 10 mL volumes of water-washed methylene chloride, discarding the washings. Transfer the solution into a glass-stoppered tube and add 5 mL of alkaline phosphatase solution, prepared by dissolving 50 mg of alkaline phosphatase in 50 mL of pH 9 buffer with magnesium. Allow to stand at 37 C for 45 minutes and extract with 25 mL of methylene chloride. Evaporate 15 mL of the methylene chloride extract in a steam-bath to dryness and dissolve the residue in 1 mL of methylene chloride and use this solution as the test solution. Separately, dissolve a portion of Dexamethasone RS in methylene chloride to contain 300 g per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography, respectively. Develop the plate with a mixture of chloroform, acetone, water (50 : 50 : 1) to a distance of about 15 cm and air-dry the plate. Spray evenly diluted sulfuric acid (1in 2) upon the plate and heat the plate at 105 C until black or brown spots appear. The spot from the test solution shows the same Rf value as the spot from the standard solution. pH Between 7.0 and 8.5. Sterility Test

and AS of Dexamethasone Phosphate of the test solution and the standard solution, respectively. Amount (mg) of dexamethasone disodium phosphate 516.41 A (C22H28FNa2O8P) = 472.45 C T A
S

C: Concentration of the Dexamethasone Phospahte RS in the standard solution (g/mL). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.5 mm in inside diameter and about 25 cm in length, packed with phenylatedsilica gel for liquid chromatography. Column temperature: A room temperature. Mobile phase: To 7.5 mL of the triethylamine, add water to make 1000 mL. Adjust by the the addition of phosphoric acid to a pH of 5.4. To 740 mL of this solution, add 260 mL of methanol. Flow rate: 1.2 mL/minute. System suitability System performance : When the procedure is run with 10 L of the standard solution under the above operating conditions, and calculate the resolution, dexamethasone and dexamethasone phosphate are eluted in this order and the resolution between the dexamethasone peak and dexamethasone phosphate peak is not less than 1.8. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area is not more than 1.0%.
Packaging and Storage Preserve in hermetic containers.

It meets the requirement.

Bacterial Endotoxins Less than 31.3 EU per mg of dexamethasone phosphate. Foreign Insoluble Matter Test ment.

It meets the requireIt

Insoluble Particulate Matter Test for Injections meets the requirement

KP 9 279
phate (C22H30FO8P: 472.45).
Method of Preparation Prepare as directed under Ophthalmic Ointments, with the Dexamethasone Disodium Phosphate. Identification Dissolve 50 mg of alkaline phosphatase to 50 mL of pH 9 buffer with magnesium. Transfer 5 mL of this solution to a stoppered test tube containing 5 mL of the test solution described in the Assay. Allow to stand at 37 C for 45 minutes, add 25 mL of dichloromethane and shake for 2 minutes to mix. Proceed with the dichloromethane extracts as directed in the Identification under Dexmethasone Disodium Phosphate Injection after evaporating 15 mL of the dichloromethane extract to residue. Sterility Test It meets the requirement. Test for Metal Particles It meets the requirement. Assay Weigh accurately a portion of Dexamethasone Disodium Phosphate Ophthalmic Ointment, equivalent to 3 mg of dexamethasone phosphate (C22H30FO8P) and transfer to a beaker. Add 65 mL of ethanol-phosphate buffer and heat the solution just to boiling. Transfer the contents to a separatory funnel containing 45 mL of isooctane, shake for 1 minute to mix and transfer the lower layer to a volumetric flask. Wash the beaker with two volumes of 15 mL of ethanol phosphate buffer, extract the rinsate with isooctane and mix the lower layer to the volumetric flask. Fill the volumetric flask with ethanol-phosphate buffer to make 100 mL, filter and use this solution as the test solution. Separately, weigh accurately a portion of dexamethasone phosphate RS, previously dried at 40 C under 0.67 kPa, dissolve in ethanolphosphate buffer to make 30 g of dexamethasone phosphate per mL and use this solution as the standard solution. Perform the test with 20 l each of the test solution and standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the dexamethasone phosphate peak area, AT

Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer an accurately measured volume of Dexmethasone Disodium Phosphate Injection, equivalent to about 8 mg of dexamethasone phosphate (C22H30FO8P), to a volumetric flask. Dilute with mobile phase to make 100 mL and mix and use this solution as test solution. Separately, weigh accurately a portion of dexamethasone phosphate RS, previously dried at 40 C under 0.67 kPa, dissolve in mobile phase to make the concentration of 80 g of dexamethasone phosphate per mL and use this solution as the standard solution. Prepare this solution at the time of use. Perform the test with 20 l each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak area, AT

and AS , of the dexamethasone phosphate of the test solution and the standard solution, respectively. Amount (mg) of dexamethasone phosphate A (C22H30FO8P) = 0.1 C T AS

C: Concentration of the dexamethasone phosphate RS in the standard solution (g/mL), Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of 0.01 mol/L monobasic potassium phosphate buffer and methanol (1 : 1). Flow rate: 1.6 mL/minute. System suitability System repeatability: When the test is repeated 5 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of dexamethasone phosphate is not more than 1.5%.
Packaging and Storage hermetic containers.

and AS , of the test solution and standard solution, respectively. Amount (mg) of dexamethasone phosphate A (C22H30FO8P) = 0.1 C T AS

Preserve in light-resistant,

Dexamethasone Disodium Phosphate Ophthalmic Ointment

Dexamethasone Disodium Phosphate Ophthalmic Ointment contains not less than 90.0% and not more than 115.0% of the labeled amount of dexamethasone phos-

C: Concentration of the dexamethasone phosphate RS in the standard solution (g/mL). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5

280 Monographs, Part I m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of methanol and 0.05 mol/L phosphate buffer(52 : 48). System suitability System Repeatability: When the test is repeated 5 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of dexamethasone phosphate is not more than 1.5%.
Packaging and Storage tight containers.

Preserve in light-resistant,

Dextran 40
Dextran 40 is a product obtained by partial decomposition of polysaccharide, which is produced by fermentation of sucrose with Leuconostoc mesenteroides van Tieghem (Lactobacillaceae) and the average molecular weight is about 40000. Dextran 40, when dried, contains not less than 98.0% and not more than 102.0% of dextran.
Description Dextran 40 is a white, amorphous powder, odorless and tasteless. Dextran 40 is practically insoluble in ethanol or in ether. Dextran 40 dissolves gradually in water. Dextran 40 is hygroscopic. Identification Take 1 mL of a solution of Dextran 40 (1 in 3000) and add 2 mL of anthrone TS: a blue-green color is observed and turns gradually dark blue-green. Then to this solution, add 1 mL of diluted sulfuric acid (1 in 2) or 1 mL of glacial acetic acid: the solution does not change in color. pH Dissolve 1.0 g of Dextran 40 in 10 mL of water: the pH of this solution is between 5.0 and 7.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Dextran 40 in 10 mL of water by warming: the solution is clear and colorless. (2) ChloridePerform the test with 2.0 g of Dextran 40. Prepare the control solution with 1.0 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.018%). (3) Heavy metalsProceed with 1.0 g of Dextran 40 according to Method 1 and perform the test (not more than 20 ppm). (4) ArsenicPrepare the test solution with 1.5 g of Dextran 40 according to Method 1 and perform the test (not more than 1.3 ppm). (5) NitrogenWeigh accurately about 2 g of Dextran 40, previously dried, and perform the test as directed under the Nitrogen Determination, where 10 mL of sulfuric acid is used for decomposition and 45 mL of a solu-

tion of sodium hydroxide (2 in 5) is added: the amount of nitrogen (N: 14.007) is not more than 0.010%. (6) Reducing substancesWeigh exactly 3.00 g of Dextran 40, previously dried, dissolve in water to make exactly 50 mL and use this solution as the test solution. Separately, weigh exactly 0.450 g of glucose, previously dried, dissolve in water to make exactly 500 mL and use this solution as the standard solution. Pipet 5.0 mL each of the test solution and the standard solutions and add water to make exactly 50 mL. Pipet 5.0 mL each of these solutions, add 5.0 mL of alkaline copper TS and heat for 15 minutes in a water-bath. After cooling, add 1 mL of a solution of potassium iodine (1 in 40) and 1.5 mL of dilute sulfuric acid and titrate with 0.005 mol/L sodium thiosulfate VS (indicator: 2 mL of starch TS). The titrant consumed for the test solution is not less than that for the standard solution.
Loss on Drying Not more than 5.0% (1 g, 105 C, 6 hours). Residue on Ignition Not more than 0.1% (1 g). Viscosity (1) Dextran 40Weigh accurately about 0.2 to 0.5 g of Dextran 40, previously dried, dissolve in water to make exactly 100 mL and use this solution as the test solution. Perform the test with the test solution and with water as directed in Method 1 under the Viscosity Determination at 25 C: the intrinsic viscosity is between 0.16 and 0.19. (2) High-molecular fractionWeigh accurately about 6 g of Dextran 40, previously dried, dissolve in water to make exactly 100 mL and transfer to a flask. Add slowly enough methanol to obtain 7% to 10% of the precipitate (usually 80 to 90 mL) at 25 1 C with stirring. Dissolve the precipitate at 35 C in a water-bath with occasional shaking and allow to stand for more than 15 hours at 25 1 C. Remove the supernatant by decantation and heat the precipitate of the lower layer to dryness on a water-bath. Dry the residue and determine the intrinsic viscosity of the dried substance as directed in (1): the value is not more than 0.27. (3) Low-molecular fractionWeigh accurately about 6 g of Dextran 40, previously dried, dissolve in water to make exactly 100 mL and transfer to a flask. Add slowly enough methanol to obtain 90% to 93% of the precipitate (usually 115 to 135 mL) at 25 1 C with stirring, centrifuge at 25 C and evaporate the supernatant to dryness on a water-bath. Dry the residue and determine the intrinsic viscosity of the dried substance as directed in (1): the value is not less than 0.09. Antigenicity Dissolve 10.0 g of Dextran 40 in Isotonic Sodium Chloride Injection to make 100 mL, sterilize and use this solution as the test solution. Inject 1.0 mL of the test solution on three occasions at intervals of 2 days into the peritoneal cavity of each of 4 well-nourished, healthy guinea pigs weighing 250 g to 300 g. Inject 0.10 mL of

KP 9 281
horse serum into the peritoneal cavity of each of 4 guinea pigs of another group as a control. Inject 0.20 mL of the test solution intravenously to each of 2 guinea pigs of the first group 14 days after the first intraperitoneal injection and each of the remaining 2 guinea pigs 21 days after the injection and inject 0.20 mL of horse serum intravenously in the same manner each guinea pig of the second group. Observe the signs of respiratory distress, collapse or death of the animals for 30 minutes after each intravenous injection and 24 hours later: the animals of the first group exhibit no signs mentioned above. All the animals of the second group exhibit symptoms of respiratory distress or collapse and not less than 3 animals are killed.
Pyrogen Test Dissolve 10.0 g of Dextran 40 in Isotonic Sodium Chloride Injection to make 100 mL and perform the test: this solution meets the requirement of the Pyrogen Test. Assay Weigh accurately about 3 g of Dextran 40, previously dried, dissolve in water to make exactly 50 mL and use this solution as the test solution. Determine the optical rotation with the test solution as directed under the Optical Rotation Determination in a 100 mm cell at 20 1 C.

color. (2) Dextran 40 Injection responds to the Qualitative Tests for sodium salt and for chloride.
pH

Between 4.5 and 7.0.

Viscosity Measure exactly 2 mL to 5 mL of Dextran 40 Injection, add Isotonic Sodium Chloride Injection to make exactly 100 mL and use this solution as the test solution. Perform the test with the test solution and with Isotonic Sodium Chloride Injection as directed in Me-

thod 1 under the Viscosity Determination at 25 C : the intrinsic viscosity is between 0.16 and 0.19. Calculate the concentration of the test solution (g/100 mL) as directed in the Assay.
Sterility Test It meets the requirement. Bacterial Endotoxins Dextran 40 Injection.

Less than 0.5 EU per mL of

Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Amount (mg) of Dextran 40 = D 253.8

Packaging and Storage Preserve in tight containers.

Determination of Volume of Injection in Containers It meets the requirement. Assay Take exactly 30 mL of Dextran 40 Injection, add water to make exactly 50 mL and use this solution as the test solution. Determine the optical rotation, D, with the test solution as directed under the Optical Rotation Determination in a 100-mm cell at 20 1 C.

Dextran 40 Injection
Dextran 40 Injection is an aqueous solution for injection. Dextran 40 Injection contains not less than 9.5 w/v% and not more than 10.5 w/v% of Dextran 40.
Method of Preparation Dextran 40 10 g Isotonic Sodium Chloride Injection a sufficient quantity To make 100 mL

Amount (mg) of Dextran 40 in 100 mL of Dextran 40 Injection = D 846.0

Packaging and Storage Preserve in hermetic containers and plastic containers for aqueous injections may be used.

Dextran 70
Dextran 70 is a product obtained by partial decomposition of polysaccharide, which is produced by fermentation of sucrose with Leuconostoc mesenteroides van Tieghem (Lactobacillaceae) and the average molecular weight is about 70000. Dextran 70, when dried, contains not less than 98.0% and not more than 102.0% of Dextran 70.
Description Dextran 70 is a white, amorphous powder and is odorless and tasteless. Dextran 70 is practically insoluble in ethanol or in ether. Dextran 70 dissolves gradually in water.

Prepare as directed under Injections, with the above ingredients. No preservative is added.
Description Dextran 40 Injection is a clear and colorless liquid, and is slightly viscous. Identification (1) Dilute 1 mL of Dextran 40 Injection with water to make 200 mL, pipet 1 mL of this solution and add 2 mL of anthrone TS: a blue-green color is observed and turns gradually dark blue-green. Then to this solution, add 1 mL of diluted sulfuric acid (1 in 2) or 1 mL of glacial acetic acid: the solution does not change in

282 Monographs, Part I

Dextran 70 is hygroscopic.
Identification Perform the test according to Identification of Dextran 40. pH Dissolve 3.0 g of Dextran 70 in 50 mL of water: the pH of this solution is between 5.0 and 7.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Dextran 70 in 10 mL of water with warming: the solution is clear and colorless. (2) ChloridePerform the test with 2.0 g of Dextran 70. Prepare the control solution with 1.0 mL of 0.01 mol/L hydrochloric acid (not more than 0.018%). (3) Heavy metalsProceed with 1.0 g of Dextran 70 according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicPrepare the test solution with 1.5 g of Dextran 70 according to Method 1 and perform the test (not more than 1.3 ppm). (5) NitrogenWeigh accurately about 2 g of Dextran 70, previously dried, perform the test as directed under the Nitrogen Determination, where 10 mL of sulfuric acid is used for decomposition and 45 mL of a solution of sodium hydroxide (2 in 5) is added: the amount of Nitrogen (N: 14.007) is not more than 0.010%. (6) Reducing substancesWeigh exactly 3.00 g of Dextran 70, previously dried, dissolve in water to make exactly 50 mL and use this solution as the test solution. Separately, weigh exactly 0.300 g of glucose, previously dried, dissolve in water to make exactly 500 mL and use this solution as the standard solution. Pipet 5.0 mL each of the test solution and the standard solution and add water to make exactly 50 mL, respectively. Pipet 5.0 mL each of these solutions, add 5.0 mL of alkaline copper TS and heat for 15 minutes in a water-bath. After cooling, add 1 mL of a solution of potassium iodine (1 in 40) and 1.5 mL of dilute sulfuric acid and titrate with 0.005 mol/L sodium thiosulfate VS (indicator: 2 mL of starch TS). The titrant consumed for the test solution is not less than that for the standard solution. Loss on Drying Not more than 5.0% (1 g, 105 C, 6 hours). Residue on Ignition Not more than 0.1% (1 g). Viscosity (1) Dextran 70Weigh accurately about 0.2 to 0.5 g of Dextran 70, previously dried, dissolve in water to make exactly 100 mL and use this solution as the test solution. Perform the test with the test solution and with water as directed in Method 1 under the Viscosity Determination at 25 C: the intrinsic viscosity is between 0.21 and 0.26. (2) High-molecular fractionWeigh accurately about 6 g of Dextran 70, previously dried, dissolve in water to make exactly 100 mL and transfer to a flask.

Add slowly enough methanol to obtain 7% to 10% of the precipitate (usually 75 to 85 mL) at 25 1 C with stirring. Dissolve the precipitate at 35 C in a water-bath with occasional shaking and allow to stand for more than 15 hours at 25 1 C. Remove the supernatant by decantation and heat the precipitate of the lower layer on a water-bath to dryness. Dry the residue and determine the intrinsic viscosity of the dried residues as directed in (1): the value is not more than 0.35. (3) Low-molecular fractionWeigh accurately about 6 g of Dextran 70, previously dried, dissolve in water to make exactly 100 mL and transfer to a flask. Add slowly enough methanol to obtain 90% to 93% of the precipitate (usually 110 to 130 mL) at 25 1 C with stirring, centrifuge at 25 C and evaporate the supernatant to dryness on a water-bath. Dry the residue and determine the intrinsic viscosity of the dried substance as directed in (1): the value is not less than 0.10.
Antigenicity Dissolve 6.0 g of Dextran 70 in Isotonic Sodium Chloride Injection to make 100 mL, sterilize and use this solution as the test solution. Perform the test according to Antigenicity under Dextran 40 Pyrogen Test Dissolve 6.0 g of Dextran 70 in Isotonic Sodium Chloride Injection to make 100 mL and perform the test: this solution meets the requirement of the Pyrogen test. Assay Weigh accurately about 3 g of Dextran 70, previously dried, dissolve in water to make exactly 50 mL and use this solution as the test solution. Determine the optical rotation, D, as directed under the Optical Rotation Determination in a 100-mm cell at 20 1 C.

Amount (mg) of Dextran 70 = D 253.8

Packaging and Storage Preserve in tight containers.

Dextran 70 Injection
Dextran 70 Injection is an aqueous solution for injection. Dextran 70 Injection contains not less than 5.7 w/v% and not more than 6.3 w/v% of Dextran 70.
Method of Preparation Dextran 70 6g Isotonic Sodium Chloride Injection a sufficient quantity To make 100 mL Prepare as directed under Injections, with the above ingredients. No preservative is added. Description Dextran 70 Injection is a clear and colorless liquid and is slightly viscous.

KP 9 283

Identification (1) Dilute 1 mL of Dextran 70 Injection with water to make 200 mL and to 1 mL of this solution, add 2 mL of anthrone TS: a blue-green color develops and turns gradually dark blue-green. Add 1 mL of diluted sulfuric acid (1 in 2) or 1 mL of glacial acetic acid: the solution does not change in color. (2) Dextran 70 Injection responds to the Qualitative Tests for sodium salt and for chloride. pH

C18H25NOHBrH2O: 370.32 Dextromethorphan Hydrobromide Hydrate contains not less than 98.0% and not more than 101.0% of dextromethorphan hydrobromide (C18H25-NOHBr), calculated on the anhydrous basis.
Description Dextromethorphan Hydrobromide Hydrate is a white crystal or crystalline powder. Dextromethorphan Hydrobromide Hydrate is very soluble in methanol, freely soluble in ethanol or in glacial acetic acid, sparingly soluble in water and practically insoluble in ether. Melting pointAbout 126 C (insert the capillary tube into the bath preheated to 116 C and continue the heating so that the temperature rises at a rate of about 3 C per minute). Identification (1) Determine the absorption spectra of solutions of Dextromethorphan Hydrobromide Hydrate and Dextromethorphan Hydrobromide Hydrate RS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Dextromethorphan Hydrobromide Hydrate and Dextromethorphan Hydrobromide Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Take 50 mL of a solution of Dextromethorphan Hydrobromide Hydrate (1 in 100), add 2 drops of phenolphthalein TS and sodium hydroxide TS until a red color is absorbed. Add 50 mL of chloroform, shake and add 5 mL of dilute nitric acid to 40 mL of the water layer. This solution responds to the Qualitative Tests for bromide. Specific Optical Rotation [ ]20 D : Between + 26 and + 30 (0.34 g, calculated on the anhydrous basis, water, 20 mL, 100 mm). pH Dissolve 1.0 g of Dextromethorphan Hydrobromide Hydrate in 100 mL of water: the pH of this solution is between 5.2 and 6.5. Purity (1) Clarity and color of solution Dissolve 0.20 g of Dextromethorphan Hydrobromide Hydrate in 20 mL of water: the solution is clear and colorless. (2) DimethylanilineTake 0.50 g of Dextromethorphan Hydrobromide Hydrate, add 20 mL of water and dissolve by heating in a water-bath. After cooling, add 2 mL of dilute acetic acid, 1 mL of sodium nitrite TS and water to make 25 mL: the solution has no more color than the following control solution.

Between 4.5 and 7.0.

Sterility Test It meets the requirement. Bacterial Endotoxins Dextran 70 Injection.

Less than 0.5 EU per mL of

Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement Viscosity Take 4 to 8 mL of Dextran 70 Injection, add Isotonic Sodium Chloride Injection to make exactly 100 mL and use this solution as the test solution. Perform the test with the test solution and with Isotonic Sodium Chloride Injection as directed in Method 1 under the Viscosity Determination at 25 C: the intrinsic viscosity is between 0.21 and 0.26. Calculate the concentration of the test solution (g/100 mL) as directed in the Assay. Assay Determine the optical rotation, D, as directed under the Optical Rotation Determination in a 100-mm cell at 20 1 C.

Amount (mg) of Dextran 70 in 100 mL of Dextran 70 Injection = D 507.6

Packaging and Storage Preserve in hermetic containers and plastic containers for aqueous injections may be used.

Dextromethorphan Hydrobromide Hydrate

CH3 H N HBr H H2O

CH3O

Control solutionDissolve 0.10 g of dimethylaniline

284 Monographs, Part I

in 400 mL of water by warming in a water-bath, cool and add water to make 500 mL. Pipet 5.0 mL of this solution and add water to make 200 mL. To 1.0 mL of this solution, add 2 mL of dilute acetic acid, 1 mL of sodium nitrite TS and water to make 25 mL. (3) Heavy metalsProceed with 1.0 g of Dextromethorphan Hydrobromide Hydrate according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Phenolic compoundsDissolve 5 mg of Dextromethorphan Hydrobromide Hydrate in 1 drop of dilute hydrochloric acid and 1 mL of water, add 2 drops of ferric chloride TS and 2 drops of potassium ferricyanide TS, shake and allow to stand for 15 minutes: no blue-green color is observed. (5) Related substancesDissolve 0.25 g of Dextromethorphan Hydrobromide Hydrate in 10 mL of methanol and use this solution as the test solution. Pipet 1.0 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of toluene, ethyl acetate, methanol, dichloromethane and 13.5 mol/L ammonia water (55 : 20 : 13 : 10 : 2) to a distance of about 15 cm and air-dry the plate. Spray evenly bismuth potassium iodide TS on the plate and then spray evenly hydrogen peroxide TS on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Water Between 4.0% and 5.5% (0.2 g, volumetric titration, reverse titration). Residue on Ignition Not more than 0.10% (1 g). Assay Weigh accurately about 0.5 g of Dextromethorphan Hydrobromide Hydrate, dissolve in 10 mL of glacial acetic acid and add 40 mL of acetic anhydride. Titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Diagnostic Sodium Citrate Solution contains not less than 3.3 w/v% and not more than 4.3 w/v% of sodium citrate hydrate (C6H5Na3O72H2O: 294.10). The requirement as described for aqueous injections under Injections are applicable.
Method of Preparation Sodium Citrate Hydrate 38 g Water for injection a sufficient quantity To make 1000 mL

Prepare as directed under Injections, with the above ingredients. No preservative is added.
Description Diagnostic Sodium Citrate Solution is a clear, colorless liquid. Identification Diagnostic Sodium Citrate Solution responds to the Qualitative Tests for sodium salt and for citrate. pH

Between 7.0 and 8.5.

Assay Pipet 5.0 mL of Diagnostic Sodium Citrate Solution and evaporate on a water-bath to dryness. Dry the residue at 180 C for 2 hours and dissolve in 30 mL of glacial acetic acid by warming. After cooling, titrate with 0.1 mol/L perchloric acid VS (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 9.803 mg of C6H5Na3O7.2H2O

Packaging and Storage Preserve in hermetic containers.

Diastase
Diastase is an enzyme mainly prepared from malt. Diastase has an amylolytic activity. Diastase contains not less than 440 starch saccharifying activity units per g. Diastase is usually diluted with suitable diluents.
Description Diastase is a pale yellow to light brown powder. Diastase is hygroscopic. Purity RancidityDiastase has no unpleasant or rancid odor and has no unpleasant or rancid taste.

Each mL of 0.1 mol/L perchloric acid VS = 35.231 mg of C18H25NOHBr

Packaging and Storage Preserve in well-closed containers.

Diagnostic Sodium Citrate Solution

Loss on Drying Not more than 4.0% (1 g, 105 C, 5 hours). Assay (i) Substrate solutionUse potato starch TS for

KP 9 285
starch digestive activity. (ii) Test solutionWeigh accurately about 0.1 g of Diastase, and dissolve in water to make exactly 100 mL. (iii) ProcedureProceed as directed in (i) Measurement of starch saccharifying activity of (1) Assay for starch digestive activity under the Digestion Test.
Packaging and Storage Preserve in tight containers at not more than 30 C.

Diazepam
CH3 O N

Cl

C16H13ClN2O: 284.74 Diazepam, when dried, contains not less than 98.0% and not more than 101.0% of diazepam (C16H13ClN2O).
Description Diazepam is a white to pale yellow, crystalline powder, is odorless and has a slightly bitter taste. Diazepam is freely soluble in acetone, soluble in acetic anhydride or in ethanol, sparingly soluble in ether, slightly soluble in dehydrated ethanol and practically insoluble in water. Identification (1) Dissolve 10 mg of Diazepam in 3 mL of sulfuric acid and observe under ultraviolet light (main wavelength: 365 nm): the solution shows a yellow-green fluorescence. (2) Dissolve 2 mg each of Diazepam and Diazepam RS in 200 mL each of solutions of sulfuric acid in dehydrated ethanol (3 in 1000) and determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Perform the test with Diazepam as directed under the Flame Coloration Test (2): a blue to blue-green color is observed. Melting Point

(2) ChlorideTake 1.0 g of Diazepam, add 50 mL of water, allow to stand for 1 hour, with occasional shaking and filter. To 25 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.20 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.014%). (3) Heavy metalsProceed with 1.0 g of Diazepam according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Related substancesDissolve 1.0 g of Diazepam in 10 mL of acetone and use this solution as the test solution. Pipet 1.0 mL of the test solution and add acetone to make exactly 100 mL. Pipet 1.0 mL of this solution, add acetone to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethylacetate and hexane (1 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than that from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.6 g of Diazepam, previously dried, dissolve in 60 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 28.474 mg of C16H13ClN2O

Packaging and Storage tight containers.

Preserve in light-resistant,

Diazepam Injection
Diazepam Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of diazepam (C16H13ClN2O: 284.74).
Method of Preparation Prepare as directed under Injections, with Diazepam. Description liquid.

Between 130 C and 134 C.

1% Absorbance E1 cm (285 nm): Between 425 and 445 [after drying , 2 mg , a solution of sulfuric acid in dehydrated ethanol (3 in 1000), 200 mL].

Purity (1) Clarity and color of solutionDissolve 0.1 g of Diazepam in 20 mL of ethanol: the solution is clear and colorless.

Diazepam Injection is a colorless, clear

286 Monographs, Part I

thanol (3 in 10000).
Identification (1) Retention time of the principal peak in the chromatogram of the test solution under the Assay corresponds to that of the standard solution. (2) Take Diazepam Injection, equivalent to 10 mg of Diazepam according to the labeled amount, transfer to a separatory funnel, add 20 mL of water. After shaking, add 20 mL of chloroform and shake extensively. Transfer the chloroform layer to a beaker through a filter with 5 g of anhydrous sodium sulfate. Wash anhydrous sodium sulfate with 20 mL of chloroform and add this washing to the filtrate. Evaporate the filtrate on a water-bath under the flow of air until the volume is reduced to 5 mL, take out the beaker from the water-bath and evaporate under the flow of air. Scratch and gather an oily membrane extensively by spatula and dry it in a desiccator with P2O5 in vacuum at 60 C for 4 hours. Determine the infrared spectra of the residue and Diazepam RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit the similiar intensities of absorption at the same wavenumbers. pH Between 6.2 and 6.9 Sterility Test It meets the requirement Bacterial Endotoxins Less than 11.6 EU per mg of Diazepam. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of methanol and water (65 : 35). Flow rate: 1.4 mL/minute. System suitability System performance: When the procedure is run with 20 L of the standard solution, as directed under the above operating conditions, the internal standard and the diazepam are eluted in this order with the resolution between their peaks being not less than 5.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the ratios of peak area of diazepam to that of the internal standard is not more than 2.0%.
Packaging and Storage hermetic containers.

Preserve in light-resistant,

Diazepam Tablets
Diazepam Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of diazepam (C16H13ClN2O: 284.74).
Method of Preparation Prepare as directed under Tablets, with Diazepam. Identification (1) The retention time of the principal peak in the chromatogram of the test solution under the Assay corresponds to that of the standard solution. (2) Weigh a portion of Diazepam Tablets, equivalent to 10 mg of Diazepam, place in a centrifuge tube, add 2 mL of acetone, shake for 5 minutes, centrifuge and use the supernatant as the test solution. Separately, weigh a portion of Diazepam RS, dissolve in acetone to make a solution containing 5 mg per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 100 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate and n-heptane (1 : 1) to a distance of about 15 cm and air-dry the plate. Expose it to an ultraviolet light (main wavelength: 254 nm). The principal spot from the test solution shows the same Rf value as that from the standard solution.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Take accurately a volume of Diazepam Injection, equivalent to about 10 mg of Diazepam, add 10.0 mL of the internal standard solution and methanol to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Diazepam RS add 10.0 mL of the internal standard solution and methanol to make exactly 50 mL and use this solution as the as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography. Calculate the ratios, QT and QS , of peak areas of Diazepam to that of the internal standard for the test solution and the standard solution, respectively.

Amount (mg) of diazepam (C16H13ClN2O) = amount (mg) of Diazepam RS

QT QS

Internal standard solutionp-tolualdehyde in me-

KP 9 287
order with the resolution between their peaks being not less than 4.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak areas of diazepam is not more than 2.0%.
Packaging and Storage tight containers.

Dissolution Test Take 1 tablet of Diazepam Tablets and perform the test using 900 mL of 0.1 mol/L hydrochloric acid as dissolution solution, at 100 revolutions per minute as directed in the Method 1 under the Dissolution Test. After 30 minutes from the start of the test, take the dissolved solution, suitably dilute with dissolution solution, if necessary and use this solution as the test solution. Separately, dissolve a suitable portion of Diazepam RS to make a solution with the same concentration as the test solution and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution at a maximum absorption wavelength of about 242 nm, as directed under the Ultraviolet-visible Spectrophotometry, using 0.1 mol/L hydrochloric acid TS as the blank. The dissolution rate of Diazepam Tablets in 30 minutes is not less than 85%. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 Diazepam Tablets and make fine powder. Transfer an accurately weighed portion of the powder, equivalent to about 10 mg of Diazepam, add 50 mL of methanol, ultrasonicate for 5 minutes and shake by mechanical means for 5 minutes. Add methanol to make exactly 100 mL, filter and use the filtrate as the test solution after discarding the first filtrate. Separately, dissolve accurately about 50 mg of Diazepam RS in methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and measure, AT and AS , of the peak area of diazepam of the test solution and the standard solution, respectively.

Preserve in light-resistant,

Dibucaine Hydrochloride
N OCH 2CH2CH2CH3 HCl

C O

NHCH2CH2N(CH2CH3)2

C20H29N3O2HCl: 379.92 Dibucaine Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of dibucaine hydrochloride (C20H29N3O2HCl).
Description Dibucaine Hydrochloride is a white crystal or crystalline powder. Dibucaine Hydrochloride is very soluble in water, in ethanol or in glacial acetic acid, freely soluble in acetic anhydride and practically insoluble in ether. Dibucaine Hydrochloride is hygroscopic. Identification (1) Determine the absorption spectra of solutions of Dibucaine Hydrochloride and Dibucaine Hydrochloride RS in 1 mol/L hydrochloric acid TS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Dibucaine Hydrochloride and Dibucaine Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Dibucaine Hydrochloride (1 in 10) responds to the Qualitative Tests for chloride. Melting Point Between 95 C and 100 C. Charge Dibucaine Hydrochloride into a capillary tube for melting point determination and dry in vacuum over P2O5 at 80 C for 5 hours. Seal immediately the open end of the tube and determine the melting point. pH Dissolve 1.0 g of Dibucaine Hydrochloride in 50 mL of water: the pH of this solution is between 5.0 and 6.0. Purity

Amount (mg) of diazepam (C16H13ClN2O) = amount (mg) of Diazepam RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of acetonitrile, water and methanol(2 : 2 : 1). Flow rate: 1 mL/minute. System suitability System performance: Dissolve suitable quantities of nordazepam RS and Diazepam RS in methanol containing about 0.1 mg each per mL. When the procedure is run with 10 L of this solution under the above operating conditions, nordazepam and Diazepam are eluted in this

(1) Clarity and color of solution Dissolve

288 Monographs, Part I

1.0 g of Dibucaine Hydrochloride in 20 mL of water: the solution is clear and colorless. Determine the absorbance of this solution at 430 nm as directed under the Ultraviolet-visible Spectrophotometry, using water as the blank: it is not more than 0.03. (2) SulfatePerform the test with 0.30 g of Dibucaine Hydrochloride. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.056%). (3) Heavy metalsProceed with 1.0 g of Dibucaine Hydrochloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Related substancesDissolve 0.20 g of Dibucaine Hydrochloride in 5 mL of ethanol and use this solution as the test solution. Pipet 1.0 mL of the test solution and add ethanol to make exactly 20 mL. Pipet 2.0 mL of this solution, add ethanol to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, water and glacial acetic acid (3 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than that from the standard solution.
Loss on Drying Not more than 2.0% (1 g, in vacuum, P2O5, 80 C, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Dibucaine Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Diclofenac Sodium, when dried, contains not less than 98.5% and not more than 101.0% of diclofenac sodium (C14H10Cl2NNaO2).
Description Diclofenac sodium is a white to pale yellowish white crystal or crystalline powder. Diclofenac sodium is freely soluble in methanol or in ethanol, sparingly soluble in water or in glacial acetic acid and practically insoluble in ether. Diclofenac sodium is hygroscopic. Identification (1) Take 1 mL of a solution of Diclofenac Sodium in methanol (1 in 250), add 1 mL of nitric acid: a dark red color is observed. (2) Perform the test with 5 mg of Diclofenac Sodium as directed under the Flame Coloration Test (2): a light green color is observed. (3) Determine the infrared spectra of Diclofenac Sodium and Diclofenac Sodium RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) A solution of Diclofenac Sodium (1 in 100) responds to the Qualitative Tests for sodium salt. Purity (1) Heavy metalsProceed with 2.0 g of Diclofenac Sodium according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Diclofenac Sodium according to Method 3 and perform the test (not more than 2 ppm). (3) Related substancesDissolve 50 mg of Diclofenac Sodium in 50 mL of the mobile phase and use this solution as the test solution. Pipet 2.0 mL of the test solution and add the mobile phase to make exactly 50 mL. Pipet 5.0 mL of this solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography under the following operating conditions. Measure each peak area of these solutions by the automatic integration method: the area of peak other than the peak of diclofenac from the test solution is not larger than that of the standard solution.

Each mL of 0.1 mol/L perchloric acid VS = 18.996 mg of C20H29N3O2HCl

Packaging and Storage tight containers.

Preserve in light-resistant,

Diclofenac Sodium
CH2CO 2Na NH Cl

Cl

C14H10Cl2NNaO2: 318.13

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column, about 4.6mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of methanol and diluted glacial acetic acid (3 in 2500) (4 : 3).

KP 9 289
Flow rate: Adjust the flow rate so that the retention time of diclofenac is about 20 minutes. System suitability System performance: Dissolve 35 mg of ethyl parahydroxybenzoate and 50 mg of propyl parahydroxybenzoate in 100 mL of the mobile phase. Take 1 mL of this solution, add the mobile phase to make exactly 50 mL. When the procedure is run with 20 L of this solution, as directed under the above operating conditions, ethyl parahydroxybenzoate and propyl parahydroxybenzoate are eluted in this order with the resolution between their peaks being not less than 5.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of peak area of Diclofenac is not more than 2.0%. Time span of measurement: About twice as long as the retention time of diclofenac beginning after the solvent peak.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Assay Weigh accurately about 0.5 g of Diclofenac Sodium, previously dried, dissolve in 40 mL of water in a separatory funnel, add 2.0 mL of dilute hydrochloric acid and extract with 50 mL of chloroform. Extract again with two 20 mL volumes of chloroform and filter the extract each time through a pledget of absorbent cotton, moistened with chloroform. Wash the tip of the separatory funnel and the absorbent cotton, with 15 mL of chloroform, combine the washing with the extracts, add 10.0 mL of a solution of 1 mol/L hydrochloric acid TS in dehydrated ethanol (1 in 100) and titrate with 0.1 mol/L potassium hydroxide-ethanol VS from the first equivalent point to the second equivalent point (potentiometric titration, Endpoint Detection Method in Titrimetry).

and not more (C6H6Cl2N2O4S2).

than

101.0%

of

diclofenamide

Description Diclofenamide is a white, crystalline powder. Diclofenamide is very soluble in dimethylformamide, soluble in ethanol, slightly soluble in ether and very slightly soluble in water. Diclofenamide dissolves in sodium hydroxide TS. Identification (1) Dissolve 10 mg each of Diclofenamide and Diclofenamide RS in 100 mL each of 0.01 mol/L sodium hydroxide TS. To 10 mL each of these solutions, add 0.1 mL each of hydrochloric acid and determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared absorption spectra of Diclofenamide and Diclofenamide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point

Between 237 C and 240 C.

Each mL of 0.1 mol/L potassium hydroxide-ethanol VS = 31.813 mg of C14H10Cl2NNaO2

Packaging and Storage Preserve in tight containers.

Diclofenamide
NH2 O S O

O Cl Cl S O NH2

C6H6Cl2N2O4S2: 305.16 Diclofenamide, when dried, contains not less than 98.0%

Purity (1) ChlorideDissolve 0.10 g of Diclofenamide in 10 mL of dimethylformamide and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.45 mL of 0.01 mol/L hydrochloric acid VS, add 10 mL of dimethylformamide, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.160%). (2) SeleniumTake 0.10 g of Diclofenamide, add 0.5 mL of a mixture of perchloric acid and sulfuric acid (1 : 1) and 2 mL of nitric acid and heat in a water-batch until no more brown gas evolves and the solution becomes to be a pale yellow clear solution. After cooling, add 4 mL of nitric acid to this solution, then add water to make exactly 50 mL and use this solution as the test solution. Separately, pipet 3 mL of Standard Selenium Solution, add 0.5 mL of a mixture of perchloric acid and sulfuric acid (1 : 1) and 6 mL of nitric acid, then add water to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Atomic Absorption Specctrophotometry according to the following conditions and determine constant absorbance, AT and AS, obtained on a recorder after rapid increasing of the absorption: AT is smaller than AS (not more than 30 ppm). Perform the test by using a hydride generating system and a thermal absorption cell. Lamp: A selenium hollow cathode lamp Wavelength: 196.0 nm Temperature of sample atomizer: When an electric Furnace is used, about 1000 C Carrier gas: Nitrogen and argon

290 Monographs, Part I

(3) Heavy metalsProceed with 2.0 g of Diclofenamide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Related substancesDissolve 0.10 g of Diclofenamide in 50 mL of the mobile phase and use this solution as the test solution. Pipet 2.0 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of these solutions by the automatic integration method: the total area of the peaks other than the peak of diclofenamide from the test solution is not larger than the peak area of diclofenamide from the standard solution. hydroxybenzoate in the mobile phase (3 in 5000).

Operating conditions Detector, column, column temperature, mobile phase and flow rate: Proceed as directed in the operating conditions under Assay. System suitability Test for required detection: To exactly 5 mL of the standard solution add the mobile phase to make exactly 100 mL. Confirm that the peak area of diclofenamide obtained from 10 L of this solution is equivalent to 3.5 to 6.5% of that of diclofenamide obtained from 10 L of the standard solution. System repeatability: When the test is repeated 6 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the peak area of diclofenamide is not more than 1.0%. Time span of measurement: About 5 times as long as the retention time of diclofenamide.
Loss on Drying Not more than 1.0% (1 g, at a pressure not exceeding 0.67 kPa, 100 C, 5 hours). Residue on Ignition

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of sodium phosphate TS and acetonitrile (1 : 1). Flow rate: Adjust the flow rate so that the retention time of diclofenamide is about 7 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, diclofenamide and the internal standard are eluted in this order with the resolution between these peaks being not less than 9. System repeatability: When the test is repeated 6 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of diclofenamide to that of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Diclofenamide Tablets
Diclofenamide Tablets contain not less than 92.0% and not more than 108.0% of the labeled amount of diclofenamide (C6H6Cl2N2O4S2: 305.16).
Method of Preparation Prepare as directed under Tablets, with Diclofenamide. Identification (1) Weigh a portion of Diclofenamide Tablets, previously powdered, equivalent to 0.2 g of Diclofenamide according to the labeled amount, add 20 mL of methanol, shake and filter. Evaporate the filtrate on a water-bath to dryness. Dissolve 10 mg of the residue in 100 mL of 0.01 mol/L sodium hydroxide TS. To 10 mL of this solution, add 0.1 mL of hydrochloric acid and determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 284 nm and 288 nm and between 293 nm and 297 nm. Dissolution Test Take 1 tablet of Diclofenamide Tablets and perform the test using 900 mL of water as a dissolution solution at 50 revolutions per minute as directed in the Method 2 under the Dissolution Test. After 60 minutes from the start of the test, take 20 mL or more of the dissolved solution and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first

Not more than 0.1% (1 g).

Assay Weigh accurately about 50 mg each of Diclofenamide and Diclofenamide RS, previously dried and dissolve in 30 mL each of the mobile phase. To each solution, add exactly 10 mL of the internal standard solution and the mobile phase to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS of the peak area of diclofenamide to that of the internal standard for the test solution and the standard solution, respectively.

Amount (mg) of diclofenamide (C6H6Cl2N2O4S2 ) QT = amount (mg) of Diclofenamide RS Q S

Internal standard solutionA solution of butyl para-

KP 9 291
10 mL of the filtrate, use the subsequent solution as the test solution. Separately, weigh accurately about 55 mg of Diclofenamide RS, previously dried at a pressure not exceeding 0.67 kPa at 100 C for 5 hours, dissolve in 10 mL of ethanol and add water to make exactly 100 mL. Pipet 10.0 mL of this solution, add water to make exactly 100 mL and use this solution as the standard solution. Measure the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 285 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Diclofenamide Tablets in 60 minutes is not less than 70.0%. Dissolution rate (%) with respect to the labeled amount of diclofenamide (C6H6Cl2N2O4S2) = WS
AT 1 90 AS C

Dicloxacillin Sodium Hydrate

O Cl O O Cl N O N H CH3 H H S N CH3 CH3 NaO H

. H2O

C19H16Cl2N3NaO5SH2O: 510.32 Dicloxacillin Sodium Hydrate contains not less than 850 g (potency) per mg of dicloxacillin (C19H16Cl2N3 NaO5S : 470.33), calculated on the anhydrous basis
Description Dicloxacillin Sodium Hydrate is a white to light yellowish white crystalline powder. Dicloxacillin Sodium Hydrate is freely soluble in water and in methanol, soluble in ethanol, and practically insoluble in ether. Identification (1) Dissolve 0.125 g of Dicloxacillin Sodium Hydrate in 5 mL of water, and add diluted hydrochloric acid to make the solution acidic, and extract with 5 mL of chloroform. Take the chloroform layer, dry without heating, add 2 mg of disodium chromotropate

WS : Amount (mg) of Diclofenamide RS, C: Labeled amount (mg) of diclofenamide (C6H6Cl2N2O4S2) in 1 tablet.

Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 tablets of Diclofenamide Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of diclofenamide (C6H6Cl2N2O4S2), add exactly 25 mL of the mobile phase and shake for 15 minutes and centrifuge. Pipet 10.0 mL of the supernatant, add 4.0 mL of the internal standard solution and the mobile phase to make exactly 20 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Diclofenamide RS, previously dried at 100 C at a pressure not exceeding 0.67 kPa for 5 hours, dissolve in 30 mL of the mobile phase, add 10.0 mL of the internal standard solution and the mobile phase to make exactly 50 mL and use this solution as the standard solution. Proceed as directed in the Assay under Diclofenamide.

dehydrate and 2 mL of sulfuric acid and heat to 150 C in oil bath with shaking: a light yellow color develops at first and the color turns to purple after 2 or 3 minutes. (2) Determine the infrared spectra of Dicloxacillin Sodium Hydrate and Dicloxacillin Sodium Hydrate RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) The solution of Dicloxacillin Sodium Hydrate responds to the Qualitative test 1) for sodium salt.
pH The pH of a solution obtained by dissolving 0.1 g of Dicloxacillin Sodium Hydrate in 10 mL of water is between 4.5 and 7.5. Sterility Test It meets the requirement, when Dicloxacillin Sodium Hydrate is used in a sterile preparation. Pyrogen Test It meets the requirement, when Dicloxacillin Sodium Hydrate is used in a sterile preparation. Weigh appropriate amount of Dicloxacillin Sodium Hydrate, dissolve in Isotonic Sodium Chloride Injection, make the solution so that each mL contains 20 mg (potency), and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not less than 3.0 % and not more than 5.0 % (0.2 g, volumetric titration, direct titration). Assay

Amount (mg) of diclofenamide (C6Cl2N2O4S2) = amount (mg) of Diclofenamide RS QT

QS

Internal standard solutionA solution of butyl parahydroxybenzoate in the mobile phase (3 in 5000).
Packaging and Storage Preserve in well-closed containers.

Weigh accurately an amount of Dicloxacillin

292 Monographs, Part I

Sodium Hydrate, dissolve in 1 % phosphate buffer, pH 6.0 to make the solution containing 1 mg (potency) dicloxacillin per mL and use the solution as the test solution. Separately, weigh accurately an amount of Dicloxacillin Sodium Hydrate RS, dissolve 1 % phosphate buffer, pH 6.0 to make the solution so that each mL contains 1 mg (potency) and use the solution as the standard solution. Keep the standard solution at not exceeding 5 C and use within 7 days. Pipet exactly 1 mL of each of the test solution and the standard solution in iodine bottle, add exactly 2 mL of 1 mol/L sodium hydroxide TS, stand for 15 minutes, add 2 mL of diluted hydrochloric acid (1 in 10) and 10.0 mL of 0.01 mol/L iodine solution, stand for 15 minutes, if necessary, add 5 mL of carbon tetrachloride , mix with shaking, titrate the test solution and the standard solution with 0.01 mol/L sodium thiosulfate by using microburet (indicator: if necessary, 0.2 ~ 0.5 mL of starch TS), and determine the consumed volume (mL) of 0.01 mol/L of iodine solution, VT and VS . Separately, add exactly 10 mL of 0.01 mol/L iodine solution to the test solution and the standard solution, perform a blank determination without 15 minutes standing and make any necessary correction. Amount [g (potency)] of dicloxacillin sodium (C19H16Cl2N3NaO5S) = Amount [g (potency)] of Dicloxacillin Sodium Hydrate RS
VT VS

cipitate is formed. The precipitate does not dissolve in nitric acid and dissolves excessive amount of ammonia TS. (2) Determine the infrared spectra of Dicyclomine Hydrochloride and Dicyclomine Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibits similar intensities of absorption at the same wavenumbers.
Melting Point

Between 169 C and 174 C.

pH Dissolve 1.0 g of Dicyclomine Hydrochloride in 100 mL of water: the pH of this solution is between 5.0 and 5.5. Purify Sulfate Perform the test with 0.5 g of Dicyclomine Hydrochloride and use this solution as the test solution. The color of the test solution is not stronger than that of the control solution D. Loss on Drying Not more than 1.0% (1 g, 105 C, 4 hours). Assay Weigh accurately about 0.6 g of Dicyclomine Hydrochloride, dissolve in 70 mL of glacial acetic acid, add 10 mL of mercuric acetate TS and titrate with 0.1 mol/L perchloric acid VS until blue color develops (indicator: 1 drop of methylosanyl chloride TS). Perform a blank determination and make any necessary correction.

Packaging and Storage Preserve in tight containers.

Each mL of 0.1 mol/L perchloric acid VS = 34.595 mg of C19H35NO2HCl

Packaging and Storage Preserve in well-closed container.

Dicyclomine Hydrochloride
O CH2CH3 C O CH2CH2N CH2CH3 HCl

Diethylcarbamazine Citrate
O C N N(CH2CH3)2 CH2CO2H HO N C CO2H

C19H35NO2HCl: 345.95 Dicyclomine Hydrochloride contains not less than 99.0% and not more than 102.0% of dicyclomine hydrochloride (C19H35NO2HCl), calculated on the anhydrous basis.
Description Dicyclomine Hydrochloride is a white fine crystalline powder, is odorless and has a bitter taste. Dicyclomine Hydrochloride is freely soluble in ethanol or in chloroform, soluble in water and very slightly soluble in ether. Identification (1) Take 5 mL of a solution of Dicyclomine Hydrochloride (1 in 500) and add 2 mL of 2 mol/L nitric acid TS and 2 mL of silver nitrate TS: a white preCH3

CH2CO2H

C10H21N3OC6H8O7: 391.42 Diethylcarbamazine Citrate, when dried, contains not less than 98.0% and not more than 101.0% of diethylcarbamazine citrate (C10H21N3OC6H8O7).
Description Diethylcarbamazine Citrate is a white, crystalline powder, is odorless and has an acid and bitter taste. Diethylcarbamazine Citrate is very soluble in water, soluble in ethanol and practically insoluble in acetone, in

KP 9 293
chloroform or in ether. A solution of Diethylcarbamazine Citrate (1 in 20) is acidic. Diethylcarbamazine Citrate is hygroscopic.
Identification (1) Dissolve 0.5 g of Diethylcarbamazine Citrate in 2 mL of water, add 10 mL of sodium hydroxide TS and extract with four 5 mL volumes of chloroform. Wash the combined chloroform extracts with 10 mL of water and evaporate chloroform on a water-bath and add 1 mL of ethyl iodide to the residue and boil gently with the aid of a reflux condenser for 5 minutes. Evaporate excess amount of ethyl iodide through air and add 4 mL of ethanol. Cool the ethanol solution in an ice-bath with continuous stirring, add ether until precipitates are produced and stir until crystallization is evident. Allow to stand in the ice-bath for 30 minutes and collect the precipitate. Dissolve the precipitate in 4 mL of ethanol, repeat the recrystallization in the same manner and then dry at 105 C for 4 hours: the melting point is between 151 C and 155 C. (2) Neutralize the residue after extracting with chloroform in (1) with dilute hydrochloric acid: the solution responds to the Qualitative Tests (2) and (3) for citrate. Melting Point

Method of Preparation Prepare as directed under Tablets, with Diethylcarbamazine Citrate. Identification (1) Take a portion of powdered Diethylcarbamazine Citrate Tablets, equivalent to 0.5 g of Diethylcarbamazine Citrate, according to the labeled amount, add 10 mL of water, shake, and filter. Add 10 mL of sodium hydroxide TS to the filtrate, and proceed as directed in the Identification (1) under Diethylcarbamazine Citrate. (2) Take a portion of powdered Diethylcarbamazine Citrate Tablets, equivalent to 0.8 g of Diethylcarbamazine Citrate according to the labeled amount, add 10 mL of water, shake, centrifuge, and filter the supernatant liquid. To 5 mL of the filtrate, add 5 mL of sodium hydroxide TS, and extract with two 20 mL volumes of chloroform. Separate the aqueous layer, and neutralize with dilute hydrochloric acid: the solution responds to the Qualitative Tests (2) and (3) for citrate. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 Diethylcarbamazine Citrate Tablets and powder. Weigh accurately a portion of the powder, equivalent to about 50 mg of diethylcarbamazine citrate (C10H21N3OC6H8O7), add 10 mL of water, shake well, add 5 mL of sodium hydroxide TS, then add exactly 20 mL of the internal standard solution, and shake vigorously for 10 minutes. Centrifuge, discard the aqueous layer, and use the chloroform layer as the test solution. Separately, weigh accurately 50 mg of Diethylcarbamazine Citrate RS, previously dried at 105 C for 4 hours, dissolve in 10 mL of water, add 5 mL of sodium hydroxide TS, proceed in the same manner as the preparation of the test solution, and use the chloroform layer as the standard solution. Perform the test with 2 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following conditions, and calculate the ratios, QT and

Between 135.5 C and 138.5 C.

Purity Heavy metalsProceed with 2.0 g of Diethylcarbamazine Citrate according to Method 4 and perform the test. Prepare the control solution with 4.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 1.0% (2 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.75 g of Diethylcarbamazine Citrate, previously dried, dissolve in 50 mL of glacial acetic acid by warming, cool and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 39.142 mg of C10H21N3OC6H8O7

Packaging and Storage Preserve in tight containers.

QS , of the peak area of diethylcarbamazine to that of the internal standard for the test solution and the standard solution, respectively.
Amount (mg) of diethylcarbamazine citrate (C10H21N3OC6H8O7) = amount (mg) of Diethylcarbamazine Citrate RS QT
QS

Diethylcarbamazine Citrate Tablets

Diethylcarbamazine Citrate Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of diethylcarbamazine citrate (C10H21N3OC6H8O7: 391.42).

Internal standard solutionA solution of noctadecane in chloroform (1 in 1250). Operating conditions Detector: A hydrogen flame-ionization detector. Column: A glass tube, 3 mm in inside diameter and 1 to 2 m in length, having 35% methylphenyl silicone po-

294 Monographs, Part I

lymer coated at the ratio of 3.0 percent on silanized siliceous earth for gas chromatography (180 m to 250 m in particle diameter). Column temperature: A constant temperature of about 145 C. Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of the internal standard is 8 to 11 minutes. Selection of column: Proceed with 2 L of the standard solution under the above operating conditions and use a column from which diethylcarbamazine and the internal standard are eluted in this order with a resolution between their peaks being not less than 5.0.
Packaging and Storage Preserve in well-closed containers.

(4) A solution of Difenidol Hydrochloride (1 in 100) responds to the Qualitative Tests for chloride.
pH Dissolve 1.0 g of Difenidol Hydrochloride in 100 mL of freshly boiled and cooled water: the pH of this solution is between 4.7 and 6.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Difenidol Hydrochloride in 10 mL of methanol: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Difenidol Hydrochloride according to Method 2 and perform the test (not more than 20 ppm). (3) ArsenicPrepare the test solution with 2.0 g of Difenidol Hydrochloride according to Method 3 and perform the test (not more than 1 ppm). (4) Related substancesDissolve 0.10 g of Difenidol Hydrochloride in methanol to make exactly 10 mL and use this solution as the test solution. Separately, dissolve 10 mg of 1,1-diphenyl-4-piperidino-1-butene hydrochloride RS in methanol to make exactly 20 mL, pipet 1.0 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the test solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of toluene, methanol and glacial acetic acid (10 : 2 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than that from the standard solution. Loss on Drying Not more than 0.5% (1 g, in vacuum, silica gel, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.35 g of Difenidol Hydrochloride, previously dried, dissolve in 30 mL of glacial acetic acid by warming, if necessary, cool, add 30 mL of acetic anhydride and titrate with 0.05 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Difenidol Hydrochloride

CH2CH2CH2COH

HCl

C21H27NOHCl: 345.91 Difenidol Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of difenidol hydrochloride (C21H27NOHCl).
Description Difenidol Hydrochloride is a white crystal or crystalline powder and is odorless. Difenidol Hydrochloride is freely soluble in methanol, soluble in ethanol, sparingly soluble in water or in glacial acetic acid and practically insoluble in ether. Melting pointAbout 217 C (with decomposition). Identification (1) Dissolve 10 mg of Difenidol Hydrochloride in 1 mL of sulfuric acid: an orange-red color is observed. Take this solution, add carefully 3 drops of water: the solution becomes yellowish brown and colorless on the addition of 10 mL of water. (2) Take 5 mL of a solution of Difenidol Hydrochloride (1 in 100) and add 2 mL of Reinecke salt TS: a light red precipitate is produced. (3) Take 10 mL of a solution of Difenidol Hydrochloride (1 in 100) and add 2 mL of sodium hydroxide TS and extract with two 15 mL volumes of chloroform. Combine the extracts, wash with three 10 mL volumes of water, evaporate the chloroform on a water-bath and dry the residue in a desiccator (in vacuum, silica gel, 55 C) for 5 hours: the residue melts between 103 C and 106 C.

Each mL of 0.05 mol/L perchloric acid VS = 17.295 mg of C21H27NOHCl

Packaging and Storage Preserve in well-closed containers.

KP 9 295
spherical octadecylsilanized silica gel for liquid chromatography (3 m in particle diameter). Mobile phase: A mixture of acetonitrile and water (50:50). Time span of measurement: Twice the retention time of diflucortolone valerate peak.
Loss on Drying Not more than 0.5% (1.0 g, 105 C, 3 hours). Assay Weigh accurately 30 mg each of Diflucortolone Valerate and Diflucortolone Valerate RS, add a mixture of acetonitrile and water (75 : 25) to make exactly 100 mL each and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the operating conditions in the Related substances under the Purity and determine the area of the peak for diflucortolone valerate in the test solution, AT , and in the

Diflucortolone Valerate
O O CH3 HO CH3 H H CH3 O

CH3

F O F

C27H36F2O5: 478.57 Diflucortolone Valerate contains not less than 97.0% and not more than 102.0% of diflucortolone valerate (C27H36F2O5), calculated on the dried basis.
Description Diflucortolone Valerate is a white crystalline powder. Diflucortolone Valerate is freely soluble in dichloromethane or in dioxin, sparingly soluble in ether, slightly soluble in methanol, and practically insoluble in water. Identification Determine the absorption spectra of solutions of Diflucortolone Valerate and Diflucortolone Valerate RS in methanol (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Specific Optical Rotation [ ]20 D : Between +98 and +103 (0.2 g, after drying, dioxane, 20 mL, 100 mm). Purity Related substancesWeigh 60 mg of Diflucortolone Valerate, add a mixture of acetonitrile and water (75 : 25) to make exactly 100 mL and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add a mixture of acetonitrile and water (75 : 25) to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and determine the area of the peaks in these solutions by the automatic integration method: the area of each peak other than the principal peak obtained from the test solution is not more than the area of the principal peak obtained from the standard solution (1%) and total area of the peaks other than the principal peak from the test solution is not more than twice the area of the principal peak from the standard solution (2%).

standard solution, AS . Amount (mg) of diflucortolone valerate (C27H36F2O5) = amount (mg) of Diflucortolone Valerate RS
Packaging and Storage tight containers.
AT AS

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Digitoxin
O O CH3 CH3 H CH3 H CH3 H H H CH3 H H H HO OH H O O OH H O H H O OH H O O H H H OH

C41H64O13: 764.94 Digitoxin, when dried, contains not less than 90.0% and not more than 101.0% of digitoxin (C41H64O13).
Description Digitoxin is a white to pale yellowish white, crystalline powder and is odorless.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 238 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 10 cm in length, packed with

296 Monographs, Part I

Digitoxin is soluble in chloroform, sparingly soluble in methanol or in ethanol and practically insoluble in water or in ether.
Identification (1) Transfer 1 mg of Digitoxin to a small test tube about 10 mm in inside diameter, dissolve in 1 mL of a solution of ferric chloride in glacial acetic acid (1 in 10000) and underlay gently with 1 mL of sulfuric acid: at the zone of contact of the two liquids, a brown ring free from a reddish color is observed and the color of the upper layer near the contact zone changes to green through purple. Finally the color of the entire glacial acetic acid layer changes to green through deep blue. (2) Take 2 mg of Digitoxin, add 25 mL of a freshly prepared solution of m-dinitrobenzene in ethanol (1 in 100) and dissolve by shaking. Pipet 2 mL of this solution, add 2 mL of a solution of tetramethylammonium hydroxide in ethanol (1 in 200) and mix: a red-purple color is observed slowly and then fades. (3) Dissolve 1 mg each of Digitoxin and Digitoxin RS in a mixture of chloroform and ethanol (1 : 1) to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of dichloromethane, methanol and water (84 : 15 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly dilute sulfuric acid upon the plate and heat at 110 C for 10 minutes: the spot from the test solution shows the same Rf value as the spot from the standard solution. Specific Optical Rotation [ ]20 D : Between + 16 and + 18 (after drying, 0.50 g, chloroform, 20 mL, 200 mm). Purity DigitoninDissolve 10 mg of Digitoxin in 2 mL of ethanol in a test tube having the inner walls which are free from scratches, add 2 mL of a solution of cholesterol in ethanol (1 in 200), mix gently and allow to stand for 10 minutes: no turbidity is produced. Loss on Drying Not more than 1.5% (0.5 g, in vacuum, 100 C, 2 hours). Residue on Ignition Not more than 0.5% (0.1 g). Assay Dissolve about 20 mg each of Digitoxin and Digitoxin RS, previously dried and accurately weighed, in methanol to make exactly 200 mL. Pipet 5.0 mL each of these solutions, add 10.0 mL of the internal standard solution to each solution, add 12.5 mL of water, then add methanol to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 50 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and cal-

culate the ratios, QT and QS of the peak area of digitoxin to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of digitoxin (C41H64O13) = amount (mg) of Digitoxin RS
QT QS

Internal standard solutionA solution of acenaphthene in methanol (3 in 1000000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm to 20 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of methanol and water (3 : 1). Flow rate: Adjust the flow rate so that the retention time of digitoxin is about 5 minutes. Selection of column: Proceed with 50 L of the standard solution according to the above conditions, use a column givion elution of digitoxin and the internal standard in this order with a resolution between their peaks being not less than 6.0.
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Digitoxin Injection
Digitoxin Injection is an aqueous solution for injection. Digitoxin Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of digitoxin (C41H64O13: 764.95).
Method of Preparation Prepare as directed under Injections, with a solution of Digitoxin in 5 to 50 % ethanol. Description liquid.

Digitoxin Injection is a clear, colorless

Identification (1) Take a portion of Digitoxin Injection, equivalent to 1 mg of Digitoxin according to the labeled amount and add 10 mL of water and extract with 10 mL of chloroform. Evaporate the chloroform extract to dryness on a water-bath. Proceed with the residue as directed in the Identification (1) under Digitoxin. (2) Take a portion of Digitoxin Injection, equivalent to 0.2 mg of Digitoxin according to the labeled amount and add 10 mL of water and extract with 10 mL of chloroform. Evaporate the chloroform extract on a water-bath

KP 9 297
with the aid of a current of air to dryness. Add 10 mL of a freshly prepared m-dinitrobenzene in ethanol (1 in 100) and dissolve by shaking. Proceed with 2 mL of this solution as directed in the Identification (2) under Digitoxin. (3) Retention time of the principal peak in the chromatogram of the test solution under the Assay corresponds to that of the standard solution.
Sterility Test

It meets the requirement.

Bacterial Endotoxins Less than 111.0 EU per mg of Digitoxin. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement

It

separatory funnel, shake with 30 mL of water and shake vigorously with 30 mL of chloroform. Filter the chloroform extract with a funnel on which a small amount of anhydrous sodium sulfate is placed and transfer to a round-bottomed flask connected by a universal joint. Evaporate the solution to dryness by warming under reduced pressure and dissolve the residue in 10 mL of chloroform. Transfer 5 mL of this solution to a small test tube about 10 mm in inside diameter and evaporate to dryness on a water-bath with the aid of a current of air. Proceed with the residue as directed in the Identification (1) under Digitoxin. (2) Evaporate 4 mL of the chloroform solution obtained in (1) to dryness, by warming under reduced pressure, add 10 mL of freshly prepared solution of mdinitrobenzene in ethanol (1 in 100) to the residue and dissolve by shaking. When the procedure is run with 2 mL of this solution as directed in the Identification (2) under Digitoxin.
Dissolution Test Take 1 tablet of Digitoxin Tablets and perform the test using 500 mL of diluted hydrochloric acid (3 in 500), degassed by a suitable method, as dissolution solution at 100 revolutions per minute as directed in the Method 1 under the Dissolution Test. After 30 minutes from the start of the test, take a + 15 mL of the dissolved solution and immediately add the same volume of fresh test solution, previously warmed at 37 0.5 C, to the vessel carefully. Filter a + 15 mL of the dissolved solution through a membrane filter (less than 0.8 m in pore size). Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Pipet exactly a mL of the test solution, equivalent to about 2 g of Digitoxin (C41H64O13) according to the labeled amount, transfer to a glass-stoppered centrifuge tube T30 and warm at 37 0.5 C for 30 minutes. Further, after 60 minutes from the start the test, take a + 15 mL of the dissolved solution, proceed in the same manner, pipet exactly a mL of the test solution and transfer to a glass-stoppered centrifuge tube T60. Separately, weigh accurately 100 times the labeled amount of digitoxin RS, previously dried under reduced pressure at 100 C for 2 hours and dissolve in ethanol to make exactly 100 mL. Pipet 1.0 mL of this solution, add the test solution to make exactly 500 mL, warm at 37 0.5 C for 60 minutes and filter through a membrane filter (less than 0.8 m in pore size). Discard the first 10 mL of the filtrate and use the subsequent filtrate as the standard solution. Pipet exactly a mL each of the standard solution and the test solution, transfer to glass-stoppered centrifuge tubes, TS Tand TB , respectively. Add exactly 7 mL of chloroform to each of the glass-stoppered centrifuge tubes, T30 , T60 , TS and

Determination of Volume of Injection in Containers It meets the requirement. Assay Take accurately a volume of Digitoxin Injection, equivalent to about 0.5 mg of Digitoxin, add 10 mL of the internal standard solution, mix, add methanol to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Digitoxin RS, previously dried at 100 C under vacuum for 2 hours, dissolve in methanol to make exactly 100 mL. Pipet 5 mL of the solution, add 10 mL of the internal standard solution and methanol to make exactly 50 mL. Use this solution as the standard solution. Perform the test as directed in the Assay under Digitoxin.

Amount (g/mL) of digitoxin (C41H64O13) = Amount of Digitoxin RS (mg)

QT QS

0.05

Internal standard solutionAcenaphthene in methanol (3 in 1000000).

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Digitoxin Tablets
Digitoxin Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of digitoxin (C41H64O13: 764.95).
Method of Preparation Prepare as directed under Tablets, with Digitoxin. Identification (1) Weigh a portion of Digitoxin Tablets, previously powdered, equivalent to 2 mg of digitoxin (C41H64O13) according to the labeled amount, place in a

TB , shake vigorously for 10 minutes and centrifuge. Discard the aqueous layer, pipet 5.0 mL of the chloroform layer, transfer to brown test tubes T '30 , T'60 , T 'S and
T 'B , evaporate the chloroform, add exactly 4 mL each of

298 Monographs, Part I

0.05 g/dL ascorbic acid-hydrochloric acid TS, shake well and allow to stand for 10 minutes. Then add exactly 0.5 mL each of dilute hydrogen peroxide TS, shake well and allow to stand at a constant temperature between 25 C and 30 C for 45 minutes. Determine the fluorescence intensities, F30 , F60 , FS and FB , of these solutions at about 395 nm of the excitation wavelength and at about 560 nm of the fluorescence wavelength as directed under the Fluorometry. Dissolution rates of Digitoxin Tablets after 30 minutes and 60 minutes are not less than 60.0% and 85.0%, respectively. No retest requirement is applied to Digitoxin Tablets. Dissolution rate (%) with respect to the labeled amount of digitoxin (C41H64O13) for 30 minutes = WS
F30 F B 1 FS F B C

ethanol (4 in 5) to make exactly 100 mL and use this solution as the standard solution. Pipet 2.0 mL each of the test solution, the standard solution and the diluted ethanol (4 in 5) into brown glass-stoppered test tubes T, S and B, respectively. Add exactly 10 mL each of 0.2 mg/mL ascorbic acid-hydrochloric acid TS, shake well and immediately add exactly 1 mL each of dilute hydrogen peroxide TS. Shake vigorously and allow to stand at a constant temperature between 25 C and 30 C for 45 minutes. Determine the fluorescence intensities, FT ,

FS and FB , of the test solution, standard solution and the diluted ethanol, respectively, at 400 nm of the excitation wavelength and at about 570 nm of the fluorescence wavelength as directed under the Fluorometry.
Amount (mg) of digitoxin (C41H64O13) = amount (mg) of Digitoxin RS
FT F B V FS F B 2000

Dissolution rate (%) with respect to the labeled amount of digitoxin (C41H64O13) for 60 minutes F 60 F B F30 F B + 15 1 =
WS ( FS F B

FS F B

500

WS : Amount (mg) of Digitoxin RS

C: Labeled amount (mg) of digitoxin (C41H64O13) in 1 tablet a +15: Measured volume (mL) of dissolved solution at the specified time.
Uniformity of Dosage Units It meets the requirement when the content uniformity test is performed according to the following method. Transfer 1 tablet of Digitoxin Tablets to a 50-mL beaker, add 0.5 mL of water to disintegrate the tablet, add 5 mL of acetonitrile and warm in a water-bath for 5 minutes, covering the beaker with a watch glass. After cooling, transfer the solution to separatory funnel A, rinse the beaker with 30 mL of chloroform and then with 20 mL of water, then transfer rinsings of the beaker to separatory funnel A, shake well and extract. Transfer the extract to separatory funnel B containing 5 mL of a solution of sodium bicarbonate (1 in 100) and shake to wash. Filter the chloroform layer through a pledget of absorbent cotton, previously moistened with chloroform. Extract the water layer in separator A with two 30 mL volumes of chloroform, wash the chloroform extract with a solution of sodium bicarbonate (1 in 100) in separator B, filter in the same manner and combine the filtrate with the first one. Evaporate this filtrate to dryness under reduced pressure by warming, add diluted ethanol (4 in 5) to make exactly V mL of a solution to contain 5 g of digitoxin (C41H64O13) per mL. Shake vigorously for 20 minutes to dissolve and use this solution as the test solution. Separately, weigh accurately about 10 mg of Digitoxin RS, previously dried at 100 C for 2 hours and dissolve in diluted ethanol (4 in 5) to make exactly 100 mL. Pipet 5.0 mL of this solution, add diluted

Assay Weigh accurately not less than 20 Digitoxin Tablets and powder. Weigh accurately a portion of the powder, equivalent to about 0.5 mg of digitoxin (C41H64O13) and shake with 12.5 mL of water for 10 minutes. Add exactly 10 mL of the internal standard solution, shake for 20 minutes and add methanol to make 50 mL. Centrifuge this solution and use the supernatant as the test solution. Separately, weigh accurately about 20 mg of Digitoxin RS, previously dried in vacuum at 100 C for 2 hours, dissolve in methanol to make exactly 200 mL. Pipet 5.0 mL of the solution, add 10.0 mL of the internal standard solution and 12.5 mL of water, then methanol to make 50 mL and use this solution as the standard solution. Proceed as directed in the Assay under Digitoxin.

Amount (mg/mL) of digitoxin (C41H64O13) = amount (mg) of Digitoxin RS QT 0.025

QS

Internal standard solutionA solution of acenaphthene in methanol (3 in 1000000).

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KP 9 299
of this solution, add 10 mL of water, add dilute ethanol to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately 5.0 mg of Gitoxin RS, previously dried under reduced pressure at 105 C for 1 hour, and add a mixture of acetonitrile and water (7 : 3) to make exactly 200 mL. Pipet accurately 2 mL of this solution, add dilute ethanol to make exactly 50 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and standard solution according to the following condition as directed under the Liquid Chromatography and determine the peak are of gitoxin in the test ( AT ) and the standard ( AS ) solutions. AT is not larger than AS , and the total of the areas of the peaks other than digoxin and gitoxin, obtained by the area percentage method, is not more than 3%.

Digoxin
O O HO H CH3 H CH3 H CH3 H H H CH3 H H H HO OH H O O OH H O H H O OH H O O H H H OH CH3 H

C41H64O14: 780.94 Digoxin, when dried, contains not less than 96.0% and not more than 106.0% of digoxin (C41H64O14).
Description Digoxin is a colorless to white crystal or white, crystalline powder. Digoxin is freely soluble in pyridine, slightly soluble in ethanol, very slightly soluble in glacial acetic acid and practically insoluble in water. Identification (1) Transfer 1 mg of Digoxin to a small test tube, about 10 mm in inside diameter, dissolve in 1 mL of a solution of ferric chloride in glacial acetic acid (1 in 10000) and underlay gently with 1 mL of sulfuric acid: at the zone of contact of the two liquids, a brown ring free from a reddish color is observed and the color of the upper layer near the contact zone changes to green through purple. Finally the color of the entire glacial acetic acid layer shows a green color through a deep blue color. (2) Determine the infrared spectra of Digoxin and Digoxin RS, both previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between + 10.0 and + 13.0 (after drying, 0.2 g, pyridine, 10 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 0.10 g of Digoxin in 15 mL of diluted ethanol (4 in 5) by warming: the solution is clear and colorless. (2) Related substancesWeigh accurately about 25.0 mg of Digoxin, dissolve in 50 mL of warm ethanol, cool, add ethanol to make exactly 100 mL. Pipet 10 mL

Operating conditions Detector, column, column temperature, mobile phase and flow rate: Proceed as directed in the operating conditions in the Assay. System suitability Test for required detectability: Pipet accurately 1 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the solution for system suitability test. Pipet accurately 1 mL of this solution and add mobile phase to make exactly 10 mL. Confirm that the peak area of digoxin obtained from 10 L of this solution is equivalent to 7% to 13% of that from the solution for system suitability test. System performance: Dissolve 25 mg of Digoxin in 50 mL of warm ethanol, cool and add ethanol to make exactly 100 mL. Pipet 10 mL of this solution, add exactly 5 mL of a solution of propyl parahydroxybenzoate in ethanol (1 in 4000), 10 mL of water and dilute ethanol to make 50 mL. When the procedure is run with 10 L of this solution under the above operating conditions, digoxin and propyl p-hydroxybenzoate are eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 10 L each of the solution for system suitability test under the above operating conditions, the relative standard deviation of the peak area of digoxin is not more than 1.0%.
Loss on Drying Not more than 1.0% (0.5 g, in vacuum, 105 C, 1 hours). Residue on Ignition Not more than 0.5% (0.1 g). Assay Weigh accurately about 25 mg each of Digoxin and Digoxin RS, previously dried, dissolve in 50 mL each of warm ethanol, cool and add ethanol to make exactly 100 mL each. Pipet accurately 10 mL each of these solutions, add 5 mL each of the internal standard solution, add 10 mL each of water and dilute ethanol to make exactly 50 mL each, and use these solutions as the test and

300 Monographs, Part I

standard solutions, respectively. Perform the test with 10 L each of the test solution and standard solution as directed under the Liquid Chromatography according to the following conditions and determine the ratios, QT and QS , of the peak area of digoxin to that of the internal standard for the test and standard solutions, respectively. Amount (mg) of digoxin (C41H64O14) = amount (mg) of Digoxin RS QT
QS

Internal standard solutionA solution of propyl phydroxybenzoate in ethanol (1 in 4000) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm) Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter) Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of water and acetonitrile (7:3). Flow rate: Adjust the flow rate so that the retention time of digoxin is about 10 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, digoxin and the internal standard are eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of digoxin to that of the internal standard is not more than 1.0%.
Packaging and Storage tight containers.

Identification Dilute Digitoxin Injection, if necessary, with ethanol so that each mL contains about 2.5 mg of Digoxin according to the labeled amount and use this solution as the test solution. Separately, dissolve 0.5 mg of Digoxin RS in 2 mL of methanol and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methanol and water (7 : 3) to a distance of about 10 cm and air-dry the plate. Spray evenly the plate with a freshly prepared mixture containing one volume of sodium toluenesulfon chloroamide trihydrate solution (3 in 100) and four volumes of trichloroacetic acid in ethanol (1 in 4), heat at 110 C for 10 minutes and examine the plate under UV light (the principal wavelength of 366 nm). The Rf values of the principal spots obtained from the test solution and the standard solution are not different each other. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 200 EU per mg of Digoxin. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer an exactly measured volume of Digoxin Injection, equivalent to 2.5 mg of digoxin (C41H64O14), add 5 mL of the internal standard solution and dilute ethanol to make 50 mL, and use this solution as the test solution. Separately, weigh accurately about 25 mg of Digoxin RS, previously dried under reduced pressure at 105 C for 1 hour, dissolve in 50 mL of warm ethanol, cool, and add ethanol to make exactly 100 mL. Pipet exactly 10 mL of this solution, add exactly 5 mL of the internal standard solution, 10 mL of water and dilute ethanol to make 50 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of digoxin to that of the internal standard, obtained with the test solution and the standard solution, respectively.

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Digoxin Injection
Digoxin Injection is an aqueous solution for injection. Digoxin Injection contains not less than 90.0% and not more than 105.0% of the labeled amount of digoxin (C41H64O14: 780.94).
Method of Preparation Prepare as directed under Injections, with a solution of Digoxin in 5 to 50 vol% ethanol. Description Digoxin Injection is a clear, colorless liquid.

Amount (mg) of digoxin (C41H64O14) = amount (mg) of Digoxin RS

QT 1 Q S 10

Internal standard solutionA solution of propyl phydroxybenzoate in ethanol (1in 40000).

KP 9 301
cipal spots obtained from the test solution and the standard solution are not different each other.
Dissolution Test Take 1 tablet of Digoxin Tablets and perform the test using 500 mL of diluted hydrochloric acid (3 in 500) as dissolution solution, at 100 revolutions per minute, as directed in the Method 1 under the Dissolution Test. After 60 minutes from the start of the test, take 30 mL or more of the dissolved solution and filter through a membrane filter (less than 0.8 m in pore size). Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately about 25 mg of digoxin RS, previously dried in vacuum at 105 C for 1 hour, dissolve in a small volume of ethanol and add a mixture, containing four volumes of ethanol and one volume of water, to make exactly 500 mL. Pipet 5.0 mL of this solution, add the test solution to make exactly 500 mL and use this solution as the standard solution. Pipet 2.0 mL each of the test solution, the standard solution, the dissolution solution and transfer to brown glass-stoppered test tubes, T, S and B, respectively. Add exactly 10 mL of 0.12 mg/mL ascorbic acidhydrochloric acid TS to each of these solutions and shake. Immediately add 1.0 mL of dilute hydrogen peroxide TS, shake well and allow to stand at a constant temperature between 25 C and 30 C for 45 minutes. Determine the fluorescence intensities, FT , FS and FB , of the test solution, standard solution and the dissolution solution, respectively, at about 360 nm of the excitation wavelength and at 485 nm of the fluorescence wavelength as directed under the Fluorometry. The dissolution rate of Digoxin Tablets in 60 minutes is not less than 65.0%. No retest requirement is applied to Digoxin Tablets.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column 4.5 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of water and acetonitrile (7:3). Flow rate: Adjust the flow rate so that the retention time of digoxin is about 10 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, digoxin and the internal standard are eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of digoxin to that of the internal standard is not more than 1.0%.
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Digoxin Tablets
Digoxin Tablets contain not less than 90.0% and not more than 105.0% of the labeled amount of digoxin (C41H64O14: 780.94).
Method of Preparation Prepare as directed under Tablets, with Digoxin. Identification Weigh a portion of powdered Digitoxin Tablets, equivalent to 0.5 mg of digitoxin (C41H64O13) according to the labeled amount, add 2 mL of methanol, shake for 10 mL to mix, filter and use the filtrate as the test solution. Separately, dissolve 0.5 mg of Digoxin RS in 2 mL of methanol and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methanol and water (7 : 3) to a distance of about 10 cm and air-dry the plate. Spray evenly the plate with a freshly prepared mixture containing one volume of sodium toluenesulfon chloroamide trihydrate solution (3 in 100) and four volumes of trichloroacetic acid in ethanol (1 in 4), heat at 110 C for 10 minutes and examine the plate under UV light (the principal wavelength of 366 nm). The Rf values of the prin-

Dissolution rate (%) with respect to the labeled amount of digoxin (C41H64O14) = W S
FT F B 1 FS F B C

WS : Amount (mg) of Digoxin RS, C: Labeled amount (mg) of digoxin (C41H64O14) in 1 tablet.
Uniformity of Dosage Units It meets the requirement when the content uniformity test is performed according to the following method. To 1 tablet of Digoxin Tablets, add 0.5 mL of water to disintegrate, add exactly 0.5 mL of the internal standard solution, and add V mL of dilute ethanol to render the concentration to be about 21 g of digoxin (C41H64O14) in 1 mL. Exposure this solution to ultrasonic waves, shake for 5 minutes to mix, filter, and use the filtrate as the test solution. Separately, weigh accurately about 25 mg of Digoxin RS, previously dried under reduced pressure at 105 C for 1 hour, dissolve in 50 mL of warm ethanol , cool, and add ethanol to make exactly 100 mL. Pipet 10 mL of this solution and add ethanol to make exactly 20 mL. Pipet 1 mL of this solution, add exactly 0.5 mL of the internal standard solution,

302 Monographs, Part I

add 1.5 mL of water and (V-2) mL of dilute ethanol and use this as the standard solution. Perform the test with the test solution and standard solution as directed in the Assay. Amount (mg) of digoxin (C41H64O14) = amount (mg) of Digoxin RS
QT 1 Q S 200

eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of digoxin to that of the internal standard is not more than 1.0%.
Packaging and Storage tight containers.

Preserve in light-resistant,

Internal standard solutionA solution of propyl phydroxybenzoate in ethanol (1in 40000/V).

Assay Weigh accurately and powder not less than 20 Digoxin Tablets. Transfer an accurately weigh a portion of the powder, equivalent to about 2.5 mg of digoxin (C41H64O14), add 30 mL of dilute ethanol, exposure to ultrasonic waves for 20 minutes and shake for 5 minutes to mix. Add exactly 5 mL of the internal standard solution and dilute ethanol to make 50 mL. Centrifuge the solution and use the supernatant liquid as the test solution. Separately, weigh accurately about 25 mg of Digoxin RS, previously dried under reduced pressure at 105 C for 1 hour, dissolve in 50 mL of warm ethanol, cool, and add ethanol to make exactly 100 mL. Pipet 10 mL of this solution, add exactly 5 mL of the internal standard solution, 10 mL of water and dilute ethanol to make 50 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of digoxin to that of the internal standard, obtained with the test solution and the standard solution, respectively.

Dihydrocodeine Phosphate
CH3 H N

H3PO4

O CH3O H H OH

Hydrocodeine Phosphate

C18N23NO3H3PO4: 399.38

Dihydrocodeine Phosphate contains not less than 98.0% and not more than 101.0% of dihydrocodeine phosphate (C18N23NO3H3PO4), calculated on the dried basis.
Description Dihydrocodeine Phosphate is a white to yellowish white, crystalline powder. Dihydrocodeine Phosphate is freely soluble in water or in glacial acetic acid, slightly soluble in ethanol and practically insoluble in ether. When dissolve 1.0 g of Dihydrocodeine Phosphate in 10 mL of water, the pH of this solution is between 3.0 and 5.0. Dihydrocodeine Phosphate is affected by light. Identification (1) Determine the absorption spectra of solutions of Dihydrocodeine Phosphate and Dihydrocodeine Phosphate RS (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Dihydrocodeine Phosphate and Dihydrocodeine Phosphate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Dihydrocodeine Phosphate (1 in 20) responds to the Qualitative Tests (1) for phosphate. Purity (1) ChloridePerform the test with 0.5 g of Dihydrocodeine Phosphate. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid (not more than 0.021%). (2) SulfatePerform the test with 0.20 g of Dihy-

Amount (mg) of digoxin (C41H64O14) = amount (mg) of Digoxin RS

QT 1 Q S 10

Internal standard solutionA solution of propyl phydroxybenzoate in ethanol (1in 40000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column 4.5 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of water and acetonitrile (7:3). Flow rate: Adjust the flow rate so that the retention time of digoxin is about 10 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, digoxin and the internal standard are

KP 9 303
drocodeine Phosphate. Prepare the control solution with 1.0 mL of 5 mol/L sulfuric acid (not more than 0.240%). (3) Related substancesDissolve 0.20 g of Dihydrocodeine Phosphate in 10 mL of diluted ethanol (1 in 2) and use this solution as the test solution. Pipet 1.0 mL of the test solution, add diluted ethanol (1 in 2) to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solutions as directed under the Thin-layer Chromatography. Spot 10 L of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of dehydrated ethanol, toluene, acetone and strong ammonia water (14 : 14 : 7 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultra-violet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than that from the standard solution.
Loss on Drying hours).

trophotometry: it exhibits a maximum between 281 nm and 285 nm.

Particle Size Distribution Test for Preparations It meets the requirement. Uniformity of Dosage Units (divided) It meets the requirement. Assay Weigh accurately about 2.5 g of 10 % Dihydrocodeine Phosphate Powder and dissolve in water to make exactly 100 mL. Pipet 2.0 mL of this solution, add 10.0 mL of the internal standard solution and water to make 20 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Dihydrocodeine Phosphate RS, separately determined its loss on drying

Not more than 1.0% (0.5 g, 105 C, 4

Assay Weigh accurately about 0.5 g of Dihydrocodeine Phosphate, dissolve in 70 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue to greenish blue (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

(105 C , 4 hours) and dissolve in water to make exactly 100 mL. Pipet 10.0 mL of this solution, add exactly 10 mL of the internal standard solution and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of dihydrocodeine to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of dihydrocodeine phosphate (C18N23NO3H3PO4) = amount (mg) of Dihydrocodeine Phosphate RS, calculated on the dried basis
QT 5 QS

Each mL of 0.1 mol/L perchloric acid VS = 39.938 mg of C18N23NO3H3PO4

Packaging and Storage tight containers.

Preserve in light-resistant,

Internal standard solutionA solution of Ethylefrine Hydrochloride (3 in 10000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in 500 mL of diluted phosphoric acid (1 in 1000) and adjust the pH to 3.0 with sodium hydroxide TS. To 240 mL of this solution, add 70 mL of tetrahydrofuran. Flow rate: Adjust the flow rate so that the retention time of dihydrocodeine is about 9 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, dihydrocodeine and the internal standard are eluted in this order with the resolution between these peaks being not less than 4. System repeatability: When the test is repeated 6

10% Dihydrocodeine Phosphate Powder

10% Hydrocodeine Phosphate Powder 10% Dihydrocodeine Phosphate Powder contains not less than 9.3% and not more than 10.7% of dihydrocodeine phosphate (C18N23NO3H3PO4: 399.38).
Method of Preparation Dihydrocodeine Phosphate 100 g Lactose Hydarate a sufficient quantity To make 1000 g Prepare as directed under Powders, with the above ingredients. Identification Determine the absorption spectrum of a solution of 10 % Dihydrocodeine Phosphate Powder (1 in 1000) as directed under the Ultraviolet-visible Spec-

304 Monographs, Part I

times with 20L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of dihydrocodeine to that of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Phosphate RS, calculated on the dried basis

QT QS

Internal standard solutionA solution of Ethylefrine Hydrochloride (3 in 10000). Operating conditions Perform as directed in Assay under 10% Dihydrocodeine Phosphate Powder.
Packaging and Storage Preserve in tight containers.

1% Dihydrocodeine Phosphate Powder

1% Hydrocodeine Phosphate Powder 1% Dihydrocodeine Phosphate Powder contains not less than 0.90% and not more than 1.10% of dihydrocodeine phosphate (C18N23NO3H3PO4: 399.38).
Method of Preparation Dihydrocodeine Phosphate 100 g Lactose Hydrate a sufficient quantity To make 1000 g Prepare as directed under Powders, with the above ingredients. Identification Determine the absorption spectrum of a solution of 1 % Dihydrocodeine Phosphate Powder (1 in 100) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 281 nm and 285 nm. Particle Size Distribution Test for Preparations It meets the requirement. Uniformity of Dosage Units (divided) It meets the requirement. Assay Weigh accurately about 5 g of 1% Dihydrocodeine Phosphate Powder and dissolve in water to make exactly 100 mL. Pipet 10.0 mL of this solution, add 10.0 mL of the internal standard solution and use this solution as the test solution. Separately, weigh accurately about 0.05 g of Dihydrocodeine Phosphate RS, separately determined its loss on drying (105 C, 4 hours) and dissolve in water to make exactly 100 mL. Pipet 10.0 mL of this solution, add exactly 10 mL of the internal standard solution and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of dihydrocodeine to that of the internal standard, for the test solution and the standard solution, respectively.

Dihydroergotamine Mesilate
O H C H N CH3 O N H N N H H O CH3 O H CH2 CH3SO 3H OH

HN

C33H37N5O5CH4O3S: 679.78 Dihydroergotamine Mesilate contains not less than 97.0% and not more than 101.0% of dihydroergotamine mesilate (C33H37N5O5CH4O3S), calculated on the dried basis.
Description Dihydroergotamine Mesilate is a white to yellowish white or grayish white to reddish white powder. Dihydroergotamine Mesilate is freely soluble in glacial acetic acid, sparingly soluble in methanol or in chloroform, slightly soluble in water or in ethanol and practically insoluble in acetic anhydride or in ether. Dihydroergotamine Mesilate is gradually affected by light. Melting pointAbout 214 C (with decomposition). Identification (1) Dissolve 1 mg of Dihydroergotamine Mesilate in 5 mL of a solution of tartaric acid (1 in 100). To 1 mL of this solution, add 2 mL of pdimethylaminobenzaldehyde-ferric chloride TS and shake: a blue color is observed. (2) Take 0.1 g of Dihydroergotamine Mesilate, add 0.4 g of sodium hydroxide, stir well and incinerate by gradual ignition. After cooling, add 10 mL of water to the residue, heat to boiling, cool and filter. To the filtrate, add 0.5 mL of hydrochloric acid: the solution responds to the Qualitative Tests for sulfate. Separately, to 0.1 g of Dihydroergotamine Mesilate, add 5 mL of dilute hydrochloric acid, shake for 5 minutes, filter and to the filtrate, add 1 mL of barium chloride TS: the solution is clear.

Amount (mg) of dihydrocodeine phosphate (C18N23NO3H3PO4) = amount (mg) of Dihydrocodeine

KP 9 305
(3) Determine the absorption spectra of solutions of Dihydroergotamine Mesilate and Dihydroergotamine Mesilate RS in methanol (1 in 20000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Dihydroergotamine Mesilate and Dihydroergotamine Mesilate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
Specific Optical Rotation [ ]20 D : Between -16.7 and 22.7 [0.5 g, when dried, a mixture of dehydrated ethanol, chloroform and strong ammonia water (10 : 10 : 1), 20 mL, 100 mm]. pH Dissolve 50 mg of Dihydroergotamine Mesilate in 50 mL of water: the pH of this solution is between 4.4 and 5.4. Purity (1) Clarity and color of solution Dissolve 0.10 g of Dihydroergotamine Mesilate in 0.1 mL of a solution of methanesulfonic acid (7 in 100) and 50 mL of water: the solution is clear and has no more color than the following control solutions (1) or (2).

thane, ethylacetate, methanol and strong ammonia water (50 : 50 : 6 : 1) to a distance of about 15 cm and air-dry the plate. Spray evenly p-dimethylaminobenzaldehyde TS for spraying on the plate and dry the plate with warm wind: the spots other than the principal spot from the test solution are not more intense than that from the standard solution (1) and the spots, which are more intense than the spot from the standard solution (2), are not more than two.
Loss on Drying Not more than 4.0% (0.5 g, at a pressure not exceeding 0.67 kPa, 100 C, 6 hours). Assay Weigh accurately about 0.2 g of Dihydroergotamine Mesilate, dissolve in 170 mL of a mixture of acetic anhydride and glacial acetic acid (10 : 1) and titrate with 0.02 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.02 mol/L perchloric acid VS = 13.596 mg of C33H37N5O5CH4O3S.

Packaging and Storage tight containers.

Preserve in light-resistant,

Control solution (1)Pipet 0.60 mL of ferric chloride stock CS and 0.150 mL of cobaltous chloride stock CS, mix and add diluted hydrochloric acid (1 in 40) to make exactly 100 mL. Control solution (2)Pipet 0.60 mL of ferric chloride stock CS and 0.250 mL of cobaltous chloride stock CS and 0.1 mL of cupric sulfate CS, mix and add diluted hydrochloric acid (1 in 40) to make exactly 100 mL.
(2) Related substancesPerform this procedure without exposure to daylight, using light-resistant vessels. Dissolve 0.10 g of Dihydroergotamine Mesilate in 5 mL of a mixture of chloroform and methanol (9 : 1) and use this solution as the test solution. Pipet 1.0 mL of the test solution, add a mixture of chloroform and methanol (9 : 1) to make exactly 200 mL and use this solution as the standard solution (1). Pipet 10.0 mL of the standard solution (1), add a mixture of chloroform and methanol (9 : 1) to make exactly 25 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of dichloromethane, ethylacetate, methanol and strong ammonia water (50 : 50 : 6 : 1) to a distance of about 15 cm and dry the plate with cold wind within 1 minute. Develop the plate again immediately with a freshly prepared mixture of dichlorome-

Dilazep Hydrochloride Hydrate

CH3O O CH3O CH3O C OCH 2CH2CH2 N N O CH2CH2CH2O C OCH 3 OCH 3 2 HCl H2O OCH 3

C31H44N2O102HClH2O: 695.63 Dilazep Hydrochloride Hydrate contains not less than 98.0% and not more than 101.0% of dilazep hydrochloride (C31H44N2O102HCl), calculated on the dried basis.
Description Dilazep Hydrochloride Hydrate is a white, crystalline powder and is odorless. Dilazep Hydrochloride Hydrate is freely soluble in glacial acetic acid or in chloroform, soluble in water, slightly soluble in ethanol or in acetic anhydride and practically insoluble in ether. Melting pointBetween 200 C and 204 C. Immerse the sample in a bath of 110 C and raise the temperature at the rate of about 3 C per minute from 140 C to 150 C, about 10 C per minute from 160 C to 195 C and about 1 C per minute from 195 C. Identification (1) Take 1 mL of a solution of Dilazep Hydrochloride Hydrate (1 in 100), add 0.1 mL of a solution of hydroxylamine hydrochloride (1 in 10) and 0.1 mL of 8 mol/L potassium hydroxide TS and warm in a water-bath of 70 C for 10 minutes. After cooling, add 0.5 mL of dilute hydrochloric acid and 0.1 mL of ferric chloride TS: a purple color is observed.

306 Monographs, Part I

(2) Take 5 mL of a solution of Dilazep Hydrochloride Hydrate (3 in 500), add 0.3 mL of Reinecke salt TS: a light red precipitate is produced. (3) Determine the absorption spectra of solutions of Dilazep Hydrochloride Hydrate and Dilazep Hydrochloride Hydrate RS (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Dilazep Hydrochloride Hydrate and Dilazep Hydrochloride Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (5) A solution of Dilazep Hydrochloride Hydrate (1 in 50) responds to the Qualitative Tests for chloride.
pH Dissolve 1.0 g of Dilazep Hydrochloride Hydrate in 100 mL of water: the pH of this solution is between 3.0 and 4.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Dilazep Hydrochloride Hydrate in 20 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 0.5 g of Dilazep Hydrochloride Hydrate. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.048%). (3) Heavy metalsProceed with 2.0 g of Dilazep Hydrochloride Hydrate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Dilazep Hydrochloride Hydrate according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.40 g of Dilazep Hydrochloride Hydrate in 10 mL of chloroform and use this solution as the test solution. Pipet 1.0 mL of the test solution, add chloroform to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of methanol, ethyl acetate, dichloromethane and hydrochloric acid (500 : 200 : 100 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly Dragendorffs TS for spraying on the plate: the spots other than the principal spot from the test solution are not more intense than that from the standard solution. Loss on Drying Between 2.0% and 3.0% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Dilazep Hydrochloride Hydrate dissolve in 40 mL of a mixture of

acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 33.881 mg of C31H44N2O102HCl
Packaging and Storage Preserve in tight containers.

Diltiazem Hydrochloride
OCH3

H S O H N O CH2CH2N(CH3)2

O C CH3 HCl

C22H26N2O4SHCl: 450.98 Diltiazem Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of diltiazem hydrochloride (C22H26N2O4SHCl).
Description Diltiazem Hydrochloride is a white crystal or crystalline powder and is odorless. Diltiazem Hydrochloride is very soluble in formic acid, freely soluble in water, in methanol or in chloroform, sparingly soluble in acetonitrile, slightly soluble in acetic anhydride or in dehydrated ethanol and practically insoluble in ether. Identification (1) Dissolve 50 mg of Diltiazem Hydrochloride in 1 mL of 1 mol/L of hydrochloric TS, add 2 mL of ammonium thiocyanate-cobalt nitrate TS and 5 mL of chloroform, shake well and allow to stand slowly: a blue color is observed in the chloroform layer. (2) Proceed as directed under the Oxygen Flask Combustion Method with 30 mg of Diltiazem Hydrochloride, using 20 mL of water as the absorbing liquid and prepare the test solution: the test solution responds to the Qualitative Tests (1) for sulfate. (3) Dissolve 10 mg each of Diltiazem Hydrochloride and Diltiazem Hydrochloride RS in 0.01 mol/L hydrochloric acid TS to make 100 mL each. Pipet 2 mL each of these solutions and add 0.01 mol/L hydrochloric acid TS to make 20 mL each. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Diltiazem Hydrochloride and Diltiazem Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhi-

KP 9 307
bit similar intensities of absorption at the same wavenumbers. (5) A solution of Diltiazem Hydrochloride (1 in 50) responds to the Qualitative Tests (2) for chloride.
Melting Point Between 210 C and 215 C (with decomposition). pH Dissolve 1.0 g of Diltiazem Hydrochloride in 100 mL of water: the pH of this solution is between 4.3 and 5.3. Purity (1) Clarity and color of solution Dissolve 1.0 g of Diltiazem Hydrochloride in 20 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 1.0 g of Diltiazem Hydrochloride. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (3) Heavy metalsProceed with 2.0 g of Diltiazem Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicPlace 1.0 g of Ditiazem Hydrochloride in a decomposition flask, add 5 mL of nitric acid and 2 mL of sulfuric acid, put a small funnel on the neck of the flask and heat cautiously until white fumes are evolved. After cooling, add 2 mL of nitric acid, heat and repeat this procedure twice. Add several 2 mL volumes of strong hydrogen peroxide water and heat until the solution changes colorless to pale yellow. After cooling, add 2 mL of saturated solution of ammonium oxalate and heat again until white fumes are evolved. After cooling, add water to make 5 mL, use this solution as the test solution and perform the test: the test solution has no more color than the following control solution (not more than 2 ppm).

Control solutionProceed in the same manner as the test solution without Diltiazem Hydrochloride, add 2.0 mL of standard arsenic solution and water to make 5 mL and proceed in the same manner as the test solution.
(5) Related substancesDissolve 50 mg of Diltiazem Hydrochloride in 50 mL of diluted dehydrated ethanol (4 in 5) and use this solution as the test solution. Pipet 1.0 mL of the test solution, add diluted dehydrated ethanol (4 in 5) to make exactly 200 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of these solutions by automatic integration method: the total peak area of peaks other than the peak of diltiazem obtained from the test solution is not more than 3/5 of the peak area of diltiazem obtained from the standard solution.

Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm to 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column Temperature: A constant temperature of about 50 C. Mobile phase: Dissolve 8 g of sodium acetate and 1.5 g of d-camphorsulfonic acid in 500 mL of water and filter using a membrane filter (0.4 m in pore size). Add 250 mL each of acetonitrile and methanol to the filtrate and adjust the solution to a pH of 6.6 by adding sodium acetate. Make any necessary adjustment. Flow rate: Adjust the flow rate so that the retention time of diltiazem is about 9 minutes. System suitability Test for required detection: To exactly 2 mL of the standard solution, add diluted dehydrated ethanol (4 in 5) to make exactly 10 mL. Confirm that the peak area of diltiazem obtained from 20 L of this solution is equivalent to 15 to 25% of that of diltiazem obtained from 20 L of the standard solution. System performance: Weigh 30 mg of Diltiazem Hydrochloride, 20 mg of d-3-hydroxy-cis-2,3-dihydro-5[2-(dimethylamino)ethyl]-2-(p-methoxyphenyl)-1,5benzothiazepin-4-(5H)-one hydrochloride (i.e., deacetylated form) and 20 mg of phenylbenzoate, dissolve in 160 mL of dehydrated ethanol and add water to make 200 mL. Using 20 L of this solution, perform the test as directed under the Liquid Chromatography according to the above operating conditions: the deacetylated form, diltiazem and phenylbenzoate are eluted in this order and the resolution of the deacetylated form and diltiazem and the resolution of diltiazem and phenyl benzoate are not less than 2.5, respectively. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of diltiazem is not more than 2.0%. Time span of measurement: About twice as long as the retention time of diltiazem after the solvent peak.
Loss on Drying hours).

Not more than 0.5% (1 g, 105 C, 2

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.7 g of Diltiazem Hydrochloride, previously dried, dissolve in 2.0 mL of formic acid, add 60 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 45.10 mg of C22H26N2O4SHCl

Packaging and Storage

Operating conditions

Preserve in light-resistant,

308 Monographs, Part I

tight containers. lution as the test solution. Pipet 1.0mL of test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test and standard solutions as directed under the Thinlayer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methanol, chloroform and strong ammonia water (150 : 150 : 1) to a distance of about 10 cm and airdry the plate. Spray evenly the plate with Dragendorffs TS for spraying: the spots other than the principal spot form the test solution are not more intense than that from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Assay Weigh accurately about 0.6 g of Dimemorfan Phosphate, previously dried, dissolve in 100 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Dimemorfan Phosphate
CH3 H N H3PO4 H

H3C

C18H25NH3PO4: 353.39 Dimemorfan Phosphate, when dried, contains not less than 98.5% and not more than 101.0% of dimemorfan phosphate (C18H25NH3PO4)
Description Dimemorfan Phosphate is a white to pale yellowish white and crystal or crystalline powder. Dimemorfan Phosphate is freely soluble in glacial acetic acid, sparingly soluble in water or in methanol, slightly soluble in ethanol and practically insoluble in ether. Melting pointAbout 265 C (with decomposition). Identification (1) Determine the absorption spectra of solutions of Dimemorfan Phosphate and Dimemorfan Phosphate RS (1 in 5000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Dimemorfan Phosphate and Dimemorfan Phosphate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibits similar intensities of absorption at the same wavenumbers. (3) Take 2 mL of a solution of Dimemorfan Phosphate (1 in 100) and add 2 to 3 drops of silver nitrate TS: a yellow precipitate is formed and it dissolves on the addition of dilute nitric acid. Specific Optical Rotation [ ]20 D : Between + 25 and + 27 (after drying, 1 g, methanol, 100 mL, 100 mm). pH Dissolve 1.0 g of Dimemorfan Phosphate in 100 mL of water: the pH of this solution is between 4.0 and 5.0. Purify (1) Heavy metalsProceed with 1.0 g of Dimemorfan Phosphate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) ArsenicProceed with 1.0 g of Dimemorfan Phosphate according to Method 3 and perform the test. Use 10 mL of a solution of magnecium nitrate in ethanol (1 in 10) (not more than 2 ppm). (3) Related substancesDissolve 0.10 g of Dimemorfan Phosphate in 10 mL of methanol and use this so-

Each mL of 0.1 mol/L perchloric acid VS = 35.339 mg of C18H25NH3PO4

Packaging and Storage Preserve in tight containers.

Dimenhydrinate
O H3C N CHOCH2CH2N(CH3)2 O N CH3 N H N Cl

C17H21NOC7H7ClN4O2: 469.96 Dimenhydrinate, when dried, contains not less than 53.0% and not more than 55.5% of diphenhydramine (C17H21NO: 255.35) and not less than 44.0% and not more than 47.0% of 8-chlorotheophylline (C7H7ClN4O2: 214.61).
Description Dimenhydrinate is a white, crystalline powder, is odorless and has a bitter taste. Dimenhydrinate is very soluble in chloroform, freely soluble in ethanol and slightly soluble in water or in ether. Identification (1) Dissolve 0.5 g of Dimenhydrinate in 30 mL of dilute ethanol, add 30 mL of water and use this solution as the test solution. Transfer 30 mL of the test solution to a separatory funnel and add 2 mL of strong ammonia water. Extract with two 10 mL volumes of ether, combine the ether extracts, wash the combined extracts with 5 mL of water and then extract the combined

KP 9 309
extracts with 15 mL of diluted hydrochloric acid (1 in 100). With this water layer, perform the following tests. (i) Take 5 mL of this acid extract, add 5 drops of Reinecke salt TS: a light red precipitate is produced. (ii) Take 10 mL of this acid extract, add 10 mL of picric acid TS and allow to stand for 30 minutes. Collect the precipitate by filtrating, recrystallize from dilute ethanol and dry at 105 C for 30 minutes: the crystals melt between 128 C and 133 C. (2) Take 30 mL of the test solution obtained in the Identification (1), add 2 mL of dilute sulfuric acid and cool for 30 minutes. Scratch the inside wall of the container frequently to facilitate crystallization: the white crystals are produced. Filter and wash the white crystals with a small amount of ice-cooled water. Dry the crystals for 1 hour at 105 C: the crystals melt between 300 C and 305 C with decomposition. (3) Take 10 mg of the crystals obtained in the Identification (2), add 10 drops of hydrogen peroxide TS and 1 drop of hydrochloric acid and evaporate on a water-bath to dryness: the residue shows a yellow-red color. When the dish containing the residue is held over a vessel containing 2 to 3 drops of ammonia TS, the color changes to red-purple, which is discharged on the addition of 2 to 3 drops of sodium hydroxide TS. (4) Mix well 50 mg of crystals obtained in the Identification (2) with 0.5 g of sodium peroxide in a nickel crucible and heat until the mass melts. Cool, dissolve the melted mass in 20 mL of water and acidify with dilute nitric acid: the solution responds to the Qualitative Tests for chloride.
Melting Point

six 15 mL volumes of ether with shaking, combine the ether extracts and wash the combined ether extracts with three 50 mL volumes of water. To the ether extracts, add 25.0 mL of 0.05 mol/L sulfuric acid and 25 mL of water. Shake thoroughly and evaporate the ether gently. Cool and titrate the excess sulfuric acid with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of methyl red TS). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L sulfuric acid VS = 25.536 mg of C17H21NO (2) 8-ChlorotheophyllineWeigh accurately about 0.8 g of Dimenhydrinate, previously dried, transfer to a 200 mL volumetric flask, add 50 mL of water, 3 mL of ammonia TS and 6 mL of a solution of ammonium nitrate (1 in 10) and heat in a water-bath for 5 minutes. Add 25.0 mL of 0.1 mol/L silver nitrate VS, heat in a water-bath for 15 minutes with occasional shaking, cool and add water to make exactly 200 mL. Allow to stand overnight to settle the precipitate and filter through a dry filter paper. Discard the first 20 mL of filtrate, measure exactly 100 mL of the subsequent filtrate, acidify with nitric acid, add 3 mL of nitric acid and titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ferric ammonium sulfate TS). Perform a blank determination and make any necessary correction. Each mL of 0.1 mol/L silver nitrate VS = 21.461 mg of C7H7ClN4O2
Packaging and Storage Preserve in well-closed containers.

Between 102 C and 107 C.

Purity (1) ChlorideTransfer 50 mL of the filtrate obtained in the Assay (2) to a Nessler tube, add 1 mL of nitric acid and allow to stand for 5 minutes: the turbidity of the solution is not greater than that of the following control solution. (2) Bromide and iodidePlace 0.10 g of Dimenhydrinate in a glass-stoppered test tube and add 50 mg of sodium nitrite, 10 mL of chloroform and 10 mL of dilute hydrochloric acid. Stopper, shake well and allow to stand: the chloroform layer remains colorless.

Dimenhydrinate Injection
Dimenhydrinate Injection is a solution of Dimenhydrinate in a mixture of propylene glycol and water. Dimenhydrinate Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of dimenhydrinate (C17H21NOC7H7ClN4O2: 469.96).
Method of Preparation Prepare as directed under Injections, with Dimenhydrinate Description Dimenhydrinate Injection is a clear, colorless liquid. Identification (1) Pipet 5 mL of Dimenhydrinate Injection into a separatory funnel containing 35 mL of water. Add 3 mL of ammonium hydroxide and 10 g of sodium chloride and extract with three 50 mL volumes of ether. Wash the combined ether extracts with 25 mL of water until any color which is observed in the final washing by the addition of 2 drops of phenolphthalein TS is dis-

Control solutionDilute 0.25 mL of 0.01 mol/L hydrochloric acid VS with 6 mL of dilute nitric acid and with water to make 50 mL, add 1 mL of silver nitrate TS and allow to stand for 5 minutes (not more than 0.044%).
Loss on Drying Not more than 0.5% (3 g, in vacuum, P2O5, 24 hours). Residue on Ignition Not more than 0.3% (1 g). Assay (1) DiphenhydramineWeigh accurately about 0.5 g of Dimenhydrinate, previously dried, transfer to a separatory funnel and add 50.0 mL of water, 3 mL of ammonia TS and 10 g of sodium chloride. Extract with

310 Monographs, Part I

charged by 1 drop of diluted hydrochloric acid (1 in 100). Extract with 10 mL of water containing 0.3 mL of 3 mol/L hydrochloric acid TS: the aqueous extract obtained meets the requirement under Identification (2) under Dimenhydrinate. (2) Proceed with the precipitate of (1) as directed in the Identification (3) and (4) under Dimenhydrinate.
pH Between 6.4 and 7.2. 8-Chlorotheophylline Content Transfer an exactly measured volume of Dimenhydrinate Injection, equivalent to about 50 mg of Dimenhydrinate, add methanol to make exactly 100 mL and mix. Pipet 5.0 mL of this solution, add methanol to make exactly 50 mL, mix and use this solution as the test solution. Weigh a portion of Dimenhydrinate RS, previously dried in a desiccater (in vacuum, P2O5) for 24 hours, dissolve in methanol to make a solution contain 0.5 mg of Dimenhydrinate per mL. Pipet 5.0 mL of this solution, add methanol to make exactly 50 mL, mix and use this solution as the standard solution. Perform the test with 25 L each of the test solution and the standard solution as dircted under the Liquid Chromatography according to the following conditions and determine the peak area, AT and AS , of these solutions, respectively. The retention time of 8chlorotheophylline is about 5 minutes. Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer an exactly measured volume of Dimenhydrinate Injection, equivalent to about 50 mg of Dimenhydrinate, add methanol to make exactly 100 mL and mix. Pipet 5.0 mL of this solution, add 5.0 mL of the internal standard solution, mix and use this solution as the test solution. Separately, weigh accurately about a portion of dimenhydrinate RS, add methanol and dissolve to make the solution contain 0.5 mg per mL. Pipet 5.0 mL of this solution, add 5.0 mL of internal standard solution, mix and use this solution as the standard solution. Perform the test with 25 L each of the test solution and the standard solution, as directed under the Liquid Chromatography according to the following conditions and calculate, QT and QS, the ratio of the peak area of Dimenhydrinate to that of the internal standard for these solutions, respectively.

Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Amount (mg/mL) of 8-chlorotheophylline (C7H7ClN4O2) = 1000 C

V AT 0 .45665 AS

Amount (mg/mL) of dimenhydrinate

Q (C17H21NOC7H7ClN4O2) = 200 C T V QS

C: Concentration of the standard solution (g/mL), V: Amount of Dimenhydrinate Injection (mL).

The amount of 8-chlorotheophylline is between 43.4% and 47.9% of the Dimenhydrinate assayed.

C: Concentration of the standard solution (g/mL), V: Amount of Dimenhydrinate (mL). Internal stanadard solution2-hydroxybenzylalcohol in methanol (2.0 mg/mL). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 229 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilianized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: Dissolve 0.8 g of ammonium bicarbonate in 800 mL of water, add 200 mL of methanol and use this solution as mobile phase A. Dissolve 0.8 g of ammonium bicarbonate in 150 mL of water, add 850 mL of methanol and use this solution as mobile phase B. During the first 7 minutes, flow the mobile phase A, then 8 minutes, flow the mobile phase B and then 7 minutes, flow the mobile phase A. Flow rate: 1.5 mL/minute. System suitability System performance: When the procedure is run

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 2 mm in inside diameter and about 12.5 cm in length, packed with octadecylsilanized silica gel (30 m to 50 m) for liquid chromatography and stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilianized silica gel (3 m to 10 m) for liquid chromatography. Column temperature: A room temperature. Mobile phase: Dissolve 0.81 g of d-camphor sulfuric acid and 0.7 g of sodium acetic acid in 700 mL of water, add 300 mL of methanol and mix. Flow rate: 2 mL/minute. System suitability System repeatability: When the test is repeated 6 times with 25 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the peak areas is not more than 1.0%.

KP 9 311
with 10 L of the standard solution, as directed under the above operating conditions, 8-chlorotheophylline and internal standard are eluted in this order with the resolution of not less than 4.5. System repeatability: When the test is repeated 6 times with 10 L of the standard solution, the relative standard deviation of the peak area is not less than 2.0%.
Packaging and Storage Preserve in hermetic containers.

Internal standard solutionA solution of 2-hydroxy benzylalcohol in methanol (2.0 mg/mL).

Packaging and Storage Preserve in well-closed containers.

Dimercaprol
H HS SH OH

Dimenhydrinate Tablets
Dimenhydrinate Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of dimenhydrinate (C17H21NOC7H7ClN4O2: 469.96).
Method of Preparation Prepare as directed under Tablets, with Dimenhydrinate. Identification (1) Weigh a portion of Dimenhydrinate Tablets, previously powdered, equivalent to 0.5 g of Dimenhydrinate according to the labeled amount, with 25 mL of warm ethanol, mix with grinding and filter. Dilute the filtrate with 40 mL of water and filter again. Use the filtrate as the test solution. Transfer 30 mL of the test solution to a separatory funnel and proceed as directed in the Identification (1) under Dimenhydrinate. (2) With 30 mL of the test solution obtained in (1), proceed as directed in the Identification (2), (3) and (4) under Dimenhydrinate. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Dimenhydrinate Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of dimenhydrinate (C17H21NOC7H7ClN4O2), transfer to a flask, add 80 mL of methanol, shake well to mix, add methanol again to make exactly 100 mL and filter. Pipet exactly 5 mL of the filtrate, add 5.0 mL of the internal standard solution and use this solution as the test solution. Separately, weigh accurately about 25 mg of Dimenhydrinate RS, add methanol to make exactly 50 mL. Pipet exactly 5 mL, add 5.0 mL of the internal standard solution and use this solution as the standard solution. Perform the test with 25 L each of the test solution and standard solution as directed in the Assay under Dimenhydrinate Injection.

and enantiomer C3H8OS2: 124.23 Dimercaprol contains not less than 98.5% and not more than 101.5% of dimercaprol (C3H8OS2).
Description Dimercaprol is a colorless to pale to yellow liquid, and has a mercaptan-like, disagreeable odor. Dimercaprol is soluble in peanut oil and sparingly soluble in water. Dimercaprol is miscible with methanol or with dehydrated ethanol. Dimercaprol shows no optical rotation. Identification (1) Add 1 drop of Dimercaprol to a mixture of 1 drop of a solution of cobaltous chloride (1 in 200) and 5 mL of water: a yellow-brown color is observed. (2) Determine the infrared spectra of Dimercaprol and Dimercaprol RS as directed in the liquid film method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Refractive index Specific Gravity
20 : Between 1.570 and 1.575. nD 20 : Between 1.238 and 1.248. d 20

Amount (mg) of dimenhydrinate (C17H21NOC7H7ClN4O2) = amount (mg) of Dimenhydrinate RS

AT 2 AS

Purity (1) Clarity and color of solutionDissolve 1.0 mL of Dimercaprol in 20 mL of peanut oil: the solution is clear and colorless to pale yellow. (2) BromideTake 2.0 g of Dimercaprol, add 25 mL of dilute potassium hydroxide-ethanol TS and heat in a water-bath under a reflux condenser for 2 hours. Evaporate the ethanol with a current of warm air, add 20 mL of water and cool. Add a mixture of 10 mL of strong hydrogen peroxide and 40 mL of water, boil gently under a reflux condenser for 10 minutes and filter rapidly after cooling. Wash the residue with two 10 mL volumes of water, combine the washings with the filtrate, add 10 mL of dilute nitric acid and 5.0 mL of 0.1 mol/L silver nitrate VS and titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 2 mL of ferric ammonium sulfate TS). Perform a blank determination and make any necessary correction.

312 Monographs, Part I

Not more than 1.0 mL of 0.1 mol/L silver nitrate VS is consumed. (3) Heavy metalsProceed with 1.0 g of Dimercaprol according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm).
Assay Weigh accurately about 0.15 g of Dimercaprol into a glass-stoppered flask, dissolve in 10 mL of methanol and titrate immediately with 0.05 mol/L iodine VS until a pale yellow color is observed. Perform a blank determination and make any necessary correction.

Each mL of 0.05 mol/L iodine VS = 6.211 mg of C3H8OS2

Packaging and Storage Preserve in hermetic containers. Store in a cold place.

Dimorpholamine
O O N C N CH2CH2 N O C N O

Each mL of 0.05 mol/L iodine VS = 6.211 mg of C3H8OS2

Packaging and Storage Preserve in tight containers. Store at 5 C or less.

CH3CH2CH2CH2

CH2CH2CH2CH3

C20H38N4O4: 398.54 Dimorpholamine, when dried, contains not less than 98.0% and not more than 101.0% of dimorpholamine (C20H38N4O4).
Description Dimorpholamine is a white to pale yellow, crystalline powder, mass or syrupy liquid. Dimorpholamine is very soluble in dehydrated ethanol or in acetic anhydride and soluble in water. When dissolve 1.0 g of Dimorpholamine in 10 mL of water, pH of this solution is between 6.0 and 7.0. Dimorpholamine is hygroscopic. Identification (1) Determine the absorption spectra of solutions of Dimorpholamine and Dimorpholamine RS (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Dimorpholamine and Dimorpholamine RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Clarity and color of solution Dissolve 1.0 g of Dimorpholamine in 50 mL of water: the solution is clear and colorless to pale yellow. (2) ChlorideTake 20 mL of the solution obtained in (1), add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.036%). (3) SulfateTake 10 mL of the solution obtained in (1), add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.096%). (4) Heavy metalsProceed with 2.0 g of Dimorpholamine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) Related substancesDissolve 2.0 g of Dimorpholamine in 10 mL of dehydrated ethanol and use this

Dimercaprol Injection
Dimercaprol Injection is an oily solution for injection. Dimercaprol Injection contains not less than 95.0 percent and not more than 105.0 percent of the labeled amount of dimercaprol (C3H8OS2: 124.23).
Method of Preparation Prepare as directed under Injections, with Dimercaprol. Benzyl Benzoate or Benzyl Alcohol may be added to increase the solubility. Description Dimercaprol Injection is a clear, colorless to light yellow liquid and has an unpleasant odor. Identification Measure a volume of Dimercaprol Injection, equivalent to 30 mg of Dimercaprol according to the labeled amount, and proceed as directed in the Identification (1) under Dimercaprol. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer an exactly measured volume of Dimercaprol Injection, equivalent to about 0.1 g of dimercaprol (C3H8OS2), into a flask, rinse the pipet several times with a mixture of methanol and chloroform (3 : 1) and add the rinsings to the flask. Add a mixture of methanol and chloroform (3 : 1) to make 50 mL, and titrate with 0.05 mol/L iodine VS until a yellow color persists. Perform a blank determination, and make any necessary correction.

KP 9 313
solution as the test solution. Pipet exactly 1 mL of this solution, add dehydrated ethanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with these solutions as directed under the Thinlayer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of dehydrated methanol and water (4:1) to a distance of about 10 cm and air-dry the plate. Allow the plate to stand in iodine vapor for 10 minutes: the spot other than the principal spot from the test solution is not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, 8 hours). Residue on Ignition Not more than 0.10% (1 g). Assay Weigh accurately about 0.6 g of Dimorpholamine, previously dried, dissolve in 50 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction.

solution of acetaldehyde (1 in 20), 0.1 mL of sodium pentacyanonitrosyl ferrate (III) TS and 0.5 mL of sodium carbonate TS : a blue color develops.
Sterility Test It meets the requirement. Bacterial Endotoxins Less than 5.0 EU per mg of Dimorpholamine Injection. Perform the test with the sample diluted to 0.15 w/v% with water for bacterial endotoxins test. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Take exactly a volume of Dimorpholamine Injection, equivalent to about 30 mg of dimorpholamine (C20H38N4O4) and add water to make exactly 200 mL. Pipet 1.0 mL of this solution, shake with exactly 4 mL of the internal standard solution for 5 minutes and use this solution as the test solution. Separately, weigh accurately about 0.15 g of Dimorpholamine RS, previously dried in a desiccator (in vacuum, P2O5) for 8 hours and dissolve in water to make exactly 1000 mL. Pipet 1.0 mL of this solution, shake with exactly 4 mL of the internal standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of dimorpholamine to that of the internal standard solution for these solutions, respectively.

Each mL of 0.1 mol/L perchloric acid VS = 39.85 mg of C20H38N4O4

Packaging and Storage tight containers.

Preserve in light-resistant,

Dimorpholamine Injection
Dimorpholamine Injection is an aqueous solution for Injection. Dimorpholamine Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of dimorpholamine (C20H38N4O4: 398.54).
Method of Preparation Prepare as directed under Injections, with Dimorpholamine. Description Dimorpholamine Injection is a clear, colorless liquid. Identification (1) Take a volume of Dimorpholamine Injection, equivalent to 0.1 g of Dimorpholamine according to the labeled amount and add 3 drops of Dragendorffs TS: an orange color is observed. (2) Take a volume of Dimorpholamine Injection, equivalent to 50 mg of Dimorpholamine according to the labeled amount, add 1 mL of dilute hydrochloric acid and evaporate on a water-bath to dryness. Dissolve this residue in 2 mL of hydrohloric acid, boil for 10 minutes under a reflux condenser, and evaporate to dryness on a water bath. Dissolve the residue with 1 mL of water, neutralize with sodium hydroxide TS, and add 0.2 mL of a

Amount (mg/mL) of dimorpholamine (C20H38N4O4) = amount (mg) of Dimorpholamine RS

QT 1 QS 5

Internal standard solutionA solution of butyl parahydroxybenzoate in acetonitrile (1 in 25000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 216 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilianized silica gel for liquid chromatogrphy (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of water and acetonitrile (1 : 1). Flow rate: Adjust the flow rate so that the retention time of dimorpholamine is about 4 minutes. System suitability

314 Monographs, Part I

System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratio of the peak area of dimorpholamine to that of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in hermetic containers.

Dinoprost
H HO CO2H CH3 HO H H HO H H

use this solution as the test solution. Pipet 3.0 mL of the test solution, add diluted methanol (1 in 5) to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of these solutions by the automatic integration method: the total area of the peaks other than the peak of Dinoprost from the test solution is not larger than the peak area of Dinoprost from the standard solution.

C20H34O5: 354.48 Dinoprost contains not less than 98.5% and not more than 101.0% of dinoprost (C20H34O5), calculated on the anhydrous basis.
Description Dinoprost is white, waxy masses or powder or clear, colorless to pale yellow and viscous liquid, and is odorless. Dinoprost is very soluble in dimethylformamide, freely soluble in methanol, in dehydrated ethanol or in ether and very slightly soluble in water. Identification (1) Weigh 5 mg of Dinoprost, add 2 mL of sulfuric acid and dissolve by shaking for 5 minutes: a dark red color is observed. To this solution, add 30 mL of sulfuric acid: an orange color is observed with green fluorescence. (2) Dissolve 1 mg each of Dinoprost and Dinoprost RS in 50 mL of diluted sulfuric acid (7 in 10) and warm in a water-bath for 50 C for 40 minutes. After cooling, determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Warm separately Dinoprost and Dinoprost RS at 40 C to make a liquid and determine the infrared spectra of the liquids as directed in the liquid film method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between + 24 and + 31 (0.2 g, dehydrated ethanol, 10 mL, 100 nm). Purity (1) Clarity and color of solutionDissolve 0.20 g of Dinoprost in 5 mL of dehydrated ethanol: the solution is clear and colorless to pale yellow. (2) Related substancesDissolve 10 mg of Dinoprost in 2 mL of methanol, add water to make 10 mL and

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 205 nm). Column: A stainless steel column, about 5 mm in inside diameter and about 15 cm in length, packed with octadecylsilianized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of 0.02 mol/L monobasic potassium phosphate TS and acetonitrile (5 : 2). Flow rate: Adjust the flow rate so that the retention time of Dinoprost is about 20 minutes. System suitability System performance: Dissolve 10 mg each of isopropyl parahydroxybenzoate and propyl parahydroxybenzoate in 2 mL of methanol and add water to make 10 mL. Take 1 mL of this solution, add diluted methanol (1 in 5) to make 30 mL. When the procedure is run with 10 L of this solution as directed under the above operating conditions, isopropyl parahydroxybenzoate and propyl parahydroxybenzoate are eluted in this order with a resolution between their peaks being not less than 2.5. Detection sensitivity: Adjust the detection sensitivity so that the peak height of Dinoprost from the standard solution composes 5.0% to 15.0% of the full scale. Time span of measurement: About 1.5 times as long as the retention time of Dinoprost after the solvent peak.
Water Not more than 0.5% (0.3 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg of Dinoprost, dissolve in 30 mL of dimethylformamide and titrate with 0.02 mol/L tetramethylammonium hydroxide VS under a stream of nitrogen (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.02 mol/L tetramethylammonium hydroxide VS = 7.090 mg of C20H34O5

Packaging and Storage Preserve in light-resistant, tight containers. Store in a place not exceeding 5 C.

KP 9 315 F: Relative response factor Ai : the peak area for each related substance obtained from the test solution AS : the peak area for dinoprostone obtained from the standard solution
Table. Relative response factor and the limit Relative Related substance retention F time
15-oxo-dinoprostone 0.79 0.85 0.90 1.15 1.80 5 1.1 1.0 1.0 5 15-epi-dinosprostone 8-isodinoprostone 5,6-trans-dinoprostone (5Z,13E,15S)-15hydroxy-9-oxoprosta-5, 10, 13 -triene-1-oic acid (5Z,13E,15S)-15hydroxy-9-oxoprosta-5, 8(12), 13-triene-1-oic acid Any other related substance

Dinoprostone
COOH

CH3 HO H H

Limit
* * * Not more than 2.0% Not more than 2.0% Not more than 2.0% Not more than 0.1% of total other related substances present

OH

Prostaglandin E2

C20H32O5 : 352.47

Dinoprostone contains not less than 97.0% and not more than 103.0% of dinoprostone (C20H32O5), calculated on the anhydrous basis.
Description Dinoprostone is a white or light gray, crystalline powder and is odorless. Identification (1) Determine the infrared spectra of Dinoprostone and Dinoprostone RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) When proceed as directed in the Assay, the retention time of the principal peak from the test solution corresponds to that from the standard solution. Specific Optical Rotation [ ]20 D : Between -82 and 90 (0.1 g, ethanol 20 mL, 100 mm). Purity Related substancesWeigh 25.0 mg of Dinoprostone, add the mobile phase to make exactly 10 mL and use this solution as the test solution. Separately, weigh 25.0 mg of Dinoprostone RS, add the mobile phase to make exactly 10 mL and use this solution as the standard solution (1). To 0.5 mL of the standard solution (1), add the mobile phase to make 50 mL and use this solution as the standard solution (2). Perform the test with 20 L each of the test solution and the standard solution (2) as directed under the Liquid Chromatography according to the following operating conditions. Determine the area of each peak in these solutions by the automatic integration method and calculate the content of each related substance according to the following equation: the content of each related substance corresponds to the values given in the following table.

1.90

1.43

1.0

* The sum of these three related substances is not more than 1.0%.

Content (%) of each related substance = C 1 Ai

W F AS

C: Concentration (g/mL) of Dinoprostone RS in the standard solution (2) W: Weight (mg) of Dinoprostone to prepared the test solution

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of methanol and 0.2% acetic acid (58:42) Flow rate: 1 mL/min System suitability System performance: When the procedure is run with 20 L of the standard solution (1) under the above operating conditions, the number of theoretical plates is not less than 6000. The resolution between dinoprostone peak and any other adjacent peak in the chromatogram obtained from the injection of the test solution is not less than 1.0. System repeatability: When the test is repeated 5 times with 20 L each of the standard solution (1) under the above operating conditions, the relative standard deviation of the areas of the principal peak is not more than 2.0%.
Water Not more than 0.5% (0.5 g, volumetric titration, direct titration).

316 Monographs, Part I

Residue on Ignition Not more than 0.5% (1 g). Assay Weigh accurately about 25 mg of Dinoprostone, add the mobile phase to make exactly 10 mL and use this solution as the test solution. Separately, weigh 25 mg of Dinoprostone RS, add the mobile phase to make exactly 10 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the operating conditions directed in the Related substances under the Purity and determine the area of dinoprostone peak in the test solution, AT ,

Identification (1) Determine the infrared spectra of Diosmin and Diosmin RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) When proceed as directed in the Assay, the retention time and the size of the principal peak from the test solution corresponds to that from the standard solution. Purity (1) Heavy metalsProceed with 2.0 g of Diosmin according to Method 2, and perform the test. Prepare the control solution with 4.0 mL of standard lead solution (not more than 20 ppm). (2) IodinePrepare the test solution with 1.0 g of Diosmin as directed under the Oxygen Flask Combustion Method, using 50 mL of 0.02 w/v% hydrazine solution as an adsorbing liquid. Separately, weigh accurately 1.66 g of potassium iodide, dissolve in water to make exactly 100 mL. Pipet 2 mL of this solution, add water to make exactly 100 mL and use this solution as the control solution. Weigh 20 g of potassium nitrate and dissolve in 0.1 mol/L nitric acid to make 100 mL. Transfer 30 mL of the solution to a beaker, immerse an iodide ion selective electrode, stir for 10 minutes until the potential of the solution is stable ( nT1 ). Add 1 mL of the test solution and

and in the standard solution, AS . Amount (mg) of dinoprostone (C20H32O5) = amount (mg) of Dinoprostone RS
AT AS

System suitability System performance: When the procedure is run with 20 L of the standard solution according to the operating condition directed in related substances under Purity, the resolution between dinoprostone peak and any other adjacent peak is not less than 1.0. System repeatability: When the test is repeated 5 times with 20 L each of the standard solution according to the operating condition directed in related substances under Purity, the relative standard deviation of the areas of the principal peak is not more than 2.0%.
Packaging and Storage well-closed containers.

Preserve in light-resistant,

measure the potential ( nT 2 ). Separately, weigh 20 g of potassium nitrate, dissolve in 0.1 mol/L nitric acid to make 100 mL. Transfer 30 mL of the solution to a beaker, immerse an iodide ion selective electrode, stir for 10 minutes until the potential of the solution is stable ( n R1 ). Add 80 L of the control solution and measure the potential ( nR2 ). The absolute value, nT 2 nT 1 , is less than the absolute value, nR 2 nR1 . (3) Related substancesWeigh accurately 25 mg of Diosmin, dissolve in dimethylsulfoxide to make exactly 25 mL and use this solution as the test solution. Separately, weigh accurately 25 mg of Diosmin RS, dissolve in dimethylsulfoxide to make exactly 25 mL. Pipet 5 mL of this solution, add dimethylsulfoxide to make 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and determine the area of the peaks in these solutions by the automatic integration method. Multiply the peak areas by 0.38 and 0.61 for the related substances I and VI, respectively, for the calculation of the content of the related substances: the area of the related substance I peak obtained from the test solution is not more than 0.2 times the area of the principal peak obtained from the standard solution (1%), the area of the related substance II peak from the test solution is not more than the area of the principal peak from the standard solution (5%) and the individual area of the related substances III, V and VI peaks from the test solution is not more than 0.6 times

Diosmin
OH OCH3 OH CH3 OH OH OH O O O O O

OH OH OH O

C28H32O15:

608.55

Diosmin contains not less than 90.0% and not more than 102.0% of diosmin (C28H32O15), calculated on the anhydrous basis.
Description Diosmin is a grayish-yellow or light yellow powder. Diosmin is soluble in dimethylsulfoxide and practically insoluble in water or in ethanol. Diosmin is soluble in dilute sodium hydroxide TS. Diosmin is hygroscopic.

KP 9 317
the area of the principal peak from the standard solution. In addition, the area of the peak of any other related substance from the test solution is not more than 0.2 times the area of the principal peak from the standard solution (1%), the area of the related substance I peak and total area of other related substances from the test solution are 0.2 times the area of the principal peak from the standard solution, and total area of the related substance peaks is not more than twice the area of the principal peak from the standard solution (10%). Disregard any peak having the area not more than 0.02 times (0.1%) the area of the principal peak from the standard solution.

Diphenhydramine

CHOCH2CH2N(CH3)2

C17H21NO: 255.36 Diphenhydramine contains not less than 96.0% and not more than 101.0% of diphenhydramine (C17H21NO).
Description Diphenhydramine is a clear, pale yellow to yellow liquid, has a characteristic odor and has a burning taste at first, followed by slight sensation of numbness on the tongue. Diphenhydramine is miscible with glacial acetic acid, with acetic anhydride, with ethanol, or with ether. Diphenhydramine is very slightly soluble in water. Boiling pointAbout 162 C (in vacuum, 0.67 k Pa). 20 Refractive index nD : About 1.55. Diphenhydramine is gradually affected by light. Identification (1) Take 50 mg of Diphenhydramine and add 2 mL of sulfuric acid: an orange-red precipitate is produced immediately and its color changes to redbrown on standing. Add carefully 2 mL of water to this solution: the intensity of the color changes, but the color tone does not change. (2) Dissolve 0.1 g of Diphenhydramine in 10 mL of dilute ethanol, add an excess of a saturated solution of picric acid in dilute ethanol with stirring and cool in ice. Collect the produced crystals, recrystallize from dilute ethanol and dry at 105 C for 30 minutes: the crystals melt between 128 C and 133 C. Specific Gravity
20 : Between 1.103 and 1.020. d 20

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 275 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 10 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m in particle diameter). Column temperature: 40 C. Mobile phase: A mixture of acetonitrile, glacial acetic acid, methanol and water (2:6:28:66). Flow rate: 1.5 mL/minute. Relative retention time: The reference retention time for diosmin peak is about 4.6 min. The retention times for the related substances I, II, III, IV, V and VI are about 0.5, 0.6, 0.8, 2.2, 2.6 and 4.5 minutes, respectively. System suitability System performance: Weigh 25 mg of Diosmin and dissolve in dimethylsulfoxide to make 25 mL. When the procedure is run with 10 L of this solution under the above operating conditions, the resolution between the related substance II and the related substance III is not less than 2.5.
Water Not more than 6.0% (0.3 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately 25 mg each of Diosmin and Diosmin RS, dissolve in dimethylsulfoxide to make 25 mL each and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the operating conditions in the Related substances under the Purity and determine the area of the principal peak of the test solution, AT , and the standard solution,

AS .
Amount (mg) of diosmin (C28H32O15) = amount (mg) of Diosmin RS
AT AS

Packaging and Storage Preserve in tight containers.

Purity (1) -DimethylaminoethanolDissolve 1.0 g of Diphenhydramine in 20 mL of ether and extract with two 10 mL volumes of water with thorough shaking. Combine the water extracts and add 2 drops of phenolphthalein TS and 1.0 mL of 0.05 mol/L sulfuric acid VS: no red color is observed. (2) BenzohydrolTransfer 1.0 g of Diphenhydramine to a separatory funnel, dissolve in 20 mL of ether and extract with two 25 mL volumes of diluted hydrochloric acid (1 in 15) with thorough shaking. Separate the ether layer, evaporate slowly in a water-bath and dry in a desiccator (in vacuum, silica gel) for 2 hours: the weight of the residue is not more than 20 mg. (3) Heavy metalsProceed with 1.0 g of Diphenhydramine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm).

318 Monographs, Part I

Melting Point Between 166 C and 170 C. Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Diphenhydramine, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. pH Dissolve 1.0 g of Diphenhydramine Hydrochloride in 10 mL of water: the pH of this solution is between 4.0 and 5.0. Purity (1) Clarity and color of solution Dissolve 1.0 g of Diphenhydramine Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Diphenhydramine Hydrochloride according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 0.20 g of Diphenhydramine Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet 1.0 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of hexane, ethyl acetate, methanol and strong ammonia water (10 : 4 : 2 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly iodine TS on the plate: the spots other than the principal spot from the test solution and the spot on the original point are not more intense than that from the standard solution. Loss on Drying Not more than 0.5% (2 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.4 g of Diphenhydramine Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3). Titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 25.535 mg of C17H21NO

Packaging and Storage Preserve in light-resistant, tight and almost well-filled containers.

Diphenhydramine Hydrochloride

CHOCH2CH2N(CH3)2

HCl

C17H21NOHCl: 291.82 Diphenhydramine Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of diphenhydramine hydrochloride (C17H21NOHCl).
Description Diphenhydramine Hydrochloride is a white crystal or crystalline powder, is odorless and has a bitter taste, followed by sensation of numbness on the tongue. Diphenhydramine Hydrochloride is very soluble in methanol or in glacial acetic acid, freely soluble in water or in ethanol, sparingly soluble in acetic anhydride and practically insoluble in ether. Diphenhydramine Hydrochloride is gradually affected by light. Identification (1) Determine the absorption spectra of solutions of Diphenhydramine Hydrochloride and Diphenhydramine Hydrochloride RS in methanol (1 in 2000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Diphenhydramine Hydrochloride and Diphenhydramine Hydrochloride RS, both previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Diphenhydramine Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride.

Each mL of 0.1 mol/L perchloric acid VS = 29.182 mg of C17H21NOHCl.

Packaging and Storage tight containers.

Preserve in light-resistant,

Diphenhydramine Hydrochloride Capsules

Diphenhydramine Hydrochloride Capsules contain not less than 90.0% and not more than 110.0% of the labeled amount of diphenhydramine hydrochloride (C17H21NOHCl: 291.82).

KP 9 319
(C17H21NOHCl: 291.82).
Method of Preparation Prepare as directed under Injections, with Diphenhydramine. Description Diphenhydramine Hydrochloride Injection is a clear, colorless liquid. Identification (1) The retention of the principal peak in the chromatogram corresponds to that of the standard preparation. (2) Take a volume of Diphenhydramine Hydrochloride Injection, equivalent to about 50 mg of Diphenhydramine Hydrochloride, add 0.03 mol/L sulfuric acid TS to make 25 mL, filter if necessary and use this solution as the test solution. Separately, to 50 mg of Diphenhydramine Hydrochloride RS, add 0.01 mol/L hydrochloric acid TS to make 25 mL and use this solution as the standard solution. To each of the test solution and the standard solution, add 2 mL of 1 mol/L sodium hydroxide TS and 4 mL of carbon disulfide and shake for 2 minutes. Filter by centrifugation, if necessary and proceed as directed in the solution method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. pH Between 4.0 and 6.5. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 3.4 EU per mg of Diphenhydramine Hydrochloride. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Method of Preparation Prepare as directed under Capsules, with Diphenhydramine. Identification (1) Weigh a portion of Diphenhydramine Hydrochloride Capsules, equivalent to about 50 mg of Diphenhydramine Hydrochloride and perform the test as directed in the Identification (2) under Diphenhydramine Hydrochloride Injection. (2) The retention of the principal peak in the chromatogram corresponds to that of the standard preparation, as obtained in the Assay. Dissolution Test Take 1 capsule of Diphenhydramine Hydrochloride Capsules and perform the test using 500 mL of water as dissolution solution, at 100 revolutions per minute according to Method 1 under the Dissolution Test. After 30 minutes start from the test, take the dissolved solution and filter. Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately a portion of Diphenhydra-

mine Hydrochloride RS, previously dried at 105 C for 3 hours and dissolve in water at the same concentration as the test solution and use this solution as the standard solution. Perform the test with 50 L of the test solution and the standard solution, as directed according to Assay under Diphenhydramine Hydrochloride Injection. The dissolution rate of Diphenhydramine Hydrochloride Capsules in 30 minutes is not less than 80%.
Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 Diphenhydramine Hydrochloride Capsules. Transfer an accurately weighed portion of the powder, equivalent to about 50 mg of Diphenhydramine Hydrochloride, dissolve in water to make exactly 100 mL and filter. Perform the test as directed according to Assay under Diphenhydramine Hydrochloride Injection.

It

Amount (mg) of diphenhydramine hydrochloride (C17H21NOHCl) = amount (mg) of Diphenhydramine Hydrochloride RS

AT AS

Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer an accurately pipeted portion of Diphenhydramine Hydrochloride Injection, equivalent to about 50 mg of Diphenhydramine Hydrochloride, add water to make exactly 100 mL and use this solution as the test solution. Weigh accurately about 50 mg of Diphenhydramine Hydrochloride, previously dried at 105 C for 3 hours, dissolve in water to make 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine AT

Packaging and Storage Preserve in tight containers

Diphenhydramine Hydrochloride Injection

Diphenhydramine Hydrochloride Injection is a sterile solution of Diphenhydramine Hydrochloride in water for Injection. Diphenhydramine Hydrochloride Injection contains not less than 90.0% and not more than 110.0% at the labeled amount of diphenhydramine hydrochloride

and AS , the peak area of diphenhydramine hydrochloride. Amount (mg) of diphenhydramine hydrochloride (C17H21NOHCl) = amount (mg) of Diphenhydramine

320 Monographs, Part I

and allow to stand for 30 minutes. Collect the precipitate by filtration, recrystallize from dilute ethanol and dry at 105 C for 30 minutes: the crystals melt between 128 C and 133 C. (3) Take 1 mL of the test solution obtained in (1) and 1 drop of ferric chloride TS: a dark blue-purple color deve1ops.
Purity Heavy metalsProceed with 1.0 g of Diphenhydramine Tannate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 7.0% (1 g, 105 C, 5 hours). Residue on Ignition

Hydrochloride RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Temperature: A room temperature. Mobile Phase: Adjust pH to 6.5 by adding glacial acetic acid to a mixture of acetonitrile, water and triethylamine (50 : 50 : 0.5). Adjust, if necessary Flow rate: 1 mL/minute. System suitability System performance: Dissolve 5 mg of benzophenone in 5 mL of acetonitrile and add water to make 500 mL. To 1 mL of this solution, add 5 mg of Diphenhydramine Hydrochloride and water to make 10 mL. When the procedure is run with 10 L of this solution as directed under the above operating conditions, benzophenone and diphenhydramine are eluted in this order with the resolution between their peaks being greater than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the peak areas is not more than 2.0%.
Packaging and Storage hermetic containers.

Not more than 1.0% (1 g).

Preserve in light-resistant,

Assay Transfer about 1.7 g of Diphenhydramine Tannate, accurately weighed, to a separatory funnel, dissolve in 20 mL of water and 3.0 mL of dilute hydrochloric acid with thorough shaking, add 20 mL of a solution of sodium hydroxide (1 in 10) and exactly 25 mL of isooctane, shake vigorously for 5 minutes, dissolve 2 g of sodium chloride with shaking and allow to stand. Pipet 20.0 mL of the isooctane layer, add 80.0 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 25.535 mg of Cl7H21NO

Packaging and Storage tight containers.

Diphenhydramine Tannate
Diphenhydramine Tannate is a compound of Diphenhydramine and Tannic Acid and contains not less than 25.0% and not more than 35.0% of diphenhydramine (Cl7H21NO: 255.35).
Description Diphenhydramine Tannate is a grayish white to light brown powder, is odorless or has a slight, characteristic odor and is tasteless. Diphenhydramine Tannate is slightly soluble in ethanol and practically insoluble in water or in ether. Identification (1) Weigh 1 g of Diphenhydramine Tannate, add 15 mL of water and 0.3 mL of dilute hydrochloric acid, shake thoroughly for 1 minute, filter and use this filtrate as the test solution. Transfer 10 mL of the test solution to a separatory funnel, extract with two 20 mL volumes of chloroform, combine the chloroform extracts and evaporate on a water-bath to dryness. Take 5 mL of a solution of the residue (1 in 100), add 5 drops of Reinecke salt TS: a light red precipitate is produced. (2) Take 10 mL of a solution of the residue obtained in (1) (1 in 100), add 10 mL of picric acid TS drop-wise

Preserve in light-resistant,

Dipyridamole
N N N HOCH 2CH2 HOCH 2CH2 N N N N N CH2CH2OH

CH2CH2OH

C24H40N8O4: 504.63 Dipyridamole, when dried, contains not less than 98.5% and not more than 101.0% of dipyridamole (C24H40N8O4).
Description Dipyridamole is a yellow crystal or crystalline powder, is odorless and has a slightly bitter taste.

KP 9 321
Dipyridamole is freely soluble in chloroform, sparingly soluble in methanol or in dehydrated ethanol and practically insoluble in water or in ether.
Identification (1) Dissolve 5 mg of Dipyridamole in 2 mL of sulfuric acid, add 2 drops of nitric acid and shake: a deep purple color is observed. (2) Determine the absorption spectra of solutions of Dipyridamole and Dipyridamole RS in a mixture of methanol and hydrochloric acid (99 : 1) (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Dipyridamole and Dipyridamole RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point

System performance: Dissolve 7 mg of Dipyridamole and 3 mg of p-terphenyl in 50 mL of methanol. When the procedure is run with 20 L of this solution as directed under the above operating conditions, Dipyridamole and p-terphenyl are eluted in this order with the resolution between their peaks being not less than 5.0. Detection sensitivity: Adjust the detection sensitivity so that the peak height of Dipyridamole obtained from 20 L of the standard solution is between 2 mm and 6 mm. Time span of measurement: About 5 times as long as the retention time of Dipyridamole.
Loss on Drying Not more than 0.2% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.6 g of Dipyridamole, previously dried, dissolve in 70 mL of methanol and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Between 165 C and 169 C.

Purity (1) Clarity and color of solutionDissolve 0.5 g of Dipyridamole in 10 mL of chloroform: the solution is clear and shows a yellow color. (2) Heavy metalsProceed with 2.0 g of Dipyridamole according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicProceed with 1.0 g of Dipyridamole according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 50 mg of Dipyridamole in 50 mL of the mobile phase and use this solution as the test solution. Pipet 0.50 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of these solutions by the automatic integration method: the total area of the peak other than the peak of Dipyridamole from the test solution is not larger than the peak area of Dipyridamole from the standard solution.

Each mL of 0.1 mol/L perchloric acid VS = 50.46 mg of C24H40N8O4

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Dirithromycin
O H3CO H3C HO H3C O H3C O CH3 O CH3 O H3C O OCH3 CH3 OH CH3 OH CH3 HO O O CH3 N(CH3)2 NH

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and 15 cm to 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 0.2 g of monobasic potassium phosphate in 200 mL of water and add 800 mL of methanol. Flow rate: Adjust the flow rate so that the retention time of Dipyridamole is about 4 minutes. System suitability

C42H78N2O14: 835.07

Dirithromycin contains not less than 950 g (potency) and not more than 1020 g (potency) per mg of dirithromycin and epidirithromycin (C42H78N2O14), calculated on the anhydrous basis. Epidirithromycin is not more than 2 %.
Description Dirithromycin is whtie to grayish white powder. Dirithromycin is freely soluble in chloroform, soluble in methanol, slightly soluble in propanol or in acetonitrile,

322 Monographs, Part I

and very slightly soluble in water or in cyclohexane.
Identification (1) Determine the infrared spectra of Dirithromycin and Dirithromycin RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Determine the retention times according to the procedure of Assay: the retention time of the principal peak in the chromatogram obtained with test solution is the same as that of the principal peak in the chromatogram obtained with the standard solution Crystalliity Test It meets the requirement. Purity (1) Heavy metalsProceed with 0.1 g of Dirithromycin according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related compoundsWeigh accurately 0.1 g of Dirithromycin, dissolve in the mixture of acetonitrle and methanol (70:30), make to exactly 10 mL and use this solution as the test solution. Separately, weigh accurately 10 mg of Dirithromycin RS, dissolve in the mixture of acetonitrle and methanol (70:30), make to exactly 50 mL, and use this solution as the standard solution. Perform the test with 10 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the Assay operating conditions, 9(S)-erythromycilamine and dirithromicin are eluted in this order. Prepare the test solution and the standard solution before use. Determine the amount of 9-(S)erythromycilamine according to the following equation, the amount is not more than 1.5 %. Other related substances are not more than 1.0 %, and total amount of related substances is not more than 5.0 %.

tion WS : the potency (mg) of Dirithromycin RS WA : the amount (mg) of Dirithromycin

Water Not more than 1.0 % (1.0 g, volumetric titration, direct titration). Assay (1) Dirithromycin Weigh accurately an amount of Dirithromycin and Dirithromycin RS, equivalent to about 20 mg (potency), dissolve each in the mixture of acetonitrle and methanol (70:30) and make exactly 10 mL, and use these solutions as the test solution and the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the Assay (2) conditions, and determine the peak areas, AT and AS , of dirithromycin. (2) Epidirithromycin Weigh accurately an amount of Dirithromycin, equivalent to about 0.1 g (potency), dissolve each in the mixture of acetonitrile and methanol (70:30) and make exactly 10 mL, and use this solution as the test solution. Separately, weigh accurately an amount of Dirithromycin RS, equivalent to about 10 mg (potency), dissolve each in the mixture of acetonitrile and methanol (70:30) and make exactly 50 mL, and use this solution as the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine the peak areas, AT and AS, of epidirithromycin. Prepare the test solution and the standard solution before use.

Amount [g (potency)] of dirithromycin or epidirithromycin = Amount [g (potency)] of dirithromycin RS

AT AS

Content ratio of 9-(S)-erythromycilamine or other related substances (%) =

AT1 W S 1 100 AS W A 5

Amount [g (potency)] of Dirithromycin = Amount [g (potency)] of dirithromycin + Amount [g (potency)] of epidirithromycin

AT1 : the peak area response corresponding to 9-(S)erythromycilamine or other related substances of the test solution AS : the peak area of dirithromycin of the standard solution WS : the potency (mg) of Dirithromycin RS WA : the amount (mg) of Dirithromycin

Content ratio of total related substances (%) =

AT2 W S 1 100 AS W A 5

AT2 : the sum of the peak area of all peaks except dirithromycin and solvent, of the test solution AS : the peak area of dirithromycin of the standard solu-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 205 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 250 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A room temperature or 40 C Mobile phase: A mixture of acetonitirile, 0.05 mol/L phosphate buffer solution (pH 7.5) and ethanol (44:37:19) Flow rate: 2.0 mL per minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, dirithromycin and epidirithromycin

KP 9 323
are eluted in this order.
Packaging and Storage Preserve in tight containers.

Disopyramide

O (CH 3)2CH NCH 2CH 2 (CH 3)2CH N C C NH 2

(3) Related substancesDissolve 0.40 g of Disopyramide in 10 mL of the methanol and use this solution as the test solution. Pipet 1.0 mL of the test solution, add methanol to make exactly 400 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and strong ammonia water (45 : 4 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots are not more intense than that from the standard solution.
Loss on Drying Not more than 0.5% (0.5 g, in vacuum, 80 C, 2 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.25 g of Disopyramide, dissolve in 30 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

and enantiomer C21H29N3O: 339.47 Disopyramide contains not less than 98.5% and not more than 101.0% of disopyramide (C21H29N3O), calculated on the dried basis.
Description Disopyramide is a white crystal or crystalline powder. Disopyramide is very soluble in methanol or in ethanol, freely soluble in acetic anhydride, in glacial acetic acid or in ether and slightly soluble in water. Identification (1) Take 1 mL of a solution of Disopyramide in ethanol (1 in 20), add 10 mL of picric acid TS and warm: a yellow precipitate is produced. Filter this precipitate, wash with water and dry at 105 C for 1 hour: the residue melts between 172 C and 176 C. (2) Determine the absorption spectra of solutions of Disopyramide and Disopyramide RS in 0.05 mol/L sulfuric acid-methanol TS (1 in 25000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Disopyramide, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
1% Absorbance E1 cm (269 nm): Between 194 and 205 (10 mg, 0.05 mol/L sulfuric acid-methanol TS, 500 mL).

Each mL of 0.1 mol/L perchloric acid VS = 16.97 mg of C21H29N3O

Packaging and Storage Preserve in tight containers.

Distigmine Bromide
CH3 N CH3 O C O CH3 C O O CH3 N 2 Br-

N(CH2)6N

C22H32Br2N4O4: 576.32 Distigmine Bromide contains not less than 98.5% and not more than 101.0% of distigmine bromide (C22H32Br2N4O4), calculated on the anhydrous basis.
Description Distigmine Bromide is a white, crystalline powder and odorless. Distigmine Bromide is very soluble in water, freely soluble in methanol, in glacial acetic acid or in ethanol, slightly soluble in acetic anhydride and practically insoluble in ether. When dissolve 1.0 g of Distigmine Bromide in 100 mL of water, pH of this solution is between 5.0 and 5.5. Bromide is slightly hygroscopic. Distigmine Bromide is gradually colored by light.

Purity (1) Heavy metalsDissolve 1.0 g of Disopyramide in 10 mL of ethanol and add 2 mL of dilute acetic acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 2.0 mL of standard lead solution, add 10 mL of ethanol, 2 mL of dilute acetic acid and water to make 50 mL (not more than 20 ppm). (2) ArsenicProceed with 1.0 g of Disopyramide according to Method 3 and perform the test (not more than 2 ppm).

324 Monographs, Part I Melting pointAbout 150 C (with decomposition).

Identification (1) Determine the absorption spectra of solutions of Distigmine Bromide and Distigmine Bromide RS (1 in 25000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Distigmine Bromide and Distigmine Bromide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Take 5 mL of a solution of Distigmine Bromide (1 in 10), add 2 mL of dilute nitric acid: the solution responds to the Qualitative Tests (1) for bromide. Purity (1) Clarity and color of solutionDissolve 0.25 g of Distigmine Bromide in 5 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 0.40 g of Distigmine Bromide. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.048%) (3) Heavy metalsProceed with 2.0 g of Distigmine Bromide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Related substancesDissolve 0.040 g of Distigmine Bromide in 10 mL of methanol and use this solution as the test solution. Pipet 1.0 mL of this solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of cellulose with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water, dehydrated ethanol and glacial acetic acid (8 : 3 : 2 : 1) to a distance of about 13 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Spray evenly Dragendorgff's TS for spraying on the plate: the spots other than the principal spot from the test solution are not more intense than that from the standard solution. Water Not more than 1.0% (1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.4 g of Distigmine Bromide, dissolve in 60 mL of a mixture of acetic anhydride and glacial acetic acid (8 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry with platinum electrode). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 28.816 mg of C22H32Br2N4O4

Packaging and Storage tight containers.

Preserve in light-resistant,

Distigmine Bromide Tablets

Distigmine Bromide Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of distigmine bromide (C22H32Br2N4O4: 576.32).
Method of Preparation Prepare as directed under Tablets, with Distigmine Bromide. Identification (1) Take a portion of powdered Distigmine Bromide Tablets, equivalent to 0.1 g of Distigmine Bromide according to the labeled amount, add 25 mL of water, shake, filter and use the filtrate as the test solution. To 5 mL of the test solution, add 3 mL of picric acid TS and proceed as directed in the Identification (2) under Distigmine Bromide. (2) Determine the absorption spectrum of the solution obtained in the Assay, as directed under the Ultravioletvisible Spectrophotometry: it exhibits a maximum between 268 nm and 272 nm and a minimum between 239 nm and 243 nm. (3) With 5 mL of the test solution obtained in (1), proceed as directed in the Identification under Distigmine Bromide. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 tablets of Distigmine Bromide Tablets and powder. Weigh accurately a portion of the powder, equivalent to about 15 mg of distigmine bromide (C22H32Br2N4O4), add 30 mL of 0.1 mol/L hydrochloric acid TS, shake for 1 hour, add 0.1 mol/L hydrochloric acid TS to make exactly 50 mL and filter. Discard the first 20 mL of the filtrate, pipet 10.0 mL of the subsequent filtrate, add 0.1 mol/L hydrochloric acid TS to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 0.03 g of Distigmine Bromide RS (previously determined the water) and dissolve in 0.1 mol/ L hydrochloric acid TS to make exactly 100 mL. Pipet 10.0 mL of this solution, add 0.1 mol/L hydrochloric acid TS to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution, AT2 and AS2, at 270 nm and, ATl and AS1, at 241 nm as directed under the Ultraviolet-visible Spectrophotometry, respectively.

Amount (mg) of distigmine bromide (C22H32Br2N4O4) = amount (mg) of Distigmine Bromide RS,

KP 9 325 AT2- AT1 1 calculated on the anhydrous basis A - A 2 S2 S1

Packaging and Storage Preserve in tight containers.

er than the peak of disulfiram from the test solution is not larger than the peak area of disulfiram from the standard solution.

Disulfiram
S (CH3CH2)2N C S S S C N(CH2CH3)2

C10H20N2S4: 296.54 Disulfiram, when dried, contains not less than 99.0% and not more than 101.0% of disulfiram (C10H20N2S4).
Description Disulfiram is a white to yellowish crystal or crystalline powder. Disulfiram is freely soluble in acetone or in toluene, slightly soluble in methanol or in ethanol and practically insoluble in water. Identification (1) Determine the absorption spectra of solutions of Disulfiram and Disulfiram RS in methanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Disulfiram and Disulfiram RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 70 C and 73 C. Purity (1) Heavy metalsProceed with 2.0 g of Disulfiram according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicWeigh 1.0 g of Disulfiram according to method 4 and perform the test (not more than 2 ppm). (3) Diethyldithiocarbamic acidDissolve 0.10 g of Disulfiram in 10 mL of toluene and shake with 10 mL of diluted sodium carboxylate TS (1 in 20). Take the water layer separately, wash that with 10 mL of toluene, shake with 5 drops of a solution of cupuric sulfate (1 in 250) and 2 mL of toluene and allow to stand: a pale yellow color is not observed in the toluene layer. (4) Related substancesDissolve 50 mg of Disulfiram in 40 mL of methanol, add water to make 50mL, and use this solution as the test solution . Pipet 1mL of the test solution, add the mobile phase to make exactly 200 mL, and use this solution as the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of both solutions by the automatic integration method: the total area of the peaks oth-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column about 5 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of methanol and water (7:3). Flow rate: Adjust the flow rate so that the retention time of disulfiram is about 8 minutes. Selection of column: Dissolve 50 mg of Disulfiram and 50mg of benzophenone in 40 mL of methanol, and add water to make 50 mL. To 1 mL of this solution add the mobile phase to make 200 mL. Proceed with 10 L of this solution under the above operating conditions, and calculate the resolution. Use a column giving elution of benzophenone and disulfiram in this order with the resolution between these peaks being not less than 4. Detection sensitivity: Adjust the detection sensitivity so that the peak height of disulfiram obtained from 10 L of the standard solution is 15 - 30 mm. Time span of measurement: About 3.5 times of the retention time of disulfiram.
Loss on Drying Not more than 0.20% (2 g, silica gel, 24 hours). Residue on Ignition Not more than 0.1% (2 g). Assay Weigh accurately about 0.2 g of Disulfiram, previously dried, in an iodine bottle, dissolve in 20 mL of acetone, add 1.5 mL of water and 1.0 g of potassium iodide and dissolve by shaking thoroughly. To this solution, add 3.0 mL of hydrochloric acid, stopper the bottle tightly, shake and allow to stand in a dark place for 3 minutes. Add 70 mL of water and titrate with 0.1 mol/L sodium thiosulfate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L sodium thiosulfate VS = 14.83 mg of C10H20N2S4

Packaging and Storage Preserve in tight containers.

326 Monographs, Part I

chromatography. Develop the plate with a mixture of chloroform, methanol and formic acid (78:22:5) to a distance of about 12 cm, and air-dry the plate. Allow the plate to stand for 5 minutes in iodine vapor: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.30% (1 g, 105 C, 3 hours).

Dobutamine Hydrochloride
OH H CH3

HCl
OH

N H

HO

and enantiomer C18H23NO3.HCl: 337.84 Dobutamine Hydrochloride, when dried, contains not less than 98.0 % and not more than 101.0% of dobutaime hydrochloride (C18H23NO3.HCl).
Description Dobutamine Hydrochloride is a white to very pale orange crystalline powder or grain. It is freely soluble in methanol, sparingly soluble in water or in dehydrated ethanol, and practically insoluble in ether. A solution of Dobutamine Hydrochloride (1 in 100) shows no optical rotation. Identification (1) Determine the infrared absorption spectra of Dobutamine Hydrochloride and Dobutamine Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) A solution of Dobutamine Hydrochloride (1 in 50) responds to the Qualitative Tests (2) for chloride. Melting Point

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.1 g each of Dobutamine Hydrochloride and Dobutamine Hydrochloride RS, previously dried, dissolve in exactly 10 mL each of the internal standard solution, add diluted methanol (1 in 2) to make 50 mL each, and use these solutions as the test solution and standard solution respectively. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS , of the peak area of dobutamine to that of the internal standard for the test solution and standard solution, respectively.

Amount (mg) of dobutamine hydrochloride (C18H23NO3.HCl) = amount (mg) of Dobutamine Hydrochloride RS QT

QS
Internal standard solutionA solution of salicylamide in diluted methanol (1 in 2) (1 in 125). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column 4.0 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of tartaric acid buffer solution, pH 3.0 and methanol (7:3). Flow rate: Adjust the flow rate so that the retention time of dobutamine is about 7 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, dobutamine and internal standard are eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 5 L each of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of dobutamine to that

Between 188 C and 191 C.

pH Dissolve 1.0 g of Dobutamine Hydrochloride in 100 mL of water: the pH of this solution is between 4.0 and 5.5. Purity (1) Clarity and color of solutionDissolve 0.5 g of Dobutamine Hydrochloride in 30 mL of water: the solution is clear and colorless. (2) Heavy metalsDissolve 1.0 g of Dobutamine Hydrochloride in 40 mL of water by warming, cool, and add 2 mL of dilute acetic acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 2.0 mL of standard lead solution add water to make 50 mL (not more than 20 ppm). (3) Related substancesDissolve 0.10 g of Dobutamine Hydrochloride in 10 mL of methanol, and use this solution as the test solution. Pipet 1 mL of the test solution, add methanol to make exactly 200 mL, and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer

KP 9 327
of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Domperidone
N O N H O N H Cl N N

C22H24Cl N5O2: 425.91 Domperidone, when dried, contains not less than 99.0% and not than 101.0% of domperidone (C22H24Cl N5O2).
Description Domperidone is a white powder. Domperidone is soluble in dimethylformamide, slightly soluble in ethanol and in methanol, and practically insoluble in water. Identification (1) Dissolve 5 mg of Domperidone in 3 mL of methanol, add 0.1 mL each of 10w/v% cobaltous nitrate solution and 10w/v% calcium chloride solution, mix and add 0.1 mL of 2 mol/L sodim hydroxide TS: a purple-blue color develops and precipitate is formed. (2) Determine the infrared spectrum of Domperidone and Domperidone RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 20 mg of Domperidone in 10 mL of methanol, and use this as the test solution. Separately, dissolve 20 mg of Domperidone RS in 10 mL of methanol, and use this as the standard solution (1). Dissolve 20 mg of Domperidone RS and 20 mg of Droperidole RS in 10 mL of methanol, and use this as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution, the standard solutions (1) and (2) on a plate of octadecylsilyl silica gel for thin-layer chromatography. Develop the plate with a mixture of ammonium acetate solution, dioxan and methanol (20 : 40 : 40) to a distance of about 15 cm, and air-dry the plate. Expose the plate to iodine vapor until the spots appear and examine in daylight: the principal spot in the chromatogram obtained with the test solution is similar in size and Rf value to the principal spot from the standard solution (1). The test is not valid unless the chromatogram from the standard solution (2) shows clearly separated spots. Melting Point

less and clear. (2) Heavy metals Proceed with 1.0 g of Domperidone according to Method 4, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesPrepare the solutions for the test immediately before use. Dissolve 0.1 g of Domperidone in 10 mL of dimethylformamide, and use this solution as the test solution. Pipet 1 mL of the test solution, and add dimethylformamide to make exactly 100 mL. Pipet 5 mL of this solution, add dimethylformamide to make exactly 20 mL, and use this solution as the standard solution (1). Perform the test with 10 L each of dimethylformamide (as a blank solution), the test solution, and the standard solution (1) as directed under the Liquid Chromatography according to the following conditions. Determine each peak area from each solution by the automatic integration method: the area of peaks other than the peak of domperidone from the test solution is not larger than the peak area of domperidone from the standard solution (1) (0.25 %), and the total area of all peak other than the peak of domperidone from the test solution is not larger than twice the peak area of domperidone from the standard solution (1) (0.5 %). Disregard any peak in the chromatogram obtained with the blank solution and any peak with an area less than 0.2 times the area of the peak of domperidone from the standard solution (1).

Between 244 C and 248 C.

Purity (1) Clarity and color of solutionDissolve 0.2 g in 20 mL of dimethylformamide: the solution is color-

Operating conditions Detector: An ultraviolet absorption spectrophotometer (wavelength: 280 nm). Column: A stainless steel column, 4.6 mm in inside diameter and about 100 cm in length, packed with basedeactivated octadecylsilanized silica gel for liquid chromatography (3 m in particle diameter). Column temperature: A Room temperature Mobile phase: At a flow rate of 1.5 mL per minute a mixture of ammonium acetate (5 g/L) and methanol (70 : 30), changing by linear gradient to methanol over 10 min, followed by elution with methanol for 2 min. Flow rate: 1.5 mL/minute. Equilibrate the column for at least 30 min with methanol and then equilibrate at the initial mobile phase composition (a mixture of ammonium acetate (5 g/L) and methanol (70 : 30) for at least 5 minutes. System suitability Selection of column: Dissolve 10 mg of Domperidone RS and 15 mg of Droperidol RS in 100 mL of dimethylformamide and use this solution as the standard solution (2). Proceed with 10 L of the standard solution (2) under the above operating conditions. Use a column from which the retention time for domperidone and droperidol is 6.5 minutes and 7 minutes, respectively, with the resolution between these peaks being not less than 2.0. If necessary, adjust the concentration of methanol in the mobile phase or adjust the time program for the linear gradient. Detection sensitivity: Adjust the detection sensitiv-

328 Monographs, Part I

ity so that the peak height of domperidone from 10 L of the standard solution (1) is not less than 50.0 percent of the full scale of the recording chart.
Loss on Drying Not more than 0.5% (1 g, 100 ~ 105 C, constant weight). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Domperidone, previously dried, dissolve in 50 mL of a mixture of glacial acetic acid and methyl ethyl ketone (1 : 7), and titrate with 0.1 mol/L perchloric acid (indictor: 0.2 mL of -naphtholbenzein TS) until the color changes from orange-yellow to green.

Each mL of 0.1 mol/L perchloric acid = 42.59 mg of C22H24Cl N5O2

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Domperidone Maleate
N O N H O N H Cl N CO2H N CO2H

Maleate and Domperidone Maleate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensity of absorption at the same wavenumbers. If the spectra obtained show differences, dissolve each substance in minimum volume of isopropanol, evaporate to dryness on a water-bath and record new spectra using the residues. (3) Dissolve 20 mg of Domperidone Maleate in 10 mL of methanol, and use this solution as the test solution. Separately, dissolve 20 mg of Domperidone Maleate RS in 10 mL of methanol, and use this as the standard solution (1). Dissolve 20 mg of Domperidone Maleate RS and 20 mg of Droperidole RS in 10 mL of methanol, and use this as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution, the standard solutions (1) and (2) on a plate of silica gel for thinlayer chromatography. Develop the plate with a mixture of acetate ammonium acetate solution, dioxan, and methanol (20 : 40 : 40) to a distance of about 15 cm and airdry the plate. Expose the plate with iodine vapor until spots appear and examine in day-light: the principal spot in the chromatogram obtained with the test solution corresponds in the size and the Rf value to that with the standard solution (1) and the two spots in the chromatogram obtained with the standard solution (2) are clearly separated with each other.
Purity (1) Clarity and color of solutionDissolve 0.2 g in 20 mL of dimethylformamide. The solution is clear and not darker than the mixture of 5 mL of the mixed solution of Cobaltous Chloride Colorimetric Stock Solution, Ferric Chloride Colorimetric Stock Solution, and 1 w/v% hydrochloric acid (6 : 24 : 70) and 95 mL of 1 w/v% hydrochloric acid. (2) Heavy metals Proceed with 1.0 g of Domperidone Maleate according to Method 4, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substances Prepare the solution immediately before use. Dissolve 0.1 g of Domperidone Maleate in 10 mL of dimethylformamide, and use this solution as the test solution. Pipet 1 mL of the test solution, and add dimethylformamide to make exactly 100 mL. Pipet 5 mL of this solution, add dimethylformamide to make exactly 20 mL, and use this solution as the standard solution (1). Perform the test with 10 L each of dimethylformamide (as a blank solution), the test solution, and the standard solution (1) as directed under the Liquid Chromatography according to the following conditions. Determine each peak area from each solution by the automatic integration method: the area of peaks other than the peak of domperidone maleate from the test solution is not larger than the peak area of domperidone maleate from the standard solution (1) (0.25 %), and the total area of all peak other than the peak of domperidone maleate from the test solution is not larger than twice the peak area of domperidone maleate from the standard so-

C22H24Cl N5O2C4H4O4: 541.98 Domperidone Maleate contains not less than 99.0% and not more than 101.0% of domperidone maleate (C22H24Cl N5O2C4H4O4), calculated on the anhydrous basis.
Description Domperidone Maleate occurs as a white powder. Domperidone Maleate is sparingly soluble in dimethylformamide, slightly soluble in methanol, and very slightly soluble in water or in ethanol. It shows polymorphism. Identification (1) Triturate 0.1 g of Domperidone Maleate with a mixture of 1 mL of strong sodium hydroxide solution and 3 mL of water. Shake with three quantities, each of 5 mL, of ether. Add 0.1 mL of the aqueous layer to the solution of 10 mg of resorcinol in 3 mL of sulfuric acid. Heat on a water-bath for 15 min. No color develops. To the remainder of aqueous layer add 2 mL of bromine solution. Heat on a water-bath for 15 min and then heat to boiling. Cool. Add 0.1 mL of the solution to a solution of 10 mg of resorcinol in 3 mL of sulfuric acid. Heat on a water-bath for 15 minutes. A violet color develops. (2) Determine the infrared spectra of Domperidone

KP 9 329
lution (1) (0.5 %). Disregard any peak in the chromatogram obtained with the blank solution and any peak with an area less than 0.2 times the area of the peak of domperidone maleate from the standard solution (1).

Dopamine Hydrochloride
HO HO CH2CH2NH2 HCl

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, 4.6 mm in inside diameter and about 100 cm in length, packed with basedeactivated octadecylsilyl silica gel for liquid chromatography (3 m in particle diameter). Column temperature: A room temperature Mobile phase: At a flow rate of 1.5 mL per minute a mixture of ammonium acetate (5 in 1000) and methanol (70 : 30), changing by linear gradient to methanol over 10 min, followed by elution with methanol for 2 min. Flow rate: 1.5 mL/minute. Equilibrate the column for at least 30 min with methanol and then equilibrate at the initial mobile phase composition (a mixture of ammonium acetate (5 in 1000) and methanol (70 : 30)) for at least 5 minutes. System suitability System performance: Dissolve 10 mg of Domperidone Maleate RS and 15 mg of Droperidol RS in 100 mL of dimethylformamide and use this solution as the standard solution (2). When the procedure is run with 10 L of this solution, as directed under the above operating conditions, Domperidone maleate and Droperidol are eluted in this order with the resolution between their peaks being not less than 2. Detection sensitivity: Adjust the detection sensitivity so that the peak height of domperidone maleate from 10 L of the standard solution (1) is not less than 50.0 percent of the full scale.
Loss on Drying Not more than 0.5% (1 g, 105 C, constant mass). Residue on Ignition Not more than 0.1% (1 g) Assay Weigh accurately 0.4 g of Domperidone Maleate, previously dried, dissolve in 50 mL of a mixture of glacial acetic acid. Titrate with 0.1 mol/L perchloric acid until the color changes from orange-yellow to green (indicator: 0.2 mL of naphtholbenzein solution TS).

C8H11NO2HCl: 189.64 Dopamine Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of dopamine hydrochloride (C8H11NO2HCl).
Description Dopamine Hydrochloride occurs as a white crystal or crystalline powder. Dopamine Hydrochloride is freely soluble in water or in formic acid, sparingly soluble in ethanol. Melting pointAbout 248 C (with decomposition). Identification (1) Determine the absorption spectra of solutions of Dopamine Hydrochloride and Dopamine Hydrochloride RS in 0.1 mol/L hydrochloric acid TS (1 in 25000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Dopamine Hydrochloride and Dopamine Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers (3) A solution of Dopamine Hydrochloride (1 in 50) responds to the Qualitative Tests (1) for chloride. pH Dissolve 1.0 g of Dopamine Hydrochloride in 50mL of water: the pH of this solution is between 4.0 and 5.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Dopamine Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 0.8 g of Dopamine Hydrochloride. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.021%). (3) Heavy metalsProceed with 1.0 g of Dopamine Hydrochloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicProceed with 1.0 g of Dopamine Hydrochloride according to Method 1 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.10 g of Dopamine Hydrochloride in 10 mL of water and use this solution as the test solution. Pipet 1.0 mL of the test solution, add water to make exactly 250 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solutions as directed under the

Each mL of 0.1 mol/L perchloric acid = 54.20 mg of C26H28Cl N5O6

Packaging and Storage Preserve in well-closed containers, protected from light.

330 Monographs, Part I

Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of cellulose with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (16 : 8 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly a solution of ninhydrin in acetone (1 in 50) on the plate and heat at 90 C for 10 minutes: the spots other than the principal spot form the test solution are not more intense than that from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Dopamine Hydrochloride, previously dried, dissolve in 5 mL of formic acid, add 15.0 mL of 0.1 mol/L perchloric acid VS and heat on a water-bath for 15 minutes. After cooling, add 50 mL of glacial acetic acid and titrate the excess perchloric acid with 0.1 mol/L sodium acetate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

ether.
Identification (1) Determine the absorption spectra of solutions of Doxapram Hydrochloride Hydrate and Doxapram Hydrochloride Hydrate RS (1 in 2500) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectrum of Doxapram Hydrochloride Hydrate and Doxapram Hydrochloride Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Doxapram Hydrochloride Hydrate (1 in 50) responds to the Qualitative Tests for chloride. Melting Point

Between 218 C and 222 C.

pH Dissolve 1.0 g of Doxapram Hydrochloride Hydrate in 50 mL of water: the pH of this solution is between 3.5 and 5.0. Purity (1) Clarity and color of solution Dissolve 1.0 g of Doxapram Hydrochloride Hydrate in 50 mL of water: the solution is clear and colorless (2) SulfatePerform the test with 1.0 g of Doxapram Hydrochloride Hydrate. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (3) Heavy metalsProceed with 2.0 g of Doxapram Hydrochloride Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicProceed with 1.0 g of Doxapram Hydrochloride Hydrate according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.5 g of Doxapram Hydrochloride Hydrate in 10 mL of methanol and use this solution as the test solution. Pipet 3.0 mL of the test solution and add methanol to make exactly 100 mL. Pipet 5.0 mL of this solution, add methanol to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 6 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform, formic acid, ethyl formate and methanol (8 : 3 : 3 : 2) to a distance of about 10 cm and air-dry the plate. Allow the plate to stand in iodine vapor: the spots other than the principal spot from the test solution are not more intense than that from the standard solution. Water Between 3.5% and 4.5% (0.5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.3% (1 g).

Each mL of 0.1 mol/L perchloric acid VS = 18.964 mg of C8H11NO2HCl

Packaging and Storage tight containers.

Preserve in light-resistant,

Doxapram Hydrochloride Hydrate

CH2CH3 N O

HCl O N CH2CH2 H

H2O

and enantiomer C24H30N2O2HClH2O: 432.98 Doxapram Hydrochloride Hydrate contains not less than 98.0% and not more than 101.0% of doxapram hydrochloride (C24H30N2O2HCl: 414.97), calculated on the anhydrous basis.
Description Doxapram Hydrochloride occurs as a white crystal or crystalline powder. Doxapram Hydrochloride is freely soluble in methanol or in glacial acetic acid, sparingly soluble in water, in ethanol, or in acetic anhydride and practically insoluble in

KP 9 331
Assay Weigh accurately about 0.8 g of Doxapram Hydrochloride Hydrate, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

bicin Hydrochloride is used in a sterile preparation.

Bacterial Endotoxins Less than 2.50 EU per mg of doxorubicin hydrochloride, when Doxorubicin Hydrochloride is used in a sterile preparation. Histamine It meets the requirement, when Doxorubicin Hydrochloride is used in a sterile preparation. Weigh appropriate amount of Doxorubicin Hydrochloride, dissolve in water, make the solution so that each mL contains 2.0 mg, use the solution as the test solution, and use 0.5 mL of the solution for the test. Water Not more than 3.0 % (0.3 g, volumetric titration, direct titration). Assay Weigh accurately an amount of Doxorubicin Hydrochloride and Doxorubicin Hydrochloride RS, equivalent to about 10 mg (potency), dissolve each in water to make exactly 25 mL. Pipet 5 mL each of these solutions, add water to make exactly 100 mL, and use these solutions as the test solution and the standard solution, respectively. Determine the absorbances at 495 nm, AT and AS , of the test solution and the standard solution as directed under Ultraviolet-visible Spectrophotometry.

Each mL of 0.1 mol/L perchloric acid VS = 41.50 mg of C24H30N2O2HCl

Packaging and Storage Preserve in tight containers.

Doxorubicin Hydrochloride
O OH O OH OH

O H3C

OH

H3C

HCl
O NH2 OH

C27H29NO11HCl: 579.98 Doxorubicin Hydrochloride is the hydrochloride of a derivative of daunorubicin. Doxorubicin Hydrochloride contains not less than 900 g (potency) of per mg of doxorubicin hydrochloride (C27H29NO11HCl), calculated on the anhydrous basis.
Description Doxorubicin Hydrochloride is a red-orage crystalline powder. Doxorubicin Hydrochloride is sparingly soluble in water, slightly soluble in methanol, very slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Dissolve 1 ~ 2 mg of Doxorubicin Hydrochloride in 5 mL of 1 mol/L sodium hydroxide: a blue-purple color develops. (2) Determine the absorption spectrum of a solution of Doxorubicin Hydrochloride in methanol (1 in 100000) as directed under Ultraviolet-visible Spectrophotometry, it exhibits maxima between 231 nm and 235 nm, and between 250 nm and 254 nm, and between 286 nm and 290 nm, and between 477 nm and 481 nm, and between 493 nm and 497 nm, and between 527 nm and 531 nm. pH The pH of a solution obtained by dissolving 50 mg of Doxorubicin Hydrochloride in 10 mL of water is between 4.0 and 5.5. Absorbance (495 nm): 200 ~ 230 (10 mg calculated on the anhydrous basis, methanol, 500 mL). Sterility Test It meets the requirement, when Doxoru1% E1 cm

Amount [g (potency)] of Doxorubicin Hydrochloride (C27H29NO11HCl) = Amount [g (potency)] of Doxorubicin Hydrochloride RS

AT AS

Packaging and Storage Preserve in tight containers.

Doxycycline Hyclate Hydrate

OH O OH OH O O NH2 HCl OH H H CH3 H OH N(CH3)2

C2H6O H2O

(C22H24N2O8HCl)2C2H6OH2O: 1025.88 Doxycycline Hyclate Hydrate contains not less than 800 g (potency) and not more than 920g (potency) per mg of doxycycline (C22H24N2O8: 444.44), calculated on the anhydrous basis and corrected by the amount of ethanol.
Description Doxycycline Hyclate Hydrate is yellow to dark yellow crystal or crystalline powder. Doxycycline Hyclate Hydrate is freely soluble in water and in methanol, slightly soluble in ethanol, and practically insoluble in ether.

332 Monographs, Part I

Identification (1) To 1 mg of Doxycycline Hyclate Hydrate add one drop of sulfuric acid: a yellow color develops. (2) Dissolve 10 mg of Doxycycline Hyclate Hydrate in 10 mL of water, and add silver nitrate TS: a white turbidity is produced. (3) To 2 mL of the solution of Doxycycline Hyclate Hydrate in water (1 in 20) add two drops of chromic acid sulfuric acid TS, mix well, and heat: a smell of acetaldehyde develops. (4) Determine the infrared spectra of Doxycycline Hyclate Hydrate and Doxycycline Hyclate Hydrate RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Crystalliity Test It meets the requirement. Ethanol Weigh accurately about 0.1 g of Doxycycline Hyclate Hydrate, dissolve in the internal standard solution to make exactly 10 mL, and use this solution as the test solution. Separately, weigh accurately about 0.4 g of dehydrated ethanol, add the internal standard solution to make exactly 100 mL. Pipet 1 mL of this solution, add the internal standard solution to make exactly 10 mL, and use this solution as the standard solution. Perform the test with 1 L each of the test solution and the standard solution as directed under Gas Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of ethanol to that of the internal standard:the amount of ethanol is not less than 4.3 % and not more than 6.0 %.

tween 2.0 and 3.0.

Water Not less than 1.4 % and not more than 2.8 % (0.3 g, volumetric titration, direct titration). Assay DoxycyclineWeigh accurately an amount of Doxycycline Hyclate Hydrate and Doxycycline Hyclate Hydrate RS, equivalent to about 0.1 g (potency), dissolve each in water and make exactly 100 mL, and use these solutions as the test solution and the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT and AS, of doxycycline.

Amount [g (potency)] of doxycycline (C22H24N2O8) = Amount [g (potency)] of Doxycycline RS

AT AS

Amount [%] of ethanol = (Amount [mg] of ethanol / Amount [mg] of Doxycycline Hyclate Hydrate)
QT QS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm) Column: A stainless steel column, about 3.0 mm in inside diameter and about 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography(10 m in particle diameter) with a guard column, about 4.6 mm in inside diameter and 30 mm in length, packed with octadecylsilanized silica gel for liquid chromatography. Mobile phase: Adjust the pH of a mixture of 0.1 mol/L sodium phosphate solution, ethanol and N,Ndimethyl-n-octylamine (450:500:3) to between 6.2 and 6.3 with phosphoric acid Flow rate: 1.5 mL per minute.
Packaging and Storage Preserve in tight containers.

Internal standard solutionA solution of 1-propanol (1 in 2000). Operating conditions Detector: A hydrogen flame-ionization detector. Column: A glass column, about 3.2 mm in inside diameter and about 1.5 m in length, packed with porous ethylvinylbenzene-divinylbenzene copolymer for gas chromatography (0.0075 m in average pore size, 500 ~ 600 m2/g in specific surface area 150 ~ 180 m in particle diameter) Column temperature: A contant temperature of about 135 o C . Carrier gas: Nitrogen Flow rate: Adjust the flow rate so that the retention time of ethanol is about 5 minutes.
pH The pH of a solution obtained by suspending 0.1 g of Doxycycline Hyclate Hydrate in 10 mL of water is be-

Doxycycline Hydrate
OH O OH OH NH2 H2O OH H H CH3 H OH N(CH3)2 O O

C22H24N2O8H2O: 462.45

Doxycycline Hydrate contains not less than 880 g (potency) and not more than 980 g (potency) per mg of doxycycline (C22H24N2O8: 444.44), calculated on the anhydrous basis
Description Doxycycline Hydrate is yellow crystalline powder. Doxycycline Hydrate is sparingly soluble in ethanol,

KP 9 333
very slightly soluble in water, and practically insoluble in chloroform or in ether. Doxycycline Hydrate dissolves in dilute acid solution or alkaline solution.
Identification Determine the infrared spectra of Doxycycline Hydrate and Doxycycline RS, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Crystalliity Test It meets the requirement. pH The pH of a suspension obtained by suspending 0.1 g of Doxycycline Hydrate in 10 mL of water is between 5.0 and 6.5. Water Not less than 3.0 % and not more than 4.6 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately an amount of Doxycycline Hydrate and Doxycycline RS, equivalent to about 0.1 g (potency), dissolve each in 24 mL of 0.1 mol/L hydrochloric acid TS and mobile phase to make exactly 100 mL, and use these solutions as the test solution and the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT and AS , of doxycycline.

Drostanolone Propionate
O H3C O C H H H3C H O H H H3C H CH2CH3

C23H36O3: 360.53 Drostanolone Propionate, when dried, contains not less than 97.0% and not more than 103.0% of drostanolone propionate (C23H36O3).
Description Drostanolone Propionate is a white to yellowish white, crystalline powder and is odorless or has a faint, characteristic odor. Drostanolone Propionate is very soluble in chloroform, freely soluble in ether, sparingly soluble in ethanol and practically insoluble in water. Identification (1) Dissolve 20 mg of Drostanolone Propionate in 1 mL of ethanol, add 1 mL of alkaline hydroxylamine TS, allow to stand for 10 minutes and add 1 mL of hydrochloric acid-ethanol TS and 1 mL of ferric chloride TS: a dark red color is observed. (2) Take 10 mg of Drostanolone Propionate, add 10 mL of freshly prepared vanillin-sulfuric acid TS and dissolve by heating on a water-bath for 5 minutes: a redpurple color is observed. (3) Determine the infrared spectra of Drostanolone Propionate and Drostanolone Propionate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, dissolve each Drostanolone Propionate and Drostanolone Propionate RS in chloroform, evaporate to dryness and repeat the test on the residues. Specific Optical Rotation [ ]20 D : Between + 22 and + 28 (after drying, 0.2 g, chloroform, 10 mL, 100 mm). Melting Point

Amount [g (potency)] of doxycycline (C22H24N2O8) = Amount [g (potency)] of doxycycline RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm) Column: A stainless steel column, about 3.0 mm in inside diameter and about 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) with a guard column, about 4.6 mm in inside diameter and 30 mm in length, packed with octadecylsilanized silica gel for liquid chromatography. Mobile phase: Adjust the pH with phosphoric acid of a mixture of 0.1 mol/L sodium phosphate solution, methanol and N,N-dimethyl-n-octylamine (450:500:3) to between 6.2 and 6.3 with phosphoric acid Flow rate: 1.5 mL per minute.
Packaging and Storage tight containers.

Between 129 C and 133 C

Preserve in lisht-resistant,

Purity (1) Clarity and color of solutionDissolve 0.20 g of Drostanolone Propionate in 10 mL of chloroform: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Drostanolone Propionate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 50 mg of Drostanolone Propionate in 5 mL of chloroform and use this so-

334 Monographs, Part I

lution as the test solution. Pipet 1.0 mL of the test solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of heptane and ethyl acetate (9 : 1) to a distance of about 10 cm and air-dry the plate. Develop the plate with the same solvent again and airdry the plate. Spray vanillin-sulfuric acid TS on the plate and heat at 105 C for 5 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 50 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 25 mg of Drostanolone Propionate and Drostanolone Propionate RS, previously dried, dissolve each in exactly 5 mL of the internal standard solution, add chloroform to make 10 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 2 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of drostanolone propionate to that of the internal standard for the test solution and the standard solution, respectively.

Packaging and Storage tight containers.

Preserve in light-resistant,

Drostanolone Propionate Injection

Drostanolone Propionate Injection is an oily solution for injection. Drostanolone Propionate Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of drostanolone propionate (C23H36O3: 360.53).
Method of Preparation Prepare as directed under Injections, with Drostanolone Propionate. Description Drostanolone Propionate Injection is a clear, colorless to pale yellow, oily liquid. Identification Take a volume of Drostanolone Propionate Injection, equivalent to 50 mg of Drostanolone Propionate according to the labeled amount, add 20 mL of diluted methanol (4 in 5), shake for 5 minutes and centrifuge. Evaporate 5 mL of the supernatant liquid on a water-bath to dryness under a current of air. Dissolve the residue in 2 mL of chloroform and use this solution as the test solution. Separately, dissolve 5 mg of Drostanolone Propionate RS in 1 mL of chloroform and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of heptane and ethyl acetate (9 : 1) to a distance of about 12 cm and air-dry the plate. Spray evenly vanillin-sulfuric acid TS on the plate and heat at 105 C for 5 minutes: the principal spot from the test solution and the spot from the standard solution show the same color tone and Rf value. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Amount (mg) of drostanolone propionate(C23H36O3) = amount (mg) of Drostanolone Propionate RS

QT QS

Internal standard solutionA solution of Cholesterol in chloroform (1 in 200). Operating conditions Detector: A hydrogen flame-ionization detector. Column: A glass column, about 3 mm in inside diameter and about 1 m in length, packed with siliceous earth for gas chromatography (125 m to 150 m in particle diameter) coated with 50% phenylmethyl silicon polymer for gas chromatography at the ratio of 3%. Column temperature: A constant temperature of about 260 C. Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of drostanolone propionate is about 8 minutes. Selection of Column: Proceed with 2 L of the standard solution as directed under the above operating conditions, and calculate the resolution. Use a column giving elution of drostanlone propionate and the internal standard in this order with the resolution between their peaks being not less than 3.0.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay (1) Chromatographic tubeUse a glass tube, about 20 mm in inside diameter and about 23 cm in length. Place a small amount of absorbent cotton in the bottom of the tube and put sea sand, 5 mm in height on it. (2) Chromatographic columnMoisten 10 g of 4% hydrated alumina neutral thoroughly with 15 to 20 mL of

KP 9 335
hexane and mix gently. Wash with hexane down to the chromatographic tube and pack by flowing out the solution. Place sea sand, 5 mm in height on it and fill with hexane up to the surface of sea sand. (3) Standard solutionWeigh accurately about 25 mg of Drostanolone Propionate RS, previously dried in a desiccator (in vacuum, P2O5, 50 C) for 2 hours and dissolve in exactly 10 mL of the internal standard solution. (4) Test stock solutionPipet a volume of Drostanolone Propionate Injection, equivalent to about 50 mg of Drostanolone Propionate (C23H36O3) and add hexane to make exactly 20 mL. (5) ProcedurePipet 5 mL of the test stock solution into the previously prepared chromatographic column and elute to the surface of sea sand. Wash the inner side of the chromatographic tube with two 5 mL volumes of hexane, elute to the surface of sea sand, then elute 120 mL of a mixture of hexane and ethyl acetate (50 : 1) at the rate of 7 to 8 mL per minute and discard the effluent solution. Elute 150 mL of a mixture of hexane and ethyl acetate (20 : 1) at the rate of 7 to 8 mL per minute and collect the effluent solution. After elution, wash the bottom part of the chromatographic tube with a small volume of hexane, combine the washing with the effluent solution and evaporate the solvent at below 30 C. To the residue, add exactly 5 mL of the internal standard solution and use this solution as the test solution. When the procedure is run with 2 L each of the test solution and the standard solution as directed in the Assay under Drostanolone Propionate. Amount (mg) of drostanolone propionate (C23H36O3) = amount (mg) of Drostanolone Propionate RS
QT 2 QS

Description Dydrogesterone is a white to pale yellowish white crystal or crystalline powder and is odorless. Dydrogesterone is freely soluble in chloroform, soluble in acetonitrile, sparingly soluble in methanol or in ethanol, slightly soluble in ether and practically insoluble in water. . Identification (1) Take 5 mg of Dydrogesterone, add 5 mL of p-anisaldehyde-acetic acid TS and 2 to 3 drops of sulfuric acid and heat in a water-bath for 2 minutes: an orange-red color is observed . (2) Determine the absorption spectra of solutions of Dydrogesterone and Dydrogesterone RS in methanol (1 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Dydrogesterone and Dydrogesterone RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between - 47 and 50 (after drying, 0.1 g, chloroform, 10 mL, 100 mm). Melting Point Between 167 C and 171 C Purity (1) Heavy metalsProceed with 1.0 g of Dydrogesterone according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 10 mg of Dydrogesterone in 200 mL of the mobile phase and use this solution as the test solution. Pipet 1.0 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of these solutions by the automatic integration method: the total area of peaks other than the peak of Dydrogesterone from the test solution is not larger than that of Dydrogesterone from the standard solution.

Internal standard solutionA solution of Cholesterol in chloroform (1 in 400).

Packaging and Storage hermetic containers.

Preserve in light-resistant,

Dydrogesterone
O H3C C CH3 H H3C H

H O

C21H28O2: 312.45 Dydrogesterone when dried, contains not less than 98.0% and not more than 102.0% of dydrogesterone (C21H28O2).

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of water, ethanol and acetonitrile (53 : 26 : 21). Flow rate: Adjust the flow rate so that the retention time of Dydrogesterone is about 12 minutes. System suitability

336 Monographs, Part I

System performance: Dissolve 1 mg of Dydrogesterone and Progesterone in 20 mL of the mobile phase. When the procedure is run with 10 L each of these solutions, as directed under the above operating conditions, Dydrogesterone and Progesterone are eluted in this order with the resolution between their peaks being not less than 8.0. Wavelength is 265 nm. Detection sensitivity: Adjust the detection sensitivity so that the peak height of Dydrogesterone obtained from 10 L of the standard solution is between 5 mm and 10 mm. Time span of measurement: About twice as long as the retention time of Dydrogesterone after the solvent peak.
Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 24 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 50 mg of Dydrogesterone, previously dried and dissolve in methanol to make exactly 100 mL. Pipet 1.0 mL of this solution and add methanol to make exactly 100 mL. Determine the absorbance, A, of this solution at the wavelength of a maximum absorption at about 286 nm as directed under the Ultraviolet-visible Spectrophotometry.

Dissolution Test Take 1 tablet of Dydrogesterone Tablets and perform the test using 900 mL of water at 50 revolutions per minute according to Method 2 under the Dissolution Test. After 30 minutes from the start of the test, take 20 mL or more of the dissolved solution and filter. Discard the first 10 mL of the filtrate and use the subsequent as the test solution. Separately, weigh accurately about 50 mg of Dydrogesterone RS, previously dried in a desiccator (in vacuum, P2O5) for 24 hours and dissolve in methanol to make exactly 100 mL. Pipet 1.0 mL of this solution, add water to make exactly 100 mL and use this solution as the standard solution. Determine the absorbance, AT and AS , of the test solution and the standard solution, respectively, at 296 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Dydrogesterone Tablets in 30 minutes is not less than 80%.

Dissolution rate (%) with respect to the labeled amount of dydrogesterone (C21H28O2) = W S
AT 1 9 AS C

WS : Amount (mg) of Dydrogesterone RS

C: Labeled amount (C21H28O2) in 1 tablet.
(mg) of dydrogesterone

Amount (mg) of dydrogesterone (C21H28O2) A = 845 100000

Packaging and Storage Preserve in tight containers.

Uniformity of Dosage Units It meets the requirement Assay Weigh accurately and powder not less than 20 Dydrogesterone Tablets. Weigh accurately a portion of the powder, equivalent to about 10 mg of dydrogesterone (C21H28O2), and shake well with 50 mL of methanol and add methanol to make exactly 100 mL. Filter this solution, discard the first 20 mL of the filtrate, pipet the subsequent 5.0 mL, add methanol to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Dydrogesterone RS previously dried in a desiccator (in vacuum, P2O5) for 24 hours, proceed in the same manner as preparation of the test solution and use this solution as this standard solution. Determine the absorbance, AT and AS , of the test solution and the standard solution, respectively, at 286 nm as directed under the Ultraviolet-visible Spectrophotometry.

Dydrogesterone Tablets
Dydrogesterone Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of dydrogesterone (C21H28O2: 312.45).
Method of Preparation Prepare as directed under Tablets, with Dydrogesterone. Identification (1) Take a portion of powdered Dydrogesterone Tablets, equivalent to 50 mg of Dydrogesterone according to the labeled amount and add 50 mL of methanol, shake well and filter. Evaporate 5 mL of the filtrate on a water-bath to dryness. Proceed with the residue as directed in the Identification (1) under Dydrogesterone. (2) Take 1 mL of the filtrate obtained in (1) and add methanol to make exactly 200 mL. Determine the absorption spectra of this solution and a solution of Dydrogesterone RS in methanol (1 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths.

Amount (mg) of dydrogesterone (C21H28O2) = amount (mg) of Dydrogestrone RS

AT AS

Packaging and Storage Preserve in tight containers.

KP 9 337
than the area of the principal peak with the standard solution (2) (0.1%), the peak area of the other related substances are not more than that of the principal peak from the standard solution (2) (0.1%). total area of all related substances is not more than 4 times the area of the principal peak from the standard solution (2) (0.4%). Disregard the peak being not more than 0.5 times the area of the principal peak with the standard solution (2).

Ebastine
H3C CH3 CH3

N O O

C32H39NO2 : 469.66 Ebastine contains ano less than 99.0% and not more than 101.0% of ebastine (C32H39NO2), calculated on the anhydrous basis.
Description Ebastine is a white crystalline powder. Ebastine is very soluble in dichloromethane, slightly soluble in methanol, and practically insoluble in water. Melting pointAbout 86 C. Identification Determine the infrared spectra of Ebastine and Ebastine RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) SulfatesWeigh 2.5 g of Ebastine, add 25 ml of dilute nitric acid, boil under reflux for 10 minutes, cool and filter. Rinse the filter paper and the residue with adequate amount of water, combine with filtrate, add water to make exactly 50 mL and perform the test using this solution as the test solution. Prepare the control solution mixing 0.53 mL of 0.005 mol/L sulfuric acid VS and 25 mL of dilute nitric acid and adding water to make 50 mL. (0.01%). (2) Related substancesKeep the test solution and the standard solution of this test protected from light. Dissolve 0.125 g of Ebastine in mobile phase to make 50 mL and use this solution as the test solution. Dissolve 5.0 mg of Ebastine Related Substance III [4(Diphenylmethoxy)piperidine] RS and 5.0 mg of Ebastine Related Substance IV {1-[4-(1,1-Dimethylethyl) phenyl]-4-(4-hydroxypiperidine-1-yl)butan-1-one] RS in mobile phase to make exactly 20 mL, pipet 1.0 mL of this solution, add mobile phase to make exactly 100 mL and use this solution as the standard solution (1). To 1.0 mL of the test solution add mobile phase to make exactly 100 mL, pipet 1.0 mL of this solution, add mobile phase to make exactly 10 mL and use this solution as the standard solution (2). Perform the test with exactly 10 L each of the test solution and the standard solutions (2) as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: any peak area of related substances I, II, III, IV, V, VI, and VII in the chromatogram obtained with the test solution is not more

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with nitrile silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: A mixture of phosphate buffer and acetonitrile (65 : 35). Adjust the percentage of acetonitrile to between 30% and 40% so that the retention time of ebastine is about 110 min. Flow rate: 1 mL/minute System suitability System performance: When the procedure is run with 10 L of the test solution under the above operating condition, relative retention time of related substances I, II, III, IV, V, VI, and VII with reference to ebastine are 0.04, 0.05, 0.20, 0.22, 0.42, 0.57 and 1.14, respectively. When the procedure is run with 10 L of the standard solution (1) under the above operating condition, the resolution between the peaks of related substance IV and related substance III is not less than 2.0. Run time: 1.4 times the retention time of ebastine. Phosphate bufferAdjust pH of 0.06w/v% phosphoric acid with 4w/v% sodium hydroxide to pH 5.0.
Water Not more than 0.5% (0.5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1.0 g). Assay Weigh accurately about 0.35 g of Ebastine, add 50 ml of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 46.97 mg of C32H39NO2.

Packaging and Storage well-closed containers.

Preserve in light-resistant,

338 Monographs, Part I

(3) Related substancesDissolve 0.20 g of Ecothiopate Iodide in 10 mL of methanol and use this solution as the test solution. Pipet exactly 3 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test and the standard solution on a plate of cellulose for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (4 : 2 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly Dragendorffs TS for spraying on the plate: any spot other than the principal spot from the test solution are not more intense than that from the standard solution.
Loss on Drying Not more than 1.0% (1 g, in vacuum, P2O5, 50 C, 3 hours). Assay Weigh accurately about 0.125 g of Ecothiopate Iodide and dissolve in water to make exactly 100 mL. Pipet exactly 10 mL of this solution, add 30 mL of water, then add exactly 10 mL of phosphate buffer solution, pH 12, stopper the container and allow to stand at 25 3 C for 20 minutes. To this solution, add quickly 2 mL of glacial acetic acid and titrate with 0.002 mol/L iodine VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform the test in the same manner without phosphate buffer solution, pH 12 and make any necessary correction.

Ecothiopate Iodide
O CH3CH2O CH3CH2O P SCH2CH2 N CH3 CH3 I CH3

C9H23INO3PS: 383.23 Ecothiopate Iodide contains not less than 95.0% and not more than 101.0% of ecothiopate iodide (C9H23INO3PS), calculated on the dried basis.
Description Ecothiopate Iodide is a white crystal or crystalline powder. Ecothiopate Iodide is very soluble in water, freely soluble in methanol, slightly soluble in ethanol, and practically insoluble in ether. Identification (1) Dissolve 0.1 g of Ecothiopate Iodide in 2 mL of water, and add 1 mL of nitric acid: a brown precipitate is produced. To 1 drop of the turbid solution containing this precipitate, add 1 mL of hexane and shake: a light red color is observed in hexane. (2) Heat the suspension of the precipitate obtained in (1) until it becomes colorless, cool, add 10 mL of water and use this solution as the test solution. 2 mL of the test solution responds to the Qualitative Tests (2) for phosphate. (3) 2 mL of the test solution obtained in (2) responds to the Qualitative Tests for sulfate. pH Dissolve 0.1 g of Ecothiopate Iodide in 40 mL of water: the pH of this solution is between 3.0 and 5.0. Melting Point

Each mL of 0.002 mol/L iodine VS = 1.5329 mg of C9H23INO3PS

Packaging and Storage Preserve in light-resistant, tight containers. Store at not more than 0 C.

Between 116 C and 122 C.

Purity (1) Clarity and color of solutionDissolve 0.5 g of Ecothiopate Iodide in 5 mL of water: the solution is clear and colorless. (2) Heavy metalsTake 1.0 g of Ecothiopate Iodide in a Kjeldahl flask, add 5 mL of nitric acid and 2 mL of sulfuric acid, put a small funnel on the mouth of the flask and heat carefully until white fumes are evolved. After cooling, add 2 mL of nitric acid and heat. Repeat this procedure twice, add several 2 mL volumes of strong hydrogen peroxide water and heat until the solution becomes colorless and white fumes are evolved. After cooling, transfer the solution together with a small volume of water to a Nessler tube and add water to make about 20 mL. Adjust the solution with strong ammonia water and ammonia TS to a pH between 3.0 and 3.5, add water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution as follows: proceed in the same manner as the preparation of the test solution and add 2.0 mL of standard lead solution and water to make 50 mL (not more than 20 ppm).

Edrophonium Chloride
CH3 N CH3 HO C2H5 Cl

C10H16ClNO: 201.69 Edrophonium Chloride, when dried, contains not less than 98.0% and not more than 101.0% of edrophonium chloride (C10H16ClNO).
Description Edrophonium Chloride is a white crystal or crystalline powder and is odorless. Edrophonium Chloride is very soluble in water, freely soluble in ethanol or in glacial acetic acid, and practically insoluble in acetic anhydride or in ether. Edrophonium Chloride is hygroscopic.

KP 9 339
Edrophonium Chloride is gradually colored by light.
Identification (1) Take 5 mL of a solution of Edrophonium Chloride (1 in 100) and add 1 drop of ferric chloride TS: a light red-purple color is observed. (2) Determine absorption spectra of solutions of Edrophonium Chloride and Edrophonium Chloride RS, in 0.1 mol/L hydrochloric acid TS (1 in 20000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) A solution of Edrophonium Chloride (1 in 50) responds to the Qualitative Tests for chloride. Melting Point composition).

rection. Each mL of 0.1 mol/L perchloric acid VS = 20.169 mg of C10H16ClNO

Packaging and Storage tight containers.

Preserve in light-resistant,

Edrophonium Chloride Injection

Edrophonium Chloride Injection is an aqueous solution for injection. Edrophonium Chloride Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of edrophonium chloride (C10H16ClNO: 201.69).
Method of Preparation Prepare as directed under Injections, with Edrophonium Chloride. Description Edrophonium Chloride Injection is a clear, colorless liquid. Identification (1) Take a volume of Edrophonium Chloride Injection, equivalent to 40 mg of Edrophonium Chloride according to the labeled amount, add 4 mL of barium nitrate TS, shake and filter. Proceed with the filtrate as directed in the Identification (1) under Edrophonium Chloride. (2) Determine the absorption spectrum of the test solution obtained in the Assay as directed under the Ultraviolet-visible Spectrophtometry: it exhibits a maximum between 272 nm and 276 nm. pH Between 6.5 and 8.0. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Between 166 C and 171 C (with de-

pH Dissolve 1.0 g of Edrophonium Chloride in 10 mL of water: the pH of this solution is between 3.5 and 5.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Edrophonium Chloride in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Edrophonium Chloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Edrophonium Chloride according to Method 1 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.50 g of Edrophonium Chloride in 10 mL of ethanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add ethanol to make exactly 100 mL. Pipet exactly 3 mL of this solution, add ethanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of methanol, chloroform and strong ammonia water (16 : 4 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than that from the standard solution. Loss on Drying Not more than 0.2% (1 g, in vacuum, P2O5, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Edrophonium Chloride, previously dried, and dissolve in 100 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3). Titrate with 0.1 mol/L perchloric acid VS (potentiometic titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary cor-

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Perform this procedure without exposure to daylight, using light-resistant containers. Take exactly a volume of Edrophonium Chloride Injection, equivalent to about 50 mg of Edrophonium Chloride, place in a chromatographic column prepared by pouring 10 mL of weakly basic DEAE-bridged dextran anion exchanger (C1 type) (50 m to 150 m in particle diameter) into a chromatographic tube, about 2 cm in inside diameter and about 10 cm in length, add 25 mL of water and elute at

340 Monographs, Part I

the flow rate of 1 to 2 mL per minute. Wash the column with two 25 mL volumes of water at the flow rate of 1 to 2 mL per minute. Combine the washings with above effluent solutions and add water to make exactly 100 mL. Pipet exactly 10 mL of this solution and add 10 mL of phosphate buffer solution, pH 8.0 and 5 g of sodium chloride. Wash this solution with four 20 mL volumes of a mixture of ether and hexane (1 : 1), collect the water layer, add 0.1 mol/L hydrochloric acid TS to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Edrophonium Chloride RS, previously dried in a desiccator (in vacuum, P2O5) for 3 hours and dissolve in water to make exactly 100 mL. Pipet exactly 10 mL of this solution and prepare the standard solution in the same manner as the test solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 273 nm as directed under the Ultraviolet-visible Spectrophotometry. Amount (mg) of edrophonium chloride (C10H16ClNO) = amount (mg) of Edrophonium Chloride RS
Packaging and Storage hermetic containers.
AT AS

Preserve in light-resistant,

Constituent Amino Acids Put about 1 mg of Elcatonin in a test tube for hydrolysis, add phenol-hydrochloric acid TS to dissolve, replace the air inside with Nitrogen, seal the tube under reduced pressure, and heat at 110 2 C for 24 hours. After cooling, open the tube, evaporate the hydrolyzate to dryness under reduced pressure, dissolve the residue in 1 mL of 0.02 mol/L hydrochloric acid TS, and use this solution as the test solution. Separately, weigh exactly 1.33 mg of L-aspartic acid, 1.19 mg of L-threonine, 1.05 mg of L-serine, 1.47 mg of L-glutamic acid, 1.15 mg of L-proline, 0.75 mg of glycine, 0.89 mg of L-alanine, 1.17 mg of L-valine, 1.89 mg of L-tyrosine, 1.83 mg of L-lysine hydrochloride, 2.10 mg of Lhistidine hydrochloride monohydrate and 2.11 mg of Larginine hydrochloride, dissolve them in 0.02 mol/L hydrochloric acid TS to make exactly 50 mL, and use this solution as the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as the following conditions: 14 peaks of amino acids appear on the chromatogram obtained from the test solution, and their respective molar ratios against alanine are 1.7 2.2 for aspartic acid, 3.5 4.2 for threonine, 2.4 3.0 for serine, 2.7 3.2 for glutamic acid, 1.7 2.2 for proline, 2.7 3.2 for glycine, 1.6 2.2 for valine, 0.8 1.2 for 2-aminosuberic acid, 4.5 5.2 for leucine, 0.7 1.2 for tyrosine, 1.7 2.2 for lysine, 0.8 1.2 for histidine and 0.7 1.2 for arginine.

Elcatonin
H Val O Leu Gly Lys Leu Ser

Ser O

Asn

Leu

Ser

Thr

NH

Gln

Glu

Leu

His

Lys

Leu

Gln

Thr

Pro

Arg

Thr

Asp

Val

Gly

Ala

Gly

Thr

Pro

NH2

C148H244N42O47 : 3363.77 Elcatonin contains not less than 5000 Elcatonin Units and not more than 7000 Elcatonin Units per 1 mg of peptide, calculated on the dehydrated and de-acetic acid bases.
Description Elcatonin is a white powder. Elcatonin is very soluble in water, freely soluble in ethanol, and practically insoluble in acetonitrile. Elcatonin is hygroscopic. The pH of its solution (1 in 500) is between 4.5 and 7.0. Identification Dissolve 5 mg each of Elcatonin and Elcatonin RS in 5 mL of water. Determine the absorption spectra of both solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths.

Operating conditions Detector: A visible spectrophotometer (wavelength: 440 nm and 570 nm). Column: A stainless steel column about 4 mm in inside diameter and about 8 cm in length, packed with strongly acidic ion-exchange resin for liquid chromatography composed with a sulfonated styrenedivinylbenzene copolymer (3 m in particle diameter). Column temperature: Varied between 50 C and 65 C. Chemical reaction vessel temperature: A constant temperature of about 130 C. Color developing time: About 1 minute. Mobile phase: Buffer solutions A, B, C and D, with sodium ion concentrations of 0.10 mol/L, 0.135 mol/L, 1.26 mol/L and 0.20 mol/L, respectively. The ion concentration of the mobile phase is changed stepwise from 0.10 mol/L to 1.26 mol/L by using these buffer solutions.
Components of buffer solutions (g) Buffer solution Citric acid Sodium citrate Sodium hydroxide Sodium chloride A 8.85 3.87 3.54 B 7.72 10.05 1.87 C 6.10 26.67 2.50 54.35 D 8.00 -

KP 9 341
velength: 210 nm) Column: A stainless steel column abut 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 13.2 g of diammonium hydrogen phosphate in 900 mL of water, add phosphoric acid to adjust the pH to 2.5, and add water to make 1000 mL. Flow rate: Adjust the flow rate so that the retention time of acetic acid is about 4 minutes. System suitanility System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, acetic acid and citric acid are eluted in this order with the resolution between their peaks being not less than 2.0. (2) Related substancesDissolve 1.0 mg of Elcatonin in 1 mL of a mixture of trifluoroacetic acid TS and acetonitrile (2:1), and use this solution as the test solution. Take exactly 0.3 mL of the test solution, add a mixture of trifluoroacetic acid TS and acetonitrile (2:1) to make exactly 10 mL, and use this solution as the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the total of the peak areas other than the peak of elcatonin of the test solution is not larger than the peak of elcatonin of the standard solution, and each peak area other than the peak of elcatonin of the test solution is not larger than 1/3 of the peak area of elcatonin from the standard solution.

Ethanol Thiodiglycol Purified water Total amount

60.0 mL 5.0 mL q.s.

5.0 mL q.s.

q.s.

60.0 mL q.s.

1000 mL 1000 mL 1000 mL 1000 mL

Reaction reagent: Mix 407 g of lithium acetate dehydrate, 245 mL of glacial acetic acid and 801 mL of 1methoxy-2-propanol, add water to make 2000 mL, stir for about 20 minutes while passing Nitrogen, and use this solution as solution A. Separately, to 1957 mL of 1methoxy-2-propanol, add 77 g of ninhydrin and 0.134 g of sodium borohydride, stir for about 20 minutes while passing Nitrogen, and use this solution as solution B. Mix solution A and solution B before use. Flow rate of mobile phase: Adjust the flow rate so that the retention time of arginine is about 75 minutes. Flow rate of reaction reagent: About 0.2 mL per minute. Selection of column: Proceed with 10 L of the standard solution under the above operating conditions. Use a column from which aspartic acid, threonine, serine, glutamic acid, praline, glycine, alanine, valine, 2aminosuberic acid, leucine, tyrosine, lysine, histidine and arginine are eluted in this order, with complete separation of each peak.
Purity (1) Acetic acidWeigh accurately about 3 6 mg of Elcatonin quickly under conditions of 25 2 o C and 50 5% relative humidity, add exactly 1 mL of the internal standard solution to dissolve it, and use this solution as the test solution. Separately, weigh accurately about 0.5 g of glacial acetic acid, and add the internal standard solution to make exactly 100 mL. Pipet 5 mL of this solution, add the internal standard solution to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS , of the peak area of acetic acid to that of the internal standard: the amount of acetic acid is not more than 7.0%.

Amount (%) of acetic acid (CH3COOH) = W ST Q T 50

W SA

QS

WST : Amount (g) of glacial acetic acid WSA : Sample amount (mg)

Internal standard solutionA solution of citric acid (1 in 4000). Operating conditions Detector: An ultraviolet absorption photometer (wa-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 225 nm). Column: A stainless steel column abut 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of trifluoroacetic acid TS and acetonitrile (change the ratio linearly from 85:15 to 55:45 in 30 minutes). Flow rate: Adjust the flow rate so that the retention time of elcatonin is about 25 minutes. System suitability System performance: Dissolve 2 mg of Elcatonin in 200 L of trypsin TS for test of elcatonin, warm at 37 C for 1 hour, then add 1 drop of glacial acetic acid, and heat at 95 C for 1 minute. To 10 L of this solution, add 50 L of the test solution, and mix. Proceed with 10 L of this solution under the above operating conditions, and calculate the resolution. Use a column such that the reso-

342 Monographs, Part I

lution between the peak of elcatonin and the peak which appears immediately before the peak of elcatonin is not less than 2.0, and the retention time of elcatonin is about 25 minutes. Time span of measurement: Continue measurement until the regularly changing baseline of the chromatogram disappears, beginning after the solvent peak.
Water Weigh accurately 1 3 mg of Elcatonin quickly under conditions of 25 2 C and 50 5% relative humidity, and perform the test as directed in the coulometric titration under the Water Determination: not more than 8.0% Nitrogen Content Weigh accurately 15 20 mg of Elcatonin quickly under conditions of 25 2 C and 50 5% relative humidity, and perform the test as directed under the Nitrogen Determination: it contains not less than 16.1% and not more than 18.7% of nitrogen (N: 14.01) in the peptide, calculated on the dehydrated and de-acetic acid basis. Assay (i) Aminals: Select healthy male SpragueDawley rats each weighing between 90 g and 110 g. Keep the rats for not less than 3 days before use, providing an appropriate uniform diet and water. (ii) Diluent for elcatonin: Dissolve 2.72 g of sodium acetate in water to make 200 mL, add 0.2 g of bovine serum albumin, and adjust the pH to 6.0 with glacial acetic acid. Prepare before use. (iii) Standard solution: Dissolve Elcatonin RS in the diluent for elcatonin to make two standard solutions, one to contain exactly 0.75 Unit in each mL which is designated as the high-dose standard solution, SH , and the other to contain exactly 0.0375 Unit in each mL which is designated as the low-dose standard solution, S L . (iv) Test solution: Weigh accurately 0.5 2.0 mg of Elcatonin quickly under conditions of 25 2 C and 50 5% relative humidity, and dissolve in the diluent for elcatonin to make two test solutions, the high-dose test solution, TH , which contains the Units per mL equivalent to S H and the low-dose test solution, TL , which contains the Units per mL equivalent to S L . (v) Deproteinizing solution for elcatonin: Dissolve 160 g of trichloroacetic acid and 30.6 g of strontium chloride in water to make 3600 mL. (vi) Procedure: Divide the animals into 4 equal groups of not less than 10 animals each. Withhold all food, but not water, for 18 to 24 hours before the injection, and withhold water during the assay until the final blood sample is taken. Handle the animals with care in order to avoid undue excitement. Inject exactly 0.2 mL each of the standard solutions and the test solutions into the tail vein of each animal as indicated in the following design:

Second group

SL

Fourth group

TL

At 1 hour after the injection, take a sufficient amount blood sample to perform the test from the carotid artery and vein of each animal under ether anesthesia, centrifuge the blood samples to separate serum, and determine the serum calcium according to the following (vii). (vii) Serum calcium determination: Take exactly 0.3 mL of the serum, add the deproteinizing solution for elcatonin to make exactly 3 mL, mix well, centrifuge, and use the supernatant liquid as the test solution for calcium determination. Separately, pipet 1 L of Standard Calcium Solution for the Atomic Absorption Spectrophotometry, and add a solution of sodium chloride (17 in 2000) to make exactly 10 mL. Pipet 5 mL of this solution, add the deproteinizing solution for elcatonin to make exactly 50 mL, and use this solution as the standard solution for calcium determination. Determine the absorbances, AT and AS , of the test solution and the standard solution as directed under the Atomic Absorption Spectrophotometry according to the following conditions. Determine the absorbance, A0 , of a solution obtained in the same manner used for preparation of the standard solution, but with 1 mL of water instead of the standard solution. Amount (mg) of calcium in 100 mL of the serum = 0 .01
AT A 0 10 100 AS A0

Gas: Combustible gasAcetylene Supporting gasAir Lamp: Calcium hollow-cathode lamp Wavelength: 422.7 nm (viii) Calculation: Amounts of calcium in 100 mL of the serum obtained with SH , S L , TH and TL in (vii) are symbolized as y1 , y2 , y3 and y4 , respectively. Sum up individual y1 , y2 , y3 and y4 to obtain Y1 , Y2 , Y3 and Y4 , respectively. Units per mg of peptide, calculated on the dehydrated and de-acetic acid basis = antilog M (units per mL of SH) (b/a)
M = 0.3010 Ya Yb

Ya = Y1 Y2 + Y3 + Y4 Yb = Y1 Y2 + Y3 Y4

a: Amount (mg) of the sample

{ 100
[water content (%) + acetic acid content (%)] } 100

b: Total volume (mL) of the high-dose test solution prepared by dissolving the sample with diluent for elcatonin F computed by the following equation should be

First group

SH

Third group

TH

KP 9 343
smaller than F shown in the table against n with which s 2 is calculated. Calculate L (P = 0.95) by use of the following equation: L should be not more than 0.20. If F exceeds F, or if L exceeds 0.20, repeat the test, increasing the number of animals or arranging the assay conditions so that F is not more than F and L is not more than 0.20.

Emetine Hydrochloride
CH3 H N HN 2 HCl

(Y1 + Y2 + Y3 Y4 )2 4 fs2 f: Number of the animals of each group. y y2 f s2 = n F' =

y : The sum of squares of y1 , y2 , y3 and y4 in each group. Y = Y12 + Y22 + Y32 + Y42 n = 4( f 1)
2

H H3CO OCH3

H OCH3 OCH3

C29H40N2O42HCl: 553.56 Emetine Hydrochloride is the hydrochloride of an alkaloid obtained from Ipecac, or prepared by methylation of cephaeline, or prepared synthetically. Emetine Hydrochloride contains not less than 98.0% and not more than 101.5% of emetine hydrochloride (C29H40N2O42HCl), calculated on the dried basis.
Description Emetine Hydrochloride is a white and pale yellow crystalline powder with no odor. Emetine Hydrochloride is freely soluble in water or ethanol. Emetine Hydrochloride is affected by light. Identification (1) Determine the ultraviolet absorption spectra of solutions Emetine Hydrochloride and Emetine Hydrochloride RS in 0.25 mol/L sulfuric acid (1 in 20000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Emetine Hydrochloride and Emetine Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) An aqeous solution (1 in 20) of Emetine Hydrochloride responds to the Quantitative Tests for chloride. Purity (1) AcidityDissolve 0.1 g of Emetine Hydrochloride in 10 mL of water, add 1 drop of methyl red TS and titrate with 0.020 mol/L sodium hydroxide VS: Less than 0.5 mL is required to observe a yellow color. (2) CephaelineDissolve 0.1 g of Emetine Hydrochloride in 10 mL of methanol and use this solution as the test solution. Separately, dissolve 23 mg of cephaeline hydrobromide RS in 100 mL of methanol and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 10 L each of the test solution and the standard solution on the plate of silica gel for Thinlayer Chromatography. Develope the plate with a mixture of chloroform and diethylamine (9 : 1), until the solvent front upto about 12 cm. Remove the plate from the tank and allow the plate to

L = 2 ( C 1 )( CM C=
Yb2

+ 0.09062)

Yb2 4 fs 2 t 2

t 2 : Value shown in the following table against n used to calculate s 2 .

n 1 2 3 4 5 6 7 8 9 10 11 12

t2 = F 161.45 18.51 10.129 7.709 6.608 5.987 5.591 5.318 5.117 4.965 4.844 4.747

n 13 14 15 16 17 18 19 20 21 22 23 24

t2 = F 4.667 4.600 4.543 4.494 4.451 4.414 4.381 4.351 4.325 4.301 4.279 4.260

n 25 26 27 28 29 30 40 60 120

t2 = F 4.242 4.225 4.210 4.196 4.183 4.171 4.085 4.001 3.920 3.841

Packaging and Storage Preserve in tight containers and store at temperate not exceeding 8 C.

344 Monographs, Part I dry in air for 20 minutes. Spray evenly the dried plate with 2.5 mol/L sodium hydroxide VS and dry at 50 C for 5 minutes. Then spray the plate with developing agent: any cephaeline spot from the test solution is not larger or not more intense than that from the standard solution (not more than 2%). Developing reagentDissolve 0.3 g of p-nitroaniline in 25 mL of 2 mol/L hydrochloric acid and cool to about 4 C. Slowly add 5 mL of sodium nitrite solution (1 in 25), maintaining the temperature at about 4 o C . Freshly prepare the solution for each test.
Water Between 15.0% and 19.0% (0.1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.15 g of Emetine Hydrochloride, dissolve in 5 mL of glacial acetic acid and warm, if necessary. Allow to cool, add 10 mL of dioxane, 5 mL of mercuric acetate TS for nonaqeous titration and titrate with 0.1 N perchloric acid VS(indiator : 3 drops of methylrosaniline chloride TS). Perform a bland determination, and make any necessary correction.

Purity CephaelineEvaporate 1.0 mL of Emetine Hydrochloride Injection on a steam-bath under nitrogen to dryness. Dissolve the residue in a sufficient volume of methanol to obtain the test solution having a concentration of 10 mg of Emetine Hydrochloride per mL. Separately, weigh 23 mg of Cephaelin Bromochloride RS, add methanol to make 100 mL and use this solution as the standard solution. Proceed as directed in the Purity (2) for cepthaeline under Emetine Hydrochloride. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 5.4 EU/mg of emetine hydrochloride. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer exactly a volume of Emetine Hydrochloride Injection, equivalent to about 0.12 g of emetine

Each mL of 0.1 mol/L perchloric acid VS = 27.678 mg of C29H40N2O42HCl

Packaging and Storage tight containers.

Preserve in light-resistant,

hydrochloride (C29H40-N2O42HCl) according to the labeled amount, to a separatory funnel containing 20 mL of water. Add ammonium hydroxide TS to become strongly alkaline and to 0.5 mL of the water layer add hydrochloric acid to become weakly acidic, extract with ether until it remains unaffected by the addition of 2 to 3 drops of Meyers TS. Evaporate the combined ether extracts on a water-bath. To the residue add 2 mL of neutralized ethanol and 30.0 mL of 0.01 mol/L sulfuric acid and warm gently until the alkaloid is dissolved. Cool and titrate the excess acid with 0.02 mol/L sodium hydroxide VS (indicator: 2 to 3 drops of methyl red). Perform a bland determination, and make any necessary correction. Each mL of 0.01 mol/L sulfuric acid = 5.536 mg of C29H40N2O42HCl
Packaging and Storage hermetic containers.

Emetine Hydrochloride Injection

Emetine Hydrochloride Injection is a sterile solution of Emetine Hydrochloride in water for injection. Emetine Hydrochloride Injection contains not less than 84.0% and not more than 94.0% of the labeled amount of emetine hydrochloride (C29H40-N2O42HCl: 553.56).
Method of Preparation Emetine Hydrochloride Injection is prepared as directed under Injections, with Emetine Hydrochloride. Description Emetine Hydrochloride Injection is a clear and colorless liquid. Identification (1) Evaporate 1 mL of Emetine Hydrochloride Injection on a steam-bath to dryness: the residue responds to Identification (1) under Emetine Hydrochloride. (2) The Emetine Hydrochloride Injection responds to Identification (3) under Emetine Hydrochloride pH

Preserve in light-resistant,

Enalapril Maleate
O H N N O H3C H O H CH3 CO2H H CO2H CO2H

Between 3.0 and 5.0.

KP 9 345 C20H28N2O5C4H4O4: 492.52 Enalaprol Maleate contains not less than 98.0% and not more than 102.0% of enalapril maleate (C20H28N2O5 C4H4O4), calculated on the dried basis. . Description Enalapril Maleate is white crystal or crystalline powder. Enalapril Maleate is freely soluble in methanol, soluble in water, and sparingly soluble in dichloromethane. Enalapril Maleate dissolves in dilute sodium hydroxide TS.
Identification (1) Retention time of main peak from the test solution in the Assay is the same as that of the standard solution (2) Determine the Infrared spectrum of Enalapril Maleate and Enalapril Maleate RS, as directed in the paste method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between -41.0 and -43.5 (after drying, 0.1 g, methanol, 10 mL, 100 mm) . Purity (1) Heavy metalsProceed with 2.0 g of Enalapril Maleate according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesWeigh accurately about 30 mg of Enalapril Maleate, dissolve in a mixture of pH 2.5 phosphate buffer solution and acetonitrile (95 : 5) to make exactly 100 mL. and use this solution as the test solution. Separately, weigh accurately about 30 mg of Enalapril Maleate RS, dissolve in a mixture of pH 2.5 phosphate buffer solution and acetonitrile (95 : 5) to make exactly 100 mL Pipet exactly 1 mL of this solution, add a mixture of pH 2.5 phosphate buffer solution and acetonitrile (95 : 5) to make exactly 100 mL and use this solution as the standard solution. Perform the test with 50 L of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak area by the automatic integration method, calculate each amount of related substances: among peaks other than the principal peak one related substance is not more than 1.0%, another related substance is not more than 0.3% and the total area of peaks of the related substances is not more than 2.0%.

AT : Peak area of each related substances in test solu-

tion
AS : Peak area of each related substances in standard solution

Operating conditions Proceed as directed in the operating conditions under the Assay.
Loss on Drying Not more than 1.0 % (at a pressure not exceeding 0.67 kPa, 60 C, 2 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 30 mg of Enalapril Maleate, dissolve in a mixture of pH 2.5 phosphate buffer solution and acetonitrile (95 : 5) to make exactly 100 mL, and use this solution as the test solution. Separately, dissolve about 30 mg, accurately weighed, of Enalapril Maleate in a mixture of pH 2.5 phosphate buffer solution and acetonitrile (95 : 5) to make exactly 100 mL and use this solution as the standard solution. Perform the test with 50 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, AT and AS , of the peak area of Enalapril Maleate to that of the internal standard, for the test solution and the standard solution, respectively.

Amount (mg) of Enalapril Maleate (C20H28N2O5C4H4O4) = amount (mg) of Enalapril Maleate RS

AT AS

Operating conditions Ditector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, 4.1 mm in inside diameter and about 15 cm in length, packed with styrenedivinyl benzene for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: 70 C Flow rate: 1.5mL/minute. Mobile phase: Program as follows with the mobile phase A and the mobile phase B in isocratic or linear gradient. The Mobile phase A: A mixture of pH 6.8 phosphate buffer solution and acetonitrile (19 : 1) The Mobile phase B: A mixture of acetonitrile and pH 6.8 phosphate buffer solution (33 : 17) Time (minutes) 0 0 to 20 20 to 25 25 to 26 Solution A (%) 95 95 to 40 40 40 to 95 Solution B (%) 5 5 to 60 60 60 to 5 Elution equilibration linear gradient isocratic linear gradient

Amount (%) of related substaced = 100 C S AT

Ci

AS

C S : the concentration of the standard solution (mg/mL) Ci : the concentration of Enalapril Maleate in the test solution (mg/mL)

346 Monographs, Part I 26 to 30 95 5 isocratic quent solution as the test solution. Separately, weigh accurately about 10 mg of Enalapril Maleate RS, dissolve in pH 6.8 phosphate buffer solution to make exactly 100 mL and use this solution as the standard solution. Perform the test according to the operating conditions as directed in the Assay under Enalapril Maleate. The dissolution rate of Enalapril Maleate Tablets in 30 minutes is not less than 80%.
Uniformity of Dosage Units It meets the requirement of the Content Uniformity Test when the test is performed according to the following method. Take 1 tablet of Enalapril Maleate Tablets, add 50 mL of pH 2.2 phosphate buffer solution, if necessary, sonicate for 15 mintes, shake for 30 minutes, add pH 2.2 phosphate buffer solution to make exactly 100 mL and use this solution as the test solution. Pipet exactly V mL each of this solution, then add buffer solution to make a solution containing 0.1 mg/mL. Separately, weigh accurately about 10 mg of Enalapril Maleate RS, add pH 2.2 phosphate buffer to make exactly 100mL and use this solution as the standard solution. Perform the test according to the operating conditions as directed in the Assay under Enalapril Maleate.

System suitability System performance: Mix 1 mL of Enalapril diketopiperazine solution and 50 mL of the standard solution. When the procedure is run with 50 L of this solution, as directed under the above operating conditions, Enalapril and enalapril diketopiperazine are eluted in this order with the resolution between their peaks being not less than 3.5. System repeatability: When the test is repeated 6 times with the standard solution, as directed under the above operating conditions, the relative standard deviation of the peak area is not more than 1.0%. pH 6.8 phosphate buffer solution: Dissolve 2.8 g of monobasic sodium phosphate in about 900 mL of water in a 1000mL volumetric flask. Adjust with phosphoric acid to a pH of about 6.8, dilute with water to volume, and mix. pH 2.5 phosphate buffer solution: Dissolve 2.8 g of monobasic sodium phosphate in about 900 mL of water in a 1000mL volumetric flask. Adjust with phosphoric acid to a pH of about 2.5, dilute with water to volume, and mix. Enalapril diketopiperazine solution: Place about 20 mg of Enalapril Maleate RS in a 100-mL beaker to form a mound on the bottom of the beaker. Place the beaker on a hot plate at about one-half the maximum hot plate temperature setting. Heat for about 5 minutes to 10 minutes until the solid is melted. Immediately remove the beaker from the hot plate, and allow to cool. Add 50 ml of acetonitrile, and sonicate for a few minutes to dissolve. The solution typically contains, in each mL, between 0.2 mg and 0.4 mg of enalapril diketopiperazine.
Packaging and Storage Preserve in well-closed containers.

Amount (mg) of Enalapril Maleate (C20H28N2O5C4H4O4) = T

C AT D AS

Enalapril Maleate Tablets

Enalapril Maleate Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of enalapril maleate (C20H28N2O5C4H4O4: 492.52).
Method of Preparation Prepare as directed under Tablets, with Enalapril Maleate. Identification Retention time of main peak from the test solution in the Assay is the same as those of the standard solution Dissolution Test Perform the test with 1 tablet of Enalapril Maleate Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of pH 6.8 phosphate buffer solution as a dissolution solution. Take 20 mL or more of the dissolved solution 30 minutes after starting the test, and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet the subse-

T : labeled amount of Enalaptil Meleate in each tablet (mg). C : Concentration of the standard solution (mg/mL). D : Concentration of Enalapril Maleate in the test solution (mg/mL). AT : peak are of Enalapril Maleate in the test solution. AS : peak area of Enalapril Maleate in the standard solution pH 2.2 phosphate bufferDissolve 1.38g of monobasic sodium phosphate in about 800 mL of water in a 1000mL volumetric flask. Adjust with phosphoric acid to a pH of about 2.2, dilute with water to volume, and mix.
Related Substances The test solution, the standard solution, pH 2.2 phosphate buffer solution and enalapril diketopiperazine solution are prepared as directed in the Assay. Pipet 1.0 mL of the standard solution, add pH 2.2 phosphate buffer solution to make exactly 100mL and use this solution as the related substances standard solution. Perform the test with 50 L of each of the test solution, the standard solution, the related substances standard solution and buffer solution as directed under the Liquid Chromatography according to the assay method. Determine each peak areas by the automatic integration method, and calculate the amounts of related substances. Measure the peak areas for all of the peak from the test solution greater than 0.1% of enalapril of the response of the peaks other than are not observed in the Buffer solu-

KP 9 347 tion. Amount (%) of anhydrous enlalprilat = 492 .53 CV AT 100

348 .39
N AS L

492.53: molecular weight of enalapril maleate 348.39: molecular weight of anhydrous enalaprilat C : concentration Enalaprilat in the standard solution (mg/mL) V : nominal capacity of the test solution (mL) N : number of tablets taken for the test L : labeled amount of enalapril maleate in the tablet AT : enalaprilat peak area obtained from the test solution AS : enalaprilat peak area obtained from the standard solution Amount (%) of enalapril ketopiperazine 492 .53 C 'V 100 AT = 358 .44 N 1.25 AS L 492.53: molecular weight of enalapril maleate 358.44: molecular weight of enalapril ketopiperazine C': concentration of Enalapril Maleate RS in the related substances standard solution (mg/mL) V: nominal capacity of the test solution (mL) N: number of tablets taken for the test 1.25: peak area for enalapril diketopiperazine relative to that of enalapril L: labeled amount of enalapril maleate in the tablet (mg). Amount (%) of any other related substances = C 'V AT 100
N AS L

leate (C20H28N2O5C4H4O4), add 50 mL of pH 2.2 phosphate buffer solution. Ultrasonicate for 15 minutes, then shake for 30 minutes, add pH 2.2 phosphate buffer solution to make exactly 100 mL, sonicate for 15 minutes and filter through a membrane filter. Separately, weigh accurately about 20 mg of Enalapril Maleate RS, add 0.5mL of enlalaprilat standard solution and dissolve in 50 mL of pH 2.2 phosphate buffer solution, then sonicate, if necessary, add pH 2.2 phosphate buffer solution to make exactly 100 mL and use this solution as the standard solution. Perform the test with exactly 50 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area, AT and AS , of the test solution and the standard solution by the automatic integration method Amount (mg) of enalapril maleate (C20H28N2O5C4H4O4) = amount (mg) of Enalapril Maleate RS AT
AS

AT : peak area of any other related substances C', V, N are defined as the above.

The sum of all related substances including those from enalaprilat and enalapril diketopiperazine is not greater than 5.0%. Enalaprilate standard solution: weigh accurately a portion of Enalapril Maleate RS, dissolve in water to make about 0.4 mg/mL. Standard solution: weigh accurately about 20 mg of Enalapril Maleate RS, add 0.5mL of enalaprilate standard solution and 50 mL of buffer solution, then shake well. Add pH 2.2 phosphate buffer solution to make exactly 100mL. Related substances standard solution: Pipet exactly 1 mL of the standard solution, add pH 2.2 phosphate buffer solution to make exactly 100 mL.
Assay Weigh accurately and powder not less than 20 Enalapril Maleate Tablets, weigh accurately a portion of the powder, equivalent to about 20 mg of enalapril ma-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 215 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with hydroxypropylsilanized silica gel for liquid chromatography. Column temperature: 50 C Mobile phase: The mixture of pH 2.2 phosphate buffer solution and acetonitrile (75 : 25). Flow rate: 2mL/minute. System suitabilit System performance: Pipet 0.5mL of Enalapril diketopiperazine solution, add the standard solution to make 25mL. When the procedure is run with 50 L of this solution, as directed under the above operating conditions, Maleate, Enalaprilat, Enalapril, Enalapril diketopiperazine are eluted in this order with the resolution between their peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 50 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the peak area of enalapril is not more than 2.0%.
Packaging and Storage Preserve in well-colosed containers.

Enflurane
F F F F H H O F Cl

and enantiomer

348 Monographs, Part I Carrier gas: Helium Flow rate: Adjust the flow rate so that the retention time of enflurane is about 3 minutes. System suitability Test for required detectability: Pipet 1 mL of Enflurane and add 2-propanol to make 100 mL. Pipet 2 mL of this solution, add 10 mL of 2-propanol and use this solution as the solution for system suitability. Pipet 1 mL of this solution and dilute with 2-propanol to exactly 10 mL. Confirm that the peak area of enflurane obtained with 5 L of this solution is equivalent to 7 to 13% of that with 5 L of the solution for system suitability. System performance: Mix 5 mL each of Enflurane and 2-propanol. When the procedure is run with 5 L of this solution under the above operating conditions, enflurane, 2-propanol are eluted in this order with the resolution between the peaks is not less than 2.0. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of enflurane is not more than 2.0%. Time span of measurement: About 3 times as long as the retention time of enflurane beginning after the solvent peak. (4) Residue on evaporationPipet exactly 65 mL of Enflurane, evaporate on steam bath to dryness and dry the residue at 105 C for 1 hour: the amount of residue is not more than 1.0 mg.
Water Not more than 0.1% (10 g, volumetric titration, direct titration). Packaging and Storage Preserve in tight container and store at not more than 30 C.

C3H2ClF5O : 184.49
Description Enflurane is a colorless clear liquid. Enflurane is slightly soluble in water, miscible with ethanol or ether. Enflurane is volatile and not flammable. Enflurane has no optical activity. Boiling pointBetween 54 C and 57 C. Identification (1) Perform the test with 50 L of Enflurane as directed under the Oxygen Flask Combustion Method to obtain the test solution. The test solution responds to the Qualitative Tests for chloride and fluoride. (2) Determine the infrared spectra of Enflurane and Enflurane RS, previously dried, as directed in the liquid film method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Refractive Index Specific Gravity
20 : Between 1.302 and 1.304. nD 20 : Between 1.520 and 1.540. d 20

Purity (1) Acidity and alkalinityTo 60 mL of Enflurane add 60 mL of freshly boiled and cooled water, shake for 3 minutes to mix, allow to separate completely and use the water layer as the test solution. To 20 mL of the test solution add 1 drop of bromcresol purple TS and 0.10 mL of 0.01 mol/L of sodium hydroxide VS: the color of the solution is violet. To 20 mL of the test solution add 1 drop of bromcresol purple TS and 60 L of 0.01 mol/L hydrochloric acid: VS the color of the solution is yellow. (2) ChlorideWeigh 20 g of Enflurane, add 20 mL of water, shake for 5 minutes, allow to separate completely and use water layer. Pipet 10 mL of the solution and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.001%). (3) Related substancesPerform the test with exactly 5 L of Enflurane as directed under Gas Chromatography according to the following conditions, and determine peak area by the automatic integration method and calculate the percentage of each peak area reference to enflurane: the peak area other than enflurane is not more than 0.10%.

Enoxacin Hydrate
CH2CH3 HN N N N 1 1/2 H2O F O CO2H

C15Hl7FN4O31H2O: 347.34 Enoxacin Hydrate, when dried, contains not less than 98.5% and not more than 101.0% of enoxacin (C15H17FN4O3: 320.32).
Description Enoxacin Hydrate is a white to pale yellow-brown crystals or crystalline powder. Enoxacin Hydrate is freely soluble in glacial acetic acid, slightly soluble in methanol, very slightly soluble in chloroform, and practically insoluble in water, in ethanol or in ether.

Operating conditions Detector: A thermal conductivity detector. Column: A fused-silica column about 3 mm in inside diameter and about 3 m in length, coated the inner surface with 20% diethyleneglycolsuccinic ester for Gas Chromatography on silica for Gas Chromatography (180 to 250 m ) 5 m in thickness. Column temperature: A constant temperature of about 80 C.

KP 9 349 Enoxacin Hydrate dissolves in dilute sodium hydroxide TS. Enoxacin Hydrate is gradually colored by light.
Identification (1) Place 20 mg of Enoxacin Hydrate and 50 mg of metallic sodium in a test tube and heat gradually to ignition with precaution. After cooling, add 0.5 mL of methanol and then 5 mL of water and heat to boiling. To this solution, add 2 mL of dilute acetic acid and filter: the filtrate responds to the Qualitative Tests (2) for fluoride. (2) Dissolve 50 mg each of Enoxacin Hydrate and Enoxacin Hydrate RS, in dilute sodium hydroxide TS to make 100 mL each. To each of 1 mL of the solutions, add water to make 100 mL each and determine the absorption spectra as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Enoxacin and Enoxacin RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point ing).

more intense than that from the standard solution.

Loss on Drying Between 7.0 and 9.0% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g, platinum crucible). Assay Weigh accurately about 0.3 g of Enoxacin Hydrate, previously dried, dissolve in 30 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 32.032 mg of C15Hl7FN4O3

Packaging and Storage tight containers.

Preserve in light-resistant,

Ephedrine Hydrochloride
OH C H NHCH3 C H CH2 HCl

Between 225 C and 229 C (after dry-

Purity (1) SulfateDissolve 1.0 g of Enoxacin Hydrate in 50 mL of dilute sodium hydroxide TS, shake with 10 mL of dilute hydrochloric acid and centrifuge. Filter the supernatant and to 30 mL of the filtrate add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.50 mL of 0.005 mol/L sulfuric acid VS, add 25 mL of dilute sodium hydroxide TS, 5 mL of dilute hydrochloric acid and water to make 50 mL (not more than 0.048%). (2) Heavy metalsProceed with 1.0 g of Enoxacin Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicProceed with 1.0 g of Enoxacin Hydrate according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 50 mg Enoxacin Hydrate in 25 mL of a mixture of chloroform and methanol (7 : 3) and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add a mixture of chloroform and methanol (7 : 3) to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of nbutanol, water and glacial acetic acid (3 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not

C10H15NOHCl: 201.69 Ephedrine Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of ephedrine hydrochloride (C10H15NOHCl).
Description Ephedrine Hydrochloride is a white crystal or crystalline powder. Ephedrine Hydrochloride is freely soluble in water, soluble in ethanol, slightly soluble in glacial acetic acid, and practically insoluble in acetic anhydride or acetonitrile. Identification (1) Determine the absorption spectra of solutions of Ephedrine Hydrochloride and Ephedrine Hydrochloride RS, respectively, in water (1 in 2000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Ephedrine Hydrochloride and Ephedrine Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Ephedrine Hydrochloride (1 in 15) responds to the Qualitative Tests for chloride. Specific Optical Rotation [ ]20 D : Between - 33.0 and - 36.0 (after drying, 1 g, water, 20 mL, 100 mm).

350 Monographs, Part I under the above operating conditions, the relative standard deviation of the peak areas of ephedrine is not more than 2.0%. Time span of measurement: About three times as long as the retention time of ephedrine after the solvent peak.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.4 g of Ephedrine Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) by warming. Cool and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, endpoint detection method in titrimetry). Perform a blank determination and make any necessary correction.

Melting Point

Between 218 C and 222 C.

pH A solution of Ephedrine Hydrochloride (1 in 20) is between 4.5 and 6.5. Purity (1) Clarity and color of solutionDissolve 0.5 g of Ephedrine Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) SulfateDissolve 50 mg of Ephedrine Hydrochloride in 40 mL of water, add 1 mL of dilute hydrochloric acid and 1 mL of barium chloride TS and allow to stand for 10 minutes: no turbidity is produced. (3) Heavy MetalsProceed with 1.0 g of Ephedrine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 1.0 mL of Standard Lead Solution (not more than 10 ppm). (4) Related SubstanceDissolve 50 mg of Ephedrine Hydrochloride in 50 mL of mobile phase and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test and the standard solution as directed under the Liquid Chromatography according to the following condition and calculate the areas of each peak by the automatic integration method: the total area of all peaks other than principal peak from the test solution is not greater than that of principal peak from the standard solution.

Each mL of 0.1 mol/L perchloric acid VS = 20.169 mg of C10H15NOHCl

Packaging and Storage Preserve in well-closed containers.

Ephedrine Hydrochloride Injection

Ephedrine Hydrochloride Injection is an aqueous solution for injection. Ephedrine Hydrochloride Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of ephedrine hydrochloride (C10H15NOHCl: 201.69).
Method of Preparation Prepare as directed under Injections, with Ephedrine Hydrochloride. Description Ephedrine Hydrochloride Injection is a clear, colorless liquid. pHThe Between 4.5 and 6.5. Identification Take a volume of Ephedrine Hydrochloride Injection, equivalent to 50 mg of Ephedrine Hydrochloride according to the labeled amount, add water to make 100 mL and the determine the absorption spectrum of this solution as directed under the Ultravioletvisible Spectrophotometry: it exhibits maxima between 249 and 253 nm, between 255nm and 259 nm and between 261 nm and 265 nm. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 7.5 EU/mg of Ephedrine Hydrochloride. Foreign Insoluble Matter Test It meets the require-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 45 C. Mobile phase: A mixture of a solution of sodium lauryl sulfate (1 in 128), acetonitrile and phosphoric acid (640 : 360 : 1) Flow rate: Adjust the flow rate so that the retention time of ephedrine is about 14 minutes. System suitability Test for required detectability: Pipet exactly 1 mL of the standard solution, add mobile phase to make 20 mL. Confirm that the peak area of ephedrine obtained from 10 L of this solution is equivalent to 4 to 6% of that from the standard solution. System performance: Dissolve 1 mg of Ephedrine Hydrochloride RS and 4 mg of atropine sulfate in 100 mL of diluted methanol (1 in 2). When the procedure is run with 10 L of this solution as directed under the above operating conditions, ephedrine and atropine are eluted in this order with the resolution between these peaks being not less than 1.5. System repeatability: When the test is repeated 6 times with 10 L of the standard solution, as directed

KP 9 351 ment.
Insoluble Particulate Matter Test for Injections meets the requirement.

It

Ephedrine Hydrochloride Powder contains not less than 9.3% and not more than 10.7% of ephedrine hydrochloride (C10H15NOHCl: 201.69).
Method of Preparation Ephedrine Hydrochloride 100 g Starch, Lactose Hydrate a sufficient quantity or their mixture Total 1000 g Prepare as directed under Powders, with the above ingredients. Identification Take 0.5 g of 10% Ephedrine Hydrochloride Powder, add 100 mL of water, shake for 20 minutes and filter. Determine the absorption spectrum of the filtrate as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 249 nm and 253 nm, between 255 nm and 259 nm and between 261 nm and 265 nm. Particle Size Distribution Test for Preparations meets the requirement.

Determination of Volume of Injection in Containers It meets the requirement. Assay Take an exact volume of Ephedrine Hydrochloride Injection, equivalent to about 40 mg of ephedrine hydrochloride (C10H15NOHCl) according to the labeled amount, add exactly 10 mL of the internal standard solution and water to make 200 mL and use this solution as the test solution. Separately, weigh accurately about 40 mg of Ephedrine Hydrochloride RS, previously dried at 105 C for 3 hours, add exactly 10 mL of internal standard solution and water to make 200 mL and use this solution as the standard solution. Perform the test with 10 L each of the test and the standard solution as directed under the Liquid Chromatography according to the following condition and calculate the ratios, QT and QS , of the peak area of ephedrine to that of the internal standard of each solution.

It

Amount (mg) of ephedrine hydrochloride (C10H5NOHCl ) = amount (mg) of Ephedrine Hydrochloride RS

QT QS

Uniformity of Dosage Units (divided) It meets the requirement. Assay Weigh accurately about 0.4 g of 10% Ephedrine Hydrochloride Powder, add 150 mL of water and extract with the aid of ultrasonicator for 10 minutes with occasional shaking. Shake more for 10 minutes, add exactly 10 mL of the internal standard solution and water to make 200 mL, centrifuge and use the supernatant as the test solution. Separately, weigh accurately about 40 mg of Ephedrine Hydrochloride RS, previously dried at 105 C for 3 hours, add exactly 10 mL of internal standard solution and water to make 200 mL and use this solution as the standard solution. Perform the test with 10 L each of the test and the standard solution as directed under the Liquid Chromatography according to the following condition and calculate the ratios, QT and QS , of the peak area of ephedrine to that of the internal standard of each solution.

Internal standard solutionA solution of etilefrine hydrochloride (1 in 500). Operating conditions Detector, column, column temperature, mobile phase and flow rate: Perform as directed in the operating conditions in the Purity (4) under Ephedrine Hydrochloride. System suitability System performance: When the procedure is run with 10 L of the standard solution as directed under the above operating conditions, the internal standard and ephedrine are eluted in this order with the resolution between these peaks being not less than 1.5. System repeatability: When the test is repeated 6 times with 10 L of the standard solution as directed under the above operating conditions, the relative standard deviation of the ratios of the peak area of ephedrine to that of internal standard is not more than 1.0%.
Packaging and Storage hermetic containers.

Amount (mg) of ephedrine hydrochloride (C10H15NOHCl ) = amount (mg) of Ephedrine Hydrochloride RS

QT QS

Preserve in light-resistant,

Internal standard solutionA solution of etilefrine hydrochloride (1 in 500). Operating conditions Detector, column, column temperature, mobile phase and flow rate: Perform as directed in the operating conditions in the Purity (4) under Ephedrine Hydrochloride. System suitability

10% Ephedrine Hydrochloride Powder

Ephedrine Hydrochloride Powder

352 Monographs, Part I System performance: When the procedure is run with 10 L of the standard solution as directed under the above operating conditions, the internal standard and ephedrine are eluted in this order with the resolution between these peaks being not less than 1.5. System repeatability: When the test is repeated 6 times with 10 L of the standard solution as directed under the above operating conditions, the relative standard deviation of the ratios of the peak area of ephedrine to that of internal standard is not more than 1.0%.
Packaging and Storage Preserve in well-closed containers.

Amount (mg) of ephedrine hydrochloride (C10H15NOHCl ) = amount (mg) of Ephedrine Hydrochloride RS

QT QS

Internal standard solutionA solution of etilefrine hydrochloride (1 in 500). Operating conditions Detector, column, column temperature, mobile phase and flow rate: Perform as directed in the operating conditions in the Purity (4) under Ephedrine Hydrochloride. System suitability System performance: When the procedure is run with 10 L of the standard solution as directed under the above operating conditions, the internal standard and ephedrine are eluted in this order with the resolution between these peaks being not less than 1.5. System repeatability: When the test is repeated 6 times with 10 L of the standard solution as directed under the above operating conditions, the relative standard deviation of the ratios of the peak area of ephedrine to that of internal standard is not more than 1.0%.
Packaging and Storage Preserve in well-closed containers.

Ephedrine Hydrochloride Tablets

Ephedrine Hydrochloride Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of ephedrine hydrochloride (C10H15NOHCl: 201.69).
Method of Preparation Prepare as directed under Tablets, with Ephedrine Hydrochloride. Identification Take an amount of powdered Ephedrine Hydrochloride Tablets, equivalent to 50 mg of Ephedrine Hydrochloride according to the labeled amount, add 100 mL of water, shake for 20 minutes and filter. Determine the absorption spectrum of the filtrate as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 249 nm and 253 nm, between 255 nm and 259 nm and between 261 nm and 265 nm. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 tablets of Ephedrine Hydrochloride Tablets. Weigh accurately an amount of powder, equivalent to about 40 mg of Ephedrine Hydrochloride, add 150 mL of water and extract with the aid of ultrasonicator for 10 minutes with occasional shaking. Shake more for 10 minutes, add exactly 10 mL of the internal standard solution and water to make 200 mL, centrifuge and use the supernatant as the test solution. Separately, weigh accurately about 40 mg of Ephedrine Hydrochloride RS, previously dried at 105 C for 3 hours, add exactly 10 mL of internal standard solution and water to make 200 mL and use this solution as the standard solution. Perform the test with 10 L each of the test and the standard solution as directed under the Liquid Chromatography according to the following condition and calculate the ratios, QT and QS , of the peak area of ephedrine to that of the internal standard of each solution.

Epinephrine
OH HO C H HO CH2NHCH3

Adrenaline Epirenamine

C9H13NO3: 183.20

Epinephrine, when dried, contains not less than 98.0% and not more than 101.0% of epinephrine (C9H13NO3: 183.20).
Description Epinephrine is a white to grayish white, crystalline powder and odorless. Epinephrine is freely soluble in glacial acetic acid, very slightly soluble in water, and practically insoluble in methanol, ethanol or ether. Epinephrine dissolves in dilute hydrochloric acid. Epinephrine gradually changes to brown in color by air and by light. Identification (1) Dissolve 10 mg of Epinephrine in 10 mL of diluted acetic acid (1 in 500) and use this solution as the test solution. Take 1 mL of the test solution, add 4 mL of water and 1 drop of ferric chloride TS: a deep green color is observed and it gradually changes to red. (2) Place 1 mL each of the test solution obtained in (1) in test tubes, A and B. Add 10 mL of potassium biph-

KP 9 353 thalate buffer solution, pH 3.5, in A and add 10 mL of phosphate buffer solution, pH 6.5, in B. To each of the test tubes, add 1 mL of iodine TS, allow to stand for 5 minutes and add 2 mL each of sodium thiosulfate TS: a red color is observed in test tube A and a deep red color is observed in test tube B.
Specific Optical Rotation [ ]20 D : Between - 50.0 and - 53.5 (after drying, 1 g, 1 mol/L hydrochloric acid TS, 25 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 0.1 g of Epinephrine in 10 mL of dilute hydrochloric acid: the solution is clear and has no more color than Color Matching Fluid A. (2) AdrenaloneDissolve 50 mg of Epinephrine in 0.05 mol/L hydrochloric acid TS to make exactly 25 mL and determine the absorbance of this solution at 310 nm as directed under the Ultraviolet-visible Spectrophotometry: it is not more than 0.40. (3) NorepinephrineDissolve 10.0 mg of Epinephrine in 2.0 mL of a tartaric acid solution (1 in 200). Pipet exactly 1 mL of the solution, add 3.0 mL of pyridine, add 1.0 mL of freshly prepared sodium naphthoquinone sulfonate TS and allow to stand in a dark place for 30 minutes. Take this solution, add 5.0 mL of pyridine containing 50 mg of Ascorbic Acid: the solution has no more color than the following control solution.

Adrenaline Hydrochloride Injection Epinephrine Injection is an aqueous solution for injection. Epinephrine Injection contains not less than 0.085 w/v % and not more than 0.115 w/v % of epinephrine (C9H13NO3: 183.20).
Method of Preparation Dissolve Epinephrine in diluted hydrochloric acid (9 in 10000) and prepare as directed under Injections. Description Epinephrine Injection is a colorless, clear liquid. Epinephrine Injection changes gradually to pale red and then to brown on exposure to air and light. pHBetween 2.3 and 5.0. Identification (1) Take 1 mL of Epinephrine Injection, add 4 mL of water and 1 drop of ferric chloride TS: a deep green color is observed and it gradually changes to red. (2) Place 1 mL each of Epinephrine Injection in test tubes, A and B, and proceed as directed in the Identification (2) under Epinephrine. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Control solutionDissolve 2.0 mg of Norepinephrine Bitartrate RS and 90 mg of Epinephrine Bitartrate RS in methanol to make exactly 10 mL. Pipet exactly 1 mL of this solution and proceed in the same manner.
Loss on Drying Not more than 1.0% (2 g, in vacuum, silica gel, 18 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Epinephrine, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Pipet exactly 30 mL of Epinephrine Injection into a separatory funnel, add 25 mL of carbon tetrachloride, shake vigorously for 1 minute, allow the liquids to separate and discard the carbon tetrachloride layer. Repeat this procedure 3 times. Rinse the stopper and mouth of the separatory funnel, with a small amount of water. Add 0.2 mL of starch TS, then while shaking the separatory funnel, add iodine TS dropwise until a persistent blue color is observed and immediately add sodium thiosulfate TS to discharge the blue color. Add 2.1 g of sodium bicarbonate to the liquid in the separatory funnel, preventing it from coming in contact with the mouth of the separatory funnel and shake until most of the sodium bicarbonate dissolves. Rapidly inject 1.0 mL of acetic anhydride into the contents of the separatory funnel. Immediately stopper the separatory funnel loosely and allow to stand until the evolution of gas ceases. Shake vigorously, allow to stand for 5 minutes, extract with six 25 mL volumes of chloroform and filter each chloroform extract through a pledget of absorbent cotton. Evaporate the combined chloroform extracts in a water-bath in a current of air to 3 mL, completely transfer this residue by means of small portions of chloroform to a tared beaker

Each mL of 0.1 mol/L perchloric acid VS = 18.321 mg of C9H13NO3

Packaging and Storage Preserve in light-resistant, under nitrogen atmosphere and in a cold place, tight containers.

Epinephrine Injection
Epinephrine Hydrochloride Injection Epirenamine Hydrochloride Injection

354 Monographs, Part I and heat again to evaporate to dryness. Dry the residue at 105 C for 30 minutes, cool in a desiccator (silica gel) and accurately measure the weight, W (mg), of the dried residue. Dissolve in chloroform to make exactly 5 mL and determine the specific optical rotation D using a 100-mm cell as directed under the optical Rotation Determination . Amount (mg) of Epinephrine (C9H13NO3) 0 .5 D = 0 .5923 W (0.5 + )
93

Packaging and Storage Preserve in light-resistant, hermetic containers and colored containers may be used.

Epirizole
CH 3 H 3C N N N OCH 3 OCH 3 N

Mepirizole

C11H14N4O2: 234.25

Epirizole, when dried, contains not less than 99.0% and not more than 101.0% of epirizole (C11H14N4O2).
Description Epirizole is a white crystal or crystalline powder. Epirizole is odorless and has a bitter taste. Epirizole is very soluble in methanol or in glacial acetic acid, freely soluble in ethanol, and sparingly soluble in water or in ether. Epirizole dissolves in dilute hydrochloric acid or in sulfuric acid. The pH of a solution of Epirizole (1 in 100) is between 6.0 and 7.0. Identification (1) Take 0.1 g of Epirizole, add 0.1 g of vanillin, 5 mL of water and 2 mL of sulfuric acid and mix with shaking for a while: a yellow precipitate is produced. (2) Dissolve 0.1 g of Epirizole in 10 mL of water and add 10 mL of picric acid TS: a yellow precipitate is produced. Collect the precipitate by filtration, wash with 50 mL of water and dry at 105 C for 1 hour: it melts between 163 C and 169 C. (3) Determine the absorption spectra of solutions of Epirizole and Epirizole RS, respectively, in 0.1 mol/L hydrochloric acid VS (1 in 200000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Melting Point

clear and colorless. (2) ChlorideAdd 0.5 g of Epirizole to a ground mixture of 0.7 g of potassium nitrate and 1.2 g of anhydrous sodium carbonate, mix well, transfer little by little to a platinum crucible, previously heated, and heat until the reaction is completed. After cooling, add 15 mL of dilute sulfuric acid and 5 mL of water to the residue, boil for 5 minutes, filter, wash the insoluble matter with 10 mL of water and add 6 mL of dilute nitric acid and water to the combined filtrate and washings to make 50 mL. Perform the test with this solution as the test solution. Prepare the control solution as follows: When the procedure is run with the same quantities of the same reagents as directed for the preparation of the test solution and add 0.25 mL of 0.01 mol/L hydrochloric acid VS and water to make 50 mL (not more than 0.018%). (3) Heavy metalsProceed with 2.0 g of Epirizole according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicProceed with 1.0 g of Epirizole according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 1.0 g of Epirizole in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add methanol to make exactly 50 mL. Pipet exactly 1 mL of this solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 2 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layor chromatography. Develop the plate with a mixture of isopropyl ether, ethanol and water (23 : 10 : 2) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than that from the standard solution. Place this plate in a chamber filled with iodine vapor: any spot other than the principal spot from the test solution is not more intense than that from the standard solution. (6) Readily carbonizable substancesPerform the test with 0.10 g of Epirizole: the solution has no more color than Color Matching Fluid A.
Loss on Drying Not more than 0.5% (1 g, silica gel, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Epirizole, previously dried, dissolve in 40 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (indicator: 2 drops of methylrosaniline chloride TS) until the color of the solution changes from purple through blue-green to green. Perform a blank determination and make any necessary correction.

Between 88 C and 91 C.

Purity (1) Clarity and color of solutionDissolve 0.20 g of Epirizole in 20 mL of water: the solution is

KP 9 355 Each mL of 0.1 mol/L perchloric acid VS = 23.426 mg of C11H14N4O2

Packaging and Storage Preserve in well-closed containers.

tween 4.0 and 5.5.

Absorbance E1 cm (495 nm): 200 ~ 230 (15 mg calculated on the anhydrous basis and corrected by the amount of the residual solvent, methanol, 1000 mL). Purity Residual solventsWeigh accurately about 50 mg of Epirubicin Hydrochloride, dissolve in water to make exactly 5 mL, and use this solution as the test solution. Separately, weigh accurately about 1.0 g of npropanol, add water to make exactly 100 mL, pipet exactly 1 mL of this solution, add water to make exactly 100 mL, and use this solution as the n-propanol standard solution. Separately, weigh accurately about 1 g of acetone, add water to make exactly 100 mL, pipet exactly 1 mL of this solution, add water to make exactly 100 mL, and use this solution as the acetone standard solution. Perform the test with 2 L each of the test solution and the standard solutions as directed under the Gas Chromatography according to the following operating conditions, and determine the peak heights of n-propanol and acetone in the test solution, H1 and H2, and in the standard solutions, HS1 and HS2 (the total amount of n-propanol and acetone: not more than 5.0 %).
1%

Epirubicin Hydrochloride
O OH O OH OH

O H3 C

O H3C

OH OH

O NH2

HCl

C27H29NO11HCl: 579.98 Epirubicin Hydrochloride is the hydrochloride of a derivative of daunorubicin. Epirubicin Hydrochloride contains not less than 900 g (potency) of per mg of epirubicin hydrochloride (C27H29NO11 HCl), calculated on the anhydrous basis and corrected by the amount of the residual solvent.
Description Epirubicin Hydrochloride is a pale yellow or red-brown powder. Epirubicin Hydrochloride is soluble in water or in methanol, slightly soluble in ethanol, and practically insoluble in ether, in chloroform, or in acetonitrile. Epirubicin Hydrochloride is hygroscopic. Identification (1) Weigh 1 - 2 mg of Epirubicin Hydrochloride, dissolve in 5 mL of 1 mol/L sodium hydroxide: a blue-purple color develops. (2) Weigh 5 mg of Epirubicin Hydrochloride, dissolve in 5 mL of water, add 1 mL of nitric acid and 2 to 3 drops of silver nitrate TS and mix with shaking: the solution turns to be turbid. (3) Weigh 15 mg of Epirubicin Hydrochloride, dissolve in methanol to make 1000 mL, and use this solution as the test solution. Determine the absorption spectrum of this solution as directed under Ultraviolet-visible Spectrophotometry, it exhibits maxima between 232 nm and 236 nm, and between 250 nm and 254 nm, and between 286 nm and 290 nm, and between 477 nm and 481 nm, and between 493 nm and 497 nm, and between 527 nm and 531 nm. Specific Optical Rotation [ ] 20 D : +310 ~ +340 (10 mg calculated on the anhydrous basis and corrected by the amount of the residual solvent, methanol, 20 mL, 100 mm). pH The pH of a solution obtained by dissolving 50 mg of Epirubicin Hydrochloride in 10 mL of water is be-

Amount [%] of n-propanol in 1 g of Epirubicin Hydrochloride = Amount [g] of n - propanol taken H1 0.0005
H S1

1 100 Amount (g) of Epirubicin Hydrochlor ide

Amount [%] of acetone in 1 g of Epirubicin Hydrochloride = Amount [g] of acetone taken H 2 0.0005
HS2

1 100 Amount (g) of Epirubicin Hydrochlor ide

Operating conditions Detector: A flame-ionization detector Column: A glass column, about 3.2 mm in inside diameter and about 2 m in length, packed with porous polymerbis (50/80 mesh). Injector temperature: A constant temperature at about 170 C Column temperature: A constant temperature at about 120 C Carrier gas: Nitrogen Flow rate: Adjust the flow rate so that the retention time of n-propanol is about 12 minutes.
Sterility Test It meets the requirement, When Epirubicin Hydrochloride is used in a sterile preparation. Pyrogen Test It meets the requirement, when Epirubicin Hydrochloride is used in a sterile preparation,. Weigh

356 Monographs, Part I an appropriate amount of Epirubicin Hydrochloride, dissolve in water, make the solution so that each mL contains 2.0 mg, and use the solution as the test solution. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit.
Histamine It meets the requirement, when Epirubicin Hydrochloride is used in a sterile preparation. Weigh an appropriate amount of Epirubicin Hydrochloride, dissolve in water to make the solution so that each mL contains 15 mg, and use the solution as the test solution. Use 0.5 mL of this solution for the test. Water Not more than 8.0 % (0.2 g, volumetric titration, direct titration). Assay Weigh accurately about 10 mg each of Epirubicin Hydrochloride and Epirubicin Hydrochloride RS, add the internal standard solution to make exactly 10 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 5 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the ratios, QT and QS, of the peak area of epirubicin hydrochloride to that of the internal standard.

tios of the peak area of epirubicin to that of the internal standard is not more than 1.0 %.
Packaging and Storage Preserve in tight containers.

Ergocalciferol
H H3C H3C H H CH3 CH3

CH3

CH2

HO H

Calciferol Vitamin D2

C28H44O: 396.65

Ergocalciferol contains not less than 97.0% and not more than 103.0% of ergocalciferol (C28H44O).
Description Ergocalciferol is a white crystal, is odorless or has a faint, characteristic odor. Ergocalciferol is freely soluble in ethanol, ether, or chloroform, sparingly soluble in isooctane and practically insoluble in water. Ergocalciferol is affected colored by air and by light. Melting pointBetween 115 C and 118 C. Transfer Ergocalciferol to a capillary tube and dry for 3 hours in a desiccator (at a pressure not exceeding 2.67 kPa). Immeadiately fireseal the capillary tube, put it in a bath fluid, previously heated to a temperature about 10 C below the expected melting point and heat at a rate of rise of about 3 C per minute and read the melting point. Identification (1) Dissolve 0.5 mg of Ergocalciferol in 5 mL of chloroform, add 0.3 mL of acetic anhydride and 0.1 mL of sulfuric acid and shake: a red color is observed and rapidly changes through purple and blue to green. (2) Determine the infrared spectra of Ergocalciferol and Ergocalciferol RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers.
1% Absorbance E1 cm (265 nm): Between 445 and 485 (10 mg, ethanol, 1000 mL).

Amount [g (potency)] of epirubicin hydrochloride (C27H29NO11HCl) = amount [g (potency)] of Epirubicin Hydrochloride RS

QT QS

Internal standard solutionWeigh 1 g of sodium 2naphthalene sulfonate, and dissolve in the mixture of water and acetonitrile (60:31) to make 500 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) Column temperature: A room temperature. Mobile phase: Adjust the pH of a mixture of water and acetonitrile (69:31) to 2.0 with phosphoric acid Flow rate: Adjust the flow rate so that the retention time of epirubicin hydrochloride is about 10minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, the internal standard and epirubicin hydrochloride are eluted in this order with the resolution between these peaks being not less than 8.0. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the operating conditions, the relative standard deviation of the ra-

Specific Optical Rotation [ ]20 D : Between + 102 and + 107 (0.3 g, ethanol, 20 mL, 100 mm). Prepare the solution of Ergocalciferol within 30 minutes after the con-

KP 9 357 tainer has been opened and determine the rotation within 30 minutes after the solution has been prepared.
Purity ErgostrerolDissolve 10 mg of Ergocalciferol in 2.0 mL of diluted ethanol (9 in 10), add allow 20 mg of digitonin in 2.0 mL of ethanol (9 in 10) and allow the mixture to stand for 18 hours: no precipitate is produced. Assay Weigh accurately about 30 mg of Ergocalciferol and Ergocalciferol RS and dissolve each in isooctane to make exactly 50 mL. Pipet exactly 10 mL each of these solutions, add exactly 3 mL each of the internal standard solution, then add the mobile phase to make exactly 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 to 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Ergocalciferol to that of the internal standard, respectively. Perform the procedure rapidly avoiding contact with air or other oxidizing agents and using light-resistant containers.

being not less than 0.7 and that between ergocalciferol and tachysterol2 being not less than 1.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of ergocalciferol to that of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in light-resistant, hermetic containers (in a cold place after displacement air with nitrogen).

Ergometrine Maleate
O H H C N H N H CH3 H C CH3 H C C CO2H CO2H CH2OH

Amount (mg) of Ergocalciferol (C28H44O)

Q = amount (mg) of Ergocalciferol RS T QS

HN

Ergonovine Maleate

C19H23N3O2C4H4O4: 441.48

Internal standard solutionA solution of dimethylphthalate in isooctane (1 in 100) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 10 cm to 30 cm in length, packed with a silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 20 C. Mobile phase: A mixture of hexane and namylalcohol (997 : 3). Flow rate: Adjust the flow rate so that the retention time of Ergocalciferol is about 25 minutes. System Suitability System performance: Dissolve 15 mg of Ergocalciferol RS in 25 mL of isooctane. Transfer this solution to a flask, heat in an oil-bath under a reflux condenser for 2 hours and cool immediately to room temperature. Transfer the solution to a quartz test tube and irradiate with a short-wave lamp (main wavelength: 254 nm) and a long-wave lamp (main wavelength: 365 nm) for 3 hours. Take 10 mL of this solution, add the mobile phase to make 50 mL. When the procedure is run with 10 L of this solution, as directed under the above operating conditions. the ratios of the retention time of previtamin D2, trans-vitamin D2 and tachysterol2 to that of ergoclaciferol are about 0.5, about 0.6 and about 1.1, respectively with resolution between previtamin D2 and trans-vitamin D2

Ergometrine Maleate, when dried, contains not less than 98.0% and not more than 101.0% of ergometrine maleate (C19H23N3O2C4H4O4).
Description Ergometrine Maleate is a white to pale yellow, crystalline powder, and is odorless. Ergometrine Maleate is sparingly soluble in water, slightly soluble in methanol or in ethanol, and practically insoluble in ether. Ergometrine Maleate gradually changes to yellow in color on exposure to light. Melting pointAbout 185 C (with decomposition). Identification (1) Prepare a solution of Ergometrine Maleate (1 in 50): the solution shows a blue fluorescence. (2) Dissolve 1 mg of Ergometrine Maleate in 5 mL of water. Take 1 mL of this solution, add 2 mL of pdimethylaminobenzaldehyde-ferric chloride TS, shake, and allow to stand for 5 to 10 minutes: a deep blue color is observed. (3) Take 5 mL of a solution of Ergometrine Maleate (1 in 500), add 1 drop of potassium permanganate TS: the red color of the solution disappears immediately. Specific Optical Rotation [ ]20 D : Between +48 and +57 (after drying, 0.25 g, water, 25 mL, 100 mm). pH Dissolve 0.10 g of Ergometrine Maleate in 10 mL of water. The pH of this solution is between 3.0 and 5.0. Purity

(1) Clarity and color of solutionDissolve

358 Monographs, Part I 0.10 g of Ergometrine Maleate in 10 mL of water: the solution is clear and colorless to light yellow. (2) Ergotamine and ergotoxineTake 20 mg of Ergometrine Maleate, add 2 mL of sodium hydroxide solution (1 in 10), and heat to boiling: the gas evolved does not change moistened red litmus paper to blue. (3) Related substancesDissolve 5.0 mg each of Ergometrine Maleate and Ergometrine Maleate RS in exactly 1 mL of methanol, and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate, prepared with silica gel for thin-layer chromatography and dilute sodium hydroxide TS. Develop the plate with a mixture of chloroform and methanol (4 : 1) to a distance of about 10 cm, and air-dry the plate. Spray evenly p-dimethylaminobenzaldehyde TS on the plate: the spots obtained from the test solution and the standard solution show a red-purple color and the same Rf value, and any spot from the test solution other than the spot from the standard solution does not appear.
Loss on Drying 4 hours).

Ergometrine Maleate Injection

Ergonovine Maleate Injection Ergometrine Maleate Injection is an aqueous solution for injection. Ergometrine Maleate Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of ergometrine maleate (C19H23N3O2C4H4O4: 441.48).
Method of Preparation Prepare as directed under Injections, with Ergometrine Maleate. Description Ergometrine Maleate Injection is a clear, colorless to pale yellow liquid. pHBetween 2.7 and 3.5. Identification (1) Measure a volume of Ergometrine Maleate Injection, equivalent to 3 mg of Ergometrine Maleate according to the labeled amount, if necessary, dilute with water or evaporate on a water-bath to make 15 mL and use this solution as the test solution: the test solution shows a blue fluorescence. (2) Add 1 mL of ammonia TS to 1 mL of the test solution obtained in (1) and extract with 20 mL of ether. To the ether extract, add 1 mL of dilute sulfuric acid, shake and warm to remove ether on a water-bath. Cool, add 2 mL of p-dimethylaminobenzaldehyde-ferric chloride TS to the residue obtained and allow to stand for 5 to 10 minutes: a deep blue color is observed. (3) Add 1 drop of potassium permanganate TS to 5 mL of the test solution obtained in (1): a red color disappears immediately. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Not more than 2.0% (0.2 g, silica gel,

Assay Weigh accurately about 10 mg each of Ergometrine Maleate and Ergometrine Maleate RS, previously dried in a desiccator (silica gel) for 4 hours, dissolve separately in water to make exactly 250 mL, and use these solutions as the test solution and the standard solution. Pipet exactly 2 mL each of the solutions and put separately into a brown glass-stoppered tube. To each tube add 4 mL of p-dimethylamino-benzaldehyde-ferric chloride TS, exactly measured, while cooling in an ice-bath, then warm at 45 C for 10 minutes. Allow to stand at room temperature for 20 minutes, and perform the test with these solutions as directed under the Ultravioletvisible Spectrophotometry using a solution, prepared with 2 mL of water in the same manner, as the blank. Determine the absorbances, AT and AS , of the subsequent solution of the test solution and the standard solution at 550 nm, respectively.

It

Amount (mg) of ergometrine maleate (C19H23N3O2C4H4O4) = amount (mg) of Ergometrine Maleate RS

Packaging and Storage tight containers.
AT AS

Determination of Volume of Injection in Containers It meets the requirement. Assay Transfer an exactly measured volume of Ergometrine Maleate Injection, equivalent to about 2 mg of ergometrine maleate (C19H23N3O2C4H4O4) and add sodium chloride in a ratio of 0.3 g to 1 mL of the solution. To this mixture, add 20 mL of ether and 2 mL of ammonia TS, shake and extract. Further, extract with three 15 mL volumes of ether, combine all the extracts, add 5 g of anhydrous sodium sulfate, filter through a pledget of absorbent cotton and wash with three 5 mL volumes of ether. Add the washings to the filtrate, shake with 5 mL of dilute sulfuric acid, evaporate the ether by warming in a current of nitrogen, add water to the remaining solution

Preserve in light-resistant,

KP 9 359 to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 2 mg of Ergometrine Maleate RS, previously dried in a desiccator (silica gel) for 4 hours, dissolve in water to make exactly 50 mL and use this solution as the standard solution. Pipet exactly 2 mL each of the test solution and the standard solution, and proceed as directed in the Assay under Ergometrine Maleate. Amount (mg) of ergometrine maleate (C19H23N3O2C4H4O4) = amount (mg) of Ergometrine Maleate RS
AT AS

while cooing in an ice-bath, after shaking and allow to stand for 1 hour at room temperature. Perform the test with these solutions as directed under the Ultravioletvisible Spectrophotometry using a solution, prepared with 4 mL of water in the same manner, as the blank. Determine the absorbances, AT and AS , of the subsequent solutions of the test solution and the standard solution at 550 nm, respectively. Amount (mg) of ergometrine maleate (C19H23N3O2C4H4O4) = amount (mg) of Ergometrine Maleate RS AT V
AS

100

Packaging and Storage Preserve in light-resistant, hermetic containers at a cool place and colored containers may be used.

Ergometrine Maleate Tablets

Ergonovine Maleate Tablets Ergometrine Maleate Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of ergometrine maleate (C19H23N3O2C4H4O4: 441.48).
Method of Preparation Prepare as directed under Tablets, with Ergometrine Maleate. Identification Powder Ergometrine Maleate Tablets, weigh a portion of the powder equivalent to 3 mg of Ergometrine Maleate according to the labeled amount, add 15 mL of warm water, shake and filter: the filtrate shows a blue fluorescence. Perform the test with this solution as directed in the Identification (2) and (3) under Ergometrine Maleate. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement when the Content Uniformity Test is performed according to the following method. Transfer 1 tablet of Ergometrine Maleate Tablets to a glass-stoppered centrifuge tube and add exactly V mL of a solution of tartaric acid (1 in 100) to make of a solution containing about 40 g of ergometrine maleate (C19H23N3O2C4H4O4) per mL. Stopper the tube, shake for 30 minutes vigorously, centrifuge and use the supernatant as the test solution. Separately, weigh accurately about 4 mg of Ergometrine Maleate RS, previously dried in a desiccator (silica gel) for 4 hours, dissolve in water to make exactly 100 mL and use this solution as the standard solution. Pipet exactly 4 mL each of the test solution and the standard solution, put separately into brown glass-stoppered test tubes, add exactly 8 mL each of p-dimethylamino-benzaldehyde-ferric chloride TS

Assay Weigh accurately and powder not less than 20 Ergometrine Maleate Tablets. Weigh accurately a portion of the powder, equivalent to about 2 mg of ergometrine maleate (C19H23N3O2C4H4O4), transfer to a glass filter (G4), add 10 mL of a solution of tartaric acid (1 in 100) and filter with thorough shaking. Repeat the procedure three times, combine the filtrates, add a solution of tartaric acid (1 in 100) to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 2 mg of Ergometrine Maleate RS, previously dried in a desiccator (silica gel) for 4 hours, dissolve in a solution of tartaric acid (1 in 100) to make exactly 50 mL and use this solution as the standard solution. Pipet exactly 2 mL each of the test solution and the standard solution and proceed as directed in the Assay under Ergometrine Maleate.

Amount (mg) of ergometrine maleate (C19H23N3O2C4H4O4) = amount (mg) of Ergometrine Maleate RS

Packaging and Storage well-closed containers.
AT AS

Preserve in light-resistant,

Ergotamine Tartrate
CH3 O H CONH H N O N CH3 H H CH2 O CH(OH)CO2H CH(OH)CO2H N OH

HN

(C33H35N5O5)2C4H6O6: 1313.41 Ergotamine Tartrate contains not less than 98.0% and not

360 Monographs, Part I more than 101.0% of ergotamine tartrate [(C33H35N5O5)2 C4H6O6], calculated on the dried basis.
Description Ergotamine Tartrate is a colorless crystals, or a white to pale yellowish white or grayish white, crystalline powder. Ergotamine Tartrate is slightly soluble in water or ethanol. Melting pointAbout 180 C (with decomposition). Identification (1) Dissolve 1 mg of Ergotamine Tartrate in 10 mL of a mixture of glacial acetic acid and ethyl acetate (1 : 1). Pipet 0.5 mL of this solution, add slowly 0.5 mL of sulfuric acid, with shaking in cold water and allow to stand: a purple color develops. Add 0.1 mL of diluted ferric chloride TS (1 in 12) to this solution: the color of the solution changes to blue or to blue-purple. (2) Dissolve 1 mg of Ergotamine Tartrate in 5 mL of a solution of tartaric acid (1 in 100). To 1 mL of this solution, add 2 mL of p-dimethylaminobenzaldehyde-ferric chloride TS and shake: a blue color develops. Specific Optical Rotation Ergotamine base : Between -155 and -165. Dissolve 0.35 g of Ergotamine Tartrate in 25 mL of a solution of tartaric acid (1 in 100), add 0.5 g of sodium bicarbonate, shake gently and sufficiently and extract with four 10 mL volumes of ethanolfree chloroform. Filter the extracts successively through a small filter paper, moistened with ethanol-free chloroform, into a 50-mL volumetric flask. Allow the flask to stand in a water-bath at 20 o C for 10 minutes and add ethanol-free chloroform (20 o C ) to make 50 mL and determine the optical rotation in a 100-mm cell. Separately, pipet exactly 25 mL of this solution, evaporate to dryness under reduced pressure at a temperature not higher than 45 o C , dissolve the residue in 25 mL of glacial acetic acid and titrate with 0.05 mol/L perchloric acid VS (indicator: 1 drop of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction. Calculate the specific optical rotation of the ergotamine base from the consumed volume of 0.05 mol/L perchloric acid VS and the optical rotation.
[ ]20 D

plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of chloroform and methanol (9 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly p-dimethylamino-benzaldehyde TS on the plate: any spot other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 5.0% (0.1 g, in vacuum, 60 C, 4 hours). Assay Weigh accurately about 0.2 g of Ergotamine Tartrate, dissolve in 15 mL of a mixture of glacial acetic acid and acetic anhydride (50 : 3) and titrate with 0.05 mol/L perchloric acid VS (indicator: 1 drop of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

Each mL of 0.05 mol/L perchloric acid VS = 32.836 mg of (C33H35N5O5)2C4H6O6

Packaging and Storage Preserve in light-resistant, tight containers and almost well-filled, or under nitrogen atmosphere. Store at not exceeding 5 C.

Ergotamine Tartrate Injection

Ergotamine Tartrate Injection is an aqueous solution for injection. Total alkaloid is 0.45 to 0.55 mg per mL, ergotamine tartrate [(C33H35N5O5)2C4H6O6: 1313.41] is 52.0% to 74.0% of total alkaloid content and Ergotamininine Tartrate is not more than 45.0% of total alkaloid content.
Method of Preparation Prepare as directed under Injections, with Ergotamine Tartrate. Description Ergotamine Tartrate Injection is a clear, colorless liquid. Identification (1) Evaporate a weighed volume of Ergotamine Tartrate Injection, equivalent to 0.1 mg of Ergotamine Tartrate according to the labeled amount, on a steam-bath to dryness. Dissolve the residue in 1 mL of a mixture of glacial acetic acid and ethyl acetate (1 : 1). Proceed with 0.5 mL of this solution under Identification (1) of Ergotamine Tartrate. (2) Weigh a volume of Ergotamine Tartrate Injection, equivalent to 0.2 mg of Ergotamine Tartrate according to the labeled amount, add 2 mL of p-dimethylamino benzaldehyde-ferric chloride reagent: it turns blue. pH

Each mL of 0.05 mol/L perchloric acid VS = 29.084 mg of C33H35N5O5

Purity Related substancesPerform this procedure without exposure to daylight, using light-resistant vessels. Weigh 40 mg of Ergotamine Tartrate, add exactly 10 mL of a solution of tartaric acid in diluted methanol (1 in 2) (1 in 1000), dissolve by thorough shaking and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add a solution of tartaric acid in diluted methanol (1 in 2) (1 in 1000) to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a

Between 3.5 and 4.0.

Sterility Test It meets the requirement. Bacterial Endotoxins Less than 357.0 EU/mg of ergo-

KP 9 361 tamine tartrate.

Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Take accurately a volume of Ergotamine Tartrate Injection in a beaker, equivalent to about 5 mg of ergotamine tartrate [(C33H35N5O5)2C4H6O6] according to the labeled amount, add 5 mL of chloroform saturated with water and a portion of sodium bicarbonate approximately equivalent in mass to one tenth that of the portion of Ergotamine Tartrate Injection taken and mix well. Add sufficient chromatographic siliceous earth (about 1 g for each mL of the Ergotamine Tartrate Injection taken plus 3 g in addition). Pack the mixture in a chromatographic column, 2.5 cm in inner diameter and 30 cm in length and this column as the 1st column. Rinse the sides of the beaker with 2 mL of chloroform saturated with water and transfer it to the column. Add sufficient amount of siliceous earth to the rinsed solution and transfer it to the above 1st column. Separately, prepare column using a mixture of 9 g of the siliceous earth with 7 mL of citric acid solution (1 in 4) in the 2nd chromatographic column. Place a mixture of 2 g of the siliceous earth and 2 mL of water on top of the 2nd column. Insert pledge of glass wool and mount the tube containing the spectrum so that elute from the 1st column will drain into the 2nd column. Add a total of 90 mL of chloroform saturated with water to the 1st column and receive elute from the 2nd column in a 200-mL volumetric flask. Rinse the tip of the 1st column with chloroform saturated with water, add chloroform saturated with water to a volume and use this solution as the Ergotaminine test solution. Extrude the adsorbent from the column by means of slight air pressure into a 600-mL beaker containing 10 g of sodium bicarbonate and mix. Cautiously add 50 mL of water, with continuous stirring and wash the mixture into a 250-mL separatory funnel and extract the ergotamine with four 15 mL volumes of chloroform saturated with water. Pass the extracts through a glass wool filter, combining them in a volumetric flask, wash the filter and dilute with chloroform saturated with water to make 100 mL and use this solution as the ergotamine test solution. Separately, pipet a volume of Ergotamine Tartrate Injection, equivalent to about 2.5 mg of Ergotamine Tartrate, into a 50 mL volumetric flask, add 25 mL of ethanol, then add tartaric acid solution (1 in 100) to make 50 mL and use this solution as the total alkaloid test solution. Separately, weigh accurately about 10 mg of the Ergotamine Tartrate RS and dissolve in 50 mL dilute ethanol solution with warm if necessary. Add water until 1 mL containing 50 g of Ergotamine Tartrate and use this sodium as the standard solution. Pipet 20 mL of the Ergotamine test solution and

10 mL of Ergotaminine test solution into separate, small conical flask. To the dried residue of the each solutions, add 5.0 mL of a mixture of ethanol andtartaric acid solution (1 in 100) (1 : 1) and dissolve. Pipet 5.0 mL each of the total alkaloid test solution and the standard solution into conical flasks separately. Place 4 each flasks in an ice-bath and swirl continuously while adding, dropwise, 10.0 mL of p-dimethyl-aminobenzaldehyde TS. Allow to stand at room temperature for not less than 90 minutes and not more than 2 hours. Determine the absorbances of the four solutions at the wavelength of a maximum absorbance at about 545 nm, with a suitable spectrophotometer, against a reagent blank. Amount (mg) of the total alkaloid = 0 .05 C Amount (%) of ergotamine tartrate [(C33H35N5O5)2C4H6O6] = 50 A ' Amount (%) of ergotaminine tartrate [(C33H35N5O5)2C4H6O6] = 50 A"
AU AU AU AS

C: Concentration of Ergotamine Tartrate in the standard solution (g/mL), AS : Absorbance of the standard solution, AU : Absorbance of the total alkaloid test solution, A': Absorbance of the Ergotamine test solution A": Absorbance of the Ergotaminine test solution.
Packaging and Storage hermetic containers.

Preserve in light-resistant,

Ergotamine Tartrate Tablets

Ergotamine Tartrate Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of ergotamine tartrate [(C33H35N5O5)2C4H6O6: 1313.41].
Method of Preparation Prepare as directed under Tablets, with Ergotamine Tartrate. Identification Weigh accurately a portion of Ergotamine Tartrate Tablets, previously dried, equivalent to about 5 mg of labeled amount and make powder.. Add 10 mL of hexane and shake and mix for a while. Place and delete hexane extracts and add 10 mL of chloroform saturated with strong ammonia water and shake and mix for a while, filter. Evaporate the filtrate in water bath. Dissolve the residue in 8 ml of a mixture of glacial acetic acid and ethyl acetate (1 : 1), pipet 1 mL of this solution. Shake and mix in ice bath and at the same time drop 1 mL of sulfuric acid: the solution is violet. Add 0.1 mL of diluted ferric chloride TS (1 in 2) in this solution: the

362 Monographs, Part I color is blue and blue-violet.

Disintegration Test Disintegrate in 5 minutes for Tablets intended for sublingual use. Dissolution Test This test is not applied to Sublingual Tablets. Take 1 tablet of Ergotamine Tartrate Tablet and perform the test at 75 revolutions per minute as directed in the Method 2 under Dissolution Test using 1000 mL of a solution of tartaric acid (1 in 100) as the dissolution solution. After 30 minutes from the start of the test, take 20 mL or more of the dissolved solution, filter and use the filtrate as the test solution. Separately, weigh accurately adequate amount of Ergotamine Tartrate RS, dissolve in dissolution solution to make a solution of known concentration and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and standard solution, respectedly, at 327 nm and 427 nm as directed under the Ultraviolet-visible Spectrophotometry using dissolution solution as the control solution.. The dissolution rate (%) of Ergotamine Tartrate Tablets in 30 minutes is not less than 75%. Uniformity of Dosage Units It meets the requirement of the Content Uniformity Test when the test is performed with Ergotamine Tartrate Tablets according to the Assay. Assay Weigh accurately and powder not less than 20 Ergotamine Tartrate Tablets. Weigh accurately a portion of the powder, equivalent to about 10 mg of ergotamine tartrate [(C33H35N5O5)2C4H6O6], add 50.0 mL of the internal standard and 300 mL of a mixture of acetonitrile and water (55 : 45) and sonicate for 10 minutes. Add a mixture of acetonitrile and water (55 : 45) to make exactly 500 mL, mix and filter. Discard the first 10 mL of the filtrate and use subsequent filtrate as the test solution. Separately weigh accurately about 10 mg of Ergotamine Tartrate RS, previously dried at 60 C for 4 hours in vacuum, dissolve in a mixture of acetonitrile and water (55 : 45) to make exactly 50 mL. Pipet exactly 5 mL of this solution, add 5.0 mL of the internal standard solution and add a mixture of acetonitrile and water (55 : 45) to make exactly 50 mL. Use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and calculate the ratios, QT and QS , of the peak areas of Ergotamine Tartrate to that of the internal standard, respectively.

and water (55 : 45). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m to10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of acetonitrile: 0.01mol/L potassium dihydrogenphosphate (55 : 45). Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 20 L of the standard solution, as directed under the above operating conditions, the symmetry factor of ergotamine is not more than 2.0 and the resolution between peaks of ergotamine and internal standard is not less than 3.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of ratios of the peak areas is not more than 2.0%.
Packaging and Storage well-closed containers.

Preserve in light-resistant,

Erythromycin
O H3C CH3 H3C HO OH H3C O H3C O CH3 O CH3 O O CH3 H3C O CH3 OH O CH3 OH CH3 OH N CH3

C37H67NO13: 733.93 Erythromycin is a macrolide substance having antibacterial activity produced by the growth of Saccharopolyspora erythraea. Erythromycin contains not less than 850 g (potency) per mg of erythromycin (C37H67NO13), calculated on the anhydrous basis.
Description Erythromycin is a white to light yellowwhite powder, is odorless, and has a bitter taste. Erythromycin is freely soluble in methanol, in ethanol, or in acetone, soluble in ether, and very slightly soluble in water. Identification (1) The melting point of Erythromycin

Amount (mg) of ergotamine tartrate (C33H35N5O5)2C4H6O6 = amount (mg) of Ergotamine Tartrate RS

QT QS

Internal standard solutionDissolve 40 mg of Ergonobine Maleate in 250 mL of a mixture of acetonitrile

KP 9 363 is between 133 C and 138 C (decomposition). (2) To 5 mg of Erythromycin add 2 mL of sulfuric acid, and mix with gently shaking: the red-brown color develops. (3) To 3 mg of Erythromycin add 2 mL of acetone, and add 2 mL of hydrochloric acid: the orange color develops and the color turns immediately to red to dark purple. (4) Dissolve each of Erythromycin and Erythromycin RS in chloroform to make 1 % solution. Determine the infrared spectra of these solutions, as directed in the solution method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers.
Specific Optical Rotation : -71 ~ -78 (1.0 g calculated on the anhydrous basis, ethanol, 50 mL, 100 mm). pH The pH of a solution obtained by dissolving 0.1 g of Erythromycin in 150 mL of water is between 8.0 and 10.5. Sterility Test It meets the requirement, when Erythromycin is used in a sterile preparation. Pyrogen Test It meets the requirement, when Erythromycin is used in a sterile preparation. Weigh appropriate amount of Erythromycin, dissolve in water, make the solution so that each mL contains 1.0 mg, and use the solution as the test solution. Use 5 mL of this solution for the test. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not more than 10.0 % (0.1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.2 % (1 g). Assay Weigh accurately about 50 mg (potency) each of Erythromycin and Erythromycin RS, dissolve each in 25 mL of methanol, and add the mobile phase to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak area of erythromycin, AT and AS.
[ ] 20 D

with octadecylsilanized silica gel for liquid chromatography (5 ~ 10 m in particle diameter) Mobile phase: The mixture of methanol and 0.067 mol/L of potassium dihydrogen phosphate (3:2).
Packaging and Storage Preserve in tight containers.

Erythromycin Estolate
O H3C CH3 H3C HO OH H3C O H3 C O CH3 O CH3 O O CH3 H3C O O CH3 O OH CH3 H N CH3

O CH3 OH CH3

C12H25OSO3H

C40H71NO14C12H25OSO3H: 1056.39 Erythromycin Estolate contains not less than 600 g (potency) per mg of erythromycin (C37H67NO13: 733.93), calculated on the anhydrous basis.
Description Erythromycin Estolate is a white crystalline powder or powder, is odorless, and has a bitter taste. Erythromycin Estolate is sparingly soluble in methanol or in ethanol, and very slightly soluble in water or in benzene. Identification Dissolve each of Erythromycin Estolate and Erythromycin Estolate RS in chloroform to make 1 % solution. Determine the infrared spectra of these solutions, as directed in the solution method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. pH The pH of a suspension obtained by suspending 0.1 g of Erythromycin Estolate in 10 mL of water is between 4.5 and 7.0. Water Not more than 4.0 % (0.2 g, volumetric titration, direct titration). Assay Perform the test according to Assay in Erythromycin. Weigh accurately an appropriate amount of of Erythromycin Estolate, dissolve in 25 mL of methanol, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solution so that each mL contains 1.0 mg (potency), and allow this solution to stand in 60 C water bath for 2 hours or at room temperature for 16 ~ 18 hours, and use this solution as the test solution. Packaging and Storage Preserve in tight containers.

Amount [g (potency)] of erythromycin (C37H67NO13) = amount [g (potency)] of Erythromycin RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 ~ 300 mm in length, packed

364 Monographs, Part I pH of the solution so that it will be 7.8 to 8.0. (2) Test organism- Staphylococcus aureus ATCC 6538 P. (3) Weigh accurately about 50 mg (potency) of Erythromycin Ethylsuccinate, dissolve in 50 mL of methanol, and add 0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly 100 mL. Take exactly a suitable amount of this solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration test solution and low concentration test solution, respectively. Separately, weigh accurately about 25 mg of Erythromycin RS, dissolve in 25 mL of methanol, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly 100 mL, and use this solution as the standard stock solution. Keep the standard stock solution at not exceeding 5 C and use within 7 days. Take exactly a suitable amount of the standard stock solution before use, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics.
Packaging and Storage Preserve in tight containers.

Erythromycin Ethylsuccinate
O H3C CH3 H3C HO OH H3C O H3C O CH3 O CH3 O O CH3 H3C O O CH3 O OH CH3 H N CH3

O CH3 OH O O CH3

C43H75NO16: 862.05 Erythromycin Ethylsuccinate is a derivative of erythromycin. Erythromycin Ethylsuccinate contains not less than 765 g (potency) per mg of erythromycin (C37H67NO13: 733.93), calculated on the anhydrous basis.
Description Erythromycin Ethylsuccinate is a white powder, and is odorless. Erythromycin Ethylsuccinate is freely soluble in methanol, soluble in ethanol, sparingly soluble in acetone, slightly soluble in ether, and practically insoluble in water. Identification Determine the absorption spectra of Erythromycin Ethylsuccinate and Erythromycin Ethylsuccinate RS as directed in the paste method under Infrared Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wave numbers. pH The pH of a suspension obtained by suspending 0.1 g of Erythromycin Ethylsuccinate in 10 mL of water is between 6.0 and 8.5. Sterility Test It meets the requirement, when Erythromycin Ethylsuccinate is used in a sterile preparation. Pyrogen Test It meets the requirement, when Erythromycin Ethylsuccinate is used in a sterile preparation,. Weigh an appropriate amount of Erythromycin Ethylsuccinate, suspend in water to make the solution so that each mL contains 50 mg, and use the suspension as the test suspension. Inject 0.1 mL of this suspension into a muscle of a leg. The amount of injection is 1.0 mL of the test solution per kg of body weight of rabbit. Water Not more than 3.0 % (0.4 g, volumetric titration, direct titration). Residue on Ignition Not more than 1.0 % (1 g). Assay The Cylinder-plate method method (1) Agar media for seed and base layer- Use the medium in I 2 1) 2 under Microbial Assay for Antibiotics. Adjust the (2)

Erythromycin Lactobionate
O H3C HO H3C OH H3C O CH3 O CH3 O CH3 O H3CO CH3 OH CH3 OH CH3 O H3C OH O CH3 N HO CH3 HOH2C H H OH H C C C OH O H OH OH O OH C OH COOH

C37H67NO13C12H22O12: 1092.22 Erythromycin Lactobionate contains not less than 570 g (potency) per mg of erythromycin (C37H67NO13: 733.93), calculated on the anhydrous basis.
Description Erythromycin Lactobionate is a white powder. Erythromycin Lactobionate is sparingly soluble in ethanol, in methanol or in water, and very slightly soluble in benzene. Identification (1) To 5 mg of Erythromycin Lactobionate add 2 mL of sulfuric acid, and mix with gently shaking: the red-brown color develops. (2) To 3 mg of Erythromycin Lactobionate add 2 mL of acetone, and add 2 mL of hydrochloric acid: the

KP 9 365 orange color develops and the color turns immediately to red to dark purple. (3) Determine the absorption spectra of Erythromycin Lactobionate and Erythromycin Lactobionate RS as directed in the paste method under Infrared Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wave numbers.
pH The pH of a solution obtained by dissolving 0.5 g of Erythromycin in 10 mL of water is between 5.0 and 7.5. Sterility Test It meets the requirement, when Erythromycin Lactobionate is used in a sterile preparation. Pyrogen Test It meets the requirement, when Erythromycin Lactobionate is used in a sterile preparation. Weigh an appropriate amount of Erythromycin Lactobionate, dissolve in water, make the solution so that each mL contains 2.5 mg (potency), and use the solution as the test solution. Inject 1.2 mL of this solution per kg of rabbit body weight of rabbit. Water Not more than 5.0 % (0.2 g, volumetric titration, direct titration). Assay The Cylinder-plate method method (1) Agar media for seed and base layer- Use the medium in I 2 1) 2 under Microbial Assay for Antibiotics. Adjust the (2) pH of the solution so that it will be 7.8 to 8.0. (2) Test organism- Staphylococcus aureus ATCC 6538 P. (3) Weigh accurately about 50 mg (potency) of Erythromycin Lactobionate, dissolve in 50 mL of methanol, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly 100 mL, and if necessary, allow to stand for 3 hours at room temperature. Take exactly a suitable amount of this solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration test solution and the low concentration test solution, respectively. Separately, weigh accurately about 25 mg of Erythromycin RS, dissolve in 25 mL of methanol, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly 100 mL, and use this solution as the standard stock solution. Keep the standard stock solution at not exceeding 5 C and use within 7 days. Take exactly a suitable amount of the standard stock solution before use, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in tight containers.

Erythromycin Stearate
O H3C CH3 H3C HO OH H3C O H3C O CH3 O CH3 O O H3C O CH3 OH CH3 O CH3 OH CH3 OH N CH3

O HO

C55H103NO15: 1018.40 Erythromycin Stearate contains not less than 500 g (potency) per mg of erythromycin (C37H67NO13: 733.93), calculated on the anhydrous basis.
Description Erythromycin Stearate is a white powder, is odorless, and has a little bit of bitter taste. Erythromycin Stearate is freely soluble in ethanol, soluble in ether, sparingly soluble in acetone, slightly soluble in methanol, and practically insoluble in water. Identification (1) Determine the absorption spectra of Erythromycin Stearate and Erythromycin Stearate RS as directed in the paste method under Infrared Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wave numbers. (2) Weigh about 10 mg each of Erythromycin Stearate and Erythromycin RS, dissolve each in 5 mL of chloroform, and use these solutions as the test solution and the standard solution, respectively. Perform the test with these solutions as directed under Thin-layer Chromatography. Spot 10 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the mixture of nbutanol, acetic acid and water (3:1:1), and air-dry the plate. Then allow the plate to expose to iodine vapor or spray 10 % sulfuric acid solution. The spots obtained from the test solution are the same with the corresponding spots from the standard solution in the Rf value. pH The pH of a solution obtained by dissolving 0.1 g of Erythromycin Stearate in 10 mL of water is between 6.0 and 11.0. Water Not more than 5.0 % (0.2 g, volumetric titration, direct titration). Residue on Ignition Not more than 2.0 % (1 g). Assay The Cylinder-plate method method (1) Agar media for seed and base layer- Use the medium in I 2 1) 2 under Microbial Assay for Antibiotics. Adjust the (2) pH of the solution so that it will be 7.8 to 8.0.

366 Monographs, Part I (2) Test organism- Staphylococcus aureus ATCC 6538 P. (3) Weigh accurately about 50 mg (potency) of Erythromycin Stearate, dissolve in 50 mL of methanol, and add 0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly 100 mL. Take exactly a suitable amount of this solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration test solution and the low concentration test solution, respectively. Separately, weigh accurately about 25 mg of Erythromycin RS, dissolve in 25 mL of methanol, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make exactly 100 mL, and use this solution as the standard stock solution. Keep the standard stock solution at not exceeding 5 C and use within 7 days. Take exactly a suitable amount of the standard stock solution before use, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinderplate method (I 8) as directed under Microbial Assay for Antibiotics.
Packaging and Storage Preserve in tight containers.

tazolam and Estazolam RS, respectively, in 1 mol/L hydrochloric acid TS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Perform the test with Estazolam as directed under the flame Coloration Test (2): a green color appears.
Melting Point

Between 229 C and 233 C

Estazolam
N N N

Cl

C16H11ClN4: 294.74 Estazolam, when dried, contains not less than 98.5% and not more than 101.0% of estazolam (C16H11ClN4).
Description Estazolam is a white to pale yellowish white crystals or crystalline powder, is odorless and has a bitter taste. Estazolam is soluble in methanol or acetic anhydride, sparingly soluble in ethanol, and practically insoluble in water or ether. Identification (1) Dissolve 10 mg of Estazolam in 3 mL of sulfuric acid: the solution shows a yellow-green fluorescence under ultraviolet light (main wavelength: 365 nm). (2) Determine absorption spectra of solutions of Es-

Purity (1) Clarity and color of solutionDissolve 0.1 g of Estazolam in 10 mL of ethanol: the solution is clear and colorless. (2) ChlorideDissolve 1.0 g of Estazolam in 10 mL of ethanol by heating, add 40 mL of water, cool with shaking in ice-water, allow to stand to attain ordinary temperature and filter. To 30 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS and 6 mL of ethanol (not more than 0.015%). (3) Heavy metalsProceed with 1.0 g of Estazolam according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Arsenic Prepare the test solution with 1.0 g of Estazolam according to Method 3 and perform the test (not more than 2 ppm). Prepare the test solution with 1.0 g of Estazolam according to Method (not more than 2 ppm). (5) Related substancesDissolve 0.2 g of Estazolam in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of hexane, chloroform and methanol (5 : 3 : 1) to a distance of about 10 cm and airdry the plate. Examine the plate under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution are not more intense than the principal spot from the standard solution. Loss on Drying Not more than 1.0% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (2 g). Assay Weigh accurately about 0.25 g of Estazolam, previously dried, dissolve in 100 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, endpoint detection method in titrimetry), until the solution changes to the second equivalence point. Perform a blank determination and make any necessary correction.

KP 9 367 mL. Pipet exactly 10 mL each of these solutions, add 5.0 mL of the internal standard solution, 100 mL of methanol and add water to make exactly 200 mL and use these solutions as the standard solutions. Perform the test with 25 L of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Estradiol to that of the internal standard for the test solution and the standard solution, respectively. Amount(mg) of estradiol (C18H24O2) =5C
H

Each mL of 0.1 mol/L perchloric acid VS = 14.737 mg of Cl6H11ClN4

Packaging and Storage Preserve in well-closed containers.

Estradiol
H3C OH H H

QT QS

H HO

C18H24O2: 272.38 Estradiol, when dried, contains not less than 97.0% and not more than 103.0% of estradiol (C18H24O2).
Description Estradiol is a white crystal or crystalline powder and is odorless. Estradiol is freely soluble in dioxane, soluble in acetone, sparingly soluble in ethanol, slightly soluble in sesame oil and practically insoluble in water. Identification (1) Determine the absorption spectra of solutions of Estradiol and Estradiol RS, respectively, in ethanol (1 in 20000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. The difference of absorption maxima in each absorption spectra is not less than 3.0% at about 280 nm. (2) Determine the infrared spectra of Estradiol and Estradiol RS as directed in the paste method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between + 76 and + 83(after drying, 0.1 g, dioxane, 10 mL, 100 mm). Melting Point

C : Final concentration of Estradiol in the standard solution (g/mL) Internal standard solutionA solution of ethylparabene in methanol (0.6 in 1000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 205 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of acetonitrile and water (55 : 100). Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 25 L of the standard solution, as directed under the above operating conditions, internal standard and the Estradiol and estrone are eluted in this order with the resolution between the peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 25 L of the standard solution, as directed under the above operating conditions, the relative standard of the ratios of peak area of Estradiol is not more than 2.0%.
Packaging and Storage tight containers.

Between 173 C and 179 C.

Preserve in light-resistant,

Water Not more than 3.5% (0.5 g, volume titration, direct titration). Assay Weigh accurately about 0.1 g of Estradiol and dissolve in methanol to make exactly 250 mL. Pipet exactly 10 mL of this solution, add 5.0 mL of the internal standard solution and 100 mL of methanol and water to make exactly 200 mL and use this solution as the test solution. Separately, weigh accurately a sufficient amount each of Estradiol RS (formerly determined water contents as directed under water determination assay) and Estrone RS and dissolve separately in methanol to contain about 400 g of Estradiol and 240 g of Estrone in 1

Estradiol Benzoate
H3C OH H H

H C O O

368 Monographs, Part I intense than the principal spot from the standard solution. C25H28O3: 376.49 Estradiol Benzoate, when dried, contains not less than 97.0% and not more than 101.0% of estradiol benzoate (C25H28O3).
Description Estradiol Benzoate is a white, crystalline powder and is odorless. Estradiol Benzoate is sparingly soluble in acetone, slightly soluble in methanol, ethanol or ether, and practically insoluble in water. Identification (1) Take 2 mg of Estradiol Benzoate, add 2 mL of sulfuric acid: a yellowish green color with a blue fluorescence is produced and the color of the solution changes to light orange on the careful addition of 2 mL of water. (2) Determine the infrared spectra of Estradiol Benzoate and Estradiol Benzoate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between + 54 and + 58 (after drying, 0.1 g, acetone, 10 mL, 100 mm). Melting Point Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 4 hours). Residue on Ignition Not more than 0.2% (0.1 g). Assay Weigh accurately about 10 mg each of Estradiol Benzoate and Estradiol Benzoate RS, previously dried and dissolve each in methanol to make exactly 20 mL. Pipet exactly 5 mL each of these solutions, add 5 mL of the internal standard solution, then add methanol to make 20 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 5 L of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Estradiol Benzoate to that of the internal standard for the test solution and the standard solution, respectively.

Amount (mg) of estradiol benzoate (C25H28O3) = amount (mg) of Estradiol Benzoate RS

QT QS

Internal standard solutionA solution of Progesterone in methanol (13 in 80000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 35 C. Mobile phase: A mixture of acetonitrile and water (7 : 3). Flow rate: Adjust the flow rate so that the retention time of Estradiol Benzoate is about 10 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, the internal standard and Estradiol Benzoate are eluted in this order with the resolutions between their peaks being not less than 9. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviations of the ratios of the peak area of Estradiol Benzoate to that of the internal standard is not more than 1.0%.
Packaging and Storage tight containers.

Between 191 C and 198 C

Purity (1) 3,17-EstradiolDissolve 10.0 g each of Estradiol Benzoate and Estradiol Benzoate RS in acetone to make exactly 200 mL each and use these solutions as the test solution and the standard solution, respectively. Pipet exactly 2 mL each of the test solution and the standard solution in separate glass-stoppered test tube, add boiling stones, evaporate the acetone by heating on a water-bath and dry the residue in a desiccator (in vacuum, P2O5) for 1 hour. Add 1.0 mL of dilute iron-phenol TS to each test tube. Stopper the test tubes loosely, heat for 30 seconds in a water-bath, shake in a water-bath for several seconds and heat for 2 minutes. Cool the solutions in ice for 2 minutes, add 4.0 mL of diluted sulfuric acid (7 in 20) and mix well: the solution obtained from the test solution has no more color than that from the standard solution. (2) Related substancesDissolve 40 mg of Estradiol Benzoate in 2 mL of acetone and use this solution as the test solution. Pipet exactly 1 mL of this solution, add acetone to make exactly 100 mL and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thinlayer chromatography, develop the plate with a mixture of chloroform and diethylamine (19 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution are not more

Preserve in light-resistant,

KP 9 369 solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Estradiol Benzoate to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of estradiol benzoate (C25H28O3) = amount (mg) of Etradiol Benzoate RS QT
QS

Estradiol Benzoate Injection

Estradiol Benzoate Injection is an oily solution for injection. Estradiol Benzoate Injection contains not less than 90.0% and not more, than 110.0% of the labeled amount of estradiol benzoate (C25H28O3: 376.49).
Method of Preparation Prepare as directed under Injections, with Estradiol Benzoate. Description oily liquid.

Estradiol Benzoate Injection is a clear,

Internal standard solutionA solution of methylparaben in methanol (1 in 2000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of acetonitrile and 0.023 mol/L ammonium phosphate solution (70 : 30). Flow rate: Adjust the flow rate so that the retention time of Estradiol Benzoate is about 10 minutes. System suitability System performance: When the prodedure is run with 20 L of the standard solution, as diredcted under the above operating conditions, the internal standard and Estradiol Benzoate are eluted in this order with a resolution between their peaks being not less than 5.0 System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above conditions, relative standard deviation of the ratio of the peak area is not more than 2.0%.
Packaging and Storage Preserve in hermetic containers.

Identification To a volume of Estradiol Benzoate Injection, equivalent to 1 mg of Estradiol Benzoate according to the labeled amount, add chloroform to make 5 mL and use this solution as the test solution. Separately, dissolve 1 mg of Estradiol Benzoate RS in 5 mL of chloroform and use this solution as the standard solution Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 50 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with dichloromethane to a distance of about 15 cm and air-dry the plate. Then develop the plate with a mixture of chloroform and methanol (99 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the principal spots obtained from the test solution and the spot obtained from the standard solution show the same Rf value. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Estradiol Benzoate Injection

(Aqueous Suspension)
Estradiol Benzoate Injection (Aqueous Suspension) is an aqueous suspension for injection. Estradiol Benzoate Injection (Aqueous Suspension) contains not less than 90.0% and not more than 110.0% of the labeled amount of estradiol benzoate (C25H28O3: 376.49).
Method of Preparation Prepare as directed under Injections, with Estradiol Benzoate. Description Estradiol Benzoate Injection (Aqueous Suspension) produces a white turbidity on shaking. Identification Extract a volume of Estradiol Benzoate Injection (Aqueous Suspension), equivalent to 1 mg of

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of well-mixed Estradiol Benzoate Injection (aqueous suspension), equivalent to about 10 mg of estradiol benzoate (C25H28O3), dissolve the crystal with an appropriate volume of methanol and add methanol to make exactly 100 mL. Pipet exactly 10 mL of this solution, add exactly 20 mL of the internal standard solution, add methanol to make 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Estradiol Benzoate RS, previously dried in desiccator (in vacuum, P2O5) for 4 hours, and prepare the standard solution in the same manner as directed for the preparation of the test solution. Perform the test with 20 L of the test solution and the standard

370 Monographs, Part I Estradiol Benzoate according to the labeled amount, with 5 mL of chloroform and use this extract as the test solution. Separately, dissolve 1 mg of Estradiol Benzoate RS in 5 mL of chloroform and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 50 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of chloroform and methanol (99 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (mail wavelength: 254 nm): the principal spots obtained from the test solution and standard solution show the same Rf value.
Sterility Test It meets the requirement. Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of well-mixed Estradiol Benzoate Injection (Aqueous Suspension), equivalent to about 2 mg of estradiol benzoate (C25H28O3), dissolve the crystals with an appropriate quantity of methanol and add methanol to make exactly 20 mL. Pipet exactly 10 mL of this solution, add exactly 10 mL of the internal standard solution, add methanol to make 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Estradiol Benzoate RS, previously dried in desiccator (reduced pressure, phosphorus pentoxide) for 4 hours, add exactly 10 mL of the internal standard solution and methanol to make 100 mL and use this solution as the standard solution. Proceed with the test and standard solutions as directed in the Assay under Estradiol Benzoate.

Estradiol Cypionate contains not less than 97.0% and not more than 103.0% of estradiol cypionate (C26H36O3), calculated on the dried basis.
Description Estradiol Cypionate is a white to almost white, crystalline powder and is ordorless or has a slightly odor. Estradiol Cypionate is soluble in ethanol, acetone, chloroform or dioxane, slightly soluble in corn oil and practically insoluble in water. Identification (1) Determine the absorption spectra of Estradiol Cypionate and Estradiol Cypionate RS, respectively, in ethanol solution (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit the same maximum and minimum absorption at the same wavelengths. Absorptivities 280 nm, calculated on the dried basis, do not differ by more than 3.0%. (2) Determine the infrared spectrum of Estradiol Cypionate and Estradiol Cypionate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit the same intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]25 D : Between +39 and +44 (after drying, 0.2 g, dioxane 10 mL, 100 mm). Melting Point

Between 149 C and 153 C.

Loss on Drying Not more than 1.0% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately 10 mg of each of Estradiol Cypionate and dry Estradiol Cypionate RS, previously dried, dissolve in 10 mL of the internal standard and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Estradiol Cypionate to that of the internal standard for the test solution and the standard solution, respectively.

Amount (mg) of estradiol benzoate (C25H28O3) = amount (mg) of Estradiol Benzoate RS QT 1 QS 5 Internal standard solutionA solution of progesterone in methanol (13 in 100000).
Packaging and Storage Preserve in hermetic containers.

Estradiol Cypionate
CH3 OCOCH 2CH2 H

Amount (mg) of estradiol cypionate(C26H36O3) = amount (mg) of Estradiol Cypionate RS QT

QS

Internal standard solutionDissolve 0.2 g of testosterone benzoate in 100 mL of tetrahydrefuran.

H

H HO

C26H36O3: 396.56

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm).

KP 9 371 Column: A stainless steel column about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Columm temperature: A room temperature. Mobile phase: Dissolve 0.8 g of ammonium nitrate in 300 mL of water and add 700 mL of acetonitrile. System suitability System performance: When the prodedure is run with 10 L of the standard solution, as directed under the above conditions, the resolution between Estradiol Cypionate and the internal standard is not less than 3.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution, as diredcted under the above conditions, the relative standard deviation of the ratio of the peak area is not more than 1.5%.
Packaging and Storage tight containers.

Injection, equivalent to about 10 mg of estradiol cypionate (C26H36O3) according to the labeled amount, transfer to a 100 mL volumetric flask, rinse the pipet used with small quantity of tetrahydrofuran, add 10 mL of the internal standard solution, mix and shake and add tetrahydrofuran to make 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Estradiol Cypionate RS and transfer to a 100 mL volumetric flask, add 10 mL of the internal standard solution and tetrahydrofuran to make 100 mL and use this solution as the standard solution. With the test solution and the standard solution, proceed as directed in the Assay under Estradiol Cypionate. Amount (mg) of estradiol cypionate (C26H36O3) = amount (mg) of Estradiol Cypionate RS
QT QS

Preserve in light-resistant,

Internal standard solutionDissolve 0.2 g of testosterone benzoate in 100 mL of tetrahydrofuran.

Packaging and Storage hermetic containers.

Estradiol Cypionate Injection

Estradiol Cypionate Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of estradiol cypionate (C26H36O3: 396.56).
Method of Preparation Prepare as directed under Injections, with Estradiol Cypionate.

Preserve in light-resistant,

Estradiol Valerate
H3C OCOCH2CH2CH2CH3 H H

Identification Transfer a volume of Estradiol Cypionate Injection, equivalent to 5 mg of Estradiol Cypionate according to the labeled amount, to a glass-stoppered test tube and add 30 mL of ethanol. Shake the mixture vigorously for 5 minutes, centrifuge until the two layers have been separated and transfer 50 mL of the ethanol layer with the aid of a hypodermic syringe to a beaker. Evaporate on a steam-bath to dryness, add 5 mL of potassium hydroxide solution (1 in 10) and heat in the steam-bath for 15 minutes and use this solution as the test solution. Separately, mix 50 mg of sulfanilic acid with 2 mL of 3 mol/L hydrochloric acid, warm the mixture, then cool in ice-water and slowly add, with agitation, 0.3 mL of sodium nitrate solution (1 in 10). Add this solution to the test solution: a red color is produced. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

H HO

C23H32O3: 356.50 Estradiol Valerate contains not less than 98.0% and not more than 102.0% of estradiol valerate (C23H32O3).
Description Estradiol Valerate is a white crystalline powder, is odorless or has a slightly oily odor. Estradiol Valerate is soluble in castor oil, methanol, benzylbenzoate or dioxane, sparingly soluble in sesame oil or peanut oil and practically insoluble in water. Identification Determine the infrared spectra of Estradiol Valerate and Estradiol Valerate RS, prepared by adding chloroform and potassium bromide to Estradiol Valerate or Estradiol Valerate RS, then grinding and drying at 105 o C , respectively: both spectra exhibits similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between +41 and +47 (0.25 g, dioxane, 10 mL, 100 mm). Melting Point

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Pipet exactly a volume of Estradiol Cypionate

Between 143 C and 150 C (Method 1).

372 Monographs, Part I

Purity (1) EstradiolDissolve 50 mg of Estradiol Valerate in 10 mL of acetone and use this solution as the test solution. Separately, dissolve 5 mg of Estradiol Valerate RS in 100 mL of acetone and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for Thinlayer chromatography. Then develop the plate with a mixture of cyclohexane and ethyl acetate (7 : 3) to a distance of about 15 cm and dry the plate at 90 o C for 30 minutes. Spray evenly a solution of sulfuric acid in methanol (3 in 10) on the plate and heat at 90 o C for 30 min: any spot other than the principal spot from the test solution or the spot corresponding to the Estradiol is not more intense or not larger than the spot from the standard solution (not more than 1.0%). (2) Free acidNeutralize 25 mL of ethanol, in a conical flask, with 0.01 mol/L sodium hydroxide VS to a faint blue color, using bromothymol blue TS. Accurately weigh about 0.50 g of Estradiol Valerate and dissolve Estradiol Valerate in the neutralized ethanol. Titrate rapidly with 0.01 mol/L sodium hydroxide VS to a faint blue color. (not more than 0.5% of valeric aid)

the Liquid Chromatography according to the following conditions and calculate the ratio, QT and QS , of the peak area of Estradiol Valerate to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of estradiol valerate(C23H32O3) = amount (mg) of Estradiol Valerate RS QT
QS

Internal standard solutionTestosterone benzoate solution in tetrahydrofuran (2 in 1000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: Dissolve 0.8 g of ammonium nitrate in 300 mL of water, add 700 mL of acetonitrile and mix. Flow rate: 2 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution, as directed under the above operating conditions, Estradiol Valerate and the internal standard are eluted in this order with the resolution between their peaks being not less than 3. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area is not more than 1.5%.
Packaging and Storage tight containers.

Each mL of 0.01 mol/L sodium hydroxide VS = 1.021 mg of C5H10O2 (3) Related substancesDissolve about 0.1 g of Estradiol Valerate in 10 mL of acetone and use this solution as the test solution. Separately, weigh 10 mg of estradiol valerate RS and add acetone to make 10 mL. Pipet exactly 0.1 mL, 0.5 mL, 1 mL and 2 mL of this solution and add acetone to each to make exactly 10 mL and use these solutions as the standard solutions (1), (2), (3) and (4). Perform the test with the test solution and the standard solutions as directed under the Thin-layer chromatography. Spot 20 L each of the test solution and the standard solutions (1), (2), (3) and (4) on a plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of cyclohexane and ether (4 : 1) to a distance of about 15 cm and air-dry the plate. Spray evenly sulfuric acid in ethanol (1 in 10) on the plate, heat the plate until burn and cool. Examine under ultraviolet light (main wavelength: 254 nm and 366 nm). Compare the intensities of any spot other than the principal spot from the test solution with those from the standard solution: the relative intensity is not more than 2.0%.
Water Not more than 0.1% (5 g, volume titration, direct titration). Assay Weigh accurately about 25 mg each of Estradiol Valerate and Estradiol Valerate RS, dissolve each in the internal standard solution to make exactly 25 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under

Preserve in light-resistant,

Estradiol Valerate Injection

Estradiol Valerate Injection is an oily solution for injection. Estradiol Valerate Injection contains not less than 90.0% and not more than 115.0% of the labeled amount of estradiol valerate (C23H32O3: 356.50).
Method of Preparation Prepare as directed under Injections, with Estradiol Valerate. Identification Add 0.5 mL of Estradiol Valerate Injection in 10 mL of hexane and 10 mL of 80% methanol in a separatory funnel and shake for 2 minutes. Allow to separate two layers. Add 1 mL of phenol TS (dilute 1 mL with 2 mL of water before use) and 3 mL of sodium carbonate VS (1 in 5): a blue color is observed. Purity EstradiolWeigh accurately a portion of Es-

KP 9 373 tradiol Valerate, add acetone to make a solution containing 30% of Estradiol according to the labeled amount. To 1.0 mL of this solution add oil labeled as vehicle to make exactly 10 mL and use this solution as the Estradiol solution. Perform the test with this solution and Estradiol Valerate Injection as directed under the Thin-layer chromatography. Spot 5 L each of solutions on a plate coated with Thin-layer chromatography silica gel and proceed as directed in the Purity for Estradiol in Estradiol Valerate: not more than 3.0%.
Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Description Estriol is a white, crystalline powder and is odorless. Estriol is sparingly soluble in methanol, slightly soluble in ethanol or dioxane, and practically insoluble in water or ether. Identification (1) Dissolve 10 mg of Estriol in 100 mL of ethanol by warming and use this solution as the test solution. Evaporate 1 mL of this solution on a water-bath to dryness, add 5 mL of a solution of sodium pphenolsulfonate in phosphoric acid (1 in 50), heat at 150 o C for 10 minutes and cool: a red-purple color develops. (2) Dissolve 10 mg each of Estriol and Estriol RS separately in 100 mL ethanol by warming and determine absorption spectra as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Estriol and Estriol RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between + 54 and + 62 (after drying, 40 mg, dioxane, 10 mL, 100 mm). Melting Point

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Pipet exactly a volume of Estradiol Valerate injection, equivalent to about 20 mg of estradiol valerate (C23H32O3) according to the labeled amount. Rinse the pipet with tetrahydrofuran and add 5.0 mL of the internal standard solution, dilute with tetrahydrofuran to make exactly 25 mL and use this solution as the test solution. Separately, weigh accurately about 20 mg of Estradiol Valerate RS, add 5.0 mL of the internal standard solution and dilute with tetrahydrofuran to make exactly 25 mL and use this solution as the standard solution. Proceed as directed for the Assay under Estradiol Valerate.

Between 281 C and 286 C.

Amount (mg) of estradiol valerate (C23H32O3) = amount (mg) of Estradiol Valerate RS

QT QS

Internal standard solutionTestosterone benzoate solution in tetrahydrofuran (8 in 1000).

Packaging and Storage hermetic containers.

Preserve in light-resistant,

Estriol
CH3 OH H OH H H H HO H

Purity (1) Heavy metalsProceed with 1.0 g of Estriol according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 40 mg of Estriol in 10 mL of ethanol by warming and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add ethanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for Thinlayer chromatography. Develop the plate with a mixture of chloroform, methanol, acetone and glacial acetic acid (18 : 1 : 1 : 1) to a distance of about 15 cm and air-dry the plate. Spray evenly diluted sulfuric acid (1 in 2) on the plate and heat at 105 C for 15 minutes: any spot other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (0.5 g, 105 C, 3 hours), Residue on Ignition Not more than 0.1% (0.5 g).

C18H24O3: 288.38 Estriol, when dried, contains not less than 97.0% and not more than 102.0% of estriol (C18H24O3).

Assay Weigh accurately about 25 mg each of Estriol and Estriol RS, previously dried, and dissolve separately in methanol to make exactly 50 mL. Pipet exactly 10 mL

374 Monographs, Part I each of these solutions, add exactly 5 mL of the internal standard solution, add methanol to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Estriol to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of Estriol (C18H24O3) = amount (mg) of Estriol RS
QT QS

Description Shake Estriol Injection (Aqueous Suspension): a white turbidity is produced. Identification Shake well, take a volume of Estriol Injection (Aqueous Suspension), equivalent to 2 mg of Estriol according to the labeled amount, add ethanol to make 20 mL and use this solution as the test solution. Proceed with the test solution as directed in the Identification (1) and (2) under Estriol. Sterility Test It meets the requirement. Determination of Volume of Injection in Containers It meets the requirement. Assay Shake well, pipet a volume of Estriol Injection (Aqueous Suspension), equivalent to about 5 mg of estriol (C18H24O3) and dissolve in methanol to make exactly 20 mL. Pipet exactly 4 mL of this solution, add 5.0 mL of the internal standard solution, then add methanol to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 25 mg of Estriol RS, previously dried at 105 o C for 3 hours and dissolve in methanol to make exactly 100 mL. Pipet exactly 4 mL of this solution, add 5.0 mL of the internal standard solution, then add methanol to make 50 mL and use this solution as the standard solution. Proceed as directed in the Assay under Estriol.

Internal standard solutionA solution of methyl benzoate in methanol (1 in 1000). Operating condition Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixed solution of water-methanol (51 : 49). Flow rate: Adjust the flow rate so that the retention time of Estriol is about 10 minutes. System suitability System performance: When the test is performed with 10 L of the standard solution under the above operating conditions, estriol and the internal standard are eluted in this order and the resolution between their peaks is not less than 8. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviations of the peak area of estriol to that of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Amount (mg) of estriol (Cl8H24O3) = amount (mg) of Estriol RS QT 1

QS

Internal standard solutionA solution of methyl benzoate in ethanol (1 in 5000).

Packaging and Storage Preserve in hermetic containers.

Estriol Tablets
Estriol Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of estriol (C18H24O3: 288.38).
Method of Preparation Prepare as directed under Tablets, with Estriol. Identification Weigh a portion of powdered Estriol Tablets, equivalent to 2 mg of Estriol according to the labeled amount, add 20 mL of ethanol, shake for 10 minutes, centrifuge and use the supernatant liquid as the test solution. Proceed with the test solution as directed in the Identification (1) and (2) under Estriol.

Estriol Injection (Aqueous Suspension)

Estriol Injection (Aqueous Suspension) is an aqueous suspension for injection. Estriol Injection (Aqueous Suspension) contains not less than 90.0% and not more than 110.0% of the labeled amount of estriol (Cl8H24O3: 288.38).
Method of Preparation Prepare as directed under Injections, with Estriol.

KP 9 375
Dissolution Test Perform the test with 1 tablet of Estriol Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water. Take 20 mL or more of the dissolved solution 30 minutes after starting the test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet exactly the subsequent V mL of the filtrate, add water to make exactly V' mL so that each mL contains about 0.1 g of estriol (C18H24O3) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately about 10 mg of Estriol RS, previously dried at 105 o C for 3 hours, dissolve in methanol to make exactly 100 mL, then pipet exactly 5 mL of this solution and add water to make exactly 100 mL. Pipet exactly 2 mL of this solution, add water to make exactly 100 mL and use this solution as the standard solution. Perform the test with 100 L each of the test solution and the standard solution according to the operating conditions as directed in the Assay under Estriol and determine the peak areas of Estriol, AT and AS , from the test solution and the standard solution, respectively. The dissolution rate of Estriol Tablets in 30 minutes is not less than 80%.

ments if the deviation exceeds 15%, not more than 1 sample shows deviation within 25%, and no sample shows deviation exceeding 25%.
Assay Weigh accurately and powder not less than 20 Estriol Tablets. Weigh accurately a portion of the powder, equivalent to about 1 mg of estriol (C18H24O3), add exactly 5 mL of water, disperse the fine particles with ultrasonic wave, shake with 25 mL of methanol for 10 minutes, centrifuge and take the supernatant liquid. Add 25 mL of methanol, repeat the above procedure twice, combine the supernatant liquid, add 5.0 mL of the internal standard solution, then add methanol to make 100 mL and use this solution as the test solution. Separately, weigh accurately about 25 mg of Estriol RS, previously dried at 105 C for 3 hours and dissolve in methanol to make exactly 100 mL. Pipet exactly 4 mL of this solution, add 5.0 mL of the internal standard solution, then add methanol to make 100 mL and use this solution as the standard solution. Proceed with 20 L each of the test solution and the standard solution as directed in the Assay under Estriol.

Amount (mg) of estriol (C18H24O3) = amount (mg) of Estriol RS

QT 1 QS 25

Dissolution rate (%) with respect to the labeled amount of estriol (C18H24O3) = W S AT V ' 1 9
AS V C

10

Internal standard solutionA solution of methyl benzoate in methanol (1 in 5000).

Packaging and Storage Preserve in tight containers.

WS : Amount of Estriol RS (mg) C: Labeled amount of estriol (C18H24O3) in 1 tablet (mg)

Etacrynic Acid
CH2 O CH3CH2 C C OCH2CO2H

Uniformity of Dosage Units It meets the requirement of the Content Uniformity Test when is performed the test according to the following method. Take one tablet of Estriol Tablets, add exactly 5 mL of water, disperse the fine particles with ultrasonic wave, add exactly 15 mL of methanol and shake for 15 minutes. Centrifuge this solution for 10 minutes, pipet a definite amount of the supernatant liquid and add methanol to make exactly a definite volume of solution so that each mL of the solution contains about 5 g of estriol (C18H24O3). Pipet exactly 5 mL of this solution, add exactly 1 mL of the internal standard solution and use this solution as the test solution. Proceed with 20 L of the test solution as directed in the Assay under Estriol. Use a solution of methyl benzoate in methanol (1 in 40000) as the internal standard solution. Calculate the mean value from each ratio of peak areas of 10 samples: the samples conform to the requirements of the deviation (%) of the mean value and each ratio of peak areas is within 15%. If the deviation (%) exceeds 15% and 1 sample shows deviation within 25%, repeat the test with 20 samples. Calculate the deviation (%) of the mean value from each ratio of peak areas of the 30 samples used in the 2 tests and each ratio of peak areas: the samples conform to the require-

Cl

Cl

C13H12Cl2O4: 303.14 Etacrynic Acid, when dried, contains not less than 98.0% and not more than 101.0% of etacrynic acid (C13H12Cl2O4).
Description Etacrynic Acid is a white, crystalline powder, is odorless and has a slightly bitter taste. Etacrynic Acid is very soluble in methanol, freely soluble in ethanol, glacial acetic acid or ether, and very slightly soluble in water. Identification (1) Dissolve 0.2 g of Etacrynic Acid in 10 mL of glacial acetic acid and to 5 mL of this solution, add 0.1 mL of bromine TS: the color of the test solution disappears. To the remaining 5 mL of the solution, add 0.1 mL of potassium permanganate TS: the color of the

376 Monographs, Part I test solution changes to light orange immediately. (2) Take 10 mg of Etacrynic Acid, add 1 mL of sodium hydroxide TS and heat in a water-bath for 3 minutes. After cooling, add 1 mL of chromotropic acid TS, and heat in a water-bath for 10 minutes: a deep purple color develops. (3) Determine absorption spectra of solutions of Etacrynic Acid and Etacrynic Acid RS, respectively, in methanol (1 in 20000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Perform the test with Etacrynic Acid as directed under the Flame Coloration Test (2): a green color appears.
Melting Point

tightly at once, shake well and titrate the liberated iodine with 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of starch TS). Perform a blank determination and make any necessary correction. Each mL of 0.05 mol/L bromine VS = 15.157 mg of C13H12Cl2O4
Packaging and Storage Preserve in well-closed containers.

Etacrynic Acid Tablets

Etacrynic Acid Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of etacrynic acid (C13H12Cl2O4: 303.14).
Method of Preparation Prepare as directed under Tablets with Etacrynic Acid. Identification Weigh a portion of powdered Etacrynic Acid Tablets, equivalent to 0.3 g of Etacrynic Acid according to the labeled amount, add 25 mL of 0.1 mol/L hydrochloric acid TS and extract with 50 mL of dichloromethane. Filter the dichloromethane extract and evaporate the filtrate on a water-bath to dryness. Proceed with the residue as directed in the Identification under Etacrynic Acid. Dissolution Test Perform the test with 1 tablet of Etacrynic Acid Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water. Take 20 mL or more of the dissolved solution 45 minutes after starting the test, and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately about 55 mg of Etacrynic Acid RS for Assay, previously dried at 60 o C for 2 hours, dissolve in 10 mL of methanol, and add water to make exactly 100 mL. Pipet exactly 5 mL of this solution, add water to make exactly 100 mL, and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 277 nm as directed under the Infrared-visible Spectrophotometry. The dissolution rate of Etacrynic Acid Tablets in 45 minutes is not less than 70%.

Between 121 C and 125 C.

Purity (1) Clarity and color of solutionDissolve 1.0 g of Etacrynic Acid in 10 mL of methanol: the solution is clear and colorless.

(2) Heavy metalsProceed with 1.0 g of Etacrynic Acid according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicWeigh 1.0 g of Etacrynic Acid according to Method 3 and perform the test. Add 10 mL of a solution of magnesium nitrate in ethanol (1 in 50), then add 1.5 mL of strong hydrogen peroxide water and fire to burn (not more than 2 ppm). (4) Related substancesDissolve 0.20 g of Etacrynic Acid in 10 mL of ethanol and use this solution as the test solution. Pipet exactly 3 mL of the test solution, add ethanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of chloroform, ethyl acetate and glacial acetic acid (6 : 5 : 2) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.25% (1 g, in vacuum, 60 o C , 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.1 g of Etacrynic Acid, previously dried, place in an iodine bottle, dissolve in 20 mL of glacial acetic acid and add exactly 20 mL of 0.05 mol/L bromine VS. To this solution, add 3 mL of hydrochloric acid, stopper tightly at once, shake and allow to stand in a dark place for 60 minutes. Add carefully 50 mL of water and 15 mL of potassium iodide TS, stopper

Dissolution rate (%) with respect to the labeled amount of etacrynic acid (C13H12Cl2O4) = WS
AT 1 45 AS C

WS : Amount of Etacrynic Acid RS (mg)

KP 9 377 C: Labeled amount of etacrynic acid (C13-H12Cl2O4) in 1 tablet (mg)

Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Etacrynic Acid Tablets. Weigh accurately a portion of the powder, equivalent to about 0.1 g of etacrynic acid (C13H12Cl2O4), add 25 mL of 0.1 mol/L hydrochloric acid TS and extract with three 30 mL volumes of dichloromethane. Filter the dichloromethane extracts through a pledget of absorbent cotton into an iodine bottle. Wash the pledget of absorbent cotton with a small amount of dichloromethane and combine the washing with the extracts. Evaporate this solution on a water-bath to dryness in a current of air, to the residue, add 20 mL of glacial acetic acid and proceed as directed in the Assay under Etacrynic Acid.

Specific Optical Rotation [ ]20 D : Between +5.5 and +6.1 (after drying, 5.0 g, water, 50 mL, 200 mm). Melting Point

Between 200 o C and 204 o C .

Each mL of 0.05 mol/L bromine VS = 15.157 mg of C13H12Cl2O4

Packaging and Storage Preserve in well-closed containers.

Ethambutol Hydrochloride
H CH 3CH 2 C NHCH 2CH 2NH CH 2OH C H CH 2CH 3 2 HCl

CH 2OH

C10H24N2O22HCl: 277.23 Ethambutol Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of ethambutol hydrochloride (C10H24N2O22HCl).
Description Ethambutol Hydrochloride is a white crystal or crystalline powder, is odorless, and has a bitter taste. Ethambutol Hydrochloride is very soluble in water, soluble in methanol or ethanol, and practically insoluble in ether. The pH of a solution of Ethambutol Hydrochloride (1 in 20) is between 3.4 and 4.0. Identification (1) Take 10 mL of a solution of Ethambutol Hydrochloride (1 in 100), add 0.5 mL of cupric sulfate TS and 2 mL of sodium hydroxide TS: a deep blue color is observed. (2) Dissolve 0.1 g of Ethambutol Hydrochloride in 40 mL of water, add 20 mL of picric acid TS and allow to stand for 1 hour. Collect the precipitate, wash with 50 mL of water and dry at 105 C for 2 hours: the precipitate melts between 193 C and 197 C. (3) A solution of Ethambutol Hydrochloride (1 in 30) responds to the Qualitative Tests for chloride.

Purity (1) Clarity and color of solutionDissolve 1.0 g of Ethambutol Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Ethambutol Hydrochloride according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicWeigh 1.0 g of Ethambutol Hydrochloride according to Method 1 and perform the test. (not more than 2 ppm). (4) 2-AminobutanolDissolve about 50 mg of Ethambutol Hydrochloride, weighed accurately, in water to make exactly 100 mL and use this solution as the test solution. Separately, dissolve adequate amount of 2Amino-1-butanol RS, weighed accurately, in water and dilute sequentially to make a solution containing 5 g per mL and use this solution as the standard solution. Transfer 10 mL of the test solution into 100 mL Erlenmeyer flask with glass stopper and add 10 mL of water and 20 mL of boric acid buffer solution. Separately, mix 10.0 mL of the test solution, 10.0 mL of the standard solution, and 20 mL of Boric acid buffer solution in 100 mL Erlenmeyer flask with glass stopper. While the contents in two flasks are mixed on shaker, add quickly 10 mL of Fluorescamine solution and stopper them and mix. Perform the test after exactly 1 minute with these solutions as directed under the Fluorimetry. Determine the fluorescence intensity at 385 nm of excitation wavelength and 485 nm of emission wavelength: the fluorescence intensity obtained from the test solution is not more than that of the difference between two solutions (not more than 1.0%). Boric acid buffer solution : Weigh about 1.24 g of boric acid, dissolve it in 90 mL of water, adjust with 5 mol/L sodium hydroxide to pH 9.0, and dilute with water to exactly 100 mL and mix. Fluorescamine solution : Transfer 5 mg of fluorescamine into cylinder equipped with stopper and scale line, and dissolve in 50 mL of acetone. Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Ethambutol Hydrochloride, previously dried, add 100 mL of glacial acetic acid and 5 mL of mercury (II) acetate TS for nonaqueous titration, mix with stirring, and titrate with 0.1 mol/L Perchloric acid VS until the color of the solution changes from blue to blue-green (indicator : methylrosaniline chloride TS). Perform a blank determination, and

378 Monographs, Part I make any necessary correction. Each mL of 0.1 mol/L perchloric acid VS = 13.862 mg of C10H24N2O22HCl
Packaging and Storage Preserve in tight containers.

Ethambutol Hydrochloride Tablets

Ethambutol Hydrochloride Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of ethambutol hydrochloride (C10H24N2O22HCl: 277.23).
Method of Preparation Prepare as directed under Tablets, with Ethambutol Hydrochloride. Identification (1) Take a portion of powdered Ethambutol Hydrochloride Tablets, equivalent to 0.1 g of Ethambutol Hydrochloride according to the labeled amount, add 3 mL of methanol in a glass motor. Add 5 mL of methanol to make a suspension, then filter through a funnel lined with a filter paper previously moistened with methanol and collect the filtrate in a beaker containing 100 mL of acetone. Stir the mixture and allow crystallization to proceed for 15 minutes. Decant the liquid and gently dry the crystals with the aid of a current of air until the odor of methanol is no longer detectable. Determine the infrared spectra of this crystals and Ethambutol Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) An aqueous solution (I in 10) of the crystals obtained in (1) responds to Qualitative Tests for chloride. Purity 2-AminobutanolTransfer adequate number of Ethambutol Hydrochloride Tablets, equivalent to 400 mg of Ethambutol Hydrochloride, into beaker, add acetone upto immersion of all sample, allow to stand for 15 minutes, decant acetone, dry. Powder the tablets removed the coating, add methanol, mix, dilute to exactly 100 mL and filter. Pipet exactly 25 mL of the filtrate, dilute with water to make exactly 200 mL. After 15 minutes filter with dried filter paper, discard first turbid filtrate, use subsequent filtrate as the test solution. Then, perform the Purity (4) under Ethambutol Hydrochloride. Dissolution Test Perform the test with 1 tablet of Ethambutol Hydrochloride Tablets at 100 revolutions per minute according to Method 1 under the Dissolution Test, using 900 mL of the 1st fluid of the Disintegration Tests. Filter after 45 minutes from starting the test and use this solution as the test solution. Separately, weigh accurately a portion of Ethambutol Hydrochloride RS, previously dried at 105C for 2 hours and dissolve in water to make

0.1 mg per mL and use this solution as the standard solution. Transfer 1 mL each of the test solution and the standard solution and water to 3 separate centrifuge test tubes with glass-stopper. Add 5.0 mL of bromocresol green solution and 10.0 mL of chloroform, stoppers and shake the mixture vigorously. Allow the mixture to separate, discard the upper aqueous layer and filter the 3 chloroform layers through separate pledges of cotton. Determine the absorption spectra with these solutions as directed under the Ultraviolet-visible Spectrophotometry, at the wavelength of maximum absorbance at about 415 nm. The dissolution rate of Ethambutol Hydrochloride Tablets in 45 minutes should be not less than 75%. Phosphate bufferDissolve 38.0 g of monobasic sodium phosphate and 2.0 g of anhydrous dibasic sodium phosphate in water to obtain 1000 mL of solution. Bromocresol green solutionDissolve 0.2 g of bromocresol green in 30 mL of water and 6.5 mL of 0.1 mol/L sodium hydroxide. Dilute with phosphate buffer to 500 mL, mix and add 0.1 mol/L hydrochloric acid to adjust to a pH of 4.6 0.1.
Uniformity of Dosage Units It meets the requirement. Assay Weigh and finely powder not less than 20 Ethambutol Hydrochloride Tablets. Dissolve an accurately weighed amount of the powder, equivalent to about 30 mg of Ethambutol Hydrochloride according to the labeled amount, in water, shake to mix, add water to make exactly 100 mL and filter. Discard 10mL of first filtrate and use subsequent filtrate as the test solution. Separately, weigh accurately about 30 mg of Ethambutol Hydrochloride RS, dissolve in and dilte with water to make exactly 100 mL and use this solution as the standard solution. Perform the test with exactly 50 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area, AT and AS , respectively.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 200 nm). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Mobile phase: A mixture of the solution, prepared by mixing 1.0 mL of triethylamine with 1000 mL of water and adjusting with phosphoric acid to pH 7.0, and acetonitrile (1 : 1). Flow rate: 1.0 mL/minute System suitability System performance: When the procedure is run with 50 L of the standard solution under the above operating condition, the symmetry factor of the peak of ethambutol are not less than not more than 2.0. System repeatability: When the test is repeated 6

KP 9 379 times with 50 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of ethambutol is not more than 2.0%.
Packaging and Storage Preserve in well-closed containers.

Ethenzamide
O C NH2 OCH2CH3

(3) Heavy metalsProceed with 2.0 g of Ethenzamide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicTake 0.40 g of Ethenzamide, add 0.3 g of potassium nitrate and 0.5 g of anhydrous sodium carbonate, mix thoroughly, ignite the mixture gradually and cool. Dissolve the residue in 10 mL of dilute sulfuric acid and heat the solution until white fumes begin to evolve. After cooling, add water carefully to make 5 mL, use this solution as the test solution and perform the test (not more than 5 ppm). (5) SalicylamideDissolve 0.20 g of Ethenzamide in 15 mL of dilute ethanol (2 in 3) and add 2 to 3 drops of dilute ferric chloride TS: no purple color develops.
Loss on Drying Not more than 1.0% (1 g, silica gel, 3 hours).

Ethoxybenzamide

C9H11NO2: 165.19

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 20 mg each of Ethenzamide and Ethenzamide RS, previously dried and dissolve each in 70 mL of ethanol by warming and after cooling, add ethanol to make exactly 100 mL. Pipet 5.0 mL each of these solutions, add ethanol to make exactly 50 mL and use these solutions as the test solution and the standard solution, respectively. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 290 nm as directed under the Ultraviolet-visible Spectrophotometry, using ethanol as the blank.

Ethenzamide, when dried, contains not less than 98.0% and not more than 101.0% of ethenzamide (C9H11NO2).
Description Ethenzamide is a white crystal or crystalline powder, is odorless and tasteless. Ethenzamide is soluble in ethanol or acetone, slightly soluble in ether, and practically insoluble in water. Ethenzamide's saturated solution is neutral. Ethenzamide begins to sublime slightly at about 105 C. Identification (1) Take 0.5 g of Ethenzamide, add 5 mL of sodium hydroxide TS and heat the mixture gently: the gas evolved turns moistened red litmus paper to blue. (2) Take 0.2 g of Ethenzamide, add 10 mL of hydrobromic acid and boil the mixture gently for 1 hour under a reflux condenser. Cool in ice-water, collect the separated crystalline precipitate, wash with three 5 mL volumes of ice-water and dry in a desiccator (in vacuum, silica gel) for 2 hours: the precipitate melts between 158 C and 161 C. Melting Point Between 131 C and 134 C. Purity (1) ChlorideDissolve 0.5 g of Ethenzamide in 30 mL of acetone, add 6 mL of dilute nitric acid and dilute with water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.7 mL of 0.01 mol/L hydrochloric acid VS, add 30 mL of acetone and 6 mL of dilute nitric acid and dilute with water to make 50 mL (not more than 0.050%). (2) SulfateDissolve 0.5 g of Ethenzamide in 30 mL of acetone, add 1 mL of dilute hydrochloric acid and dilute with water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.50 mL of 0.005 mol/L sulfuric acid VS, add 30 mL of acetone and 1 mL of dilute hydrochloric acid and dilute with water to 50 mL (not more than 0.048%).

Amount (mg) of ethenzamide (C9H11NO2) = amount (mg) of Ethenzamide RS

AT AS

Packaging and Storage Preserve in well-closed containers.

Anesthetic Ether
CH3CH2OCH 2CH 3

C4H10O: 74.12 Anesthetic Ether contains not less than 96.0% and not more than 98.0% (by specific gravity) of ether (C4H10O). Anesthetic Ether contains small quantities of ethanol and water. Suitable stabilizers may be added. Anesthetic Ether is not to be used for anesthesia if it has been removed from the original container for more than 24 hours.
Description Anesthetic Ether is a colorless, clear, mobile liquid, having a characteristic odor. Anesthetic Ether is miscible with ethanol. Anesthetic Ether is soluble in

380 Monographs, Part I water. Anesthetic Ether is highly volatile and flammable. Anesthetic Ether is slowly oxidized by the action of air and light, with the formation of peroxides. The vapor of Anesthetic Ether, when mixed with air and ignited, may explode violently, Boiling pointBetween 35 C and 37 C.
Specific Gravity
20 d 20

C20H24O2: 296.40 Ethinylestradiol, when dried, contains not less than 98.0% and not more than 101.0% of ethinyestradiol (C20H24O2).
Description Ethinylestradiol is a white to pale yellow crystal or crystalline powder and is odorless. Ethinylestradiol is freely soluble in pyridine or tetrahydrofuran, soluble in ethanol or ether, and practically insoluble in water. Ethinylestradiol dissolves in sodium hydroxide TS. Identification (1) Dissolve 2 mg of Ethinylestradiol in 1 mL of a mixture of ethanol and sulfuric acid (1 : 1): a purplish red color develops with a yellow-green fluorescence. Add carefully 2 mL of water to this solution: the color of the solution changes to red-purple. (2) Transfer 20 mg of Ethinylestradiol to a test tube with glass-stopper, dissolve in 10 mL of a solution of potassium hydroxide (1 in 20), add 0.1 g of benzoyl chloride and shake. Collect the resulting precipitate, recrystallize from methanol and dry in a desiccator (in vacuum, P2O5): the precipitate melts between 200 C and 202 C. Specific Optical Rotation [ ]20 D : Between 26 and 31 (after drying, 0.1 g, pyridine, 25 mL, 200 mm). Melting Point Between 180 C and 186 C or Between 142 C and 146 C. Purity EstroneDissolve 5 mg of Ethinylestradiol in 0.5 mL of ethanol and add 50 mg of m-dinitrobenzene. Add 0.5 mL of freshly prepared dilute potassium hydroxide-ethanol TS, allow to stand in a dark place for 1 hour and add 10 mL of ethanol: the solution has no more color than the following control solution.

: Between 0.718 and 0.721.

Purity (1) Foreign odorPlace 10 mL of Anesthetic Ether in an evaporating dish and allow to evaporate spontaneously to a volume of about 1 mL: no foreign odor is perceptible. Drop this residue onto a piece of clean, odorless filter paper to evaporate the ether: no foreign odor is perceptible. (2) AcidPlace 10 mL of diluted ethanol (4 in 5) and 0.5 mL of phenolphthalein TS in a glass-stoppered flask and add 0.02 mol/L sodium hydroxide VS dropwise to produce a red color which persists after shaking for 30 seconds. Add 25 mL of Anesthetic Ether, stopper the flask, shake gently and add 0.40 mL of 0.02 mol/L sodium hydroxide VS with shaking: a red color develops. (3) AldehydeTo 100 mL of water in a glassstoppered flask add 10 mL of Anesthetic Ether and 1 mL of a solution of sodium bisulfite (1 in 1000), stopper tightly, shake vigorously for 10 seconds and allow the mixture to stand in a cool place for 30 minutes, protected from light. Add 2 mL of starch TS and add dropwise 0.01 mol/L iodine VS until a pale blue color develops. Shake with about 2 g of sodium bicarbonate to decolorize the solution and add 1 mL of diluted 0.01 mol/L iodine VS (9 in 40): a blue color develops. Keep the temperature of the solution below 18 o C during the procedure. (4) PeroxidePlace 10 mL of Anesthetic Ether in a Nessler tube, add 1 mL of a freshly prepared solution of potassium iodide (1 in 10), shake occasionally for 1 hour, protecting from light, then add 1 mL of starch TS and shake well: no color is produced in the aqueous layer and in the ether layer. (5) Residue on evaporationEvaporate 50 mL of Anesthetic Ether and dry the residue at 105 C for 1 hour: the weight of the residue is not more than 1.0 mg. Packaging and Storage Preserve in light-resistant, tight containers with avoiding from filling up and fire. Store at not exceeding 25 C.

Control solutionProceed in the same manner as mentioned above, omitting Ethinylestradiol.

Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 4 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately about 0.2 g of Ethinylestradiol, previously dried, and dissolve in 40 mL of tetrahydrofurane. Add 10 mL of a solution of silver nitrate (1 in 20) and titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry).

Ethinylestradiol
H3C C CH OH

Each mL of 0.1 mol/L sodium hydroxide VS = 29.640 mg of C20H24O2

H

H HO

Packaging and Storage tight containers.

Preserve in light-resistant,

KP 9 381

Ethinylestradiol Tablets
Ethinylestradiol Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of ethinylestradiol (C20H24O2: 296.40).
Method of Preparation Prepare as directed under Tablets, with Ethinylestradiol. Identification (1) Evaporate to dryness 5 mL of the test solution obtained in Assay and add 2 mL of a mixture of sulfuric acid and ethanol (2 : 1) to the residue: a light red color with a yellow fluorescence develops. To the solution, add carefully 4 mL of water: the color of the solution changes to red-purple. (2) Evaporate to dryness 10 mL of the test solution obtained in Assay, add 0.2 mL of acetic acid and 2 mL of phosphoric acid to the residue and heat in a water-bath for 5 minutes: a red color with a yellow-green fluorescence develops. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement of the Content Uniformity Test when the test is performed according to the following method. Place 1 tablet of Ethinylestradiol Tablets in a separator, add 10 mL of the 2nd fluid of test fluids under the Disintegration Test and shake until the tablet is disintegrated. Add 10 mL of dilute sulfuric acid and 20 mL of chloroform, shake vigorously for 5 minutes and filter the chloroform layer into a conical flask through filter paper on which 5 g of anhydrous sodium sulfate is placed. Extract the aqueous layer with two 20 mL volumes of chloroform, proceed with the extracts in the same manner as before and combine the filtrates with the previous one. Evaporate gently the combined filtrate in a water-bath with the aid of a current of nitrogen, dissolve the residue in exactly 100 mL of methanol and centrifuge, if necessary. Pipet x mL of the supernatant liquid, add methanol to make exactly V mL of a solution containing about 0.04 g of ethinylestradiol (C20H24O2) per mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Ethinylestradiol RS, previously dried in a desiccator (in vacuum, P2O5) for 4 hours, dissolve in methanol, dilute to a volume containing about 0.04 g of ethinylestradiol (C20H24O2) per mL, and use this solution as the standard solution. Pipet exactly 4 mL of sulfuric acidmethanol TS into three test tubes with glass-stopper, T, S and B, cool in ice, to each tube, add exactly 1 mL each of the test solution, the standard solution and methanol, shake immediately and allow to stand in a water-bath at 30 C for 40 minutes. And allow to stand in a water-bath at 20 o C for 5 minutes. Perform the test with the test solution and the standard solution as directed under the Fluorometry. Determine the fluorescence intensities, FT ,

FS and FB , of the test solution, the standard solution and the blank solution, respectively, using the fluorophotometer at about 460 nm of the excitation and at about 493 nm of the fluorescence.

Amount (mg) of ethinylestradiol (C20H24O2) = amount (mg) of Ethinylestradiol RS

FT F B V 1 F S F B 2500 x

Assay Weigh accurately and powder not less than 20 Ethinylestradiol Tablets. Weigh accurately a portion of the powder, equivalent to about 0.5 mg of ethinylestradiol (C20H24O2), place in a 50 mL beaker, add 2 mL of water, shake well, add 3 mL of chloroform and shake well again. Add 4 g of siliceous earth for chromatography, mix well until the contents do not stick to the inner wall of the beaker and use the substance as the sample. Take the chromatographic column, add the sample with a funnel and pack in proper hardness. Mix well the sample sticking to the beaker with 0.5 g of siliceous earth for chromatography and place in the chromatographic tube. Wipe off the test solution sticking to the beaker and the tamping rod with glass wool and place in the chromatographic tube. Push down the sample and press lightly on the chromatographic column to make the height of the column, 110 mm to 130 mm. Take 70 mL of chloroform, rinse the inner wall of the chromatographic tube with a portion of the chloroform and transfer the remaining portion to the chromatographic tube. Collect the efficient solution at a flow rate not more than 0.8 mL per minute. After completing the elution, rinse the lower end of the chromatographic tube with a small quantity of chloroform, add chloroform to make exactly 100 mL and use this solution as the test solution. Weigh accurately about 10 mg of Ethinylestradiol RS, previously dried in a desiccator (in vacuum, P2O5) for 4 hours and dissolve in chloroform to make exactly 100 mL. Pipet exactly 5 mL of this solution and add chloroform to make exactly 100 mL. Use this solution as the standard solution. Transfer exactly 6 mL each of the test solution and the standard solution to each separators and add 20 mL of isooctane. Add exactly 10 mL of a mixture of sulfuric acid and methanol (7 : 3), shake vigorously for 5 minutes, allow to stand in a dark place for 15 minutes and centrifuge. Perform the test with the resulting color solutions as directed under the Ultraviolet-visible Spectrophotometry, using a solution, prepared with 6 mL of chloroform in the same manner, as the blank. Determine the absorbances, AT and AS , of the subsequent solutions obtained from the test solution and the standard solution at 540 nm, respectively. Chromatographic column: Pack a pledget of glass wool in the bottom of a column 25 mm in inside diameter and 300 mm in length and place 5 g of anhydrous sodium sulfate on the glass wool. Separately, place 5 g of siliceous earth for chromatography in a 200-mL beaker, soak well in 4 mL of 1 mol/L hydrochloric acid TS and mix uniformly. Put the siliceous earth into the chromato-

382 Monographs, Part I graphic column in small portions to make 60 to 80 mm in length in proper hardness with 1 tamping rod. Amount (mg) of ethinylestradiol (C20H24O2) = amount (mg) of Ethinylestradiol RS
AT 1 A S 20

Packaging and Storage Preserve in tight containers.

Ethionamide
N CH2CH3

C S

NH2

C8H10N2S: 166.24 Ethionamide, when dried, contains not less than 98.5% and not more than 101.0% of ethionamide (C8H10N2S).
Description Ethionamide is yellow crystals or crystalline powder, having a characteristic odor and taste. Ethionamide is soluble in methanol or glacial acetic acid, sparingly soluble in ethanol, slightly soluble in ether, and practically insoluble in water. Identification (1) Determine the absorption spectra of solutions of Ethionamide and Ethionamide RS, respectively, in methanol (3 in 160000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Ethionamide and Ethionamide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point

without exposure to light, using light-resistant vessels. Dissolve 0.20 g of Ethionamide in 10 mL of methanol and use this solution as the test solution. Pipet exactly 0.5 mL of test solution, add acetone to make exactly 100 mL and use this solution as the standard solution (1). Separately, pipet exactly 0.2 mL of the test solution, add acetone to make exactly 100 mL, and use this solution as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, hexane and methanol (6 : 2 : 1) to a distance of about 15 cm and air-dry the plate. Examine the plate under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot obtained with the test solution is not more intense than the corresponding spot with the standard solution (1), and the number of the spots other than the principal spot obtained with the test solution being more intense than that with the standard solution (2) is not more than one.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Ethionamide, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from orange-red to dark orange-brown (indicator: 2 mL of -naphtholbenzeine TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 16.624 mg of C8H10N2S

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Between 161 C and 165 C.

Purity (1) AcidDissolve 3.0 g of Ethionamide in 30 mL of methanol by warming, add 90 mL of water, allow to stand in ice-water for 1 hour and filter. Take 80 mL of the filtrate, add 0.8 mL of cresol red TS and 0.20 mL of 0.1 mol/L sodium hydroxide VS: a red color develops. (2) Heavy metalsProceed with 1.0 g of Ethionamide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicWeigh 1.0 g of Ethionamide and perform according to Method 3. Add 10 mL of a solution of magnesium nitrate in ethanol (1 in 50), then add 1.5 mL of strong hydrogen peroxide water and fire to burn (not more than 2 ppm). (4) Related substancesProceed this procedure

Ethionamide Tablets
Ethionamide Tablets contain not less than 95.0% and not more than 110.0% of the labeled amount of ethionamide (C8H10N2S: 166.24).
Method of Preparation Prepare as directed under Tablets, with Ethionamide. Identification (1) The test solution used in Assay shows maximum absorption at 290 nm. (2) Weigh a sufficient portion of powdered Ethionamide Tablets, equivalent to 1 g of Ethionamide, add 50 mL of methanol. After shaking, filter with porous glass filter and evaporate the filtrate on a water-bath to dry-

KP 9 383 ness: the melting point of the residue is not less than 155 C and not more than 164 C.
Dissolution Test Take 1 Tablet of Ethionamide Tablet, perform the test as directed in Method 1 under the Dissolution Test at 100 revolutions per minute, and using 900 mL of 0.1 mol/L hydrochloric acid as the dissolution medium. After 45 minutes from starting of the test, take the dissolved solution, and filter through a membrane filter and dilute the filtrate with the dissolution medium. The diluted filterate used as the test solution. Separately weigh accurately the suitable weight of Ethionamide standard, and dissolve in the dissolution medium to make exactly the same concentration of the test solution, and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution, respectively, at the maximum absorption wavelength of about 274 nm as directed under the Ultraviolet-visible Spectrophotometry using dissolution medium as a blank. The dissolution rate of Ethionamide Tablets after 45 minutes should be not less than 75%. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Ethionamide Tablets. Weigh accurately a portion of the powder, equivalent to about 0.1 g of ethionamide (C8H10N2S: 166.24) and put into porous filter bottle with vacuum and extract with ten 10 mL of methanol (complete every filtration). Collect the filtrates and add methanol to make 250 mL. Pipet 5.0 mL of this solution and add methanol to make 200 mL and use this solution as the test solution. Separately weigh accurately 0.05g of Ethionamide RS and add methanol to make 100 mL. Pipet 2.0 mL of this solution and add methanol to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 290 nm as directed under the Ultraviolet-visible Spectrophotometry, using methanol as the blank.
Amount (mg) of ethionamide (C8H10 N 2S) = amount (mg) of Ethionamide RS
AT 2 AS

C7H11NO2: 141.17 Ethosuximide contains not less than 98.5% and not more than 101.0% of ethosuximide (C7H11NO2) calculated on the anhydrous basis.
Description Ethosuximide is a white, paraffin-like solid or powder, is odorless or has a slight, characteristic odor. Ethosuximide is very soluble in methanol, ethanol, ether, or dimethylformamide, and freely soluble in water. Melting pointAbout 48 C. Identification (1) Take 0.2 g of Ethosuximide, add 10 mL of sodium hydroxide TS and boil: the gas evolved turns a moistened red litmus paper blue. (2) Dissolve 50 mg of Ethosuximide in 1 mL of ethanol, add 3 drops of a solution of cupric acetate (1 in 100), warm slightly and add 1 to 2 drops of sodium hydroxide TS: a purple color is produced. (3) Determine the absorption spectra of the solutions of Ethosuximide and Ethosuximide RS, respectively, in ethanol (1 in 2000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Purity (1) Clarity and color of solutionDissolve 1.0 g of Ethosuximide in 10 mL of water: the solution is clear and colorless. (2) ChlorideWith 1.0 g of Ethosuximide, perform the test. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.011%). (3) Heavy metalsProceed with 1.0 g of Ethosuximide according to Method 1. and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Arsenic Weigh 1.0 g of Ethosuximide and perform the test according to Method 1(not more than 2 ppm). (5) Acid anhydrideDissolve 0.50 g of Ethosuximide in 1 mL of ethanol, add 1 mL of hydroxylamine hydrochloride-ferric chloride TS and allow to stand for 5 minutes. Add 3 mL of water, mix and allow to stand for 5 minutes: the red to red-purple color of this solution is not more intense than that of the following control solution.

Packaging and Storage Preserve in a cool place, wellclosed containers.

Control solutionDissolve 70 mg of succinic anhydride in ethanol to make exactly 100 mL. To 1.0 mL of this solution, add 1 mL of hydroxylamine hydrochlorideferric chloride TS and proceed in the same manner. (6) CyanideDissolve 1.0 g of Ethosuximide in 10 mL of ethanol and add 3 drops of ferrous sulfate TS, 1 mL of sodium hydroxide TS and 2 to 3 drops of ferric chloride TS. Warm gently and acidify with dilute sulfuric acid: not a blue precipitate and a blue color are produced within 15 minutes.

Ethosuximide
O H N O CH2CH3 CH3

and enantiomer
Water

Not more than 0.5% (2 g, volumetric titra-

384 Monographs, Part I tion,direct titration).

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Ethosuximide, dissolve in 20 mL of dimethyl-formamide and titrate with 0.1 mol/L tetramethylammonium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary connection.

Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 14.117 mg of C7H11NO2

Packaging and Storage Preserve in tight containers.

water, 2 drops of phenolphthalein TS and 0.50 mL of 0.01 mol/L sodium hydroxide VS: a red color is produced. (2) ChlorideDissolve 0.20 g of Ethyl Aminobenzoate in 5 mL of ethanol, add 2 to 3 drops each of dilute nitric acid and of silver nitrate TS: no change occurs immediately. (3) Heavy metalsDissolve 2.0 g of Ethyl Aminobenzoate in 20 mL of ethanol, add 2 mL of dilute acetic acid and ethanol to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution as follows: to 2.0 mL of standard lead solution, add 2 mL of dilute acetic acid and sufficient ethanol to make 50 mL (not more than 10 ppm). (4) Readily carbonizable substancesPerform the test with 0.5 g of Ethyl Aminobenzoate: the solution has no more color than Matching Fluid A.
Loss on Drying Not more than 1.0% (1 g, silica gel, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weight accurately about 0.25 g of Ethyl Aminobenzoate, previously dried, dissolved in 10 mL of hydrochloric acid and 70 mL of water, add 10 mL of a solution of potassium bromide (3 in 10) and cool to a temperature below 15 C. Then titrate with 0.1 mol/L sodium nitrite VS (potentiometric titration or Amperometric titration, Endpoint Detection Methods in Titrimetry).

Ethyl Aminobenzoate
O C OCH2CH3

NH2

Benzocaine Anesthezine

C9H11NO2: 165.19

Ethyl Aminobenzoate, when dried, contains not less than 99.0% and not more than 101.0% of ethyl aminobenzoate (C9H11NO2).
Description Ethyl Aminobenzoate is a white crystal or crystalline powder and is odorless. Ethyl Aminobenzoate has a slightly bitter taste, numbing the tongue. Ethyl Aminobenzoate is freely soluble in ethanol or in ether, and very slightly soluble in water. Ethyl Aminobenzoate dissolves in dilute hydrochloric acid. Identification (1) Dissolve 10 mg of Ethyl Aminobenzoate in 1 mL of dilute hydrochloric acid and 4 mL of water. This solution responds to the Qualitative Tests for primary aromatic amines. (2) Dissolve 0.1 g of Ethyl Aminobenzoate in 5 mL of water with the aid of dilute hydrochloric acid added drop-wise and add iodine TS drop-wise: a brown precipitate is produced. (3) Warm 50 mg of Ethyl Aminobenzoate with 2 drops of acetic acid and 5 drops of sulfuric acid: the odor of ethyl acetate is perceptible. Melting Point Between 89 C and 91 C. Purity (1) AcidDissolve 1.0 g of Ethyl Aminobenzoate in 10 mL of neutralized ethanol and add 10 mL of

Each mL of 0.1 mol/L sodium nitrite VS = 16.519 mg of C9H11NO2

Packaging and Storage Preserve in well-closed containers.

Ethyl Cysteine Hydrochloride

H HS CH2 C NH2 O C OCH 2CH3 HCl

C5H11NO2SHCl: 185.67 Ethyl Cysteine Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of ethyl cysteine hydrochloride (C5H11NO2SHCl).
Description Ethyl Cysteine Hydrochloride is a white crystal or crystalline powder, has a characteristic odor, and has a bitter taste at first with a burning after taste. Ethyl Cysteine Hydrochloride is very soluble in water, freely soluble in ethanol, and practically insoluble in ether. Melting pointAbout 126 C (with decomposition).

KP 9 385
Identification (1) Determine the infrared spectra of Ethyl Cysteine Hydrochloride and Ethyl Cysteine Hydrochloride RS, respectively, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) A solution of Ethyl Cysteine Hydrochloride (1 in 20) responds to the Qualitative Tests (1) for chloride. Specific Optical Rotation [ ]20 D : Between -10.0 and 13.0 (after drying, 2.0 g, 1 mol/L hydrochloric acid TS, 25 mL, 100 mm) Purity (1) SulfatePerform the test with 0.6 g of Ethyl Cysteine Hydrochloride. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.011%). (2) Heavy metalsProceed with 1.0 g of Ethyl Cysteine Hydrochloride according to Method 1 and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesPerform this procedure rapidly. Dissolve 50 mg each of Ethyl Cysteine Hydrochloride and N-ethylmaleimide in 5 mL of mobile phase, allow to stand for 30 minutes and use this solution as the test solution. Pipet exactly 3 mL of the test solution, add the mobile phase to make exactly 200 mL and use this solution as the standard solution. Perform the test with 2 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution and the standard solution by the automatic integration method: a peak area from the test solution with the ratio of the retention time to ethyl cysteine hydrochloride-N-ethylmaleimide complex from the standard solution being about 0.7 is not larger than the peak area of ethyl cysteine hydrochloride-N-ethyl-maleimide complex from the standard solution. Each area of all peaks other than the peaks of ethyl cysteine hydrochloride-N-ethylmaleimide complex and N-ethylmaleimide from the test solution is not larger than 1/3 of the peak area of ethyl cysteine hydrochloride-N-ethylmaleimide complex from the standard solution.

System suitability System performance: Dissolve 50 mg of Ethyl Cysteine Hydrochloride, 10 mg of L-cysteine hydrochloride and 50 mg of N-ethyl-maleimide in 25 mL of the mobile phase and allow to stand for 30 minutes. When the procedure is run with 2 L of this solution, as directed under the above conditions, L-cysteine hydrochloride-N-ethylmaleimide complex, ethyl cysteine hydrochloride-L-ethylmaleimide complex and N-ethylmaleimide are eluted in this order and after complete resolution of each component, with the resolution of the peaks of Lcysteine hydrochloride-N-ethylmaleimide complex and ethyl cysteine hydrochloride-N-ethylmaleimide complex being not less than 3. Detection sensitivity: Adjust the detection sensitivity so that the peak height of ethyl cysteine hydrochlorideN-ethylmaleimide complex obtained from 2 L of the standard solution is between 10 mm and 20 mm. Time span of measurement: About 3 times as long as the retention time of ethyl cysteine hydrochloride-Nethylmaleimide complex.
Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.25 g of Ethyl Cysteine Hydrochloride, previously dried, transfer into a glassstoppered flask and dissolve in 10 mL of water previously freshly boiled and cooled to a temperature not exceeding 5 C in a stream of nitrogen. Add exactly 20 mL of 50 mmol/L iodine VS, previously cooled to a temperature not exceeding 5 C and allow to stand for 30 seconds, then titrate with 0.1 mol/L sodium thiosulfate VS, on cooling below 5 C (indicator: 1 mL of starch TS). Perform a blank determination and make any necessary correction.

Each mL of 0.05 mol/L iodine VS = 18.567 mg of C5H11NO2SHCl

Packaging and Storage Preserve in tight containers.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 250 nm). Column: A stainless steel column, about 6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of 0.02 mol/L monobasic potassium phosphate TS and acetonitrile (2 : 1). Flow rate: Adjust the flow rate so that the retention time of ethyl cysteine hydrochloride-N-ethylmaleimide complex is about 4 minutes.

Ethylmorphine Hydrochloride Hydrate

CH3 H N

HCl

2H2O

O CH3CH2O H H OH 2

386 Monographs, Part I C19H23NO3HCl2 H2O: 385.88 Ethylmorphine Hydrochloride Hydrate, when dried, contains not less than 98.0% and not more than 101.0% of ethylmorphine hydrochloride (C19H23NO3HCl : 349.85).
Description Ethylmorphine Hydrochloride Hydrate is a white to pale yellow crystal or crystalline powder. Ethylmorphine Hydrochloride Hydrate is very soluble in methanol or glacial acetic acid, freely soluble in water, soluble in ethanol, sparingly soluble in acetic anhydride, and practically insoluble in ether. Ethylmorphine Hydrochloride Hydrate is colored by light. Melting pointAbout 123 C (with decomposition). Identification (1) Determine the absorption spectra of solutions of Ethylmorphine Hydrochloride Hydrate and Ethylmorphine Hydrochloride Hydrate RS, separatey, in water (1 in 10000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Ethylmorphine Hydrochloride Hydrate and Ethylmorphine Hydrochloride Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Ethylmorphine Hydrochloride Hydrate (1 in 50) responds to the Qualitative Tests (2) for chloride. Specific Optical Rotation [ ]20 D : Between -103 and 106 (0.4 g calculated on the anhydrous basis, water, 20 mL, 100 mm). pH Dissolve 0.10 g of Ethylmorphine Hydrochloride Hydrate in 10 mL of water: the pH of this solution is between 4.0 and 6.0. Purity Related substancesDissolve 0.20 g of Ethylmorphine Hydrochloride Hydrate in 10 mL of diluted ethanol (1 in 2) and use this solution as the test solution. Pipet exactly 0.5 mL of the test solution, add diluted ethanol (1 in 2) to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for Thinlayer chromatography. Develop the plate with a mixture of dehydrated ethanol, toluene, acetone and strong ammonia water (14 : 14 : 7 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Water Between 8.0% and 10.0% (0.25 g, volume titration, direct titration). Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately about 0.5 g of Ethylmorphine Hydrochloride Hydrate and dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 34.985 mg of C19H23NO3HCl

Packaging and Storage tight containers.

Preserve in light-resistant,

Etilefrine Hydrochloride
OH C H HO CH2NHCH2CH3 HCl

and enantiomer C10H15NO2HCl: 217.69 Etilefrine Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of etilefrine hydrochloride (C10H15NO2HCl).
Description Etilefrine Hydrochloride is a white crystal or crystalline powder, is odorless and has a bitter taste. Etilefrine Hydrochloride is very soluble in water, freely soluble in ethanol, and sparingly soluble in glacial acetic acid. Etilefrine Hydrochloride is gradually colored to yellowbrown by light. pHThe pH of a solution of Etilefrine Hydrochloride in water (1 in 10) is between 3.8 and 5.8. Identification (1) Dissolve 5 mg each of Etilefrine Hydrochloride and Etilefrine Hydrochloride RS, separately in 100 mL of diluted hydrochloric acid (1 in 1000). Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Etilefrine Hydrochloride and Etilefrine Hydrochloride RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Etilefrine Hydrochloride (1 in 1000)

KP 9 387 responds to the Qualitative Tests for chloride.

Melting Point

Between 118 C and 122 C.

directed under the Ultraviolet-visible Spectrophotometry, using diluted hydrochloric acid (1 in 1000) as the blank: it exhibits a maximum between 271 nm and 275nm.
Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement of the Content Uniformity Test when the test is performed according to the following method. To 1 tablet of Etilefrine Hydrochloride Tablets add 60 mL of diluted hydrochloric acid (1 in 1000), and proceed as directed in the assay.

Purity (1) Clarity and color of solutionDissolve 0.5 g of Etilefrine Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) Acidity or alkalinityTo 10 mL of a solution of Etilefrine Hydrochloride (1 in 50) add 0.1 mL of methylred TS for acid or alkali test and 0.2 mL of 0.01 mol/L sodium hydroxide VS: a yellow color develops, and the necessary volume of 0.01 mol/L hydrochloric acid VS to change the color to red is not more than 0.4 mL. (3) SulfatePerform the test with 0.85 g of Etilefrine Hydrochloride. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.020%). (4) Heavy metalsDissolve 1.0 g of Etilefrine Hydrochloride in 30 mL of water and 2 mL of glacial acetic acid, adjust with 0.1 mol/L sodium hydroxide TS to a pH of 3.3, add water to make 50 mL and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.15 g of Etilefrine Hydrochloride, previously dried, dissolve in 20 mL of glacial acetic acid, add 50 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, endpoint detection method in titrimetry). Perform a blank determination and make any necessary correction.

Amount (mg) of etilefrine hydrochloride (C10H15NO2HCl) = W S AT 1

AS

10

WS : Amount of Etilefrine Hydrochloride RS (calculated on anhydrouse basis).

Each mL of 0.1 mol/L perchloric acid VS = 21.769 mg of C10H15NO2HCl

Packaging and Storage tight containers.

Preserve in light-resistant,

Assay Weigh accurately and powder not less than 20 Etilefrine Hydrochloride Tablets. Weigh accurately a portion of the powder, equivalent to about 5 mg of etilefrine hydrochloride (C10H15NO2HCl), add 60 mL of diluted hydrochloric acid (1 in 1000) to make exactly 100 mL and filter. Discard the first 20 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately about 50 mg of Etilefrine Hydrochloride RS (previously determined the loss on drying), dissolve in diluted hydrochloride acid (1 in 1000) to make exactly 100 mL, dilute 10 mL of this solution with diluted hydrochloric acid (1 in 1000) to exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine the peak areas, AT and AS , of etilefrine.

Amount (mg) of etilefrine hydrochloride (C10H15NO2HCl) = amount (mg) of Etilefrine Hydroch-

Etilefrine Hydrochloride Tablets

Etilefrine Hydrochloride Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of etilefrine hydrochloride (C10H15NO2HCl: 217.69).
Method of Preparation Prepare as directed under Tablets, with Etilefrine Hydrochloride. Identification Take a portion of powdered Etilefrine Hydrochloride Tablets, equivalent to 5 mg of Etilefrine Hydrochloride according to the labeled amount, add 60 mL of diluted hydrochloric acid (1 in 1000), shake well, add 40 mL of diluted hydrochloric acid (1 in 1000) and filter. Determine the absorption spectrum of the filtrate as

loride RS AT 1
AS

10

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 5 g of sodium lauryl sulfate in 940 mL of water and 500 mL of acetonitrile, and adjust the pH to 2.3 with phosphoric acid. Flow rate: Adjust the flow rate so that the retention

388 Monographs, Part I time of etilefrine is about 6 minutes. System suitability System performance: Dissolve 4 mg of bamethan sulfate and 4 mg of etilefrine hydrochloride in the mobile phase to make 50 mL. When the procedure is run with 20 L of this solution under the above operating conditions, etilefrine and bamethan are eluted in this order with the resolution between their peaks being not less than 5. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of etilefrine is not more than 1.0%.
Packaging and Storage tight containers.

lution as the standard stock solution Pipet 10.0 mL of the standard stock solution, add 5.0 mL of the internal standard, dilute with dimethylformamide to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 1 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following conditions, and calculate the ratios, QT and QS , of the peak area of Etodolac to that of the internal standard for the test solution and the standard solution, respectively. Amount of methanol and ethanol in the portion of Etodolac is not more than 0.1%, respectively. Amount (%) of methanol or ethanol = 500
C QT W QS

Preserve in light-resistant,

Etodolac
O N H CH2CH3 CH2 CH2CH3 C OH

C: Concentration of ethanol or methanol in the standard solution (mg/mL). W: Weight of Etodolac in the test solution (mg). Internal standard solutionDissolve a suitable quantity of isopropanol in dimethylformamide to make a solution having a concentration of 2.5 L/mL. Pipet 5.0 mL of this solution, and dilute this solution with dimethylformamide to make exactly 100 mL. Operating conditions Detector: A flame-ionization detector. Column: A fused silica capillary column, 0.32 mm in inside diameter and 25 m in length, coated with a 5 m film of 1% vinyl5% phenylmethylpolysiloxane for gas chromatography. Carrier gas: Helium. Flow rate: 50 mL/minute. Injection port temperature: 200 C. Detector temperature: 300 C. Column temperature: Maintain the temperature of 45 C for 5 minutes, then raise at the rate of 30 C per minute to 280 C and maintain this temperature for 27 minutes. System suitability System performance: When the procedure is run with 1 L of the standard solution, as directed under the above operating conditions, methanol, ethanol, and isopropanol are eluted in this order with the resolution between their peaks being not less than 1.0. (4) Related substancesDissolve 25 mg of Etodolac in acetonitrile to make exactly 250 mL, and use this solution as the test solution. Perform the test with the test solution as directed under the Liquid Chromatography according to the following conditions. Each peak area of any peak other than the principal peak from the test solution is not more than 0.5 percent to the total area of all peaks, and the total area is not more than 2.0 percent. Content (%) of related substances= 100 AT

and enantiomer C17H21NO3: 287.35

Etodolac contains not less than 98.0% and not more than 102.0% of etodolac (C17H21NO3), calculated on the anhydrous basis.
Description Etodolac is a white crystalline powder. Etodolac is freely soluble in acetone or ethanol, and practically insoluble in water. Identification Determine the infrared spectra of Etodolac and Etodolac RS, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensity of absorption at the same wavenumbers. Purity (1) ChlorideDissolve 0.5 g of Etodolac in 30 mL of methanol, add water to make exactly 50 mL, and use this solution as the test solution. Dissolve 0.42 mL of 0.01 mol/L hydrochloric acid VS in 30 mL of methanol, add water to make exactly 50 mL, and use this solution as a control solution (not more than 0.03%). (2) Heavy metalsProceed with 1.0 g of Etodolac according to Method 2, and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (3) Limit of methanol and ethanolDissolve about 0.5 g of Etodolac, weighed accurately, in 5.0 mL of the internal standard solution, and use this solution as the test solution. Separately, pipet exactly 5 mL each of methanol and ethanol, add dimethylformamide to make exactly 200 mL, pipet 5.0 mL of this solution, add dimethylformamide to make exactly 100 mL, and use this so-

AS

KP 9 389

AT : Each peak area of the peaks other than the principle peak. AS : Total area of all peaks. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 214 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (3 to 10 m in particle diameter). Mobile phase: Use variable mixtures of Solution A and Solution B as a mobile phase. First 5 minutes, use mixture of 60 percent solution A and 40 percent solution B as the mobile phase. For next 30 minutes, change the mixture ratio as linear gradient to finally make mixture of 20 percent solution A and 80 percent solution B. Adjust mobile phase to 40 percent of solution B before injection of the test solution and the standard solution, and keep re-equilibrium. Mobile phase A: Mix 0.6 mL of phosphoric acid with 100 mL of water. Mobile phase B: Mix 0.6 mL of phosphoric acid with 100 mL of acetonitrile. Flow rate: 1 mL/min. System suitability System performance: Dissolve suitable amount each of Etodolac Related Substance I RS and Etodolac RS in acetonitrile to make solutions containing 10 g of Etodolac Related Substance I RS and 0.2 mg of Etodolac RS per mL, respectively. When the procedure is run with 20 L of this solution, as directed under the above operating conditions, etodolac related substance I and Etodolac are eluted in this order with the resolution between their peaks being not less than 3.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the ratio of the peak area is not more than 3%.
Water Not more than 0.5% (2 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.23 g of Etodolac, dissolve in 60 mL of methanol, and titrate with 0.1 mol/L tetrabutylammonium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction.

Etodolac Tablets
Etodolac Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of etodolac (C17H21NO3: 287.36)
Method of Preparation Prepare as directed under Tablets, with Etodolac. Identification Test The retention time of major peak of the test solution corresponds to that of the standard solution as obtained in the Assay. Dissolution Test Take 1 tablet of Etodolac Tablets, perform the test as directed in Method 1 under the Dissolution Test at 100 revolutions per minute, and using 1000 mL of pH 6.8 phosphate buffer as the dissolution medium. After 30 minutes from starting of the test, take 20 mL of the dissolved solution, and filter through a membrane filter with a pore size of 0.8 m or less. Discard the first 10 mL of the filtrate, and use the subsequent filtrate as the test solution. Seperately weigh accurately 25 mg of etodolac reference standard, and dissolve in the dissolution medium to make exactly 100 mL. Pipet 5 mL of this solution, add the dissolution medium to make exactly 50 mL, and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at the maximum wavelength at about 274 nm as directed under the Ultraviolet-visible Spectrophotometry using the dissolution medium as a blank. The dissolution rate of Etodolac Tablets after 30 minutes should be not less than 80%.

Dissolutio n rate (%) with respect tothe labeledamount of etodolac(C17H21NO3 ) = WS AT 100 AS C

WS : Amount of Etodolac RS (mg) C: Labeled amount of etodolac (C17H21NO3) in each tablet (mg).

Each mL of 0.1 mol/L tetrabutylammonium hydroxide VS = 28.736 mg of C17H21NO3

Packaging and Storage Preserve in tight containers.

Uniformity of Dosage Units It meets the requirement when the content uniformity test is performed according to the following method. Take 1 tablet of Etodolac Tablets, and shake with 10 mL of phosphate buffer until the tablet is disintegrated. Add phosphate buffer to make exactly 100 mL, and centrifuge this solution. Pipet x mL of the supernatant liquid, add phosphate buffer to make exactly V mL to provide a solution that contains about 25 g of etodolac (C17H21NO3) per mL, and use this solution as the test solution. Separately, weigh accurately 25 mg of etodolac reference standard, and dissolve in phosphate buffer to make exactly 100 mL. Pipet 10 mL of this solution, add phosphate

390 Monographs, Part I buffer to make exactly 100 mL, and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at maximum wavelength at about 274 nm as directed under the Ultraviolet-visible spectrophotometry.
Amount (mg) of etodolac (C17 H 21 NO 3 ) = amount (mg) of Etodolac RS
AT V 1 AS 10 x
NH

Packaging and Storage Preserve in tight containers.

Etofenamate
O O O OH

Assay Weigh accurately and powder not less than 20 Etodolac Tablets. Weigh accurately a portion of the powder, equivalent to about 0.1 g of etodolac (C17H21NO3), and add 30 mL of a mobile phase. Shake for 15 minutes, and sonicate 5 minutes for integration. After cooling, add the mobile phase to make exactly 50 mL. After leaving 10 minutes, pipet 10.0 mL of this solution, then add the mobile phase to make exactly 100 mL, filter, and use this filtrate as the test solution. Separately, weigh exactly 20 mg etodolac reference standard and dissolve in the mobile phase to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak areas, AT and AS , of the test solution and the standard solution, respectively.
Amount (mg) of etodolac (C17 H 21NO3 ) = amount (mg) of Etodolac RS
AT 5 AS

CF3

C18H18F3NO4 : 369.34 Etofenamate contains not less than 98.5% and not more than 101.5% of etofenamate (C18H18F3NO4), calculated on the anhydrous basis.
Description Etofenamate is a yellow viscous liquid. Etofenamate is practically insoluble in water. Etofenamate is miscible with ethanol or with ethyl acetate. Identification Determine the infrared spectra of Etofenamate and Etofenamate RS as directed in the liquid film method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Heavy metalsProceed with 2.0 g of Etofenamate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) DiethyleneglycolWeigh accurately about 0.2 g of Etofenamate, add 10 L of internal standard solution and 2 mL of N-methyltrimethylsilyltrifluoroacetamide, heat at 50 o C for 30 minutes and use this solution as the test solution. Separately, pipet 6.0 mg of diethylene glycol, transfer into a 10 mL volumetric flask, add 3 mL of N-methyltrimethylsilyltrifluroacetamide, heat at 50 o C for 30 minutes, allow to cool and add Nmethyltrimethylsilyl-trifluoroacetamide to make 10 mL. Mix 10 L of this solution, 10 L of internal standard solution and 2 mL of N-methyltrimethylsilyltrifluoroacetamide and use this solution as the standard solution. Perform the test with exactly 0.2 L each of the test solution and the standard solutions as directed under Gas Chromatography according to the following conditions, and determine the percentages of peak heights of diethylene glycol, HT and HS, reference to the peak height of the internal standard, respectively. (0.1%)

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 274 nm). Column: A stainless steel column, about 4.6 nm in inside diameter and about 25 cm in length, packed with 3 ~ 10 m in particle diameter octadecylsilanized silicagel for liquid chromatography. Mobile phase: acetonitrile water phosphate mixture (500 : 500 : 0.25). Flow rate: 1.5 mL/minute. System suitability System performance: Dissolve Etodolac related substance RS and Etodolac RS in acetonitrile to make Etodolac related substance RS 10 g and Etodolac RS about 0.2 mg per mL. When the precedure is run with 20 L of this solution, as directed under the above operating conditions, Etodolac related substance and Etodolac are eluted in this order with the resolution between their peaks being not less than 3.0. System repeatability: When the test is repeated 6 times with about 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the peak areas is not more than 3%.

KP 9 391 Internal standard solutionDissolve 6.0 mg of tetracain in hexane to make 10 mL. Operating conditions Detector: A hydrogen flame-ionization detector Column: A fused-silica column about 0.20 mm in inside diameter and about 25 m in length, coated the inner surface with polydimethyldiphenylsiloxane for Gas Chromatography 0.33 m in thickness. Column temperature: Time Temperature Rate (minutes) (C) (C /minute) column 0~13 60150 7 13~19 150300 25 19~34 300 Injector 150 detector 300 Carrier gas: Hydrogen Flow rate: 0.9 mL/minute (3) Related substancesWeigh accurately 50.0 mg of Etofenamate, dissolve in 30 mL of methanol, add water to make exactly 50 mL and use this solution as the test solution. Dissolve 10.0 mg of Etofenamate Related Substance I RS {2-Hydroxyethyl-2-[[3-(trifluoromethyl) phenyl]amino]benzoate} in methanol to make exactly 20 mL. To 0.2 mL of this solution add a mixture solution of water and methanol (40 : 60) to exactly 100 mL and use this solution as the standard solution (1). To 0.2 mL of the test solution add a mixture solution of water and methanol (40 : 60) to make exactly 100 mL and use this solution as the standard solution (2). Mix 5.0 mL each of the standard solution (1) and the standard solution (2) and use this solution as the standard solution (3). Dissolve 10.0 mg of Etofenamate RS for peak identification (contains etofenamate spiked with about 1% each of related substances II, III, IV, V and VI) in 6.0 mL of methanol and add water to make exactly 10 mL and use this solution as the standard solution (4). Perform the test with exactly 20 L each of the test and standard solutions (1), (2), (3), and (4) as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method. For the calculation of contents of related substances II, IV and VI, multiply each peak area by the corresponding correction factor, 0.62, 0.45 and 0.77, respectively: the area of the related substance II in the chromatogram from the test solution is not more than 1.25 times the area of the principal peak from the standard solution (2) (0.25%), each peak area of related substance III, IV, and VI is not more than the area of the principal peak from the standard solution (2) (each 0.2%), the area of the related substance I is not more than the area of the principal peak from the standard solution (1) (0.2%), the area of the related substance V is not more than 2.5 times the area of the principal peak in the chromatogram obtained from reference solution (b) (0.5%), the peak area of any other related substance is not more than half the area of the principal peak with the standard solution (2) (0.1%), the total area of all peaks other than the principal peak is not more than 6 times the area of the principal peak from the standard solution (2) (1.2%). Disregard peaks being not more than 0.25 times the area of the principal peak from the standard solution (2) (0.05%). Retention time of etofenamate peak is about 13 minutes and relative retention time of related substances II, IV, I, VI, III and V with reference to etofenamate are 0.2, 0.7, 0.85, 1.5, 1.6 and 1.7, respectively. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 286 nm). Column: A stainless steel column 4.0 mm in inside diameter and 10 cm in length, packed with octadecylsilyl silica gel for Liquid Chromatography (3 m in particle diameter). Column temperature: A constant temperature of about 40 o C . Mobile phase: Program with mobile phase A and mobile phase B in isocratic or linear gradient as following. mobile phase A: Dissolve 1.3 g of ammonium phosphate and 4.0 g of tetrabutylammonium hydroxide in 900 mL of water. Adjust pH to 8.0 with dilute phosphoric acid and add water to make 1000 mL. mobile phase B: methanol Mobile phase A Mobile phase B Time (vol%) (vol%) 0~13 40 60 13~20 4010 6090 20~25 10 90 25~26 1040 9060 26~31 40 60 Flow rate: 1.2 mL/minute System suitability System performance: Dissolve 10.0 mg of Etofenamate Related Substance I RS in methanol to make 20 mL. To 0.2 mL of this solution add a mixture of water and methanol (40 : 60) to make 100 mL. To 5.0 mL of this solution add 5.0 mL of the solution prepared by dilution of 0.2 mL of the test solution with a mixture of water and methanol (40 : 60) to make 100 mL. When the procedure is run with 20 L of this solution under the above operating condition, resolution between the peaks of etofenamate and related substance I is not less than 2.3.
Water Not more than 0.5% (1 g, volumetric titration, direct titration) Assay Weigh accurately about 3g of Etofenamate, add 20 mL of 2-propanol and 20 mL of 1 mol/L sodium hydroxide VS, heat under reflux for 2 h and cool. Titrate with 1 mol/L hydrochloric acid VS (indicator : 0.1 mL of bromthymol blue ). Perform a blank determination and make any necessary correction.

Each mL of 1 mol/L sodium hydroxide VS

392 Monographs, Part I = 369.35 mg of C18H18F3NO4.

Packaging and Storage Preserve in tight containers.

Etoposide
H3CO O H HO O H O O O CH 3 H3CO HO OH O O O H

than etoposide obtained with the test solution is not greater than 1/5 times the peak area of etoposide obtained with the standard solution, and the total area of all peaks other than the peak of etoposide obtained with the test solution is not larger than 1/2 times the peak area of etoposide obtained with the standard solution. Operating conditions Detector, column, column temperature, mobile phase, and flow rate: Proceed as directed in the operating conditions in the Assay. System suitability System performance: Proceed as directed in the system suitability in the Assay. Test for the required detectability: Measure exactly 1 mL of the standard solution, and add the mobile phase to make exactly 10 mL. Conform that the peak area of etoposide obtained with 50 L of this solution is equivalent to 7 to 13% of that of etoposide obtained with 50 L of the standard solution. System repeatability: When the test is repeated 6 times with 50 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of etoposide is not more than 2.0%. Time span of measurement: About 3 times as long as the retention time of etoposide beginning after the solvent peak.
Water Not more than 4.0% (0.5 g, volumetric titration, direct titration). Residue on ignition Not more than 0.1% (1 g) Assay Weigh accurately about 25 mg each of Etoposide and Etoposide RS (determined previously the water), dissolve separately in and dilute with methanol to make exactly 25 mL each. Pipet 10 mL each of these solutions, add exactly 5 mL of the internal standard solution and the mobile phase to make exactly 50 mL and use this solution as the test solution and standard solution, respectively. Perform the test with 50 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine the ratios, QT and QS , of the peak area of etoposide to that of the internal standard.

C29H32O13: 588.56 Etoposide contains not less than 98.0% and not more than 102.0% of etoposide (C29H32O13), calculated on the anhydrous basis.
Description Etoposide is a white crystals or crystalline powder. Etoposide is sparingly soluble in methanol, slightly soluble in ethanol, and very slightly in water. Melting pointabout 260 o C Identification (1) Determine the absorption spectra of solutions of Etoposide and Etoposide RS, respectively, in methanol (1 in 10000) as directed under Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Etoposide and Etoposide RS, according to the paste method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between 100 and 105 [0.1 g calculated on the anhydrous basis, methanol, 20 mL, 100 mm]. Purity (1) Heavy metalsProceed with 2.0 g of Etoposide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesDissolve 50 mg of Etoposide in 10 mL of methanol and add mobile solution to make 50 mL and use this solution as the test solution. Pipet 2 mL of the test solution, add the mobile phase to make 200 mL, and use this solution as the standard solution. Perform the test with exactly with exactly 50 L each of the test solution and standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of any peak other

Amount (mg) of C29H32O13 = WS

QT QS

WS : Amount of the Etoposide RS, calculated on the anhydrous basis (mg)

Internal standard solutionA solution dichlorophenol in methanol (3 in 2500). Operating conditions

of

2,6-

KP 9 393 Detector: An ultraviolet absorption photometer (wavelength: 290 nm). Column: A stainless steel column, about 3.9 mm in inside diameter and about 30 cm in length, packed with phenylsilyl silica gel for liquid chromatography (10 m particle diameter). Column temperature: A constant temperature of about 35 C Mobile phase: Dissolve 6.44 g of sodium sulfate decahydrate in diluted glacial acetic acid (1 in 100) to make 1000 mL, and add 250 mL of acetonitrile. Flow rate: Adjust the flow rate so that the retentiontime of etoposide is about 20 minutes. System suitability System performance: Dissolve 10 mg of Etoposide in 2 mL of methanol, add 8 mL of the mobile phase, and mix well. Add 0.1 mL of diluted glacial acetic acid (1 in 25) and 0.1 mL of phenolphthalein TS, and add sodium hydroxide TS until the color of the solution changes to faintly red. After allowing to stand for 15 minutes, add 0.1 mL of diluted glacial acetic acid (1 in 25). When the procedure is run with 10 L of this solution under the above operating conditions, the resolution between the peak having the relative retention time of about 1.3 with respect to etoposide is not less than 3.0. System repeatability: When the test is repeated 6 times with 50 L of the standard solution under the above operating conditions, the relative standard deviation of the peak areas of etoposide to that of internal standard is not more than 1.0%.
Packaging and Storage tight containers.

at the same wavelengths. (2) Determine the infrared spectra of Famotidine and Famotidine RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
Purity (1) Clarity and color of solutionDissolve 0.5 g of Famotidine in 10 mL of 0.5 mol/L hydrochloric acid TS: the solution is clear and colorless to pale yellow. (2) Heavy metalsProceed with 2.0 g of Famotidine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve 0.20 g of Famotidine in 10 mL of glacial acetic acid and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add glacial acetic acid to make exactly 100 mL. Pipet exactly 1 mL, 2 mL and 3 mL of this solution, add glacial acetic acid to make exactly 10 mL, respectively and use these solutions as the standard solutions (1), (2) and (3). Perform the test with the test solution and the standard solutions (1), (2) and (3) as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solutions (1), (2) and (3) on a plate of silica gel (5 to 7 m in particle diameter) with fluorescent indicator for thin-layer chromatography and dry in a stream of nitrogen. Develop the plate with a mixture of ethyl acetate, methanol, toluene and strong ammonia water (40 : 25 : 20 : 2) to a distance of about 8 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot and other than the spot of the starting point from the test solution are not more intense than the spot from the standard solution (3). Total intensity of the spots other than the principal spot and other than the spot of the starting point from the test solution is not more than 0.5% calculated on the basis of intensities of the spots from the standard solutions (1) and (2) (each spot is equivalent to 0.1% and 0.2%, respectively). Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, 80 C, 4 hours). Residue on Ignition Not more than 0.10% (1 g). Assay Weigh accurately about 0.3 g of Famotidine, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration). Perform a blank determination and make any necessary correction.

Preserve in light-resistant,

Famotidine
H2N C H2N N S N CH2SCH 2CH2 NH2 C N O S O NH2

C8H15N7O2S3: 337.45 Famotidine, when dried, contains not less than 98.5% and not more than 101.0% of famotidine (C8H15N7O2S3).
Description Famotidine is a white to yellowish white crystal. Famotidine is freely soluble in glacial acetic acid, slightly soluble in ethanol, and very slightly soluble in water. Melting pointAbout 164 C (with decomposition). Identification (1) Determine the absorption spectra of solutions of Famotidine and Famotidine RS in 0.05 mol/L monobasic potassium phosphate TS (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption

Each mL of 0.1 mol/L perchloric acid VS = 16.873 mg of C8H15N7O2S3

Packaging and Storage tight containers.

Preserve in light-resistant,

394 Monographs, Part I and filter through a membrane filter (pore size of not more than 0.8 m). Discard the first 10 mL of the filtrate, and use the subsequent filtrate as the test solution. Separately, weigh accurately 20 mg of famotidine RS, add buffer solution to make exactly 100 mL. Pipet 5.0 mL of this solution, add the buffer solution to make exactly 50 mL, and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution at an absorption maximum near 265 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Famotidine Tablets in 30 minutes should be not less than 75%. Dissolution rate (%) with respect to the labeled amount of famotidine (C8H15N7O2S3) 90 AT = amount (mg) of Famotidine RS A C S C: labeled amount (mg) of famotidine (C8H15N7O2S3) in 1 tablet.
Assay Take a number of Famotidine Tablets, equivalent to 0.2 g of famotidine (C8H15N7O2S3), add 50 mL of water and disintegrate by shaking well. Then add 100 mL of methanol, shake well, add methanol to make exactly 200 mL, and centrifuge. Pipet exactly 5 mL of the surpernatant liquid, add exactly 5 mL of the internal standard solution, add the mobile phase to make exactly 50 mL, and use this solution as the test solution. Separately, weigh accurately about 0.1 g of Famotidine RS, previously dried in vacuum with P2O5 at 80 C for 4 hours, and dissolve in methanol to make exactly 100 mL. Pipet exactly 5 mL of this solution, add exactly 5 mL of the internal standard solution, add the mobile phase to make exactly 50 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS, of the peak area of famotidine to that of the internal standard.

Famotidine Tablets
Famotidine Tablets contain not less than 94.0% and not more than 106.0% of the labeled amount of famotidine (C8H15N7O2S3: 337.45).
Method of Preperation Prepare as directed under Tablets, with Famotidine. Identification Weigh a portion of powdered Famotidine Tablets, equivalent to 10 mg of famotidine according to the labeled amount, add 50 mL of 0.05 mol/L potassium dihydrogenphosphate TS, shake well and centrifuge. Take 5 mL of the supernatant liquid, add 0.05 mol/L potassium dihydrogen phosphate TS to make 50 mL, and determine the spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits an absorbance maximum between 263 nm and 267 nm. Uniformity of Dosage Units It meets the requirement when the Content Uniformity Test is performed according to the following method Take 1 tablet of Famotidine Tablets, add 2 mL of water, shake to disintegrate. Add a suitable amount of methanol and shake well, then add methanol to make exactly V mL of a solution containing about 0.2 mg of famotidine (C8H15N7O2S3) per mL and centrifuge. Pipet exactly 10 mL of the supernatant liquid, add 2.0 mL of the internal standard solution, add the mobile phase to make exactly 20 mL, and use this solution as the test solution. Separately, weigh accurately about 0.1 g of Famotidine RS, previously dried in vacuum with P2O5 at 50 C for 4 hours, and dissolve in methanol to make exactly 100 mL. Pipet exactly 10 mL of this solution, add 2.0 mL of the internal standard solution, add the mobile phase to make exactly 20 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the operating conditions described in the Assay and calculate the ratios, QT and QS, of the peak area of famotidine to that of the internal standard.

Amount (mg) of famotidine (C8H15N7O2S3) QT = amount (mg) of Famotidine RS Q 2 S Internal standard solutionTake 5 mL of a solution of methyl paraoxybenzoate in methanol (1 in 500) add water to make 50 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C.

Amount (mg) of famotidine (C8H15N7O2S3) QT V = amount (mg) of Famotidine RS Q 500 S Internal standard solutionTake 5 mL of a solution of methyl paraoxybenzoate in methanol (1 in 500) add water to make 50 mL.
Dissolution Test Perform the test with 1 tablet of Famotidine Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of 0.1 mol/L phosphate buffer (pH 4.5). Take 20 mL of the dissolved solution at 30 minutes after starting the test,

KP 9 395 Mobile phase: Dissolve 2 g of sodium 1-heptane sulfonate in 900 mL of water, adjust the pH to 3.0 with anhydrous acetic acid, and add water to make 1000 mL. To this solution, add 240 mL of acetonitrile and 40 mL of methanol. Flow rate: Adjust the flow rate so that the retention time of famotidine is about 6 minutes. System suitability System performance: When the precedure is run with 5 L of the standard solution, as directed under the above operating conditions, famotidine and internal standard are eluted in this order with the resolution between their peaks being not less than 11. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of famotidine is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

of the contents with the washings, add water to the combined solution to make exactly 100 mL, and use this solution as the test solution. Pipet exactly 1 mL of this solution, add water to make exactly 100 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine each peak area of these solutions by the automatic integration method: the total area of the peaks other than the major peak from the test solution is not larger than the major peak from the standard solution. Operating conditions Proceed with a Detector, column, column temperature, mobile phase, flow rate, and selection of column as directed in the Assay. Detection sensitivity: Adjust the detection sensitivity so that the peak height of famotidine obtained from 5 L of the standard solution is between 5 mm and 10 mm. Time span of measurement: About 2 times as long as the retention time of famotidine after the solvent peak.
Water Not more than 1.5 percent (0.1 g, coulometric titration). Sterility Test It meets the requirement. Baterial Endotoxins Less than 15 EU/mg of Famotidine for injection. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Famotidine for Injection

Famotidine for injection is a solution for injection which is dissolved before use. Famotidine for injection contains not less than 94.0% and not more than 106.0% of the labeled amount of famotidine (C8H15N7O2S3: 337.45).
Method of Preperation Prepare as directed under Injections, with Famotidine. Description Famotidine for injection occurs as porous masses or powder. Identification Weigh a portion of Famotidine for injection, equivalent to 10 mg of Famotidine according to the labeled amount, dissolve in 50 mL of 0.05 mol/L potassium dihydrogenphosphate TS, to 5 mL of this solution add 0.05 mol/L potassium dihydrogen phosphate TS to make 50 mL, and determine the spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits the maximum absorbance between 263 nm and 267 nm. pH Weigh a portion of Famotidine for injection, equivalent to 20 mg of famotidine according to the labeled amount, dissolve in 1 mL of water: the pH of this solution is between 4.9 and 5.5. Purity (1) Clarity and color of solution Weigh a portion of Famotidine for injection, equivalent to 20 mg of famotidine according to the labeled amount, dissolve in 1 mL of water; the solution is clear and colorless. (2) Related substancesTake a number of Famotidine for injection, equivalent to about 0.1 g of famotidine (C8H15N7O2S3), dissolve each content in water, wash the inside of the container with water, combine the solutions

It

Uniformity of Dosage Units It meets the requirement. Assay Take a number of Famotidine for injection, equivalent to about 0.1 g of famotidine (C8H15N7O2S3), dissolve each content in water, wash the inside of the container with water, combine the solutions of the contents with the washings, add water to the combined solution to make exactly 100 mL. Pipet exactly 5 mL of this solution, add 5.0 mL of the internal standard solution, add the mobile phase to make exactly 50 mL, and use this solution as the test solution. Separately, weigh accurately about 50 mg of Famotidine RS, previously dried in vacuum with P2O5 at 80 C for 4 hours, dissolve in the mobile phase to make exactly 50 mL. Pipet exactly 5 mL of this solution add 5.0 mL of the internal standard solution, add the mobile phase to make exactly 50 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and calculate the ratios, QT and QS, of the peak area of famotidine to that of the internal standard.

396 Monographs, Part I

Amount (mg) of famotidine (C8H15N7O2S3) QT = amount (mg) of Famotidine RS Q 2 S Internal standard solutionTake 5 mL of a solution of methyl paraoxybenzoate in methanol (1 in 500) add water to make 50 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Dissolve 2 g of sodium 1-heptane sulfonate in 900 mL of water, adjust the pH to 3.0 with glacial acetic acid, and add water to make 1000 mL. To this solution, add 240 mL of acetonitrile and 40 mL of methanol. Flow rate: Adjust the flow rate so that the retention time of famotidine is about 6 minutes. System suitability System performance: When the precedure is run with 5 L of the standard solution, as directed under the above operating conditions, famotidine and internal standard are eluted in this order with the resolution between their peaks being not less than 11. System repeatability: When the test is repeated 6 times with 5 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the peak area of famotidine is not more than 1.0%.
Packaging and Storage Preserve in hermetic containers.

Description Felodipine is a light yellow to yellow crystalline powder. Felodipine is freely soluble in acetone or in ethanol, slightly soluble in heptane, and practically insoluble in water. Identification (1) Determine the infrared spectra of Felodipine and Felodipine RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensity of absorption at the same wavenumbers. (2) The retention time of the major peak in the chromatogram of the test solutoin is same as that of the standard solution, as obtained in the Assay. Purity (1) Clarity and color of solutionDissolve 1.0 g of Felodipine in methanol to make exactly 50 mL. Perform the test with this solution as directed under the Ultraviolet-visible Spectrophotometry, using methanol as the blank: the absorbance at 440 nm is not more than 0.2. (2) Heavy metalsProceed with 1.0 g of Felodipine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesPerform the test with 40 L of the test solution prepared as in the Assay as directed under the Liquid Chromatography according to the following conditions, and determine each peak area of the test solution: each peak area except major peak to that of major peak area is not more than 1.0%, and the sum of all peak area is not more than 1.5%.

Ai Amount (%) of related substances = 100 A s Ai : each peak area except major peak As : the sum of all peak area Operating conditions Proceed as directed in the Assay. Time span of measurement: About 2 times as long as the retention time of felodipine.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours).

Felodipine
H3C H N CH3

O H3C H O O Cl

CH3

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 30 mg each of Felodipine and Felodipine RS, add the mobile phase to make exactly 100 mL, and use these solutions as the test solution and the standard solution. The test solution and the standard solution are prepared before use. Perform the test with 40 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine AT and AS, of the peak area of felodipine of each solution.

Cl

and enantiomer C18H19Cl2NO4: 384.25

Felodipine contains not less than 98.0% and not more than 101.0% of felodipine (C18H19Cl2NO4), calculated on the dried basis.

KP 9 397 Amount (mg) of felodipine (C18H19C12NO4) AT = amount (mg) of Felodipine RS A S Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of phosphate buffer, acetonitrile, and methanol (40 : 40 : 20). Flow rate: 1 mL/minute. System suitability System performance: Dissolve 0.15 g of Felodipine in a mixture of 25 mL of tertiary butyl alcohol and 25 mL of 1 mol/L perchloric acid, add 10 mL of 0.1 mol/L ceric sulfate, mix, and allow to stand for 15 minutes. Add 3.5 mL of 10 mol/L sodium hydroxide TS, and neutralize with 2 mol/L sodium hydroxide TS. Shake the mixture with 25 mL of methylene chloride in a sparator. Draw off the lower layer, and evaporate it to dryness under a stream of nitrogen on a water-bath. Dissolve 10 mg of the residue (felodipine oxidation product) and 5 mg of Felodipine RS in the mobile phase to make 100 mL. Pipet 1.0 mL of this solution, and add mobile phase to make 100 mL. When the procedure is run with 20 L of this solution, as directed under the above operating conditions, felodipine oxidation product and felodipine are eluted in this order with the resolution between their peaks being not less than 2.5. And perform the test with 40 L of the standard solution, as directed under the above operating conditions, the tailing factor is not greater than 1.5. Detection sensitivity: Adjust the detection sensitivity so that the heights of the two peaks in the chromatogram are not less than 20.0% of the full scale. Phosphate buffer: Dissolve 6.9 g of monobasic sodium phosphate in 400 mL of water, add 8 mL of 1 mol/L phosphoric acid, and add water to make 1000 mL.
Packaging and Storage tight containers.

and has a bitter taste. Fenbufen is sparingly soluble in acetone, slightly soluble in methanol, in ethanol or in ether and practically insoluble in water. Melting pointAbout 188 C (with decomposition).
Identification (1) Determine the absorption spectra of solutions of Fenbufen and Fenbufen RS in ethanol (1 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectrum of Fenbufen and Fenbufen RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Heavy metalsTake 2.0 g of Fenbufen, add 2 mL of sulfuric acid and carbonize by gentle heating, proceed according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Fenbufen according to Method 3 and perform the test (not more than 2 ppm). (3) Related substancesDissolve 0.1 g of Fenbufen in 20 mL of acetone and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add acetone to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of dichloromethane, methanol and water (80 : 20 : 3) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.3% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Fenbufen, previously dried, dissolve in 100 mL of dehydrated ethanol and titrate with 0.1 mol/L potassium hydroxide-ethanol VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

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Fenbufen
O C CH 2CH 2CO 2H

C16H14O3: 254.28 Fenbufen, when dried, contains not less than 98.0% and not more than 101.0% of fenbufen (C16H14O3).
Description

Each mL of 0.1 mol/L potassium hydroxide-ethanol VS = 25.428 mg of C16H14O3

Packaging and Storage Preserve in tight containers.

Fenbufen is a white crystalline powder

398 Monographs, Part I And separately, dissolve 10.0 mg of fenofibrate RS, 10.0 mg of fenofibrate related substance I RS [(4chlorophenyl)(4-hydroxyphenyl)methanone], 10.0 mg of of fenofibrate related substance II RS [2-[4-(4-chloro benzoyl)phenoxy]-2-methylpropanoic acid (fenofibric acid)] and 20.0 mg of fenofibrate related substance III RS [1-methylethyl 2-[[2-[4-(4-chlorobenzoyl)phenoxy]2-methylpropanoyl]oxy]2-methylpropanoate] in the mobile phase to make exactly 100 mL. To 1.0 mL of this solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution (2). Perform the test with 20 L each of the test solution and the standard solutions as directed under the Liquid Chromatography according to the following conditions: the areas of the peaks of fenofibrate related substance I, II and III from the test solution is not larger than the areas of the corresponding peaks from the standard solution (2) (0.1% each of fenofibrate related substance I and II; 0.2% of fenofibrate related substance III). The areas of peaks other than the major peak and peaks of fenofibrate related substance I, II and III from the test solution is not larger than the the area of peak of fenofibrate from the standard solution (2) (0.1%). The total area of all the peaks other than the major peak from the test solution is not larger than 5 times area of the peak of fenofibrate from the standard solution (2) (0.5%). Disregard any peak with an area less than 0.l times that of the peak of fenofibrate from the standard solution (2). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 286 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of acetonitrile and water (adjust the pH to 2.5 with phosphoric acid) (70 : 30). Flow rate: 1 mL/minute. System suitability System performance: Adjust the detection sensitivity with the standard solution (2) so that the heights of the peaks in the chromatogram are not less than 20.0% of the full scale. The relative retention times of fenofibrate related substances I, II, IV, V, VI, VII and III are about 0.34, 0.36, 0.50, 0.65, 0.80, 0.85 and 1.35, respectively and the resolution between the peaks of fenofibrate related substances I and II is not less than 1.5. Time span of measurement: About 2 times as long as the retention time of fenofibrate, beginning after the solvent peak.
Loss on Drying Not more than 0.5% (0.5 g, 60 C, in vacuum, constant mass). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh acculately about 25 mg each of Fenofibrate and Fenofibrate RS, dissolve in exactly 25 mL of

Fenofibrate
O Cl O O H3C CH3 CH3 CH3

C20H21ClO4: 360.83 Fenofibrate contains not less than 98.5% and not more than 101.0% of fenofibrate (C20H21ClO4), calculated on the dried basis.
Description Fenofibrate is a white crystalline powder. Fenofibrate is very soluble in dichloromethane, slightly soluable in ethanol and practically insoluable in water. Identification Determine the infrared spectra of Fenofibrate and Fenofibrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point

Between 79 C and 82 C.

Purity (1) Clarity and color of solutionDissolve 0.50 g of Fenofibrate in aceton to make exactly 10 mL: the solution is clear. (2) AcidDissolve 1.0 g of Fenofibrate in 50 mL of ethanol, previously neutralized using 0.2 mL of phenolphthalein solution TS: not more than 0.2 mL of 0.1 mol/L sodium hydroxide VS is required to change the color of the indicator to pink. (3) ChlorideTo 0.5 g of Fenofibrate, add 25 mL of water, heat at 50 C for 10 minutes, cool and filter. To 5 mL of the filtrate, add 6 mL of dilute nitric acid TS and add water to make 50 mL. Prepare the control solution with 6 mL of dilute nitric acid and 0.30 mL of 0.01 mol/L hydrochloric acid VS, adding water to make 50 mL (not more than 0.01%). (4) Sulfate To 0.5 g of Fenofibrate, add 25 mL of water, heat at 50 C for 10 minutes, cool and filter. To 10 mL of the filtrate, add 1 mL of dilute hydrochloric acid TS and add water to make 50 mL. Prepare the control solution with 1 mL of dilute hydrochloric acid and 0.42 mL of 0.005 mol/L sufuric acid VS, adding water to make 50 mL (not more than 0.01%). (5) Heavy metalsProceed with 1.0 g of Felodipine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (6) Related substancesDisolve 0.100 g of Fenofibrate in the mobile phase to make exactly 100 mL and use this solution as the test solution. Separately, dissolve 25.0 mg of Fenofibrate RS in the mobile phase to make exactly 25 mL and use this solution as the standard solution (1).

KP 9 399 the mobile phase and use these solutions as the test solution and the standard solution (1), respectively. Seperately, weigh 10.0 mg of Fenofibrate RS, 10.0 mg of fenofibrate related compound II RS and 20.0 mg of fenofibrate related compound III RS, dissolve in the mobile phase to make exactly 100 mL, take 1.0 mL of this solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution (2). Perform the test with 5 L each of the test solution and the standard solution (1) as directed under the Liquid Chromatography according to the following conditions of the Related substances, and determine AT and AS, of the peak area of fenofibrate of each solution. Amount (mg) of fenofibrate (C20H21ClO4) AT = amount (mg) of Fenofibrate RS A S System suitability System repeatability: When the test is repeated 6 times with 5 L of the standard solution (1), as directed under the above operating conditions, the relative standard deviation of the peak area of fenofibrate is not more than 1.0%. Detection sensitivity: Adjust the detection sensitivity so that the heights of the peaks in the chromatogram are not less than 50.0% of the full scale.
Packaging and Storage tight containers.

TS. (2) Determine the infrared spectra of Fenoprofen Calcium Hydrate and of Fenoprofen Calcium Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
Purity (1) CalciumWeigh accurately about 0.75 g of Fenoprofen Calcium Hydrate, dissolve in ethanol by heating, if necessary, add ethanol to make exactly 50 mL and use this solution as the test solution. Put 70 mL of water, 2 mL of a solution of sodium hydroxide (1 in 10) and 0.3 g of hydroxynaphtol, mix, add about 1 mL of the test solution and titrate with 0.05 mol/L disodium ehthylenediaminetetraacetate VS until blue color appears (7.3 to 8.0%, calculated on the anhydrous basis)

Each mL of 0.05 mol/L disodium ethylenediaminetetraacetate = 2.044 mg of Ca (2) Heavy metalsProceed with 1.0 g of Fenoprofen Calcium Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve about 0.2 g of Fenoprofen Calcium Hydrate, accurately weighed, in a mixture of water-acetonitrile (1 : 1) to make exactly 100 mL and use this solution as the test solution. Separately, dissolve about 20 mg of Fenoprofen Calcium Hydrate RS, accurately weighed, in a mixture of wateracetonitrile (1 : 1) to make exactly 100 mL, take 5.0 mL of this solution, add a mixture of water-acetonitrile (1 : 1) to make exactly 50 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, determine each peak area of the test solution and the standard solution by the automatic integration and calculate the amount of each related compound: the amount of each related compound is not more than 0.5% and the total amount of related compounds is not more than 2.0%. Amount (%) of a related comound Ai C = 10000 W A S C : concentration (mg/mL) of Fenoprofen Calcium Hydrate RS in the standard solution W : amount (mg) of Fenoprofen Calcium Hydrate taken to prepare the test solution Ai : peak area of each related compound obtained from the test solution As : peak area of fenoprofen obtained from the standard solution Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 270 nm).

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Fenoprofen Calcium Hydrate

O H3C O Ca O O 2H2O CH3

C30H26CaO62H2O: 558.63 Fenoprofen Calcium Hydrate contains not less than 97.0% and not more than 103.0% of fenoprofen calcium (C30H26CaO6: 522.60), calculated on the anhydous basis.
Description Fenoprofen Calcium Hydrate is white crystalline powder. Fenoprofen Calcium Hydrate is slightly soluble in nhexane, in methanol, or in water and practically insoluble in chloroform. Identification (1) Mix 1 g of Fenoprofen Calcium Hydrate with 50 mL of acetic acid, heat and filter. Add 2 mL of ammonium oxalate TS to the filtrate: white precipitate is formed and it dissolves in 3 mol/L hydrochloric acid

400 Monographs, Part I Column : A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase : With the mobile phase A and the mobile B, control the composition stepwise or gradiently as follows. Mobile phase A: A mixture of water and acetic acid (98 : 2) Mobile phase B: A mixture of acetonitrile and acetic aicd (98 : 2) Time (minutes) 0 0~3 3 ~ 41 41 ~ 42 42 ~ 43 43 ~ 55 Mobile phase A (vol%) 70 70 70 10 10 10 70 70 Mobile phase B (vol%) 30 30 30 90 90 90 30 30 Elution condition Equilibriation Isocratic Linear gradient Isocratic Linear gradient Reequilibriation

= 100 C AT
AS

C : Amount (mg) of fenoprofen calcium in Fenoprofen Calcium Hydrate RS, calculated on the anhydrous basis Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 272 nm). Column : A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase : A mixture of acetonitrile, water and phosphoric acid (50 : 49.6 : 0.4) Flow rate: 2 mL/minute System suitability System performance: Dissolve 5 mg of Fenoprofen Calcium Hydrate RS and 5 mg of gemfibrozil in a mixture of methanol and water (1 : 1) to make 5 mL. When the procedure is run with 20 L of this solution under the above operating conditions, relative retention times are about 0.5 for fenoprofen and 1.0 for gemfibrozil, the resolution between them is not less than 8 and the column efficiency determined from the peak of fenoprofen is not less than 3000 theoretical plates. System reproducibility: When the test is repeated 5 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of fenoprofen is not more than 2.0%.
Packaging and Storage Preserve in tight containers.

Flow rate: 1.5 mL/minute System suitability System performance: Dissolve 2 mg of 3phenoxybenzoic acid and Fenoprofen Calcium Hydrate RS in a mixture of water and acetonitrile (1 : 1) to make 100 mL. When the procedure is run with 20 L of this solution under the above operating conditions, relative retention times are about 0.89 for 3-phenoxybenzoic acid and 1.00 for fenoprofen, the resolution between them is not less than 9.0 and the symmetry factor of fenoprofen is not more than 2.0. System reproducibility: When the test is repeated 5 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of fenoprofen is not more than 2.0%.
Water Between 5.0% and 8.0% (1 g, volumetric titration, direct titration) Assay Weigh accurately about 70 mg each of Fenoprofen Calcium Hydrate and Fenoprofen Calcium Hydrate RS, add 0.5 mL of 0.5 mol/L hydrochloric acid TS and 2 mL of ethanol, add a mixture of methanol and water (7 : 3) to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, measure the peak areas of fenopropen obtained from each solutions, AT and AS .

Fenoterol Hydrobromide
H HO OH H N H HBr

CH3 OH

OH

C17H21NO4HBr: 384.27 Fenoterol Hydrobromide contains not less than 99.0% and not more than 101.0% of fenoterol hydrobromide (C17H21NO4HBr), calculated on the dried basis.
Description Fenoterol Hydrobromide is a white crystalline powder. Fenoterol Hydrobromide is soluble in ethanol. Identification (1) Dissolve 10 mg of Fenoterol Hydrobromide in 2 w/v% solution of disodium tetraborate to make 50 mL. Add 1 mL of 1 w/v% solution of aminopyrazolone, 10 mL of 2 w/v% solution of potassium ferri-

Amount (mg) of fenoprofen calcium [(C15H13O3)2Ca]

KP 9 401 cyanide and 10 mL of dichloromethane, mix and allow to stand: a reddish-brown color develops in the lower layer. (2) Prepare 0.037 w/v% solutions in diluted hydrochloric acid (1 in 10000) of Fenoterol Hydrobromide and Fenoterol Hydrobromide RS, respectively. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Fenoterol Hydrobromide and Fenoterol Hydrobromide RS as directed in potassium bromide disk method under the Infrared absorption Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) Dissolve 10 mg each of Fenoterol Hydrobromide and Fenoterol Hydrobromide RS in ethanol to make 10 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 2 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methanol, water and ammonia solution (28) (90 : 10 : 1.5) to a distance of about 15 cm and air-dry the plate. Spray evenly 1 w/v% potassium permanganate solution on the plate: the principal spot from the test solution shows the same Rf value as the principal spot from the standard solution. (5) A solution of Fenoterol Hydrobromide (1 in 100) responds to the Qualitative Test (1) for bromides.
pH Dissolve 2.0 g of Fenoterol Hydrobromide in water to make exactly 50: the pH of this solution is between 4.2 and 5.2. Purity (1) Clarity and color of solutionDissolve 2.0 g of Fenoterol Hydrobomide in water to make exactly 50 mL: the solution is clear and colorless. (2) IronProceed with 1.0 g of Fenoterol Hydrobomide according to Method 3 and perform the test according to Method A. Prepare the control solution with 0.5 mL of standard iron solution (not more than 5 ppm). (3) PhenoneDissolve 2.0 g of Fenoterol Hydrobomide in water to make exactly 50 mL and Determine the absorbance of this solution at 330 nm, as directed under the Ultraviolet-visible Spectrophotometry: the absorbance is not more than 0.42 (not more than 0.2%) (4) IsomerDissolve 20 mg each of Fenoterol Hydrobromide and Fenoterol Hydrobromide RS in water to make 10 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak heights, HT and HS of peak eluted right after the principal peak, for the test solution and the standard solution and calculate the amount of isomer: the amount of isomer is not more than 4.0%.

Amount(%) of isomer = amount(%) of isomer labeled on Reference Standard H T

HS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 280 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Mobile phase : Mix 10 mL of 0.9 w/v% potassium dihydrogen phosphate and 690 mL of 2.5 w/v% sodim hydrogen phosphate dodecahydrate and add phosphoric acid to adjust pH to 8.5, add 300 mL of methanol and mix. Flow rate: 1.0 mL/minute. Adjust the flow rate so that the retention time of the principal peak is not more than 20 minutes. System suitability System performance: When the procedure is run with 20 L of this solution under the above operating conditions, adjust the sensitivity so that the height of peak eluted right after the principal peak is not less than 10% of the full scale and the height of valley between the principal peak and the isomer peak is not more than 4% of the full scale.
Loss on Drying Not more than 0.5% (0.5 g, 105 C, constant mass). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.6 g of Fenoterol Hydrobromide, dissolve in 50 mL of water, add 5 mL of dilute nitric acid TS and 25 mL of 0.1 mol/L silver nitrate VS, shake and titrate with 0.1 mol/L ammonium thiocyanate VS until an orange color is obtained (indicator: 2 mL of ferric ammonium sulfate solution TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L silver nitrate VS = 38.48 mg of C17H21NO4

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Fentanyl Citrate
O C CH2CH2 N N CH2CH3 HO CH2CO2H C CO2H

CH2CO2H

C22H28N2OC6H8O7: 528.60

402 Monographs, Part I tion, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction. Each mL of 0.02 mol/L perchloric acid VS = 10.572 mg of C22H28N2OC6H8O7
Packaging and Storage tight containers.

Fentanyl Citrate contains not less than 98.0% and not less than 101.0% of fentanyl citrate (C22H28N2OC6H8O7), calculated on the dried basis.
Description Fentanyl Citrate is a white crystal or crystalline powder. Fentanyl Citrate is freely soluble in methanol or in glacial acetic acid, sparingly soluble in water or in ethanol and very slightly soluble in ether. Identification (1) Dissolve 50 mg each of Fentanyl Citrate and Fentanyl Citrate RS in 10 mL of 0.1 mol/L hydrochloric acid TS and ethanol to make 100 mL and determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Fentanyl Citrate and Fentanyl Citrate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Fentanyl Citrate (1 in 100) responds to the Qualitative Tests (1) for citrate. pH Dissolve 0.10 g of Fentanyl Citrate in 10 mL of water: the pH of this solution is between 3.0 and 5.0. Melting Point Between 150 C and 154 C. Purity (1) Heavy metalsProceed with 0.5 g of Fentanyl Citrate according to Method 2 and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 0.10 g of Fentanyl Citrate in 5 mL of methanol and use this solution as the test solution. Pipet 1.0 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thinlayer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (3 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly Dragendorffs TS for spraying on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (0.2 g, in vacuum, silica gel, 60 C, 2 hours). Residue on Ignition Not more than 0.2% (0.5 g). Assay Weigh accurately about 75 mg of Fentanyl Citrate, dissolve in 50 mL of glacial acetic acid and titrate with 0.02 mol/L perchloric acid VS (potentiometric titra-

Preserve in light-resistant,

Fenticonazole Nitrate
Cl

Cl H O HNO3 N S N

and enantiomer

C24H20Cl2N2OSHNO3: 518.41 Fenticonazole Nitrate contains not less than 99.0% and not more than 101.0% of fenticonazole nitrate (C24H20Cl2N2OSHNO3), calculated on the dried basis.
Description Fenticonazole Nitrate is a white powder. Fenticonazole Nitrate is freely soluble in methanol and in dimethylformamide, sparingly soluble in ethanol, and practically insoluble in water. Identification (1) Determine the absorption spectra of solutions of Fenticonazole Nitrate and Fenticonazole Nitrate RS in ethanol (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Fenticonazole Nitrate and Fenticonazole Nitrate RS, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Weigh a portion of Fenticonazole Nitrate equivalent to about 1 mg of nitrate ion, mix with a mixture of 0.1 mL of nitrobenzene and 0.2 mL of sulfuric acid, stand for 5 minutes, cool in ice-water, add gently 5 mL of water while stirring. Add 5 mL of 10 mol/L sodium hydroxide TS and 5 mL of of aceton, shake and stand: a color of deep violet develops in the upper layer. Meting Point Between 134 C and 137 C Purity (1) TolueneWeigh exactly 0.2 g of Fenticonazole Nitrate in a 10-mL vial, add exactly 5 mL of water to disperse and use this solution as the test solution, Separately, add 4.0 mg of toluene to water to make 1000 mL.

KP 9 403 Take 5 mL of this solution to a 10-mL vial and use it as the standard solution. Perform the test with the test solution and the standard solution as directed in the standard addition method under the Gas Chromatography according to the following operating conditions. Use a headspace sample introduction device to measure the amount of toluene by the standard addition method: the amount of toluene is not more than 100 ppm. Operating conditions Detector: A hydrogen flame-ionization detector. Column: A column, about 0.32 mm in inside diameter and about 25 m in length, coated with poly(cyanopropyl)(7)phenyl(7)methyl(86)siloxane for gas chromatography (1.2 m in film thickness). Column temperature: 80 C. Inlet temperature: 180 C. Detector temperature: 220 C. Carrier gas: Helium. Split ratio: about 1 : 25 Column head pressure: 40 kPa. Injection volume: Maintain each solution at 90 C for 1 hour and transfer 1 mL of the vapor phase onto the column. (2) Related substancesDissolve about 25 mg of Fenticonazole Nitrate, accurately weighed, in the mobile phase to make exactly 25 mL and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add the mobile phase to make exactly 200 mL, and use this solution as the standard solution (1). Pipet exactly 10 mL of the standard solution (1), add the mobile phase to make exactly 25 mL, and use this solution as the standard solution (2). Pipet exactly 1 mL of the standard solution (1), add the mobile phase to make exactly 10 mL, and use this solution as the standard solution (3). Pipet exactly 5 mL of the test solution, add 5.0 mg of fenticonazole related substance I RS {(RS)-1-[2-(2,4dichlorophenyl)-2-hydroxyethyl]-3-[4-(phenylsulphanyl) benzyl]imidazolium nitrate} add the mobile phase to make exactly 100 mL, pipet exactly 2 mL of this solution, add the mobile phase to make exactly 10 mL and use this solution as the standard solution (4). Perform the test with 10 L each of the test solution and the standard solution (1) as directed under the Gas Chromatography according to the following operating conditions and determine the area of each peak by the automatic integration method: the area of any peak except the principal peak and the nitrate ion peak (which correspond to the dead volume of the column) obtained from the test solution is not greater than the area of the principal peak obtained from the standard solution (2) (0.2%) and the sum of of the areas of such peaks is not greater than the area of the principal peak obtained from the standard solution (1) (0.5%). Disregard any peak with an area less than that of the principal peak from the standard solution (3). Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 210 nm). Column : A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Mobile phase : A mixture of acetonitrilethe and phosphate buffer (70 : 30) Flow rate: 1.0 mL/minute System suitability The procedure is run with 10 L of the standard solution (2) under the above operating conditions, and adjust the sensitivity of the system so that the height of fenticonazole peak is not less than 10% of the full scale of the recorder. When the procedure is run with 10 L each of the standard solution (3) and the standard solution (4) under the above operating conditions, the resolution between the peaks of fenticonazole related substance I and fenticonazole is not less than 2, and the signal-tonoise ratio of the chromatogram obtained with the standard solution (3) is not less than 5. Phosphate bufferDissolve 3.4 g of potassium dihydrogen phosphare in 900 mL of water, adjusting to pH 3.0 with phosphoric acid, and add water to make 1000 mL.
Loss on Drying Not more than 0.5% (1 g, in vacuum, 60 C, constant mass). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.45 g of Fenticonazole Nitrate, dissolve in 50 mL of a mixture of methyl ethyl ketone and glacial acetic acid (1 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 51.84 mg of C24H20Cl2N2OSHNO3

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Ferrous Fumarate
HC O C O CH Fe C O O

C4H2FeO4: 169.91 Ferrous Fumarate, when dried, contains not less than 97.0% and not more than 101.0% of ferrous fumarate (C4H2FeO4).
Description Ferrous Fumarate is a reddish-orange to

404 Monographs, Part I reddish-brown powder and is odorless. Ferrous Fumarate is slightly soluble in water and very slightly soluble in ethanol. The solubility of Ferrous Fumarate in dilute hydrochloric acid is decreased by liberation of fumaric acid.
Identification (1) To 1.5 g of Ferrous Fumarate, add 25 mL of diluted hydrochloric acid (1 in 2), dilute with water to make 50 mL, heat to obtain complete solution, then cool, filter on a glass filter, wash the precipitate with diluted hydrochloric acid (3 in 100) and dry the precipitate at 105 C. Determine the infrared absorption spectra of the dried precipitate and Fumaric Acid RS, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The filtrate of (1) responds to the Qualitative Tests for iron. Purity (1) SulfateTransfer 1.0 g of Ferrous Fumarate to a beaker, add 100 mL of water and heat on a water-bath, adding hydrochloric acid drop-wise until complete solution is obtained (about 2 mL of the acid is required). Filter the solution, if necessary, and dilute the filtrate with water to make 100 mL. Heat this solution to boiling, add 10 mL of barium chloride TS, warm on a water-bath for 2 hours, cover and allow to stand for 16 hours. If crystals of ferrous fumarate form, warm the solution on a water-bath to dissolve them. Filter the solution through weighing filter paper, add ammonium sulfide TS to the filtrate, wash the residue with hot water until no more black precipitate is produced and transfer the paper containing the residue to a previously tared crucible. Char the paper, without burning and ignite the crucible and its contents at 600 C to a constant mass: each mg of residue is equivalent to 0.412 mg of sulfate (SO4) (not more than 0.2%). (2) ArsenicTransfer about 2.0 g of Ferrous Fumarate to a beaker and add 10 mL of water and 10 mL of sulfuric acid. Warm to precipitate fumaric acid completely, cool, add 20 mL of water and filter into a 50 mL volumetric flask. Wash the precipitate with water, add the washings to the flask, add water to to the mark. Perform the test with 25 mL of this solution. Prepare the control solution with 3.0 mL of standard arsenic solution (not more than 3 ppm). (3) Ferric ionTransfer about 2.0 g of Ferrous Fumarate, accurately weighed, to a glass-stoppered, Erlenmyer flask, add 25 mL of water and 4 mL of hydrochloric acid and heat on a hot plate until complete solution is obtained. Stopper the flask and cool to room temperature. Add 3 g of potassium iodide, stopper the flask, swirl to mix and allow to stand in the dark for 5 minutes. Remove the stopper, add 75 mL of water and titrate with 0.1 mol/L sodium thiosulfate VS (indicator: 3 mL of starch TS). Not more than 7.16 mL of 0.1 mol/L sodium thiosulfate is consumed (not more than 2.0%). (4) MercuryPerform the test with light-resistant container. Dissolve about 1 g of Ferrous Fumarate, accu-

rately weighed, in 30 mL of diluted nitric acid (1 in 10), by heating on a water-bath. Cool quickly by immersion in an ice-bath and filter through a filter (G4), previously washed with diluted nitric acid (1 in 10) and water. To the filtrate, add 20 mL of sodium citrate solution (1 in 4) and 1 mL of hydroxylamine hydrochloride TS and use this solution as the test solution. Separately, prepare a control solution consisting of 3.0 mL of standard mercury solution, 30 mL of diluted nitric acid (1 in 10), 5 mL of sodium citrate solution (1 in 4) and 1 mL of hydroxylamine hydrochloride TS. To the control solution, add ammonium hydroxide TS to adjust to a pH of 1.8, add sulfuric acid to the test solution to adjust to a pH of 1.8 and transfer to a separator, respectively. Perform the test with the test solution and the control solution as follows. Extract with two 5 mL volumes of dithizone solution for extraction and 5 mL of chloroform and transfer the chloroform extracts to a second separator. Add 10 mL of diluted hydrochloric acid (1 in 2), shake to mix, allow the layers to separate and discard the chloroform layer. Wash the acid extract with 3 mL of chloroform and discard the washing. Add 0.1 mL of disodium ethylenediamine tetraacetate solution (1 in 50) and 2 mL of 6 mol/L acetic acid, mix and add slowly 5 mL of ammonia TS. Stopper the separator and cool under cold running water. Remove the stopper and pour the contents into a beaker. Adjust the test solution and the control solution to a pH of 1.8 in the same manner as before and return the solution to its separator, respectively. Add 5.0 mL of diluted dithizone solution for extraction, shake vigorously and allow the layers to separate. At this point, compare the colors observed in the chloroform layers of the two solutions that have been treated in parallel: the color observed by the test solution is not more intense than that observed by the control solution (not more than 3 ppm). Mercury stock solutionTransfer 135.4 mg of mercuric chloride to a volumetric flask, dissolve in 0.5 mol/L sulfuric acid and add 0.5 mol/L sulfuric acid to make 100 mL and mix. This solution contains 0.1 g of mercury (Hg) in 100 mL. Mercury standard solutionTransfer 1.0 mL of mercury stock solution to a volumetric flask before use and add 0.5 mol/L sulfuric acid to make 1000 mL and mix. This solution contains 1 g of mercury (Hg) in 1 mL. Diluted dithizone solution for extractionDilute 5 mL of dithizone solution for extraction with 25 mL of chloroform before use. (5) LeadAdd 1.0 g of Ferrous Fumarate to a beaker, add 6 mL of nitric acid and 10 mL of perchloric acid, cover with a watch glass and heat until complete dryness. Cool, dissolve the residue in 10 mL of 9 mol/L hydrochloric acid and transfer with the aid of about 10 mL of water to a volumetric flask. Add 20 mL of ascorbic acid-sodium iodide solution and 5.0 mL of trioctylphosphine oxide solution, shake for 30 seconds and allow to

KP 9 405 separate. Add water to bring the organic solvent layer into the neck of the flask, shake again and allow to separate. Use the organic solvent layer as the test solution. Separately, transfer 5.0 mL of lead stock solution and add water to make exactly 100 mL. Transfer exactly 2 mL of the resulting solution to a beaker. To this beaker and to a second empty beaker, add 6 mL of nitric acid and 10 mL of perchloric acid and evaporate to dryness. Cool, dissolve the residue in 10 mL of 9 mol/L hydrochloric acid and transfer with the aid of about 10 mL of water to a volumetric flask. To each flask, add 20 mL of ascorbic acid-sodium iodide solution and 5.0 mL of trioctylphosphine oxide solution, shake for 30 seconds and allow to separate. Add water to bring the organic solvent layer into the neck of each flask, shake again and allow to separate. Use the organic solvent layers as the standard solution (2.0 g/mL) and the blank solution, respectively. Determine the absorbances of the blank solution, the test solution and the standard solution as directed under the Atomic Absorption Spectrophotometer according to the following conditions: the absorbance of the test solution doses not exceed that of the standard solution (not more than 0.001%). But, using 4-methyl-2-pentanone to set the instrument to zero, the absorbance of the blank solution is not greater than 20% of the difference between the absorbance of the standard preparation and that of the blank solution. Gas used: Combustible gas- Acetylene. Supporting gas: Air. Lamp: A lead hollow-cathode lamp. Wavelength: 283.3 nm. Trioctylphosphine oxide solutionDissolve 5.0 g of trioctylphosphine oxide in 4-methyl-2-pentanone to make 100 mL.
Loss on Drying Not more than 1.5% (1 g, 105 C, 16 hours). Assay Transfer about 0.5 g of Ferrous Fumarate, accurately weighed, to an Erlenmyer flask, add 25 mL of diluted hydrochloric acid (2 in 5) and heat to boiling. Add a solution of 5.6 g of stannous chloride in 50 mL of diluted hydrochloric acid (3 in 10) until yellow color disappears, then add 2 drops in excess. Cool the solution in an ice-bath to room temperature, add 10 mL of mercuric chloride solution (1 in 20) and allow to stand for 5 minutes. Add 200 mL of water, 25 mL of diluted sulfuric acid (1 in 2) and 4 mL of phosphoric acid and then titrate with 0.1 mol/L ceric ammonium sulfate VS (indicator: ophenanthroline TS). Perform a blank determination and make any necessary correction.

Ferrous Fumarate Tablets

Ferrous Fumarate Tablets contain not less than 95.0% and not more than 110.0% of the labeled amount of ferrous fumarate (C4H2FeO4: 169.90).
Method of Preparation Prepared as directed under Tablets, with Ferrous Fumarate. Identification Shake well a portion of powdered Ferrous Fumarate Tablets, equivalent to 1 g of Ferrous Fumarate according to the labeled amount, with 25 mL of diluted hydrochloric acid (1 in 2) and add 25 mL of water. Boil the solution for a few minutes, cool and filter: the filtrate responds to the Qualitative Tests for iron. Dissolution Test Perform the test with 1 tablet of Ferrous Fumarate Tablets at 75 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL out of 1000 mL solution of 5 g of lauryl sodium sulfate in 0.1 mol/L hydrochloric acid. Take the dissolved solution at 45 minutes after starting the test and filter. Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, dry and weigh accurately a portion of Ferrous Fumarate RS, dissolve in the same solvent to make a constant concentration and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution at 248.3 nm as directed under the Ultraviolet-visible Spectrophotometry using the dissolution solution as the blank.

The dissolution rate of Ferrous Fumarate Tablets in 45 minutes is not less than 75%.
Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Ferrous Fumarate Tablets, transfer an accurately weighed portion of the powder, equivalent to about 0.5 g of ferrous fumarate (C4H2FeO4), to a beaker and add 25 mL of water, 25 mL of nitric acid and 7.5 mL of perchloric acid. Cover with a watch glass and heat until strong fumes are produced. Cool, rinse the watch glass and the sides of the beaker with water and then evaporate in a hood to neardryness. Wash down the watch glass and the sides of the beaker with hydrochloric acid 2 mL and then with a small volume of water. Warm slightly, if necessary, to dissolve the residue, transfer to a glass-stoppered Erlenmyer flask, wash the beaker with 2 mL of hydrochloric acid and transfer the washing to the flask. Completely wash the beaker with 20 to 25 mL of water for the transfer, add 4 g of potassium iodide to the flask, stopper and allow to stand in the dark for 5 minutes. Add 75 mL of water and titrate with 0.1 mol/L sodium thiosulfate VS (indicator: 3 mL of starch TS, add it as the endpoint is approached.)

Each mL of 0.1 mol/L ceric ammonium sulfate VS = 16.990 mg of C4H2FeO4

Packaging and Storage Preserve in well-closed containers.

406 Monographs, Part I Each mL of 0.1 mol/L sodium thiosulfate VS = 16.990 mg of C4H2FeO4
Packaging and Storage Preserve in tight containers

Ferrous Gluconate Hydrate

OH OH HOH2C C H C H H C OH OH C H CO2
-

Fe 2

2+

2H2O

C12H22FeO142H2O: 482.17 Ferrous Gluconate Hydrate, when dried, contains not less than 97.0% and not more than 102.0% of ferrous gluconate (C12H22FeO14: 446.14).
Description Ferrous Gluconate Hydrate is a yellowish gray or pale yellow green fine powder or granule and has a slight odor like sulfur-burning. One gram of Ferrous Gluconate Hydrate is soluble in 10 mL of hot water and very slightly soluble in ethanol. An aqueous solution of ferrous gluconate (1 in 20) is acidic. Identification (1) Dissolve about 10 mg of Ferrous Gluconate Hydrate in 10 mL of water, if necessary, heat on a water-bath at 60 o C and use this solution as the test solution. Separately, dissolve 10 mg of Ferrous Gluconate Hydrate RS in 10 mL of water and use this solution as the standard solution. Apply separately 5 L volume of the test solution and the standard solution in a suitable thin-layer chromatographic plate coated with chromatographic silica gel. Develop the chromatogram in a solvent system consisting of a mixture of ethanol, water, strong ammonium hydroxide solution and ethyl acetate (50 : 30 : 10 : 10) until the solvent front has moved about 15 cm of the plate. Remove the plate from the chamber and dry at 110 o C for 20 minutes. Allow to cool, spray with a spray reagent prepared as follow; dissolve 2.5 g of ammonium molibdenate in about 50 mL of 1 mol/L sulfuric acid TS in a volumetric flask, add 1.0 g of ceric sulfate, swirl to dissolve, add 1 mol/L sulfuric acid TS to make 100 mL and mix. Heat the plate at 110 o C for about 10 minutes: the color, size and Rf value of the principal spot obtained from the test solution should be the same as those obtained from the standard solution. (2) Addition of potassium ferricyanide TS to an aqueous solution of Ferrous Gluconate Hydrate (1 in 200): dark blue precipitation is produced. Purity (1) ChlorideProceed with 1.0 g of Ferrouse Gluconate Hydrate. Prepare the control solution with 1.0 mL of 0.02 mol/L hydrochloric acid VS (not more than 0.07%).

(2) SulfateProceed with 1.0 g of Ferrous Gluconate Hydrate. Prepare the control solution with 1.0 mL of 0.01 mol/L sulfuric acid VS (not more than 0.1%). (3) Oxalic acidDissolve 1.0 g of Ferrous Gluconate Hydrate in 10 mL of water, add 2 mL of hydrochloric acid and transfer to a separator. Extract successively with 50 mL and 20 mL volumes of ether. Combine the ether extracts, add 10 mL of water and evaporate the ether on a steam-bath. Add 1 drop of 6 mol/L acetic acid and 1 mL of calcium acetate solution (1 in 20): no turbidity is produced within 5 minutes. (4) LeadAdd 1.0 g of Ferrous Gluconate Hydrate, 10 mL of 9 mol/L hydrochloric acid TS, about 10 mL of water, 20 mL of ascorbic acid, sodium iodide solution and 5.0 mL of triocytylphosphine oxide mythyl isobytylketone solution to a volumetric flask, shake for 30 seconds and allow to separate. Add water into the neck of the flask, shake again and allow to separate. The organic solvent layer collected is used as the test solution. Separately, pipet 5.0 mL of lead nitrate standard stock solution into a volumetric flask, dilute with water to make 100 mL, pipet 2.0 mL of the solution to a volumetric flask and then prepare the standard solution, as directed under the test solution. Determine the absorbances of the test solution and the standard solution according to the following conditions, as directed under the Ultravioletvisible Spectrophotometry: the absorbance of the solution obtained from the test solution is not greater than that from the standard solution. Gas: Flammable Acetylene gas. Delayed gas: Air. Lamp: Lead hollow-cathode lamp. Wavelength: 283.8 nm. (5) ArsenicWeigh 1.0 g of Ferrous Gluconate Hydrate in a round-bottom flask and add 40 mL of 4.5 mol/L sulfuric acid and 2 mL of potassium bromide solution (3 in 10). Immediately collect to a suitable distillation apparatus having a reservoir with a water jacket, cooled with circulating icewater and heat to dissolve the test specimen. Prepare 25 mL of distillate as the test solution and perform the test (not more than 3 ppm). (6) Ferric ionDissolve about 5 g of Ferrous Gluconate Hydrate, exactly weighed, in a mixture of 100 mL of water and 10 mL of hydrochloric acid and add 3 g of potassium iodide. Shake and allow to stand in the dark place for 5 minutes. Titrate any liberated iodine with 0.1 mol/L sodium thiosulfate VS (indicator: 3 mL of starch TS). Perform a blank determination and make any necessary correction (not more than 2.0%). Each mL of 0.1 mol/L sodium thiosulfate VS = 5.585 mg of Fe+3 (7) Reducing sugarsDissolve about 500 mg of Ferrous Gluconate Hydrate in 10 mL of water, warm and render alkaline with 1 mL of 6 mol/L ammonium hydroxide TS. Pass hydrogen sulfide gas into the solution to precipitate the iron and allow to stand for 30 minutes.

KP 9 407 Filter and wash the precipitate with two successive 5 mL volumes of water. Acidify the combined filtrate and washings with hydrochloric acid and add 2 mL of dilute hydrochloric acid. Boil the solution until the vapors no longer darken lead acetate paper and continue to boil, if necessary, until concentrated to be about 10 mL. Cool, add 5 mL of sodium carbonate TS and 20 mL of water, filter and add water to make 100 mL. Take 5 mL of the filtrate, add 2 mL of Fehling TS and boil for 1 minute: no red precipitate is produced within 1 minute.
Loss on Drying Between 6.5% and 10.0% (1 g, 105 C, 16 hours). Assay Dissolve about 1.5 g of Ferrous Gluconate Hydrate, accurately weighed, in a mixture of 75 mL of water and 15 mL of dilute sulfuric acid in a cornical-flask. Add 250 mg of zinc powder, close the flask with a stopper containing a Bunsen valve and allow to stand at room temperature for 20 minutes or until the solution becomes colorless. Filter the solution through a filtering crucible containing an asbestos mat coated with a thin-layer of zinc dust and wash the crucible and contents with 10 mL of dilute sulfuric acid, followed by 10 mL of water. Add o-phenanthroline TS and titrate the filtrate in the suction flask immediately with 0.1 mol/L ammonium ceric sulfate VS. Perform a blank determination and make any necessary correction.

solution 80 minutes after the start of the test, filter through a membrane filter and use the filtrate solution as the test solution. If necessary, dilute the test solution as a suitable concentration using the test solution. Separately, weigh accurately a portion of Ferrous Gluconate Hydrate RS, add the test solution to dissolve and prepare the standard solution containing the same iron concentration as the test solution. Determine the absorbances of the test solution and the standard solution according to the following conditions, as directed under the Ultravioletvisible Spectrophotometry. Calculate the content of iron using the calibration curve obtained from the absorbance of standard solution. Gas used: Combustible gas- Acetylene. Supporting gas: Air. Lamp: Iron hollow-cathode lamp. Wavelength: 248.3 nm The dissolution rate of Ferrous Gluconate Tablets in 80 minutes is not less than 80.0%.
Uniformity of Dosage Units It meets the requirement. Assay Weigh and finely powder not less than 20 Ferrous Gluconate Tablets. Weigh accurately a portion of the powder, equivalent to about 1.5 g of Ferrous Gluconate Hydrate and dissolve in a mixture of 75 mL of water and 15 mL of dilute sulfuric acid in a cornical-flask. Proceed as directed in the Assay under Ferrous Gluconate Hydrate.

Each mL of 0.1 mol/L ammonium ceric sulfate VS = 44.61 mg of C12H22FeO14

Packaging and Storage Preserve in tight containers.

Each mL of 0.1 mol/L ammonium ceric sulfate VS = 48.22 mg of C12H22FeO14.2H2O

Packaging and Storage Preserve in tight containers.

Ferrous Gluconate Tablets

C12H22FeO142 H2O: 482.17 Ferrous Gluconate Tablets contain not less than 93.0% and not more than 107.0% of ferrous gluconate hydrate (C12H22FeO142 H2O: 482.17).
Method of Preparation Prepare as directed under Tablets, with Ferrous Gluconate Hydrate. Identification Take a portion of powdered Ferrous Gluconate Tablets, equivalent to about 1 g of Ferrous Gluconate Hydrate according to the labeled amount, add 100 mL of water and filter: the suitable volume of the filtrate is diluted with water, to a designated concentration. Perform the test with the solution as directed in the Identification under Ferrous Gluconate Hydrate. Dissolution Test Perform the test with 1 tablet of Ferrous Gluconate Tablets at 150 revolutions per minute according to Method 1 under the Dissolution Test, using 900 mL of artificial gastric solution. Take the dissolved

Ferrous Sulfate Hydrate

FeSO47H2O: 278.02 Ferrous Sulfate Hydrate contains not less than 98.0% and not more than 104.0% of ferrous sulfate (FeSO47H2O).
Description Ferrous Sulfate Hydrate is a pale green crystal or crystalline powder, is odorless and has an astringent taste. Ferrous Sulfate Hydrate is freely soluble in water and practically insoluble in ethanol. Ferrous Sulfate Hydrate is efflorescent in dry air and its crytalline surface becomes yellowish brown in moist air. Identification A solution of Ferrous Sulfate Hydrate (1 in 10) responds to the Qualitative Tests for ferrous salt and for sulfate. Purity (1) Clarity of solutionDissolve 1.0 g of Ferrous Sulfate Hydrate in 20 mL of water and 1 mL of di-

408 Monographs, Part I lute sulfuric acid: the solution is clear. (2) AcidTake 5.0 g of powdered Ferrous Sulfate Hydrate, add 50 mL of ethanol, shake well for 2 minutes and filter the mixture. To 25 mL of the filtrate, add 50 mL of water, 3 drops of bromothymol blue TS and 0.5 mL of dilute sodium hydroxide TS: a blue color is observed. (3) MercuryPerform the test with light-resistant container. Dissolve about 1 g of Ferrous Sulfate Hydrate, accurately weighed, in 30 mL of diluted nitric acid (1 in 10), by heating on a water-bath. Cool quickly by immersion in an ice-bath and filter through a filter (G4), previously washed with diluted nitric acid (1 in 10) and water. To the filtrate, add 20 mL of sodium citrate solution (1 in 4) and 1 mL of hydroxylamine hydrochloride TS and use this solution as the test solution. Separately, prepare a control solution consisting of 3.0 mL of standard mercury solution, 30 mL of diluted nitric acid (1 in 10), 5 mL of sodium citrate solution (1 in 4) and 1 mL of hydroxylamine hydrochloride TS. To the control solution, add ammonium hydroxide TS to adjust to a pH of 1.8, add sulfuric acid to the test solution to adjust to a pH of 1.8 and transfer to a separator, respectively. Perform the test with the test solution and the control solution as follows. Extract with two 5 mL volumes of dithizone solution for extraction and 5 mL of chloroform and transfer the chloroform extracts to a second separator. Add 10 mL of diluted hydrochloric acid (1 in 2), shake to mix, allow the layers to separate and discard the chloroform layer. Wash the acid extract with 3 mL of chloroform and discard the washing. Add 0.1 mL of disodium ethylenediamine tetraacetate solution (1 in 50) and 2 mL of 6 mol/L acetic acid, mix and add slowly 5 mL of ammonia TS. Stopper the separator and cool under cold running water. Remove the stopper and pour the contents into a beaker. Adjust the test solution and the control solution to a pH of 1.8 in the same manner as before and return the solution to its separator, respectively. Add 5.0 mL of diluted dithizone solution for extraction, shake vigorously and allow the layers to separate. At this point, compare the colors observed in the chloroform layers of the two solutions that have been treated in parallel: the color observed by the test solution is not more intense than that observed by the control solution (not more than 3 ppm). Mercury stock solutionTransfer 135.4 mg of mercuric chloride to a volumetric flask, dissolve in 0.5 mol/L sulfuric acid and add 0.5 mol/L sulfuric acid to make 100 mL and mix. This solution contains 0.1 g of mercury (Hg) in 100 mL. Mercury standard solutionTransfer 1.0 mL of mercury stock solution to a volumetric flask before use and add 0.5 mol/L sulfuric acid to make 1000 mL and mix. This solution contains 1 g of mercury (Hg) in 1 mL. Diluted dithizone solution for extractionDilute 5 mL of dithizone solution for extraction with 25 mL of chloroform before use.

(5) LeadTake 1.0 g of Ferrous Sulfate Hydrate in a 50 mL volumetric beaker, add 10 mL of 9 mol/L hydrochloric acid about 10 mL of water, 20 mL of ascorbic acid-sodium iodide solution and 5 mL of trioctylphosphine oxide solution in methylisobutylketone (5 in 100), shake for 30 seconds and allow to separate. Add water to bring the organic solvent layer into the neck of the flask, shake again and allow to separate. Use the organic solvent layer as the test solution. Separately, transfer exactly 5 mL of lead stock solution and add water to make exactly 100 mL. Transfer exactly 2 mL of the resulting solution to a 50 mL volumetric flask and proceed as the test solution and use this soluteon as the standard solution. Determine the absorbances of the the test solution and the standard solution as directed under the Atomic Absorption Spectrophotometer according to the following conditions: the absorbance of the test solution doses not exceed that of the standard solution. Gas used: Combustible gas: Acetylene. Supporting gas: Air. Lamp: A lead hollow-cathode lamp. Wavelength: 283.3 nm. (5) ArsenicTake 1.0 g of Ferrous Sulfate Hydrate in 100 mL round-bottomed flask, add 40 mL of 4.5 mol/L sulfuric acid and 2 mL of potassium bromide solution (1 in 10) and connect the flask to a distillation apparatus with ice-water cooled condenser. Perform the test using 25 mL of the distillate as the test solution (not more than 3 ppm).
Assay Dissolve about 0.5 g of Ferrous Sulfate Hydrate, accurately weighed, in a mixture of 20 mL of 1 mol/L sulfuric acid TS and 80 mL of freshly boiled and cooled water and then titrate with 0.1 mol/L ceric ammonium sulfate VS (indicator: ortho-phenanthroline TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L ceric ammonium sulfate VS = 27.802 mg of FeSO47H2O

Packaging and Storage Preserve in tight containers.

Ferrous Sulfate Tablets

Ferrous Sulfate Tablets contain not less than 95.0% and not more than 110.0% of the labeled amount of ferrous sulfate hydrate (FeSO47H2O: 278.02).
Method of Preparation Prepared as directed under Tablets, with Ferrous Sulfate. Identification Weigh a portion of finely powdered Ferrous Sulfate Tablets, equivalent to 0.25 g of Ferrous Sulfate according to the labeled amount, add water, aci-

KP 9 409 dify with hydrochloride and add water again to make 25 mL: the solution responds to the Qualitative Tests for ferrous salts and for sulfate.
Dissolution Test Perform the test with 1 tablet of Ferrous Sulfate Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of 0.1 mol/L hydrochloric acid. Take the dissolved solution at 45 minutes after starting the test and filter. Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, dry and weigh accurately a portion of ferrous sulfate RS, dissolve in the same solvent to make a constant concentration and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution as directed under the Atomic Absorption Spectrophotometry.

Finasteride
O CH3 H N H CH3 CH3 CH3

CH3

H O N H

C23H36N2O2 : 372.54 Finasteride contains not less than 98.5% and not more than 101.0% of finasterid (C23H36N2O2), calculated on the anhydrous basis.
Description Finasteride is a white to grayish white, crystalline powder. Finateride is freely soluble in ethanol, or in chloroform, and very slightly soluble in water. Melting pointabout 257 C Identification (1) Determine the infrared spectra of Finateride and Finasteride RS, as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The retention time of the principal peak obtained from the test solution under Assay is equal to that of the principal peak obtained from the standard solution. Specific Optical Rotation [] 25 405 nm : Between -56.0 and -60.0 (0.1 g, methanol, 10 mL, 100 mm) Purity (1) Heavy metalsProceed with 1.0 g of Finasteride according to Method 2 and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesDissolve about 0.1 g of Finasteride, accurately weighed, in a mixture of water and acetonitrile (1 : 1) to make exactly 100 mL and use this solution as the test solution. Perform the test with 15 L of the test solution as directed under the Liquid Chromatography according to the following operating conditions and determine the area of each peak and calculate the amount of each related substance: the amount of any related subtances is not more than 0.5% and the total amount of related substances is not more than 1.0%.

Gas used: Combustible gas: Acetylene. Supporting gas: Air. Lamp: A iron hollow-cathode lamp. Wavelength: 248.3 nm. The dissolution rate of Ferrous Sulfate Tablets in 45 minutes is not less than 75% (Q).
Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Ferrous Sulfate Tablets, weigh accurately a portion of the powder, equivalent to about 0.5 g of Ferrous Sulfate (FeSO47H2O), dissolve in a mixture of 20 mL of 1 mol/L sulfuric acid and 80 mL of freshly boiled and cooled water. Filter the solution as soon as all soluble ingredients in the tablets are dissolved and wash the residue in the container and the filter with small portions of a mixture of 20 mL of 1 mol/L sulfuric acid and 80 mL of freshly boiled and cooled water. Immediately titrate the combined filtrate and washings with 0.1 mol/L ceric ammonium sulfate VS (indicator: ortho-phenanthroline TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L ceric ammonium sulfate VS = 27.802 mg of FeSO47H2O

Packaging and Storage Preserve in tight containers.

Ai Amount (%) of each related substance = 100 A s Ai : peak area of each related substance As : the sum of all peak area obtained from the test solution

410 Monographs, Part I Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 210 nm). Column : A stainless steel column, about 4.6 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (4 m in particle diameter). Mobile phase : A mixture of water, tetrahydrofuran and acetonitrile (8 : 1 : 1) Column temperature: A constant temperature of about 60 C Flow rate: 1.5 mL/minute System suitability System performance: Dissolve 10 mg of Finasteride RS in a mixture of water and acetonitrile (1 : 1) to make 10 mL. When the procedure is run with 15 L of this solution under the above operating conditions, the column efficiency is not less than 10000 theoretical plates and the symmetry factor is not more than 1.3.
Water Not more than 0.3% (1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve about 20 mg each of Finateride and Finasteride RS, accurately weighed, in a mixture of water and acetonitrile (1 : 1) to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and determine the peak areas, AT and AS, of the principal peak for the test solution and the standard solution, respectively.

peak area is not more than 1.0%.

Packaging and Storage Preserve in tight containers.

Flavin Adenine Dinucleotide Sodium

NH2 N N

H H2C C OH H3C N

H C OH N

H C OH O CH2O

ONa P O O

ONa P O OCH 2

O H H H OH OH

NH H3C N O

C27H31N9Na2O15P2: 829.51 Flavin Adenine Dinucleotide Sodium contains not less than 93.0% and not more than 101.0% of flavin adenine dinucleotide sodium (C27H31N9Na2O15P2), calculated on the anhydrous basis.
Description Flavin Adenine Dinucleotide Sodium is an orange-yellow to pale yellow-brown powder, is odorless or has a slight, characteristic odor and has a slightly bitter taste. Flavin Adenine Dinucleotide Sodium is freely soluble in water and practically insoluble in methanol, in ethanol, in ethylene glycol or in ether. Flavin Adenine Dinucleotide Sodium is hygroscopic. Flavin Adenine Dinucleotide Sodium is decomposed by light. Identification (1) A solution of Flavin Adenine Dinucleotide Sodium (1 in 100000) is pale yellow-green in color and shows a strong yellow-green fluorescence. Take 5 mL of the solution, and add 20 mg of hydrosulfite sodium: the color and the fluorescence of the solution disappear and gradually reappear when the solution is shaken in air. Add dilute hydrochloric acid or sodium hydroxide TS drop-wise: the fluorescence of the solution disappears. (2) Determine the infrared spectrum of Flavin Adenine Dinucleotide Sodium and Flavin Adenine Dinucleotide Sodium RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Take 0.1 g of Flavin Adenine Dinucleotide Sodium, add 10 mL of nitric acid, evaporate on a waterbath to dryness and ignite. To the residue, add 10 mL of diluted nitric acid (1 in 50), boil for 5 minutes and after cooling, neutralize with ammonia TS, then filter the solution, if necessary: the solution responds to the Qualitative

Amount (mg) of f finasteride (C23H36N2O2) AT = amount (mg) of Finasteride RS A S Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 215 nm). Column : A stainless steel column, about 3.0 mm in inside diameter and about 30 cm in length, packed with octylsilanized silica gel for liquid chromatography (3 m in particle diameter). Mobile phase : A mixture of water and tetrahydrofuran (4 : 1) Flow rate: about 3 mL/minute System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the column efficiency is not less than 1800 theoretical plates and the symmetry factor is not more than 1.3. System reproducibility: When the test is repeated 5 times with 10 L of the standard solution under the above operating conditions, the relative deviation of the

KP 9 411 Tests for sodium salt and the Qualitative Tests (1) and (3) for phosphate.
Specific Optical Rotation [ ]20 D : Between -21.0 and 25.5 (0.3 g, calculated on the anhydrous basis, water, 20 mL, 100 mm). pH Dissolve 1.0 g of Flavin Adenine Dinucleotide Sodium in 100 mL of water: the pH of this solution is between 5.5 and 6.5 Purity (1) Clarity and color of solutionDissolve 0.20 g of Flavin Adenine Dinucleotide Sodium in 10 mL of water: the solution is clear and orange-yellow in color. (2) Free phosphoric acidWeigh accurately about 20 mg of Flavin Adenine Dinucleotide Sodium, dissolve in 10 mL of water and use this solution as the test solution. Separately, measure exactly 2 mL of standard phosphoric acid solution, add 10 mL of water and use this solution as the standard solution. To each of the test solution and the standard solution, add 2 mL of diluted perchloric acid (100 in 117), then add 1 mL of ammonium molybdate TS and 2 ml of 2,4-diaminophenol hydrochloride TS, respectively, shake, add water to make exactly 25 mL and allow to stand at 20 1 o C for 30 minutes. Perform the test with the test solution and the standard solution as directed under the Ultraviolet-visible Spectrophotometry, using a solution prepared in the same manner with 2 mL of water, as the blank and determine the absorbances, AT and AS , of the test solution and the standard solution at 730 nm, respectively: the amount of free phosphoric acid is less than 0.25%.

Detector: An ultraviolet absorption photometer (wavelength: 260 nm). Column, column temperature, mobile phase, flow rate, selection of column: Proceed as directed in operating conditions of Procedure (ii) under Assay (1). System suitability System performance: Proceed as directed in the system suitability in Procedure (ii) under Assay (1). Test for required detection: To exactly 2 mL of the test solution, add the mobile phase to make exactly 20 mL, and use this solution as the solution for system suitability test. Confirm that the peak area of flavin adenine dinucleotide obtained from 20 L of this solution is equivalent to 8 to 12% of that of flavin adenine dinucleotide obtained from 20 L of the test solution. System repeatability: When the test is repeated 6 times with 20 L of the solution for system suitability test under the above operating conditions, the relative standard deviation of the peak area of flavin adenine dinucleotide is not more than 1.0%.
Water Take 50 mL of a mixture of methanol for water determination and ethylene glycol for water determination (1 : 1) into a dry titration flask and titrate with Karl Fischer TS water determination until end point. Weigh accurately about 0.1 g of Flavin Adenine Dinucleotide Sodium, transfer quickly to the titration flask, add an excess and constant volume of Karl Fischer TS water determination, dissolve by stirring for 10 minutes and perform the test: the water content is not more than 10.0%. Assay Procedure (i) Total Flavin content: Conduct this procedure without exposure to daylight, using lightresistant vessels. Weigh accurately about 0.1 g of Flavin Adenine Dinucleotide Sodium and dissolve in water to make exactly 200 mL. Pipet 5.0 mL of this solution, add 5 mL of zinc chloride TS and heat on a water-bath for 30 minutes. After cooling, add water to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Riboflavin RS, previously dried at 105 C for 2 hours, dissolve in 200 mL of diluted acetic acid (1 in 100) by warming, cool and add water to make exactly 500 mL. Measure exactly 10 mL of this solution, add water to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution at 450 nm, respectively, as directed under the Ultraviolet-visible Spectrophotometry, using water as the blank.

Amount (%) of free phosphoric acid (H3PO4) =

AT 1 5 .16 AS W

W: Amount (mg) of Flavin Adenine Dinucleotide Sodium, calculated on the anhydrous basis. (3) Heavy metalsProceed with 1.0 g of Flavin Adenine Dinucleotide Sodium according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicPrepare the test solution With 2.0 g of Flavin Adenine Dinucleotide Sodium according to Method 3 and perform the test (not more than 1 ppm). (5) Related substancesDissolve 0.10 g of Flavin Adenine Dinucleotide Sodium in 200 mL of the mobile phase and use this solution as the test solution. Perform the test with 20 L of the test solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak area, A, of Flavin Adenine Dinucleotide and the area, S, of peaks other than the peak of Flavin Adenine Dinucleotide by the automatic integration method: S/(A+S) is not more than 0.10. Operating conditions

Total amount (mg) of Flavin = amount (mg) of Riboflavin RS

AT 4 AS 5

(ii) Peak area ratio of Flavin Adenine Dinucleotide: Dissolve 0.1 g of Flavin Adenine Dinucleotide Sodium in 200 mL of water and use this solution as the test solution. Perform the test with 5 L of this solution as directed

412 Monographs, Part I under the Liquid Chromatography according to the following conditions. Determine the peak area, A, of Flavin Adenine Dinucleotide and the total area, S, of the peaks other than Flavin Adenine Dinucleotide by the automatic integration method. Peak area ratio of Flavin Adenine Dinucleotide = 1.08
1.08 A 10 .8 A + S

Packaging and Storage tight containers.

Preserve in light-resistant,

Flavoxate Hydrochloride
O N CH2CH2O C O HCl CH3 O

Operating conditions Detector: A visible spectrophotometer (wavelength: 450 nm). Column: A stainless steel column, about 4 mm in inside diameter and 15 to 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 35 o C . Mobile phase: A mixture of a solution of monobasic potassium phosphate (1 in 500) and methanol (4 : 1). Flow rate: Adjust the flow rate so that the retention time of Flavin Adenine Dinucleotide is about 10 minutes. System suitability Test for required detectability: To exactly 2 mL of the test solution, add water to make exactly 20 mL, and use this solution as the solution for system suitability test. Pipet 2 mL of the solution, and add water to make exactly 20 mL. Confirm that the peak area of flavin adenine dinucleotide obtained from 5 L of this solution is equivalent to 8 to 12% of that of flavin adenine dinucleotide obtained from 5 L of the solution for system suitability test. System performance: Dissolve about 20 mg each of Flavin Adenine Dinucleotide Sodium and riboflavin sodium phosphate in 100 mL of water. When the procedure is run with 5 L of this solution under the above operating conditions and calculate the resolution, flavin adenine dinucleotide and riboflavin phosphate in this order are eluted in this order with the resolution between their peaks being not less than 2.0. System repeatibility: When the test is repeated 6 times with 5 L of the solution for system suitability test under the above operating conditions, the relative standard deviation of the peak area of flavin adenine dinucleotide is not more than 1.0%. Time span of measurement: About 4.5 times as long as the retention time of flavin adenine dinucleotide. Amount (mg) of flavin adenine dinucleotide sodium (C27H31N9Na2O15P2) = f T f R 2.2040
f T : Total amount (mg) of Flavin in Flavin Adenine Dinucleotide Sodium obtained from the procedure (i), f R : Peak area ratio of Flavin Adenine Dinucleotide in Flavin Adenine Dinucleotide Sodium obtained from the procedure (ii).

C24H25NO4HCl: 427.92 Flavoxate Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of flavoxate hydrochloried (C24H25NO4HCl).
Description Flavoxate Hydrochloride is a white crystal or crystalline powder. Flavoxate Hydrochloride is sparingly soluble in glacial acetic acid or chloroform, slightly soluble in water or ethanol and practically insoluble in acetonitrile or ether. Identification (1) Determine the absorption spectra of the solutions of Flavoxate Hydrochloride and Flavoxate Hydrochloride RS in 0.01 mol/L hydrochloric acid TS (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectrum of Flavoxate Hydrochloride and Flavoxate Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Flavoxate Hydrochloride (1 in 100) responds to the Qualitative Tests for chloride. Purity (1) Heavy metalsProceed with 2.0 g of Flavoxate Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicPrepare the test solution with 2.0 g of Flavoxate Hydrochloride according to Method 4 and perform the test (not more than 1 ppm). (3) Related substancesDissolve 80 mg of Flavoxate Hydrochloride in 10 mL of chloroform and use this solution as the test solution. Pipet 1.0 mL of the test solution and add chloroform to make exactly 20 mL, then pipet 1.0 mL of this solution, add chloroform to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solu-

KP 9 413 tion on a plate of silica gel with a fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (3 : 1 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 1.0% (1 g, in vaccum, silica gel, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.6 g of Flavoxate Hydrochloride, previously dried, add 10 mL of glacial acetic acid and 40 mL of acetonitrile to dissolve, add 50 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectrum of Floctafenine and Floctafenine RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
Melting Point Between 176 C and 180 C. Purity (1) Heavy metalsProceed with 2.0 g of Floctafenine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Floctafenine according to Method 4 and perform the test (not more than 2 ppm). (3) Related substancesDissolve 20 mg of Floctafenine in 50 mL of the mobile phase and use this solution as the test solution. Pipet 1.0 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area from both solutions by the automatic integration method: the total area of the peaks other than the peak of Floctafenine from the test solution is not larger than the peak area of Floctafenine from the standard solution.

Each mL of 0.1 mol/L perchloric acid VS = 42.79 mg of C24H25NO4.HCl

Packaging and Storage Preserve in tight containers.

Floctafenine
CF3 N

NH O C OH OCH2CCH2OH H

and enantiomer C20H17F3N2O4: 406.36

Floctafenine, when dried, contains not less than 98.5% and not more than 101.0% of floctafenine (C20H17F3N2O4).
Description Floctafenine is a white to pale yellowish white crystalline powder. Floctafenine is freely soluble in glacial acetic acid, slightly soluble in methanol or in ethanol, very slightly soluble in ether and practically insoluble in water. A solution of Floctafenine in 0.1 mol/L hydrochloric acid TS (1 in 100) shows no specific rotation. Identification (1) Determine the absorption spectra of the solutions of Floctafenine and Floctafenine RS in 0.1 mol/L hydrochloric acid TS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 224 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: Adjust the pH of a mixture of methanol, water and phosphoric acid (240 : 160 : 1) to 3.5 with sodium hydroxide TS. Flow rate: Adjust the flow rate so that the retention time of Floctafenine is about 6 minutes. System suitability System performance: Dissolve 10 mg of Floctafenine and 50 mg of ethyl parahydroxy-benzoate in 250 mL of the mobile phase. When the procedure is run with 20 L of this solution according to the above operating conditions, Floctafenine and ethyl parahydroxybenzoate are eluted in this order with the resolution between their peaks being not less than 7. Detection sensitivity: Adjust the detection sensitivity so that the peak height of Floctafenine from 20 L of the standard solution is between 5% and 15% of the full scale. Time span of measurement: About four times as long as the retention time of Floctafenine after the solvent peak.

414 Monographs, Part I pione according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Flopropione according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.10 g of Flopropione in 10 mL of dehydrated ethanol and use this solution as the test solution. Pipet 1.0 mL of the test solution, add dehydrated ethanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of hexane, dehydrated ethanol and glacial acetic acid (40 : 20 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly pnitrobenzene-diazonium TS for spraying on the plate and dry in cold wind for about 5 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Water Not more than 4.0% (0.5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g).
OH

Loss on Drying Not more than 1.0% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g, platinum crucible). Assay Weigh accurately about 0.6 g of Floctafenine, previously dried, dissolve in 30 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination above operating method and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 40.64 mg of C20H17F3N2O4

Packaging and Storage Preserve in tight containers.

Flopropione
COC2H5 HO OH

C9Hl0O4: 182.17 Flopropione contains not less than 98.0% and not more than 101.0% of flopropione (C9Hl0O4), calculated on the anhydrous basis.
Description Flopropione is a white to pale yellowbrown, crystalline powder. Flopropione is odorless and has a bitter taste. Flopropione is very soluble in dimethylformamide, freely soluble in methanol, in dehydrated ethanol or in ether and practically insoluble in water. Identification (1) Take 1 mL of a solution of Flopropione in dehydrated ethanol (1 in 200), add 4 mL of water and 1 mL of ferric nitrate TS: a red-purple color is observed. (2) Determine the absorption spectra of the solutions of Flopropione and Flopropione RS in dehydrated ethanol (1 in 200000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Melting Point Between 177 C and 181 C. Purity (1) Clarity and color of solutionDissolve 1.0 g of Flopropione in 10 mL of dehydrated ethanol: the solution is clear and has no more color than Matching Fluid H. (2) Heavy metalsProceed with 1.0 g of Flopro-

Assay Weigh accurately about 0.3 g of Flopropione, dissolve in 30 mL of dimethyl-formamide and titrate with 0.1 mol/L tetra-methylammonium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 18.217 mg of C9Hl0O4

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Flubendazole
O F H N N H N O O CH3

C16H12FN3O3 : 313.28 Flubendazole contains not less than 99.0% and not more than 101.0% of flubendazole (C16H12FN3O3), calculated on the dried basis.
Description Flubendazole is white powder. Flubendazole is practically insoluble in water, in dichlo-

KP 9 415 romethane or in ethanol. Flubendazole shows polymorphism.

Identification Determine the infrared spectra of Flubendazole and Flubendazole RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity Related substancesDissolve 0.10 g of Flubendazole in dimethylformamide to make exactly 100 mL and use this solution as the test solution. Pipet exactly 1 mL of the test solutionn, add dimethylformamide to make exactly 100 mL. Pipet exactly 5 mL of the this solutionn, add dimethylformamide to make exactly 25 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions: peak area of any related substance with relative retention time between 1.2 and 1.3 is not more than that of the principal peak from the standard solution (0.25%), and sum of areas of peaks from the test solution except the principal peak is not more than 6 times the area of the principal peak from the standard solution (1.5%). Disregard peaks from the test solution with areas of 0.2 times of the area of the principal peak from the standard solution. Assay Weigh accurately about 0.25 g of Flubendazole, dissolve in 3 mL of formic acid, add 50 mL of a mixture of methyl ethyl ketone and glacial acetic acid (7 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 31.330 mg of C16H12FN3O3

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Flucytosine
H N O NH F NH2

C4H4FN3O: 129.09 Flucytosine, when dried, contains not less than 98.5% and not more than 101.0% of flucytosine (C4H4FN3O) and not less than 14.0% and not more than 15.5% of Fluorine (F: 19.00).
Description Flucytosine is a white, crystalline powder and is odorless. Flucytosine is sparingly soluble in water, slightly soluble in methanol, in acetic anhydride, in glacial acetic acid or in ethanol and practically insoluble in ether. Flucytosine dissolves in 0.1 mol/L hydrochloric acid TS. The pH of a solution of Flucytosine (1 in 100) is between 5.5 and 7.5. Flucytosine is slightly hygroscopic. Melting point About 295 C (with decomposition). Identification (1) Take 5 mL of a solution of Flucytosine (1 in 500), add 0.2 mL of bromine TS: a yellowbrown color of bromine TS is immediately discharged. Further add 2 mL of barium hydroxide TS: a purple precipitate is produced. (2) Proceed with 0.1 g of Flucytosine as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid. The solution responds to the Qualitative Tests (2) for fluoride. (3) Determine the absorption spectra of the solutions of Flucytosine and Flucytosine RS in 0.l mol/L hydrochloric acid TS (1 in 125,000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Purity (1) Clarity and color of solutionDissolve 1.0

Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 270 nm). Column : A stainless steel column, about 4.6 mm in inside diameter and about 10 cm in length, packed with base-deactivated octadecylsilanized silica gel for liquid chromatography (3 m in particle diameter). Mobile phase : With the mobile phase A and the mobile B, control the composition stepwise or gradiently as follows. Mobile phase A: Dissolve 7.5 g of ammonium acetate in water to make 1000 mL. Mobile phase B: Acetonitrile Time (minutes) 0 ~ 15 15 ~ 30 30 ~ 32 32 ~ 37 37 ~ 38 38 ~ 42 Mobile phase A (vol%) 90 75 75 45 45 10 10 10 90 90 Mobile phase B (vol%) 10 25 25 55 55 90 90 90 10 10

Flow rate: 1.2 mL/minute

Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g).

416 Monographs, Part I g of Flucytosine in 100 mL of water: the solution is clear and colorless. (2) ChlorideDissolve 1.0 g of Flucytosine in 80 mL of water by heating on a water-bath. After cooling, to 40 mL of this solution, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.20 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.014%). (3) FluorideDissolve 0.10 g of Flucytosine in 10.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20). Transfer 5.0 mL of this solution to a volumetric flask, add 10 mL of a mixture of alizarin complexone TS, acetic acid-potassium acetate buffer solution, pH 4.3 and cerrous nitrate TS (1 : 1 : 1) and add water to make 20 mL. Allow the mixture to stand for 1 hour and use this solution as the test solution. Separately, transfer 4.0 mL of standard fluorine solution to a volumetric flask, add 5.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20), add 10 mL of a mixture of alizarin complexone TS, acetic acid-potassium acetate buffer solution, pH 4.3 and cerrous nitrate TS (1 : 1 : 1). Proceed in the same manner as directed in the preparation of the test solution and use this solution as the standard solution. Transfer 5.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20) to a volumetric flask, proceed in the same manner as directed in the preparation of the standard solution and use this solution as the blank solution. Determine the absorbances, AT and AS , of the test solution and the standard solution at 600 nm, respectively, using the blank solution as the control as directed under Ultraviolet-visible Spectrophotometry: AT is not larger than AS (not more than 0.048%). (4) Heavy metalsProceed with 1.0 g of Flucytosine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (5) ArsenicPrepare the test solution with 1.0 g of Flucytosine according to Method 2 and perform the test (not more than 2 ppm). (6) Related substancesDissolve 50 mg of Flucytosine in 5 mL of a diluted methanol (1 to 2) and use this solution as the test solution. Pipet 1.0 mL of this solution, add a diluted methanol (1 to 2) to make exactly 25 mL. Pipet exactly 1 mL of this solution, add a diluted methanol (1 in 2) to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the chromatogram with a mixture of ethyl acetate, methanol and water (5 : 3 : 2) to a distance of about 12 cm, air-dry the plate and observe the spots under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 1.0% (1 g, 105 C, 4

hours).
Residue on Ignition Not more than 0.1% (1 g). Assay (1) FlucytosineWeigh accurately about 0.2 g of Flucytosine, previously dried, dissolve in 40 mL of glacial acetic acid, add 100 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 12.909 mg of C4H4FN3O (2) FluorineWeigh accurately about 10 mg of Flucytosine, previously dried and proceed as directed in the determination of fluorine under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide VS and 20 mL of water as the absorbing liquid.
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Fludiazepam
CH3 O N

Cl

N F

C16H12ClFN2O: 302.73 Fludiazepam, when dried, contains not less than 99.0% and not more than 101.0% of fludiazepam (C16H12ClF N2O).
Description Fludiazepam is a white to pale yellow crystal or crystalline powder. Fludiazepam is very soluble in chloroform, freely soluble in methanol, in ethanol, in glacial acetic acid or in ether and practically insoluble in water. Identification (1) Prepare the test solution with 10 mg of Fludiazepam as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid: the test solution responds to the Qualitative Tests (2) for fluoride. (2) Determine the absorption spectra of the solutions of Fludiazepam and Fludiazepam RS in methanol (1 in 200000) as directed under the Ultraviolet-visible Spec-

KP 9 417 trophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. And determine spectra of the solutions of Flueliazepam and Fludiazepam RS in methonol (1 in 20000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths (3) Determine the infrared spectrum of Fludiazepam and Fludiazepam RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) Perform the test with Fludiazepam as directed under the Flame Coloration Test (2): a green color is observed.
Melting Point Between 91 C and 94 C. Purity (1) ChlorideDissolve 1.0 g of Fludiazepam in 50 mL of ether, add 50 mL of water and shake. Separate the water layer, wash with two 20 mL volumes of ether and filter the water layer. To 20 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.036%). (2) Heavy metalsProceed with 2.0 g of Fludiazepam according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve 0.10 g of Fludiazepam in 20 mL of chloroform and use this solution as the test solution. Pipet 1.0 mL of the test solution and add chloroform to make exactly 50 mL. Pipet 2.0 mL of this solution, add chloroform to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thinlayer chromatography. Develop the plate with a mixture of chloroform and ethyl acetate (10 : 7) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.3% (1 g, in vacuum, 60 o C , 3 hours). Residue on Ignition Not more than 0.1% (1 g, platinum crucible). Assay Weigh accurately about 0.5 g of Fludiazepam, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 30.273 mg of C16H12ClFN2O

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Fludrocortisone Acetate
O OH H CH3 H H3C HO O CH3

O H

F O

C23H31FO6: 422.49 Fludrocortisone Acetate, when dried, contains not less than 97.0% and not more than103.0% of fludrocortisone acetate (C23H31FO6).
Description Fludrocortisone Acetate is a white to pale yellow crystal or crystalline powder, is odorless or has slight odor. Fludrocortisone Acetate is hygroscopic. Fludrocortisone Acetate is hardly soluble in ether, slightly soluble in ethanol or in chloroform and practically insoluble in water. Identification Determine the infrared spectra of Fludrocortisone Acetate and Fludrocortisone Acetate RS, as directed in the paste method under the Infrared Spectrophotometry: both spectra similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between + 126 and + 138 (dried, 50 mg, acetone, 10 mL, 100 mm). Purity Related substancesWeigh accurately about 0.1 g of Fludrocortisone Acetate and dissolve in 5 mL of chloroform and 1 mL of acetone and dilute with chloroform to make 10 mL. Use this solution as the test solution. Dilute 1 mL of this solution with chloroform to make 100 mL. Use this solution as the standard solution. Proceed with the test solution as the standard solution and perform the test directed under Thin Layer Chromatography. Spot 10 L each of the test solution and the standard solution on a silica gel plate. Develop the plates with developing solution of a mixture of chloroform, methanol and water (85 : 14 : 1) to the distance of about 15 cm and air-drying. When infrared line (wavelength : 254 nm) was lighted on these plates, black spots of the test solution, except main black spot, are smaller and less darker than that of the standard solution (not more than 1.0%). Loss on Drying Not more than 3.0% (1 g, 100 C, in

418 Monographs, Part I vacuum, magnesium perchlorate, 2 hours).

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 25 mg of Fludrocortisone Acetate and Fludrocortisone Acetate for Assay, previously dried at 100 C for 2 hours in vacuum and dissolve in chloroform to make 250 mL. Pipet 10.0 mL of this solution and dilute with chloroform to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Pipet 10.0 mL each of the test solution and the standard solution into a volumetric flask and add 1 mL of methanol solution of bluetetrazolium (dissolve 50 mg of bluetetrazolium in 10 mL of methanol) and mix. Add 1.0 mL of a mixture of tetramethylammonium hydroxide solution and methanol (1 : 4) and stand for 10 minutes. Add a mixture of hydrochloric acid and methanol (1 in 100) to make exact 25 mL. Perform the test with 10 mL each of the test solution and the standard solution as directed under the Ultraviolet-visible Spectrophotometry. Determine absorbances, AT and AS , of Fludrocortisone Acetate of the test solution and standard solution, respectively, at a maximum wavelength near 525 nm. Identification (1) Mix 5 mg of Flumequine with 45 mg of magnesium oxide and ignite for about 5 minutes until an almost white residue is obtained. After cooling, add 1 mL of water and 2 drops of phenolphthalein TS, add about 2 mL of dilute hydrochloric acid to render the solution colorless. Filter and use the filtrate as the test solution. Add to the test solution a freshly prepared mixture of 0.1 mL of alizarin S TS and 0.1 mL of the zirconyl nitrate solution, mix, allow to stand 5 minutes and compare the color of the solution with that of the blank solution prepared in the same manner: the color of the test solution changes from red to yellow, while the color of the blank solution remains red.

Zirconyl nitrate solutionDissolve 0.1 g of zirconyl nitrate in a mixture of 60 mL of hydrochloric acid and 40 mL of water. (2) Determine the infrared absorption spectra of Flumequine and Flumequine RS as directed in the potassiumn bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 5 mg of Flumequine in dichloromethane to make 10 mL and use this solution as the test solution. Separately, dissolve 5 mg of Flumequine RS in dichloromethane to make 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethanol, water and 9 mol/L ammonia solution TS (90 : 10 : 10) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the principal spots obtained from the test solution and the standard solution has the same Rf value. 9 mol/L ammonia solutionTo 67 g of 13.5 mol/L ammonia solution, add water to make 100 mL.
Specific Optical Rotation [ ]20 D : Between -0.10 and +0.10 (5.0 g, sodium hydroxide TS, 50 mL, 100mm). Purity (1) Clarity and color of solutionDissolve 5.0 g of Flumequine in 0.5 mol/L sodium hydroxide TS to make 50 mL: the solution is clear. (2) Heavy metalsProceed with 2.0 g of Flumequine according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of Standard Lead Solution (not more than 10 ppm). (3) Related substancesDissolve 35.0 g of Flumequine in dimethylformamide to make exactly 100 mL and use this solution as the test solution. Separately, dissolve 5.0 mg each of flumequine RS and flumequine related substance I RS [(RS)-9-fluoro-5-methyl-1-oxo-6,7dihydro-1H,5H-benzo[i.j]quinolizine-2-carboxylate

Amount (mg) of fludrocortisone acetate (C23H31FO6) = amount of Fludrocortisone Acetate RS

AT AS

C: Concentration of the standard solution (L/mL).

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Flumequine
H CH3 N

F O

COOH

and enantiomer C14H12FNO3 : 261.25

Flumequine containes not less than 99.0% and not more than 101.0% of flumequine (C14H12FNO3), calculated on the dried basis.
Description Flumequine is a white, fine crystalline powder. Flumequine is sparingly soluble in dichloromethane, very slightly soluble in methanol, and practically insoluble in water. Flumequine dissolves in dilute sodium hydroxide TS.

KP 9 419 (flumequine ethyl ester)] in dimethylformamide to make exactly 100 mL and use this solution as the standard solution (1). To 1.0 mL of the test solution, add dimethylformamide to make exactly 200 mL and use this solution as the standard solution (2). Perform the test with 10 L each of the blank solution (dimethylformamide), the test solution and the standard solutions (1) and (2) as directed under the Liquid Chromatography according to the following conditions: area of any peak from the test solution except the principal peak is not more than that of the principal peak from the standard solution (2) (0.5%), and sum of areas of peaks from the test solution except the principal peak is not more than 2 times the area of the principal peak from the standard solution (2) (1.0%). Disregard peaks from dimetylformamide and the test solution with areas of 0.1 times of the area of the principal peak from the standard solution (2). Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 250 nm). Column : A stainless steel column, about 4.6 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase : A mixture of the potassium dihydrogen phosphate buffer and methanol (51 : 49) Flow rate : 0.8 mL/minute System suitability System performance: When the procedure is formed accoding to the above operating conditions, retention times of flumequine related substance I RS and flumequine are about 11 minutes and 13 minutes, respectively and the resolution between these peaks is not less than 2.0. Potassium dihydrogen phosphate bufferDissolve 1.36 g of potassium dihydrogen phosphate in water to make 1000 mL.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.5 g, platinum crucible). Assay Weigh accurately about 0.5 g of Flumequine, dissolve in 50 mL of dimethylformamide and titrate with 0.1 mol/L tetrabutylammonium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Flunitrazepam
CH3 O N

O2N

N F

Cl6H12FN3O3: 313.28 Flunitrazepam, when dried, contains not less than 99.0% and not more than 101.0% of flunitrazepam (Cl6H12FN3O3).
Description Flunitrazepam is a white to pale yellow crystalline powder. Flunitrazepam is freely soluble in glacial acetic acid, soluble in acetic anhydride or in acetone, slightly soluble in dehydrated ethanol or in ether and practically insoluble in water. Identification (1) Determine the absorption spectra of the solutions of Flunitrazepam and Flunitrazepam RS in dehydrated ethanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectrum of Flunitrazepam and Flunitrazepam RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 168 C and 172 C. Purity (1) ChlorideTake 1.0 g of Flunitrazepam, add 50 mL of water, allow to stand for 1 hour with occasional stirring and filter. To 20 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test with this solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.022%). (2) Heavy metalsProceed with 2.0 g of Flunitrazepam according to Method 4 using a platinum crucible and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve 50 mg of Flunitrazepam in 10 mL of acetone and use this solution as the test solution. Pipet 2.0 mL of the test solution and add acetone to make exactly 20 mL. Pipet 1.0 mL of this solution, add acetone to make exactly 25 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of

Each mL of 0.1 mol/L tetrabutylammonium hydroxide VS = 26.126 mg of C14H12FNO3

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420 Monographs, Part I silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of 1,2dichloroethane, ether and strong ammonia water (200 : 100 : 3) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): numbers of the spots other than the principal spot from the test solution are not more than 2 and they are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g, platinum crucible). Assay Weigh accurately about 0.5 g of Flunitrazepam, previously dried, dissolve in 20 mL of glacial acetic acid, add 50 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

(2) Dissolve 10 mg of Fluocinolone Acetonide in 1 mL of methanol, add 1 mL of Fehling TS and heat: a red precipitate is produced. (3) Proceed with 10 mg of Fluocinolone Acetonide as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as an absorbing liquid. The test solution responds to the Qualitative Tests for fluoride. (4) Determine the infrared spectra of Fluocinolone Acetonide and Fluocinolone Acetonide RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit the similar intensity of absorption at the same wavenumbers. If any difference appears in the absorption spectra, dissolve the test and the RS in acetone, respectively, evaporate acetone and perform the test with the residue in the same manner.
Specific Optical Rotation [ ]20 D : Between +98 and +108 (after drying, 0.1 g, methanol, 10 mL, 100 mm). Purity Related substancesDissolve about 15 mg of Fluocinolone Acetonide in 25 mL of mobile phase and use this solution as the test solution. Pipet exactly 2 mL of this solution, add mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution and the standard solution by the automatic integration method: the total area of all peaks other than the peak of Fluocinolone Acetonide from the test solution is not larger than the peak area of Fluocinolone Acetonide from the standard solution.

Each mL of 0.1 mol/L perchloric acid VS = 31.328 mg of Cl6H12FN3O3

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Fluocinolone Acetonide
O H HO H3C H H F O H F H H3C C CH2OH O O CH3 CH3

C24H30F2O6: 452.49 Fluocinolone Acetonide, when dried, contains not less than 97.0% and not more than 102.0% of fluocinolone acetonide (C24H30F2O6).
Description Fluocinolone Acetonide is a white crystal or crystalline powder. Fluocinolone Acetonide is odorless. Fluocinolone Acetonide is freely soluble in glacial acetic acid or acetone, soluble in ethanol or dehydrated ethanol, sparingly soluble in methanol or chloroform, slightly soluble in acetonitrile, very slightly soluble in ether, and practically insoluble in water. Melting pointBetween 266 C and 274 C (with decomposition). Identification (1) Take 2 mg of Fluocinolone Acetonide, add 2 mL of sulfuric acid: a yellow color is observed.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of chloroform saturated with water, methanol and glacial acetic acid (200 : 3 : 2). Flow rate: Adjust the flow rate so that the retention time of Fluocinolone Acetonide is about 12 minutes. System suitability System performance: Dissolve each 15 mg of Fluocinolone Acetonide and Triamcinolone Acetonide in 25 mL of the mobile phase. Take 5 mL of this solution, add mobile phase to make 20 mL. When the procedure is run with 20 L of this solution, as directed under the above operating conditions, Triamcinolone Acetonide and Fluocinolone Acetonide are eluted in this order with a resolution between their peaks being not less than 1.9. System repeatability: When the test is repeated 6

KP 9 421 times with 20 L of the standard solution, as directed under the above operating conditions, the relative deviation of the peak area of Fluocinolone Acetonide is not more than 1.0%. Test for required detection: Take exactly 5 mL of the standard solution add the mobile phase to make exactly 100 mL. Confirm that the peak area of Fluocinolone acetonide obtained from 20 L of this solution is equivalent to 4 to 6% of that of Fluocinolone acetonide obtained from 20 L of the standard solution. Time span of measurement: About twice as long as the retention time of Fluocinolone Acetonide.
Loss on Drying Not more than 1.0% (0.2 g, in vacuum, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.2 g, platinum crucible). Assay Weigh accurately about 20 mg each of Fluocinolone Acetonide and Fluocinolone Acetonide RS, previously dried, dissolve in 40 mL of methanol, add exactly 10 mL of the internal standard solution, add water to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Fluocinolone Acetonide to that of the internal standard for the test solution and the standard solution, respectively.

ditions, isopropyl p-hydroxybenzoate and propyl phydroxybenzoate are eluted in this order with a resolution between their peaks being not less than 1.9. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative deviation of the peak area of Fluocinolone Acetonide is not more than 1.0%.
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Fluocinolone Acetonide Cream

Fluocinolone Acetonide Cream contains not less than 90.0% and not more than 110.0% of the labeled amount of fluocinolone acetonide (C24H30F2O6: 452.49).
Method of Preparation Prepare as directed under Creams, with Fluocinolone Acetonide. Identification Weigh accurately a portion of Fluocinolone Acetonide Cream, equivalent to about 0.5 mg of fluocinolone acetonide (C24H30F2O6) according to the labeled amount, transfer to a centrifugal tube, suspend with 5 mL of water, add 10 mL of chloroform, shake and centrifuge. Discard the water layer, add 10 mL of water, shake and centrifuge. Dehydrate 2 mL of chloroform extracts with about 0.2 g of anhydrous sodium sulfate and use this solution as the test solution. Separately, dissolve 5 mg of Fluocinolone Acetonide RS in 100 mL of chloroform and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 50 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the chromatogram with a mixture of chloroform and diethylamine (2 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the principal spot from the test solution shows the same Rf value and color as the principal spot from the standard solution. Microbiology Limit When the test is performed, Staphylococcus aureus and Pseudomonas aeruginosa are not detected. Assay Weigh accurately a portion of Fluocinolone Acetonide Cream, equivalent to about 0.75 mg of fluocinolone acetonide (C24H30F2O6), add about 10 mL of acetonitrile and warm in the water-bath until dissolved. Transfer to a 25 mL volumetric flask with aid of three 2 mL volumes of acetonitrile. To this solution, add 3.0 mL of the internal standard solution and 5.0 mL of water, shake, add acetonitrile to make 25 mL, cool in the ice-bath, centrifuge and use the supernatant as the test solution. Sepa-

Amount (mg) of fluocinolone acetonide (C24H30F2O6) = amount (mg) of Fluocinolone Acetonide RS

QT QS

Internal standard solutionA solution of ethyl parahydroxybenzoate in methanol (1 in 2500). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, having octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of water and acetonitrile (7 : 3). Flow rate: Adjust the flow rate so that the retention time of Fluocinolone Acetonide is about 20 minutes. System suitability System performance: Dissolve 5 mg each of isopropyl parahydroxybenzoate and propyl parahydroxybenzoate in 50 mL of acetonitrile and add water to make 100 mL. When the precedure is run with 20 L of this solution, as directed under the above operating con-

422 Monographs, Part I rately, weigh accurately a portion of Fluocinolone Acetonide RS, previously dried at 105 C for 3 hours, dissolve in acetonitrile to obtain a solution having a known concentration of about 300 g per mL. Pipet exactly 5 mL of this solution, add 6.0 mL of the internal standard solution and 15.0 mL of water, add acetonitrile to make exactly 50 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Fluocinolone Acetonide to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of fluocinolone acetonide (C24H30F2O6) = amount (mg) of Fluocinolone Acetonide RS
QT QS

Identification Evaporate 10 mL of the test solution in the Assay to dryness, dissolve the residue in 1 mL of chloroform and use this solution as the test solution. Separately, dissolve 5 mg of Fluocinolone Acetonide RS in 100 mL of chloroform and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 50 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the chromatogram with a mixture of chloroform and diethylamine (2 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the principal spot from the test solution shows the same Rf value and color as the principal spot from the standard solution. Microbiology Limit When the test is performed, Staphylococcus aureus and Pseudomonas aeruginosa are not detected. Assay Weigh accurately a portion of Fluocinolone Acetonide Ointment, equivalent to about 0.7 mg of fluocinolone acetonide (C24H30F2O6), transfer to a 50 mL centrifugal tube, suspend with 35.0 mL of diluted internal standard solution using sonicator, centrifuge and use the supernatant as the test solution. Separately, weigh accurately a portion of Fluocinolone Acetonide RS, previously dried at 105 o C for 3 hours, dissolve in acetonitrile to obtain a solution having a known concentration of about 200 g/mL. Pipet exactly 10 mL of this solution, add 2.0 mL of the internal standard solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Fluocinolone Acetonide to that of the internal standard for the test solution and the standard solution, respectively.

Internal standard solutionA solution of norethindrone in acetonitrile (2 in 10000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, having octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of acetonitrile and water (3 : 5). Flow rate: 2 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution, as directed under the above operating conditions, the resolution between peaks of the internal standard and fluocinolone acetonide being not less than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution, as diredcted under the above operating conditions, the relative deviation of the peak area of Fluocinolone Acetonide is not more than 1.5%.
Packaging and Storage Preserve in tight containers.

Amount (mg) of fluocinolone acetonide (C24H30F2O6) = amount (mg) of Fluocinolone Acetonide RS QT

QS

Fluocinolone Acetonide Ointment

Fluocinolone Acetonide Ointment contains not less than 90.0% and not more than 110.0% of the labeled amount of fluocinolone acetonide (C24H30F2O6: 452.49).
Method of Preparation Prepare as directed under Ointments, with Fluocinolone Acetonide.

Internal standard solutionA solution of norethindrone in methanol (1.7 in 200000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 50 cm in length, having octadecylsilanized silica gel for liquid chromatography (5 m to10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of acetonitrile and water (1 :

KP 9 423 1). Flow rate: 2 mL/minute. System suitability System performance: When the test procedure is run with 10 L of the standard solution as directed under the above operating conditions, the resolution between their peaks being not less than 2.0. System repeatability: When the test is repeated 6 times and the relative standard deviation of the ratios of the peak area is not more than 1.5%. Detection of sensitivity: Adjust the peaks of each component to be less than 50% and not more than 90% of full scale. Diluted internal standard solutionDilute 5.0 mL of the internal standard solution with methanol to make 250 mL.
Packaging and Storage Preserve in tight containers.

(4) Determine the infrared spectra of Fluocinonide and Fluocinonide RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears in the absorption spectra, dissolve the test and the RS in ethyl acetate, respectively, evaporate the ethyl acetate and perform the test with the residue in the same manner.
Specific Optical Rotation [ ]20 D : Between +81 and +89 (after drying, 0.2 g, chloroform, 20 mL, 100 mm). Purity Related substancesDissolve 10 mg of Fluocinonide in 2 mL of chloroform and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of chloroform and methanol (97 : 3) to a distance of about 12 cm and air-dry the plate. Spray evenly alkaline blue tetrazolium TS on the plate: any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Loss on Drying Not more than 1.0% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.5 g, platinum crucible). Assay Weigh accurately about 20 mg of Fluocinonide and fluocinonide RS, previously dried, dissolve each in 50 mL of acetonitrile, to each add exactly 8 mL of the internal standard solution and water to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Fluocinonide to that of the internal standard for the test solution and the standard solution, respectively.

Fluocinonide
O H HO H3C H H F O H F H H3C C CH2O O O O C CH3 CH3 CH3

C26H32F2O7: 494.53 Fluocinonide, when dried, contains not less than 97.0% and not more than 103.0% of fluocinonide (C26H32F2O7).
Description Fluocinonide is a white crystal or crystalline powder. Fluocinonide is sparingly soluble in chloroform, slightly soluble in acetonitrile, methanol, ethanol or ethyl acetate, very slightly soluble in ether, and practically insoluble in water. Identification (1) Take 10 mg of Fluocinonide, add 4 mL of water and 1 mL of Fehling's TS and heat: a red precipitate is produced. (2) Prepare the test solution with 10 mg of Fluocinonide as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as an absorbing liquid: the test solution responds to the Qualitative Tests for fluoride. (3) Determine the absorption spectra of solutions of Fluocinonide and Fluocinonide RS, respectively, in methanol (1 in 10000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths.

Amount (mg) of fluocinonide (C26H32F2O7) = amount (mg) of Fluocinonide RS

QT QS

Internal standard solutionA solution of propyl benzoate in acetonitrile (1 in 100). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in

424 Monographs, Part I inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of acetonitrile and water (1 : 1). Flow rate: Adjust the flow rate so that the retention time of Fluocinonide is about 8 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution, as directed under the above operating conditions, Fluocinonide and the internal standard are eluted in this order with a resolution between their peaks being not less than 6. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions and the relative deviation of the peak area of Fluocinonide is not more than 1.0%.
Packaging and Storage Preserve in well-closed containers.

(3) Char 0.5 g of Fluorescein Sodium by ignition, cool, mix the residue with 20 mL of water and filter: the filtrate responds to the Qualitative Tests for sodium salt.
Purity (1) Clarity and color of solutionDissolve 1.0 g of Fluorescein Sodium in 10 mL of water: the solution is clear and shows a red color. (2) ChlorideDissolve 0.15 g of Fluorescein Sodium in 20 mL of water, add 6 mL of dilute nitric acid and water to make 30 mL and filter. Take 20 mL of the filtrate, add 2 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 1.0 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.355%). (3) SulfateDissolve 0.20 g of Fluorescein Sodium in 30 mL of water, add 2.5 mL of dilute hydrochloric acid and water to make 40 mL and filter. To 20 mL of the filtrate, add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 1.0 mL of 5 mmol/L sulfuric acid VS (not more than 0.480%). (4) ZincDissolve 0.1 g of Fluorescein Sodium in 10 mL of water, add 2 mL of hydrochloric acid and filter. To the filtrate, add 0.1 mL of potassium ferrocyanide TS: no turbidity is produced immediately. (5) Related substancesDissolve 0.20 g of Fluorescein Sodium in 10 mL of methanol and use this solution as the test solution. Perform the test with this solution as directed under the Thin-layer chromatography. Spot 5 L of the test solution on a plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of chloroform, methanol and strong ammonia water (30 : 15 : 1) to a distance of about 10 cm and airdry the plate: any colored spot other than the principal spot does not appear. Loss on Drying Not more than 10.0% (1 g, 105 C, constant weight). Assay Transfer 0.5 g of Fluorescein Sodium, accurately weighed, to a separator. Dissolve in 20 mL of water, add 5 mL of dilute hydrochloric acid and extract the solution with four 20 mL volumes of a mixture of isobutanol and chloroform (1 : 1). Wash each extract with 10 mL of water. Evaporate the combined extracts on a waterbath with the aid of a current of air. Dissolve the residue in 10 mL of dehydrated ethanol, evaporate the solution on a water-bath to dryness, dry the residue at 105 o C for 1 hour and weigh as fluorescein (C20H12O5: 332.31).

Fluorescein Sodium
NaO O O

CO2Na

C20H10Na2O5: 376.27 Fluorescein Sodium contains not less than 98.5% and not more than 101.0% of fluorescein sodium (C20H10Na2O5), calculated on the dried basis.
Description Fluorescein Sodium is an orange powder, is odorless and tasteless. Fluorescein Sodium is freely soluble in water, in methanol or in ethanol and practically insoluble in ether. Fluorescein Sodium is hygroscopic. Identification (1) Take a solution of Fluorescein Sodium (1 in 100) having a strong green fluorescence, add a large volume of water: the fluorescence remains. Acidify the solution with hydrochloric acid: the fluorescence disappears. Then render the solution alkaline with sodium hydroxide TS: the fluorescence reappears. (2) Place 1 drop of a solution of Fluorescein Sodium (1 in 2000) on a piece of filter paper: a yellow spot develops. Expose the spot, while moist, to the vapor of bromine for 1 minute and then to ammonia vapor: the yellow color of the spot changes to red.

Amount (mg) of C20H10Na2O5 = amount (mg) of fluorescein (C20H12O5) 1.1323

Packaging and Storage Preserve in tight containers.

KP 9 425 fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of dichloromethane, acetone and methanol (45 : 5 : 1) to a distance of about 12 cm, and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 1.0% (0.2 g, in vacuum, P2O5, 60 C, 3 hours). Residue on Ignition Not more than 0.2% (0.2 g, platinum crucible).

Fluorometholone
O H HO CH3 H CH3 CH3 OH

F O H CH3

C22H29FO4 : 376.46 Fluorometholone, when dried, contains not less than 97.0% and not more than 103.0% of fluorometholone (C22H29FO4).
Description Fluorometholone is as a white to light yellowish white, crystalline powder and is odorless. Fluorometholone is freely soluble in pyridine, slightly soluble in methanol, in ethanol or in tetrahydrofuran, and practically insoluble in water or in ether. Identification (1) Proceed with 7 mg of Fluorometholone as directed under Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as an absorbing liquid: the liquid responds to the Qualitative Tests (2) for fluoride. (2) Determine the absorption spectra of solutions of Fluorometholone and Fluorometholone RS in methanol (1 in 100,000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared absorption spectra of Fluorometholone and Fluorometholone RS, previously dried, as directed in the potassiumn bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between +52 and +60 (after drying, 0.1 g, pyridine, l0 mL, 100mm). Purity (1) Heavy metalsProceed with l.0 g of Fluorornetholone according to Method 3, and performn the test. Prepare the control solution with 2.0 mL of Standard Lead Solution (not more than 20 ppm). (2) Related substancesDissolve 20 mg of Fluorometholone in 10 mL of tetrahydrofuran, and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add tetrahydrofuran to make exactly 100 mL, and use this solution as the standard solution. Perform the test with these solutions as directed under the Thinlayer Chromatography. Spot 25 L each of the test solution and the standard solution on a plate of silica gel with

Assay Weigh accurately about 0.1 g each of Fluorometholone and Fluorometholone RS, previously dried, and dissolve each in methanol to make exactly 100 mL. Pipet exactly 5 mL each of these solutions, and add diluted methanol (7 in 10) to make exactly 50 mL. Pipet exactly 10 mL each of these solutions, add exactly 10 mL of the internal standard solution and diluted methanol (7 in 10) to make 100 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 25 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine the ratios, QT and QS, of the peak area of fluorometholone to that of the internal standard.

Amount (mg) of fluorometholone (C22H29FO4) = amount QT (mg) of Fluorometholone RS Q S Internal standard solutionA solution of butyl parahydroxybenzoate in methanol (1 in 10000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nmn). Column: A stainless steel column about 4 nim in inside diameter and 25 to 30 cm in length, packed with ociadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 35 C. Mobile phase: Diluted methanol (7 in 10). Flow rate: Adjust the flow rate so that the retention time of fluorometholone is about 8 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, fluorometholone and the internal standard in this order with the resolution between these peaks being not less than 4.
Packaging and Storage well-closed containers.

Preserve in light-resistant,

426 Monographs, Part I mol/L sodium hydroxide TS (1 in 20) in the same manner, as the blank: the absorbance of the test solution at 600 nm is not larger than that of the standard solution (not more than 0.012%). (3) Heavy metalsProceed with 1.0 g of Fluorouracil according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicTo 1.0 g of Fluorouracil in a crucible, add 10 mL of a solution of magnesium nitrate in ethanol (1 in 10), ignite the ethanol to burn and incinerate by strong heating at 750 C to 850 C. If a carbonized substance remains in this method, moisten with a small amount of nitric acid and incinerate by strong heating. Cool, add 10 mL of dilute hydrochloric acid to the residue, dissolve by warming on a water-bath, use this solution as the test solution and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.10 g of Fluorouracil in 10 mL of water and use this solution as the test solution. Measure exactly 1 mL of this solution, add water to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, acetone and water (7 : 4 : 1) to a distance of about 12 cm, air-dry the plate and examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, in vacuum, 80 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay (1) FluorouracilWeigh accurately about 0.2 g of Fluorouracil, previously dried, dissolve in 20 mL of dimethylformamide and titrate with 0.1 mol/L tetramethylammonium hydroxide VS until the color of the solution changes from yellow through blue-green to blue (indicator: 3 drops of thymol blue-dimethylformamide TS). Perform a blank determination and make any necessary correction.

Fluorouracil
H N O

NH F O

C4H3FN2O2: 130.08 Fluorouracil, when dried, contains not less than 98.5% and not more than 101.0% of fluorouracil (C4H3FN2O2) and not less than 13.1% and not more than 16.1% of fluorine (F: 19.00).
Description Fluorouracil is a white crystal or crystalline powder and is odorless. Fluorouracil is freely soluble in dimethylformamide, sparingly soluble in water, slightly soluble in ethanol and practically insoluble in ether. Melting pointAbout 282 C (with decomposition). Identification (1) Take 5 mL of a solution of Fluorouracil (1 in 500), add 0.2 mL of bromine TS: the color of bromine TS is discharged. Further add 2 mL of barium hydroxide TS: a purple precipitate is produced. (2) Proceed with 10 mg of Fluorouracil as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid. The test solution responds to the Qualitative Tests for fluoride. (3) Determine the absorption spectra of the solutions of Fluorouracil and Fluorouracil RS in 0.1 mol/L hydrochloric acid VS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. Purity (1) Clarity and color of solutionAdd 20 mL of water to 0.20 g of Fluorouracil and dissolve by warming: the solution is clear and colorless. (2) FluorideDissolve 0.10 g of Fluorouracil in 10.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20). Transfer 5.0 mL of this solution to a volumetric flask, add 10 mL of a mixture of alizarin complexone TS, acetic acid-potassium acetate buffer solution, pH 4.3 and cerous nitrate TS (1 : 1 : 1) and add water to make 20 mL. Allow to stand for 1 hour and use this solution as the test solution. Separately, transfer 1.0 mL of standard fluorine solution to a volumetric flask, add 5.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20) and add 10 mL of a mixture of alizarin complexone TS, acetic acidpotassium acetate buffer solution, pH 4.3 and cerous nitrate TS (1 : 1 : 1). Proceed in the same manner as directed for the preparation of the test solution and use this solution as the standard solution. Perform the test as directed under the Ultraviolet-visible Spectrophotometry, using a solution, prepared with 5.0 mL of diluted 0.01

Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 13.008 mg of C4H3FN2O2 (2) FluorineWeigh accurately about 4 mg of Fluorouracil, previously dried and proceed as directed in the determination of fluorine under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid.
Packaging and Storage Preserve in tight containers.

KP 9 427

Fluorouracil Cream
Fluorouracil Cream contains not less than 90.0% and not more than 110.0% of the labeled amount of fluorouracil (C4H3FN2O2: 130.08).
Method of Preparation Prepare as directed under Creams, with Fluorouracil. Sodium hydroxide may be added to adjust the pH. Identification Transfer, accurately weighed, a portion of fluorouracil Cream, equivalent to about 5 mg of Fluorouracil, to a stoppered conical flask, add 50 mL of ethanol, shake until dissolved and use this solution as the test solution. Separately, weigh 5 mg of fluorouracil RS, dissolve in 50 mL of ethanol and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 100 L each of the test solution and the standard solution (5 times with 20 L) on a line about 3 cm from the bottom edge of a thin-layer chromatographic plate coated with a 25-m layer of silica gel, develop the chromatogram with a mixture of ethyl acetate, methanol and strong ammonia water (75 : 25 : 1) to a distance of about 15 cm and remove and air-dry the plate. Observe the spots under ultraviolet light (main wavelength: 254 nm): the spots from the test solution and the standard solution show the same Rf value. Assay Weigh accurately a portion of Fluorouracil Cream, equivalent to about 10 mg of fluorouracil (C4H3FN2O2), add 20 mL of methanol, shake and add water to make exactly 100 mL. Pipet 1.0 mL of this solution, add water to make 100 mL, filter and use this filtrate as the test solution. Separately, weigh accurately about 10 mg of Fluorouracil RS, previously dried, dissolve in water to make 100 mL, pipet 1.0 mL of this solution, add water to make 10 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions: determine the peak areas, AT and AS , of Fluorouracil, for the test solution and the standard solution, respectively.

Fluorouracil Injection
Fluorouracil Injection is an aqueous solution for injection. Fluorouracil Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of fluorouracil (C4H3FN2O2: 130.08).
Method of Preparation Prepare as directed under Injections, with Fluorouracil by adding sodium hydroxide. Description Fluorouracil Injection is a colorless, clear liquid. Identification (1) The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that of the standard preparation, both relative to the internal standard, as obtained in the Assay. (2) Measure a volume of Fluorouracil Injection, equivalent to about 0.1 g of Fluorouracil according to the labeled amount, acidify with glacial acetic acid carefully, shake and precipitate the Fluorouracil. Collect the precipitate, wash with 1 mL of water and dry in vacuum over P2O5 at 80 C for 4 hours. Determine the infrared spectra of the precipitate and fluorouracil RS, respectively, as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Proceed as directed in the Identification (1) under Fluorouracil. pH Between 8.6 and 9.4. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 0.33 Endotoxin unit per mg of Fluorouracil. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injection meets the requirement.

It

Determination of Volume of Injection in Container It meets the requirement. Assay Measure exactly a volume of Fluorouracil Injection, equivalent to about 50 mg of fluorouracil (C4H3FN2O2) according to the labeled amount and add water to make 100 mL. Pipet 5.0 mL of this solution, add water to make 250 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of fluorouracil RS, previously dried, dissolve in water to make 100 mL, pipet 5.0 mL of this solution, add water to make 50 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions:

Amount (mg) of fluorouracil (C4H3FN2O2) = amount (mg) of Fluorouracil RS

AT AS

Operating conditions Proceed as directed in the Operating conditions under Fluorouracil Injection.
Packaging and Storage Preserve in tight containers.

428 Monographs, Part I absorption at the same wavenumbers. (2) A solution of Fluoxetine Hydrochloride (1 in 50) uid responds to the Qualitative Tests for chloride.
Purity (1) Heavy metalsProceed with l.0 g of Fluoxetine Hydrochloride according to Method 2, and perform the test. Prepare the control solution with 3.0 mL of Standard Lead Solution (not more than 30 ppm). (2) Related substancesDissolve about 56 mg of Fluoxetine Hydrochloride, accurately weighed, in the mobile phase to make exactly 10 mL and use this solution as the test solution (1). To exactly 2 mL of the test solution (1), add the mobile phase to make exactly 10 mL and use this solution as the test solution (2). Perform the test with 10 L each of the test solution (1) and the test solution (2) as directed under the Liquid Chromatography according to the following conditions. Measure the area of the peak corresponding to fluoxetine related substance I {N-methyl-3-pheny-[(,,-(trufluoro-mtolyl)oxy)propylamine hydrochloride], AT and the area of peak of fluoxetine, AU, from the test solution (2) and calculate the area of peak of each related substances, Ai, and total area of peaks of related substances excelpt the principal peak form the test solution (1): the amount of the fluoxetine related substance I is not more than 0.15%, the amount of -[2-(methylamino)ethyl]benzenemethanol is not more than 0.25%, the amount of the fluoxetine related substance II {N-methyl-3-phenylpropylamine} is not more than 0.25% and the total amount of related substances is not more than 0.5%.

determine the peak areas, AT and AS , of Fluorouracil for the test solution and the standard solution, respectively. Amount (mg) of fluorouracil (C4H3FN2O2)
A = amount (mg) of Fluorouracil RS T 5 AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, having octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Mobile phase: Water. Flow rate: 1.0 mL/minute. System suitability System performance: When the procedure is run with the standard solution, as directed under the above operating conditions, theoretical plate number being not less than 2500. System repeatability: When the test is repeated 6 times and the relative standard deviation of the ratio of the peak area is not more than 2.0%.
Packaging and Storage Preserve in light-resistant, hermetic containers. Store at a dark place and avoid freezing.

Fluoxetine Hydrochloride
O H F3C HCl H N CH3

Amount (%) of fluoxetine related substance I AT = 100 AT + AU Amount (%) of each related substance = 100
Ai AS + 5 AU

C17H17F3NOHCl : 344.78 Fluoxetine Hydrochloride contains not less than 98.0% and not more than 102.0% of fluoxetine hydrochloride (C17H17F3NOHCl), calculated on the anhydrous basis.
Description Fluoxetine Hydrochloride is a white, crystalline powder. Fluoxetine Hydrochloride is freely soluble in ethanol or in methanol, sparingly soluble in dichloromethane, and practically insoluble in diethyl ether. Identification (1) Determine the infrared absorption spectra of Fluoxetine Hydrochloride and Fluoxetine Hydrochloride RS, previously dried, as directed in the potassiumn bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 215 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with base-deactivated octylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of the triethylamine buffer, stabilizer-free tetrahydrofuran, and methanol (6 : 3 : 1). Flow rate: 1 mL/minute. System suitability System performance: Dissolve about 22 mg of Fluoxetine Hydrochloride RS, accurately weighed, in 10 mL of 1 mol/L sulfuric acid TS, heat at 80 C for 3 hours, cool and transfer 0.4 mL of this solution to a 25-mL volumetric flask. Add 28 mg of Fluoxetine Hydrochloride RS, 1 mg of fluoxetine related substance I and 1 mg of fluoxetine related substance II, and add the mobile phase to make exactly 25 mL. When the procedure is run with

KP 9 429 10 L of this solution under the above operating conditions, relative retention times of -[2-(methylamino) ethyl]benzenemethanol, fluoxetine related substance II, fluoxetine related substance I, fluoxetine and 4trifluoromethylphenol are about 0.24, 0.27, 0.94, 1.0 and 2.17, respectively, and the ratio of the height of the fluoxetine related substance I peak to the depth of the valley between the fluoxetine and fluoxetine related substance I peaks measured from the fluoxetine related substance I peak height is not more than 1.1. Time span of measurement: Three times as long as the retention time of Fosfestrol. Triethylamine bufferAdd 10 mL of triethylamine to 980 mL of water and adjust to a pH of 6.0 with phosphoric acid.
Water Not more than 0.5% (1 g, volumetric titration, direct titration). Assay Dissolve about 10 mg each of Fluoxetine Hydrochloride and Fluoxetine Hydrochloride RS, accurately weighed, in the mobile phase to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and measure the areas of the pricipal peaks obtained from each solutions, AT and AS .

phoric acid.
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Fluoxymesterone
H HO H3C H H3C CH3 OH

F O

C20H29FO3: 336.44 Fluoxymesterone, when dried, contains not less than 97.0% and not more than 102.0% of fluoxymesterone (C20H29FO3).
Description Fluoxymesterone is a white crystal or crystalline powder. Fluoxymesterone is odorless. Fluoxymesterone is sparingly soluble in methanol, slightly soluble in ethanol or chloroform, very slightly soluble in ether, and practically insoluble in water. Identification (1) Dissolve 5 mg of Fluoxymesterone in 2 mL of sulfuric acid: a yellow color is observed. (2) Prepare the test solution with 10 mg of Fluoxymesterone as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as an absorbing liquid: the test solution responds to the Qualitative Tests (2) for fluoride. (3) Determine the absorption spectra of solutions of Fluoxymesterone and Fluoxymesterone RS, respectively, in ethanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Fluoxymesterone and Fluoxymesterone RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit the similar intensity of absorption at the same wavenumbers. If any difference appears in the absorption spectra, dissolve the test and the references standard in dehydrated ethanol, respectively, evaporate the dehydrated ethanol and perform the test with the residue in the same manner. Specific Optical Rotation [ ]20 D : Between +104 and +112 (after drying, 0.1 g, ethanol, 10 mL, 100 mm). Purity (1) Heavy metalsProceed with 0.5 g of Fluoxymesterone according to Method 2 and perform the test. Prepare the control solution with 1.5 mL of standard lead solution (not more than 30 ppm). (2) Related substancesDissolve 30 mg of Fluo-

Amount (mg) of fluoxetine hydrochloride (C17H17F3NOHCl) = amount (mg) of Fluoxetine Hydrochloride RS

AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 215 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with base-deactivated octylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of the triethylamine buffer, stabilizer-free tetrahydrofuran, and methanol (6 : 3 : 1). Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the symmetry factor of the fluoxetine peak is not more than 2.0. System reapetibility: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of fluoxetine is not more than 2.0%. Triethylamine bufferAdd 10 mL of triethylamine to 980 mL of water and adjust to a pH of 6.0 with phos-

430 Monographs, Part I xymesterone in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of toluene, ethanol and ethyl acetate (3 : 1 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 1.0% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.2% (0.5 g, platinum crucible). Assay Weigh accurately about 25 mg each of Fluoxymesterone and Fluoxymesterone RS, previously dried, dissolve each in the internal standard solution to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Fluoxymesterone to that of the internal standard for the test solution and the standard solution, respectively.

above operating conditions, fluoxymesterone and the internal standard are eluted in this order with a resolution between their peaks being not less than 6. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative deviation of the peak area of fluoxymesterone is not more than 1.5%.
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Fluoxymesterone Tablets
Fluoxymesterone Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of fluoxymesterone (C20H29FO3: 336.44).
Method of Preparation Prepare as directed under Tablets, with Fluoxymesterone. Identification Weigh equivalent to about 20 mg of fluoxymesterone (C20H29FO3) to a portion of powdered Fluoxymesterone Tablets, add 20 mL of hot chloroform, shake and decant the supernatant clear liquid through a filter. Repeat the extraction with two 20 mL volumes of hot chloroform, combine the extracts, evaporate on the water-bath to dryness, dissolve the residue in 5 mL of acetone, decant the supernatant liquid and filter the precipitate produced after adding 20 mL of water. Dissolve the precipitate in 5 mL of acetone and filter the precipitate produced after adding 20 mL of water. Perform the test with the precipitate, dried at 105 C for 3 hours, as directed in the Identification (4) under Fluoxymesterone. Dissolution Test Perform the test with 1 tablet of Fluoxymesterone Tablets at 75 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of 0.10 mol/L hydrochloric acid. Take the dissolved solution after 60 minutes from starting of the test, filter, take 20 mL of the filtrate, add 2.0 mL of the internal standard solution and 0.10 mol/L of hydrochloric acid to make exactly 25 mL and use this solution as the test solution. Separately, weigh accurately about 28 mg of Fluoxymesterone RS, previously dried at 105 C for 3 hours and add ethanol to make exactly 25 mL. Pipet exactly 5 mL of this solution, add 2.0 mL of the internal standard solution exactly, add 0.10 mol/L hydrochloric acid to make exactly 25 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Fluoxymesterone to that of the internal standard for the test solution and the standard solution, respectively. The dissolution rate of Fluoxymesterone Tablets in 60

Amount (mg) of fluoxymesterone (C20H29FO3) = amount (mg) of Fluoxymesterone RS

QT QS

Internal standard solutionA solution of methylprednisolone in a mixture of chloroform and methanol (19 : 1) (1 in 5000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 30 cm in length, packed with silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 o C . Mobile phase: A mixture of n-butyl chloride, watersaturated n-butyl chloride, tetrahydrofuran, methanol and glacial acetic acid (95 : 95 : 14 : 7 : 6). Flow rate: Adjust the flow rate so that the retention time of Fluoxymesterone is about 9 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution, as directed under the

KP 9 431 minutes is not less than 70%. Internal standard solutionA solution of Norethindrone in ethanol (4.6 in 100000).
CH2CH2CH2 N N CH2CH2O

Fluphenazine Enanthate
O C (CH2)5CH3

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, having octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of water and acetonitrile (58 : 42). Flow rate: 3 mL/minute. System suitability System performance: When the procedure is run with 20 L of the standard solution, as directed under the above operating conditions, Fluoxymesterone and norethindrone are eluted in this order with the resolution between their peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 20L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the ratio of the peak area is not more than 2.0%.
Uniformity of Dosage Units It meets the requirement of the Content Uniformity Test when the test is performed according to the following method. Transfer 1 Fluoxymesterone Tablets to a suitable container, add 2 mL of water and sonicate for about 30 minutes until the tablet completely disintegrates. Add the internal standard solution to obtain a solution having a known concentration of 250 g per mL of fluoxymesterone (C20H29FO3) and proceed as directed in the Assay under Fluoxymesterone. Assay Weigh accurately and powder not less than 20 Fluoxymesterone Tablets. Weigh accurately a portion of the powder, equivalent to about 5 mg of fluoxymesterone (C20H29FO3), add 20 mL of the internal standard solution. Sonicate for 10 minutes and mix for 15 minutes. Filter this solution and use this solution as the test solution. Proceed as directed in the Assay under Fluoxymesterone.

N CF3

C29H38F3N3O2S: 549.69 Fluphenazine Enanthate, when dried, contains not less than 98.5% and not more than 101.0% of fluphenazine enanthate (C29H38F3N3O2S).
Description Fluphenazine Enanthate is a pale yellow to yellowish orange, viscous liquid. Fluphenazine Enanthate is generally clear and can be opaque by producing crystal. Fluphenazine Enanthate is freely soluble in methanol or ether, soluble in glacial acetic acid or ethanol, and practically insoluble in water. Identification (1) Prepare the test solution with 10 mg of Fluphenazine Enanthate as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as the absorbing liquid: the test solution responds to the Qualitative Tests for fluoride. (2) Dissolve 2 mg each of Fluphenazine Enanthate and Fluphenazine Enanthate RS separately in 200 mL of a solution of hydrochloric acid in methanol (17 in 2000) and determine the absorption spectra as directed under the Ultraviolet-visible Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectra of Fluphenazine Enanthate and Fluphenazine Enanthate RS as directed in the liquid method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Heavy metalsProceed with 1.0 g of Fluphenazine Enanthate according to Method 2 and perform the test. Prepare the control solution with 3.0 mL of standard lead solution (not more than 30 ppm). (2) Related substancesDissolve 0.25 g of Fluphenazine Enanthate in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thinlayer chromatography. Develop the plate with a mixture of acetone, hexane and strong ammonia water (16 : 6 : 1) to a distance of about 15 cm and air-dry the plate. Ex-

Amount (mg) of fluoxymesterone (C20H29FO3) = amount (mg) of Fluoxymesterone RS

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QT QS

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432 Monographs, Part I amine under ultraviolet light (main wavelength: 254 nm): any spot other than the principal spot from the test solution is not more intense than the spot from the standard solution. Then spray evenly diluted sulfuric acid (1 in 2) on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 1.0% (1 g, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.5 g of Fluphenazine Enanthate, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

(2) Determine the infrared spectra of Fluphenazine Hydrochloride and Fluphenazine Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Fluphenazine Hydrochloride responds to the Qualitative Tests for chloride.
Purity (1) Heavy metalsWeigh about 1.0 g of Fluphenazine Hydrochloride and perform a test according to the Method 2. The control solution contains 3.0 mL of standard lead solution (not more than 30 ppm). (2) Related substancesDissolve about 0.1 g of Fluphenazine Hydrochloride in a mixture of Sodium Hydroxide and methanol to make 10 mL and use this solution as the test solution. Separately, dissolve 10 mg of Fluphenazine Hydrochloride RS in a mixture of Sodium Hydroxide and methanol to make 10 mL. Pipet exactly 0.1 mL, 0.5 mL, 1 mL and 2 mL of test solution, add sodium hydroxide and methanol to make exactly 10 mL and use this solution as the standard solution (1), standard solution (2), standard solution (3), standard solution (4). Perform the test with the test solution and the standard solutions as directed under the Thin-layer chromatography. Spot 20 L each of the test solution and the standard solution (1), standard solution (2), standard solution (3), standard solution (4) on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of acetone, hexane and strong ammonia water (40 : 15 : 1) to a distance of about 15 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): prepare intensities of the spots other than the principal spot from the test solution and the spot from the standard solutions, the relative intensities is not less than 2.0%. Loss on Drying Not more than 1.0% (1 g, 65 C, 3 hours). Residue on Ignition

Each mL of 0.1 mol/L perchloric acid VS = 27.485 mg of C29H38F3N3O2S

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Fluphenazine Hydrochloride
CH2CH2CH2 N N CF3 N CH2CH2OH 2 HCl

C22H26F3N3OS2HCl: 510.44 Fluphenazine Hydrochloride contains not less than 97.0% and not more than 103.0% of fluphenazine hydrochloride (C22H26F3N3OS2HCl), calculated on the dried basis.
Description Fluphenazine Hydrochloride is a white or nearly white crystalline powder and has no odor. Fluphenazine Hydrochloride is freely soluble in water, sparingly soluble in acetone, ethanol or chloroform, and practically insoluble in benzene or ether. Melting point225 C. Identification (1) The ultraviolet-visible absorption spectrum of a solution of Fluphenazine Hydrochloride in methanol (1 in 100000) exhibits maxima and minima at the same wavelengths as that of a similar preparation of Fluphenazine Hydrochloride RS, concomitantly measured and the respective absorbance, calculated on the dried basis, at the wavelength of a maximum absorbance at about 259 nm do not differ by more than 2.5%.

Not more than 0.5% (1 g).

Assay Dissolve about 0.12 g of Fluphenazine Hydrochloride, accurately weighed, in a mobile phase without triethylamine to make 100 mL exactly. Pipet exactly 5 mL of this solution, dilute in mobile phase without triethylamine to make 100 mL exactly. Filter and use this solution as the test solution. Separately, dissolve about 0.12 g of Fluphenazine Hydrochloride RS, accurately weighed, in a mobile phase without triethylamine to make 100 mL exactly. Pipet exactly 5 mL of this solution, dilute in mobile phase without triethylamine to make 100 mL exactly. Filter and use this solution as the standard solution. Perform the test with 25 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions and calculate the ratios, AT and AS , of the peak area of fluphenazine.

KP 9 433 Amount (mg) of fluphenazine hydrochloride (C22H26F3N3OS) = amount (mg) of Fluphenazine Hydrochloride RS
AT AS

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 12.5 cm in length, having octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: Filter a mixture of 0.05 mol/L of potassium dihydrogen phosphoric acid (controlled by phosphoric acid to pH 2.5), acetonitrile and methanol (40 : 30 : 30) and add triethylamine to make 0.2%. Flow rate: 1 mL/minute. System suitability System performance: When the procedure is run with 25 L of the standard solution, as directed under the above operating conditions, symmetry coefficient is not less than 2.0. System repeatability: When the test is repeated 6 times with 25 L of the standard solution and the relative standard deviation of the ratio of the peak area is not more than 2.0%.
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test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of acetone, cyclohexane, diethylamine (40 : 15 : 1) to a distance of about 15 cm and air-dry the plate. Spray evenly on the plate with a solution of sulfuric acid in methanol (2 in 5): the Rf value and color of the principal spot obtained from the test solution correspond to those obtained from the standard solution.
Dissolution Test Perform the test with 1 tablet of Fluphenazine Hydrochloride Tablets at 100 revolutions per minute according to Method 1 under the Dissolution Test, using 900 mL of 0.01 mol/L hydrochloric acid VS. Take the dissolved solution after 45 minutes from the start of the test and perform the Assay with following differences: dilute the amount of the test solution to be withdrawn with an equal volume of mobile phase without triethylamine; inject a volume of 100 L, in the Standard preparation, use a concentration and composition similar to that of the test solution; in the Mobile phase, use 0.3% triethylamine; in the Chromatographic system, use a flow rate of about 2.0 mL per minute. The dissolution rate of Fluphenazine Hydrochloride Tablets in 45 minutes should be not less than 75%. Uniformity of Dosage Units It meets the requirement. Assay Weigh and finely powder not less than 20 Fluphenazine Hydrochloride Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 6 mg of Fluphenazine Hydrochloride, to a separator, add 80 mL of mobile phase without triethylamine, shake well for 1 hour. Then sonicate for 10 minutes to make a fine colloid solution. Filter and use this solution test solution. Separately, place about 6 mg of Fluphenazine Hydrochloride RS, accurately weighed to a volumetric flask, add mobile phase without triethylamine to dissolve and make 100 mL exactly and use this solution as the standard solution. Separately inject equal volumes (about 25 L) of the test solution and standard solution and determine the absorbances of the solutions as directed under the Liquid Chromatographic method, calculate the absorbances, AT and AS , for the test solution and the standard solution, respectively.

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Fluphenazine Hydrochloride Tablets

Fluphenazine Hydrochloride Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of fluphenazine hydrochloride (C22H26F3N3OS2HCl: 510.44).
Method of Preparation Prepare as directed under Tablets, with Fluphenazine Hydrochloride. Identification Transfer a portion of finely powdered Tablets, equivalent to about 10 mg of Fluphenazine Hydrochloride according to the labeled amount, and 10 mg of Fluphenazine Hydrochloride RS separately into two separators. Add 5 mL of water and 20 mL of diluted hydrochloric acid (1 in 120) to each separator, shake for 10 minutes and to each mixture, add 20 mL of chloroform saturated with sodium carbonate solution (1 in 10). Extracts each mixture with five 20 mL volumes of chloroform-washed cotton filters into separate beakers. Evaporate the extracts on a steam-bath to dryness and dissolve the residues separately in 0.5 mL of a mixture of methanol and water (4 : 1) and use the solutions as the test solution and the standard solution. Perform the test with the

Amount (mg) of fluphenazine (C22H26F3N3OS2HCl) = amount (mg) of Fluphenazine Hydrochloride RS

AT AS

Operating conditions Detector, column, column temperature, mobile phase and system suitability conform to the requirements of Fluphenazine Hydrochloride in the Requirements for operating conditions of Assay.
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434 Monographs, Part I Neutralize 20 mL of the filtrate with dilute nitric acid and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.036%) (3) SulfateNeurtralize 20 mL of the filtrate obtained in (2) with dilute hydrochloric acid and add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.048%). (4) Heavy metalsProceed with 2.0 g of Flurazepam according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) ArsenicPrepare the test solution with 1.0 g of Flurazepam according to Method 3 and perform the test (not more than 2 ppm). (6) Related substancesDissolve 0.20 g of Flurazepam in 20 mL of chloroform and use this solution as the test solution. Pipet 1.0 mL of the test solution and add chloroform to make exactly 20 mL. Pipet 3.0 mL of this solution, add chloroform to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with flurescence indicator for Thinlayer chromatography. Develop the plate with a mixture of cyclohexane, acetone and strong ammonia water (60 : 40 : 1) to a distance of about 12 cm and air-dry the plate. Examine under the ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.2% (1 g, in vacuum, 60 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g, platinum crucible). Assay Weigh accurately 0.3 g of Flurazepam, previously dried, dissolve in 50 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS to the second equivalence point (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Flurazepam
C2H5 CH2CH2N O N C2H5

Cl

N F

C21H23ClFN3O: 387.88 Flurazepam, when dried, contains not less than 99.0% and not more than 101.0% of flurazepam (C21H23ClFN3O).
Description Flurazepam occurs as white to pale yellow crystals or crystalline powder. Flurazepam is very soluble in chlorform, freely soluble in methanol, in ethanol, in acetic anhydride, or in ether and practically insoluble in water. Identification (1) Dissolve 0.01 g of Flurazepam in 3 mL of sulfuric acid: the solution shows a greenish yellow flurescence under ultraviolet light (main wavelength: 365 nm). (2) Dissolve 0.01 g of Flurazepam in 3 mL of citric acid acetic acid TS and heat in a water-bath for 4 minutes: a dark red color develops. (3) Prepare the test solution with 0.01 g of Flurazepam as directed under the Oxygen Flask Combustion Method, using a mixture of 0.5 mL of 0.01 mol/L sodium hydroxide TS and 20 mL of water as an absorbing liquid: the test solution responds to the Qualitative Tests (2) for fluoride. (4) Determine the absorption spectra of the solutions of Flurazepam and Flurazepam RS in methanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. And determine the spectra of the solutions of Flurazepam and Flurazepam RS in methanol (1 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths (5) Perform the test with Flurazepam as directed under the Flame Coloration Test (2): a green color appears. Melting Point Between 79 C and 83 C. Purity (1) Clarity and color of solutionDissolve 1.0 g of Flurazepam in 10 mL of ethanol: the solution is clear and colorless to pale yellow. (2) Chloride Dissolve 1.0 g of Flurazepam in 50 mL of ether, add 46 mL of water and 4 mL of sodium carbonate TS, shake, separate the water layer, wash with two 20-mL volumes of ether and filter the water layer.

Each mL of 0.1 mol/L perchloric acid VS = 19.394 mg of C21H23ClFN3O

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KP 9 435
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Flurazepam Capsules
Flurazepam Capsules contain not less than 93.0% and not more than 107.0% of the labeled amount of flurazepam (C21H23ClFN3O: 387.88).
Method of Preparation Prepare as directed under Capsules, with Flurazepam. Identification (1) Powder the contents of Flurazepam Capsules. To a quantity of the powder, equivalent to 0.1 g of Flurazepam according to the labeled amount, add 100 mL of 0.1 mol/L hydrochloric acid TS, stir and filter. To 40 mL of the filtrate, add 80 mL of a solution of sodium hydroxide (1 in 250) and 100 mL of hexane, extract by shaking well and use the hexane layer as the test solution. Evaporate 25 mL of the test solution on a waterbath to dryness. Dissolve the residue in 3 mL of sulfuric acid: the solution shows a greenish yellow fluorescence under ultraviolet light. (2) Evaporate 25 mL of the test solution obtained in (1) on a water-bath to dryness. Dissolve the residue in 3 mL of citric acid-acetic acid TS and heat in a water-bath for 4 minutes: a dark red color develops. (3) Determine the absorption spectrum of the test solution obtained in the Assay as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 315 nm and 319 nm and a minimum between 297 nm and 301 nm. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately the contents of not less than 20 Flurazepam Capsules and powder the combined contents. Weigh accurately a portion of the powder, equivalent to about 0.05 g of flurazepam (C21H23ClFN3O), add 30 mL of methanol, stir well for 10 minutes and add methanol to make exactly 50 mL. Filter this solution, discard the first 20 mL of the filtrate, pipet 6.0 mL of the subsequent filtrate, add methanol to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 0.05 g of Flurazepam RS, previously dried in vacuum at 60 C for 2 hours and dissolve in methanol to make exactly 50 mL. Pipet 6.0 mL of this solution, add methanol to make exactly 50 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of test solution and the standard solution at 317 nm as directed under the Ultravioletvisible Spectrophotometry, respectively.

Flurazepam Hydrochloride
C2H5 CH2CH2N O N C2H5

Cl

N F

HCl

C21H23ClFN3OHCl: 424.34 Flurazepam Hydrochloride, when dried, contains not less than 99.0% and not more than 101.0% of flurazepam hydrochloride (C21H23ClFN3OHCl).
Description Flurazepam Hydrochloride occurs as a white to yellowish white crystal or crystalline powder. Flurazepam Hydrochloride is freely soluble in water, in ethanol, in dehydrated ethanol, or in glacial acetic acid. Melting pointAbout 197 C (with decomposition). Identification (1) Determine the absorption spectra of the solutions of Flurazepam Hydrochloride and Flurazepam Hydrochloride RS in sulfuric acid ethanol TS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibits similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectrum of Flurazepam Hydrochloride and Flurazepam Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Flurazepam Hydrochloride (1 in 20) responds to the Qualitative Tests for chloride. pH Dissolve 1.0 g of Flurazepam Hydrochloride in 20 mL of water: the pH of this solution is between 5.0 and 6.0. Purity (1) Clarity and color of solutionDissolve 1.0 g of Flurazepam Hydrochloride in 10 mL of water: the solution is clear and colorless to pale yellow. (2) SulfatePerform the test with 1.5 g of Flurazepam Hydrochloride. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.011%). (3) Heavy metalsProceed with 1.0 g of Flurazepam Hydrochloride in a platinum crucible according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Related substancesDissolve 50 mg of Flurazepam Hydrochloride in 5 mL of ethanol and use this solu-

Amount (mg) of flurazepam (C21H23ClFN3O) = amount (mg) of Flurazepam RS

AT AS

436 Monographs, Part I tion as the test solution. Pipet 1.0 mL of this solution, add ethanol to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test and standard solutions as directed under the Thin-layer chromatography. Spot 20 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Place the plate in a chamber filled with ammonia vapor, allow to stand for about 15 minutes and immediately develop the plate with a mixture of ether and diethylamine (39 : 1) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): not more than 3 spots other than the principal spot and the spot on the starting point from the test solution appear and are not more intense than the spot from the standard solution
Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Identification (1) Determine the absorption spectra of the solutions of Flurbiprofen and Flurbiprofen in methanol (1 in 200000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectrum of Flurbiprofen and Flurbiprofen RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 114 C and 117 C. Purity (1) ChlorideDissolve 0.6 g of Flurbiprofen in 40 mL of acetone and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.25 mL of 0.01 mol/L hydrochloric acid VS, add 40 mL of acetone, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.015%). (2) Heavy metalsDissolve 2.0 g of Flurbiprofen in 30 mL of acetone and add 2 mL of dilute acetic acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 2.0 mL of standard lead solution, add 30 mL of acetone, add 2 mL of dilute acetic acid and water to make 50 mL (not more than 10 ppm). (3) Related substances-Dissolve 20 mg of Flurbiprofen in 10 mL of a mixture of water and acetonitrile (11 : 9) and use this solution as the test solution. Pipet 1.0 mL of the test solution and add a mixture of water and acetonitrile (11:9) to make exactly 200 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution and the standard solution by the automatic integration method: each area of the peaks other than the peak of Flurbiprofen from the test solution is not larger than the peak area of Flurbiprofen from the standard solution and the total area of these peaks is not larger than twice the peak area of Flurbiprofen from the standard solution.

Not more than 0.1% (1 g).

Assay Weigh accurately about 0.3 g of Flurazepam Hydrochloride, previously dried, dissolve in 10 mL of glacial acetic acid, add 40 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 21.217 mg of C21H23ClFN3OHCl

Packaging and Storage Preserve in tight containers.

Flurbiprofen
H F CO2H CH3

and enantiomer C15H13FO2: 244.26 Flurbiprofen, when dried, contains not less than 98.0% and not more than 101.0% of flurbiprofen (C15H13FO2).
Description Flurbiprofen is a white, crystalline powder and has a slightly irritating odor. Flurbiprofen is freely soluble in methanol, in ethanol, in acetone or in ether, soluble in acetonitrile and practically insoluble in water. A solution of Flurbiprofen in ethanol (1 in 50) shows no optical rotation.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 30 C. Mobile phase: A mixture of water, acetonitrile and glacial acetic acid (12 : 7 : 1). Flow rate: Adjust the flow rate so that the retention time of Flurbiprofen is about 20 minutes. System suitability Test for required detectability: To exactly 5 mL of

KP 9 437 the standard solution, add a mixture of water and acetonitrile (11:9) to make exactly 25 mL. Confirm that the peak area of flurbiprofen from 20 L of this solution is equivalent to 16 to 24% of that of flurbiprofen from 20 L of the standard solution. System performance: Dissolve 0.04 g of flurbiprofen and 0.02 g of butyl parahydroxybenzoate in 100 mL of a mixture of water and acetonitrile (11:9). To 5 mL of this solution, add a mixture of water and acetonitrile (11:9) to make 50 mL. When the procedure is run with 20 L of this solution under the above operating conditions, butyl parahydroxybenzoate and flurbiprofen are eluted in this order with the resolution between these peaks being not less than 12. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of flurbiprofen is not more than 2.0%. Time span of measurement: About twice as long as the retention time of Flurbiprofen.
Loss on Drying Not more than 0.1% (1 g, in vacuum at a pressure not exceeding 0.67 kPa, silica gel, 4 hours). Residue on Ignition Not more than 0.1% (1 g, platinum crucible). Assay Weigh accurately about 0.6 g of Flurbiprofen, previously dried, dissolve in 50 mL of ethanol and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Perform a blank determination and make any necessary correction.

(C25H31F3O5S), calculated on the anhydrous and soventfree basis.

Description Fluticasone Propionate is as a white, fine powder. Fluticasone Propionate is sparingly soluble in dichloromethane, slightly soluble in ethanol, and practically insoluble in water. Identification (1) Determine the infrared absorption spectra of Fluticasone Propionate and Fluticasone Propionate RS as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The retention time of the principal peak from the test solution is same as that from the standard solution obtained in the Assay. Specific Optical Rotation [ ]20 D : Between +32 and +36 (calculated on the anhydrous and solvent-free basis, 0.1 g, dichloromethane, 20 mL, 100 mm). Purity (1) Related substancesDissolve 20 mg of Fluticasone Propionate in 5.0 mL of the mobile phase A by sonication, add 5.0 mL of the mobile phase C and use this solution as the test solution. Perform the test with 50 L of the test solution as directed in the area percentage method under the Liquid Chromatography according to the following conditions, and measure the area of each realted substance and the total area of all of the related substances, Ai and AS : the limit of the related substances are as shown in Table I.

Each mL of 0.1 mol/L sodium hydroxide VS = 24.426 mg of C15H13FO2

Packaging and Storage Preserve in well-closed containers.

Table 1 Related substances Relative retention time Limit (%) Fluticasone propionate re0.5 0.2 lated substance I Fluticasone propionate re0.75 0.1 lated substance II Fluticasone propionate re0.8 0.1 lated substance III Fluticasone propionate re0.95 0.3 lated substance IV Fluticasone propionate 1.0 Fluticasone propionate re1.3 0.3 lated substance V Other related substances 0.1 Total related substances 1.0 Any related substances of less than 0.5% are disregarded from the total amount of related substances. Ai Amount(%) of each related substance = 100 A S

Fluticasone Propionate
F S O O CH3 HO CH3 H O CH3

F O F

C25H31F3O5S : 500.57 Fluticasone Propionate contains not less than 98.0% and not more than 100.5% of fluticasone propionate

Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 239 nm).

438 Monographs, Part I Column : A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Control the gradient by mixing the mobile phases A, B and C as directed in the following table. Mobile phase A: Mix 0.5 mL of phosphoric acid with 1000 mL of acetonitrile Mobile phase B: Mix 0.5 mL of phosphoric acid with 1000 mL of methanol Mobile phase C: Mix 0.5 mL of phosphoric acid with 1000 mL of water Table 2
Time after injection (minutes) 0 0 to 40 40 to 60 60 to 70 70 to 75 Mobile phase A (vol%) 42 42 53 53 87 87 87 42 Mobile phase B (vol%) 3 3 3 3 3 Mobile phase C (vol%) 55 55 44 44 10 10 10 55 Elution condition Equalibriation Linear gradient Linear gradient Isocratic Reequalibriation

Column : A capillary column, about 0.35 mm in inside diameter and about 25 m in length, coated with 5% phenyl-95% methylpolysiloxane with the thickness of 5 m. Column temperature: Initially the temperature of the column is maintained at 40 C for 3.5 minutes, then the temperature is increased at a rate of 30 C per minute to 200 C, and maintained at 200 C for 10 minutes. Carrier gas: Nitrogen Flow rate: 2.8 mL/minute Inlet temperature: 85 C Split ratio: about 70 : 1 Detector temperature: 250 C (3) AcetoneDissolve 0.5 g of Fluticasone Propionate, accurately weighed, in the internal standard solution to make exactly 10 mL and use this solution as the test solution. Separately, take exactly 50 L of acetone, add the internal standard solution to make exactly 100 mL and use this solution as the standard solution. Perform the test with 0.1 L each of the test solution and the standard solution as directed under Gas Chromatography according to the following conditions, and calculate the ratios, QT and QS, of the peak height of acetone to that of the internal standard, for the test solution and the standard solution, respectively. (not more than 1.0%) QT D Amount (%) of acetone = 0.05 C Q S D: density of acetone at 20 C C: concentration (g/mL) of Fluticasone Propionate in the test solution Internal standard solutionTo 50 L of tetrahydrofuran, add dimethylformamide to make 100 mL. Operating conditions Detector : A hydrogen flame ionization detector. Column : A capillary column, about 0.53 mm in inside diameter and about 25 m in length, coated with polyethylene glycol (mean molecular mass is between 3000 and 3700) with the thickness of 2 m. Column temperature: Initially the temperature of the column is maintained at 60 C for 3.5 minutes, then the temperature is increased at a rate of 30 C per minute to 180 C, and maintained at 180 for 3 minutes. Carrier gas: Nitrogen or helium Flow rate: 5.5 mL/minute Inlet temperature: 150 C Detector temperature: 250 C System suitability System repeatability: When the test is repeated 6 times with 0.1 L of the standard solution under the above operating conditions, the relative standard deviation of the peak height of acetone is not more than 5.0%.
Water Not more than 0.2% (1 g, volumetric titration,

Flow rate : 1.5 mL/minute System suitability System performance: Weigh about 2.0 mg og fluticasone propionate in 5.0 mL of the mobile phase A by sonication, add 5.0 mL of the mobile phase C and use this solution as the system suitability solution. When the procedure is performed with 50 L of this solution accoding to the above operating conditions, the resolution between peaks of fluticasone propionate related substance II and fluticasone propionate related substance III is not less than 1.5 and their relative retion times are as shown in Table I. (2) BromofluoromethaneDissolve 0.2 g of Fluticasone Propionate in 1 mL of dimethylformamide and use this solution as the test solution. Separately, measure 20 L of bromofluoromethane, add dimethylformamide to make 10 mL, take 10 L of this solution, and add dimethylformamide to make 1 mL. Take 10 L of this solution, add dimethylformamide to make 1 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under Gas Chromatography according to the following conditions, and determine heights of peaks of bromofluoromethane: the height of the bromofluoromethane obtained from the test solution is less than that from the standard solution. Operating conditions Detector : An electron capture detector.

KP 9 439 direct titration).

Assay Dissolve about 40 mg each of Fluticasone Propionate and Fluticasone Propionate RS, accurately weighed, in the mobile phase to make exactly 100 mL. Pipet exactly 5 mL of this solution and add the mobile phase to make exactly 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and measure the areas of fluticasone propionate obtained from each solutions, AT and AS .

Folic Acid
OH N N CO2H H2N N N CH2NH O C NH H C CH2CH2CO2H

C19H19N7O6: 441.40 Folic Acid, when dried, contains not less than 98.0% and not more than 102.0% of folic acid (C19H19N7O6).
Description Folic Acid is a yellow to orange-yellow, crystalline powder and is odorless. Folic Acid is practically insoluble in water, in methanol, in ethanol, in pyridine or in ether. Folic Acid dissolves in hydrochloric acid, in sulfuric acid, in dilute sodium hydroxide TS or in a solution of sodium carbonate (1 in 100) and these solutions are yellow in color. Folic Acid is affected gradually by light. Identification (1) Dissolve 1.5 mg each of Folic Acid and Folic Acid RS in dilute sodium hydroxide TS to make 100 mL. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Take 10 mL of the solution obtained with Folic Acid in (1), add 1 drop of potassium permanganate TS and mix well until the color changes to blue and immediately observe under ultraviolet light (main wavelength: 365 nm): a blue fluorescence is produced. Purity (1) Clarity and color of solutionDissolve 0.10 g of Folic Acid in 10 mL of dilute sodium hydroxide TS: the solution is clear and yellow in color. (2) Free aminesPipet exactly 30 mL of the test solution obtained in the Assay, add 20 mL of dilute hydrochloric acid and water to make accurately 100 mL and use this solution as the test solution. Weigh accurately about 50 mg of p-aminobenzoylglutamic acid RS, previously dried in a desicator (in vacuum, silica gel) for 4 hours, dissolve in diluted ethanol (2 in 5) to make exactly 100 mL. Pipet exactly 3 mL of this solution, add water to make exactly 1000 mL and use this solution as the standard solution. Pipet exactly 4 mL each of the test solution and the standard solution, proceed as directed in the Assay and perform the test as directed under the Ultraviolet-visible Spectrophotometry. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at 550 nm: the content of free amines is not more than 1.0%.

Amount (mg) of fluticasone propionate (C25H31F3O5S) = amount (mg) of A Fluticasone Propionate RS T AS Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 239 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: A mixture of methanol, 0.01 mol/L ammonium dihydrogen phosphate buffer, and acetonitrile (50 : 35 : 15). Flow rate: 1.5 mL/minute. System suitability System performance: Dissolve 2.0 mg of fluticasone propionate related substance IV RS in 50 mL of the mobile phase. When the procedure is run with 20 L each of this solution and the standard solution under the above operating conditions, relative retention times peaks of fluticasone propionate related substance IV and fluticasone propionate are 1.1 and 1.0, respectively, and the resolution between them is not less than 1.5. System reapetibility: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of fluticasone propionate is not more than 2.0%. 0.01 mol/L ammonium dihydrogen phosphate buffer Dissolve 11.5 g of ammonium dihydrogen phosphate in 1000 mL of water and adjust to a pH of 3.5 0.05 with phosphoric acid.
Packaging and Storage Preserve in light-resistant, tight containers and store at the temperature of not more than 30 C.

W' AT Content (%) of free amines = A W S W: Amount (mg) of Folic Acid, calculated on the an-

440 Monographs, Part I hydrous basis, W: Amount (mg) of p-aminobenzoyl-glutamic acid RS.
Water Not more than 8.5% (10 mg, coulometric titration). Residue on Ignition Not more than 0.5% (1 g). Assay Weigh accurately about 50 mg each of Folic Acid and Folic Acid RS. To each, add 50 mL of dilute sodium hydroxide TS, mix well to dissolve, add dilute sodium hydroxide TS to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. To 30 mL each of the test solution and the standard solution, accurately measured, add 20 mL of dilute hydrochloric acid and water to make exactly 100 mL. Take 60 mL each of these solutions, add 0.5 g of zinc powder and allow to stand with frequent shaking for 20 minutes. Filter each mixture through a dry filter paper and discard the first 10 mL of the filtrate. Pipet exactly 10 mL each of the subsequent filtrate and add water to make exactly 100 mL. To 4 mL each of solutions, accurately measured, add 1 mL of water, 1 mL of dilute hydrochloric acid and 1 mL of a solution of sodium nitrite (1 in 1000), mix well and allow to stand for 2 minutes. To each solution, add 1 mL of a solution of ammonium sulfate (1 in 200), mix thoroughly and allow to stand for 2 minutes. To each of these solution, add 1 mL of a solution of N-(1-naphthyl)-N-diethylethlyen ediamine oxalate (1 in 1000), shake, allow to stand for 10 minutes and add water to make exactly 20 mL. Separately, to 30 mL of the test solution, accurately measured, add 20 mL of dilute hydrochloric acid and water to make exactly 100 mL. Pipet exactly 10 mL of this solution and add 18 mL of dilute hydrochloric acid and water to make exactly 100 mL. Pipet exactly 4 mL of this solution and prepare the blank solution in the same manner as the test solution. Perform the test with these solutions as directed under the Ultraviolet-visible Spectrophotometry, using a solution prepared with 4 mL of water in the same manner as a blank. Determine the absorbances, AT, AS and AC, of the test solution, the standard solution and the blank solution, respectively, at 550 nm.

more than 115.0% of the labeled amount of folic acid (C19H19N7O6: 441.40).
Method of Preparation Dissolve Folic Acid in water with the aid of Sodium Hydroxide or Sodium Carbonate and prepare as directed under Injections. Description Folic Acid Injection is a yellow to orangeyellow, clear liquid. pH Between 8.0 and 11.0. Identification (1) Take a volume of Folic Acid Injection, equivalent to 1.5 mg of folic acid according to the labeled amount, add dilute sodium hydroxide TS to make 100 mL. Perform the test with this solution as directed in the Identification (2) under Folic Acid. (2) Perform the test with the solution obtained in (1) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 255 nm and 257 nm, between 281 nm and 285 nm, and between 361 nm and 369 nm and when A1 is the absorbance at the absorption maximum between 255 nm and 257 nm, and A2 is the absorbance at the absorption maximum between 361 nm and 369 nm, the ratio, A2/A1, is between 2.80 and 3.00. (3) Folic Acid Injection responds to the Qualitative Tests (1) for sodium salt. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay To an exactly measured volume of Folic Acid Injection, equivalent to about 50 mg of folic acid (C19H19N7O6: 441.40), add dilute sodium hydroxide TS to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Folic Acid RS, dissolve in dilute sodium hydroxide TS to make exactly 100 mL and use this solution as the standard solution. Proceed with 30 mL each of the test solution and the standard solution, exactly measured, as directed in the Assay under Folic Acid.

Amount (mg) of folic acid (C19H19N7O6) = amount (mg) of Folic Acid RS, AT - AC calculated on the anhydrous basis A S
Packaging and Storage tight containers.

Preserve in light-resistant,

Amount (mg) of folic acid (C19H19N7O6) = amount (mg) of Folic Acid RS, AT - AC calculated on the anhydrous basis A S
Packaging and Storage hermetic containers.

Folic Acid Injection

Folic Acid Injection is an aqueous solution for injection. Folic Acid Injection contains not less than 95.0% and not

Preserve in light-resistant,

KP 9 441

Folic Acid Tablets

Folic Acid Tablets contain not less than 90.0% and not more than 115.0% of the labeled amount of folic acid (C19H19N7O6: 441.40).

Formoterol Fumarate Hydrate

H H N H CH3O CH3 OH 2 OH NHCHO HO2C C 2H2O C H CO2H H

Method of Preparation Prepare as directed under Tablets, with Folic Acid. Identification (1) Take a portion of powdered Folic Acid Tablets, equivalent to 1.5 mg of Folic Acid according to the labeled amount, add 100 mL of dilute sodium hydroxide TS, shake well and filter. Discard the first 10 mL of the filtrate, use the subsequent filtrate as the test solution and perform the test as directed in the Identification (2) under Folic Acid. (2) Perform the test with the solution obtained in (1) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 255 nm and 257 nm, between 281 nm and 285 nm, and between 361 nm and 369 nm and when A1 is the absorbance at the absorption maximum between 255 nm and 257 nm, and A2 is the absorbance at the absorption maximum between 361 nm and 369 nm, the ratio, A2/A1, is between 2.80 and 3.00. Disintegration Test It meets the requirement. Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately and powder not less than 20 Folic Acid Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of folic acid (C19H19N7O6: 441.40). Add 50 mL of dilute sodium hydroxide TS, shake frequently, then filter into a volumetric flask and wash with dilute sodium hydroxide TS. Take the combined filtrate and washings, add dilute sodium hydroxide TS to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Folic Acid RS, dissolve in dilute sodium hydroxide TS to make exactly 100 mL and use this solution as the standard solution. Take 30 mL each of the test solution and the standard solution, exactly measured and proceed as directed in the Assay under Folic Acid.

(C19H24N2O4)2C4H4O42H2O: 840. 91 Formoterol Fumarate Hydrate contains not less than 98.5% and not more than 101.0% of formoterol fumarate [(C19H24N2O4)2C4H4O4 : 804.882], calculated on the anhydrous basis.
Description Formoterol Fumarate Hydrate is a white to yellowish white, crystalline powder. Formoterol Fumarate Hydrate is freely soluble in glacial acetic acid, soluble in methanol, very slightly soluble in water or in ethanol and practically insoluble in ether. A solution of Formoterol Fumarate in methanol (1 in 100) shows no optical rotation. Meting pointAbout 138 C (with decomposition). Identification (1) Dissolve 0.5 g of Formoterol Fumarate Hydrate in 20 mL of 0.5 mol/L sulfuric acid TS and extract with three 25 mL volumes of ether. Wash the combined ether extracts with 10 mL of 0.5 mol/L sulfuric acid TS and evaporate the ether layer under reduced pressure and dry the residue at 105 C for 3 hours: the residue melts at about 290 C (with decomposition, in a sealed tube). (2) Determine the absorption spectra of solutions of Formoterol Fumarate Hydrate and Formoterol Fumarate Hydrate RS in methanol (1 in 40000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectrum of Formoterol Fumarate and Formoterol Fumarate Hydrate RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Heavy metalsProceed with 1.0 g of Formoterol Fumarate Hydrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 0.20 g of Formoterol Fumarate Hydrate in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for Thin-layer chromatography. Devel-

Amount (mg) of Folic Acid (C19H19N7O6) = amount (mg) of Folic Acid RS, AT - AC calculated on the anhydrous basis A S
Packaging and Storage well-closed containers.

Preserve in light-resistant,

442 Monographs, Part I op the plate with a mixture of chloroform, dioxane, dehydrated ethanol and strong ammonia water (20 : 20 : 10 : 3) to a distance of about 12 cm and air-dry the plate. Allow the plate to stand for 5 minutes in iodine vapor: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Water Between 4.0% and 5.0% (0.5 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.7 g of Formoterol Fumarate Hydrate, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Fosfestrol RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
pH Dissolve 0.10 g of Fosfestrol in 30 mL of water: the pH of this solution is between 1.0 and 2.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Fosfestrol in 15 mL of sodium hydroxide TS: the solution is clear and colorless. (2) ChlorideDissolve 0.10 g of Fosfestrol in 30 mL of ethanol, add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.70 mL of 0.01 mol/L hydrochloric acid VS, add 6 mL of dilute nitric acid, 30 mL of ethanol and water to make 50 mL (not more than 0.248%). (3) Heavy metalsProceed with 1.0 g of Fosfestrol according to Method 4 and perform the test. Use a solution of magnesium nitrate in ethanol (1 in 5). Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicWeigh about 1.0 g of Fosfestrol and perform the test according to Method 3. Use a solution of magnesium nitrate in ethanol (1 in 5) (not more than 2 ppm). (5) Free phosphoric acidWeigh accurately about 0.4 g of Fosfestrol, dissolve in a mixture of water and formamide (1 : 1) to make exactly 200 mL and use this solution as the test solution. Separately, weigh accurately 0.112 g of monobasic potassium phosphate, previously dried in a desicator (silica gel) to constant weight, dissolve in 10 mL of diluted sulfuric acid (1 in 10) and water to make exactly 1000 mL. Take exactly 10 mL of this solution, add 100 mL of formamide and water to make exactly 200 mL and use this solution as the standard solution. Take exactly 10 mL each of the test solution and the standard solution and place in a volumetric flask, respectively. To each of these solutions, add 2.5 mL of ammonium molybdate-sulfuric acid TS and 1 mL of 1amino-2-naphthol-4-sulfonic acid TS, shake, add water to make 22 mL and allow to stand at 20 1 C for 30 minutes. Perform the test with these solutions, using a solution obtained in the same manner with 10 mL of a mixture of water and formamide (1 : 1) as the blank, as directed under the Ultraviolet-visible Spectrophotometry. Determine the absorbances, AT and AS, of the solutions obtained from the test solution and the standard solution, respectively, at 740 nm: the amount of free phosphoric acid is not more than 0.2%.

Each mL of 0.1 mol/L perchloric acid VS = 40.24 mg [(C19H24N2O4)2C4H4O4]

Packaging and Storage Preserve in tight containers

Fosfestrol
OPO3H2 CH3CH2

CH2CH3 H2O3PO

Diethylstilbestrol Diphosphate

C18H22O8P2: 428.31

Fosfestrol, when dried, contains not less than 98.5% and not more than 101.0% of fosfestrol (C18H22O8P2).
Description Fosfestrol is a white, crystalline powder and is odorless. Fosfestrol is freely soluble in ethanol, soluble in formamide, slightly soluble in water and practically insoluble in acetonitrile or in ether. Fosfestrol dissolves in sodium hydroxide TS. Meting pointAbout 234 C (with decomposition). Identification (1) Dissolve 15 mg of Fosfestrol in 1 mL of sulfuric acid: a yellow to orange color is observed. To this solution, add 10 mL of water: the color of the solution disappears. (2) Place 0.4 g of Fosfestrol in a crucible, wet by adding 0.1 mL of sulfuric acid and heat to carbonize. Add 10 mL of water to the residue, stir well and filter. Add 0.1 mL of nitric acid to the filtrate and heat in a water-bath for 15 minutes: this solution responds to the Qualitative Tests for phosphate. (3) Determine the infrared spectrum of Fosfestrol and

Amount (%) of free phosphoric acid (H3PO4) 1 AT = A W 80.65 S W: Amount (mg) of Fosfestrol. (6) Related substancesDissolve 20 mg of Fosfe-

KP 9 443 strol in 100 mL of the mobile phase and use this solution as the test solution. Pipet exactly 5 mL of this solution and add the mobile phase to make exactly 50 mL. Pipet exactly 3 mL of this solution, add the mobile phase to make exactly 20 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of the test solution and the standard solution by the automatic integration method: the total area of the peaks other than the principal peak from the test solution is not larger than the peak area of the principal peak from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of a solution of potassium dihydrogen phosphate (1 in 500), acetonitrile and tetrabutyl-ammonium hydroxide TS (70 : 30 : 1). Flow rate: Adjust the flow rate so that the retention time of fosfestrol is about 8 minutes. System suitability System performance: Dissolve 20 mg of Fosfestrol and 8 mg of methyl p-hydroxy-benzoate in 100 mL of the mobile phase. When the precedure is run with 10 L of this solution, as directed under the above operating condition, methyl p-hydroxybenzoate and fosfestrol are eluted in this order with a resolution between their peaks being not less than 3. Detection sensitivity: Adjust the detection sensitivity so that the peak height of fosfestrol obtained from 10 L of the standard solution is between 5 mm and 15 mm. Time span of measurement: Three times as long as the retention time of fosfestrol.
Loss on Drying Not more than 1.0% (1 g, 105 C, 4 hours). Assay Weigh accurately about 0.2 g of Fosfestrol, previously dried, dissolve in 60 mL of water and titrate with 0.1 mol/L sodium hydroxide VS (potentiometric titration, Endpoint Detection Method in Titrimetry). The end point is the second equivalent point. Perform a blank determination and make any necessary correction.

Fosfestrol Tablets
Diethylstilbestrol Diphosphate Tablets Fosfestrol Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of fosfestrol (C18H22O8P2: 428.31).
Method of Preparation Prepare as directed under the Tablets, with Fosfestrol. Identification (1) Take a portion of powdered Fosfestrol Tablets, equivalent to 0.5 g of fosfestrol according to the labeled amount, add 50 mL of 0.1 mol/L hydrochloric acid TS, shake well and filter. To the filtrate, add 100 mL of ether, extract and evaporate carefully the ether extract in a water-bath to dryness. Proceed with 15 mg of the residue as directed in the Identification (1) under Fosfestrol. (2) Dry 0.01 g of the residue obtained in (1) at 105 C for 4 hours and determine the infrared absorption spectrum as directed in the potassium bromide disk method under the Infrared Spectrophotometry: it exhibits absorption at the wavenumbers of about 2970 cm-1, 1605 cm-1, 1505 cm-1, 1207 cm-1 and 1006 cm-1. Dissolution Perform the test with 1 tablet of Fosfestrol Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water. Take 20 mL or more of the dissolved solution after 20 minutes from starting of the test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet exactly 2 mL of the subsequent, add a solution of sodium hydroxide (1 in 250) to make exactly 20 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Fosfestrol RS, previously dried at 105 C for 4 hours and dissolve in a solution of sodium hydroxide (1 in 250) to make exactly 100 mL. Pipet exactly 2 mL of this solution, add a solution of sodium hydroxide (1 in 250) to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at 242 nm as directed under the Ultraviolet-visible Spectrophotometry.

The dissolution rate of Fosfestrol Tablets in 20 minutes is not less than 80%. Dissolution rate (%) with respect to the labeled amount AT 1 of fosfestrol (C18H22O8P2) = WS A C 180 S WS: Amount (mg) of Fosfestrol RS C: Labeled amount (mg) of fosfestrol (C18H22O8P2) in 1 tablet.

Each mL of 0.1 mol/L sodium hydroxide VS = 10.708 mg of C18H22O8P2

Packaging and Storage Preserve in tight containers.

444 Monographs, Part I

Uniformaity of Dosage Unit It meets the requirement. Assay Weigh accurately and powder not less than 20 Fosfestrol Tablets. Weigh accurately a portion of the powder, equivalent to about 1 g of fosfestrol (C18H22O8P2) according to the labeled amount, add 100 mL of a solution of sodium hydroxide (1 in 125), shake well, add water to make exactly 500 mL and filter. Discard the first 30 mL of the filtrate, pipet exactly 2 mL of the subsequent, add 30 mL of a solution of sodium hydroxide (1 in 125) and water to make exactly 250 mL and use this solution as the test solution. Separately, weigh accurately about 80 mg of Fosfestrol RS, previously dried at 105 C for 4 hours and dissolve in a solution of sodium hydroxide (1 in 125) to make exactly 50 mL. Pipet exactly 1 mL of this solution, add 10 mL of a solution of sodium hydroxide (1 in 125) and water to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at 242 nm as directed under the Ultraviolet-visible Spectrophotometry.

of water, neutralize with a saturated solution of sodium hydrogen carbonate. To this solution add 1 mL of potassium iodide: a red color does not develop. Test with 3 mL of perchloric acid (1 in 4) in the same manner as the above for a blank test solution: a red color develops. (2) To 2 mL of an aqueous solution of Fosfomycin Calcium Hydrate (1 in 500), add 1 mL of perchloric acid and 2 mL of 0.1 mol/L sodium periodate TS, heat for 10 minutes in water bath, allow to cool, add 1 mL of ammonium molybdate sulfuric acid TS and 1 mL of 1-amino2-naphthol-4-sulfonic acid TS and allow to stand for 30 minutes: a blue color develops. (3) To 10 mL of an aqueous solution of Fosfomycin Calcium Hydrate (1 in 500), and add 1 mL of ammonium oxalate TS: A white precipitate develops. Filter the precipitate, and add dilute acetic acid to the precipitate: the precipitate is not dissolved. To this suspension add dilute hydrochloric acid: the precipitate is dissolved. (4) Fosfomycin Calcium Hydrate responds to the Qualitative Tests 1) for calcium salt.
Specific Optical Rotation [ ] 20 D : -3 ~ -5 (0.5 g, 0.4 mol/L disodium dihydrogen ethylenediamine tetraacetate TS, pH 8.5, 10 mL, 100 mm). pH Weigh 40 mg of Fosfomycin Calcium Hydrate, suspend in 10 mL of water, allow to cool to about 5 o C , and raise the temperature to room temperature. The pH of the susepension is between 8.0 and 10.0. Purity Heavy metalsProceed with 1.0 g of Fosfomycin Calcium Hydrate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Water Not more than 12.0 % (0.1 g, volumetric titration, direct titration). Use a mixture of formamide for water determination and methanol for water determination (2 : 1) instead of methanol for water determination Phosphorus Content Weigh accurately about 0.1 g of Fosfomycin Calcium Hydrate, transfer into 100 mL Erlenmeyer flask, add 40 mL of 0.05 mol/L sodium periodate TS and 2 mL of pechloric acid, heat for 1 hour in water bath and allow to cool, and add water to make exactly 200 mL. Pipet exactly 10 mL of this solution and transfer into 100 mL volumetric flask, and add 1 mL of potassium iodide TS: the color of the solution is brown. To this solution add sodium thiosulfate TS until the solution is colorless, add water to make exactly 100 mL, and use this solution as the test solution. Separately, weigh accurately 70 mg of potassium dihydrogenphosphate, proceed with this solution in the same manner as directed for the preparation of the test solution, and use the solution so obtained as the standard solution. Proceed and prepare a solution in the same manner for the preparation of the test solution without using Fosfomycin Sodium, and use the solution so obtained as the blank solution.

Amount (mg) of fosfestrol (C18H22O8P2) AT 25 = amount (mg) of Fosfestrol RS A 2 S

Packaging and Storage Preserve in tight containers.

Fosfomycin Calcium Hydrate

H H

H3C O

PO3Ca H2O

C3H5CaO4PH2O: 194.14 Fosfomycin Calcium Hydrate is the calcium salt of a substance having antibacterial activity produced by the growth of Streptomyces fradiae or by the chemical synthesis. Fosfomycin Calcium Hydrate contains not less than 725 g (potency) per mg of fosfomycin (C3H7O4P: 138.06), calculated on the anhydrous basis.
Description Fosfomycin Calcium Hydrate is a white crystalline powder, and is tasteless. Fosfomycin Calcium Hydrate is slightly soluble in water, and practically insoluble in methanol, in acetone, in ethylacetate, in chloroform, or in ether. Identification (1) Dissolve about 0.1 g of Fosfomycin Calcium Hydrate in 3 mL of perchloric acid (1 in 4), add 1 mL of 0.1 mol/L sodium periodate TS, and heat for 30 minutes in 60 o C water bath. Allow to cool, add 50 mL

KP 9 445 Pipet 5 mL of each of the test solution, the standard solution, and the blank solution, transfer each into a 25 mL volumetric flask, add 2.5 mL of ammonium molybdatesulfuric acid TS and 1 mL of 1-amino-2-naphthol-4sulfonic acid TS, mix with shaking, and add water to make exactly 25 mL. After allowing these solutions to stand for 30 minutes at 20 1 o C , perform the test with these solutions as directed under Ultraviolet-visible Spectrophotometry, using water as a blank, and determine the absorbances at 740 nm, A1, A2, and A3, of the test solution, the standard solution and the blank solution (not less than 15.2 % and not more than 16.7 %). Amount [%] of phosphorus =
A1 A3 W2 22 .76 A2 A3 W1

lution. Keep the standard stock solution below 5 o C , and use within 7 days. Take exactly a suitable amount of the standard stock solution before use, add 0.05 mol/L Tris buffer solution, pH 7.0 to make solutions so that each mL contains 10.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics.
Packaging and Storage tight containers.

Preserve in light-resistant,

Fosfomycin Sodium
H H

A1: Absorbance of the test solution A2: Absorbance of the standard solution A3: Absorbance of the blank solution W1: Amount (mg) of Fosfomycin Sodium W2: Amount (mg) of potassium dihydrogenphosphate 22.76: Content (%) of phosphorus in potassium dihydrogenphosphate
Calcium Content Weigh accurately about 0.2 g of Fosfomycin Calcium Hydrate, transfer into a 250 mL flask, add 4 mL of 1 mol/L hydrochloric acid TS, and shake well until the sample is completely dissolved. To this solution add 100 mL of water, 9 mL of sodium hydroxide TS and 0.1 g of methylthymol bluesodium chloride indicator, and titrate with 0.05 mol/L disodium dihydrogen ethylenediamine tetraacetate VS until the color of the solution changes from clear to gray or gray-purple (not less than 19.6 % and not more than 21.7 %).

H3C O

PO3Na2

C3H5Na2O4P: 182.02 Fosfomycin Sodium is the sodium salt of a substance having antibacterial activity produced by the growth of Streptomyces fradiae or by the chemical synthesis. Fosfomycin Sodium contains not less than 700 g (potency) per mg of fosfomycin (C3H7O4P: 138.06), calculated on the anhydrous basis.
Description Fosfomycin Sodium is a white crystalline powder, and has a little bit of salty taste. Fosfomycin Sodium is very soluble in water, sparingly soluble in methanol, and practically insoluble in ethanol or in ether. Identification (1) Dissolve about 0.1 g of Fosfomycin Sodium in 3 mL of perchloric acid (1 in 4), add 1 mL of 0.1 mol/L sodium periodate TS, and heat for 30 minutes in 60 C water bath. Allow to cool, add 50 mL of water, neutralize with sodium hydrogen carbonate solution, and add 1 mL of potassium iodide: a red color does not develop. Test with 3 mL of perchloric acid (1 in 4) in the same manner as the above for a blank test solution: a red color develops. (2) To 2 mL of an aqueous solution of Fosfomycin Sodium (1 in 250), add 1 mL of perchloric acid and 2 mL of 0.1 mol/L sodium periodate TS, heat for 10 minutes in water bath, allow to cool, add 1 mL of ammonium molybdate sulfuric acid TS and 1 mL of 1-amino-2naphthol-4-sulfonic acid TS and allow to stand for 30 minutes: a blue color develops. (3) Fosfomycin Sodium responds to the Qualitative Tests 1) for sodium salt. Specific Optical Rotation
[ ] 20 D : -3.5 ~ -5.5 (0.5 g,

Each mL of 0.05 mol/L disodium dihydrogen ethylenediamine tetraacetate VS = 2.004 mg of Ca

Assay The Cylinder-plate method (1) Agar media for seed and base layer- Use the media (1) under the Assay in Fosfomycin Sodium. (2) Test organism- Proteus sp. MB 838. (3) Test organism suspension- Prepare according to (3) under the Assay in Fosfomycin Sodium. (4) Weigh accurately about 50 mg (potency) of Fosfomycin Calcium Hydrate, and dissolve in 0.05 mol/L Tris buffer solution, pH 7.0 to make the solution so that each mL contains 1 mg (potency). Take exactly a suitable amount of this solution, add 0.05 mol/L Tris buffer solution, pH 7.0 to make solutions so that each mL contains 10.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration test solution and the low concentration test solution, respectively. Separately, weigh accurately about 10 mg (potency) of Fosfomycin RS, and dissolve in 0.05 mol/L Tris buffer solution, pH 7.0 to make the solution so that each mL contains 500 g (potency), and use the solution as the standard stock so-

446 Monographs, Part I water, 10 mL, 100 mm).

pH The pH of a solution obtained by dissolving 70 mg of Fosfomycin Sodium in 1 mL of water is between 8.5 and 10.5. Purity Heavy metalsWeigh 2.0 g of Fosfomycin Sodium, dissolve in 4 mL of acetic acid and water to make 50 mL, and use this solution as the test solution. Perform the test with this solution. Prepare the control solution with a mixture of 2.0 mL of standard lead solution, 2 mL of dilute acetic acid and 46 mL of water (not more than 10 ppm). Sterility Test It meets the requirement, when Fosfomycin Sodium is used in a sterile preparation. Bacterial Endotoxins Less than 0.125 EU per mg of fosfomycin, when Fosfomycin Sodium is used in a sterile preparation. Water Not more than 3.0 % (0.2 g, volumetric titration, direct titration). Phosphorus Content Weigh accurately about 0.1 g of Fosfomycin Sodium, transfer into 100 mL Erlenmeyer flask, add 40 mL of 0.05 mol/L sodium periodate TS and 2 mL of pechloric acid, heat for 1 hour in water bath and allow to cool, and add water to make exactly 200 mL. Pipet exactly 10 mL of this solution and transfer into 100 mL volumetric flask, and add 1 mL of potassium iodide TS: the color of the solution is brown. To this solution add sodium thiosulfate TS until the solution is colorless, add water to make exactly 100 mL, and use this solution as the test solution. Separately, weigh accurately 70 mg of potassium dihydrogenphosphate, proceed with this solution in the same manner as directed for the preparation of the test solution, and use the solution so obtained as the standard solution. Proceed and prepare a solution in the same manner for the preparation of the test solution without using Fosfomycin Sodium, and use the solution so obtained as the blank solution. Pipet 5 mL of each of the test solution, the standard solution, and the blank solution, transfer each into a 25 mL volumetric flask, add 2.5 mL of ammonium molybdatesulfuric acid TS and 1 mL of 1-amino-2-naphthol-4-sulfonic acid TS, mix with shaking, and add water to make exactly 25 mL. After allowing these solutions to stand for 30 minutes at 20 1 C , perform the test with these solutions as directed under Ultraviolet-visible Spectrophotometry, using water as a blank, and determine the absorbances at 740 nm, A1, A2, and A3, of the test solution, the standard solution and the blank solution (not less than 16.2 % and not more than 17.9 %).

A1: Absorbance of the test solution A2: Absorbance of the standard solution A3: Absorbance of the blank solution W1: Amount (mg) of Fosfomycin Sodium W2: Amount (mg) of potassium dihydrogenphosphate 22.76: Content (%) of phosphorus in potassium dihydrogenphosphate
Assay The Cylinder-plate method (1) Agar media for seed and base layerPeptone 5.0 g Yeast extract 2.0 g Meat extract 3.0 g Agar 15.0 ~ 20.0 g Water 1000 mL Mix all the ingredients, and sterilize. Adjust the pH of the solution so that it will be between 6.5 and 6.6 after sterilization. (2) Test organism- Proteus sp. MB 838. (3) Test organism suspension- Incubate the test organism on the slant of the agar medium for transferring test organism at 37 C for 40 ~ 48 hours. Sub-culture at least three times. Inoculate the grown organisms onto the surface of 300 mL of the agar medium for transferring test organism in a Roux bottle, incubate at 37 C for 40 ~ 48 hours, and suspend the grown organisms in about 30 mL of water. To the suspension add water to dilute 10fold so that the percent transmission at 560 nm of the suspension is 17 %. Keep the suspension of test organism at 10 C or below and use within 7 days. Add 1.0 ~ 2 mL of the suspension of test organism to 100 mL of the agar medium for seed layer previously kept at 48 C, mix with shaking, and use this as the seeded agar layer. (4) Weigh accurately about 50 mg (potency) of Fosfomycin Sodium, and dissolve in 0.05 mol/L Tris buffer solution, pH 7.0 to make the solution so that each mL contains 1 mg (potency). Take exactly a suitable amount of this solution, add 0.05 mol/L Tris buffer solution, pH 7.0 to make solutions so that each mL contains 10.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration test solution and the low concentration test solution, respectively. Separately, weigh accurately about 10 mg (potency) of Fosfomycin RS, and dissolve in 0.05 mol/L Tris buffer solution, pH 7.0 to make the solution so that each mL contains 500 g (potency), and use the solution as the standard stock solution. Keep the standard stock solution below 5 o C , and use within 7 days. Take exactly a suitable amount of the standard stock solution, add 0.05 mol/L Tris buffer solution, pH 7.0 to make solutions so that each mL contains 10.0 g (potency) and 5.0 g (potency), and use these solutions as the high concentration standard solution and the low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method (I 8) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in tight containers.

Amount [%] of phosphorus =

A1 A3 W2 22 .76 A2 A3 W1

KP 9 447 (not more than 4 ppm). (7) CalciumDissolve 0.5 g of Fructose in 5 mL of water, add 2 to 3 drops of ammonia TS and 1 mL of ammonium oxalate TS and allow to stand for 1 minute: the solution is clear. (8) ArsenicDissolve 1.5 g of Fructose in 5 mL of water, heat with 5 mL of dilute sulfuric acid and 1 mL of bromine TS on a water-bath for 5 minutes, concentrate to 5 mL and cool. Perform the test with this solution as the test solution (not more than 1.3 ppm). (9) 5-HydroxymethylfurfuralsDissolve 5.0 g of Fructose in 100 mL of water and read the absorbance at 284 nm as directed under the Ultraviolet-visible Spectrophotometry: the absorbance is not more than 0.32.
Loss on Drying Not more than 0.5% (1 g, in vacuum, silica gel, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 4 g of Fructose, previously dried, dissolve in 0.2 mL of ammonia TS and 80 mL of water and after standing for 30 minutes, add water to make exactly 100 mL and determine the optical rotation, D , in a 100 mm cell at 20 1 C as directed under the Optical Rotation Determination.

Fructose
H O OH H HO OH H HO CH2OH

C6H12O6: 180.16 Fructose, when dried, contains not less than 98.0% and not more than 101.0% of fructose (C6H12O6).
Description Fructose is a colorless to white crystal or crystalline powder, is odorless and has a sweet taste. Fructose is very soluble in water, sparingly soluble in ethanol and practically insoluble in ether. Fructose is hygroscopic. Identification (1) Add 2 to 3 drops of a solution of Fructose (1 in 20) to 5 mL of boiling Fehlings TS: a red precipitate is produced. (2) Determine the infrared spectrum of Fructose and Fructose RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. pH Dissolve 4.0 g of Fructose in 20 mL of water: the pH of the solution is between 4.0 and 6.5. Purity (1) Clarity and color of solutionDissolve 25.0 g of Fructose in 50 mL of water: the solution is clear and has no more color than the following control solution.

Amount (mg) of fructose (C6H12O6) = | D | 1087.0

Packaging and Storage Preserve in tight containers.

Fructose Injection
Fructose Injection is an aqueous solution for injection. Fructose Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of fructose (C6H12O6: 180.16).
Method of Preparation Prepare as directed under Injections, with Fructose. No preservative is added. Description Fructose is a colorless to white crystal or crystalline powder, is odorless and has a sweet taste. Identification (1) Take a volume of Fructose Injection, equivalent to 1 g of Fructose according to the labeled amount, dilute with water or concentrate on a water-bath to make 20 mL, if necessary and use this solution as the test solution. Add 2 to 3 drops of the test solution to 5 mL of boiling Fehlings TS: a red precipitate is produced. (2) Take 10 mL of the test solution obtained in (1), add 0.1 g of resorcin and 1 mL of hydrochloric acid and warm on a water-bath for 3 minutes: a red color is observed. pH

Control solutionTo a mixture of 1 mL of cobaltous chloride stock CS, 3 mL of ferric chloride stock CS and 2 mL of cupric sulfate stock CS and add water to make 10 mL. To 3 mL of the solution, add water to make 50 mL. (2) AcidDissolve 5 g of Fructose in 50 mL of freshly boiled and cooled water and add 3 drops of phenolphthalein TS and 0.6 mL of 0.01 mol/L sodium hydroxide VS: a red color is observed. (3) ChloridePerform the test with 2.0 g of Fructose. Prepare the control solution with 1.0 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.018%). (4) SulfatePerform the test with 2.0 g of Fructose. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (5) SulfiteDissolve 0.5 g of Fructose in 5 mL of water and add 0.25 mL of 0.01 mol/L iodine: the color of the solution is yellow. (6) Heavy metalsProceed with 5.0 g of Fructose according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution

Between 3.0 and 6.5.

448 Monographs, Part I

COOH

Purity (1) Heavy metalsTake a volume of Fructose Injection, equivalent to 5.0 g of Fructose, according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution. (2) ArsenicTake a volume of Fructose Injection, equivalent to 1.5 g of Fructose according to the labeled amount, dilute with water or concentrate on a water-bath to make 5 mL, if necessary and add 5 mL of dilute sulfuric acid and 1 mL of bromine TS. Proceed as directed in the Purity (8) under Fructose. Residue on Ignition Measure exactly a volume of Fructose Injection, equivalent to about 2.0 g of Fructose according to the labeled amount, evaporate on a waterbath to dryness and perform the test: the residue weighs not more than 2.0 mg. Sterility Test It meets the requirement. Pyrogen Test Perform the test with Fructose Injection stored in a container in a volume exceeding 10 mL: it meets the requirement of the Pyrogen Test. When the Fructose concentration is greater than 10 w/v%, dilute with the Fructose solution with water for injection to make 10 w/v% and perform the test. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

NHCH2

H2NO2S Cl

C12H11ClN2O5S: 330.74 Furosemide, when dried, contains not less than 98.0% and not more than 101.0% of furosemide (C12H11ClN2O5S).
Description Furosemide is a white crystal or crystalline powder. Furosemide is freely soluble in dimethylformamide, soluble in methanol, sparingly soluble in ethanol, slightly soluble in glacial acetic acid and practically insoluble in water. Furosemide dissolves in sodium hydroxide TS. Furosemide is gradually colored by light. Melting pointAbout 205 C (with decomposition). Identification (1) Dissolve 25 mg of Furosemide in 10 mL of methanol. To 1 mL of this solution, add 10 mL of 2 mol/L hydrochloric acid TS. Heat the solution in a water-bath under a reflux condenser for 15 minutes, cool and add 18 mL of sodium hydroxide TS to make weakly acidic: this solution responds to the Qualitative Tests for primary aromatic amines. A red to red-purple color is observed. (2) Determine the absorption spectra of solutions of Furosemide and Furosemide RS in dilute sodium hydroxide TS (1 in 25,000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared spectrum of Furosemide and Furosemide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Clarity and color of solutionDissolve 0.5 g of Furosemide in 10 mL of a solution of sodium hydroxide (1 in 50): the solution is colorless and clear. (2) ChlorideDissolve 2.6 g of Furosemide in 90 mL of dilute sodium hydroxide TS, add 2 mL of nitric acid and filter. To 25 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.020%). (3) SulfateTo 20 mL of the filtrate in (2), add 1 mL of dilute hydrochloric acid and perform the test using this solution as the test solution. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS, 1 mL of hydrochloric acid and water to make 50 mL (not more than 0.030%).

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of Fructose Injection, equivalent to about 4 g of fructose (C6H12O6: 180.16), add 0.2 mL of ammonia TS, dilute with water to make exactly 100 mL, shake well and after allowing to stand for 30 minutes, determine the optical rotation, D , in a 100 mm cell at 20 1 C as directed under the Optical Rotation Determination.

Amount (mg) of fructose (C6H12O6) = | D | 1087.0

Packaging and Storage Preserve in hermetic containers and plastic containers for aqueous injections may be used.

Furosemide

KP 9 449 (4) Heavy metalsProceed with 2.0 g of Furosemide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (5) Related substances Dissolve 25 mg of Furosemide in 25 ml. of the dissolving solution, and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add the dissolving solution to make exactly 200 mL, and use this solution as the standard solution. Perform the test with exactly 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of each peak appeared ahead of the peak of furosemide is not more than 2/5 times the peak area of furosemide from the standard solution, the area of each peak appeared behind the peak of furosemide is not more than 1/4 times the peak area of furosemide from the standard solution, and the total area of these peaks is not more than 2 times the peak area of furosemide from the standard solution. Dissolving solutionTo 22 mL of anhydrous acetic acid, add a mixture of water and acetonitrile (1 : 1) to make l000 mL. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 272 nm). Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of water, tetrahydrofuran and glacial acetic acid (70 : 30 : 1). Flow rate: Adjust the flow rate so that the retention time of furosemide is about 18 minutes. System suitability Test for required detectability: Measure exactly 2 mL of the standard solution, and add the dissolving solution to make exactly 50 mL: the peak area of furosemide obtained from 20 L of this solution is equivalent to 3.2 to 4.8% of that obtained from 20 L of the standard solution. System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, the number of theoretical plates and the symmetrry factor of the peak of furosemide are not less than 7000 and not more than 1.5, respectively. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of furosemide is not more than 2.0%. Time span of measurement: About 2.5 times as long as the retention rime of furosemide beginning after the solvent peak.

Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Furosemide, previously dried, dissolve in 50 mL of dimethylformamide and titrate with 0.1 mol/L sodium hydroxide VS until the color of the solution changes from yellow to blue (indicator: 3 drops of bromothymol blue TS). Perform a blank determination with a mixture of 50 mL of dimethylformamide and 15 mL of water and make any necessary correction.

Each mL of 0.1 mol/L sodium hydroxide VS = 33.074 mg of C12H11ClN2O5S

Packaging and Storage tight containers.

Preserve in light-resistant,

Furosemide Tablets
Furosemide Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of furosemide (C12H11ClN2O5S: 330.74).
Method of Preperation Prepare as directed under Tablets, with Furosemide. Identification (1) Shake well a quantity of powdered Furosemide Tablets, equivalent to 0.2 g of furosemide according to the labeled amount, with 40 mL of acetone, and filter. To 0.5 mL of the filtrate add 10 ml of 2 mol/L hydrochloric acid TS and heat under a reflux condenser on a water bath for 15 minutes. After cooling, add 18 mL of sodium hydroxide TS to make the solution slightly acidic: the solution responds to the Qualitative Tests for Primary Aromatic Amines, producing a red to red-purple color. (2) Determine the absorption spectrum of the test solution obtained in the Assay as directed under Ultravioletvisible Spectrophotometry : it exhibits maxima between 227 nm and 231 nm, between 269 nm and 273 nn, and between 330 nm and 336 nm. Purity To a quantiy of powdered Furosemide Tablets, equivalent to 40 mg of furosemide according to the labeled amount, add about 30 mL of acetone, shake well, and add acetone to make exactly 50 mL. Centrifuge the solution, add 3.0 mL of water to 1.0 mL of the supenlatant liquid, cool in ice, add 3.0 mL of dilute hydrochloric acid and 0.15 mL of sodium nitrite TS, shake, and allow to stand for 1 minute. Add 1.0 ml. of ammonium amidosulfate TS, shake well, allow to stand for 3 minutes, add 1.0 mL of N,N-diethyl-N-1-naphthylethylenediamine oxalate TS, shake well, and allow to stand for 5 minutes.

450 Monographs, Part I Perform the test with this solution as directed under Ultraviolet-visible Spcctrophotometry, using a solution prepared in the same manner with 1.0 mL of acetone as the blank: the absorbance at 530 nm is not more than 0.10.
Dissolution Test Perform the test with 1 tablet of Furosemide Tablets at 50 revolutions per minute according to the Method 2, using 900 mL of 2nd fluid for dissolution test as the dissolution medium. Withdraw 20 mL or more of the dissolution medium 15 minutes after starting the test for a 20-mg tablet or 30 minutes after for a 40-mg tablet, and filter through a membrane filter with pore size of not more than 0.45 m. Discard the first 10 mL of the filtrate, pipet V mL of the subsequent filtrate, add 2nd fluid for dissolution test to make exactly V mL so that each mL contains about l0 g of furosemide (C12H11ClN2OS) according to the labeled amount, and use this solution as the test solution. Separately, weigh accurately about 20 mg of Furosemide RS, previously dried at 105 C for 4 hours, and dissolve in 5 mL of methanol, and add 2nd fluid for dissolution test to make exactly 100 mL. Pipet 5 mL of this solution, add 2nd fluid for dissolution test to make exactly 100 mL, and use this solution as the standard solution. Determine the absorbances, AT and AS of the test solution and the standard solution at 277 nm as directed under Ultraviolet-visible Spectrophotometry.

tion of the powder, equivalent to about 40 mg of furoscmide (C12H11ClN2O5S), add about 70 mL of 0.05 mol/L sodium hydroxide TS, shake well, and add 0.05 mol/L sodium hydroxide TS to make exactly 100 mL. Filter, discard the first 10 mL of the filtrate, pipet exactly 2 mL of the subsequent filtrate, add 0.05 mol/L sodium hydroxide TS to make exactly 100 mL, and use this solution as the test solution. Separately, weigh accurately about 20 mg of Furosemide RS, previously dried at 105 C for 4 hours, and dissolve in 0.05 mol/L sodium hydroxide TS to make exactly 50 mL. Pipet exactly 2 mL of this solution, add 0.05 mol/l. sodium hydroxide TS to make exactly 100 mL, and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution at 271 nm as directed under the Ultraviolet-visible Spectrophotometry. Amount (mg) of furosemide (C12H11ClN2O5S) AT = amount (mg) of Furosemide RS A S
Packaging and Storage Preserve well-closed containers.

Fursultiamine Hydrochloride
H3C N NH2 N CH2N H3C CHO S SCH2 O H HCl

The dissolution rate of Furosemide Tablets in 60 minutes is not less than 80%. Dissolution rate (%) with respect to the labeled amount of furosemide (C12H11ClN2O5S) = amount (mg) of Furosemide RS AT V 1 45
AS V C

CH2CH2OH

and enantiomer C17H26N4O3S2HCl: 435.00

C : Labeled amount (C12H11ClN2O5S) in 1 tablet

(mg)

of

furosemide

Uniformity of Dosage Units Perform the test according to the following method: it meets the requirement. To 1 tablet of Furosemide Tablets add a suitable amount of 0.05 mol/L sodium hydroxide TS, shake to disintegrate, then add 0.05 mol/L sodium hydroxide TS to make exactly V mL, so that each mL contains about 0.4 mg of furosemide (C12H11ClN2O5S). Filter the solution, discard the first 10 mL of the filtrate, pipet the subsequent 2 mL of the filtrate, add 0.05 mol/L sodium hydroxide TS to make exactly 100 mL, and use this solution as the test solution. Proceed as directed in the Assay.

Fursultiamine Hydrochloride contains not less than 98.5% and not more than 101.0% of fursultiamine hydrochloride (C17H26N4O3S2HCl), calculated on the anhydroys basis.
Description Fursultiamine Hydrochloride is a white crystal or crystalline powder, is odorless or has a characteristic odor and has a bitter taste. Fursultiamine Hydrochloride is freely soluble in water, in methanol or in ethanol and practically insoluble in ether. Identification (1) Dissolve 5 mg of Fursultiamine Hydrochloride in 6 mL of 0.1 mol/L hydrochloric acid TS, add 0.1 g of zinc dust, allow to stand for several minutes and filter. To 3 mL of the filtrate, add 3 mL of sodium hydroxide TS and 0.5 mL of potassium ferricyanide TS, then add 5 mL of isobutanol, shake vigorously for 2 minutes, allow to stand to separate the isobutanol layer and examine under ultraviolet light (main wavelength: 365 nm): the isobutanol layer shows a blue-purple fluorescence. The fluorescence disappears by acidifying and appears again by alkalifying.

Amount (mg) of furosemide (C12H11ClN2O5S) = amount (mg) of Furosemide RS AT V

AS

50

Assay Weigh accurately the mass of not less than 20 Furosemide Tablets, and powder. Weigh accurately a por-

KP 9 451 (2) Determine the infrared absorption spectra of Fursultiamine Hydrochloride and Fursultiamine Hydrochloride RS, previously dried in a desiccator (in vacuum, P2O5) for 24 hours, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any differences appear, dissolve the Fursultiamine Hydrochloride in water, evaporate the water and dry the residue in a desiccator (in vacuum, P2O5) for 24 hours and repeat the test. (3) A solution of Fursultiamine Hydrochloride (1 in 50) responds to the Qualitative Tests (2) for chloride.
Purity (1) Clarity and color of solutionDissolve 1.0 g of Fursultiamine Hydrochloride in 20 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 1.5 g of Fursultiamine Hydrochloride. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS: not more than 0.011%. (3) Heavy metalsProceed with 1.0 g of Fursultiamine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Related substancesDissolve 0.10 g of Fursultiamine Hydrochloride in 100 mL of the mobile phase and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add the mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography. Determine peak area of each solution by the automatic integration method: the total area of the peaks other than the peak of fursultiamine from the test solution is not larger than the peak area of fursultiamine from the standard solution.

standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS, of the peak area of fursultiamine to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of fursultiamine hydrochloride (C17H26N4O3S2HCl) = amount (mg) of Fursultiamine Hydrochloride RS, calculated QT on the anhydrous basis Q S Internal standard solutionA solution of isopropyl p-aminobenzoate in ethanol (3 in 400) Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 50 C. Mobile phase: Dissolve 1.01 g of sodium 1-heptane sulfonate in 1000 mL of diluted glacial acetic acid (1 in 100). Take 675 mL of this solution, add 325 mL of a mixture of methanol and acetonitrile (3 : 2). Flow rate: Adjust the flow rate so that the retention time of fursultiamine is about 9 minutes. Selection of the column: Proceed with 10 L of the standard solution under the above operating conditions and calculate the resolution. Use a column giving elution of fursultiamine and the internal standard in this order with the resolution between their peaks being not less than 10.
Packaging and Storage Preserve in tight containers.

Operating conditions Detector, column, column temperature, mobile phase, flow rate and selection of column: Proceed as directed in the operating conditions under the Assay. Detector sensitivity: Adjust the detection sensitivity so that the peak height of fursultiamine from 10 L of the standard solution is between 20 mm and 30 mm. Time span of measurement: About 3 times as long as the retention time of fursultiamine.
Water Not more than 5.0% (0.3 g, volume titration, direct titration). Residue on Ignition Not more than 0.10% (1 g). Assay Weigh accurately about 55 mg each of Fursultiamine Hydrochloride and Fursultiamine Hydrochloride RS, separately determined for water, dissolve each in 50 mL of water, add exactly 10 mL each of the internal standard solution and then add water to make exactly 100 mL. Take 8 mL each of the solutions, add water to make 50 mL and use these solutions as the test solution and the

Gabexate Mesilate
NH H 2N C NH(CH 2)5 O C O O C OCH 2CH 3 CH 3SO 3H

C16H23N3O4CH4O3S: 417.48 Gabexate Mesilate, when dried, contains not less than 98.5% and not more than 101.0% of gabexate mesilate (C16H23N3O4CH4O3S).
Description Gabexate Mesilate is a white crystal or crystalline powder. Gabexate Mesilate is very soluble in water, freely soluble in ethanol and practically insoluble in ether.

452 Monographs, Part I

Identification (1) Take 4 mL of a solution of Gabexate Mesilate (1 in 2000), add 2 mL of -naphthol TS and 1 mL of diacetyl TS and allow to stand for 10 minutes: a red color is observed. (2) Dissolve 1 g of Gabexate Mesilate in 5 mL of water, add 2 mL of sodium hydroxide TS and heat on a water-bath for 5 minutes. After cooling, add 2 mL of dilute nitric acid and 5 mL of ethanol, shake, add 5 drops of ferric chloride TS and shake: a purple color is observed. (3) Determine the absorption spectra of solutions of Gabexate Mesilate and Gabexate Mesilate RS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Take 0.1 g of Gabexate Mesilate, add 0.2 g of sodium hydroxide, fuse by heating gently and continue the heating for 20 to 30 seconds. After cooling, add 0.5 mL of water and 3 mL of dilute hydrochloric acid and warm: the gas evolved changes a potassium iodate-starch paper to blue. Melting Point Between 90 C and 93 C pH Dissolve 1.0 g of Gabexate Mesilate in 10 mL of water: the pH of this solution is between 4.5 and 5.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Gabexate Mesilate in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Gabexate Mesilate according to Method 1 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) ArsenicDissolve 2.0 g of Gabexate Mesilate in 20 mL of 1 mol/L hydrochloric acid by heating on a water-bath and continue the heating for 20 minutes. After cooling, centrifuge and use 10 mL of the supernatant liquid as the test solution. Perform the test (not more than 2 ppm). (4) Ethyl parahydroxybenzoateWeigh 50 mg of Gabexate Mesilate, previously dried and dissolve in dilute ethanol to make exactly 100 mL. Pipet 5.0 mL of this solution, add exactly 5 mL of the internal standard solution and use this solution as the test solution. Separately, dissolve 5.0 mg of ethyl parahydroxybenzoate in dilute ethanol to make exactly 100 mL. Pipet 1.0 mL of this solution and add dilute ethanol to make exactly 20 mL. Take exactly 5 mL of this solution, add exactly 5 mL of the internal standard solution and use this solution as the standard solution. Perform the test with 3 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the operating conditions in the Assay and calculate the ratios, QT and QS , of the peak area of ethyl parahydroxybenzoate to that of the internal standard, respectively: QT is not larger than QS .

hydroxybenzoate in diluted ethanol (1 in 5000). (5) Related substancesDissolve 0.20 g of Gabexate Mesilate in 5 mL of ethanol and use this solution as the test solution. Pipet 1.0 mL of the test solution, add ethanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for Thinlayer chromatography. Develop the plate with a mixture of ethyl acetate, water and glacial acetic acid (3 : 1 : 1) to a distance of about 10 cm and air-dry the plate until it has no acetic odor. Spray evenly a solution of 8-oxyquinoline in acetone (1 in 1000) on the plate and after air-drying, spray evenly bromine-sodium hydroxide TS: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.3% (1 g, in vacuum, silica gel, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 50 mg of Gabexate Mesilate and Gabexate Mesilate RS, previously dried and dissolve each in dilute ethanol to make exactly 100 mL. Pipet 5.0 mL each of these solutions, add exactly 5 mL each of the internal standard solution and use these solutions as the test solution and the standard solution, respectively. Perform the test with 3 L each of the test solution and the standard solution as directed under the Thin-layer chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of gabexate to that of the internal standard, for the test solution and the standard solution, respectively.

Amount (mg) of gabexate mesilate (C16H23N3O4.CH4O3S) = amount (mg) of Gabexate Mesilate RS

QT QS

Internal standard solutionA solution of butyl parahydroxybenzoate in diluted ethanol (1 in 5000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 245 nm) Column: A stainless steel column, about 5 mm in inside diameter and about 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 o C . Mobile phase: A mixture of methanol, a solution of sodium lauryl sulfate (1 in 1000), a solution of sodium 1heptane sulfonate (1 in 200) and glacial acetic acid (540 : 200 : 20 : 1).

Internal standard solutionA solution of butyl para-

KP 9 453 Flow rate: Adjust the flow rate so that the retention time of Gabexate is about 13 minutes. System suitability System performance: When the procedure is run with 3 L of the standard solution, as directed under the above operating condition, the internal standard and Gabexate are eluted in this order with the resolution between their peaks being not less than 5. System repeatability: When the test is repeated 6 times with 3 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the ratios of the peak area of Gabexate to that of the internal standard is not more than 1.0%
Packaging and Storage Preserve in tight containers.

Gallamine Triethiodide (1 in 100) is colorless and clear. (2) Heavy metalsProceed with 1.0 g of Gallamine Triethiodide according to Method 1, and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm).
Loss on Drying Not more than 1.5% (1 g, 100 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 25 mg each of Gallamine Triethiodide and Gallamine Triethiodide RS, dissolve in the mobile phase to make exactly 25 mL each and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and determine the area of the peak of gallamine triethiodide in the test solution, AT , and in the standard solution, AS , respectively.

Gallamine Triethiodide
CH3 CH3 N O O N CH3 O N CH3 CH3 CH3 CH3 CH3

Amount (mg) of gallamine triethiodide (C30H60I3N3O3) = 25 C

I

AT AS

C: Concentration (mg/mL) of gallamine triethiodide in the standard solution.

I

CH3

C30H60I3N3O3 : 891.53 Gallamine Triethiodide contains not less than 98% and not more than 101.0% of gallamine triethiodide (C30H60I3N3O3), calculated on the dried basis.
Description Gallamine Triethiodide is a white, amorphous power and is odorless. Gallamine Triethiodide is very soluble in water, sparingly soluble in ethanol and very slightly soluble in chloroform. Gallamine Triethiodide is hygroscopic. Identification (1) Determine the infrared spectra of Gallamine Triethiodide and Gallamine Triethiodide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) A solution of Gallamine Triethiodide (1 in 100) responds to the Qualitative Tests for iodide. pH Dissolve 1 g of Gallamine Triethiodide in 50 mL of water: the pH of this solution is between 5.3 and 7.0. Purity (1) Clarity and color of solutionA solution of

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 200 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3-10 m in particle diameter). Mobile phase: A mixture of sodium perchlorate buffer and acetonitrile (69:31). Flow rate: 1.0 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the number of theoretical plates is not less than 5000 with the symmetry factor being not more than 1.4. System repeatability: When the test is repeated 5 times with 10 L each of the standard solution under the above operating conditions, the relative standard deviation of the areas of the peak of gallamine triethiodide is not more than 2.0%. Sodium perchlorate bufferDissolve sodium perchlorate in water to render the concentration of 0.14 mol/L and adjust the pH to 3.0 by addition of 10 mol/L sodium hydroxide TS or of 0.05 mol phosphoric acid.
Packaging and Storage tight containers.

Preserve in light-resistant,

454 Monographs, Part I graphy. Spot 50 L of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with the developing solvent which is a mixture of chloroform, ammonia solution (28) and methanol (2:1:1), and air-dry the plate. Then allow the plate to stand in iodine chamber containing 2 ~ 4 g of iodine for 3 ~ 5 minutes. The spots obtained from the test solution are the same with the corresponding spots from the standard solution in color tone and the Rf value, respectively.
Specific Optical Rotation [ ] 20 +107 ~ +121 D : (0.25 g calculated on the dried basis, water, 25 mL, 200 mm) pH The pH of a solution obtained by dissolving 0.4 g of Gentamicin Sulfate in 10 mL of water is between 3.5 and 5.5. Sterility Test It meets the requirement, when Gentamicin Sulfate is used in a sterile preparation.
O NH2 OH HN CH3 OH O CH3 HO xH2SO4 NH2

Gallium Citrate ( Ga) Injection

Gallium (67Ga) Citrate Injection is an aqueous solution for injection containing Gallium (67Ga) Citrate Injection. It conforms to the requirements of Gallium (67Ga) Citrate Injection specified in the Korean Pharmaceutical Codex(Third Edition, 2007). The Insoluble Particulate Matter Test for Injections is not applied to this injection.
Description Gallium (67Ga) Citrate Injection is a clear, colorless or light red liquid.

67

Gentamicin Sulfate
R1 HN R2 R3 O

Bacterial Endotoxins Less than 0.50 EU per mg of gentamicin, when Gentamicin Sulfate is used in a sterile preparation. Loss on Drying Not more than 18.0 % (0.151 g, reduced pressure not exceeding 0.67 kPa, 110 C, 3 hours).

O NH2

Gentamicin C1: R1 = CH3 Gentamicin C1a: R1 = H Gentamicin C2: R1 = H Gentamicin C2a: R1 = H Gentamicin C2b: R1 = CH3

R2 = CH3 R2 = H R2 = CH3 R2 = H R2 = H

R3 = H R3 = H R3 = H R3 = CH3 R3 = H

Gentamicin Sulfate is the sulfate of a mixture of aminoglycoside substances having antibacterial activity produced by the growth of Micromonospora purpurea or Micromonospora echinospora. Gentamicin Sulfate contains not less than 590 g (potency) per mg of gentamicin C1 (C21H43N5O7: 477.60), calculated on the dried basis
Description Gentamicin Sulfate is a white to light yellowish white powder. Gentamicin Sulfate is very soluble in water, and practically insoluble in ethanol or in ether. Identification (1) Dissolve 50 mg of Gentamicin Sulfate in 1 mL of water, and add 2 drops of the solution of 1-naphthol in ethanol(1 in 500). Gently superimpose the solution on 1 mL of sulfuric acid: a purple-blue color develops at the zone of contact. (2) Dissolve 0.1 g each of Gentamicin Sulfate and Gentamicin Sulfate RS in 100 mL of 0.1 mol/L phosphate buffer (pH 8.0), and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under Thin-layer Chromato-

Content Ratio of Gentamicins Weigh accurately an amount of Gentamicin Sulfate and Gentamicin Sulfate RS, dissolve in water and make a solution so that each mL contains 0.65 mg (potency). To 10 mL of each of these solutions add 5 mL of isopropanol and 4 mL of the O-Phthalaldehyde solution, mix, and add isopropanol to obtain 25 mL of solutions. Heat at 60 C in water bath for 15 minutes, and cool. The resulting solutions are used as the test solution and the standard solution. Perform the test with each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and obtain the peak areas, Af of gentamicins (gentamicin C1: 25 50 %, gentamicin C1a : 10 - 35 %, gentamicin C2 and C2a : 25 55 %).

Content ratio of gentamicin C1, C1a, C2 and C2a (%) Af = 100 AS Af : the peak area response corresponding to the particular gentamicin AS : the sum of the area responses of all four peaks
Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 330 nm) Column: A stainless steel column, about 5 mm in inside diameter and about 100 mm in length, packed with

KP 9 455 octadecylsilanized silica gel or fine ceramic particle for liquid chromatography(5 ~ 10 m in particle diameter) Mobile phase: Mix 700 mL of methanol, 250 mL of water and 50 mL of glacial acetic acid. Dissolve 5 g of sodium 1-heptanesulfonate in this solution. Flow rate: about 1.5 mL per minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, the capacity factor determined from the gentamicin C1 peak is between 2 and 7, the column efficiency determined from the gentamicin C2 peak is mot less than 1200 theoretical plates, and the resolution, R, between any two peaks is not less than 1.25. The elution order is gentanicin C1, gentanicin C1a, gentamicin C2a, and gentamicin C2. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the operating conditions, the relative standard deviation of any peak area is not more than 2.0 %.
Assay The Cylinder-plate method (1) Agar media for seed and base layerPeptone 6.0 g Glucose 2.0 g Yeast extract 3.0 g Sodium chloride 10.0 g Meat extract 1.5 g Agar 13.0 20.0 g Water 1000 mL Mix all the ingredients, and sterilize. Adjust the pH of the solution so that it will be 7.8 to 8.0 after sterilization. (2) Agar medium for transferring test organisms- Use 2 under Microbial Assay for the medium in I 2 1) (2) Antibiotics. (3) Test organism- Staphylococcus epidermidis ATCC 12228 (4) Weigh accurately an amount of Gentamicin Sulfate, equivalent to about 25 mg (potency) and dissolve in 0.1 mol/L phosphate buffer solution, pH 8.0 to make the solution containing 1 mg gentamicin per mL (potency) and use the solution as the test stock solution. Take exactly a suitable amount of the test stock solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 4 g (potency) and 1 g (potency), and use these solutions as the high concentration test solution and low concentration test solution, respectively. Separately, weigh accurately an amount of Gentamicin Sulfate RS, equivalent to about 25 mg (potency) and dissolve in 0.1 mol/L phosphate buffer solution, pH 8.0 to make the solution containing 1 mg gentamicin per mL (potency) and use the solution as the standard stock solution. Take exactly a suitable amount of the standard stock solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 4 g (potency) and 1 g (potency), and use these solutions as the high concentration standard solution and low concentration standard solution, respectively. Perform the test with these solutions according to the Cylinder-plate method as directed under Microbial Assay for Antibiotics.

Packaging and Storage Preserve in tight containers.

Glibenclamide
Cl O C NHCH2CH2 O S O OCH3 H N O C H N

C23H28ClN3O5S: 494.00 Glibenclamide, when dried, contains not less than 98.5% and not more than 101.0% of glibenclamide (C23H28ClN3O5S).
Description Glibenclamide is a white to pale yellowish white crystalline powder. Glibenclamide is freely soluble in dimethylformamide, sparingly soluble in chloroform, slightly soluble in methanol or in ethanol and practically insoluble in water or in ether. Identification (1) Determine the absorption spectra of solutions of Glinbenclamide and Glibenclamide RS in methanol (1 in 10000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared absorption spectra of Glibenclamide and Glibenclamide RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Perform the test with Glibenclamide as directed under the Flame Coloration Test (2): a green color is observed. Melting Point Between 168 C and 173 C. Purity (1) Heavy metalsProceed with 1.0 g of Glibenclamide according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 0.20 g of Glibenclamide in 20 mL of chloroform and use this solution as the test solution. Pipet 1.0 mL of the test solution and add chloroform to make exactly 50 mL. Pipet 2.5 mL of this solution, add chloroform to make exactly 10 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-

456 Monographs, Part I layer chromatoghraphy. Develop the plate with a mixture of n-propanol, chloroform and diluted ammonia TS (4 in 5) (11 : 7 : 2) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Assay Weigh accurately about 0.9 g of Glibenclamide, previously dried, dissolve in 50 mL of dimethylformamide and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Perform a black determination with a solution prepared by adding 18 mL of water to 50 mL of dimethylformamide and make any necessary correction.

Each mL of 0.1 mol/L sodium hydroxide VS = 49.40 mg of C23H28ClN3O5S

Packaging and Storage Preserve in tight containers.

Gliclazide
O N N H H3C N H

O S

C15H21N3O3S: 323.41 Gliclazide contains not less than 99.0% and not more than 101.0% of gliclazide (C15H21N3O3S), calculated on the dried basis.
Description Gliclazide is a white power. Gliclazide is freely soluble in dichloromethane, sparingly soluble in acetone, slightly soluble in alcohol and practically insoluble in water. Identification Determine the infrared spectra of Gliclazide and Gliclazide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Heavy metalsProceed with 1.5 g of Gliclazide according to Method 2, and perform the test. Prepare the control solution with 1.5 mL of standard lead solution (not more than 10 ppm). (2) Related substance IWeigh accurately 0.4 g of Gliclazide, dissolve in 2.5 mL of dimethylsulfoxide, add

water to make exactly 10 mL, mix by shaking for 10 minutes, allow the solution to stand at 4 C for 30 minutes, filter and use the filtrate as the test solution. Weigh 20.0 mg of gliclazide related substance I RS [2-nitrosooctahydrocyclopenta[c]pyrrole] and add dimethylsulfoxide to make exactly 100 mL. To 1.0 mL of this solution, add 12 mL of dimethylsulfoxide and water to make exactly 50 mL, and use this solution as the standard solution (1). To 1.0 mL of the standard solution (1), add 12 mL of dimethylsulfoxide and water to make exactly 50 mL, and use this solution as the standard solution (2). Perform the test with 50 L each of the test solution and the standard solution (2) as directed under the Liquid Chromatography according to the operating conditions directed in the Related substance under the Purity: the area of the related substance I peak obtained from the test solution is not greater than that from the standard solution (2) (2 ppm). (3) Related substancesWeigh accurately 50 mg of Gliclazide, dissolve in 23 mL of acetonitrile, add water to make exactly 50 mL, and use this solution as the test solution. To 1.0 mL of the test solution and add a mixture of water and acetonitrile (55 : 45) to make exactly 100 mL. Pipet 10.0 mL of the solution, add a mixture of water and acetonitrile (55 : 45) to make exactly 100 mL, and use this solution as the standard solution (1). Dissolve 5 mg of Gliclazide and 15 mg of gliclazide related substance II RS [1-(hexahydrocyclopenta[c] pyrrol2(1H)-yl)-3-[(2-methylphenyl)sulfonyl]urea] in 23 mL of acetonitrile, add water to make 50 mL, pipet 1.0 mL of this solution, add a mixture of water and acetonitrile (55 : 45) to make exactly 20 mL and use this solution as the standard solution (2). Dissolve 10.0 mg of gliclazide related substance II RS in 45 mL of acetonitrile and add water to make exactly 100 mL, pipet 1.0 mL of this solution, add a mixture of water and acetonitrile (55 : 45) to make exactly 100 mL and use this solution as the standard solution (3). Perform the test with 20 L each of the test solution, the standard solution (1) and (3) as directed under the Liquid Chromatography according to the following operating conditions and determine the area of peaks from the these solutions: the peak area corresponding to the related substance II obtained from the test solution is not greater than the area of the principal peak obtained from the standard solution (3) (0.1%), the area of any peak other than the principal peak or the related substance II peak from the test solution is not greater than the area of the principal peak from the standard solution (1) (0.1%), and total area of these other peaks from the test solution is not greater than twice the area of the principal peak from the standard solution (1) (0.2%). Disregard any peak having the area not more than 0.2 times the area of the principal peak from the standard solution (1). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 235 nm). Column: A stainless steel column about 4 mm in in-

KP 9 457 side diameter and about 25 cm in length, packed with octylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of water, acetonitrile, trifluoroacetic acid and triethylamine (55:45:0.1:0.1). Flow rate: 0.9 mL/minute. System suitability System performance: Proceed with 20 L of the standard solution (2) under the above operating conditions and adjust the sensitivity of the system so that the heights of the two principal peaks obtained from the standard solution (2) are not less than 50 % of the full scale of the recorder. Under this operating condition, the resolution between the two principal peaks is not less than 1.8.
Loss on Drying Not more than 0.25% (1 g, 105 C, 2 hours). Residue on Ignition

RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) When proceed as directed in the Related substances under the Purity, the Rf value of the principal spot from the test solution corresponds that from the standard solution (1).
Melting Point

Between 176 C and 181 C.

Not more than 0.1% (1 g).

Assay Weigh accurately about 0.25 g of Gliclazide, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchlroic acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of perchloric acid VS = 32.34 mg of C15H21N3O3S

Packaging and Storage Preserve in well-closed containers.

Gliquidone
O S O NH O

MeO
N O

NH

Me

Me

Purity (1) Heavy metalsProceed with 1.0 g of Gliquidone according to Method 2, and perform the test. Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesDissolve 1.0 g each of Gliquidone and Gliquidone RS in 10 mL each of a mixture of methanol and dichloromethane (1 : 1) and use these solutions as the test solution and the standard solution (1), respectively. Dilute the standard solution (1) with a mixture of methanol and dichloromethane (1 : 1) to render the concentration of 0.0030w/v% and use this solution as the standard solution (2). Separately, dissolve 3.0 mg of gliquidone sulfonamide RS in 100 mL of a mixture of methanol and dichloromethane (1 : 1) and use this solution as the standard solution (3). Mix equal volumes of the standard solution (2) and (3), and use the mixture as the standard solution (4). Perform the test with the test solution and the standard solutions as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution, the standard solutions (1), (2), (3) and (4) on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of cyclohexane, chloroform, ethanol and glacial acetic acid (45 : 45 : 5 : 5) to a distance of about 10 cm and air-dry the plate. Examine the plate under ultraviolet light: the spot corresponding to gliquidone sulfonamide from the test solution is not more intense than that from the standard solution (3) (0.3%) and any other secondary spot from the test solution is not more intense than that from the standard solution (2) (0.3%). The test is valid when two principal spots are clearly separated with each other in the chromatogram obtained from the standard solution (4). Loss on Drying Not more than 1.0% (1 g, 105 C, constant mass). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately 0.3 g of Gliquidone, previously dried, dissolve in 70 mL of dimethylformamide and titrate immediately with 0.1 mol/L of tetrabutyl ammonium hydroxide VS (indicator: 0.05 mL of 0.3w/v% thymol blue in methanol) until the color of the solution changes to blue. Perform a blank determination and make any necessany correction.

C27H33N3O6S: 527.63 Gliquidone contains, when dried, not less than 98.5% and not more than 101.5% of gliquidone (C27H33N3O6S).
Description Gliquidone is a white powder. Gliquidone is freely soluble in dimethylformamide, soluble in acetone, slightly soluble in ethanol or in methanol, and practically insoluble in water. Identification (1) Dissolve 30 mg of Gliquidone in methanol, evaporate the solvent using a rotary evaporator and dry the residue at 50 C under vacuum. Determine the infrared spectra of the dried residue and Gliquidone

Each ml of 0.1 mol/L tetrabutyl ammonium hydroxide

458 Monographs, Part I VS = 52.76 mg of C27H33N3O6S.

Packaging and Storage Preserve in tight containers.

KP 9 459 ethanol and boil under a reflux condenser: the solution is clear. (8) Soluble starch and sulfiteDissolve 1.0 g of Glucose in 10 mL of water and add 1 drop of iodine TS: a yellow color is observed.
Loss on Drying Not more than 1.0% (1 g, 105 C, 6 hours).

Glucose
CH2OH H HO H OH O H OH H OH H

D-Glucopyranose

C6H12O6: 180.16

Residue on Ignition Not more than 0.1% (2 g). Assay Weigh accurately about 10 g of Glucose, previously dried, dissolve in 0.2 mL of ammonia TS and water to make exactly 100 mL, allow to stand for 30 minutes and determine the optical rotation, D, of this solution at 20 1 C in a 100-mm cell as directed under the Optical Rotation Determination.

Glucose is -D-glucopyranose, -D-glucopyranose or a mixture of them. Glucose, when dried, contains not less than 99.5% and not more than 101.0% of glucose [D-glucopyranose (C6H12O6)].
Description Glucose is a white crystals or crystalline powder, is odorless and has a sweet taste. Glucose is freely soluble in water, soluble in hot ethanol, slightly soluble in ethanol and practically insoluble in ether. Identification Add 2 to 3 drops of a solution of Glucose (1 in 20) to 5 mL of boiling Fehling's TS: a red precipitate is produced. Purity (1) Clarity and color of solutionAdd 25.0 g of Glucose to 30 mL of water in a Nessler tube, warm at 60 C in a water-bath until solution is obtained, cool and add water to make 50 mL: the solution is clear and has no more color than the following control solution.

Amount (mg) of C6H12O6 = D 1895.4

Packaging and Storage Preserve in tight containers.

Glucose Injection
Glucose Injection is an aqueous solution for injection. Glucose Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of glucose (C6H12O6: 180.16).
Method of Preparation Prepare as directed under Injections, with Glucose. No preservative is added. Description Glucose Injection is a clear, colorless liquid. Glucose has a sweet taste. It occurs as a colorless to pale yellow, clear liquid when its labeled concentration exceeds 40%. Identification Measure a volume of Glucose Injection, equivalent to 0.1 g of glucose according to the labeled amount and, if necessary, add water or evaporate on a water-bath to make of 2 mL. Add 2 to 3 drops of this solution to 5 mL of boiling Fehling's TS: a red precipitate is produced. pH Between 3.5 and 6.5. In the case where the labeled concentration of the injection exceeds 5%, dilute to 5% with water before the test. Purity 5-Hydroxymethylfurfural and related substancesMeasure exactly a volume of Glucose Injection, equivalent to 2.5 g of glucose according to the labeled amount and add water to make exactly 100 mL. Determine the absorbance of this solution at 284 nm as directed under the Ultraviolet-visible Spectrophotometry: it is not more than 0.80.

Control solutionTake a mixture of 1.0 mL of cobaltous chloride stock CS, 3.0 mL of ferric chloride stock CS and 2.0 mL of cupric sulfate stock CS, add water to make 10.0 mL. Pipet 3.0 mL of this solution and add water to make 50 mL. (2) AcidDissolve 5.0 g of Glucose in 50 mL of freshly boiled and cooled water and add 3 drops of phenolphthalein TS and 0.60 mL of 0.01 mol/L sodium hydroxide VS: a red color is observed. (3) ChloridePerform the test with 2.0 g of Glucose. Prepare the control solution with 1.0 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.018%). (4) SulfatePerform the test with 2.0 g of Glucose. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (5) Heavy metalsProceed with 5.0 g of Glucose according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 4 ppm). (6) ArsenicDissolve 1.5 g of Glucose in 5 mL of water, add 5 mL of dilute sulfuric acid and 1 mL of bromine TS, heat in a water-bath for 5 minutes and concentrate to 5 mL. After cooling, perform the test with this solution as the test solution (not more than 1.3 ppm). (7) DextrinTake 1.0 g of Glucose, add 20 mL of

460 Monographs, Part I

Sterility Test It meets the requirement. Baterial Endotoxins Glucose Injection. pH Dissolve 1 g of Glutamine in 50 mL of water: the pH of this solution is between 4.0 and 6.0. Purity (1) ChlorideProceed with 0.35 g of Glutamine and perform the test. Use 0.50 mL of 0.01 mol/L hydrochloric acid VS as the control solution (not more than 0.05%). (2) SulfateProceed with 0.2 g of Glutamine and perform the test. Use 0.25 mL of 0.005 mol/L sulfuric acid VS as the control solution (not more than 0.03%). (3) IronDissolve 0.333 g of Glutamine in water to make 45 mL, add 2 mL of hydrochloric acid as the test solution. To 1 mL of standard iron solution, add water to make 45 mL, add 2 mL of hydrochloric acid and use this solution as the standard solution. Add 50 mg each of ammonium peroxydisulfate crystal and 3 mL of 30% ammonium thiocyanate solution to the test solution and the standard solution: the color obtained from the test solution is not darker than that from the control solution (30 ppm). (4) Heavy metalsProceed with 1.0 g of Glutamine according to Method 1, and perform the test. Prepare the control solution with 1.5 mL of standard lead solution (not more than 15 ppm). (5) Related substancesDissolve 0.10 g of Glutamine in 10 mL of water and use this solution as the test solution. Dissolve 50 mg of Glutamine RS in water to make exactly 100 mL, pipet 5.0 mL of this solution, add water to make exactly 50 mL and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and standard solution on a plate of silica gel for thin-layer chromatography. Develop the ploate with a mixture of n-butanol, water and glacial acetic acid (3:1:1) to a distance of about 10 cm, and dry the plate at 80 C for 30 minutes. Spray the plate evenly with a solution, prepared by dissolving 0.2 g of ninhydrin in 100 mL of a mixture of n-butanol and 2 mol/L acetic acid (95:5), and heat the plate at 80 C for 10 minutes: the spots other than the principal spot obtained from the test solution is not more intense than that from the standard solution. Loss on Drying Not more than 0.3% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.3% (1 g). Assay Weigh accurately about 0.15 g of Glutamine, add 3 mL of formic acid and 50 mL of glacial acetic acid to dissolve and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Less than 0.50 EU per mL of

Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Measure accurately a volume of Glucose Injection, equivalent to about 4 g of glucose (C6H12O6) and add 0.2 mL of ammonia TS and water to make exactly 100 mL. Shake the solution well, allow to stand for 30 minutes and determine the optical rotation, D, at 20 1 C in a 100-mm cell as directed under the optical Rotation Determination.

Amount (mg) of glucose (C6H12O6) = D 1895.4

Packaging and Storage Preserve in hermetic containers. Plastic containers for aqueous injections may be used.

Glutamine
O O

H2N H NH2

OH

L-glutamine

C5H10N2O3: 146.15

Glutamine contains not less than 98.5% and not more than 101.5% of L-glutamine (C5H10N2O3), calculated on the dried basis.
Description Glutamine is a white crystal or crystalline powder, is odorless and has a faint, characteristic taste. Glutamine is soluble in water and practically insoluble in ethanol or in ether. Identification Determine the infrared spectra of Glutamine and Glutamine RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between +6.3 and + 7.3 (2 g, after drying, water, 50 mL, 100 mm).

Each mL of 0.1 mol/L perchloric acid = 14.615 mg of C5H10N2O3

Packaging and Storage Preserve in well-closed con-

KP 9 461 tainers. tion has no more color than the following control solution. Control solutionPipet 0.40 mL of ferric chloride colorimetric stock solution into a Nessler tube and add water to make 50 mL. (2) Acidity or alkalinityTo 2 mL of Concentrated Glycerin, add 8 mL of water and mix: the solution is neutral. (3) ChlorideTake 10.0 g of Concentrated Glycerin and perform the test. Prepare the control solution with 0.30 mL of 10 mmol/L hydrochloric acid VS (not more than 0.001%). (4) SulfateTake 10.0 g of Concentrated Glycerin and perform the test. Prepare the control solution with 0.40 mL of 5 mmol/L sulfuric acid VS (not more than 0.002%). (5) AmmoniumTake 5 mL of Concentrated Glycerin, add 5 mL of a solution of sodium hydroxide (1 in 10) and boil: the gas evolved does not change moistened red litmus paper to blue. (6) Heavy metalsProceed with 5.0 g of Concentrated Glycerin according to Method 1 and perform the test. Prepare the control solution with 2.5 mL of standard lead solution (not more than 5 ppm). (7) CalciumTake 5 mL of the solution obtained in (2), add 3 drops of ammonium oxalate TS: the solution remains unchanged. (8) ArsenicPrepare the test solution with 1.0 g of Concentrated Glycerin according to Method 1 and perform the test (not more than 2 ppm). (9) Acrolein, glucose or other reducing substancesTake 1.0 g of Concentrated Glycerin, add 1 mL of ammonia TS, mix and warm in a water-bath at 60 for 5 minutes: no yellow color is produced. Take the solution out of the water-bath, add 3 drops of silver nitrate TS immediately and allow to stand in a dark place for 5 minutes: the color of the solution does not change and no turbidity is observed. (10) Diethylene glycol and related substances Weigh accurately about 5 g of Concentrated Glycerin, add water to make 100 mL and use this solution as the test solution. Separately, dissolve suitable amount of Diethylene Glycol RS in water to render the concentration of 0.05 mg in 1 mL and use this solution as the standard solution. Perform the test with exactly 0.5 L of the test and standard solution by the following condition as directed under the Gas Chromatography. Determine the peak areas diethylene glycol from the test solution, AT , and from the standard solution, AS . Amount of diethylene glycol, as calculated by the following equation, is not more than 0.1%: Amount (%) of diethylene glycol (C4H10O3)
=
C S AT 100 CT AS

Glycerin
Glycerol C3H8O3: 92.09

Glycerin contains not less than 84.0% and not more than 87.0% of glycerin (C3H8O3) (by specific gravity).
Description Glycerin is a clear, colorless, viscous liquid, is odorless and has a sweet taste. Glycerin is miscible with water or with ethanol. Glycerin is very slightly soluble in ether. Glycerin is hygroscopic. Identification Proceed as directed in the Identification under Concentrated Glycerin. Refractive index Specific Gravity
20 nD : Between 1.449 and 1.454. 20 d 20 : Between 1.221 and 1.230.

Purity Proceed as directed in the Purity under Concentrated Glycerin. Residue on Ignition Proceed as directed in the Residue on ignition under Concentrated Glycerin. Packaging and Storage Preserve in tight containers.

Concentrated Glycerin
Concentrated Glycerol C3H8O3: 92.09

Concentrated Glycerin contains not less than 98.0% and not more than 101.0% of glycerin (C3H8O3) (by specific gravity).
Description Concentrated Glycerin is a clear, colorless and viscous liquid, is odorless and has a sweet taste. Concentrated Glycerin is miscible with water or with ethanol. Concentrated Glycerin is very slightly soluble in ether. Concentrated Glycerin is hygroscopic. Identification Heat 2 to 3 drops of Concentrated Glycerin with 0.5 g of potassium bisulfate: the odor of acrolein is perceptible. Refractive index Specific Gravity
20 : Not less than 1.470 nD 20 : Not less than 1.258. d 20

Purity (1) ColorPlace 50 mL of Concentrated Glycerin in a Nessler tube and observe downward: the solu-

CS : Concentration of diethylene glycol in the stan-

462 Monographs, Part I dard solution (mg/mL) CT : Concentration of diethylene glycol in the test solution (mg/mL) In addition, determine the peak area ( Ai ) of diethylene glycol or individual related substance, other than those detected from the residual solvent in the test solution, and the sum of all peak areas ( ATS ) in the test solution. Amounts of individual related substances other than diethylene glycol, as calculated by the following equation, are not more than 0.1% each and the sum of amount of related substances, including diethylene glycol, is not more than 1.0%. Amount (%) of individual related substance
=
Ai 100 ATS

drops of phenolphthalein TS). Perform a blank determination and make any necessary correction. (12) Readily carbonizable substancesTake 5 mL of Concentrated Glycerin, add carefully 5 mL of sulfuric acid, mix gently at a temperature between 18 and 20 and allow to stand for 1 hour at room temperature: the solution has no more color than Matching Fluid H.
Residue on Ignition Weigh accurately about 10 g of Concentrated Glycerin in a tared crucible, heat to boiling and fire to burn immediately. Cool, moisten the residue with 1 to 2 drops of sulfuric acid and ignite cautiously to constant weight: the weight of the residue is not more than 0.01% . Packaging and Storage Preserve in tight containers.

Ai : Peak area of diethylene glycol or individual related substance ( ATS ) other than those detected from the residual solvent in the test solution ATS : Total peak area in the test solution

Gramicidin
Gramicidin is the mixture of peptide substances having antibacterial activity produced by the growth of Bacillus brevis Dubos. Gramicidin contains not less than 900 g (potency) per mg of gramicidin, calculated on the dried basis.
Description Gramicidin occurs as a white to light yellowish white crystal or crystalline powder. Gramicidin is freely soluble in ethanol or in methanol, soluble in acetone or in dioxane, and practically insoluble in water. Identification (1) To 10 mg of Gramicidin add 2mL of 6 mol/L hydrochloric acid TS, and heat in a water bath for 30 minutes with occasional stirring. After cooling, neutralize with 6 mol/L sodium hydroxide TS, add 1 mL of ninhydrinacetic acid TS and 0.5 mL of pyridine, and heat for 2 minutes: a blue-purple to red-purple color develops. (2) Determine the absorption spectra of the solutions of Gramicidin and Gramicidin RS in methanol (1 in 25000), as directed under Ultraviolet-visible Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wavelengths. Melting Point Not less than 229 C (after drying) pH The pH of a solution obtained by dissolving 0.4 g of Gentamicin Sulfate in 10 mL of water is between 3.5 and 5.5. Loss on Drying Not more than 3.0 % (0.1 g, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 1.0 % (1 g).

Operating conditions Detector: A hydrogen flame ionization detector Column: Coat the inside wall of a quartz tube, 0.53 mm in inside diameter and 30 m in length, to 3.0 m thickness with 6% cyanopropylphenyl- and 94% dimethylpolysiloxan for gas chromatography Column temperature: Start at 100 , increase to 220 C at 7.5 C per minute and keep at 220 for 4 minutes Injection port temperature: A constant temperature of about 220 C Detector temperature: A constant temperature of about 250 C Carrier gas: Helium Flow rate: 38 cm/sec Split ratio: About 1 : 10 System suitability System performance: Take suitable amount of Glycerin RS and diethylene glycol and dissolve in water to render the concentration of 0.5 mg in 1 mL each. Perform the test with 0.5 L of the solution under the above operating conditions. The resolution between the peaks of glycerin and diethylene glycol is not less than 7.0. System repeatability: When the test is repeated 3 times with 0.5 L of the standard solution under the above operating conditions, the relative standard deviation of the peak of glycerin is not more than 15.0%. (11) Fatty acids and estersMix 50 g of Concentrated Glycerin with 50 mL of freshly boiled and cooled water, add 10 mL of 0.1 mol/L sodium hydroxide VS, accurately measured, boil the mixture for 15 minutes, cool and titrate the excess sodium hydroxide with 0.1 mol/L hydrochloric acid VS: not more than 3.0 mL of 0.1 mol/L sodium hydroxide VS is consumed (indicator: 3

KP 9 463
Assay The Turbidimetric method (1) Agar medium for transferring testorganismPeptone 5.0 g Potassium dihydrogen phosphate 2.0 g Yeast extract 20.0 g Polysorbate 80 0.1 g Glucose 10.0 g Agar 15.0 20.0 g Water 1000 mL Mix all the ingredients, and sterilize. Adjust the pH of the solution so that it will be 6.7 to 6.9 after sterilization. (2) Liquid medium for suspending test organismsUse the culture medium in III 1 2) under Microbial Assay for Antibiotics. (3) Test organism and preparation of the test organism suspension- Use Streptococcus faecium ATCC 10541 as test organism. Puncture the test organism in the agar medium for transferring test organism, incubate, and keep below 5 C. Transfering the organism is carried out every week. Transfer the organism so obtained in 10 mL of the liquid medium for suspending test organism, incubate at 37 C for 20 to 24 hours, and use this medium as the test organism suspension. Before use, mix one mL of this suspension and 100 mL of the liquid medium for suspending test organism, and use this as the test organism suspension. (4) Weigh accurately an amount of Gramicidin, dissolve, and dilute in ethanol(95 %) to make the test solution containing 0.0400 g (potency) per mL, and use this solution as the test solution. Separately, weigh accurately an amount of Gramicidin RS, dissolve in ethanol (95 %) to make the standard stock solution containing 1 mg (potency) per mL. Keep the standard solution at not exceeding 5 C and use within 30 days. Take exactly a suitable amount of the standard stock solution, dilute in ethanol (95 %) to make the standard solutions so that each mL contains 0.0320, 0.0350, 0.0400, 0.0448 and 0.0500 g (potency) respectively. To each of the test tube add 0.1 mL of the test solution and each of the standard solutions, and perform the test according to the Turbidimetric method (III 6) as directed under Microbial Assay for Antibiotics. Packaging and Storage Preserve in tight containers.

Griseofulvin contains not less than 900 g (potency) per mg of griseofulvin (C17H17ClO6), calculated on the dried basis.
Description Griseofulvin is a white crystal or crystalline powder. Griseofulvin is soluble in N,N-dimethylformamide, slightly soluble in methanol and in ethanol, very slightly soluble in ether, and practically insoluble in water. Identification Determine the absorption spectra of the solutions of Griseofulvin and Griseofulvin RS in ethanol (1 in 100000), as directed under Ultraviolet-visible Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wavelengths. Specific Optical Rotation [ ]D : +348 ~ +364(0.25 g calculated on the dried basis, N,N-dimethylformamide, 25 mL, 200 mm) Melting Point Between 217 and 224 C. Purity Heavy metalsProceed with 1.0 g of Griseofulvin according to Method 1 and perform the test. Prepare the control solution with 2.5 mL of standard lead solution (not more than 25 ppm). Loss on Drying Not more than 1.0 % (0.1 g, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 0.2 % (1 g). Assay Weigh accurately an amount of Griseofulvin and Griseofulvin RS, equivalent to about 25 mg (potency), dissolve each in 25 mL of methanol, add methanol to 5 mL of each solution to make exactly 50 mL and use these solutions as the test solution and the standard solution. Perform the test with each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and obtain the peak areas of griseofulvin, AT and AS.
25

Amount [g (potency)] of griseofulvin (C17H17ClO6) = Amount [g (potency)] of Griseofulvin RS

AT AS

Griseofulvin
Cl H3CO H3CO O O

O OCH3

CH3

C17H17ClO6HCl: 352.77 Griseofulvin is a substance having antifungal activity produced by the growth of Penicillium griseofulvum.

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 250 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 150 ~ 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography(5 ~ 10 m in particle diameter) Mobile phase: 60 % methanol Flow rate: Adjust the flow rate so that the retention time of griseofulvin is about 6 minutes.
Packaging and Storage Preserve in tight containers.

464 Monographs, Part I (5) Free guaiacolTake 1.0 g of Guaifenesin, add exactly 25 mL of water, dissolve by warming, cool and use this solution as the test solution. Separately, dissolve 0.10 g of guaiacol in water to make exactly 1000 mL. Pipet 3.0 mL of this solution, add exactly 22 mL of water and use this solution as the standard solution. To each of the test solution and the standard solution, add 1.0 mL of potassium ferricyanide TS and 5.0 mL of a solution of 4aminoantipyrine (1 in 200) and immediately after shaking for exactly 5 seconds, add a solution of sodium bicarbonate (1 in 1200) to make exactly 100 mL. Determine the absorbances of the test solution and the standard solution at 500 nm exactly 15 minutes after the addition of the 4-aminoantipyrin solution as directed under the Ultraviolet-visible Spectrophotometry, using a solution, prepared in the same manner with 25 mL of water, as the blank: the absorbance of the solution obtained from the test solution is not greater than that from the standard solution. (6) Related substancesDissolve 1.0 g of Guaifenesin in 100 mL of ethanol and use this solution as the test solution. Pipet 1.0 mL of the test solution, add water to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for Thinlayer chromatography. Develop the plate with a mixture of ether, ethanol and strong ammonia water (40 : 10 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly p-dimethylaminobenzaldehyde TS for spraying on the plate and heat at 110 o C for 10 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, in vacuum, 60 o C , 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 60 mg of Guaifenesin, previously dried, dissolve in water to make exactly 100 mL. Pipet 5.0 mL of the solution and add water to make exactly 100 mL. Determine the absorbance, A, of the solution at 273 nm as directed under the Ultraviolet-visible Spectrophotometry.

Guaifenesin
H OH OCH 2CCH 2OH OCH 3

and enantiomer Guaiacol Glyceryl Ether C10H14O4: 198.22

Guaifenesin, when dried, contains not less than 98.0% and not more than 102.0% of guaifenesin (C10H14O4).
Description Guaifenesin is a white crystal or crystalline powder. Guaifenesin is freely soluble in hot water or in ethanol, soluble in chloroform, sparingly soluble in water and slightly soluble in ether. A solution of ethanol (1 in 20) shows no optical rotation. Identification (1) Take 5 mL of Guaifenesin, add 1 mL of formalin-sulfuric acid TS: a red-purple color is observed. (2) Determine the absorption spectra of solutions of Guifenesin and Guaifenesin RS (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Determine the infrared absorption spectra of Guaifenesin and Guaifenesin RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers . Melting Point Between 80 and 83 . pH Dissolve 1.0 g of Guaifenesin in 100 mL of water: the pH of the solution is between 5.0 and 7.0. Purity (1) Clarity and color of solutionDissolve 0.20 g of Guaifenesin in 10 mL of water: the solution is clear and colorless. (2) ChlorideDissolve 0.7 g of Guaifenesin in 25 mL of water by warming. Cool, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.40 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.020%). (3) ArsenicPrepare the test solution with 1.0 g of Guaifenesin according to Method 3 and perform the test (not more than 2 ppm). (4) Heavy metalsDissolve 2.0 g of Guaifenesin in 25 mL of water by warming. Cool, add 2 mL of dilute acetic acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm).

Amount (mg) of guaifenesin (C10H14O4) A = 20000 117

Packaging and Storage Preserve in tight containers.

KP 9 465

Guanethidine Sulfate
NH N CH2CH2NHC NH2 H2SO4

Assay Weigh accurately about 0.5 g of Guanethidine Sulfate, previously dried, dissolve in 100 mL of formic acid and add 70 mL of a mixture of acetic anhydride and glacial acetic acid (6 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

C10H22N4H2SO4: 296.39 Guanethidine Sulfate, when dried, contains not less than 98.5% and not more than 101.0% of guanethidine sulfate (C10H22N4H2SO4).
Description Guanethidine Sulfate is a white crystal or crystalline powder, is odorless or has a slight, characteristic odor and a bitter taste. Guanethidine Sulfate is very soluble in formic acid, freely soluble in water and practically insoluble in ethanol or in ether. Melting pointBetween 251 C and 256 C (an evacuated sealed capillary tube, with decomposition). Identification (1) Take 4 mL of a solution of Guanethidine Sulfate (1 in 4000), add 2 mL of -naphthol TS, 1 mL of diacetyl TS and 15 mL of water and allow to stand for 30 minutes: a red color is observed. (2) Determine the infrared absorption spectra of Guanethidine Sulfate and Guanethidine Sulfate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers . (3) A solution of Guanethidine Sulfate (1 in 10) responds to the Qualitative Tests for sulfate. pH Dissolve 1.0 g of Guanethidine Sulfate in 50 mL of water: the pH of the solution is between 4.7 and 5.7. Purity (1) Clarity and color of solutionDissolve 1.0 g of Guanethidine Sulfate in 50 mL of water: the solution is clear and colorless. (2) Methylisothiourea sulfateDissolve 2.0 g of Guanethidine Sulfate in 80 mL of sodium hydroxide TS and allow to stand for 10 minutes. Add 60 mL of hydrochloric acid, 2 g of sodium bromide and water to make 200 mL. Then, to this solution, add 0.70 mL of 1/60 mol/L potassium bromate VS and 2 mL of zinc iodide-starch paste TS: a blue color is observed. (3) Heavy metalsProceed with 2.0 g of Guanethidine Sulfate according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition

Each mL of 0.1 mol/L perchloric acid VS = 29.639 mg of C10H22N4H2SO4

Packaging and Storage tight containers.

Preserve in light-resistant,

Haloperidol
OH F COCH 2 CH 2 CH 2 N Cl

C21H23ClFNO2: 375.86 Haloperidol, when dried, contains not less than 99.0% and not more than 101.0% of haloperidol (C21H23ClFNO2).
Description Haloperidol is a white to pale yellow crystal or powder. Haloperidol is freely soluble in glacial acetic acid, soluble in chloroform, sparingly soluble in methanol, slightly soluble in isopropanol or in dehydrated ethanol and practically insoluble in water. Identification (1) Dissolve 30 mg each of Haloperidol and Haloperidol RS in 100 mL of isopropanol. Add 10 mL of 0.1 mol/L hydrochloric acid TS and isopropanol in 5 mL of these solutions to make 100 mL. Determine the absorption spectra of these solutions as directed under Ultraviolet-visible absorption Spectrophotometry according to the operating conditions: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Haloperidol and Haloperidol RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry according to the operating conditions: both spectra exhibit similar intensities of absorption at the same wave numbers. Melting Point Between 149 C and 153 C. Purity (1) SulfateTake 1.0 g of Haloperidol, add 50 mL of water, shake and filter. Take 25 mL of the filtrate, add 1 mL of dilute hydrochloric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution with 0.50 mL of 5 mmol/L sulfuric acid VS (not more than 0.048%). (2) Heavy metalsProceed with 1.0 g of Haloperi-

Not more than 0.2% (1 g).

466 Monographs, Part I dol according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substanceDissolve 25 mg of Haloperidol in 50 mL of mobile phase, use this solution as the test solution. Pipet exactly 1 mL of this solution, add the mobile phase to make exactly 200 mL, use this solution as the standard solution. Pipet exactly 10 L each of test solution and standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of both solutions by the automatic integration method: the area of peak other than the area of Haloperidol from the test solution is not larger than 2 times the peak area of the Haloperidol from the standard solution. Peak areas of about 0.15, 1.2 and 2.6 in the relative retention time to Haloperidol are the values multiplied the area determined according to the automatic integration method by sensitivity factor, 0.75, 1.47 and 0.76, respectively. Operating conditions Detector: Ultraviolet-visible absorption Photometry (wavelength: 220 nm). Column: A stainless steel column, 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsylanized silica gel for Liquid Chromatography (5 m in particle diameter). Column temperature: A constant temperature at about 40 C. Mobile phase: Dissolve 2.95 g of sodium citrate in 900 mL of water, add dilute hydrochloric acid to adjust pH 3.3, add water to make 1000 mL. Add 700 mL of methanol in 300 mL of this solution, add 1 g of sodium lauryl sulfate, and dissolve. Flow rate: Adjust to about 9 minutes of the retention time of Haloperidol. System suitability Test for required detection: Weigh exactly 5 mL of standard solution, add the mobile phase to make 25 mL. The peak area of Haloperidol obtained from 10 L of this solution is between 15 and 25% of the peak area of Haloperidol obtained from the standard solution. System performance: Perform the test with 10 uL of the standard solution according to the above operating conditions: the theoretical plate numbers is not less than 4000 and the symmetric factor is not more than 2.0. System repeatability: Perform the test 6 times with 10 uL of the standard solution according to the above operating conditions: the relative standard deviation of Haloperidol peak area is not more than 2.0%. Time span of measurement: About 2 times as long as the retention time of Haloperidol after the solvent peak.
Loss on Drying Not more than 0.5% (1 g, in vacuum, 60 C, P2O5, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.6 g of Haloperidol, previously dried and dissolve in 40 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (indicator: 1 drop of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 37.587 mg of C21H23ClFNO2

Packaging and Storage tight containers.

Preserve in light-resistant,

Haloperidol Oral Solution

Haloperidol Oral Solution contains not less than 90.0% and not more than 110.0% of the labeled amount of haloperidol (C21H23ClFNO2: 375.86).
Method of Preparation Prepare as directed under Solutions, with Haloperidol. Haloperidol Oral Solution contains lactic acid as a solubilizing agents. Identification Determine the ultraviolet absorption spectrum of the test solution, prepared in the Assay: it exhibit maxima at 245 2 nm. pH

Between 2.75 and 3.75.

Assay Transfer an accurately measured volume of Haloperidol Oral Solution, equivalent to about 10 mg of haloperidol (C21H23ClFNO2), to a separator, add 20 mL of dilute hydrochloric acid (1 in 20), extract with four 20 mL volumes of ether, combine the all extracts and wash the ether layer with four 50 mL volumes of dilute hydrochloric acid (1 in 20). Discard the ether layer, add the acid washing to the aqueous phase, filter the aqueous phase through a pledget of cotton, add the dilute hydrocholoric acid (1 in 20) to the filtrate to make exactly 50 mL and mix. Pipet 10.0 mL of this solution, dilute with methanol to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Haloperidol RS, add dilute hydrochloric acid (1 in 20) to make exactly 50 mL. Pipet 10.0 mL of this solution, dilute with methanol to make exactly 100 mL, use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at absorption maximum wavelength of about 245 nm as directed under the Ultraviolet-visible Spectrophotometry using a solution added 1 mL of dilute hydrochloric acid (1 in 20) in 10 mL of methanol as the blank.

Amount (mg) of haloperidol (C21H23ClFNO2) = Amount (mg) of Haloperidol RS

AT AS

KP 9 467 Preserve in light-resistant, at 105C for 2 hours: the amount of the residue is not more than 1.0 mg. (5) Volatile related substancesTake 100 mL of Halothane, add exactly 5.0 L of the internal standard and use this solution as the test solution. Perform the test with 5 L of the test solution as directed under the Gas Chromatography and determine each peak area by the automatic integration method: the total area of the peaks other than those of Halothane and the internal standard is not larger than the peak area of the internal standard. Internal standard substance1,1,2-trichloro-1,2,2trifluoro-ethane. Operating conditions Detector: A hydrogen flame-ionization detector. Column: A column, about 3 mm in inside diameter and 3 m in length, at the first 2 m from the injection port, having macrogol 400 coated in the ratio of 30% on siliceous earth for gas chromatography (180 m to 250 m in particle diameter) and at the remaining 1 m, having dinonyl phthalate coated in the ratio of 30% on siliceous earth for gas chromatography (180 m to 250 m in particle diameter). Column temperature: A constant temperature of about 50 o C . Carrier gas: Nitrogen. Flow rate: Adjust the flow rate so that the retention time of the internal standard is 2 minutes to 3 minutes. System suitability System performance: Mix 3 mL of Halothane and 1 mL of the internal standard. When the procedure is run with 1 L of this solution, as directed under the above operating conditions, the internal standard and Halothane are eluted in this order with a resolution between their peaks being not less than 10. Detection sensitivity: Adjust the detection sensitivity so that the peak height of the internal standard obtained from 5 L of the test solution composes 30% to 70% of the full scale. Time span of measurement: About 3 times as long as the retention time of Halothane.
Distilling Range Not less than 95 vol% distils within a 1 C range between 49 C and 51 C. Thymol To 0.50 mL of Halothane, add 5.0 mL of isooctane and 5.0 mL of titanium dioxide TS, shake vigorously for 30 seconds and allow to stand: the separated upper layer has more color than the following control solution A and has no more color than the following control solution B.

Packaging and Storage tight containers.

Halothane
F F C F Cl C Br H

and enantiomer C2HBrClF3: 197.38

Halothane contains not less than 0.008% and not more than 0.012% of Thymol as a stabilizer.
Description Halothane is a clear, colorless and mobile liquid. Halothane is miscible with ethanol, with ether or with isooctane. Halothane is slightly soluble in water. Halothane is a volatile, nonflammable liquid and setting fire to its heated vapor does not support combustion. Halothane is affected by light. 20 : Between 1.369 and 1.371. Refractive index nD Identification Transfer about 3 L each of Halothane and Halothane RS to gas cells having light path, 10 cm in length and determine the infrared absorption spectra as directed in the gas sampling method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Gravity
20 : Between 1.872 and 1.877. d 20

Purity (1) Acid or alkaliShake 60 mL of Halothane with 60 mL of freshly boiled and cooled water vigorously for 3 minutes. Separate the water layer and use this solution as the test solution. To 20 mL of the test solution, add 1 drop of bromocresol purple TS and 0.10 mL of 0.01 mol/L sodium hydroxide VS: a red-purple color is observed. To 20 mL of the test solution, add 1 drop of bromocresol purple TS and 0.6 mL of 0.01 mol/L hydrochloric acid VS: a yellow color is observed. (2) Halide and halogenTo 5 mL of the test solution obtained in (1), add 1 drop of nitric acid and 0.20 mL of silver nitrate TS: no turbidity is produced. To 10 mL of the test solution obtained in (1), add 1 mL of potassium iodide TS and 2 drops of starch TS and allow to stand for 5 minutes: a blue color is not observed. (3) PhosgeneTransfer 50 mL of Halothane to a dried conical flask, suspend a strip of phosgene test paper vertically inside the flask with the lower end about 10 mm above the surface of the liquid, insert the stopper and allow to stand in a dark place for 20 to 24 hours: the test paper shows no yellow color. (4) Residue on evaporationPipet exactly 50 mL of Halothane, evaporate on a water-bath and dry the residue

Control solutionDissolve 0.225 g of Thymol RS in isooctane to make exactly 100 mL. Take 10 mL each of this solution, accurately measured, add isooctane to make exactly 150 mL and 100 mL, respectively. Proceed with 0.50 mL each of these solutions in the same manner as Halothane and use the separated upper layers obtained

468 Monographs, Part I from the control solutions A and B, respectively.

Packaging and Storage Preserve in light-resistant, tight containers. Store at a temperature not exceeding 30 C. Purity (1) Clarity and color of solutionDissolve 0.10 g of Haloxazolam in 20 mL of dehydrated ethanol: the solution is clear and colorless. (2) Soluble halidesTake 1.0 g of Haloxazolam, add 50 mL of water, allow to stand for 1 hour with occasional shaking and filter. To 25 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and use this solution as the test solution. Perform the test with this solution as directed under the Chloride Limit Test. Prepare the control solution with 0.10 mL of 0.01 mol/L hydrochloric acid (Cl: not more than 0.0071%). (3) Heavy metalsProceed with 1.0 g of Haloxazolam according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicTake 1.0 g of Haloxazolam in a decomposition flask, add 5 mL of nitric acid and 2 mL of sulfuric acid, place a small funnel on the mouth of the flask and heat carefully until white fumes are evolved. After cooling, add 2 mL of nitric acid, heat, repeat this procedure twice, add several 2 mL of strong hydrogen peroxide water and heat until the solution is colorless to pale yellow. After cooling, add 2 mL of a saturated solution of ammonium oxalate and heat until white fumes are evolved. After cooling, add water to make 5 mL and perform the test with this solution: the solution has no more color than the following control solution (not more than 2 ppm).

Haloxazolam
H N O

Br F O

and enantiomer C17H14BrFN2O2: 377.21 Haloxazolam, when dried, contains not less than 99.0% and not more than 101.0% of haloxazolam (C17H14BrFN2O2).
Description Haloxazolam is a white crystal or crystalline powder and is odorless and tasteless. Haloxazolam is freely soluble in glacial acetic acid, sparingly soluble in acetonitrile, in methanol or in dehydrated ethanol, slightly soluble in ether and practically insoluble in water. Melting pointAbout 183 C (with decomposition). Identification (1) Dissolve 10 mg of Haloxazolam in 10 mL of methanol, add 1 drop of hydrochloric acid under ultraviolet light (main wavelength: 365 nm): the solution shows a yellow-green fluorescence. To this solution, add 1 mL of sodium hydroxide TS: the fluorescence disappears immediately. (2) Prepare the test solution with 50 mg of Haloxazolam as directed under the Oxygen Flask Combustion Method, using a mixture of 20 mL of dilute sodium hydroxide TS and 1 mL of strong hydrogen peroxide water as an absorbing liquid: the test solution responds to the Qualitative Tests for bromide and for fluoride. (3) Determine the absorption spectra of solutions of Haloxazolam and Haloxazolam RS in methanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Haloxazolam and Haloxazolam RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
1% Absorbance E1 cm (247 nm) (10 mg, methanol, 1000 mL).

Control solution-Proceed in the same manner as above without using Haloxazolam, add 2.0 mL of standard arsenic solution and water to make 5 mL and proceed in the same manner as the test solution. (5) Related substancesDissolve 0.10 g of Haloxazolam in 100 mL of acetonitrile and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add acetonitrile to make exactly 100 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine each peak area of both solutions by the automatic integration method: the total area of all peaks other than the area of the Haloxazolam from the test solution is not larger than the peak area of the Haloxazolam from the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 250 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature.` Mobile phase: Dissolve 6.2 g of boric acid and 7.5 g of potassium chloride in 900 mL of water, adjust the pH with triethylamine to 8.5 and add water to make 1000 mL. To 3 volumes of this solution, add 2 volumes of ace-

Between 390 and 410

KP 9 469 tonitrile. Flow rate: Adjust the flow rate so that the retention time of Haloxazolam is about 10 minutes. System suitability Test for required detection: Weigh exactly 5 mL of the standard solution, add acetonitrile to make exactly 50 mL. The peak area of Haloxazolam obtained from 10 L of this solution is between 8 and 12% of the peak area of Haloxazolam obtained from the standard solution. System performance: Dissolve 10 mg each of Haloxazolam and Cloxazolam in 200 mL of acetonitrile. When the precedure is run with 10 L of this solutions, as directed under the above operating conditions, haloxazolam and cloxazolam are eluted in this order with a resolution between their peaks being not less than 1.5. System repeatability: When the test is repeated 6 times with 10 mL of standard solution according to the above conditions: the relative deviation of the peak area of haloxazolam is not more than 1.0%. Time span of measurement: About 3 times as long as the retention time of Haloxazolam after the solvent peak.
Loss on Drying Not more than 0.5% (1 g, 105 C, 3 hours). Residue on Ignition num crucible).

powder or grains and is odorless. Heparin Sodium is soluble in water and practically insoluble in ethanol or in ether. Heparin Sodium is hygroscopic.
pH The pH of a solution of 1.0 g of Heparin Sodium in 100 mL of water is between 6.0 and 8.0. Purity (1) Clarity and color of solutionDissolve 0.5 g of Heparin Sodium in 20 mL of water: the solution is clear and colorless to pale yellow. (2) BariumDissolve 30 mg of Heparin Sodium in 3.0 mL of water and use this solution as the test solution. To 1.0 mL of the test solution, add 3 drops of dilute sulfuric acid and allow to stand for 10 minutes: no turbidity is produced. (3) Total nitrogenWeigh accurately about 100 mg of Heparin Sodium, previously dried at 60 C for 3 hours under reduced pressure and perform the test as directed under the Nitrogen Determination: the amount of nitrogen (N: 14.01) is not more than 3.0%. (4) ProteinTake 1.0 mL of the test solution obtained in (2), add 5 drops of a solution of trichloroacetic acid (1 in 5): neither a precipitate nor turbidity is produced. Loss on Drying Not more than 10% (20 mg, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 40% (after drying, 20 mg). Pyrogen Test Dissolve Heparin Sodium in isotonic sodium chloride solution so as to contain 1000 units per ml according to the labeled units. Inject 2.0 mL of this solution per kg of the body weight of rabbits and perform the test: it meets the requirement of the Pyrogen Test. Assay (1) Substrate solutionDissolve 15 mg of Nbenzoyl-1-isoleucyl-Lglutamyl (r-OR)-glycyl-Larginyl-p-nitroanilide hydrochloride acid in 20 mL of water. (2) Antithrombin III solutionDissolve human antithrombin III in water to make a solution containing 1 unit per mL. (3) Activated blood coagulation factor X solution Dissolve bovine activated blood coagulation factor X in water to make a solution containing 0.426 units per mL. (4) Buffer solutionDissolve 6.06 g of 2-amino-2hydroxymethyl-1,3-propanediol in 750 mL of water, adjust the pH to 8.4 with 1 mol/L hydrochloric acid TS, and add water to make 1000 mL. (5) Reaction stop solutionTo 20 mL of glacial acetic acid add water to make 40 mL. (6) Heparin standard solutionsDissolve Heparin Sodium RS in isotonic sodium chloride solution to make a solution containing 10 units per mL, and use this solution as the standard stock solution. To 100 L of the standard stock solution add the buffer solution to make

Not more than 0.1% (1 g, plati-

Assay Weigh accurately about 0.5 g of Haloxazolam, previously dried, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 37.721 mg of C17H14BrFN2O2

Packaging and Storage tight containers.

Preserve in light-resistant,

Heparin Sodium
Heparin Sodium is obtained from the livers, the lungs and the intestinal mucosa of healthy edible animals and prolongs the clotting time of blood. Heparin Sodium obtained from the livers and the lungs contains not less than 110 heparin units per mg and that obtained from the intestinal mucosa contains not less than 130 heparin units per mg. Heparin Sodium, calculated on the dried basis, contains not less than 90.0% and not more than 110.0% of the labeled units. Label the name of the organ used as the starting material.
Description Heparin Sodium is white to grayish brown

470 Monographs, Part I exactly 5 mL, and use this solution as the standard solution. Prepare the heparin standard solutions (1), (2), (3), (4) and (5) by addition of antithrombin III solution, human normal plasma and the buffer solution to the standard solution as directed in the following table.
Heparin Standard Solutions Buffer So- AntithHeparin lutions rombins concentra(L) (L) No. tion (Unit/mL) Human normal blood (L)

Heparin Sodium Injection

Heparin Sodium Injection is an aqueous solution for injection. Heparin Sodium Injection contains not less than 90.0% and not more than 110.0% of the labeled heparin units. Label the name of organ used as the starting material of Heparin Sodium supplied for preparing Heparin Sodium Injection.
Method of Preparation Dissolve Heparin Sodium in isotonic sodium chloride solution and prepare as directed under Injections. Description Heparin Sodium Injection is a clear, colorless to pale yellow liquid. pH Between 5.5 and 8.0. Purity (1) BariumMeasure exactly a volume of Heparin Sodium Injection, equivalent to 3000 units of Heparin Sodium according to the labeled unit. Add water to make 3.0 mL and use this solution as the test solution. To 1.0 mL of the test solution, add 3 drops of dilute sulfuric acid and allow to stand for 10 minutes: no turbidity is produced. (2) ProteinProceed as directed in the Purity (4) under Heparin Sodium. Sterility Test It meets the requirement. Bacterial Endotoxins Less than 0.0030 EU/unit of heparin. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter for Injections the requirement.

Standard Solutions (L)

(1) (2) (3) (4) (5)

0 0.02 0.04 0.06 0.08

800 700 600 500 400

100 100 100 100 100

100 100 100 100 100

0 100 200 300 400

(7) Test solutionWeigh accurately an adequate amount of Heparin Sodium, dissolve in isotonic sodium chloride solution, so that each mL contains about 0.5 units according to the labeled amount. To 100 L of this solution add 100 L of antithrombin III solution, 100 L of human normal plasma and 700 L of the buffer solution, and use this solution as the test solution. (8) ProcedureTransfer 400 L of the test solution to a test tube, and warm at 37 C for 4 minutes. Add 200 L of the activated blood coagulation factor X solution, mix well, warm at 37 C for exactly 30 seconds, add 400 L of the substrate solution, previously warmed at 37, and mix well. Allow the tube to stand at 37 C for exactly 3 minutes, add 600 L of the reaction stop solution, mix immediately, and determine the absorbance at 405 nm, using the blank solution prepared by addition of 600 L of the reaction stop solution and 600 L of water to 400 L of the test solution. Proceed the same way with the heparin standard solution (1), the heparin standard solution (2), the heparin standard solution (3), the heparin standard solution (4) and the heparin standard solution (5), and determine their absorbances at 405 nm. (9) CalculationPlot the absorbances of the standard solutions on the vertical axis and their heparin concentrations on the horizontal axis to prepare a calibration curve. Determine the heparin concentration, C, to the test solution from its absorbance by using the calibration curve, and calculate heparin units per mg of Heparin Sodium from the following formula. Units per mg of Heparin Sodium = C10 (b/a) a : Amount (mg) of Heparin Sodium taken. b: Total volume (mL) of isotonic sodium chloride solution used to dissolve the sample to make the solution containing about 0.5 units per mL.
Packaging and Storage Preserve in tight containers.

It meets

Determination of Volume of Injection in Containers It meets the requirement. Assay Proceed as directed in the Assay under Heparin Sodium, replacing the test solution indicated in (7) and the calculation in (9) with the following. (7) Test solutionMeasure exactly an adeqsuate portion of Heparin Sodium Injection according to the labeled units, dilute it with isotonic sodium chloride solution so that each mL contains about 0.5 units. To 100 L of this solution add 100 L of antithrombin III solution, 100 L of human normal plasma and 700 L of the buffer solution, and use this solution as the test solution. (9) CalculationPlot the absorbances of the standard solutions on thevertical axis and their heparin concentrations on the horizontal axis to prepare a calibration curve. Determine the heparin concentration, C, of the test solution from its absorbance by using the curve, and calcu-

KP 9 471 late heparin units per mL of Heparin Sodium Injection from the following formula. Units per mL of Heparin Sodium Injection = C x 10 x (b/a) a: Amount of sample (mL) b: Total volume (mL) of isotonic sodium chloride solution used to dilute the sample to make the solution containing about 0.5 units per mL.
Packaging and Storage hermetic containers.

no red-purple color is produced. (3) Other alkaloidsDissolve 0.15 g of Homatropine Hydrobromide in 3 mL of water and use this solution as the test solution. (i) Take 1 mL of the test solution, add 2 to 3 drops of tannic acid TS: no precipitate is produced. (ii) Take 1 mL of the test solution, add 2 to 3 drops each of dilute hydrochloric acid and platinic chloride TS: no precipitate is produced.
Loss on Drying Not more than 1.5% (0.5 g, 105 C, 2 hours). Residue on Ignition Not more than 0.25% (0.2 g).

Preserve in light-resistant,

Homatropine Hydrobromide
H O NCH3 H H O C CH OH HBr

Assay Dissolve by warming about 0.4 g of Homatropine Hydrobromide in 60 mL of a mixture of acetic anhydride and glacial acetic acid (7 : 3). Cool and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detdction Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 35.626 mg of C16H21NO3.HBr C16H21NO3HBr: 356.26

Packaging and Storage tight containers.

Preserve in light-resistant,

Homatropine Hydrobromide contains not less than 99.0% and not more than 101.0% of homatropine hydrobromide (Cl6H21NO3.HBr) calculated on the dried basis.
Description Homatropine Hydrobromide is a white crystal or crystalline powder and is odorless. Homatropine is freely soluble in water, sparingly soluble in ethanol, slightly soluble in glacial acetic acid, very slightly soluble in acetic anhydride and practically insoluble in ether. Homatropine Hydrobromide is affected by light. Melting pointAbout 214 C (with decomposition). Identification (1) Take 5 mL of a solution of Homatropine Hydrobromide (1 in 20), add 2 to 3 drops of iodine TS: a brown precipitate is produced. (2) Dissolve 50 mg of Homatropine Hydrobromide in 5 mL of water and add 3 mL of picric acid TS: a yellow precipitate is produced. Filter the precipitate, wash with five 10 mL volumes of water and dry at 105 C for 2 hours: it melts between 184 C and 187 C. (3) A solution of Homatropine Hydrobromide (1 in 20) responds to the Qualitative Tests for bromide. Purity (1) AcidDissolve 1.0 g of Homatropine Hydrobromide in 20 mL of water and add 0.40 mL of 0.01 mol/L sodium hydroxide VS and 1 drop of methyl redmethylene blue TS: a green color develops. (2) Atropine, hyoscyamine and scopolamineTake 10 mg of Homatropine Hydrobromide, add 5 drops of nitric acid, evaporate on a water-bath to dryness and cool. Dissolve the residue in 1 mL of dimethylformamide and add 5 to 6 drops of tetraethylammonium hydroxide TS:

Homochlorocyclizine Hydrochloride
H C N Cl 2 HCl

N CH3

and enantiomer C19H23ClN22HCl: 387.77

Homochlorocyclizine Hydrochloride, when dried, contains not less than 98.0 and not more than 101.0% of homochlorocyclizine hydrochloride (C19H23ClN22HCl).
Description Homochlorcyclizine Hydrochloride is a white to pale brown crystal or powder, and is odorless. Homochlorcyclizine Hydrochloride is very soluble in water, freely soluble in glacial acetic acid, soluble in methanol or in ethanol, very slightly soluble in acetic anhydride and practically insoluble in ether. Homochlorcyclizine Hydrochloride dissolves in 0.1 mol/L hydrochloric acid TS. Homochlorcyclizine Hydrochloride is hygroscopic. Homochlorcyclizine Hydrochloride is colored slightly by light.

472 Monographs, Part I The water solution (I in 10) of Homochlorcyclizine Hydrochloride shows not optical rotation. Melting pointAbout 227 C (with decomposition).
Identification (1) Determine the absorption spectra of solutions of Homochlorocyclizine Hydrochloride and Homochlorocyclizine Hydrochloride RS in 0.1 mol/L hydrochloric acid TS (1 in 100000) as directed under Ultraviolet-visible Spectrophotometry: both spectra exhibit similr intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Homochlorocyclizine Hydrochloride and Homochlorocyclizine Hydrochloride RS, previously dried, as directed in the potassium chloride disk method under Infrared Spectrophotometry: both spectra exhibit similr intensities of absorption at the same wavenumbers. (3) A solution of Homochlorocyclizine Hydrochloride (1 in 100) responds to the Qualitative Tests for chloride. Purity (1) Heavy metalsProceed with 1.0 g of Homochlorocyclizine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 0.10 g of Homochlorcyclizine Hydrochloride in 100 mL of mobile phase and use this solution as the test solution. Measure exactly 1 mL of the test solution and add mobile phase to make exactly 100 mL and use this solution as the standard solution. Perform the test with exactly 10 L each of the test solution and the standard solution as directed under Liquid Chromatography according to the following conditions, and determine the peak area by the automatic integration method: the area of the peaks other than homochlorcyclizine obtained from the test solution are not more than1/2 times the peak area of homochlorcyclizine from the standard solution, and the total area of the peaks other than homochlorcyclizine from the test solution is not more than the peak area of homochlorcyclizine from the standard solution.

7 to 13% of that of homochlorcyclizine obtained from 10 L of the standard solution. System performance: Dissolve 5 mg of Homochlorcyclizine Hydrochloride and 5 mg of methyl parahydroxybenzoate acid in 100 mL of the mobile phase. When the procedure is run with 10 L of this solution under the above operating conditions, methyl parahydroxybenzoate acid and homochlorcyclizine are eluted in this order with the resolution between these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of homochlorcyclizine is not more than 1.0%. Time span of measurement: About 2 times as long as the retention time of homochlorcyclizine.
Loss on Drying Not more than 2.0% (1 g, 110 C, 4 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.3 g of Homochlorcyclizine Hydrochloride, previously dried, dissolve in 50 mL of a mixture of anhydride and glacial acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid (potentiometric titration, Endpoint Detdection Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 19.389 mg of C19H23ClN22HCl

Packaging and Storage tight containers.

Preserve in light-resistant,

Hydralazine Hydrochloride
NHNH 2 N HCl N

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 223 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 um in particle diameter). Column temperature: A constant temperature of about 40. Mobile phase: A mixture of water, acetonitrile and perchloric acid (134 : 66 : 1). Flow rate: Adjust the flow rate so that the retention time of homochlorcyclizine is about 10 minutes. System suitability Test for required detection: To exactly 5 mL of the standard solution add the mobile phase to make exactly 50 mL. Confirm that the peak area of homochlorcyclizine obtained from 10 L of this solution is equivalent to

C8H8N4HCl: 196.64 Hydralazine Hydrochloride, when dried, contains not less than 98.0% and not more than 101.0% of hydralazine hydrochloride (C8H8N4HCl).
Description Hydrazine Hydrochloride is a white, crystalline powder and is odorless and has a bitter taste. Hydrazine Hydrochloride is soluble in water, slightly soluble in ethanol and practically insoluble in ether. Melting pointAbout 275 C (with decomposition). Identification (1) Determine the absorption spectra of

KP 9 473 solutions of Hydralazine Hydrochloride and Hydralazine Hydrochloride RS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Hydralazine Hydrochloride and Hydralazine Hydrochloride RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Hydralazine Hydrochloride (1 in 50) responds to the Qualitative Tests for chloride.
pH Dissolve 1.0 g of Hydralazine Hydrochloride in 50 mL of water: the pH of the solution is between 3.5 and 4.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Hydralazine Hydrochloride in 50 mL of water: the solution is clear, and colorless or pale yellow. (2) Heavy metalsProceed with 1.0 g of Hydralazine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 0.5% (0.5 g, in vacuum, P2O5, 8 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.15 g of Hydralazine Hydrochloride, previously dried, transfer to a glassstoppered flask, dissolve in 25 mL of water, add 25 mL of hydrochloric acid, cool to room temperature, add 5 mL of chloroform and titrate with 0.05 mol/L potassium iodide VS while shaking until the purple color of the chloroform layer disappears. The end point is reached when the red-purple color no more reappears in the chloroform layer within 5 minutes after the layer has been decolorized. Method of Preparation Prepare as directed under Injections, with Hydralazine Hydrochloride. Description Hydralazine Hydrochloride for Injection is a white to pale yellow powder of mass and is odorless and has a bitter taste. Identification Determine the absorption spectrum of a solution of Hydralazine Hydrochloride for Injection (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 238 nm and 242 nm, between 258 nm and 262 nm, between 301 nm and 305 nm and between 313 nm and 317 nm. pH Dissolve 1.0 g of Hydralazine Hydrochloride for Injection in 50 mL of water: the pH of this solution is between 3.5 and 4.5. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

It

Uniformity of Dosage Unit It meets the requirement when the Mass Variation test is performed. Assay Weigh accurately a volume (not less than 10 samples) of Hydralazine Hydrochloride for Injection, equivalent to about 0.15 g of Hydralazine Hydrochloride, transfer to a stoppered flask, dissolve in 25 mL of water, add 25 mL of hydrochloric acid, cool to room temperature, add 5 mL of chloroform and titrate with 0.05 mol/L potassium iodate VS while shaking until the purple color of the chloroform layer disappears. The end point is reached when the red-purple color no more reappears in the chloroform layer within 5 minutes after the layer has been decolorized.

Each mL of 0.05 mol/L potassium iodate VS = 9.832 mg of C8H8N4HCl

Packaging and Storage Preserve in tight containers.

Each mL of 0.05 mol/L potassium iodate VS = 9.832 mg of C8H8N4HCl

Packaging and Storage Preserve in hermetic containers.

Hydralazine Hydrochloride for Injection

Hydralazine Hydrochloride for Injection is a preparation for injection which is reconstituted before use. Hydralazine Hydrochloride contains not less than 99.0% and not more than 113.0% of the labeled amount of hydralazine hydrochloride (C8H8N4HCl: 196.64).

Hydralazine Hydrochloride Powder

Hydralazine Hydrochloride Powder contains not less than 95.0% and not more than 105.0% of the labeled amount of hydralazine hydrochloride (C8H8N4HCl: 196.64).
Method of Preparation

Prepare as directed under

474 Monographs, Part I Powders, with Hydralazine Hydrochloride.

Identification Weigh a portion of Hydralazine Hydrochloride Powder, equivalent to 25 mg of Hydralazine Hydrochloride according to the labeled amount, add 100 mL of water, shake well and filter, if necessary. Add water to 2 mL of the filtrate to make 50 mL and determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 238 nm and 242 nm, between 258 nm and 262 nm, between 301 nm and 305 nm and between 313 nm and 317 nm. Particle Size Distribution Test for Preparations meets the requirement. Uniformity of Dosage Units (divided) quirement.

It

It meets the re-

solved solution after 30 minutes from the start of the Dissolution test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet the subsequent V mL, add water to make exactly V mL, so that each mL of the filtrate contains about 11 g of hydralazine hydrochloride (C8H8N4HCl) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately about 50 mg of Hydralazine Hydrochloride RS, previously dried at 105C for 3 hours and dissolve in water to make exactly 50 mL. Pipet exactly 1 mL of this solution, add water to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively at 260 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Hydralazine Hydrochloride Tablets in 45 minutes should be not less than 80%. Dissolution rate (%) with respect to the labeled amount of hydralazine hydrochloride (C8H8N4HCl) AT V' 1 = WS A V C 18 S WS: Amount (mg) of Hydralazine Hydrochloride RS, C: Labeled amount (mg) of hydralazine hydrochloride (C8H8N4HCl) in 1 tablet.
Uniformit of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 Hydralazine Hydrochloride Tablets and powder. Weigh accurately a portion of the powder, equivalent to about 0.15 g of hydralazine hydrochloride (C8H8N4HCl), transfer to a stoppered flask and proceed as directed in the Assay under Hydralazine Hydrochloride.

Assay Weigh accurately a portion of Hydralazine Hydrochloride Powder, equivalent to about 0.15 g of Hydralazine Hydrochloride, transfer to a glass-stoppered flask, add 25 mL of water, shake well, add 25 L of hydrochloric acid, cool to room temperature and proceed as directed in the Assay under Hydralazine Hydrochloride.

Each mL of 0.05 mol/L potassium iodate VS = 9.832 mg of C8H8N4HCl

Packaging and Storage Preserve in tight containers.

Hydralazine Hydrochloride Tablets

Hydralazine Hydrochloride Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of hydralazine hydrochloride (C8H8N4HCl: 196.64).
Method of Preparation Prepare as directed under Tablets, with Hydralazine Hydrochloride. Identification Weigh a portion of powdered Hydralazine Hydrochloride Tablets, equivalent to 25 mg of Hydralazine Hydrochloride according to the labeled amount, add 100 mL of water, mix well and filter, if necessary. Take 2 mL of this solution, add water to make 50 mL and determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits maxima between 238 nm and 242 nm, between 258 nm and 262 nm, between 301 nm and 305 nm and between 313 nm and 317 nm. Dissolution Test Perform the test with 1 tablet of Hydralazine Hydrochloride Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water. Take 30 mL or more of the dis-

Each mL of 0.05 mol/L potassium iodate VS = 9.832 of C8H8N4HCl

Packaging and Storage Preserve in tight containers.

Hydrochloric Acid
HCl: 36.46 Hydrochloric Acid contains not less than 35.0% and not more than 38.0% of hydrogen chloride (HCl).
Description Hydrochloric Acid is colorless liquid having a pungent odor. Hydrochloric Acid is fuming but ceases to fume when it is diluted with 2 volumes of water. 20 About 1.18. Specific gravity d 20 Identification (1) Allow a glass stick wet with ammo-

KP 9 475 nia water to come near the surface of Hydrochloric Acid: a remarkable white smoke evolves. (2) A solution of Hydrochloric Acid (1 in 100) changes blue litmus paper to red and responds to the Qualitative Tests for chloride.
Purity (1) SulfateTake 15 mL of Hydrochloric Acid, add water to make 50 mL and use this solution as the test solution. To 3 mL of the test solution, add 5 mL of water and 5 drops of barium chloride TS and allow to stand for 1 hour: no turbidity is produced. (2) SulfiteTake 3 mL of the test solution obtained in (1), add 5 mL of water and 1 drop of iodine TS: the color of iodine TS does not disappear. (3) Bromide or iodidePlace 10 mL of the test solution obtained in (1) in a glass-stoppered test tube, add 1 mL of chloroform and 1 drop of 0.002 mol/L potassium permanganate VS and shake well: the chloroform layer remains colorless. (4) Bromide or chlorinePlace 10 mL of the test solution obtained in (1) in a glass-stoppered test tube, add 5 drops of potassium iodide TS and 1 mL of chloroform and shake for 1 minute: the chloroform layer remains free from a purple color. (5) Heavy metalsEvaporate 5 mL of Hydrochloric Acid on a water-bath to dryness and add 2 mL of dilute acetic acid and water to the residue to make 50 mL. Perform the test using this solution the test solution. Prepare the control solution as follows: to 3.0 mL of standard lead solution, add 2 mL of dilute acetic acid and water to make 50 mL (not more than 5 ppm). (6) MercuryDilute 20 mL of hydrochloric Acid with water to make exactly 100 mL and use the solution as the test solution. Perform the test with this test solution as directed under the Atomic Absorption Spectrophotometry (cold vapor type). Place the test solution in a sample bottle of the atomic absorption spectrophotometer, add 10 mL of stannous chloride-sulfuric acid TS, connect the bottle immediately to the spectrophotometer, circulate air and determine the absorbance, AT, of the test solution after the recorder reading has risen rapidly and becomes constant at a wavelength of 253.7 nm. On the other hand, to 8 mL of standard mercury solution, add water to make exactly 100 mL and determine the absorbance, AS, of the solution obtained by the same procedure as used for the test solution: AT is smaller than AS (not more than 0.04 ppm). (7) ArsenicPipet 1.7 mL of Hydrochloric Acid, make the test solution according to Method 1 and perform the test (not more than 1 ppm). Residue on Ignition Pipet exactly 10 mL of Hydrochloric Acid, add 2 drops of sulfuric acid, evaporate to dryness and ignite: not more than 1.0 mg of residue remains. Assay Weigh accurately a glass-stoppered flask containing 20 mL of water, add about 3 mL of Hydrochloric Acid and weigh accurately again. Dilute with 25 mL of water and titrate with 1 mol/L sodium hydroxide VS (in-

dicator: 2 to 3 drops of methyl red TS). Each mL of 1 mol/L sodium hydroxide VS = 36.461 mg of HCl
Packaging and Storage Preserve in tight containers.

Dilute Hydrochloric Acid

Dilute Hydrochloric Acid contains not less than 9.5 w/v% and not more than 10.5 w/v% of hydrogen chloride (HCl: 36.46).
Description Dilute Hydrochloric Acid is a colorless liquid. Dilute Hydrochloric Acid is odorless and has a strong acid taste. 20 Specific gravity d 20 About 1.05. Identification A solution of Dilute Hydrochloric Acid (1 in 30) changes blue litmus paper to red and responds to the Qualitative Tests for chloride. Purity (1) SulfateTake 3.0 mL of Dilute Hydrochloric Acid, add 5 mL of water and 5 drops of barium chloride TS and allow to stand for 1 hour: no turbidity is produced. (2) SulfiteTake 3.0 mL of Dilute Hydrochloric Acid, add 5 mL of water and 1 drop of iodine TS: the color of iodine TS does not disappear. (3) Bromide or iodidePlace 10 mL of Dilute Hydrochloric Acid in a glass-stoppered test tube, add 1 mL of chloroform and 1 drop of 0.002 mol/L potassium permanganate VS and shake well: the chloroform layer remains colorless. (4) Bromine or chlorinePlace 10 mL of Dilute Hydrochloric Acid in a glass-stoppered test tube, add 5 drops of potassium iodide TS and 1 mL of chloroform and shake for 1 minute: the chloroform layer remains free from a purple color. (5) Heavy metalsEvaporate 9.5 mL of Dilute Hydrochloric Acid in a water-bath to dryness, add 2 mL of dilute acetic acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution as follows: to 3.0 mL of standard lead solution, add 2 mL of dilute acetic acid and water to make 50 mL (not more than 3 ppm). (6) MercuryDilute 80 mL of Dilute Hydrochloric Acid with water to make exactly 100 mL and use this solution as the test solution. Perform the test with this solution according to the Atomic Absorption Spectrophotometry (cold vapor type). Place the test solution in a sample bottle of the atomic absorption spectrophotometer, add 10 mL of stannous chloride-sulfuric acid TS, connect the bottle immediately to the spectrophotometer, circulate air and read the absorbance, AT , of the test solution after the recorder reading has risen rapidly and become

476 Monographs, Part I constant at a wavelength of 253.7 nm. Separately, to 8 mL of standard mercury solution, add water to make exactly 100 mL and read the absorbance, AS , of the solution obtained by the same procedure as used for the test solution: AT is smaller than AS (not more than 0.01 ppm). (7) ArsenicProceed with 4.0 mL of Dilute Hydrochloric Acid according to Method 1 and perform the test (not more than 0.5 ppm).
Residue on Ignition Pipet exactly 10 mL of Dilute Hydrochloric Acid, add 2 drops of sulfuric acid, evaporate to dryness and ignite: the weight of the residue is not more than 1.0 mg. Assay Measure exactly 10 mL of Dilute Hydrochloric Acid and dilute with 20 mL of water. Titrate with 1 mol/L sodium hydroxide VS (indicator: 2 to 3 drops of methyl red TS).

ride TS: a white precipitate is produced, (3) Take 4 mL of the filtrate obtained in (2), add 5 mL of dilute nitric acid and 3 drops of silver nitrate TS: a white precipitate is produced. (4) Dissolve 12 mg each of Hydrochlorothiazide and Hydrochlorothiazide RS in 100 mL of sodium hydroxide TS. To 10 mL each of these solutions, add water to make 100 mL and determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths.
Purity (1) ChlorideDissolve 1.0 g of Hydrochlorothiazide in 30 mL of acetone, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution as follows: to 1.0 mL of 0.01 mol/L hydrochloric acid VS, add 30 mL of acetone, 6 mL of dilute nitric acid and water to make 50 mL (not more than 0.036%). (2) SulfateDissolve 1.0 g of Hydrochlorothiazide in 30 mL of acetone, add 1 mL of dilute hydrochloric acid and water to make 50 mL and perform the test using this solution as the test solution. Prepare the control solution as follows: to 1.0 mL of 0.005 mol/L sulfuric acid VS, add 30 mL of acetone, 1 mL of dilute hydrochloric acid and water to make 50 mL (not more than 0.048%). (3) Heavy metalsProceed with 1.0 g of Hydrochlorothiazide according to Method 2 and perform the test. Prepare the test solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) Primary aromatic aminesDissolve 80 mg of Hydrochlorothiazide in acetone to make exactly 100 mL. Measure exactly 1 mL of the solution, add 3.0 mL of dilute hydrochloric acid, 3.0 mL of water and 0.15 mL of sodium nitrite TS, shake and allow to stand for 1 minute. Shake this solution with 1.0 mL of ammonium sulfamate TS, allow to stand for 3 minutes, then add 1.0 mL of N(l-naphthyl)-N-diethylethylenediamine oxalate TS, shake and allow to stand for 5 minutes. Perform the test with this solution as directed under the Ultraviolet-visible Spectrophotometry, using a solution prepared with 1.0 mL of acetone in the same manner as the blank: the absorbance at 525 nm is not more than 0.10. Loss on Drying Not more than 1.0% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 30 mg each of Hydrochlorothiazide and hydrochlorothiazide RS, previously dried and dissolve in 150 mL of the mobile phase, add exactly 10 mL each of the internal standard solution, then add the mobile phase to make 200 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS, of the peak

Each mL of 1 mol/L sodium hydroxide VS = 36.461 mg of HCl

Packaging and Storage Preserve in tight containers.

Hydrochlorothiazide
O H2N S O Cl O S NH O

N H

C7H8CIN3O4S2: 297.74 Hydrochlorothiazide, when dried, contains not less than 99.0% and not more than 101.0% of hydrochlorothiazide (C7H8CIN3O4S2).
Description Hydrochlorothiazide is a white crystal or crystalline powder, is odorless and has a slightly bitter taste. Hydrochlorothiazide is freely soluble in acetone, sparingly soluble in methanol, very slightly soluble in water or in ethanol and practically insoluble in ether. Hydrochlorothiazide dissolves in sodium hydroxide TS. Melting pointAbout 267 C (with decomposition). Identification (1) Take 5 mg of Hydrochlorothiazide, add 5 mL of chromotropic acid TS and allow to stand for 5 minutes: a purple color is observed. (2) Fuse a mixture of 0.1 g of Hydrochlorothiazide and 0.5 g of sodium carbonate cautiously: the gas evolved changes moistened red litmus paper to blue. After cooling, crush with a glass rod, add 10 mL of water, stir and filter. Take 4 mL of the filtrate, add 2 drops of hydrogen peroxide (28) solution, 5 mL of diluted hydrochloric acid (1 in 5) and 2 to 3 drops of barium chlo-

KP 9 477 area of Hydrochlorothiazide to that of the internal standard for the test solution and the standard solution, respectively. Amount (mg) of hydrochlorothiazide (C7H8CIN3O4S2) QT = amount (mg) of Hydrochlorothiazide RS Q S Internal standard solutionA solution of 8aminoacetophenone in acetonitrile (9 in 2000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: pH 3.0, a mixture of 0.1 mol/L monobasic sodium phosphate TS and acetonitrile (9 : 1). Flow rate: Adjust the flow rate so that the retention time of Hydrochlorothiazide is about 10 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, hydrochlorothiazide and the internal standard are eluted in this order with a resolution between their peaks being not less than 4.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of hydrochlorothiazide to that of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in well-closed containers.

Hydrocortisone is sparingly soluble in methanol, in ethanol and in dioxane, slightly soluble in chloroform and very slightly soluble in ether and in water. Melting pointBetween 212 C and 220 C (with decomposition).
Identification (1) Take 2 mg of Hydrocortisone, add 2 mL of sulfuric acid: the solution shows a yellow-green fluorescence immediately and the color of the solution changes gradually from orange to dark red. Dilute carefully the solution with 10 mL of water: the color changes through yellow to orange-yellow with green fluorescence and a small amount of a flocculent precipitate is produced. (2) Dissolve 10 mg of Hydrocortisone in 1 mL of methanol, add 1 mL of Fehling's TS and heat: a red precipitate is produced. (3) Determine the infrared absorption spectra of Hydrocortisone and Hydrocortisone RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, dissolve the test and RS in ethanol, respectively, evaporate to dryness and repeat the test on the residues. Specific Optical Rotation [ ]20 D : Between +150 and +156 (after drying, 0.1 g, dioxane, 10 mL, 100 mm). Purity Related substancesDissolve 20 mg of Hydrocortisone in 10 mL of a mixture of chloroform and methanol (9 : 1) and use this solution as the test solution. Pipet exactly 1 mL of this solution, add a mixture of chloroform and methanol (9 : 1) to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thinlayer chromatography. Develop the plate with a mixture of chloroform and ethanol (17 : 3) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 1.0% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.5 g).

Hydrocortisone
O H HO H3C H H3C C CH2OH OH

H O

C21H30O5: 362.47 Hydrocortisone, when dried, contains not less than 97.0% and not more than 102.0% of hydrocortisone (C21H30O5).
Description Hydrocortisone is a white, crystalline powder and is odorless.

Assay Dissolve about 20 mg each of Hydrocortisone and Hydrocortisone RS, previously dried and accurately weighed, in 20 mL each of a mixture of chloroform and methanol (9 : 1), add 10 mL each of the internal standard solution, then add a mixture of chloroform and methanol (9 : 1) to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 5 L each of the test solution and the stan-

478 Monographs, Part I dard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS, of the peak area of Hydrocortisone to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of hydrocortisone (C21H30O5) QT = amount (mg) of Hydrocortisone RS Q S Internal standard solutionA solution of prednisone in a mixture of chloroform and methanol (9 : 1) (9 in 10000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 20 C. Mobile phase: A mixture of chloroform, methanol and anhydrous acetic acid (1000 : 20 : 1). Flow rate: Adjust the flow rate so that the retention time of Hydrocortisone is about 15 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution, as directed under the above operating conditions, the internal standard and hydrocortisone are eluted in this order with a resolution between their peaks being not less than 7. System repeatability: When the test is repeated 6 times with 5 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the ratios of the peak area of hydrocortisone to that of the internal standard is not more than 1.0.
Packaging and Storage Preserve in tight containers.

of anhydrous ethanol, shake for 15 minutes, filter and evaporate the filtrate in a water-bath to dryness. Proceed with the residue as directed in the Identification (3) under Hydrocortisone.
Dissolution Test Perform the test with 1 tablet of Hydrocortisone Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water. Take the dissolved solution after 30 minutes from starting of the test, filter, dilute with the dissolution medium, if necessary and use this solution as the test solution. Separately, weigh accurately a portion of Hydrocortisone RS, dried at 105 C for 3 hours, add the dissolution medium to make the same concentration as the test solution and use this solution as the standard solution. Determine the absorbances of the test solution and the standard solution at 248 nm as directed under the Ultraviolet-visible Spectrophotometry using the test solution as the blank. The dissolution rate of Hydrocortisone Tablets in 30 minutes is not less than 70%. Uniformity of Dosage Units It meets the requirement when the content uniformity test is performed according to the following procedure. Transfer 1 tablet of Hydrocortisone Tablets to a suitable container and add about 0.3 mL of water directly on the tablet. Allow the tablet to stand for about 5 minutes. Shake the container to break up the tablet and sonicate to ensure complete disintegration. Add 4 to 5 small glass beads and 50.0 mL of the internal standard solution to the container. Shake the container for about 30 minutes. Dilute a V mL of the clear supernatant liquid, accurately measured, with a known, accurately measured volume of the internal standard solution to obtain a solution having a known concentration of 0.1 mg per mL and use this solution as the test solution. Separately, weigh accurately a portion of Hydrocortisone RS, previously dried at 105 C for 3 hours, dissolve in the internal standard solution to obtain a solution having a known concentration of 0.1 mg per mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS, of the peak area of Hydrocortisone to that of the internal standard for the test solution and the standard solution, respectively.

Hydrocortisone Tablets
Hydrocortisone Tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of hydrocortisone (C21H30O5: 362.46).
Method of Preparation Prepare as directed under Tablets, with Hydrocortisone. Identification Weigh a portion of powdered Hydrocortisone Tablets, equivalent to about 50 mg of Hydrocortisone according to the labeled amount, add 15 mL of hexane, shake for 15 minutes and remove the hexane. Take the residue, add 10 mL of hexane again, shake for 15 minutes, remove the hexane, add 10 mL of peroxide-free ether, shake for 15 minutes, remove the ether, add 25 mL

Amount (mg) of hydrocortisone (C21H30O5) V' QT = 50 V C Q S V: Final volume (mL) of the test solution, C: Concentration (mg/mL) of standard solution. Internal standard solutionA solution of prednisone in a saturated chloroform in water (6 in 100000). Operating conditions

KP 9 479 Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, having porous silica gel for liquid chromatography (3 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of butyl chloride, a saturated butyl chloride in water, tetrahydrofuran, methanol and anhydrous acetic acid (95 : 95 : 4 : 7 : 6). System suitability System performance: When the procedure is run with 10 L of the standard solution, as directed under the above operating conditions, the resolution between their peaks being not less than 3.0. System repeatability: When the test is repeated 6 times, as directed under the above conditions, the relative standard deviation of the ratios of the peak area is not more than 2.0%.
Assay Weigh accurately and powder not less than 20 Hydrocortisone Tablets, weigh accurately a portion of the powder, equivalent to about 5 mg of hydrocortisone (C21H30O5), transfer to a centrifuge and add 50 mL of the internal standard solution. Shake vigorously for 30 minutes, centrifuge and use the clear supernatant liquid as the test solution. Proceed as directed in the Uniformity of Dosage Units under Hydrocortisone

water. Melting pointAbout 220 C (with decomposition).

Identification (1) Add 2 mL of sulfuric acid to 2 mg of Hydrocortisone Acetate: the solution shows a yellowish green fluorescence immediately and the color of the solution gradually changes through orange-yellow to dark red. This solution shows a strong light green fluorescence under ultraviolet light. Add carefully 10 mL of water to this solution: the color changes from yellow to orange-yellow with a light green fluorescence and a yellow-brown, flocculent precipitate is formed. (2) Dissolve 10 mg of Hydrocortisone Acetate in 1 mL of methanol by warming, add 1 mL of Fehling's TS and heat: an orange to red precipitate is formed. (3) Take 50 mg of Hydrocortisone Acetate, add 2 mL of potassium hydroxide-ethanol TS and heat in a waterbath for 5 minutes. Cool, add 2 mL of diluted sulfuric acid (2 in 7) and boil gently for 1 minute: the odor of ethyl acetate is perceptible. (4) Determine the infrared spectra of Hydrocortisone Acetate and Hydrocortisone Acetate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, dissolve the test sample and RS in ethanol, respectively, evaporate to dryness and repeat the test on the residues. Specific Optical Rotation [ ]20 D : Between + 158 and + 165 (after drying, 50 mg, dioxane, 10 mL, 100 mm). Purity Related substancesDissolve 0.040 g of Hydrocortisone Acetate in 25 mL of a mixture of chloroform and methanol (9 : 1) and use this solution as the test solution. Pipet exactly 2 mL of the test solution, add a mixture of chloroform and methanol (9 : 1) to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solutions as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of dichloromethane, ether, methanol and water (160 : 30 : 8 : 1) to a distance of about 12 cm and air-dry the plate. Spray evenly alkaline blue tetrazolium TS on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 1.0% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Dissolve about 20 mg each of Hydrocortisone Acetate and Hydrocortisone acetate RS, previously dried and accurately weighed, in methanol, add exactly 10 mL

Amount (mg) of hydrocortisone (C21H30O5) QT = 50 C Q S

Packaging and Storage Preserve in well-closed containers.

Hydrocortisone Acetate
O H HO H3C H H3C C CH2O OH O C CH3

H O

C23H32O6: 404.50 Hydrocortisone Acetate, when dried, contains not less than 97.0% and not more than 102.0% of hydrocortisone acetate (C23H32O6).
Description Hydrocortisone Acetate is a white crystals or crystalline powder and is odorless. Hydrocortisone Acetate is sparingly soluble in dioxane, slightly soluble in methanol, in ethanol or in chloroform, very slightly soluble in ether and practically insoluble in

480 Monographs, Part I each of the internal standard solution, then add methanol to make 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and calculate the ratios, QT and QS, of the peak area of Hydrocortisone Acetate to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of hydrocortisone acetate (C23H32O6) QT = amount (mg) of Hydrocortisone Acetate RS Q S Internal standard solutionA solution of benzyl phydroxybenzoate in methanol (1 in 1000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of water and acetonitrile (13 : 7). Flow rate: Adjust the flow rate so that the retention time of Hydrocortisone Acetate is about 8 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions, hydrocortisone acetate and the internal standard are eluted in this order with a resolution between their peaks being not less than 4. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of hydrocortisone acetate to that of the internal standard is not more than l.0%.
Packing and storage Preserve in tight containers.

(Aqueous Suspension) shows a white turbidity on shaking.

Identification Shake well and transfer a volume of Hydrocortisone Acetate Injection (Aqueous Suspension), equivalent to 50 mg of Hydrocortisone Acetate according to the labeled amount, to a separator. Extract with two 10 mL volumes of ether, remove the ether extracts, filter by suction, wash the filter with small amount of water, dry at 105 C for 1 hour and proceed with the residue as directed in the Identification (4) under Hydrocortisone Acetate. pH

Between 5.0 and 7.0

Sterility Test It meets the requirement. Determination of Volume of Injection in Containers It meets the requirement. Assay Shake well, measure exactly a volume of Hydrocortisone Acetate Injection (Aqueous Suspension), equivalent to about 50 mg of hydrocortisone acetate (C23H32O6) in separatory funnel, add 15 mL of water, four 25 mL volumes of chloroform. Filter each extracts with absorbent cotton, previously moistened with chloroform, add chloroform to make exactly 100 mL. Pipet exactly 10 mL to 50-mL glass stopper flask, dry in water bath, and cool. Dissolve the residue in 20.0 mL of ethanol and use this solution as the test solution. Separately, weigh accurately about 50 mg of Hydrocortisone Acetate RS, dissolve in ethanol to make exactly 250 mL. Pipet exactly 5 mL of this solution, add ethanol to make exactly 100 mL and use this solution as the standard solution. Take 2 flasks each with the test solution and the standard solution and a flask with 20.0 mL of ethanol for blank test, add a 2.0 mL solution of 50 mg of bluetetrazol in 20 mL of methanol and mix. Add 2.0 mL of ethanoltetramethylammoniumhydroxide (9 : 1) to each flask and mix. Place them in dark place for 90 minutes. Perform the test with the test solution and the standard solution as directed under the Ultraviolet-visible Spectrophotometry at the 525 nm and measure the absorbance, AT and AS, for the test solution and the standard solution, respectively, using the blank solution as a blank.

Hydrocortisone Acetate Injection (Aqueous Suspension)

Hydrocortisone Acetate Injection (Aqueous Suspension) is an aqueous suspension for injection. Hydrocortisone Acetate Injection contains not less than 90% and not more than 110% of the labeled amount of hydrocortisone acetate (C23H32O6: 404.50).
Method of Preparation Prepare as directed under Injections, with Hydrocortisone Acetate. Description

Amount (mg) of hydrocortisone acetate (C23H32O6) AT = amount (mg) of Hydrocortisone Acetate RS A S

Packaging and Storage hermetic containers.

Preserve in light-resistant,

Hydrocortisone

Acetate

Injection

KP 9 481 this solution as the test solution. Pipet exactly 2 mL of this solution and add tetrahydrofuran to make exactly 50 mL. Pipet exactly 5 mL of this solution, add tetrahydrofuran to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of 1,2-dichloroethane, methanol and water (470 : 30 : 1) to a distance of about 15 cm and air-dry the plate. Spray evenly alkaline blue tetrazolium TS on the plate: the spots other than the principal spot from the test solution are not more than two in number and not more intense than those from the standard solution in color.
Loss on Drying Not more than 1.0% (1 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 50 mg of Hydrocortisone Butyrate, previously dried and dissolve in dehydrated ethanol to make exactly 100 mL. Pipet exactly 2 mL of this solution and add dehydrated ethanol to make exactly 50 mL. Determine the absorbance, A, of this solution at the wavelength of a maximum absorption at about 241 nm as directed under the Ultraviolet-visible Spectrophotometry.

Hydrocortisone Butyrate
O H HO O H3C H C CH2CH2CH3 H3C C CH2OH O

H O

C25H36O6: 432.55 Hydrocortisone Butyrate, when dried, contains not less than 96.0% and not more than 104.0% of hydrocortisone butyrate (C25H36O6).
Description Hydrocortisone Butyrate is a white powder and is odorless. Hydrocortisone Butyrate is freely soluble in tetrahydrofuran, in chloroform or in 1,2-dichlroethane, soluble in methanol, sparingly soluble in dehydrated ethanol, slightly soluble in ether and practically insoluble in water. Melting pointAbout 200 C (with decomposition). Identification (1) Add 2 mL of sulfuric acid to 2 mg of Hydrocortisone Butyrate: the solution shows a yellowish green fluorescence immediately and the color of the solution gradually changes through orange-yellow to dark red. This solution shows a strong light green fluorescence under ultraviolet light (main wavelength: 254 nm). Add carefully 10 mL of water to this solution: the color changes from yellow to orange-yellow with a light green fluorescence and a yellow-brown, flocculent precipitate is produced. (2) Dissolve 10 mg of Hydrocortisone Butyrate in 1 mL of methanol by warming, add 1 mL of Fehlings TS and heat: an orange to red precipitate is formed. (3) Take 50 mg of Hydrocortisone Butyrate, add 2 mL of potassium hydroxide-ethanol TS and heat on a water-bath for 5 minutes. Cool, add 2 mL of diluted sulfuric acid (2 in 7) and boil gently for 1 minute: the odor of ethyl butyrate is perceptible. (4) Determine the infrared absorption spectra of Hydrocortisone Butyrate and Hydrocortisone Butyrate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers Specific Optical Rotation [ ]20 D : Between +48 and +52 (after drying, 0.1 g, chloroform, 10 mL, 100 mm). Purity (1) Heavy metalsProceed with 1.0 g of Hydrocortisone Butyrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesDissolve 25 mg of Hydrocortisone Butyrate in 5 mL of tetrahydrofuran and use

Amount (mg) of hydrocortisone butyrate (C25H36O6) A = 375 25000

Packaging and Storage Preserve in tight containers.

Hydrocortisone Sodium Phosphate

O H HO H3C H H3C C CH2OPO 3Na2 OH

H O

C21H29Na2O8P: 486.40 Hydrocortisone Sodium Phosphate contains not less than 96.0% and not more than 102.0% of Hydrocortisone sodium phosphate (C21H29Na2O8P), calculated on the dried basis.
Description Hydrocortisone Sodium Phosphate is a white to pale yellow powder and is odorless.

482 Monographs, Part I Hydrocortisone Sodium phosphate is freely soluble in water, sparingly soluble in methanol, very slightly soluble in ethanol and practically insoluble in ether. Hydrocortisone Sodium Phosphate is hygroscopic.
Identification (1) Take 2 mg of Hydrocortisone Sodium Phosphate, add 2 mL of sulfuric acid: a yellowish green fluorescence is exhibited initially, then gradually changes through orange-yellow to dark red. Examine the solution under ultraviolet light (main wavelength: 254 nm): an intense, light green fluorescence is exhibited. Take this solution, add carefully 10 mL of water: the color changes from yellow to orange-yellow with a light green fluorescence and a yellow-brown, flocculent floating substance is formed. (2) Determine the infrared absorption spectra of Hydrocortisone Sodium Phosphate and Hydrocortisone Sodium Phosphate RS as directed in the paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, dissolve each of Hydrocortisone Sodium Phosphate and Hydrocortisone Sodium Phosphate RS in methanol, evaporate to dryness and repeat the test on the residues. (3) Moisten 1.0 g of Hydrocortisone Sodium Phosphate with a small volume of sulfuric acid and incinerate by gradual heating. After cooling, dissolve the residue in 10 mL of dilute nitric acid and heat in a water-bath for 30 minutes. After cooling, filter, if necessary: this solution responds to the Qualitative Tests for sodium salt and for phosphate. Specific Optical Rotation [ ]20 D : Between + 121 and + 129 (1 g, calculated on the dried basis, phosphate buffer solution, pH 7.0, 100 mL, 100 mm). pH Dissolve 1.0 g of Hydrocortisone Sodium Phosphate in 100 mL of water: the pH of this solution is between 7.5 and 9.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Hydrocortisone Sodium Phosphate in 10 mL of water: the solution is clear and colorless to pale yellow. (2) ChlorideDissolve 0.30 g of Hydrocortisone Sodium Phosphate in 20 mL of water and add 6 mL of dilute nitric acid and water to make 100 mL. To 5 mL of this solution, add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.600%). (3) Heavy metalsProceed with 0.5 g of Hydrocortisone Sodium Phosphate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 40 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Hydrocortisone Sodium Phosphate according to Method 3 and perform the test (not more than 2 ppm). (5) Free phosphoric acidWeigh accurately about 0.25 g of Hydrocortisone Sodium Phosphate, dissolve in

water to make exactly 100 mL and use this solution as the test solution. Pipet exactly 5 mL each of the test solution and phosphoric acid solution VS into separate volumetric flasks, add 2.5 mL of ammonium molybdatesulfuric acid TS and 1 mL of 1-amino-2-naphthol-4sulfonic acid TS, shake, add water to make exactly 25 mL and allow to stand at 20 1C for 30 minutes. Perform the test with the test solution and the standard solutions as directed under the Ultraviolet-visible Spectrophotometry, using a solution prepared with 5 mL of water in the same manner as a blank. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at 740 nm: the amount of free phosphoric acid is not more than 1.0%. Content (%) of free phosphoric acid (H3PO4) 1 AT = A W 257.8 S W: Amount (mg) of Hydrocortisone Sodium Phosphate, calculated on the dried basis. (6) Free hydrocortisoneDissolve 25 mg of Hydrocortisone Sodium Phosphate in the mobile phase to make exactly 20 mL and use this solution as the test solution. Separately, weigh 25 mg of Hydrocortisone RS, previously dried at 105 C for 3 hours and dissolve in the mobile phase to make exactly 100 mL. Pipet exactly 10 mL of this solution, add the mobile phase to make exactly 200 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak areas, AT and As, of hydrocortisone from each solution: AT is not larger than AS. Operating conditions Proceed as directed under the operating conditions in the Assay. System suitability System performance: Proceed as directed under the system performance in the Assay. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of hydrocortisone is not more than 1.0%.
Loss on Drying Not more than 5.0% (1 g, in vacuum, 80 C, 5 hours). Assay Weigh accurately about 20 mg each of Hydrocortisone Sodium Phosphate and Hydrocortisone Sodium Phosphate RS (determine its loss on drying before use), dissolve each in 50 mL of the mobile phase, add exactly 10 mL of the internal standard solution, then add the mobile phase to make 200 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 20 L each of the test solution and the

KP 9 483 standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS, of the peak area of hydrocortisone phosphate to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of hydrocortisone sodium phosphate (C21H29Na2O8P) = amount (mg) of Hydrocortisone Sodium Phosphate RS, QT calculated on the dried basis Q S Internal standard solutionA solution of isopropyl parahydroxybenzoate in the mobile phase (3 in 5000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of 0.05 mol/L sodium dihydrogen phosphate TS, pH 2.6 and methanol (1 : 1). Flow rate: Adjust the flow rate so that the retention time of hydrocortisone phosphate is about 10 minutes. System suitability System performance: When the procedure is run with 20 L of the standard solution under the above operating conditions and calculate the resolution, hydrocortisone phosphate and isopropyl parahydroxybenzoate are eluted in this order with the resolution between their peaks being not less than 8. System repeatability: When the test is repeated 6 rimes with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of hydrocortisone phosphate to thai of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Hydrocortisone Sodium Succinate contains not less than 97.0% and not more than 103.0% of hydrocortisone sodium succinate (C25H33NaO8), calculated on the dried basis.
Description Hydrocortisone Sodium Succinate is a white powder or mass and is odorless. Hydrocortisone Sodium Succinate is freely soluble in water, in methanol or in ethanol and practically insoluble in ether. Hydrocortisone Sodium Succinate is hygroscopic. Hydrocortisone Sodium Succinate is gradually colored by light. Identification (1) Dissolve 0.2 g of Hydrocortisone Sodium Succinate in 20 mL of water. and add 0.5 mL of dilute hydrochloric acid with stirring: a white precipitate is formed. Collect the precipitate. wash it with two 10mL portions of water, and dry at 105 C for 3 hours. To 3 mg of this dried matter add 2 mL of sulfuric acid: the solution shows a yellowish green fluorescence immediately, and the color of the solution gradually changes through orange-yellow to dark red. This solution shows a strong light green fluorescence under ultraviolet light. Add carefully 10 ml of water to this solution: the color changes from yellow to orange-yellow with a light green fluorescence, and a yellow-brown flocculent precipitate is formed. (2) Dissolve 10 mg of the dried matter obtained in (1) in 1 mL of methanol, add 1 mL of Fehling's TS, and heat: an orange to red precipitate is formed. (3) To 0.1 g of the dried matter obtained in (1) add 2 mL of sodium hydroxide TS, and allow to stand for 10 minutes. Filter the solution to remove the precipitate formed, mix the filtrate with 1 mL of dilute hydrochloric acid, filter if necessary, then adjust the solution to a pH of about 6 with diluted ammonia TS (1 in 10), and add 2 to 3 drops of iron (III) chloride TS: a brown precipitate is formed. (4) Determine the infrared absorption spectra of the dried matter obtained in (1) and Hydrocortisone Sodium Succinate RS as directed in the potassium bromide disk method under Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears between the specera, dissolve Hydrocortisone Sodium Succinate and Hydrocortisone Sodium Succinate RS in methanol, respectively, then evaporate the methanol to dryness, and repeal the test on the residues. (5) Hydrocortisone Sodium Succinate responds to the Qualitative Tests (1) for sodium salt. Specific Optical Rotation [ ]20 D : Between +135 and +145 (0.1 g, calculated on the dried basis, ethanol, 10 mL, 100 mm).

Hydrocortisone Sodium Succinate

O H HO H3C H H3C C CH2O OH O C CH2CH2CO2Na

H O

C25H33NaO8: 484.51

Purity (1) Clarity and color of solutionDissolve 0.5 g of Hydrocortisone Sodium Succinate in 10 mL of water: the solution is clear and colorless.

484 Monographs, Part I (2) Related substancesDissolve 25 mg of Hydrocortisone Sodium Succinate in methanol to make exactly 10 mL and use this solution as the test solution. Separately, dissolve 25 mg of hydrocortisone RS in methanol to make exactly 10 mL. Pipet exactly 1 mL of this solution, add methanol to make exactly 20 mL and use this solution as the standard solution (1). Pipet exactly 6 mL of the standard solution (1), add methanol to make exactly 10 mL and use this solution as the standard solution (2). Perform the test with the test solution and the standard solutions (1) and (2) as directed under the Thin-layer chromatography. Spot 3 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chloroform, dehydrated ethanol and formic acid (150 : 10 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spot from the test solution corresponding to the spot from the standard solution (1) is not more intense than the spot from the standard solution (1). Any spot other than the principal spot and the above spot obtained from the test solution is not more than one and is not more intense than the spot from the standard solution (2).
Loss on Drying Not more than 2.0% (0.5 g, 105 C, 3 hours). Assay Weigh accurately about 10 mg of Hydrocortisone Sodium Succinate and dissolve in methanol to make exactly 100 mL. Pipet exactly 5 mL of this solution, add methanol to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Hydrocortisone Succinate RS, previously dried at 105 C for 3 hours, proceed in the same manner as directed for the test solution and use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at 240 nm as directed under the Ultraviolet-visible Spectrophotometry.

O H HO H3C H H3C C CH2O OH

O C CH2CH2CO 2H

H O

C25H34O8: 462.53 Hydrocortisone Succinate, when dried, contains not less than 97.0% and not more than 103.0% of hydrocortisone succinate (C25H34O8.)
Description Hydrocortisone Succinate is a white, crystalline powder and is odorless. Hydrocortisone Succinate is very soluble in methanol, freely soluble in dehydrated ethanol, sparingly soluble in ethanol, very slightly soluble in ether and practically insoluble in water. Identification (1) Take 3 mg of Hydrocortisone Succinate, add 2 mL of sulfuric acid: the solution shows a yellowish green fluorescence immediately and the color of the solution gradually changes through orange-yellow to dark red. This solution shows a strong light green fluorescence under ultraviolet light. Add carefully 10 mL of water to this solution: the color changes from yellow to orange-yellow with a light green fluorescence and a yellow-brown flocculent precipitate is produced. (2) Determine the infrared absorption spectra of Hydrocortisone Succinate and Hydrocortisone Succinate RS, previously dried, according to the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, dissolve Hydrocortisone Succinate and Hydrocortisone Succinate RS in methanol, respectively, evaporate to dryness and perform the test on the residues. Specific Optical Rotation [ ]20 D : Between +147 and +153 (after drying, 0.1 g, dehydrated ethanol, 10 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 0.5 g of Hydrocortisone Succinate in 10 mL of methanol: the solution is clear and colorless. (2) Related substancesDissolve 25 mg of Hydrocortisone Succinate in methanol to make exactly 10 mL and use this solution as the test solution. Separately, dissolve 25 mg of Hydrocortisone RS in methanol to make exactly 10 mL. Pipet exactly 1 mL of this solution, add methanol to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 3 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Devel-

Amount (mg) of hydrocortisone sodium succinate (C25H33NaO8) = amount (mg) of Hydrocortisone AT Sodium Succinate RS A 1.0475 S
Packaging and Storage tight containers.

Preserve in light-resistant,

Hydrocortisone Succinate

KP 9 485 op the plate with a mixture of chloroform, dehydrated ethanol and formic acid (150 : 10 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 2.0% (0.5 g, 105 C, 3 hours). Residue on Ignition Not more than 0.1% (0.5 g). Assay Weigh accurately about 50 mg each of Hydrocortisone Succinate and Hydrocortisone Succinate RS, previously dried and dissolve in methanol to make exactly 50 mL. Pipet exactly 5 mL each of these solutions, add exactly 5 mL each of the internal standard solution, then add methanol to make 50 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions and calculate the ratios, QT and QS, of the peak area of hydrocortisone succinate to that of the internal standard for the test solution and the standard solution, respectively.

Packaging and Storage tight containers.

Preserve in light-resistant,

Hydrogen Peroxide Solution

Hydrogen Peroxide Solution contains not less than 2.5% and not more than 3.5% of hydrogen peroxide (H2O2: 34.02). Hydrogen Peroxide Solution contains not more than 0.5 w/v% of suitable preservatives.
Description Hydrogen Peroxide Solution is a colorless, clear liquid and is odorless or has ozone-like odor. Hydrogen Peroxide Solution is acidic and produce bubbles in the opening of the container. Hydrogen Peroxide Solutions decomposition is accelerated by contacting oxidants or reductants. Hydrogen Peroxide Solution can be degraded by heat. Hydrogen Peroxide Solution needs to be protected from light. 20 About 1.01. Specific gravity d 20 Identification Shake 1 mL of Hydrogen Peroxide Solution with 10 mL of water containing 1 drop of dilute sulfuric acid, and add 2 mL of ether. Subsequently, 1 drop of potassium chromate TS is added to produce an evanescent blue color in water layer which, upon agitation and standing, passes into the ether layer. Purity (1) AcidityTake 25 mL of Hydrogen Peroxide Solution, add 2 drops of phenolphthalein and 2.5 mL of 0.1 mol/L sodium hydroxide: a red color develops. (2) Heavy metalsTake 5 mL of Hydrogen Peroxide Solution, add 20 mL of water and 2 mL of Ammonia TS and evaporate on a water-bath to dryness. Dissolve the residue in 2 mL of dilute acetic acid by heating and add water to make 50 mL. Perform the test with this solution as a test solution. Pipet 2.5 mL of standard lead solution and add 2 mL of dilute acetic acid and water to make 50 mL and use this solution as a reference solution (not more than 5 ppm). (3) BariumTo 1.0 mL add two drops of 2 N sulfuric acid: no turbidity or precipitate is produced within 10 minutes. (4) Organic preservativesPipet 100.0 mL of Hydrogen Peroxide Solution and extract sequentially with 50 mL, 25 mL and 25 mL volumes of a mixture of chloroform and ether (3 : 2), place the combined extracts in the container, previously weighed, evaporate at room temperature and dry the residue in a desiccator with silica gel for 2 hours: not more than 50 mg. (5) Non volatile residuePipet 2.0 mL of Hydrogen

Amount (mg) of hydrocortisone succinate (C25H34O8) QT = amount (mg) of Hydrocortisone Succinate RS Q S Internal standard solutionA solution of butyl parahydroxy benzoate in methanol (1 in 2500). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel (5 m to 10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of acetic acid-sodium acetate buffer solution, pH 4.0 and acetonitrile (3 : 2). Flow rate: Adjust the flow rate so that the retention time of hydrocortisone succinate is about 5 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution, as directed under the above operating conditions, hydrocortisone succinate and the internal standard in this order with the resolution between their peaks being not less than 9. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative deviation of the peak area of hydrocortisone succinate is not more than 1.0%.

486 Monographs, Part I Peroxide Solution, evaporate on a water-bath to dryness and dry residue at 105 o C for 1 hour: not more than 30 mg.
Assay Place accurately about 2 mL of Hydrogen Peroxide Solution to a flask containing 20 mL of water, add 20 mL of dilute sulfuric acid and titrate with 0.02 mol/L potassium permanganate VS.

Each mL of 0.02 mol/L potassium permanganate VS = 1.701 mg of H2O2

Packaging and Storage Preserve in light-resistant, tight containers. Store at 30 C or less.

mg of potassium hydrogen sulfate, and fuse by igniting. Cool, break up the mass with a glass rod, add 3 mL of water, and dissolve by boiling. Add 1 drop of phenolphthalein TS, and add dropwise sodium hydroxide TS until the solution devellops a light red. Then add 0.5 g of sodium acetate trihydrate, 0.5 mL of dilute acetic acid and 0.5 mL of a solution of disodium l-nitroso-2-naphthol3,6-disulfonate (1 in 500): a red to orange-red color develops immediately. Then add 0.5 mL of hydrochloric acid, and boil for 1 minute: the red color does not disappear. (3) Add 0.5 mL of dehydrated ethanol and 1 mL of sulfuric acid to 20 mg of Hydroxocobalamin Acetate and heat the mixture: the odor of ethyl acetate is perceptible.
Purity Cyanocobalamin and colored related substancesDissolve 50 mg of Hydroxocobalamin Acetate in exactly 5 mL each of acetic acid-sodium acetate buffer solution, pH 5.0, in two tubes. Take one tube, add 0.15 mL of potassium thiocyanate TS, allow to stand for 30 minutes and use this solution as the test solution (1). To the other tube, add 0.10 mL of potassium cyanide TS, allow to stand for 30 minutes and use this solution as the test solution (2). Separately, dissolve 3.0 mg of Cyanocobalamin RS in exactly 10 mL of acetic acid-sodium acetate buffer solution, pH 5.0 and use this solution as the standard solution. Perform the test with the test solutions (1) and (2) and the standard solution as directed under the Thin-layer chromatography. Apply 20 L each of the test solutions (1) and (2) and the standard solution, 25 mm in length along the starting line, 10 mm apart from each other, on a plate of silica gel for thin-layer chromatography. Develop the plate for 18 hours with 2butanol saturated with water, while supporting the plate at an angle of about 15 to a horizontal plane and air-dry the plate: the spot from the test solution (1) corresponding to that from the standard solution is not more intense than the spot from the standard solution and the spots other than the principal spot from the test solution (2) are not more intense than the spot from the standard solution. Loss on Drying Not more than 12% (0.05 g, in vacuum, at a pressure not exceeding 0.67 kPa, P2O5, l00 C, 6 hours). Assay Weigh accurately about 20 mg of Hydroxocobalamin Acetate and dissolve in acetic acid-sodium acetate buffer solution, pH 5.0, to make exactly 50 mL. Pipet exactly 2 mL of this solution into a 50-mL volumetric flask, add 1 mL of a solution of potassium cyanide (1 in 1000) and allow to stand for 30 minutes at ordinary temperature. Add acetic acid-sodium acetate buffer solution, pH 5.0, to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 20 mg of cyanocobalamin RS after determining the loss on drying in the same manner as for Cyanocobalamin and dissolve in water to make exactly 50 mL. To 2 mL of this solution, exactly measured, add acetic acid-sodium acetate buffer solution, pH 5.0, to make exactly 50 mL and

Hydroxocobalamin Acetate
O H2N H2N C C CH2CH2 CH2 OH N Co N N CH3 CH3 H CH2CH2 O C NH2 CH3CO2H H N CH2CH2 H CH3 H3C CH2 H O C NH2 O C NH2

O CH3 O H2N C H3C H2C H H3C O NH C CH2CH2

H3C

CH3

O H2C C H CH3 O H H O CH2OH H O P O

N OH

CH3

C62H89CoNl3O15PC2H4O2: 1406.41 Hydroxocobalamin Acetate contains not less than 95.0% and not more than 101.0% of hydroxocobalamin acetate (C62H89CoNl3O15P C2H4O2), calculated on the dried basis.
Description Hydroxocobalamin Acetate is a dark red crystal or powder and is odorless. Hydroxocobalamin Acetate is freely soluble in water, slightly soluble in ethanol and practically insoluble in ether. Hydroxocobalamin Acetate is hygroscopic. Identification (1) Determine the absorption spectra of solutions of Hydroxocobalamin Acetate and Hydroxocobalamin Acetate RS in acetic acid-sodium acetate buffer solution, pH 4.5, (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Mix 1 mg of Hydroxocobalamin Acetate with 50

KP 9 487 use this solution as the standard solution. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at 361 nm as directed under the Ultraviolet-visible Spectrophotometry. Amount (mg) of hydroxocobalamin acetate (C62H89CoNl3O15PC2H4O2) = amount (mg) of cyanocobalamin RS, calculated on AT the dried basis A 1.0377 S
Packaging and Storage Preserve in light-resistant, tight containers in a cold place.

tion of Hydroxychloroquine Sulfate RS, dissolve in a mixture of methanol and water (90 : 10) to make solutions containing 0.01, 0.05, 0.1, and 0.2 mg per mL, respectively, and use these solutions as the standard solutions. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 20 L each of the test solution and standard solutions on a plate of silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of ethanol, water and ammonia solution (28) (80 : 16 : 4) to a distance of about 15 cm, and air-dry the plate. Examine under ultraviolet light (main wavelengths: 254 nm and 366 nm): the spots other than the principal spot from the test solution are not more intense than the spots from the standard solution.
Loss on Drying Not more than 2.0% (105 C, 2 hours). Assay Weigh accurately about 0.1 g each of Hydroxychloroquine Sulfate and Hydroxychloroquine Sulfate RS, dissolve in 5 mL of water, and add diluted hydrochloric acid (1 in 100) to make exactly 100 mL. Pipet exactly 1.0 mL each of these solutions, add diluted hydrochloric acid (1 in 100) to make exactly 100 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Ultraviolet-visible Spectrophotometry. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at the wavelength of maximum absorbance near 343 nm.

Hydroxychloroquine Sulfate
Cl H N N N CH3 CH3 OH H2SO4

C18H26ClN3OH2SO4 : 433.95 Hydroxychloroquine Sulfate contains not less than 98.0% and not more than 102.0% of hydroxychloroquine sulfate (C18H26ClN3OH2SO4), calculated on the anhydrous basis.
Description Hydroxychloroquine Sulfate is a white, crystalline powder, is odorless, and has bitter taste. Hydroxychloroquine Sulfate is freely soluble in water, and practically insoluble in ethanol, in chloroform, and in ether. Hydroxychloroquine Sulfate shows polymorphism. The ordinary form of Hydroxychloroquine Sulfate melts at about 240 C, while other forms melt at about 198 C. Identification (1) Determine the absorption spectra of solutions of Hydroxychloroquine Sulfate and Hydroxychloroquine Sulfate RS in diluted hydrochloric acid (1 in 100) (1 in 100000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (2) Determine the infrared absorption spectra of Hydroxychloroquine Sulfate and Hydroxychloroquine Sulfate RS, previously dried, as directed in the potassium bromide disk method under lnfrared Spectrophotornetry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) A solution of Hydroxychloroquine Sulfate (1 100) responds to the Qualitative Tests for sulfate. Purity Related substancesDissolve 0.10 g of Hydroxychloroquine Sulfate in a mixture of methanol and water (90 : 10) to make exactly 10 mL, and use this solution as the test solution. Separately, weigh exactly a por-

Amount (mg) of hydroxychloroquine sulfate AT (C18H26ClN3OH2SO4) = 10 C A S C: Concentration of Hydroxychloroquine Sulfate RS in the standard solution (g/mL).
Packaging and Storage well-closed containers.

Preserve in light-resistant,

Hydroxyprogesterone Caproate
H3C H3C O O O H CH3

H3C

H O

C27H40O4: 428.60 Hydroxyprogesterone Caproate, contains not less than 97.0% and not more than 103.0% of hydroxyprogesterone caproate (C27H40O4), calculated on the anhydrous

488 Monographs, Part I basis.

Description Hydroxyprogesterone Caproate is a white to milky white crystalline powder and is odorless or has slight odor. Hydroxyprogesterone Caproate is soluble in ether, slightly soluble in benzene and practically insoluble in water. Identification Determine the infrared absorption spectra of Hydroxyprogesterone Caproate and hydroxyprogesterone caproate RS, previously dried in vacuum for 4 hours, according to the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Points Between 120 C and 124 C. Specific Optical Rotation [ ]20 D : Between + 58 and + 64 (0.1 g, calculated on the anhydrous basis, chloroform, 10 mL, 100 mm). Purity (1) Free n-caproic acidWeigh about 0.20 g of Hydroxyprogesterone Caproate and dissolve in 25 mL of neutralized ethanol. Titrate with 0.02 mol/L sodium hydroxide VS: not more than 0.50 mL of 0.02 mol/L sodium hydroxide is required (0.58%). (2) Related compoundsWeigh accurately about 0.1 g of Hydroxyprogesterone Caproate, dissolve in 10 mL of chloroform and use this solution is the test solution. Separately, weigh accurately about 10 mg of Hydroxyprogesterone Caproate RS, previously dried for 4 hours in vacuum (silica gel) and dissolve in chloroform to make 10 mL. Pipet exactly 0.1 mL, 0.5 mL, 1.0 mL and 2.0 mL of this solution, add 10 mL of acetone exactly and use these solutions as standard solutions (1), (2), (3) and (4), respectively. Perform the test with the test solution and the standard solutions (1), (2), (3) and (4), as directed under Thin Layer Chromatography. Spot 20 L each of the test solution and the standard solutions (1), (2), (3) and (4) on silica gel plate for the thin layer chromatography. Develop the plates with a mixture chloroform and ethyl acetate (3:1) to a distance about 15 cm and air-dry the plate. Spray a mixture of sulfuric acid and ethanol (1 : 9), prepared by adding sulfuric acid slowly and carefully to ethonal on an ice-bath, on the plates thoroughly and heat the plates until it charr. Using ultraviolet light (main wavelength 366 nm), the relative intensities of the spots from the test solution, except the principal spot, compared to that of each standard solution is not more than 2.0%. Water Not more than 0.1% (5 g, volumetric titration, direct titration). Assay Weigh accurately about 50 mg each of Hydroxyprogesterone Caproate and Hydroxyprogesterone Caproate RS, previously dried in vacuum in a dessicator (si-

lica gel) for 4 hours, dissolve in ethanol to make exactly 100 mL and mix by shaking. Pipet exactly 2 mL each of these solutions, add ethanol to make exactly 100 mL and use these solutions as the test solution and the standard solution, respectively. Perform the test with the test solution and the standard solution as directed under the Ultraviolet-visible Spectrophotometry. Determine the absorbances, AT and AS, of the test solution and the standard solution, respectively, at the wavelength of maximum absorbance near 240 nm. Amount (mg) of Hydroxyprogesterone Caproate AT (C27H40O4) = 5 C A S C: Concentration of the standard solution (g/mL).
Packaging and Storage Preserve in well-closed containers.

Hydroxyprogesterone Caproate Injection

Hydroxyprogesterone Caproate Injection is an oily injection. Hydroxyprogesterone Caproate Injection contains not less than 90.0% and not more than 110.0% of the labeled amount of hydroxyprogesterone caproate (C27H40O4: 428.60).
Method of Preparation Prepare as directed under Injections, with Hydroxyprogesterone Caproate. Identification (1) Transfer a volume of Hydroxyprogesterone Caproate Injection, equivalent to 0.125 g of Hydroxyprogesterone Caproate according to the labeled amount, to a separatory funnel containing 10 mL of hexane, 8 mL of methanol and 2 mL of water and shake for 2 minutes and stand to separate. Titrate 3 mL of lower layer with sulfuric acid until the color of solution appears. Add 3 mL of methanol: violet color appears. Light with long wavelength ultraviolet line shows a pale yellowish fluorescence. (2) Evaporate 4 mL of the test solution of the Assay to dryness by heating in a water-bath. Dissolve the residue in 0.5 mL of chloroform and use this solution as the test solution. Dissolve a suitable amount of Hydroxyprogesterone Caproate RS in a suitable amount of chloroform so as to contain 400 g of hydroxyprogesterone caproate per mL. Use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin Layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel. Develop the plates with a mixture of chloroform and ethyl acetate (3 : 1) at a distance about 10 cm and air-dry. Spray a mixture of sulfuric acid and ethanol (1 : 3) on these plates tho-

KP 9 489 roughly and heat the plates at 105 C for 5 minutes: the Rf value of the principal yellowish green spot obtained from the test solution corresponds to that from the standard solution.
Water Not more than 0.2% (5 g, volumetric titration, direct titration). Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections meets the requirement.

Cl

N C H

CH2CH2OCH2CH2OH 2 HCl

and enantiomer C21H27ClN2O22HCl: 447.83 Hydroxyzine Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of hydroxyzine hydrochloride (C21H27ClN2O22HCl).
Description Hydroxyzine Hydrochloride is a white, crystalline powder, is odorless and has a bitter taste. Hydroxyzine Hydrochloride is very soluble in water, freely soluble in methanol, in ethanol or in anhydrous acetic acid, very slightly soluble in acetic anhydride and practically insoluble in ether. Melting pointAbout 200 C (with decomposition). Identification (1) Take 5 mL of a solution of Hydroxyzine Hydrochloride (1 in 100), add 2 to 3 drops of ammonium thiocyanate-cobaltous nitrate TS: a blue precipitate is produced. (2) Determine the absorption spectra of solutions of Hydroxyzine Hydrochloride and Hydroxyzine Hydrochloride RS in methanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) A solution of Hydroxyzine Hydrochloride (1 in 10) responds to the Qualitative Tests for chloride. pH Dissolve 1.0 g of Hydroxyzine Hydrochloride in 20 mL of water: the pH of this solution is between 1.3 and 2.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Hydroxyzine Hydrochloride in 10 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Hydroxyzine Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Related substancesDissolve 0.20 g of Hydroxyzine Hydrochloride in 10 mL of methanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution, add methanol to make exactly 200 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, ethanol and

It

Determination of Volume of Injection in Containers It meets the requirement. Assay Take accurately a volume of Hydroxyprogesterone Caproate Injection, equivalent to 0.25 g of hydroxyprogesterone caproate (C27H40O4) according to the labeled amount, add methanol to make 250 mL and mix. Pipet exactly 5 mL of this solution, add methanol to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Hydroxyprogesterone Caproate RS, previously dried in vacuum in a desicator for 4 hours, add methanol to make exactly 50 mL. Pipet exactly 5 mL of this solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Pipet exactly 5 mL each of the test solution and the standard solution to a stopered flask and add 10.0 mL of the isonicotinic acid hydrazide solution. Mix and allow to stand at 30 C on a water-bath for about 45 minutes. Determine the absorbances of these solutions as directed under the Ultraviolet-visible Spectrophotometry, AT and AS, at the wavelength of maximum absorbance near 380 nm, using a mixture of methanol and the isonicotinic acid hydrazide solution (1 : 2) as a blank.

Amount (mg) of hydroxyprogesterone caproate (C27H40O4) = amount (mg) of Hydroxyprogesterone AT Caproate RS A 5 S Isonicotinic acid hydrazide solutionDissolve 0.375 g of Isonicotinic Acid Hydrazide in 0.47 mL of hydrochloric acid and 500 mL of methanol.
Packaging and Storage Preserve in hermetic containers.

Hydroxyzine Hydrochloride

490 Monographs, Part I ammonia water (28) (150 : 95 : 1) to a distance of about 10 cm and air-dry the plate. Allow the plate to stand in iodine vapor: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 3.0% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.2% (1 g). Assay Weigh accurately about 0.1 g of Hydroxyzine Hydrochloride, previously dried, dissolve in 60 mL of a mixture of acetic anhydride and anhydrous acetic acid (7 : 3) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection method in Titrimetry). Perform a blank determination and make any necessary correction.

(2) Take 1 mL of the water layer obtained in (1), add 2 mL of 1 mol/L hydrochloric acid TS: a yellow precipitate is produced. Collect the precipitate, dissolve the precipitate in 5 mL of methanol and add 1 drop of ferric chloride TS: a green color is observed. (3) Take 2 mL of the test solution obtained in test (1), evaporate the test solution on a water-bath to dryness, dissolve the residue in 0.1 mol/L hydrochloric acid TS to make 500 mL. Determine the absorption spectrum of the solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 231 nm and 233 nm. (4) Perform the test with Hydroxyzine Pamoate as directed under the Flame Coloration Test (2): a green color is observed.
Purity (1) Clarity and color of solutionDissolve 1.0 g of Hydroxyzine Pamoate in 10 mL of dimethylformamide: the solution is clear and shows a slightly greenish, pale yellow-brown color. (2) ChlorideTake 0.3 g of Hydroxyzine Pamoate, add 6 mL of dilute nitric acid and 10 mL of water, shake for 5 minutes and filter. Wash the residue with two 10 mL volumes of water, combine the washings with the filtrate and add water to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution with 0.80 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.095%). (3) Heavy metalsProceed with 1.0 g of Hydroxyzine Pamoate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (4) ArsenicPrepare the test solution with 2.0 g of Hydroxyzine Pamoate according to Method 3 and perform the test (not more than 1 ppm). (5) Related substancesDissolve 0.40 g of Hydroxyzine Pamoate in 10 mL of a mixture of sodium hydroxide TS and acetone (1 : 1) and use this solution as the test solution. Pipet exactly 1 mL of this solution, add a mixture of sodium hydroxide TS and acetone (1 : 1) to make exactly 20 mL. Pipet exactly 5 mL of this solution, add a mixture of sodium hydroxide TS and acetone (1 : 1) to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, ethanol and ammonia water (150 : 95 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly chloroplatinic acid-potassium iodide TS on the plate: the spots other than the spots from hydroxyzine and pamoic acid obtained from the test solution are not more intense than the spots from the standard solution. Water Not more than 3.0% (1 g, volumetric titration, direct titration). Residue on Ignition Not more than 0.5% (1 g).

Each mL of 0.1 mol/L perchloric acid VS = 22.392 mg of C21H27ClN2O22HCl

Package and Storage Preserve in tight containers.

Hydroxyzine Pamoate
CO2H Cl N C H N CH2CH2OCH2CH2OH CH2 OH OH

CO2H

and enantiomer C2lH27ClN2O2C23H16O6: 763.27 Hydroxyzine Pamoate contains not less than 98.0% and not more than 101.0% of Hydroxyzine Pamoate (C2lH27ClN2O2C23H16O6), calculated on the anhydrous basis.
Description Hydroxyzine Pamoate is a pale yellow, crystalline powder, is odorless and has a slightly bitter taste. Hydroxyzine Pamoate is freely soluble in dimethylformamide, slightly soluble in acetone and practically insoluble in water, in methanol, in ethanol or in ether. Identification (1) Take 0.1 g of Hydroxyzine Pamoate, add 25 mL of sodium hydroxide TS and shake vigorously. Extract with 20 mL of chloroform and use the chloroform layer as the test solution. Reserve the water layer for (2). Take 5 mL of the test solution, add 2 mL of ammonium thiocyanate-cobaltous nitrate TS, shake and allow to stand: a blue color is observed in the chloroform layer.

KP 9 491

Assay Weigh accurately about 0.6 g of Hydroxyzine Pamoate, add 25 mL of sodium hydrolride TS, shake and extract with six 25 mL volumes of chloroform. Filter each extract through 5 g of anhydrous sodium sulfate on a pledget of absorbent cotton. Combine the chloroform extracts and evaporate the combined chloroform extracts on a water-bath to about 30 mL. Add 30 mL of anhydrous acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue to blue-green (indicator: 2 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary connection.

Melting Point Between 187 C and 191 C. Purity (1) ChlorideDissolve 0.8 g of Hymecromone in 40 mL of a mixture of acetone and water (2 : 1) and add 6 mL of dilute nitric acid and a mixture of acetone and water (2 : 1) to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.25 mL of 0.01 mol/L hydrochloric acid VS, add 6 mL of dilute nitric acid and a mixture of acetone and water (2 : 1) to make 50 mL (not more than 0.011%). (2) SulfateDissolve 0.8 g of Hymecromone in 40 mL of a mixture of acetone and water (2 : 1) and add 1 mL of dilute hydrochloric acid and a mixture of acetone and water (2 : 1) to make 50 mL. Perform the test using this solution as the test solution. Prepare the control solution as follows: to 0.40 mL of 0.005 mol/L sulfuric acid VS, add 1 mL of dilute hydrochloric acid and a mixture of acetone and water (2:1) to make 50 mL (not more than 0.024%). (3) Heavy metalsProceed with 2.0 g of Hymecromone according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) Arsenic Prepare the test solution with 1.0 g of Hymecromone according to Method 3 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 80 mg of Hymecromone in 10 mL of ethanol and use this solution as the test solution. Pipet exactly 1 mL of the test solution and add ethanol to make exactly 50 mL. Pipet exactly 1 mL of this solution, add ethanol to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of chloroform and ethanol (10 : 1) to a distance of about 10 cm and air-dry the plate. Allow the plate to stand in iodine vapor for 5 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.25 g of Hymecromone, previously dried, dissolve in 90 mL of dimethylformamide and titrate with 0.1 mol/L tetramethylammonium hydroxide VS (potentiometric titration, Endpoint Detection method in Titrimetry). Separately, perform a blank determination with a solution prepared by adding 14 mL of water to 90 mL of dimethylformamide and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 38.164 mg of C2lH27ClN2O2C23H16O6

Packaging and Storage Preserve in tight containers.

Hymecromone
HO O O

CH3

C10H8O3: 176.17 Hymecromone, when dried, contains not less than 98.0% and not more than 101.0% of hymecromone (C10H8O3).
Description Hymecromone is a white crystal or crystalline powder, is odorless and tasteless. Hymecromone is freely soluble in dimethylformamide, sparingly soluble in ethanol, in dehydrated ethanol or in acetone, slightly soluble in ether and practically insoluble in water. Identification (1) Dissolve 2 mg of Hymecromone in 5 mL of ammonia-ammonium chloride buffer solution, pH 11.0: the solution shows an intense blue-purple fluorescence. (2) Dissolve 25 mg of Hymecromone in 5 mL of diluted ethanol (1 in 2) and add 1 drop of ferric chloride TS: initially a blackish brown color is observed and when allowed to stand the color changes to yellowbrown. (3) Determine the absorption spectra of solutions of Hymecromone and hymecromone RS in dehydrated ethanol (1 in 250000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (4) Determine the infrared spectra of Hymecromone and hymecromone RS, previously dried, as directed in the potassium bromide disc method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.

492 Monographs, Part I Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 17.617 mg of C10H8O3
Packaging and Storage Preserve in tight containers.

than the spot from the standard solution.

Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, not more than 0.67 kPa, 4 hours). Residue on Ignition Not more than 0.1% (1 g).

Ibuprofen
CH3 CHCH2 CH3 CH3 CCO 2H H

and enantiomer C13H18O2: 206.28

Assay Weigh accurately about 0.5 g of Ibuprofen, previously dried, dissolve in 50 mL of ethanol and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Perform a blank determination and make any necessary correction.

Ibuprofen, when dried, contains not less than 98.5% and not more than 101.0% of ibuprofen (C13H18O2).
Description Ibuprofen is a white crystalline powder. and has slightly characteristic smell and taste. Ibuprofen is very soluble in ethanol, in absolute ethanol, in acetone and sparingly soluble in water. Ibuprofen is soluble in sodium hydroxide. Identification (1) Determine the absorption specta of solutions of Ibuprofen and Ibuprofen RS in diluted sodium hydroxide TS (1.5 in 10000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibits similar intensities of absorption at the same wavelengths. (2) Determine the Infrared absorption specta of Ibuprofen and Ibuprofen RS, previously dried, as detected in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 75 C and 77 C. Purity (1) Heavy metalsProceed with 3.0 g of Ibuprofen according to Method 2 and perform the test. Prepare the control solution with 3.0 mL of standard lead solution (not more than 10 ppm). (2) ArsenicPrepare the test solution with 1.0 g of Ibuprofen according to Method 3 and perform the test (not more than 2 ppm). (3) Related substancesDissolve 0.50 g of Ibuprofen in 5 mL of chloroform and use this solution as the test solution. Pipet 1.0 mL of the test solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thinlayer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of hexane, ethyl acetate and glacial acetic acid (15 : 5 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense

Each mL of 0.1 mol/L sodium hydroxide VS = 20.628 mg of C13H18O2

Packaging and Storage Preserve in well-closed containers

Ichthammol
Ichthammol, calculated on the dried basis, contains not less than 2.5% of ammonia NH3: 17.030), not more not more than 8.0% of ammonium sulfate [(NH4)2SO4: 132.14] and not less than 10.0% of total sulfur (as S: 32.07).
Description Ichthammol is a red-brown to blackish brown, viscous fluid and has a characteristic odor. Ichthammol is miscible with water and is partially soluble in ethanol or in ether. Identification (1) Take 4 mL of a solution of Ichthammol (3 in 10), add 8 mL of hydrochloric acid: a yellow-brown to blackish brown, oily or resinous mass is produced. Cool the mass with ice to solidify and discard the water layer. Wash the residue with ether: a part of the mass dissolves but it does not dissolve completely even when it is washed until almost no color develops in the washing. Perform the following tests with this residue. (i) Take 0.1 g of the residue, add 1 mL of a mixture of ether and ethanol (1 : 1): it dissolves. (ii) Take 0.1 g of the residue, add 2 mL of water: it dissolves. To 1 mL of this solution, add 0.4 mL of hydrochloric acid: a yellow-brown to blackish brown oily or resinous substance is produced. (iii) Take 1 mL of the solution obtained in (ii), add 0.3 g of sodium chloride: a yellow-brown or blackish brown oily or resinous substance is produced. (2) Boil 2 mL of a solution of Ichthammol (1 in 10) with 2 mL of sodium hydroxide TS: the gas evolved changes moistened red litmus paper to blue. Loss on Drying Not more than 50% (0.5 g, 105 C, 6 hours). Residue on Ignition Not more than 0.5% (1 g).

KP 9 493

Assay (1) AmmoniaWeigh accurately about 5 g of Ichthammol, transfer to a Kjeldahl flask and add 60 mL of water, 1 mL of octyl alcohol and 4.5 mL of a solution of sodium hydroxide (2 in 5). Connect the flask to a distilling tube with a spray trap and a condenser and immerse the lower outlet of the condenser in the receiver containing exactly 30 mL of 0.25 mol/L sulfuric acid VS. Distil slowly, collect about 50 mL of the distillate and titrate the excess sulfuric acid with 0.5 mol/L sodium hydroxide VS (indicator: 3 drops of methyl red TS). Perform a blank determination and make any necessary correction.

I NH

N HOH 2C H H OH H O

C9H11IN2O5: 354.10 Idoxuridine, when dried, contains 98.0% and not more than 101.0% of idoxuridine (C9H11IN2O5).
Description Idoxuridine is a colorless, crystal or a white, crystalline powder. Idoxuridine is odorless. Idoxuridine is freely soluble in dimethylformamide, slightly soluble in water, very slightly soluble in ethanol and practically insoluble in ether. Idoxuridine dissolves in sodium hydroxide TS. Melting pointAbout 176 C (with decomposition). Identification (1) Dissolve 10 mg of Idoxuridine in 5 mL of water by warming, add 5 mL of diphenylamineglacial acetic acid TS and heat for 5 minutes: a blue color is observed. (2) Heat 0.1 g of Idoxuridine: a purple gas evolves. (3) Dissolve 2 mg of Idoxuridine and Idoxuridine TS in 50 mL of 0.01 mol/L sodium hydroxide VS and determine the absorption specta of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelength . Specific Optical Rotation [ ]20 D : Between +28 and +31 (after drying, 0.20 g, sodium hydroxide TS, 20 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 0.20 g of Idoxuridine in 5 mL of a solution of sodium hydroxide (1 in 200): the solution is clear and colorless. (2) Heavy metalsProceed with 2.0 g of Idoxuridine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (3) Related substancesDissolve about 0.10 g of Idoxuridine in exactly 10 mL of a mixture of dilute ethanol and strong ammonia water (99 : 1) and use this solution as the test solution. Perform the test with the test solution as directed under the Thin-layer chromatography. Spot 50 L of the test solution on a plate of silica gel with fluorescent indicator for Thin-layer chromatography. Develop the plate with a mixture of ethyl acetate and diluted isopropanol (2 in 3) (4 : 1) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): no spot other than principal spot appears.

Each mL of 0.25 mol/L sulfuric acid VS = 8.515 mg of NH3 (2) Ammonium sulfateWeigh accurately about 1 g of Ichthammol, add 25 mL of ethanol, stir thoroughly and filter. Wash with a mixture of ether and ethanol (1:1) until the washings are clear and colorless. Dry the filter paper and the residue in air, dissolve the residue in 200 mL of hot water acidified slightly with hydrochloric aicd and filter. Boil the filtrate, add 30 mL of barium chloride TS slowly, heat for 30 minutes on a water bath and filter. Wash the precipitate with water, dry and ignite to constant weight. Weigh the residue as barium sulfate (BaSO4: 233.39). Amount (mg) of ammonium sulfate [(NH4)2SO4] = amount (mg) of barium sulfate (BaSO4) 0.5662 (3) Total sulfurWeigh accurately about 0.6 g of Ichthammol, transfer to a 200-mL Kjeldahl flask and add 30 mL of water and 5 g of potassium chlorate, then add slowly 30 mL of nitric acid and evaporate the mixture to about 5 mL. Transfer the residue to a 300-mL beaker with the aid of 25 mL of hydrochloric acid and evaporate again to 5 mL. Add 100 mL of water, boil, filter and wash with water. Heat the combined filtrate and washings to boil, add gradually 30 mL of barium chloride TS, heat the mixture on a water-bath for 30 minutes and filter. Wash the precipitate with water, dry and ignite to constant weight. Weigh the residue as barium sulfate (BaSO4). Amount (mg) of total sulfur (S) = amount (mg) of barium sulfate (BaSO4) 0.13739
Packaging and Storage Preserve in tight containers.

Idoxuridine

494 Monographs, Part I (4) Iodine and iodideDissolve 0.10 g of Idoxuridine in 20 mL of water and 5 mL of sodium hydroxide TS and add immediately 5 mL of dilute sulfuric acid under ice-cooling. Allow to stand for 10 minutes with occasional shaking and filter. Transfer the filtrate into a Nessler tube, add 10 mL of chloroform and 3 drops of a solution of potassium iodate (1 in 100), shake for 30 seconds and allow to stand: the chloroform layer has no more color than the following control solution. Control solutionWeigh accurately 0.111 g of potassium iodide and dissolve in water to make 1000 mL. To exactly 1 mL of this solution, add 19 mL of water, 5 mL of sodium hydroxide TS and 5 mL of dilute sulfuric acid, mix and filter. Transfer the filtrate to a Nessler tube and proceed in the same manner.
Loss on Drying Not more than 0.5% (2 g, in vacuum, 60 C, 3 hours). Residue on Ignition Not more than 0.3% (1 g). Assay Weigh accurately about 0.7 g of Idoxuridine, previously dried, dissolve in 80 mL of dimethylformamide and titrate with 0.1 mol/L tetramethylammonium hydroxide VS until the color of the solution changes from yellow through yellow-green to blue (indicator: 5 drops of thymol blue-dimethylformamide TS). Perform a blank determination and make any necessary correction.

hydrous sodium carbonate, heat slowly, evaporate to dryness and ignite until the residue is incinerated. Dissolve the residue in 5 mL of water, acidify with hydrochloric acid and add 2 to 3 drops of sodium nitrate TS: a yellow-brown color is observed. Then add 2 to 3 drops of starch TS: a deep blue color is observed. (3) Take a volume of Idoxuridine Ophthalmic Solution, equivalent to 2 mg of Idoxuridine according to the labeled amount, add 0.01 mol/L sodium hydroxide TS to make 50 mL. Determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 277 nm and 281 nm.
pH Between 4.5 and 7.0. Purity 5-Iodouracil and 2-deoxyuridineTake a volume of Idoxuridine Ophthalmic Solution, equivalent to 4.0 mg of Idoxuridine according to the labeled amount, add water to make exactly 5 mL and use this solution as the test solution. Separately, dissolve 12.0 mg of 5iodouracil and 4.0 mg of 2-deoxyuridine RS in water to make exactly 200 mL. Measure exactly 5 mL of this solution, add water to make exactly 25 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and determine the peak areas of 5-iodouracil and 2-deoxyuridine: the peak areas of 5iodouracil and 2-deoxyuridine of the test solution are not more than the peak areas of 5-iodouracil and 2deoxyuridine of the standard solution.

Each mL of 0.1 mol/L tetramethylammonium hydroxide VS = 35.410 mg of C9H11IN2O5

Packaging and Storage tight containers.

Preserve in light-resistant,

Idoxuridine Ophthalmic Solution

Idoxuridine Opthalmic Solution contains not less than 90.0% and not more than 110.0% of the labeled amount of idoxuridine (C9H11IN2O5: 354.10).
Method of Preparation Prepare as directed under Ophthalmic Solutions, with Idoxuridine. Description Idoxuridine Opthalmic Solution is a clear, colorless liquid. Identification (1) Take a volume of Idoxuridine Ophthalmic Solution, equivalent to 5 mg of Idoxuridine according to the labeled amount, add 5 mL of diphenylamine-glacial acetic acid TS and heat for 20 minutes: a pale blue color is observed. (2) Place a volume of Idoxuridine Ophthalmic Solution, equivalent to 5 mg of Idoxuridine according to the labeled amount, in a porcelain crucible, add 0.1 g of an-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 15 to 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: Constant temperature of apploximately 25 C Mobile phase: A mixture of water and methanol (24 : 1). Flow rate: Adjust the flow rate so that the retention time of 2-deoxyuridine is about 6 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, 2-deoxyuridine and 5-iodouracil are eluted in this order with the resolution between these peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of 2-deoxyuridine is not more than 1.0%.

KP 9 495
Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement Insoluble Particulate Matter for Ophthalmic Solutions It meets the requirement Assay Measure exactly a volume of Idoxuridine Ophthalmic Solution, equivalent to 3 mg of Idoxuridine (C9H11IN2O5) according to the labeled amount, add exactly 2 mL of the internal standard solution, then add water to make 10 mL and use this solution as the test solution. Separately, weigh accurately about 10 mg of Idoxuridine RS, previously dried at 60 C for 3 hours and dissolve in water to make exactly 10 mL. Measure exactly 3 mL of this solution, add exactly 2 mL of the internal standard solution, then add water to make 10 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Idoxuridine to that of the internal standard, for the test solution and the standard solution, respectively. Packaging and Storage Preserve in light-resistant, tight containers in a cold place and avoid freezing.

Ifenprodil Tartrate
H CH 2 N C CH 3 OH C H OH HOOC 2 H OH H OH CO 2H

(C21H27NO2)2C4H6O6: 800.99 Ifenprodil Tartrate contains not less than 98.5% and not more than 101.0% of ifenprodil tartrate [(C21H27NO2)2C4H6O6], calculated on the anhydrous basis.
Description Ifenprodil Tartrate occurs as a white crystalline powder and is odorless. Ifenprodil Tartrate is freely soluble in glacial acetic acid, soluble in ethanol, slightly soluble in water or in methanol and practically insoluble in ether.

Amount (mg) of idoxuridine (C9H11IN2O5) = amount (mg) of Idoxuridine RS

QT 3 QS 10

Melting pointAbout 148 C (with decomposition). Specific Optical Rotation [ ]20 D Between +11 and +15 (1.0 g, calculated on the anhydrous basis, ethanol, 20 mL, 100 mm).
Identification (1) Determine the absorption spectra of solutions of Ifenprodil Tartrate and Ifenprodil Tartrate RS in methanol (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelenght. (2) Determine the infrared spectra of Ifenprodil Tartrate and Ifenprodil Tartrate RS as directed in the potassium bromide disc method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (3) Dissolve 0.4 g of Ifenprodil Tartrate in 40 mL of water by warming. After cooling, add 0.5 mL of ammonia TS to this solution, extract with two 40-mL volumes of chloroform and collect the water layer. Evaporate 30 mL of the water layer on a water-bath to dryness and after cooling, dissolve the residue in 6 mL of water: the solution responds to the Qualitative Tests for tartrate. Purity (1) Heavy metalsProceed with 2.0 g of Ifenprodil Tartrate according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (2) Related substancesDissolve 0.30 g of Ifenprodil Tartrate in 10 mL of diluted ethanol (3 in 4) and use this solution as the test solution. Pipet 1.0 mL of the test solution, add diluted ethanol (3 in 4) to make exactly 200 mL and use this solution as the standard solution. Perform the test solution with the test and standard solutions as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution

Internal standard solutionA solution of sulfathiazole in mobile phase (1 in 4000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and 15 to 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: Constant temperature of apploximately 25 C Mobile phase: A mixture of water and methanol (87 : 13). Flow rate: Adjust the flow rate so that the retention time of Idoxuridine is about 9 minutes. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, idoxuridine and the internal standard are eluted in this order with the resolution between these peaks being not less than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area of idoxuridine to that of the internal standard is not more than 1.0%.

496 Monographs, Part I on a plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, hexane, n-butanol and strong ammonia water (140 : 40 : 20 : 1) to a distance of about 10 cm and air-dry the plate. Spray chloroplantinic acid-potassium iodide TS evenly on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Water Not more than 4.0% (0.5 g, volume titration, direct titration). Residue on Ignition

tion, pH 7.0, 10 mL, 100 mm).

Sterility Test It meets the requirement, when Imipenem Hydrate is used in a sterile preparation. Bacterial Endotoxins Less than 0.25 EU per mg of imipenem, when Imipenem Hydrate is used in a sterile preparation. Water Perform the test according to Method 2 under Heat Analysis Method. Weigh about 5 to 10 mg of Imipenem Hydrate in vacuum, and heat to about 200 C with increasing temperature by 20 C per minute. Weigh accurately the mass of the sample about at 150 C, calculate the mass difference between before and after, and determine the water content (not less than 5.0 % and not more than 8.0 %). Assay Weigh accurately about 25 mg (potency) each of Imipenem Hydrate and Imipenem RS, add 10 mL of 0.9 % sodium chloride solution and 1 mL of 0.1 % of sodium bicarbonate solution, add 0.001 mol/L phosphate buffer solution to make exactly 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 10 L of each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the peak areas, AT, and AS of imipenem,.

Not more than 0.1% (1 g).

Assay Weigh accurately about 0.5 g of Ifenprodil Tartrate, dissolve in 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Determination Method in Titrimetry). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 40.05 mg of (C21H27NO2) 2C4H6O6

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Imipenem Hydrate
HO O O N H HO H3C H H S HN NH

Amount [g (potency)] of imipenem (C12H17N3O4S)

H2O

= Amount [g (potency)] of Imipenem RS

AT AS

C12H17N3O4SH2O: 317.36 Imipenem Hydrate contains not less than 849 g (potency) and not more than 990 g (potency) of per mg of imipenem (C12H17N3O4S: 299.35), calculated on the anhydrous basis.
Description Imipenem Hydrate is a whtie powder. Imipenem Hydrate is freely soluble in water, and sparingly soluble in methanol. Identification Determine the absorption spectra of Imipenem Hydrate and Imipenem RS as directed in the paste method under Infrared Spectrophotometry, both spectra exhibit similar intensities of absorption at the same wave numbers. Crystalliity Test It meets the requirement. Specific Optical Rotation [ ] 20 D : +85 ~ +95(50 mg calculated on the anhydrous basis, phosphate buffer solu-

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm) Column: A stainless steel column, about 3.9 mm in inside diameter and about 300 mm in length, packed with octadecylsilanized silica gel for liquid chromatography (10 m in particle diameter) Mobile phase: Weigh 2.0 g of sodium 1hexanesulfonate, dissolve in 800 mL of 0.001 mol/L phosphate buffer solution, adjust the pH of the solution to 6.8 with phosphoric acid or 5 mol/L sodium hydroxide, and add 0.001 mol/L phosphate buffer solution to make 1000 mL. Flow rate: 2.0 mL per minute.
Packaging and Storage Preserve in tight containers (2 ~ 5 C).

Imipramine Hydrochloride

KP 9 497 mine Hydrochloride in 10 mL of ethanol and use this solution as the test solution. Pipet 1.0 mL of this solution and add ethanol to make exactly 50 mL. Pipet 5.0 mL of this solution, add ethanol to make exactly 50 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for Thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, glacial acetic acid, hydrochloric acid and water (11 : 7 : 1 : 1) to a distance of about 12 cm and air-dry the plate. Spray evenly potassium dichromate-sulfuric acid TS on the plate: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Imipramine Hydrochloride, previously dried and dissolve in 20 mL of water. Add 5 mL of sodium hydroxide TS and extract with three 20 mL volumes of chloroform. Filter each extract through a pledget of absorbent cotton on which a small quantity of anhydrous sodium sulfate is placed. Combine the chloroform extracts and titrate with 0.1 mol/L perchloric acid VS until the yellow solution changes to red-purple (indicator: 10 drops of metanil yellow TS). Perform a blank determination and make any necessary correction.

HCl N CH3 CH2CH2CH2N CH3

C19H24N2HCl: 316.87 Imipramine Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of imipramine hydrochloride (C19H24N2HCl).
Description Imipramine Hydrochloride is a white to pale yellowish white, crystalline powder and is odorless. Imipramine Hydrochloride is freely soluble in water or in ethanol and practically insoluble in ether. Imipramine Hydrochloride is gradually affected by light. Identification (1) Dissolve 5 mg of Imipramine Hydrochloride in 2 mL of nitric acid: a deep blue color is observed. (2) Dissolve 5 mg of Imipramine Hydrochloride and Imipramine Hydrochloride TS in 250 mL of 0.01 mol/L hydrochloric acid TS and determine the absorption spectrum of this as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths. (3) Dissolve about 50 mg of Imipramine Hydrochloride in 5 mL of water, add 1 mL of ammonia TS, allow to stand for 5 minutes, filter and acidify the filtrate with dilute nitric acid: it responds to the Qualitative Tests (2) for chloride. Melting Point Between 170 C and 174 C (with decomposition). Purity (1) Clarity and color of solutionDissolve 1.0 g of Imipramine Hydrochloride in 10 mL of water: the solution is clear and has no more color than the following control solution.

Each mL of 0.1 mol/L perchloric acid VS = 31.687 mg of C19H24N2HCl

Packaging and Storage tight containers.

Preserve in light-resistant,

Control solutionTake exactly 1.0 mL of cobaltous chloride colorimetric stock solution, 2.4 mL of ferric chloride CS, 0.4 mL of cupric sulfate colorimetric stock solution and 6.2 mL of diluted hydrochloric acid (1 in 40) and mix. Pipet 0.5 mL of this solution and add exactly 9.5 mL of water. (2) IminodibenzylDissolve 50 mg of Imipramine Hydrochloride in 10 mL of a mixture of hydrochloric acid and ethanol (1 : 1) in a brown volumetric flask. Cool the flask in ice-water, add 5 mL of an ethanol solution of furfural (1 in 250) and 5 mL of hydrochloric acid and allow to stand at 25 C for 3 hours. Add a mixture of hydrochloric acid and ethanol (1 : 1) to make 25 mL and determine the absorbance of this solution at 565 nm as directed under the Ultraviolet-visible Spectrophotometry: not more than 0.16. (3) Related substancesDissolve 0.20 g of Imipra-

Imipramine Hydrochloride Tablets

Imipramine Hydrochloride Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of the imipramine hydrochloride (C19H24N2HCl: 316.87).
Method of Preparation Prepare as directed under Tablets, with Imipramine Hydrochloride. Identification (1) Weigh a quantity of powered Imipramine Hydrochloride Tablets, equivalent to 0.25 g of Imipramine Hydrochloride according to the labeled amount, add 25 mL of chloroform and filter after shaking well. Evaporate the filtrate on a water-bath. Proceed with the residue as directed in the Identification (1) under Im-

498 Monographs, Part I ipramine Hydrochloride. (2) Dissolve an amount of the residue obtained in (1), equivalent to 5 mg of Imipramine Hydrochloride, in 250 mL of 0.01 mol/L hydrochloric acid TS and determine the absorption spectrum as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 249 nm and 253 nm and a shoulder between 270 nm and 280 nm. (3) Dry the residue obtained in (1) at 105 for 2 hours: the residue melts between 170 and 174 (with decomposition)
Dissolution Test Perform the test with 1 tablet of Imipramine Hydrochloride Tablets at 75 revolutions per minute according to Method 2 under the Dissolution Test, using pH 6.8 phosphate buffer solution (1 in 2) as the dissolution solution. Take more than 20 mL of the dissolved solution after 60 minutes from the start of the dissolution test and filter through a membrane filter with pore size of not more than 0.8 m. Discard the first 10 mL of the filtrate, pipet the subsequent V mL, add the diluted pH 6.8 phosphate buffer solution (1 in 2) to make exactly V mL so that each mL of the filtrate contains about 10 g of imipramine hydrochloride (C19H24N2HCl) according to the labeled amount and use this solution as the test solution. Separately, weigh accurately 0.025 g of Imipramine Hydrochloride RS, previously dried at 105 C for 2 hours, dissolve in the diluted pH 6.8 phosphate buffer solution (1 in 2) to make exactly 100 mL. Pipet 4.0 mL of this solution, add the 1st fluid to make exactly 100 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution at the wavelength of 250 nm as directed under the Ultravioletvisible Spectrophotometry, respectively. The dissolution rate of Imipramine Hydrochloride Tablets in 60 minutes should be not less than 75%.

ipramine Hydrochloride for Assay, previously dried at 105 C for 2 hours, dissolve in 0.01 mol/L hydrochloric acid TS to make exactly 100 mL and use this solution as the standard solution. Pipet 3.0 mL each of the test solution and the standard solutions into separators which contain 15 mL of potassium biphthalate buffer solution, pH 5.6, 8 mL of bromocresol green-sodium hydroxide TS and 30 mL of chloroform and shake. Filter the chloroform layer through a pledget of absorbent cotton into a 100 mL volumetric flask. Repeat the extraction with two 30-mL portions of chloroform, combine the chloroform layers in the 100-mL volumetric flask and add chloroform to make exactly 100 mL. Perform the test with these solutions as directed under the Ultraviolet-visible Spectrophotometry, using a solution obtained by proceeding with 3 mL of 0.01 mol/L hydrochloric acid TS in the same manner as blank. Determine the absorbances, AT and AS , for the test solution and the standard solution, respectively, at 416 nm . Amount (mg) of imipramine hydrochloride (C19H24N2HCl) = amount (mg) of Imipramine Hydrochloride for Assay
AT AS

Packaging and Storage Preserve in tight containers.

Indapamide
O N N H CH3

O S

O NH2

Cl

Dissolution rate (%) with respect to the labeled amount of imipramine hydrochloride (C19H24N2HCl) = WS
AT V ' 1 36 AS V C

C16H16ClN3O3S : 365.84 Indapamide contains not less than 98.0% and not more than 101.0% of indapamide (C16H16ClN3O3S), calculated on the dried basis.
Description Indamaide is a white, crystalline powder. Indamaide is soluble in methanol, in ethanol, in acetonitril, in glacial acetic acid or in ethyl acetate, slightly soluble in ether or in chloroform, and practically insoluble in water. Melting point between 167 and 170 (decomposition). Identification (1) Determine the absorption spectra of solution of Indapamide and Indapamide RS in methnol (1 in 200000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhit similar intensities of absorption at the same wavelength. (2) Determine the infrared spectra of Indapamide and

WS : Amount (mg) of Imipramine Hydrochloride RS, C: Labeled amount (mg) of imipramine hydrochloride (C19H24N2HCl) in 1 tablet.

Uniformity of Dosage Units It meets the requirement. Assay Take 20 Imipramine Hydrochloride Tablets, add exactly 200 mL of 0.01 mol/L hydrochloric acid TS and shake well until the tablets are completely disintegrated. After centrifuging the solution, pipet a volume of the superantant liquid, equivalent to about 0.025 g of imipramine hydrochloride (C19H24N2HCl) according to the labeled amount, add 0.01 mol/L hydrochloric acid TS to make exactly 100 mL and use this solution as the test solution. Separately, weigh accurately about 0.025 g of Im-

KP 9 499 Indapamide RS as directed in the potassium bromide disk method under the spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers.
Purity Related substancesPerform the test without exposure to daylight. Dissolve about 0.3 g of Indapamide in methanol to make exactly 10 mL and use this solution as the test solution. Separately, dissolve 30 mg of Indapamide RS in methanol to make exactly 100 mL, and use this solution as the standard solution (1). Add methanol to 10 mL of the standard solution (1) to make exactly 20 mL, and use this solution as the standard solution (2). And, add methanol to 10 mL of the standard solution (2) to make exactly 20 mL, and use this solution as the standard solution (3). Perform the test with these solutions as directed under the Thin-layer Chromatography. Spots separately, 10 L of the test solution, 10 L each of the standard solution (2) and the standard solution (3) on a plate of silica gel with fluorescent indicator for the Thinlayer Chromatography. Develop the plate with a mixture of toluene, ethyl acetate and glacial acetic acid (70:30:1) to a distance of about 15 cm. Remove the plate from the developing chamber, and air-dry the plate. Examine under ultraviolet light(main wavelength: 254 m), and compare the intensities of any secondary spots observed in the chromatogram of the test solution with those of the principal spots in the chromatograms of the standard solution: secondary spot from the chromatograms of the test solution is not larger or more intense than the principal spot obtained from standard solution (2) (0.5%), and the sum of the intensities of the secondary spots obtained from the test solution is not more than 2.0%. Loss on Drying 4hours).

the standard solution. Internal standard solutionWeigh exactly 50 mg of p-chloroacetanilide, dissolve in methanol to make exactly 10 mL. Operating conditions Detector : An ultraviolet absorption photometer (wave length: 254 nm). Column : A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with between 3 and 10 m in particle diameter octadecylsilanized silica gel for liquid chromatography. Mobile phase : A mixture of water, acetonitrile, methanol and glacial acetic acid (650:175:175:1). Flow rate: 2.0 mL per minute. System suitability System performance: When the procedure is run with 5 L of the standard solution, the resolution of pchloroacetanilide peak and Indapamide peak is not less than 2.0, and symmetry factor of Indapamide peak is not more than 2.0. System repeatability: When the test is repeated 5 times with 5 L of the standard solution under the above operating conditions: the relative standard deviation of the peak area of Indapamide is not more than 2.0%.
Packaging and Storage Preserve in well-closed containers.

Indenolol Hydrochloride
OH OCH2CCH2NHCH H CH3 HCl H OCH2CCH2NHCH OH CH3 CH3 HCl CH3

Not more than 3.0% (1g, 105 C, Not more than 0.1%.

Residue on Ignition

Assay Weigh about 0.1 g of Indapamide, accurately weighed, dissolve in 5.0 mL of the internal standard solution, add the mobile phase to make exactly 100 mL, and use this solution as the test solution. Separately, weigh accurately about 20 mg of Indapamide RS, dissolve in 1.0 mL of internal standard solution, add the mobile phase to make exactly 20 mL, and use this solution as the standard solution. Perform the test with 5 L each of the test solution and the standard solution as directed under the Liquid chromatography according to the following operating conditions. Determine the ratios of peak areas of Indapamide, QT and QS to the peak area of the internal standard substance of each solution.

and enantiomer C15H21NO2HCl: 283.80 Indenolol Hydrochloride is a mixture of 1-(7-indenyl oxy)-3-iospropylamino-2-propanolmonohydrochloride and 1-(4-indenyloxy)-3-iospropylamino-2-propanol monohydrochloride. Indenolol Hydrochloride, when dried, contains not less than 98.5% and not more than 101.0% of indenolol hydrochloride (C15H21NO2HCl).
Description Indenolol Hydrochloride is a white to pale yellow crystal or crystalline powder. Hydrochloride is freely soluble in water or in glacial acetic acid, soluble in ethanol, or in chloroform, very slightly soluble in acetic anhydride, slightly soluble in ethyl acetate and practically insoluble in ether. Dissolve 1.0 g of Indenolol Hydrochloride in 10 mL of water: the pH of this solution is between 3.5 and 5.5.

Amount (mg) of indapamide (C16H16ClN3O3S) = 100 C QT

QS

C : Concentration, in mg per mL, Indapamide RS in

500 Monographs, Part I Indenolol Hydrochloride is affected in color by light

Identification (1) Dissolve 0.1 g of Indenolol Hydrochloride in 1 to 2 drops of dilute hydrochloric acid and 5 mL of water and add 1 mL of Reinecke salt TS: a red-purple precipitate is produced. (2) Determine the absorption spectra of solutions of Indenolol Hydrochloride and Indenolol Hydrochloride RS (1 in 50000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelength. (3) Determine the infrared absorption spectra of Indenolol Hydrochloride and Indenolol Hydrochloride RS, previously dried, as directed in the potassium bromide disc method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the wavenumbers. (4) A solution of Indenolol Hydrochloride (1 in 10) responds to the Qualitative Tests for chloride. Melting Point Between 140 C and 143 C.
1% Absorbance E1 cm (250 nm): Between 330 and 340 (after drying, 10 mg, water, 1000 mL).

drated trifluoroacetic acid (9 : 1) and use this solution as the test solution. Perform the test with 2 L of the test solution as directed under the Gas Chromatography according to the following conditions. Determine the areas of two adjacent peaks, Aa and Ab , having the retention times of about 16 minutes, where Aa is the peak area of shorter retention time and Ab is the peak area of longer retention time: the ratio, Aa /( Aa + Ab ) , is between 0.6 and 0.7. Operating conditions Detector: A hydrogen flame-ionization detector. Column: A glass column, about 2 mm in inside diameter and about 2 m in length, packed with siliceous earth for gas chromatography (150 to 180 m in particle diameter) coated with 65% phenyl-methyl silicon polymer for gas chromatography at the ratio of 2%. Column temperature: A constant temperature between 150 C and 170 C. Carrier gas: Helium. Flow rate: Adjust the flow rate so that the retention time of the peak showing earlier elution of the two peaks of Indenolol Hydrochloride is about 16 minutes. Selection of column: Proceed with 2 L of the test solution under the above operating conditions and calculate the resolution. Use a column with the resolution between the two peaks being not less than 1.1.
Assay Weigh accurately about 0.5 g of Indenolol Hydrochloride, previously dried, dissolve in 50 mL of a mixture of acetic anhydride and glacial acetic acid (4 : 1) and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from purple through blue to green (indicator: 3 drops of methylrosaniline chloride TS). Perform a blank determination and make any necessary correction.

Purity (1) Clarity and color of solutionDissolve 1.0 g of Indenolol Hydrochloride in 10 mL of water: the solution is clear and colorless to pale yellow. (2) Heavy metalsProceed with 1.0 g of Indenolol Hydrochloride according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Indenolol Hydrochloride according to Method 1 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.20 g of Indenolol Hydrochloride in 10 mL of chloroform and use this solution as the test solution. Pipet 1.0 mL of the test solution, add chloroform to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel with a fluorescent indicator for Thinlayer chromatography. Develop the plate with a mixture of 1,2-dichloroethane, dehydrated ethanol and strong ammonia water (70 : 15 : 2) to a distance of about 12 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution. Loss on Drying Not more than 0.5% (1 g, in vacuum, P2O5, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Isomer Ratio Dissolve 5 mg of Indenolol Hydrochloride in 1.0 mL of a mixture of ethyl acetate and dehy-

Each mL of 0.1 mol/L perchloric acid VS = 28.379 mg of C15H21NO2HCl

Packaging and Storage well-closed containers.

Preserve in light-resistant,

Indigocarmine
O NaO3S H N

N H O

SO3Na

Cl6H8N2Na2O8S2: 466.35 Indigocarmine, when dried, contains not less than 95.0% and not more than 101.0% of indigocarmine (Cl6H8N2Na2O8S2).

KP 9 501 of carbon dioxide and titrate, while being hot, with 0.1 mol/L titanium trichloride VS until the color of the solution changes from blue through yellow to orange. Each mL of 0.1 mol/L titanium trichloride VS = 23.318 mg of C16H8N2Na2O8S2
Packaging and Storage tight containers.

Description Indigocarmine is blue to dark blue powder or granules and is odorless. Indigocarmine is sparingly soluble in water and practically insoluble in ethanol or in ether. Indigocarmine is hygroscopic. When compressed, Indigocarmine has a coppery luster. Identification (1) A solution of Indigocarmine (1 in 100) is dark blue in color. Perform the following tests with this solution as the test solution: the dark blue color of each solution disappears. (i) Add 1 mL of nitric acid to 2 mL of the test solution, (ii) Add 1 mL of bromine TS to 2 mL of the test solution, (iii) Add 1 mL of chlorine TS to 2 mL of the test solution. and (iv) Add 2 mL of sodium hydroxide TS and 0.2 g of zinc dust to 2 mL of the test solution and warm. (2) Dissolve 0.1 g of Indigocarmine and Indigocarmine RS in 100 mL of an ammonium acetate solution (1 in 650). To 1 mL of this solution, add an ammonium acetate solution (1 in 650) to make 100 mL and determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 610 nm and 614 nm. (3) Ignite 1 g of Indigocarmine to carbonize. After cooling, add 20 mL of water to the residue, shake and filter the mixture: the filtrate responds to the Qualitative Tests for sodium salt and for sulfate. pH Dissolve 0.10 g of Indigocarmine in 20 mL of water: the pH of the solution is between 5.0 and 6.0. Purity (1) Water-insoluble substancesTake 1.00 g of Indigocarmine, add 200 mL of water, shake and filter through a tared glass filter (G4). Wash the residue with water until the blue color of the filtrate becomes practically colorless and dry the residue at 105 o C for 4 hours: the weight of the residue does not exceed 5.0 mg. (2) ArsenicPlace 0.8 g of Indigocarmine in a Kjeldahl flask, add 5 mL of sulfuric acid and 5 mL of nitric acid and ignite gently. Repeat the addition of 2 to 3 mL of nitric acid occasionally and continue to heat until a colorless to pale yellow solution is obtained. After cooling, add 15 mL of a saturated ammonium oxalate solution, heat the solution until dense white fumes are evolved and concentrate to 2 to 3 mL. After cooling, dilute with water to make 10 mL and perform the test with 5 mL of this solution as the test solution (not more than 5 ppm). Loss on Drying hours).

Preserve in light-resistant,

Indigocarmine Injection
Indigocarmine Injection is an aqueous solution for injection. Indigocarmine Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of indigocarmine (Cl6H8N2Na2O8S2: 466.35).
Method of Preparation Prepare as directed under Injections, with Indigocarmine. Description Indigocarmine Injection is a dark blue liquid. pHBetween 3.0 and 5.0. Identification (1) Take a volume of Indigocarmine Injection, equivalent to 0.02 g of Indigocarmine according to the labeled amount, add 1 mL of nitric acid: the dark blue color of the liquid disappears and a yellow-brown color develops. (2) Take a volume of Indigocarmine Injection, equivalent to 0.02 g of Indigocarmine according to the labeled amount, add 1 mL of bromine TS: the dark blue color disappears and a yellow-brown color develops. (3) Take a volume of Indigocarmine Injection, equivalent to 0.02 g of Indigocarmine according to the labeled amount, add 1 mL of chlorine TS: the dark blue color disappears and a yellow-brown color develops. (4) Take a volume of Indigocarmine Injection, equivalent to 0.01 g of Indigocarmine according to the labeled amount, add ammonium acetate solution (1 in 650) to make 1000 mL and determine the absorbance of the solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 610 nm and 614 nm. Sterility Test It meets the requirement. Insoluble Particulate Matter Test quirement.

It meets the re-

Not more than 10.0% (1 g, 105 C , 2

Residue on Ignition Not less than 28.0% and not more than 38.0% (after drying, 1 g). Assay Weigh accurately about 0.5 g of Indigocarmine, previously dried, add 15 g of sodium bitartrate and dissolve in 200 mL of water, boil with bubbling of a stream

Insoluble Particulate Matter Test for Injections It meets the requirement Determination of Volume of Injection in Containers It meets the requirement. Assay Measure exactly a volume of Indigocarmine In-

502 Monographs, Part I jection, equivalent to about 0.2 g of indigocarmine (Cl6H8N2Na2O8S2), add 6 g of sodium bitartrate and dissolve in water to make 200 mL. Then boil under a carbon dioxide stream and proceed as directed in the Assay under Indigocarmine. Each mL of 0.1 mol/L titanium trichloride VS = 23.318 mg of Cl6H8N2Na2O8S2
Packaging and Storage Preserve in light-resistant, hermetic, colored containers may be used.

KP 9 503 with the test solution and the standard solutions as directed under the Thin-layer chromatography. Spot 25 L each of the test solution and the standard solution on a plate of silica gel with fluorescent indicator for Thinlayer chromatography. Develop the plate with a mixture of dehydrated ether and glacial acetic acid (100 : 3) to a distance of about 10 cm and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.7 g of Indomethacin, previously dried, dissolve in 60 mL of methanol, add 30 mL of water and titrate with 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phenolphthalein TS). Perform a blank determination and make any necessary correction.

Indomethacin
O C Cl

CH3

CH3O

CH2COOH

C19H16ClNO4: 357.79 Indometacin, when dried, contains not less than 98.0% and not more than 101.0% of indometacin (C19H16ClNO4).
Description Indometacin is a white to pale yellowish white, very fine crystalline powder. Indometacin is sparingly soluble in methanol, in ethanol or in ether and practically insoluble in water. Indometacin dissolves in sodium hydroxide TS. Indometacin is colored by light Melting pointBetween 155 C and 162 C. Identification (1) Dissolve 2 mg of Indomethacin in 100 mL of methanol and determine the absorption spectrum of this solution as directed under the Ultravioletvisible Spectrophotometry: it exhibits a maximum between 317 nm and 321 nm. (2) Determine the infrared spectra of Indometacin and Indometacin RS, as directed in the potassium bromide disc method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears between the spectra, recrystallize the test and the RS with ether, filter and dry the crystals and perform the test with the crystals. (3) Perform the test with Indomethacin as directed under the Flame Coloration Test (2): a green color appears. Purity (1) AcidTake 1.0 g of Indomethacin, add 50 mL of water, shake for 5 minutes and filter. To the filtrate, add 0.20 mL of 0.1 mol/L sodium hydroxide VS and 1 drop of phenolphthalein TS: a red color develops. (2) Heavy metalsProceed with 1.0 g of Indometacin according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 1.0 g of Indomethacin according to Method 3 and perform the test (not more than 2 ppm). (4) Related substancesDissolve 0.10 g of Indomethacin in 10 mL of methanol and use this solution as the test solution. Pipet 1.0 mL of the test solution and add methanol to make exactly 50 mL. Pipet 5.0 mL of this solution, add methanol to make exactly 20 mL and use this solution as the standard solution, Perform the test

Each mL of 0.1 mol/L sodium hydroxide VS = 35.779 mg of C19H16ClNO4

Packaging and Storage tight containers.

Preserve in light-resistant,

Indomethacin Capsules
Indomethacin Capsules contain not less than 90.0% and not more than 110.0%of the labeled amount of indomethacin (C19H16CINO4: 357.79).
Method of Preparation Prepare as directed under Capsules, with Indometacin. Identification Powder the contents of Indomethacin Capsules. Take a portion of the powder, equivalent to 0.1 g of Indomethacin according to the labeled amount, add 20 mL of chloroform, shake well and centrifuge. Filter the supernatant liquid and evaporate the filtrate to dryness. After cooling, dissolve the residue in 20 mL of methanol. To 10 mL of this solution, add methanol to make 50 mL, then to 2 mL of this solution, add methanol to make 100 mL and use this solution as the test solution. Determine the absorption spectrum of the test solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 317 nm and 321 nm. Purity Related substancesPowder the content of Indomethacin Capsules. Take a portion of the powder, equivalent to 0.10 g of Indometacin according to the labeled amount, add exactly 10 mL of methanol, shake well, filter and use the filtrate as the test solution. Dissolve 0.025 g of Indometacin RS in methanol to make

504 Monographs, Part I exactly 50 mL. Pipet 1.0 mL of the solution, add methanol to make exactly 10 mL and use this solution as the standard solution. Proceed as directed in the Purity (4) under Indomethacin.
Dissolution Test Take 1 capsule of Indomethacin Capsules and perform the test using 900 mL of a mixture of water and phosphate buffer solution, pH 7.2, (4 : 1) at 100 revolutions per minute as directed in Method 1 under the Dissolution Test. Take 20 mL or more of the dissolved solution at 20 minutes after starting the test and filter through a membrane filter (less than 0.8 nm in pore size). Discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately about 0.03 g of Indomethacin RS, previously dried at 105 C for 4 hours, dissolve in a mixture of water and phosphate buffer solution, pH 7.2, (4 : 1) to make exactly 1000 mL and use this as the standard solution, Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 320 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Indometacin Capsules in 20 minutes should be not less than 75%.

Amount (mg) of indomethacin (C19H16ClNO4) = amount (mg) of Indomethacin RS

QT QS

Internal standard solutionA solution of butyl parahydroxybenzoate in methanol (1 in 1000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, 4.6 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (7 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of methanol and diluted phosphoric acid (1 in 1000) (7 : 3). Flow rate: Adjust the flow rate so that the retention time of Indometacin is about 8 minutes. System suitability System performance: Dissolve 50 mg of 4chhlorobenzoic acid, 30 mg of butyl p-hydroxybenzoate and 50 mg of Indometacin in 50 mL of methanol. Take 5 mL of this solution add the mobile phase to make 100 mL. When the procedure is run with 20 L of this solution, as directed under the above operating conditions, 4chlorobenzoic acid, butyl p-hydroxybenzoate and Indomethacin are eluted in this order, with the resolution between the peaks of 4-chlorobenzoic acid and butyl phydroxybenzoate being not less than 2.0 and between the peaks of butyl p-hydroxybenzoate and Indomethacin being not less than 5. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the ratios of the peak area of Indometacin to that of the internal standard is not more than 1.0%.
Packaging and Storage Preserve in tight containers.

Dissolution rate (%) with respect to the labeled amount of indometacin (C19H16ClNO4) = WS
AT 90 AS C

WS : Amount (mg) of Indomethacin RS, C: Labeled amount (mg) of indomethacin (C19H16ClNO4) in 1 capsule.

Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately the contents of not less than 20 Indomethacin Capsules. Powder the combined contents and weigh accurately a portion of the powder, equivalent to about 0.05 g of indomethacin (C19H16ClNO4). Dissolve in 40 mL of methanol and add methanol to make exactly 50 mL. Filter this solution, discarding the first 10 mL of the filtrate. Pipet the subsequent 5.0 mL of the filtrate, add exactly 3 mL of the internal standard solution, add the mobile phase to make 100 mL and use this solution as the test solution. Separately, weigh accurately about 0.05 g of Indomethacin RS, previously dried at 105 C for 4 hours and dissolve in methanol to make exactly 50 mL. Pipet 5.0 mL of the solution, add exactly 3 mL of the internal standard solution, add the mobile phase to make 100 mL and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Indometacin to that of the internal standard, for the test solution and the standard solution, respectively.

Indomethacin Suppositories
Indomethacin Suppositories contain not less than 90.0% and not more than 110.0% of the labeled amount of indometacin (C19H16ClNO4: 357.79).
Method of Preparation Prepare as directed under Suppositories, with Indomethacin. Identification Dissolve a quantity of Indomethacin Suppositories, equivalent to 0.05 g of Indometacin according to the labeled amount, in 20 mL of methanol by warming, add methanol to make 50 mL and filter, if necessary. Take 2 mL of this solution, add methanol to make 100 mL and use this solution as the test solution.

KP 9 505 Determine the absorption spectrum of the test solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 317 nm and 321 nm.
Dissolution Test Take 1 capsule of Indometacin Capsules, and perform the test using 900 mL of a mixture of water and phosphate buffer solution, pH 7.2, (4 : 1) as the test solution at 50 revolutions per minute as directed in the method 2. Take 20 mL or more of the dissolved solution at 60 minutes after startion the test and filter through a membrane filter (less than 0.8 m in pore size). Discard the first 10 mL of the filtrate, and use the subsequent filtrate as the test solution. Separately, weigh accurately about 30 mg of indometacin RS, previously dried at 105 for 4 hours, dissolve in a mixture of water and phosphate buffer solution, pH 7.2, (4 : 1) to make exactly 1000 mL, and use this as the standard solution. Determine the absorbances, AT and AS of the sample solution and the standard solution at 300 nm as directed under Ultraviolet visible Spectrophotometry. The dissolution rate of Indometacin Capsules in 20 minutes should be not less than 75%.

to that of the internal standard, for the test solution and the standard solution, respectively. Amount (mg) of indometacin (C19H16ClNO4) = amount (mg) of Indometacin RS
QT QS

Internal standard solutionA solution of butyl parahydroxybenzoate in methanol (1 in 1000). Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, 4 mm in inside diameter and 10 to 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 to 10 m in particle diameter). Column temperature: A constant temperature of about 25 C. Mobile phase: A mixture of methanol and diluted phosphoric acid (1 in 1000) (7 : 3). Flow rate: Adjust the flow rate so that the retention time of Indometacin is about 8 minutes. System suitability System performance: Dissolve 50 mg of 4chlorobenzoic acid, 30 ml of butyl p-hydroxybenzoate and 50 mg of Indometacin in 50 mL of methanol. Take 5 mL of this solution, and add 95 mLof the mobile phase, mix. When the procedure is run with 20 L of this solution, as directed under the above operating conditions, 4chlorobenzoic acid, butyl parahydroxybenzoate and Indomethacin are eluted in this order, with the resolution between the peaks of 4-chlorobenzoic acid and butyl parahydroxybenzoate being not less than 2.0 and beteen the peaks of butyl p-hydroxybenzoate and Indometacin being not less than 5.0. System repeatability: When the test is repeated 6 times with 20 L of the standard solution, as directed under the above operating conditions, the relative standard deviation of the ratios of peak area of Indometacin to that of the internal standard is not more than 1.0%.
Packaging and Storage well-closed containers.

Dissolution rate (%) with respect to the labeled amount of indometacin (C19H16ClNO4) = WS
AT 90 AS C

WS : Amount (mg) of Indometacin Reference standard. C : Labeled amount (mg) of indometacin (C19H16ClNO4) in 1 capsule

Uniformity of Dosage Units It meets the requirement. Assay Weigh accurately not less than 20 Indomethacin Suppositories, cut into small pieces carefully and mix well. Weigh accurately a portion of the mass, equivalent to about 0.05 g of indometacin (C19H16ClNO4), add 40 mL of tetrahydrofuran, warm at 40 C, dissolve by shaking, cool and add tetrahydrofuran to make exactly 50 mL. Filter the solution, discard the first 10 mL of the filtrate, pipet the subsequent 5.0 mL of the filtrate, add exactly 3 mL of the internal standard solution and add the mobile phase to make 100 mL. Allow the solution to stand for 30 minutes, filter through a membrane filter (0.5 m in pore size), discard the first 10 mL of the filtrate and use the subsequent filtrate as the test solution. Separately, weigh accurately about 0.05 g of Indometacin RS, previously dried at 105 o C for 4 hours and dissolve in tetrahydrofuran to make exactly 50 mL. Pipet 5.0 mL of the solution, proceed in the same manner as the test solution and use this solution as the standard solution. Perform the test with 20 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS , of the peak area of Indometacin

Preserve in light-resistant,

Inositol
HO OH HO OH

HO

OH

Inosit

C6H12O6: 180.16

Inositol, when dried, contains not less than 97.0% and

506 Monographs, Part I not more than 101.0% of inositol (C6H12O6).

Description Inositol is a white crystal or crystalline powder, is odorless and has a sweet taste. Inositol is very soluble in water, and practically insoluble in ethanol, ether or chloroform. Inositol shows no specific optical rotation. pHThe pH of a solution of Inositol is neutral. Identification (1) Add 6 mL of nitric acid to 1 mL of Inositol solution in water (1 in 50). Evaporate in a waterbath and add 0.5 mL of strontium nitrate solution (1 in 10) to the residue. Evaporate in a water-bath again: a redpurple color is observed. (2) Add 1 mL of lead acetate TS to 4 mL of Inositol solution in water (1 in 100), shake and mix. Heat in a water-bath for 5 minutes: the solution changes to a halftransparent gel. (3) The melting point of hexaacetylinositol obtained from the Assay is between 212 C and 216 C. Melting Point Between 223 C and 227 C. Purity (1) Clarity and color of solutionDissolve about 1.0 g of Inositol in 10 mL of water: the solution is clear and colorless. (2) ChloridePerform the test with about 2.0 g of Inositol. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.005%). (3) SulfatePerform the test with about 4.0 g of Inositol. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.006%). (4) Heavy metalsProceed with 1.0 g of Inositol according to Method 1 and perform the test. Prepare the control solution with 2.5 mL of standard lead solution (not more than 25 ppm). (5) IronDissolve about 2.0 g of Inositol in 40 mL of water, add 2 mL of hydrochloric acid, 40 mg of ammonium persulfate and 2 mL of Ammonium thiocyanate TS: the solution is not more intense than the following control solution.

viously dried, in a beaker. Add 5 mL of the mixture of 1 mL of dilute sulfuric acid and 50 mL of acetic anhydride, heat the covered beaker in a water-bath for 20 minutes and cool in ice. Add 100 mL of water and boil for 20 minutes. After cooling, transfer the substance in a beaker to a separating funnel, wash the wall of the beaker with a small volume of water and combine. Extract with 30 mL, 25 mL, 20 mL, 15 mL, 10 mL and 10 mL volumes of chloroform. Combine all chloroform extracts and wash with 10 mL of water. Filter the chloroform layer using a pledget of absorbant cotton, with 10 mL of chloroform. After combine the filtrate and the washing solution, evaporate on a water-bath and dry at 105 C for 2 hours. After cooling, weigh the substance and it is the weight of hexaacetylinositol (C18H24O12: 432.38). Amount (mg) of inositol (C6H12O6) = amount (mg) of hexaacetylinositol (C18H24O12) 0.4167
Packaging and Storage Preserve in well-closed containers.

Insulin
Insulin is obtained from the pancreas of healthy bovine or porcine, that has blood sugar-decreasing activity. Potency of Insulin, calculated on the dried basis, is not less than 26 Insulin units in each mg. Insulin is labeled to indicate the animal species from which it is derived.
Description Insulin is a white, crystalline powder and is odorless. Insulin is practically insoluble in water, in ethanol or in ether. Insulin dissolves in diluted hydrochloric acid (1 in 360) or in ether. Insulin is hygroscopic. Identification Dissolve 0.01 g of Insulin in 10 mL of 0.1 mol/L hydrochloric acid TS and use this solution as the test solution. Proceed as the Identification for Insulin Injection with the test solution. Purity Clarity and color of solutionDissolve 0.10 g of Insulin in 10 mL of diluted hydrochloric acid (1 in 360): the solution is clear and colorless to pale yellow. Zinc Content Weigh accurately about 0.01 g of Insulin, dissolve in 5 mL of 0.1 mol/L hydrochloric acid TS and water to make exactly 50 mL. If necessary, dilute the solution with water so as to contain 0.4 to 1.0 g of zinc (Zn: 65.41) per mL and use this solution as the test solution. Add water to an accurately measured volume of standard zinc solution for Atomic Absorption Spectrophotometry to make a solution containing 0.3 to 1.2 g of zinc (Zn: 65.41) per mL and use this solution as the

Control solutionPrepare in the same manner, using 1.0 mL of standard iron solution instead of Inositol. (6) CalciumDissolve about 1.0 g of Inositol in 10 mL of water, add 1 mL of Ammonium hydroxide TS and allow to stand for 1 minute: the solution is transparent. (7) Reducible substance to Fehlings TSDissolve about 0.5 g of Inositol in 10 mL of water, add 5 mL of Fehlings TS, boil for 3 minutes and allow to stand for 30 minutes: no orange-red precipitate is observed.
Loss on Drying Not more than 0.5% (1 g, 105 C, 4 ours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Inositol, pre-

KP 9 507 standard solution. Perform the test with the test solution and the standard solution as directed under the Atomic Absorption Spectrophotometry according to the following conditions and determine the amount of zinc in the test solution using the calibration curve obtained from the absorbance of the standard solution: the amount of zinc is not less than 0.27% and not more than 1.08%, calculated on the dried basis. Gas: Combustible gas-Acetylene gas. Supporting gas-Air. Lamp: Zinc hollow-cathode lamp. Wavelength: 213.9 nm.
Loss on Drying Not more than 10.0% (0.2 g, 105 C, 16 hours). Residue on Ignition Weigh accurately 0.02 to 0.04 g of Insulin in a tared platinum dish, add 2 drops of nitric acid and heat the dish at first very gently and then strongly to incinerate. Place the dish in a muffle furnace, heat at 600C for 15 minutes, cool in a desiccator (silica gel) and weigh: the weight of the residue is not more than 2.5%. Nitrogen Content Weigh accurately about 0.02 g of Insulin, dissolve in 10 mL of 0.1 mol/L hydrochloric acid TS and perform the test as directed under the Nitrogen Determination: not less than 14.5% and not more than 16.5% of nitrogen (N: 14.01) is found, calculated on the dried basis. Assay (1) Animals: Select healthy rabbits weighing not less than 1.8 kg. Keep the rabbits in the laboratory not less than 1 week before use in the Assay by feeding them with an appropriate uniform diet and water. (2) Diluent for insulin: Dissolve 1.0 to 2.5 g of phenol or n-cresol in 500 mL of 0.01 mol/L hydrochloric acid VS and add 14 to 18 g of glycerin and 0.01 mol/L hydrochloric acid VS to make 1000 mL. (3) Standard stock solution: Weigh accurately about 0.02 g of insulin RS and dissolve in the diluent for Insulin to make a standard stock solution containing exactly 20.0 units in each mL. Preserve this solution between 1 C and 15 C and use within 6 months.

(4) Standard solution: Dilute two portions of the standard stock solution to make two standard solutions with the diluent for Insulin, one to contain exactly 2.0 units in each mL which is designated as the high-dose standard solution, SH and the other to contain exactly 1.0 unit in each mL which is designated as the low-dose standard solution, SL. (5) Test solution: weigh accurately about 0.02g of Insulin according to the labeled units, dissolve with the diluents for Insulin to make two different test solutions, one to contain exactly 2.0 units in each mL which is designated as the high-dose test solution, TH and the other to contain exactly 1.0 unit in each mL which is designated as the low-dose test solution, TL. (6) Dose for injection: Select the dose for injection on the basis of trial or experience. Inject an identical volume, usually 0.3 to 0.5 mL, of the standard solutions and the test solutions throughout the whole run. (7) Procedure: Divide the animals into 4 equal groups of not less than 6 animals each, with least difference in body weight. Withhold all food, except water, for not less than 14 hours before the injections and withhold water during the Assay until the final blood test is taken. Handle the animals with care in order to avoid undue excitement. Inject into each of the animals subcutaneously the dose of the standard solutions and the test solutions indicated in the following design. First group Second group SH SL Third group TH Fourth group TL

The second injection should be made on the day after the first injection or within 1 week, using the dose of the standard solutions and the test solutions indicated in the following design. First group Second group TL TH Third group Fourth group SL SH

At 1 hour and 2.5 hours after the time of injection, obtain a sufficient blood to perform the test from a marginal ear vein of each animal and determine the blood sugar content of the blood samples according to (8).

Conversion Table for the blood sugar content (%) mL* 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.385 0.355 0.331 0.310 0.290 0.270 0.251 1 0.382 0.352 0.329 0.308 0.288 0.268 0.249 2 0.379 0.350 0.327 0.306 0.286 0.266 0.247 3 0.376 0.348 0.325 0.304 0.284 0.264 0.245 4 0.373 0.345 0.323 0.302 0.282 0.262 0.243 5 0.370 0.343 0.321 0.300 0.280 0.260 0.241 6 0.367 0.341 0.318 0.298 0.278 0.259 0.240 7 0.364 0.338 0.316 0.296 0.276 0.257 0.238 8 0.361 0.336 0.314 0.294 0.274 0.255 0.236 9 0.356 0.333 0.312 0.292 0.272 0.253 0.234

508 Monographs, Part I

0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

0.232 0.213 0.195 0.177 0.159 0.141 0.124 0.106 0.088 0.070 0.052 0.034 0.017

0.230 0.211 0.193 0.175 0.157 0.139 0.122 0.104 0.084 0.068 0.050 0.032 0.015

0.228 0.209 0.191 0.173 0.155 0.138 0.120 0.102 0.084 0.066 0.048 0.031 0.014

0.226 0.208 0.190 0.172 0.154 0.136 0.119 0.101 0.083 0.065 0.047 0.029 0.012

0.222 0.206 0.188 0.170 0.152 0.134 0.117 0.099 0.081 0.063 0.045 0.027 0.010

0.222 0.204 0.186 0.168 0.150 0.132 0.115 0.097 0.079 0.061 0.043 0.025 0.008

0.221 0.202 0.184 0.166 0.148 0.131 0.113 0.093 0.077 0.059 0.041 0.024 0.007

0.219 0.200 0.182 0.164 0.146 0.119 0.111 0.093 0.075 0.057 0.039 0.022 0.005

0.217 0.199 0.181 0.163 0.145 0.127 0.110 0.092 0.074 0.056 0.038 0.020 0.003

0.215 0.197 0.179 0.161 0.143 0.125 0.108 0.090 0.072 0.054 0.036 0.019 0.002

*Indicates the volume of 0.005 mol/L sodium thiosulfate VS required in titration. For example, if the amount was 1.28 mL, the blood sugar content would be 0.127% from the above table. (8) Blood sugar determination: Place 5.0 mL of a solution of zinc sulfate (9 in 2000) in a test tube, 18 mm in outside diameter and 165 mm in length, add 1.0 mL of a solution of sodium hydroxide (1 in 250) and add gently 0.10 mL of the blood sample to the mixture in the test tube using a blood sugar pipet. Suck up the supernatant liquid into the pipet, wash out the remaining blood in the inner wall of the pipet and repeat this procedure. Shake thoroughly the contents in the test tube and heat the test tube in a water- bath for 3 minutes. Filter the mixture through a funnel, 30 to 40 mm in diameter in which a pledget of absorbent cotton, previously washed with two 3 mL volumes of warm water, has been placed, receive the filtrate into a test tube, 30 mm in inside diameter and 90 mm in length, wash the test tube and the funnel with two 3 mL volumes of water and combine the washings with the filtrate. Add 2.0 mL of alkaline potassium ferricyanide TS, heat in a water-bath for 15 minutes, cool immediately, add 3.0 mL of potassium iodide-zinc sulfate TS and 2.0 mL of diluted glacial acetic acid (3 in 100) and titrate the liberated iodine with 0.005 mol/L sodium thiosulfate VS (indicator: 2 to 4 drops of starch-sodium chloride TS). Perform a blank determination and make any necessary correction. From the consumed volume (mL) of 0.005 mol/L sodium thiosulfate VS, obtain the blood sugar content (%) according to the table of the previous page.. (9) Calculation: Sum up the two blood sugar values of each animal after each injection. Subtract the blood sugar value effected by the first injection from that effected by the second injection of each animal in the first group and the third group. The differences are symbolized as y1 and y3, respectively. Subtract the blood sugar value effected by the second injection from that effected by the first injection of each animal in the second group and the fourth group. The differences are symbolized as y2 and y4, respectively. Sum up not less than 6 values of individual differences in the blood sugar values, y1, y2, y3 and y4 to obtain Y1, Y2, Y3 and Y4, respectively. Units in each mg of Insulin = antilog M units in each mL of the high-dose b standard solution a Yb M = 0.301 x Y a Ya = -Y1+Y2+Y3-Y4 Yb = Y1+Y2+Y3+Y4 a: Mass (mg) of the sample, b: Total volume (mL) of the high-dose test solution prepared by diluting the volume of the test with diluent for Insulin. Compute L (p = 0.95) by using the following equation: L should be not more than 0.1212. If L exceeds 0.1212, repeat the Assay by increasing the number of animals or improving the Assay conditions in a better way until L becomes not more than 0.1212.

L = 2 (C 1)(CM 2 + 0.09062)

C=

Yb2 Yb2 4 fs 2 t 2

KP 9 509

s2 =
Y = Y12

y2 f
n
2

+ Y2 + Y32 + Y42

n = 4( f 1)
f : Number of the animals of each group. y2 : The sum of spuares of y1, y2, y3 and y4in each group. t2 : Value shown in the following table against n for which s2 is calculated. n 1 2 3 4 5 6 7 8 9 10 11 12 t2 = F1 161.45 18.51 10.129 7.709 6.608 5.987 5.591 5.318 5.117 4.965 4.844 4.747 n 13 14 15 16 17 18 19 20 21 22 23 24 t2 = F1 4.667 4.600 4.543 4.494 4.451 4.414 4.381 4.351 4.325 4.301 4.279 4.260 n 25 26 27 28 29 30 40 60 120 t2 = F1 4.242 4.225 4.210 4.196 4.183 4.171 4.085 4.001 3.920 3.841

Insulin Injection, equivalent to 500 to 1000 units according to the labeled units, in a tared platinum dish and evaporate slowly by heating on a water-bath to dryness. Add 2 drops of nitric acid to the residue and heat at first very gently, then strongly to incinerate. Place in a muffle furnace and heat at 600 C for 15 minutes, cool in a desiccator (silica gel) and weigh: the weight of the residue is not more than 1.0 mg for each labeled 1000 units.
Nitrogen Content Perform the test as directed under the Nitrogen Determination: not less than 0.50 mg and not more than 0.64 mg of nitrogen (N: 14.01) is found for each labeled 100 units. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections It meets the requirement Determination of Volume of Injection in Containers It meets the requirement Assay Proceed with Insulin Injectionn as directed in the Assay under Insulin, and (5) Test solution and (9) Calculation are as follows. (5) Test solution: Aaccording to the labeled units, dilute Insulin Injection with the diluent for Insulin to make two different test solutions, one to contain exactly 2.0 units in each mL which is designated as the highdose test solution, TH and the other to contain exactly 1.0 unit in each mL which is designated as the low-dose test solution, TL. (9) Calculation: Proceed as directed in the Assay (9) Calculation under Insulin, using the following equation.

Packaging and Storage Preserve in tight containers not more than 8 C.

Insulin Injection
Insulin Injection is an aqueous solution for injection. Insulin Injection contains not less than 95.0% and not more than 105.0% of the labeled Insulin units.
Method of Preparation Suspend Insulin in water for injection, dissolve by adding hydrochloric acid and prepare as directed under Injections. Insulin Injection contains 0.10 to 0.25 g of phenol or cresol and 1.4 to 1.8 g of concentrated glycerin for each 100 mL of Insulin Injection. Insulin Injection should not contain sodium chloride. Description Insulin Injection is a clear, colorless or pale yellow liquid. Identification Adjust Insulin Injection to pH between 5.1 and 5.3 with a solution of sodium hydroxide (1 in 100): a precipitate is produced. Adjust the solution to a pH between 2.5 and 3.5 with dilute hydrochloric acid: the precipitate dissolves. pH

Units in each mg of Insulin = antilog M x units in b each mL of the high-dose standard solution a a: Volume (mL) of the sample. instead of following equation Units in each mg of Insulin = antilog M units in b each mL of the high-dose standard solution a a: Mass (mg) of the sample
Packaging and Storage Preserve in hermetic containers, Avoiding freezing in cold place. Expiration Date 24 months after preparation.

Between 2.5 and 3.5

Residue on Ignition Measure exactly a volume of

510 Monographs, Part I

Insulin Zinc Injection (Aqueous Suspension)

Insulin Zinc Injection (Aqueous Suspension) is an aqueous suspension for injection. Insulin Zinc Injection (Aqueous Suspension) contains not less than 90.0% and not more than 110.0% of the labeled Insulin units and not less than 0.12 mg and not more than 0.30 mg of zinc (Zn: 65.41) for each labeled 100 units.
Method of Preparation Prepare as directed under Injections, with Insulin and Zinc Chloride. Each 100 mL of Insulin Zinc Injection (Aqueous Suspension) contains 0.15 to 0.17 g of sodium acetate, 0.65 to 0.75 g of sodium chloride and 0.09 to 0.11 g of methyl parahydroxybenzoate. Description Insulin Zinc Injection (Aqueous Suspension) is a white suspension. When allowed to stand, it separates into a white precipitate and colorless supernatant liquid and it readily becomes a suspension again on gentle shaking. When it is examined microscopically, most part of the particles in the suspension are crystals, the dimension of which is mostly 10 to 40 m. Others are amorphous with less than 2 m. Identification Adjust the pH of Insulin Zinc Injection (Aqueous Suspension) to between 2.5 and 3.5 with dilute hydrochloric acid: the particles dissolve and the solution is clear and colorless. pH

Zinc Injection (Aqueous Suspension), equivalent to about 600 units according to the labeled units, centrifuge, discard the supernatant liquid, suspend the residue in 5 mL of water, add 10 mL of sodium acetateacetone TS, shake for 3 minutes and centrifuge. Discard the supernatant liquid and repeat the above treatment on the residue. Wash down the residue into a Kjeldahl flask with 15 mL of sulfuric acid and perform the test as directed under the Nitrogen Determination: the amount of nitrogen (N: 14.01) is not less than 55.0 and not more than 70.0% of the total nitrogen content. Calculate the total nitrogen content for insulin units of a sample from the values of nitrogen obtained in the Nitrogen content.
Packaging and Storage Preserve in hermetic containers, avoiding freezing in cold place. Expiration Date 24 months after preparation.

Insulin Zinc Protamine Injection (Aqueous Suspension)

Insulin Zinc Protamine Injection (Aqueous Suspension) is an aqueous suspension for injection. Insulin Zinc Protamine Injection contains not less than 90.0% and not more than 110.0% of the labeled Insulin units and not less than 0.20 mg and not more than 0.30 mg of zinc (Zn: 65.41) for each labeled 100 units.
Method of Preparation Prepare as directed under Injections, with Insulin, Protamine Sulfate and Zinc Chloride. Insulin Zinc Protamine Injection contains 0.38 to 0.63 g of dibasic sodium phosphate, 1.4 to 1.8 g of concentrated Glycerin and 0.18 to 0.22 g of Cresol or 0.22 to 0.28 g of Phenol for each 100 mL of Insulin Zinc Protamine Injection (Aqueous Suspension). Description Insulin Zinc Protamine Injection (Aqueous Suspension) is a white suspension. When allowed to stand, it separates into a white precipitate and a colorless, supernatant liquid and it readily becomes suspension again on gentle shaking. When it is examined microscopically, no large particles are seen. Identification Adjust the pH of Insulin Zinc Protamine Injection (Aqueous Suspension) to between 2.5 and 3.5 with dilute hydrochloric acid: the particles dissolve and the solution is clear and colorless. pH

Between 7.1 and 7.5.

Purity Dissolved insulinPerform the test according to Purity (2) of Insulin Zinc Protamine Injection (Aqueous Suspension). Nitrogen content Perform the test as directed under the nitrogen determination: not less than 0.5 mg and not more than 0.64 mg of nitrogen (N: mol.wt. 14.01) is found for each labeled 100 Units. Sterility Test It meets the requirement.

Determination of Volume of Injection in Containers It meets the requirement. Assay (1) InsulinProceed as directed in the Assay under the Insulin Injection with the clear liquid obtained from Crystalline Insulin Zinc Injection (Aqueous Suspension) by adjusting the pH to about 2.5 with diluted hydrochloric acid (1 in 100). (2) ZincPerform the test according to Assay (2) of Insulin Zinc Protamine Injection (Aqueous Suspension). (3) Crystalline insulinMeasure accurately Insulin

Between 7.0 and 7.4.

Purity (1) ProteinPerform the test as directed under the Nitrogen Determination: not exceeding 1.25 mg of nitrogen (N: 14.01) is found for each labeled 100 units.

KP 9 511 (2) Dissolved InsulinPerform the following test with the clear liquid obtained by centrifuging Insulin Zinc Protamine Injection (Aqueous Suspension): not more than 2.5% of the labeled units is found. Use a clear liquid of Insulin Zinc Protamine Injection (Aqueous Suspension) as the test solution and prepare the standard solution by proceeding as directed in the Assay (4) under Insulin Injection to adjust its concentration to 2.5% of the labeled units of Insulin Zinc Protamine Injection (Aqueous Suspension). Divide the healthy rabbits weighing not less than 1.8 kg, fasted for not less than 14 hours before injection, into 2 equal groups of not less than 3. Inject subcutaneously an amount of the test solution or the standard solution equivalent to 0.3 units per kg of body weight. Collect blood before and 1 hour and 2.5 hours after injection, proceed as directed in the Assay (8) under Insulin Injection and calculate the ratios of the average blood sugar content in each rabbit measured 1 hour and 2.5 hours after injection to the content before injection: the mean value for the group injected with the test solution is not less than the mean value for the group injected with the standard solution.
Sterility Test It meets the requirement. Determination of Volume of Injection in Containers It meet the requirement. Assay (1) InsulinProceed as directed in the Assay under Insulin with the clear liquid obtained by adjusting the pH to 2.5 with diluted hydrochloric acid (1 in 1000) with alterations v) test solution and ix) calculation as follows. v) Test solution According to labeled units, dilute two portions of Insurin Zinc Protamine Injection to make two test solutions with diluents for Insurin, one to contain exactly 2.0 units in each mL which is designated as the high-dose test solution, TM and the other to contain exactl 1.0 unit in each mL which is designated as the low-dose test solution,TL. ix) Calculation Proceed as directed in the Assay ix) Calculation under Insurin Injection, using the following equation.

Zinc Protamine Injection (Aqueous Suspension), equivalent to about 200 units according to the labeled units, add 1 mL of 0.1 mol/L hydrochloric acid TS and sufficient water to make exactly 200 mL, dilute with water to contain 0.6 to 1.0 g of zinc in 1 mL and use this solution as the test solution. Separately, pipet a volume of standard zinc solution for the Atomic Absorption Spectrophotometry, dilute with water to contain 0.4 to 1.2 g of zinc per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution according to the Atomic Absorption Spectrophotometry under the following conditions and determine the amount of zinc in the test solution using the analytical curve obtained from the absorbance of the standard solution. Gas: Combustible gas - Acetylene gas. Supporting gas - Air. Lamp: Zinc hollow-cathode lamp. Wavelength: 213.9 nm.
Packaging and Storage Preserve in hermetic containers. Store at a cold place and avoid freezing. Expiration Date 24 months after preparation.

Neutral Insulin Injection

Neutral Insulin Injection is an aqueous solution for injection. Neutral Insulin Injection contains not less than 90.0% and not more than 110.0% of the labeled Insulin units.
Method of Preparation Prepare as directed under the Injections, with Insulin. Neutral Insulin Injection contains a buffering agent, preservative and isotonizer. Description Neutral Insulin Injection is a clear, colorless liquid. Identification Adjust the pH of Neutral Insulin Injection to between 5.1 and 5.3 with dilute hydrochloric acid: a precipitate is produced, dissolves when the pH is adjusted to between 2.5 and 3.5 with additional dilute hydrochloric acid: a precipitate is dissolved. pH

Units in each mg of Insulin = antilog M x units in each mL of the high-dose standard solution x b/a a: Volume (mL) of the sample. instead of following equation Unit in each mg of Insurin = antilog M x units in each mL of the high-dose standard solution x b/a a: Mass (mg) of the sample (2) ZincMeasure accurately a volume of Insulin

Between 6.9 and 7.8

Purity ZincMeasure exactly a volume of Neutral Insulin Injection, equivalent to about 200 units according to the labeled units, add 1 mL of 0.1 mol/L hydrochloric acid TS and sufficient water to make exactly 200 mL, dilute with water to make a solution containing 0.4 g, 0.8 g, 1.0 g, 1.2 g and 1.6 g of zinc (Zn: 65.41) per mL and use this solution as the test solution. Separately, pipet a volume of standard zinc solution for atomic absorption spectrophotometry, dilute with water to make solutions containing 0.4 g, 0.8 g,

512 Monographs, Part I 1.0 g, 1.2 g and 1.6 g of zinc (Zn: 65.41) per mL, respectively, and use these solutions as the standard solutions. Perform the test with the test solution and the standard solutions as directed under the Atomic Absorption Spectrophotometry according to the following conditions and determine the amount of zinc in the test solution using the calibration curve obtained from the absorbances of the standard solutions: the amount of zinc (Zn: 65.41) is not more than 40.0 g for each labeled 100 units. Gas: Combustible gas-Acetylene gas. Supporting gas-Air. Lamp: Zinc hollow-cathode lamp. Wavelength: 213.9 nm.
Nitrogen Content Measure exactly a volume of Neutral Insulin Injection, equivalent to about 400 units according to the labeled units and perform the test as directed under the Nitrogen Determination: not less than 0.50 mg and not more than 0.64 mg of nitrogen (N: 14.01) is found for each labeled 100 units. Sterility Test It meets the requirement. Foreign Insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections It meets the requirement. Determination of Volume of Injection in Containers It meets the requirement. Assay Proceed as directed in the Assay under Insulin Injection. Packaging and Storage Preserve in hermetic containers in a cold place and avoid freezing. Expiration Date 24 months after preparation.

Iodamide is slightly soluble in water or ethanol, and practically insoluble in ether. Iodamide dissolves in sodium hydroxide TS or sodium carbonate TS. The color of Iodamide is gradually colored by light.
Identification (1) Take 10 mg of Iodamide, add 5 mL of hydrochloric acid and heat in a water-bath for 5 minutes: the solution responds to the Qualitative Tests for primary aromatic amines. (2) Heat about 0.1 g of Iodamide over a flame: a purple gas evolves. (3) Determine the infrared spectra of Iodamide and Iodamide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. If any difference appears, weigh 1 g of Iodamide, add 100 mL of water and dissolve by heating, boil gently and concentrate the solution to about 30 mL. After cooling, collect the separated crystals by filtration, dry and perform the test in the same manner. Purity (1) Clarity and color of solutionDissolve 1.0 g of Iodamide in 10 mL of diluted sodium hydroxide TS (1 in 5): the solution is clear and colorless. (2) Primary aromatic aminesDissolve 0.20 g of Iodamide in 5 mL of water and 1 mL of sodium hydroxide TS, add 4 mL of a sodium nitrite solution (1 in 100) and 10 mL of 1 mol/L hydrochloric acid TS, shake well and allow to stand for 2 minutes. To this solution, add 5 mL of ammonium sulfamate TS, shake thoroughly, allow to stand for 1 minute, add 0.4 mL of a solution of -naphthol in ethanol (1 in 10), 15 mL of sodium hydroxide TS and water to make exactly 50 mL and determine the absorbance at 485 nm as directed under the Ultraviolet-visible Spectrophotometry, using a solution, prepared in the same manner, as the blank: the absorbance of the solution is not more than 0.12. (3) Soluble halideDissolve 2.5 g of Iodamide in 20 mL of water and 2.5 mL of ammonia TS and then add 20 mL of dilute nitric acid and water to make 100 mL. Allow to stand for 15 minutes with occasional shaking and filter. Discard the first 10 mL of the filtrate, transfer 25 mL of the subsequent filtrate to a Nessler tube and add ethanol to make 50 mL. Use this solution as the test solution and proceed as directed under the Chloride Limit Test. Prepare the control solution with 0.10 mL of 0.1 mol/L hydrochloric acid VS and 6 mL of dilute nitric acid and dilute with water to make 25 mL, then with ethanol to 50 mL. (4) IodineDissolve 0.20 g of Iodamide in 2 mL of sodium hydroxide TS, add 2.5 mL of 1 mol/L sulfuric acid TS, allow to stand for 10 minutes with occasional shaking, then add 5 mL of chloroform, shake vigorously and allow to stand: the chloroform layer remains colorless. (5) Heavy metalsProceed with 2.0 g of Iodamide according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution

Iodamide
COOH I I

CH3COHN I

CH2NHCOCH 3

C12H11I3N2O4: 627.94 Iodamide contains not less than 98.5% and not more than 101.0% of iodamide (C12H11I3N2O4), calculated on the dried basis.
Description Iodamide is a white, crystalline powder and is odorless.

KP 9 513 (not more than 10 ppm). (6) ArsenicPrepare the test solution with 1.0 g of Iodamide according to Method 3 and perform the test (not more than 2 ppm).
Loss on Drying Not more than 3.0% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Iodamide in a saponification flask, dissolve in 40 mL of sodium hydroxide TS, add 1 g of zinc dust, connect the flask with a reflux condenser, boil for 30 minutes, cool and filter. Wash the flask and filter paper with 50 mL of water and combine the washings with the filtrate. Add 5 mL of glacial acetic acid and titrate with 0.1 mol/L silver nitrate VS until the color of the precipitate changes from yellow to green (indicator: 1 mL of tetrabromophenolphthalein ethyl ester TS).

dered iodine with 20 mL of water and filter the mixture. To 10 mL of the filtrate, add diluted sulfurous acid (1 in 5) drop-wise until the yellow color disappears. Add 1 mL of ammonia TS, followed by 1 mL of silver nitrate TS in small portions and add water to make 20 mL. Shake well, filter and after discarding the first 2 mL of the filtrate, take 10 mL of the subsequent filtrate. To the filtrate, add 2.0 mL of nitric acid and water to make 20 mL: the solution so obtained has no more turbidity than the following control solution. Control solutionTo 0.20 mL of 10 mmol/L hydrochloric acid VS, add 5 mL of water, 2.5 mL of ammonia TS, 1 mL of silver nitrate TS, 2.0 mL of nitric acid and water to make 20 mL.
Assay Place 1 g of potassium iodide and 1 mL of water in a glass-stoppered flask, weigh accurately, add 0.3 g of Iodine to the flask and weigh accurately again. Dissolve the iodine by gentle shaking, add 20 mL of water and 1 mL of dilute hydrochloric acid and titrate with 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of starch TS).

Each mL of 0.1 mol/L silver nitrate VS = 20.931 mg of C12H11I3N2O4

Packaging and Storage tight containers.

Preserve in light-resistant,

Each mL of 0.1 mol/L sodium thiosulfate VS = 12.690 mg of I

Packaging and Storage Preserve in tight containers.

Iodine
I: 126.90
OH

Iodixanol
OH H N O O H N OH HO

Iodine contains not less than 99.5% and not more than 101.0% of iodine (I).
Description Iodine is a grayish black plate or heavy, granular crystal and has a metallic luster and a characteristic odor. Iodine is freely soluble in ether, soluble in ethanol, sparingly soluble in chloroform and very slightly soluble in water. Iodine dissolves in potassium iodide TS. Iodine sublimes at room temperature. Identification (1) A solution of Iodine in ethanol (1 in 50) shows a red-brown color. (2) A solution of Iodine in chloroform (1 in 1000) shows a red-purple to purple color. (3) Add 0.5 mL of starch TS to 10 mL of a saturated solution of Iodine: a dark blue color is observed. When the mixture is boiled, the color disappears and it reappears on cooling. Purity (1) Non-volatile residueSublime 2.0 g of Iodine in a water-bath and dry the residue at 105C for 1 hour: the weight of the residue is not more than 1.0 mg.
HO

I OH H N

I OH H N OH

N O O CH3 OH H3C

N O O

C35H44I6N6O15 : 1550.18 Iodixanol contains not less than 98.6% and not more than 101.0% of iodixanol (C35H44I6N6O15), calculated on the anhydrous basis.
Description Iodixanol is a white, amorphous powder and is odorless. Iodixanol is very soluble in water. Iodixanol is hygroscopic. Identification (1) Determine the infrared spectra of Iodixanol and Iodixanol RS, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) The retention times of the two principal peaks obtained from the test solution of the related substance (2) corresponds to these obtained from the standard solution (2) under purity. A third isomer may appear as a

(2) Chloride or bromideMix 1.0 g of finely pow-

514 Monographs, Part I minor peak. (3) Take about 0.5 g of Iodixanol, in the crucible, heat: violet vapors are evolved.
Specific Optical Rotation [ ]25 D : Between -0.5 and +0.5 (1 g, water, 20 mL, 100 mm). Purity (1) Heavy metalsProceed with 1.0 g of Iodixanol according to Method 1 and perform the test (not more than 10 ppm). Prepare the control solution with 1.0 mL of standard lead solution (not more than 10 ppm). (2) Free iodineWeigh 2.0 g of Iodixanol to a glass-stoppered tube, add 20 mL of water, 5 mL of toluene, and 5 mL of 1mol/L sulfuric acid TS, shake vigorously, and allow to stand: the toluene layer shows no red or pink color. (3) Free iodide Weigh 5.0 g of Iodixanol, add about 30 mL of water, dissolve and titrate with 0.001mol/L silver nitrate VS. (potentiometric titration, Endpoint Detection Method in Titrimetry). Not more than 10 g of iodide per g is found.

C AT AB W AS AB

C: Concentration of Iohexol Related Compound II RS in the standard solution (g /mL). W: Mass of Iodixanol (mg) taken to prepare the test solution. (5) CalciumWeigh accurately about 2.0 g of Iodixanol, dissolve in 10 mL of water, add 2 mL of internal standard solution, add water to make exactly 20 mL, and use this solution as the test solution. Perpare a solution containing 10 g of Calcium per 1 mL with the standard Calcium solution, pipet 0.5, 2.5, 5.0, and 10.0 mL of this solution, add 5.0 mL each of the Internal standard solution, add water to make exactly 50 mL, and use these solutions as the standard solution (1), (2), (3) and (4). Pipet 5.0 mL of the internal standard solution, add water to make 50 mL, and use this solution as the blank test solution. Perform the test with the test solution and the standard solution (1), (2), (3) and (4) as directed under Atomic absorption spectrophotometry. Concomitantly determine the absorbances of each standard solution and the test solution at 393.37 nm, the calcium emission line, and at 361.38 nm, the scandium emission line, using the blank solution as the blank. Prepare a calibration line of the ratio of the calcium absorption to the scandium absorption versus the respective calcium concentrations. From the calibration line so obtained, determine the calcium concentration, C, in g per mL, in the test solution. Calculate the content of calcium, in g per g (not more than 5 g per g): Amount (g /g) of calcium = 20
C W

Each mL of 0.001N silver nitrate VS = 126.9 g of I. (4) Free aromatic amine Weigh accurately about 0.2 g of Iodixanol, dissolve in 15 mL of water, and use this solution as the test solution. Dissolve an accurately weighed quantity of Iohexol related substance RS in water, and prepare a solution containing 10 g per mL, Add 5 mL of water to 10.0 mL of this solution, and use this solution as the standard solution. Separately, use 15 mL of water as the blank solution. Place the standard solution, the test solution, and the blank solution, in an ice bath for 5 minutes. Add 1.5 mL each of 6 mol/L hydrochloric acid, mix by swirling, add 1.0 mL each of sodium nitrite solution (2 in 100), mix, and allow to stand in the ice bath for 4 minutes. Remove each solution from the ice bath, add 1.0 mL of 4% sulfamic acid solution, and swirl gently until gas evolution ceases. Add 1.0 mL of N-(1-Naphthyl) ethylenediamine dihydrochloride solution, a freshly prepared solution of that in a mixture of propylene goycol and water (70 : 30) mix, add water to make exactly 25 mL, and allow to stand for 5 minutes. And compare the color: the color obtained from the test solution is lighter than the color obtained from the standard solution (not more than 0.05%). If the color obtained from the test solution is about the same color or darker than the color obtained from the standard solution, concomitantly determine the absorbances of the solutions obtained from the blank solution, the test solution and the standard solution, AB , AT and AS , as directed under the Ultraviolet Spectrophotometry at the wavelength of maximum absorption of about 495 nm using the water as blank and 5-cm cells in length (not more than 0.05 %). Amount (%) of free aromatic amine

W : Mass of Iodixanol (g) taken to prepare the test solution. Internal standard solutionAccurately weigh about 3.067g of scandium oxide, and dissolve in 1000 mL of water, add water to make 1000 mL. (6) Ionic substancesUse all gloass upparatus washed with water. Determine the specific conductance of a solution of Iodixanol (2 in 100), the conductance of a solution of ionic substancd is not more than a solution of Sodium Cholride (4 g per mL) (not more than 0.02%). (7) Methanol, isopropyl alcohol, and methoxyethanolTransfer about 0.25 g of Iodixanol, accurately weighed, to a headspace vial. Add 1.0 mL of the Internal standard solution, seal the vial, mix until dissolved, and use this solution as the test solution. Weigh accurately about 0.5 g of methanol, about 1.0 g each of isopropanol and methoxyethanol, add water to make exactly 500 mL. Add water to 5.0 mL of this solution to make exactly 100 mL. Pipet 10.0 mL of this solution and 1.0 mL of internal standard solution, add water to

KP 9 515 make exactly 100 mL. Pipet exactly 1.0 mL of this solution, seal, and use this solution as the standard solution. This solution contains about 0.005 mg of methanol, 0.01 mg each of isopropanol and methoxyethanol. Perform the test with 1 mg each of test solution and standard solution as directed under the Gas Chromatography according to the following operating conditions. Calculate the ratio of each peak area of methanol Isopropanol and methoxyethanol to the peak area of the internal standard, QT and QS , respectively. Amount of methanol, isopropanol and methoxyethanol per1 g of Iodixanol are not more than 50 g, respectively. Amount (%) of methanol, isopropanol and methoxyethanol
= 100
C QT W QS

C : Concentration (mg/mL) of each related substance. W : Taken amount (mg) of Iodixanol. Internal standard stock solutionWeigh about 0.5 g of secondary butyl alcohol, add water to make 500-mL, and add water to 1.0 mL of this solution to make 100 mL. Operating conditions Detector: A hydrogen flame ionization detector (FID) Column: A capillaray column about 0.54 mm in inside diameter and about 30 m in length, coated with 1 m thickness of polyehtyleneglycol compound (carbowax 20M). Column temperature: Maintain at 40 for 3 minutes, then it is increased linearly at a rate of 8 C per minute to 100 C, and is maintained at 100 C for 1 minute. Carrier gas: Helium Flow rate: 11 mL/minute Injection port temperature: 150 C Detector temperature: 200 C System suitability System performance-When the procedure is run with 1 mL of the standard solution according to the above operating conditions, methanol, isopropanol, 2butanol and methoxy ethanol are eluted in this order, with the resolution between methanol peak and isopropanol peak being not less than 1.0. System repeatability-When the test is repeated 6 times with 1 mL of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak area for methanol and for isopropanol is not more than 0.5%, and the relative standard deviation of the ratio of the peak area for methoxy ethanol is not more than 10%. (8) Related substances () Weigh exactly an amount of Iodixanol, equivalent to about 0.5 g of anhydrous Iodixanol, dissolve in water make exactly 20

mL, use this solution as the test solution (1). Add water to 5.0 mL of the test solution (1) make esactly 50 mL, and use this solution as the test solution (2). Separately, quantitatively, dissolve accurately weighed quantity of Iodixanol RS, in water to obtain a solution having a know concentration of about 12.5 mg of anhydrate Iodixanol per mL, and use this solution as standard stock solution (1). Quantitatively dissolve an accurately weighed quantity of Iodixanol related substances RS in water to obtain a solution having a known concentration of about 0.25 mg of anhydrate iodixanol related substance per mL, and use this solution as the standard stock solution (2). Quantitatively dissolve an accurately weighed quantity of Iodixanol related substances II RS in water to obtain a solution having a known concentration of about 0.025 mg of anhydrate iodixanol related substance II per mL, and use this solution as the standard stock solution (3). Pipet exactly 2.0 mL of the standard stock solution (1), add water to make exactly 10 mL, and use this solution as the standard solution (1). Pipet exactly 5.0 mL of the standard stock solution (1), 2.5 mL of the standard stock solution (2) and 2.5 mL of the standard stock solition (3), add water to make exactly 25 mL, use this solution as the standard solution (2). Pipet exactly 5.0 mL of the test solution (1) and 2.5 mL of the standard stock solution (2), add water to make exactly 50 mL, and use this solution as the control solution. Use water as the blank solution. Perform the test with 10 L each of the blank solution, the test solution (1) and the test solution (2) as directed under the Liquid Chromatography according to the following operating conditions. When it is specified to proceed as directed for Nigh-low chromatography obtained from test solution (1) and test solution (2), calculate the amount (%) of each related substance according to the following formula. Amount (%) of related substance = 10 X (1) 0.1Y + Z X : Peak area of the specified related substance obtained from the test solution(1). Y : Total area of all the peaks eluted before and after Iodixanol peak obtained from test solution (1), disgarding any peaks due to injection noise or solvent. Z : Sum of peak area of the peak of all related substances and the principal peak of Iodixanol obtained from test solution (2). IohexolIf Iohexol is present, it exhibits two peaks with retention times of about 0.37 and 0.39 relative to the principal iodixanol peak, in the chromatogram obtained from test solution (1). Draw a baseline at the height of the baseline obtained from the blank solution. Calculate the total area of the two peaks and the amount of iohexol according to the equation(1) (not more than 0.6%). Iodixanol related substance IIIIf iodixanol re-

516 Monographs, Part I lated substance III is present, it elutes as a single peak with a retention time of about 0.34 relative to the principal iodixanol peak, in the chromatogram obtained from test solution (1). Determine the peak area of Iodixanol revolted substaned III and calculate the amount of Iodixanol related substance III according to the equation(1) (not more than 0.2%). Iodixanol related substance IIf iodixanol related substance I is present, only the first and larger peak, with a retention time of about 1.07 relative to the principal iodixanol peak, is seen between the two principal iodixanol peaks in the chromatogram obtained from test solution (1); the second iodixanol related substance I peak co-elutes with iodixanol. The area of the first is about 80 % of the total area of related substance I, determine the area of the first peak, X2. Calculate the amount of related substance I by formula (not more than 0.4%). Amount (%) of Iodixanol related substance I = 12.5 X 2 (2) 0.1Y + Z Y and Z are: See formula(1).
Iodixanol related substance IVIf iodixanol related compound IV is present, only the first peak with a retention time of about 0.8 relative to the principal iodixanol peak, can be seen in the chromatogram obtained from test solution (1); the second peak co-elutes with Iodixanol. The area of the first peak corresponds to about 25% of the total area of Iodixanol related substance IV. Determine the area of the first peak, calculate the amount of iodixanol related substance IV by following formula (not more than 0.2%). Overalkylated related substancesThese compounds exhibit after iodixanol related compound V, with a retention time greater than 1.18 relative to the last iodixanol peak in the chromatogram of the test solution(1). Calculate the amount of overalkylated related substances by the formula (1) determining the amount of the related substances (not more than 1.0%). Unspecified related substance 1Determine the peak area other than the above peak among the peak be shown before or after iodixanol prineipal peak in the chromatogram obtained from test solution (1), calculate the amount of unspecified related substance 1 according to the formula (1). Other unspecified related substance 2 Determine the peak area other than the above peak among the peak be shown between principal peaks of iodixanol in the chromatogram obtained from the test solution (1), calculate the amount of other unspecified related substance 2. Amounts of each related substances of and are not more than 2%, and amount of total unspecified related substances is not more than 0.5%. Total related substancesDetermine total area of all peaks shown between the principal peaks of iodixanol in the chromatogram obtained from the test solution (1), calculate the amount of total related substance (not more than 1.5%).

Amount (%) of total related substances

X 3 X1 X + 2 + 100 Y X 1 X 3 + 0.25 0.8 0.85 = 10(0.1Y + Z )

in which the variables are as defined above. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5m in particle diameter). Mobile phase: Adjust with mobile phase A and mobile phase B according to step-wise or the slope of concentration as follows. mobile phase A - A mixture of acetonitrile and water (1 : 1) mobile phase B - water Time (minute) 0 0 ~ 30 30 ~ 70 70 ~ 80 80 ~ 81 Mobile phase A (vol%) 6 6 20 Mobile phase B (vol%) 94 94 80 Elution condition solvent equilibrium (about 20 minute) linear concentration slope linear concentration slope A constant formation linear concentration

Amount (%) of Iodixanol related substance IV = 40 X 1 (3) 0.1Y + Z Y and Z are: See formula(1).
Iodixanol related substance V If iodixanol related compound V is present, the second peak only, with a retention time of about 1.18 relative to the last iodixanol peak, is seen in the chromatogram obtained from test solution (1); the first peak co-elutes with iodixanol. The area of the second peak corresponds to about 85% of the total area of iodixanol related substance V, determine the area of the second peak, X3, calculate the amount of the related substance V by the following formula (not more than 0.2 %).

Amount (%) of Iodixanol related substance V =

10 X 3 0.85 (0.1Y + Z ) (4)

20 100 80 0 100 100 6 0 0 94

Y and Z are: See formula(1).

KP 9 517 slope 81 ~ 90 6 94 A constant formation this solution as the standard solution (3). Pipet 2.0 mL of standard stock solution (1), add water to make exactly 10 mL, and use this solution as the standard solution (1). Pipet 5.0 mL of the standard stock solution (1) and 2.5 mL of the standard stock solution (2), add water to make exactly 25 mL, and use this solution as the standard solution (2). Pipet 1.0 mL of standard solution (1) and 1.0 mL of standard stock solution (3), add water to make 3.0 mL, and use this solution as the standard solution (3). Use water as the blank solution. Perform the test with 10 L each of the test solution, the standard solution (1), the standard solution (2) and the standard solution (3) as directed under the Liquid Chromatography according to the following operating conditions. Inject 3 times repeatly for the standard solution (2). Compare the retention times of the peaks obtained from the standard solution (3) to those obtained from the test solution. Iodixanol related substance VI exhibits two peaks, the second of which may partly overlap with another peak, and use only the area of the first and larger peak, corresponding to about 60% of the total area of Iodixanol related substance VI. Draw baseline at the height of the baseline obtained from the blank solution. Calculate the area percent of Iodixanol related substance VI, {5-[[3-[[3-[[2,3-dihydroxypropyl)amino] carbonyl]-5-[[amino]carbonyl]-2,4,6-triiodphenyl] (acetylimino]-2-hydroxypropyl]-(acetylimino)]-N,N'bis(2,3-dihydroxypropyl)-2,4,6-triiod-1,3-benzenedicar boxide}, by dividing the area obtained from the test solution 0.6 is not more than 0.3%. Calculate the amount of the related substance VII shown as a single peak, with a shoulder, on the tail of the Iodixanol peak is not more than 0.6%. Operating conditions Detector: An ultraviolet absorption photometry (wavelength: 254 nm). Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with monomolecular layer aminopropylsilanized silica gel for the liquid chromatography (5 m particle diameter). Mobile phase: Adjust with mobile phase A and mobile phase B according to step-wise or the slope of concentration as follows.. mobile phase A - Acetonitrile mobile phase B - Water Time (minute) 0 0 ~ 25 Mobile phase A (vol%) 85 85 66 Mobile phase B (vol%) 15 15 34 Elution condition solvent equilibrium (about 20 minute) linear concentration slope

Flow rate: 1.0 mL per minute System suitability Chromatograph the solutions asdirected for procedure, in the following injection sequence : Blank solution, standard solution(1), control solution and at least three replicates of standard solution(2). System performance: When the procedure is run with 10 L of the standard solution (1) according to the above operating conditions, the chromatogram obtained from the standard solution (1) exhibits two or three principal unresolved peaks. If the chromatogram exhibits two principal peaks, their relative areas are about 60% and 40%. If the chromatogram exhibits three principal peaks, their relative areas are about 60%, 38%, and 2%. When the procedure is run with 10 L of the standard solution (2) according to the above operating conditions, the chromatogram obtained from standard solution (2) exhibits two resolved peaks due to Iodixanol related substance II that elute before the Iodixanol peaks and one Iodixanol related substance I peak between the two principal Iodixanol peaks. The area of the two Iodixanol related substance II peaks is between 0.075% and 0.125% of the total area. Disregard any peak due to the solvent front and any peak corresponding to those obtained from the blank solution. System repeatability : When the test is repeated 3 times with 10 L of the standard solution (2) according to the above operating conditions, the relative standard deviation of sum of areas of two isomer peaks obtained from Iodixanol related substance II is not more than 5%. Measure the height of the Iodixanol related substance I peak, and adjust the sensitivity of the amplfier to obtain a peak height between 80% and 100% of the full scale. Measure the height, A, above the baseline of the Iodixanol related substance I peak and the height, B, above the baseline of the lowest part of the curve separating this peak from the first principal Iodixanol peak: A is not less than 1.3B. Perform the test with 10 L of the control solution according to the above operating conditions: the peak of Iodixanol related substance I measurable. () Dissolve an accurately weighed quantity of Iodixanol, equivalent to about 0.125 g of anhydrous Iodixanol, in water to make exactly 50 mL, and use this solution as the test solution. Quantitatively dissolve an accurately weighed quantity of Iodixanol RS, equivalent to about 0.125 g of anhydrous Iodixanol, in water to make 10 mL, and use this solution as the standard stock solution (1). Weigh accurately quantity of Iodixanol related substance II RS, equivqlent to about 0.125 g of anhydrous Iodixanol, dissolve in water to make exactly 100 mL, add water to 2.0 mL of this solution to make exactly 100 mL, and use this solution as the standard stock solution (2). Quantitatively dissolve an accurately weighed about 25 mg of Iodixanol related substance VI RS in water to make exactly 10 mL, and use

Flow rate: 2.5 mL per minute System suitability System performance: When the procedure is run with 10 L of the standard solution (1) according to the above operating conditions: the chromatogram obtained

518 Monographs, Part I from standard solution (1) exhibits three principal unresolved peaks,the relative peak areas are about 62%, 35% and 3%; and the retention time of the last Iodixanol peak is not more than 14 minutes. When the procedure is run 10 L of the standard solution (2) according to the above operating conditions: the chromatogram obtained from standard solution (2) exhibits two partially unresolved peaks due to Iodixanol related substance II with relative retention times of about 0.33 and 0.39, the peak area of Iodixanol related substance II is between 0.075% and 0.125% of the total area. When the procedure is run with 10 L of the standard solution (3) according to the above operating conditions: the chromatogram obtained from standard solution (3) exhibits two unresolved peaks due to Iodixanol related compound E, with relative retention times of about 0.67 and 0.72. The resolution, between the first peak of Iodixanol related substance VI and the first principal peak of Iodixanol is not less than 5.0. System repeatability: When the test is repeated 3 times with 10 L of standard solution (2) according to the above conditions: the relative standard deviation of total peak areas of two isomers of the related substance II is not more than 5%.
Microbial Limit Its total viable aeronic microbial count does not exceed 100 per mL. Water Not more than 4.0% (0.5 g, volumetric titration, direct titration) Assay Weigh accurately about 0.5 g of Iodixanol, add 25 mL of a sodium hydroxide solution (1 in 20) and 0.5 g of powdered zinc, connect the flask to a reflux condenser, and reflux for 1 hour. Cool the flask to room temperature, and wash the condenser with 20 mL of water, combine the washing with the reaction mixture, filter. Wash the flask and the filter with several small portions of water, filter, and add the filtered washings to the filterate. Add 5 mL of glacial acetic acid, and titrate with 0.1 mol/L silver nitrate VS (potentiometric titration, Endpoint Detection Method in Titrimetry).

Iodoform is freely soluble in ether, sparingly soluble in ethanol, and practically insoluble in water. Iodoform is slightly volatile at ordinary temperature. Melting pointAbout 120 o C (with decomposition).
Identification Heat 0.1 g of Iodoform: a purple gas is evolved. Purity

(1) Water-soluble colored substances and

acidity or alkalinityShake well 2.0 g of Iodoform, previously powdered, with 5 mL of water for 1 minute, allow to stand and filter the supernatant liquid: the filtrate is colorless and neutral.

(2) ChlorideShake well 3.0 g of Iodoform, previously powdered, with 75 mL of water for 1 minute, allow to stand and filter the supernatant liquid. Take 25 mL of the filtrate, add 6 mL of dilute nitric acid and water to make 50 mL and perform the test with this solution as the test solution. Prepare the control solution with 0.30 mL of 0.1 mol/L hydrochloric acid VS (not more than 0.011%). (3) SulfateTo 25 mL of the filtrate obtained in (2), add 1 mL of dilute hydrochloric acid and water to make 50 mL and perform the test with this solution as the test solution. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.017%).
Loss on Drying Not more than 0.5% (1 g, silica gel, 24 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.2 g of Iodoform, previously dried, in a glass-stoppered flask and dissolve in 20 mL of ethanol. Add exactly 30 mL of 0.1 mol/L silver nitrate VS and 10 mL of nitric acid, stopper the flask, shake well and allow to stand in a dark place over 16 hours. Add 150 mL of water and titrate the excess silver nitrate with 0.1 mol/L ammonium thiocyanate VS (indicator: 5 mL of ferric ammonium sulfate TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 N silver nitrate = 25.84 mg of C35H44I6N6O15.

Packaing and Storage containers.

Light-resistant, well-closed

Each mL of 0.1 mol/L silver nitrate VS = 13.124 mg of CHI3

Packaging and Storage tight containers.

Iodoform
CHI3: 393.73 Iodoform, when dried, contains not less than 99.0% and not more than 101.0% of iodoform (CHI3).
Description Iodoform is a lustrous, yellow crystal or crystalline powder. Iodoform has a characteristic odor.

Preserve in light-resistant,

KP 9 519
Purity (1) Clarity and color of solutionWeigh 16.18 g of Iohexol, dissolve in water, to make exactly 25 mL, filter through a filter membrane having a porosity of 0.22 m. Determine the absorbanas of this filterate as directed under the Ultraviolet-visible Spectrophotometry and using water as the blank, determine the absorbances of this solution at 400, 420 and 450 nm are not more than than 0.180, 0.030, and 0.015, respectively. (2) Heavy metals Proceed with 1.0 g of Iohexol according to Method 1 and perform the test. Preare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) Free aromatic amine Transfer 0.2 g of Iohexol to a 50-mL volumetric flask, add 15 mL of water, and mix to dissolve. To a second 50-mL volumetric flask, add 5 mL of water and 10 mL of a solution containing 10 g per mL by dissolving Iohexol related substance 1 {5-(amino)-N,N,-bis(2,3-dihydroxypropyl)2,4,6-triiod-1,3-benzenedicarboxyamide)} RS in water. To a third 50-mL volumetric flask, add 15 mL of water. Place these solutions in an ice bath, and cool for 5 minutes. Add 3.0 mL each of 5 mol/L hydrochloric acid TS and swirl to mix. Add 2.0 mL of sodium nitrite solution (1 in 50) and allow to stand for 4 minutes. Add 2.0 mL each of sulfamic acd solution (1 in 25), shake, and allow to stand for 1 minute. Remove these solutions from the ice bath. To each flask, add 2 mL each, of a solution of N-(1-naphthyl)ehtylenediamine dihydrochloride in dilute propylene glycol (7 in 10) (1 in 1000), mix, add water to make 50 mL, allow to stand for 5 minutes, and use these solutions as test solution, standard solution and blank solution. Perform the test with these solutions as the directed under Ultravioletvisible spectrophotometry: the absorbance of the test solution is not larger than the absorbance of the standard solution (not more than 0.05%). (4) Free iodineTransfer 2.1 g of Iohexol to a 50mL centrifuge tube with a stopper, add about 20 mL of water, and shake vigorously to dissolve. Add 5 mL of toluene and 5 mL of 1 mol/L sulfuric acid TS, shake, and centrifuge at high speed: the toluene layer shows no red or pink color. (5) Free iodideWeigh 5.0 g of Iohexol, dissolve in about 20 mL of water, transfer to a centrifuge tube with a stopper, add 20 mL of water, and shake vigorously or heating weakly to dissolve, and titrate with 0.001 mol/L silver nitrate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform a blank determination, and make any necessary correction (0.001%).

Iohexol
OH O H N OH

I OH

HO N I HO H3C O O

H N

OH

C19H26I3N3O9 : 821.14 Iohexol contains not less than 98.0% and not more than 102.0% of iohexol (C19H26I3N3O9), calculated on the anhydrous basis.
Description Iohexol is a white or off-white powder and is odorless. Iohexol is freely soluble in water and in methanol, and practically insoluble in ether or in chloroform. Iohexol is hygroscopic. Identification (1) Heat about 0.5 g in a crucible: violet vapors are evolved. (2) Determine the absorption spectra of solutions of Iohexol and Iohexol RS (1 in 100000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit a maximum and a minimum of absorption at the same wavelengths. (3) Determine the infrared spectra of Iohexol and Iohexol RS as directed in the potassium bromide disk method under the Infrared Spectrometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (4) Weigh 0.1 g each of Iohexol and Iohexol RS, dissolve in 10 mL of methanol, and use these solutions as the test solution and the standard solution. Perform the test with these solutions as directed under the Thin-

layer Chromatography. Spots 10 L each of the test solution and the standard solution a silica gel with fluorescent indicator for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (50 : 25 : 11) to a distance of about 15 cm, and air-dry the plates. Examine under ultraviolet (254 nm): the presence of exo- and endo- isomers in the test solution and the standard solution is shown by the appearance of two spots, each of which correspond to the corresponding principal spot. The Rf values obtained from the test solution and the standard solution are the same.
Specific Optical Rotation [ ]25 D : between -0.5 and +0.5 C (0.5 g, water 10 mL and 100 mL).

Each mL of 0.001 mol/L AgNO3 = 126.9 g of I (6) Ionic substancesRinse all glassware 5 times with distilled water. Determine the specific resistance, ( RSP ), at 20 with a solution of Iohexol (1 in 50), calculate the specific conductance, k, taken by the formula: this specific conductance is not larger than the conductance of 0.0002% sodium chloride (not more

520 Monographs, Part I than 0.01%). Specific conductance =

1 106 R SP

butanol in water containing about 0.05 mg per each mL. Operating conditions Detector : A hydrogen flame-ionization detector Column : About 0.53 mm in inside diameter and 30 m in length fused silica capillary column bonded with a 3 m layer of 6% cyanopropylphenyl and 94% dimethylpolysiloxane. Column temperature : It is held at 40 C for 5 minutes and is programmed to increase at a rate of 10 C per minute to 100 C, maintain at 100 for 1 minute. Carrier gas : helium Flow rate : 14 mL/minute Inject port temperature : 140 C Detector temperature : 250 C System suitability System performance : When the procedure is run with standard solution (5) according to the above operating conditions, the relative retention times of methanol, isopropanol, 2-butanol and methoxy ethanol are about 0.3, 0.5, 1.0 and 1.3, respectively, with the resolution between methanol peak and isopropanol peak being not less than 2.5. System repeatability: When the test is repeated 6 times with standard solution (5) according to the above operating conditions, the relative standard deviation of each peak area is not more than 5%. (8) 3-chloro-1,2-propanediol Dissolve about 1 g of Iohexol, accurately weighed, in 1.0 mL of water, extract 4 times with 2 mL of ethyl acetate, and combine the extracts. Dry the combined extracts with anhydrous sodium sulfate. Filter, and wash the filter with a small amount of ethyl acetate. Combine the wash with the filtrate, and concentrate to the volume of 2.0 mL, using a warm water bath and a stream of nitrogen. Pass this solution through a membrane filter, and use the clear filtrate as the the test solution. Separatry, dissolve an accurately weighed quantity of 3-chloro-1,2-propanediol quantitatively in ethyl acetate to obtain the solution having a known concentration of about 20 g per mL, and use this solution as the standard solution. Perform the test with about 2 L each of the test solution and the standard solution as directed under the Gas Chromatography according to the following operating conditions: 3-chloro-1,2-propanediol, a mixture of two isomers, exhibits two principal peaks at 12 and 12.5 minutes, respectively. Measure the sums of two peak areas obtained from 3-chloro-1,2-propanediol, AST and ASA , calculate the amount of 3-chloro-1,2propanediol (not more than 0.0025 %). Amount (g ) of 3-chloro-1,2-propanediol = 100
AST 2CST ASA R

(7) Methanol, isopropanol, methoxy ethanol Transfer about 6.25 g of Iohexenol exactly weighed, add 5 mL of the internal standard solution, add water to make 25 mL, and use this solution as the test solution (1). Transfer 5.0 mL of the test solution (1) and 1 mL of water to a vial with gum stopper. Heat the sealed vial at 95 for 15 minutes, use this solution as the test solution (2). Transfer 5.0 mL of the test solution (1) and 1 mL of s the tandard solution (2) to a vial with gum stopper. Heat the sealed vial at 95 C for 15 minutes, and use this solution as the test solution (3). Transfer 5.0 mL of t the test solution (1) and 1.0 mL of the standard solution (3) to a vial with gum stopper. Heat the sealed vial at 95 C for 15 minutes, and use this solution as the test solution (4). Transfer 5.0 mL of the test solution (1) and 1.0 mL of the standard solution (4) to a vial with gum stopper. Heat the sealed vial at 95 C for 15 minutes, and use this solution as the test solution (5). Separately, transfer about 0.6 g of methanol accurately weighed, add about 100 mL of water, mix, add about 0.6 g of methoxy ethanol in this solution, and mix, add water to make 1000 mL, and use this solution as the standard solution (1). Pipet about 10.0 mL of standard solution (1), add water to make exactly 50 mL, pipet 10.0 mL of this solution, add water to make 100 mL, and use this solution as the standard solution (2). Pipet about 5.0 mL of standard solution (1), add water to make exactly 100 mL, and use this solution as the standard solution (3). Add water in 10.0 mL of standard solution (1) to make exactly 100 mL, and use this solution as the standard solution (4). Add water in 10.0 mL of the standard solution (4) and 10.0 mL of the internal standard solution to make exactly 50 mL, transfer 6.0 mL of this solution to a vial fitted with a septum and crimp cap, and seal, heat the sealed vial at 95 C for 15 minutes, and use this solution as the standard solution (5). Perform the test with 2 mL each of the test solution (2), the test solution (3), the test solution (4), the test solution (5) and the standard solution (5) as directed under the Gas Chromatography according to the following operating conditions using the injection port apparatus for the headspace, calculate the peak areas of the principal peak from each chromatograms. Plot the peak area ratios obtained from the test solutions against the quatity of amount of methanol, amount of isopropanol and amount of methoxyethanol added per g of Iohexol. Extrapolate the line joining the points until it intercepts the concentration axis. The distance between this point and the intersection of the axis is the amount (mg/g) of methanol, isopropanol and methoxyethanol. Amounts (mg/g) of methanol and isopropanol are not more than 0.005%, respectively, the amount of methoxyethanol is not more than 0.002%. Internal standard solutionPrepare a solution of 2-

CST : Concentration (g/mL) of 3-chloro-1,2propanediol in the standard solution. R : The recovery ratio (%) measured in the system

KP 9 521 suitability test. Operating conditions Detector : A hydrogen flame-ionization detector. Column : A about 0.32 mm in inside diameter and about 30 m fused-silica capillary column bonded with a 1 m layer of phase G46 with 14 % cyanopropylphenyl-86 % methylpolysiloxane. Column temperature : At first, allow to stand at 50 C for 2 minutes and is programmed to increase at a rate of 10 C per minute to 200 C. Carrier gas : helium Injection port temperature : About 230 C Detector temperature : About 250 C System suitability System performance: When the procedure is run with system suitability solution according to the above operating conditions, the recovery rate of 3-chloro-1,2propanediol obtained from iohexanol is between 60 and 90 %. Recovery rate (%) of 3-chloro-1,2-propanediol = 100 ARC CST
AST C RS

10 L of this solution as directed under the Liquid Chromatography according to the following operating conditions, and determine the amount of each the related substances: the peaks other than the principal peak are not more than 0.1%, respectively, the peaks of O-alkylated substances are not more than 0.6 %, the sum of all the related substances other than O-alkylated substances is not more than 0.3%. Amount (%) of the related substance = 100
Ai AS

Ai : Each peak area other than the principal peak A s : Sum of the area of all peaks

CRS: Concentration (g/mL) of 3-chloro-1,2propanediol obtained from the system suitability solution. CST : Concentration (g/mL) of 3-chloro-1,2propanediol obtained from the standard solution. ARC : Peak area of 3-chloro-1,2-propanediol obtained from the system suitability solution. AST : Peak area of 3-chloro-1,2-propanediol obtained from the standard solution. System repeatability: When the test is repeated 6 times with the standard solution according to the above condotions: the relative standard deviation is not more than 10 %. System suitability solution: Dissolve 1 g of Iohexol containing less than 5 g of chloropropanediol in 1 mL of water. Quantitatively dissolve an accurately weighed quantity of 3-chloro-1,2-propanediol in ethyl acetate to obtain a solution having a concentration of about 25 g per mL, Add 2.0 mL of ethyl acet ate solution to the aqueous solution of Iohexol in a separator, amd mix. Transfer the ethyl acetate layer to a separate containers, and extract the aqueous layer three additional times with 2 mL of ethyl acetate, combining all four extracts. Dry the combined extracts using anhydrous sodium sulfate, filter, and wash the filter with a small amount of ethyl acetate. combine the wash, concentrate to 2.0 mL under nitrgen stream, filter with membrane filter, and use the filterate as the system suitability solution. (9) Related substancesWeigh accurately 75 mg of Iohexol, add water, dissolve to make 50 mL, and use this solution as the test solution. Perform the test with

Operating conditions Detector : An ultraviolet absorption Photometer (wavelength: 254 nm) Column: A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (3 m to 10 m in particle size diameter). Mobile phase : Change the rate of acetonitrile and water, the chromatograph is programmed to provide variable mixtures of these solvents, the percentage of acetonitrile increases from 1% to 13% at an increase rate of 0.2% per minute. Flow rate : 1.0 mL/minute System suitability System performance : When the procedure is run with 10 L of system suitability solution according to the above conditions, the relative retention time for the O-alkylated substances is between 1.1 and 1.4 to 1.0 of the retention time for the exo-isomer of iohexol with the resolution between iohexol related substance II and iohexol related substance III being not less than 20.0, and the peak rea of iohexol related substance III is 0.5 % 0.1 % by comparison to the total area of all of the peaks in the chromatogram. System suitability solutionDissolve acculately weighed quantities of Iohexol RS, Iohexol related substance I RS, and Iohexol related substance III RS in water to obtain a solution having known concentrations of about 1.5, 0.0075 and 0.0069 mg per mL, repectively, and use this solution as the system suitability solution.
Water Not more than 4.0 % (0.3 g, volumetric titration, direct titration). Assay Weigh accurately about 0.5 g of Iohexol, add 25 mL of 1.25 mol/L sodium hydroxide TS and 0.5 g of powdered zinc, connect the flask to a reflux condenser, and reflux the mixture for 1 hour. Cool the flask to room temperature, rinse the condenser with 20 mL of water, add the washing in the reaction mixture, mix, and filter the mixture. Rinse the flask and the filter throughly with small portions of water, sum the rinsings to the filtrate. Add 5 mL of glacial acetic acid, and

522 Monographs, Part I titrate with 0.1 mol/L silver nitrate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Each mL of 0.1 mol/L silver nitrate = 27.37 mg of C19H26I3N3O9
Packaging and Storage well-closed containers.

Preserve in light-resistant,

Iopamidol
HO O NH OH

I O HO H OH N H I

H N OH O OH

fate (1 in 10), shake well, allow to stand for 1 minute, and add 1 mL of naphthylethylenediamine TS and water to make exactly 50 mL. Determine the absorbance of this solution at 495 nm as directed under Ultravioletvisible Spectrophotometry using a solution, prepared in the same manner without Iopamidol, as the blank: the absorbance is not more than 0.12 (not more than 0.020%). (3) Iodine Dissolve 2.0 g of Iopamidol in 25 mL of water, add 5 mL of 1 mol/L sulfuric acid and 5 mL of toluene, shake well, and allow to stand:the toluene layer is colorless. (4) Free iodine ion Weigh accurately about 5.0 g of Iopamidol, dissolve in 70 mL of water, and adjust the pH to about 4.5 with dilute acetic acid . To this solution add 2 mL of 0.1 mol/L sodium chloride TS, and titratewith 0.001 mol/L silver nitrate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Content of iodine ion in Iopamidol is not more than 0.001% Each mL of 0.001 mol/L silver nitrate VS = 0.1269 mg of I (5) Heavy metalsMoisten 1.0 g of Iopamidol with a small quantity of sulfuric acid, heat gradually to almost incinerate by a possibly lower temperature. After cooling, moisten again with a small quantity of sulfuric acid, heat gradually until white fumes no longer are evolved, and incinerate by ignition 450 to 550 C. Proceed as directed in Method 2, and perform the test. Prepare the control solution with 1.0 mL of Standard Lead Solution (not more than 10 ppm). (6) Related substancesDissolve 0.10 g of Iopamidol in water to make exactly 10 mL, and use this solution as the test solution. Separately, dissolve 10 mg of N,N-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5hydroxyacetylamino2,4,6-triiodoisophthalamide in water to make exactly 100 mL. Pipet 5 mL of this solution, add water to make exactly 50 mL, and use this solution as the standard solution. Perform the test with exactly 20 L each of the test solution and standard solution as directed under Liquid Chromatography according to the following conditions, and determine each peak area of the both solutions by the automatic integration method: each area of the peaks other than the peak of iopamidol from the test solution is not larger than the peak area of the standard solution, and the total of these areas is not larger than 2.5 times of the peak area of the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength; 240 nm). Column: A stainless steel column about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of

C17H22I3N3O8 : 777.09 Iopamidol, when dried, contains not less than 99.0% and not more the 101.0% of iopamidol ( C17H22I3N3O8).
Description Iopamidol is a white, crystalline powder. Iopamidol is very soluble in water, sparingly soluble in methanol, and very slightly soluble in dehydrated ethanol. Identifiation (1) To 0.05 g of Iopamidol add 5 mL of hydrochloric acid, heat for 10 minutes in a water bath: the test solution responds to the Qualitative Tests for primary aromatic amines. (2) Heat 0.1 g of Iopamidol over a flame: a purple gas is evolved. (3) Determine the infrared absorption spectrum of Iopamidol and Iopamidol RS, previously dried, as directed in the potassium bromide disk method under Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers. Specific Optical Rotation [ ]20 436 nm : Between 4.6 and 5.2 (after drying, 4 g, water, warm, after cooling, 10 mL, 100 mm). Purity (1) Clarity and color of solution Dissolve 1.0 g of Iopamidol in 10 mL of water: the solution is clear and colorless (2) Primary aromatic amines Dissolve 0.60 g of Iopamidol in 8 mL of water add 1 mL of a solution of sodium nitrite (1 in 50 ) and 12 mL of 2 mol / L hydrochloric acid TS, shake, and allow to stand for 2 minutes. Add 1 mL of a solution of ammonium amidosul-

KP 9 523 about 35C. Mobile phase: Use water as the mobile phase A, and a mixture of water and methanol (3:1) as the mobile phase B. Change the mixed ratios of the mobile phase A and mobile phase B stepwise as follows:
Time (minute) 0~6 6 ~ 18 18 ~ 30 30 ~ 34 Mobile phase A (vol%) 92 92 65 65 8 8 Mobile phase B (vol%) 8 8 35 35 92 92

Iopanoic Acid
COOH CH2CC2H5 I H I

NH2 I

C11Hl2I3NO2: 570.93 Iopanoic Acid, when dried, contains not less than 97.0% and not more than 101.0% of iopanoic acid (C11Hl2I3NO2).
Description Iopanoic Acid is a pale yellowish white, crystalline powder and has a flint, characteristic odor. Iopanoic Acid is soluble in ethanol or in acetone, sparingly soluble in glacial acetic acid or in ether and practically insoluble in water. Iopanoic Acid dissolves in sodium hydroxide TS. Iopanoic Acid is gradually colored by light. Identification Mix about 100 mg of Iopanoic acid with 500 mg of sodium carbonate in a crucible, and heat until thoroughly charred. Cool, add 5 ml of hot water, heat on a steam bath for 5 minutes, and filter: the solution responds to the tests for Iodide Melting Point composition).

Flow rate:Adjust the flow rate to 1.5 mL per minute. Time span of measurement: About 4.3 times as long as the retention time of iopamidol. System suitability System performance: Dissolve 1 mL of the test solution and 10 mg of N,Nbis[2-hydroxy-1(hydroxymethyl)ethyl]-5-hydroxyacetylamino-2,4,6triiodoisophthalamide in water to make 100 mL. When the procedure is run with 20 L of this solution under the above operating conditions, N,Nbis[2-hydroxy-1(hydroxymethyl)ethyl]-5-hydroxyacetylamino-2,4,6triiodoisophthalamide and iopamidol are eluted in this order with the resolution between these peaks being not less than 7. System repeatability: When the test is repeated 6 times with 20 L of the standard solution under the above operating conditions, the relative standard deviation of the peak areas of N,Nbis[2-hydroxy-1(hydroxymethyl)ethyl]-5-hydroxyacetylamino-2,4,6triiodoisophthalamide is not more than 1.0%.
Loss on Drying Not more than 0.3% (1 g, 105C, 3 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.5 g of Iopamidol, previousiy dried, transfer to a saponification flask, dissolve in 40 mL of sodium hydroxide TS, add 1 g of zinc powder, boil for 30 minutes under a reflux confenser, cool, and filter. Wash the flask and the filter paper with 50 mL of water, and combine the washing with the filtrate. Add 5 mL of acetic acid (100) to this solution, and titrate with 0.1 mol/L silver nitrate VS (potentiometric titration, Endpoint Detection Method in Titrimetry).

Between 152 C and 158 C (with de-

Each mL of 0.1 mol/L silver nitrate VS = 25.90 mg of C17H22I3N3O8

Packaging and Storage Light-resistant, well-closed containers.

Purity (1) Soluble halidesTransfer 0.5 g of Iopanoic Acid to the stoppered cylinder, add 10 mL of 2 mol/L nitric acid and 15 mL of water, mix stirring for 5 minutes, filter with filter pater, perform the test with 10 mL of the filtrate as the test solution according to the Chloride Limit test, this solution is not dark than the following control solution. Control solution Take 0.1 mL of 0.01 mol.L hydrochloric acid and 6 mL of dilute nitric acid, and mix. Add water to this solution to make 10 mL. (2) Free iodineDissolve 0.20 g of Iopanoic Acid in 2.0 mL of distilled water and 2.0 mL of chloroform, and allow to stand for 1 minute with shaking, The chloroform layer exhibit not purple color.. (3) Heavy metalsProceed with 1.0 g of Iopanoic Acid according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). Loss on Drying Not more than 1.0% (1 g, 105 C, 1 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.25 g of lopanoic Acid, previously dried, and add 0.5 g of zinc powder and

524 Monographs, Part I 30 mL of sodium hydroxide TS. Boil for 30 minutes with a reflux condenser, add 20 mL of water through the condenser and filter through absorbent cotton. Wash the flask and filter with a little volume of water and combine the filtrate and washings. After cooling, add 5 mL of acetic acid anhydride, and titrate with 0.05 mol/L silver nitrate VS until the color of the precipitate changes from yellow to green (indicator: 1 mL of tetrabromophenol-phthalein ethyl ester TS). Each mL of 0.1 mol/L silver nitrate VS = 9.516 mg of C11Hl2I3NO2
Packaging and Storage tight containers.

trate with 0.1 mol/L sodium hydroxide VS. Each mL of 0.1 mol/L sodium hydroxide VS = 57.09 mg of C11Hl2I3NO2
Packaging and Storage tight containers.

Preserve in light-resistant,

Iophendylate
H I C(CH 2)8COOC 2H5 CH 3

Preserve in light-resistant,

and enantiomer C19H29IO2: 416.34 Iophenylate, a mixture of isomers of ethyl iodophenylundecanoate, consists of ethyl 10-(iodophenyl) undecanoate and contains not less than 98.0% and not more than 102.0% of iophenylate (C19H29IO2).
Description Iophendylate is a colorless or pale yellow colored syrupy liquid and is odorless or has etherlike odor. Iophendylate is freely soluble in ethanol, in benzene, in chloroform or in ether and slightly soluble in water. Iophendylate is gradually colored in the air. Identification Place about 1 mL of Iophendylate, 15 mL of water and 7 g of potassium dichromate in a round-bottom flask. Carefully add 10 mL of sulfuric acid, moderating the vigorous reaction by cooling the flask with tap water. When the reaction has subsided, reflux the mixture for 2 hours. After cooling, pour the contents of the flask into the 25 mL of water, filter the mixture with suction and wash the precipitate with a small quantity of cold water. Crystallize the precipitate from 10 mL of diluted alcohol: the sublimate of piodobenzoic acid melts between 268 C and 272 C. Refractive Index
20 : Between 1.524 and 1.526. nD

Iopanoic Acid Tablets

Iopanoic Acid Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of iopanoic acid (C11Hl2I3NO2: 570.93).
Method of Preparation Prepare as directed under Tablets, with Iopanoic Acid. Identification Triturate a quantity of finely powdered Tablets, equivalent to about 1 g of Iopanoic Acid, with two 10 mL portions of hexane, and decant and discard the liquid. Allow the residue to dry spontaneously, triturate with 15 mL of acetone, and filter. Repeat the trituration with another 15 mL portion of acetone, evaporate the combined filtrates on a steam bath to a volume of not more than 1 mL, add, with constant stirring, 20 mL of water, filter, wash the precipitate with two 5 mL portions of water, and dry at 105 C for 2 hour: the Iopanoic Acid so obtained melts between 150 C and 158 C, with decomposition, and responds to the Identification test of Iopanoic Acid. Purity Soluble halidesPrepare the powder of Iopanoic Acid Tablet, equivalent to 0.5 g of Iopanoic Acid according to the labeled amount, proceed as directed in the Purity(1) of Iopanoic Acid. Disintegration Test Not more than 30 minutes. Uniformity of Dosage Units ment.

Saponification Value Between 132 and 142.

It meets the require-

Specific Gravity

20 : Between 1.248 and 1.257. d 20

Assay Weigh accurately and powder not less than 20 Iopanoic Acid Tablets. Weigh accurately a portion of the powder, equivalent to about 1 g of iopanoic acid (C11Hl2I3NO2), and add 10 mL of hexane, triturate and filter. Repeat the same procedure, then add the neutralized ethanol to residue, warm to 70 o C and filter. Wash four times the residue with 10 mL of neutralized ethanol, cool combined filtrate and washings, to room temperature, add 3 ~ 5 drops of thymol blue TS and ti-

Purity (1) Free acidTransfer about 4 g of Iophendylate, accurately weighed, to a small flask, add 20 mL of alcohol and dissolve. Add 5 drops of phenolphthalein TS and titrate with 0.05 mol/L alcoholic potassium hydroxide VS to a pink color that persists for 30 seconds: not more than 0.60 mL of 0.05 mol/L alcoholic potassium hydroxide VS is required for neutralization. Perform the blank determination and make any necessary correction (not more than 0.3% as iophenylundecanoic acid). (2) Free iodineDetermine absorbance of Iophen-

KP 9 525 dylate in a 40 mm cell, at a 485 nm, as directed in the Ultraviolet-visible Spectrophotometry, using water as the blank: the absorbance is not greater than 0.16 (not more than 7.5 ppm).
Residue on Ignition Not more than 0.1% (1 g). Assay Dissolve about 50 mg of Iophendylate, accurately weighed, in 5 mL of toluene contained in a suitable separatory funnel. Add 15 mL of sodium biphenyl and shake vigorously for 2 minutes. Extract with three 10 mL volumes of 5 mol/L phosphoric acid. Combine the lower phases and add 1 mol/L sodium hypochlorite TS drop-wise until the solution turns brown. Add an additional 0.5 mL of 1 mol/L sodium hypochlorite TS and shake for 3 minutes. Add 5 mL of saturated phenol solution, freshly prepared and stand for 1 minutes, accurately timed. Add 1 g of potassium iodide, shake 30 seconds and titrate rapidly with 0.1 mol/L sodium thiosulfate VS, adding 3 mL of starch TS as the endpoint is approached. Bacterial Endotoxins Less than 0.9 EU per 1 mg of Iophendylate. Foreign insoluble Matter Test It meets the requirement. Insoluble Particulate Matter Test for Injections It meets the requirement. Determination of Volume of Injection in Containers It meets the requirement. Assay Proceed as directed in the Assay under Iophendylate. Packaging and Storage tight containers.

(2) under Iophendylate.

Residue on Ignition Not more than 0.1% (1 g). Sterility Test It meets the requirement.

Preserve in light-resistant,

Each mL of 0.1 mol/L sodium thiosulfate VS = 6.939 mg of C19H29IO2

Packaging and Storage tight Containers.

Preserve in light-resistant,

Iopromide
CH3 OH OH O N

Iophendylate Injection
I

I OH H N OH

Iophendylate Injection contains not less than 98.0 % and not more than 102.0% of the iophendylate (C19H29IO2: 416.34).
Method of Preparation Prepare as directed under the Injections, with Iophendylate. Description Iophendylate Injection is colorless or pale yellow colored syrupy liquid, is odorless or has ether-like odor. Iophendylate Injection is freely soluble in ethanol, in benzene, in chloroform or in ether and very slightly soluble in water. Iophendylate Injection is gradually colored in the air. Identification Proceed as directed in the Identification under Iophendylate. Refractive Index
20 : Between 1.524 and 1.526. nD

O H3CO N H I O

C18H24I3N3O8 : 791.11 Iopromide contains not less than 97.0% and not more than 102.5% of iopromide (C18H24I3N3O8), calculated on the anhydrous and solvent-free basis.
Description White to slightly yellow powder. Iopromide is freely soluble in water and in dimethylsulfoxide, and practically insoluble in acetone, in ethanol or in ether. Identification (1) Determine the infrared spectra of Iopromide and Iopromide RS, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Perform the test with Iopromide according to the related substances under the Purity: the principal sports obtained from the test solution and the standard solution show the same Rf values. Purity (1) Heavy metalsWeigh about 1.0 g of Iopromide, dissolve in water to make exactly 20 mL. Add

Saponification Value Between 132 and 142. Specific Gravity

20 : Between 1.248 and 1.257. d 20

Purity (1) Free acid Proceed as directed in the Purity (1) under Iophendylate. (2) Free iodine Proceed as directed in the Purity

526 Monographs, Part I 2.0 mL of pH 3.5 Acetate buffer to 12.0 mL of this solution, and use this solution as the test solution. Separately, pipet 1.0 mL of standard lead solution, add 9.0 mL of water, 2.0 mL of the test solution and 2.0 mL of acetic acidammonium acetate buffer, and use this solution as the standard solution. Add 5 mL of water to 15 mL of 1 mol/L sodium hydroxide, mix, and add 20 mL of glycerin. Pipet each 1.0 mL of this solution and each 0.2 mL of 4% thioacetamide TS into two nessler tube, and heat in water bath for 20 seconds. Immediately add the test solution and standard solution to each solution, allow to stand for 2 minutes, and view downward over a white surface: the color of the solution from the test solution is not darker than that of the solution from the standard solution (Not more than 20 ppm). pH 3.5 acetic acidammonium acetate bufferDissolve 25.0 g of ammonium acetate in 25 mL of water, add 38.0 mL of 6 mol/L hydrochloric acid TS, and mix. If necessary, add 6 mol/L ammonia TS or 6 mol/L hydrochloric acid TS to adjust a pH 3.5, and add water to make exactly 100 mL. (2) Free iodineWeigh 2.0 g of Iopromide, and dissolve in 20 mL of water. Add 2 mL of toluene and 2 mL of diluted surfuric acid, and shake vigorously: the toluene layer shows no red color. (3) Free iodideWeigh 10.0 g of Iopromide, and dissolve in 70 mL of water. Adjust with 0.05 mol/L surfuric acid VS to a pH of 3.5 0.5, titrate with 0.001 mol/L silve nitrate VS (potentiometric titration, Endpoint Detection Method in Titrimetry). Perform the blank determination and make any necessary correction (not more than 0.002%). Each mL of 0.001 mol/L silver nitrate VS = 126.9 g of I. (4) Free aromatic amineTransfer 0.5 g of Iopromide to a 25-mL volumetric flask, add 20 mL of water, and dissolve, and use this solution as the test solution. Dissolve a suitable quantity of Iopromide related substance I RS in water, and dilute with water to make exactly 100 mL. Transfer 2.0 mL of this solution to a 25-mL volumetric flask, add 18.0 mL of water, and mix, and use this solution as the standard solution. Transfer 20 mL of water to a 25-mL volumetric flask, and use this solution as the blank solution. Place the flasks in an ice bath, and protect from light. Add slowly 1.0 mL of 8 mol/L hydrochloric acid TS, mix, and allow to stand for 5 minutes. Add 1.0 mL of sodium nitrite solution (1 in 50), mix, and allow to stand for 5 minutes. Add 0.50 mL of freshly prepared sulfamic acid solution, (8 in 100). Shake each flask vigorously several times within the next 5 minutes, venting off the gas that evolves. Add 1.0 mL of freshly prepared solution of N-(1naphthyl)ethylenediamine dihydrochloride in a mixture of propylene glycol and water (70:30) (1 in 1000), and shake. Remove the flasks from the ice bath, and allow to stand in a water bath at about 25 for 10 minutes. Dilute with water to make exactly 25 mL, and degas with the aid of sonication for 1 minute. Perform the test with these solutions as directed under the Ultravioletvisible Spectrophotometry. Determine the absorbances, AT and AS , of the test solution and the standard solution at the wavelength maximum absorbance at about 495 nm using the blank solution. Calculate the percentage of aromatic amine in the portion of Iopromide taken by the following formula: it is not more than 0.10%. The absorbance of the standard solution is not less than 0.40.
= 10
WS AU W U WS

WS : The quantity, in mg, of Iopromide related substance I RS taken to prepare the standard solution. WU : The quantity, in mg, of the Iopromide taken to prepare the test solution.

(5) EthanolDissolve an exactly weighed 0.5 g of Iopromide in dimethylformamide to make exactly 10 mL, and use this solution as the test solution. Separately, weigh accurately a suitable volume of ethanol, add dimethylformamide, mix, prepare a solution having a known concentration of about 0.050 mg of ethanol (C2H5OH) per mL, and use this solution as the standard solution. Use dimethylformamide as the blank solution. Transfer 2.0 mL each of the test solution, the standard solution and the blank solution to separate headspace vials, add 10 L each of 1 mol/L hydrochloric acid TS to each vial, then seal the vials using a flanged cap. Perform the test as directed under the Gas Chromatography according to the following operating conditions, and determine the peak area of ethanol, AT and AS (not more than 0.4%). Concentration (mg/mL) of ethanol =
C AT I AS

C : Concentration of Ethanol in the standard solution (mg/mL). I : Concentration of Iopromide in the test solution (mg/mL). Operating conditions Detector : A hydrogen flame ionization detector Column : A capillary column is about 0.25 mm in inside diameter and about 30 cm in length, packed with a 1.4 m film of liquid phase 6% cyanopropylphenyl94% dimethylpolysiloxane. Column temperature : Programmed according to the following steps: it is held at 40 C for 10 minutes, then increased at a rate of 5 C per minute to 70 C; it is then increased at a rate of 30 C per minute to 220 C. Inject port temperature : 160 C Headspace sampler temperature : 80 C

KP 9 527 Detector temperature : maintained at 250 C. Carrier gas : helium Flow rate : 27 mm/second System suitability System performance: When the procedure is run with 2.0 mL of the standard solution as directed under the above operating conditions, the retention time of ethanol is about 3 minutes. System repeatability: When the test is repeated 3 times with 2.0 mL of the standard solution, the relative standard deviation of the peak area is not more than 4.0%. When the procedure is run with the blank solution according to the above operating conditions, the chromatogram shows no peak responses at the retention time for alcohol. (6) N-acetyl compound (Iopromide related compound I)Using the chromatograms obtained in the Assay, calculate the percentage of N-acetyl compound in the Iopromide taken by the formula: Amount (%) of N-acetyl compound = ( A + AY2 ) W 20 B Y1 W1 ( RY1 + RY2 )
WB : Quantity, in mg, of Iopromide related substance I RS taken to prepare the related substance I standard solution. W 1 : Quantity, in mg of Iopromide taken to prepare the test preparation. AY1 , AY2 : Peak responses for the Iopromide related substance I Y1 - and Y2 -isomers, respectively, in the chromatogram obtained from the test preparation. RY1 , RY2 : Peak responses for the Iopromide related substance I Y1 - and Y2 -isomers, respectively, in the chromatogram obtained from the Related compound I standard solution.

vapors 10 to 30 minutes and air-dry the plates. Both plates are exposed to UV light until the principal spots appear yellow. Spray evenly with visusalization solution, and examine the plates under ambient light. Determine total concentration of all secondary spots, except those due to free aromatic amine and to the Nacetyl compound obtained in the test solution for limit of N-acetyl compound, is not more than 3.0%. Acidic eluantchloroform : methanol : water : formic acid (62 : 32 : 6 : 2) Basic eluantdioxane : water : ethanol (85 : 15 : 4) Visualization solutionDissolve 2.7 g of ferric chloride in a 100 mL of 2.4 mol/L hydrochloric acid, use this solution as the solution A. Store this solution in a refrigerator. Dissolve 3.5 g of potassium ferricyanide in 100 mL of water, use this solution as the solution B. Store this solution in a refrigerator. Dissolve 5.0 g of sodium arsenite in 30 mL of 1 mol/L sodium hydroxide solution that has been cooled to 0. While stirring mix with 65 mL of 2.4 mol/L hydrochloric acid, use this solution as the solution C, and store at room temparature. Use the clear supernatant. Mix 10 mL of solution A, 10 mL of solution B and 2.0 mL of solution C, use this solution within 30 minutes. (8) IsomerUsing the chromatogram obtained from the test solution in the Assay, calculate the amounts of E 1 - and Z1 -isomers of Iopromide according to the following equation formula, is between 49.0 and 60.0%. Amounts (%) of E1- and Z1 -isomers of Iopromide = 100(rB1 + rZ1) /(rB1 + rB2 + rZ1 + rZ2 ) Amounts (%) of E2 - and Z2 -isomers of Iopromide = 100(rE2 + rZ2 ) /(rE1 + rE2 + rZ1 + rZ2 )
rE1 , rE2 , rZ1 , and rZ2 : the peak areas of E 1 , E2 , Z1 and Z 2 - isomers of Iopromide.

(7) Other related substancesDissolve about 0.1 g of Iopromide in a mixture of methanol and water (1:1) to make exactly 10 mL and use this solution as test solution. Separately, dissolve dilute appropriately a suitable amount of Iopromide RS in a mixture of methanol and water (1:1) to make exactly 0.01, 0.05, 0.1 and 0.2 mg per each mL, and use this solution as the standard solutions. Perform the test with these solutions as directed under the Thin-layer chromatography. Spot 1 L and 2 L of the test solution and 1 L of each of the standard solutions on two separate thin-layer chromatographic plates of silica gel with fluorescent indicator for thin-layer chromatography. Place one plate in a development chamber containing the acidic eluant, and the second plate in a development chamber containing the basic eluant. Develop the plates to a distance about 15 cm, remove the plates from the chambers, and airdry the plates. Both plates are exposed to UV light (main wavelength: 254 nm). Separately, the plate developed with the acetic eluant is exposed to ammonia

Water Not less than 1.5% (1 g, volumetric titration method, direct titration). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 38 mg of Iopromide and Iopromide RS and dissolve in a mixture of methanol and water (1 : 1) to make exactly 20 mL, and use this solution as the test solution. Separately, weigh accurately about 38 mg of Iopromide RS (previously measure the water), dissolve in a mixture of methanol and water (1 : 1), to make exactly 20 mL, and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operatiing conditions and calculate the peak area of Iopromide. The test for the peak

528 Monographs, Part I identification of E-isomer is run as follows. Add a constant volume of standard solution to a vial, seal, heat for 15 minutes at 121, and cool. Perform the test with the cooled solution according to the following operating conditions, and compare with the peak of standard solution unheated: identify with the retention time of two isomer peaks increasing the size after heating. Amount (mg) of iopromide (C18H24I3N3O8) = amount (mg) of Iopromide RS AT
AS

Packaging and Storage well-closed containners.

Preserve in light-resistant,

Iotalamic Acid
COOH I I

CH3COHN I

CONHCH 3

C : Concentration (mg/mL) of Iopromide RS in the standard solution. AT : Sum of peak area of E 1 , E 2 , Z1 and Z 2 isomers obtained from the test solution. AS : Sum of peak area of E 1 , E 2 , Z1 and Z 2 isomers obtained from the standard solution. Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 254 nm) Column : A stainless steel column, about 4.6 mm in inside diameter and about 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Mobile phase : Mix gently 6 g of chloroform with 59 g of methanol, then add 900 g of water, and do not stir or sparge the mobile phase during use. Allow the mobile phase to flow for not less than 60 minutes between each injection. Flow rate : About 1.2 mL/minute Column temperature: A constant temperature of about 20. System suitability System performance: When the procedure is run with 10 L of the standard solution as directed under the above operating conditions, the relative retention times for Iopromide E 1 -isomer, Iopromide E 2 -isomer, Iopromide Z1 -isomer, and Iopromide Z 2 -isomer are about 0.70, 0.75, 0.85, and 1.0, respectively with the resolution between Iopromide isomers Z1 and Z 2 being not less than 2.0; Separately, weigh accurately about 1.9 mg of Iopromide related substance I RS. Add the mixture of methanol and water (1 : 1), and dissolve to make exactly 100 mL.. When the procedure is run with 10 L of this solution as directed under the above operating conditions, the relative retention times of Y1 and Y2 -isomers of the Iopromide related compound I are about 0.28 and 0.31, respectively, and the signal-tonoise ratio for Y2 -isomer peak is not less than 20. System repeatability: When the test is repeated 5 times with 10 L of standard solution according to the above operating conditions: the relative standard deviation of the peak areas of Iopromide is not more than 2.0%.

C11H9I3N2O4: 613.91 Iotalamic Acid, when dried, contains not less than 99.0% and not more than 101.0% of iotalamic acid (C11H9I3N2O4).
Description Iotalamic Acid is a white powder and is odorless. Iotalamic Acid sparingly soluble in ethanol, very slightly soluble in water and practically insoluble in ether. Iotalamic Acid dissolves in sodium hydroxide TS. Iotalamic Acid is gradually colored by light. Identification (1) Heat 0.1 g of Iotalamic Acid over a flame: a purple gas is evolved. (2) Determine the infrared spectra of Iotalamic Acid and Iotalamic Acid RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Purity (1) Clarity and color of solutionDissolve 2.0 g of Iotalamic Acid in 10 mL of sodium hydroxide TS: the solution is clear and colorless. (2) Primary aromatic aminesTo 0.50 g of Iotalamic Acid, add 15 mL of water and dissolve in 1 mL of sodium hydroxide TS while ice-cooling. Add 4 mL of a solution of sodium nitrite (1 in 100) to the solution, immediately add 12 mL of 1 mol/L hydrochloric acid TS and shake gently. Then allow the mixture to stand for exactly 2 minutes, add 8 mL of ammonium sulfamate TS and shake occasionally for 5 minutes. Add 3 drops of a solution of -naphthol in ethanol (1 in 10), allow to stand for 1 minute, add 3.5 mL of ammoniaammonium chloride buffer solution, pH 10.7, mix and immediately add water to make 50 mL. Determine within 20 minutes the absorbance of this solution at 485 nm as directed under the Ultraviolet-visible Spectrophotometry, using a solution prepared in the same manner, as the blank: the absorbance is not more than 0.25. (3) Soluble halidesDissolve 0.5 g of Iotalamic Acid in 20 mL of diluted ammonia TS (1 in 40), add 6 mL of dilute nitric acid, shake, allow to stand for 5 minutes and filter. Transfer the filtrate to a Nessler tube, wash the residue with 20 mL of water, combine the fil-

KP 9 529 trate and the washings and add water to make 50 mL. Proceed as directed for the Chloride Limit Test using this solution as the test solution. Prepare the control solution as follows: to 0.10 mL of 0.01 mol/L hydrochloric acid VS and add 20 mL of diluted ammonia TS (1 in 40), 6 mL of dilute nitric acid and water to make 50 mL. (4) IodineDissolve 0.20 g of Iotalamic Acid in 2.0 mL of sodium hydroxide TS, add 2.5 mL of 0.5 mo1/L sulfuric acid TS and allow to stand for 10 minutes with occasional shaking. Add 5 mL of chloroform, shake well and allow to stand: the chloroform layer remains colorless. (5) Heavy metalsProceed with 1.0 g of Iotalamic Acid according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (6) ArsenicPrepare the test solution with 0.6 g of Iotalamic Acid according to Method 3 and perform the test (not more than 3.3 ppm).
Loss on Drying Not more than 0.5% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.4 g of Iotalamic Acid, previously dried, place in a flask, dissolve in 40 mL of sodium hydroxide TS, add 1 g of zinc powder and heat for 30 minutes under a reflux condenser. Cool, filter, wash the flask and the filter paper with 50 mL of water and combine the washings and the filtrate. Add 5 mL of glacial acetic acid to this solution and titrate with 0.1 mol/L silver nitrate VS, until the color of the precipitate changes from yellow to green (indicator: 1 mL of tetrabromophenolphthalein ethyl ester TS). Description Ipratropium Bromide is a white, crystalline powder. Ipratropium Bromide is freely soluble in water, soluble in dehydrated ethanol, slightly soluble in acetonitrile or in glacial acetic acid and practically insoluble in ether. pHThe pH of a solution of Ipratropium Bromide (1 in 20) is between 5.0 and 7.5. Melting pointAbout 223 C (with decomposition, after drying). Identification (1) To 5 mg of Ipratropium Bromide, add 0.5 mL of fuming nitric acid and evaporate on a water-bath to dryness. After cooling, dissolve the residue in 5 mL of acetone and add 2 drops of potassium hydroxide-ethanol TS: a purple color develops. (2) Determine the absorption spectra of solutions of Ipratropium Bromide and Ipratropium Bromide RS in 0.01 mol/L hydrochloric acid TS (3 in 2000) as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelength. maxima . (3) Determine the infrared spectra of Ipratropium Bromide and Ipratropium Bromide RS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumber. (4) The solution of Ipratropium Bromide (1 in 100) responds to the Qualitative Tests for bromide. Purity (1) Clarity and color of solutionDissolve 1.0 g of Ipratropium Bromide in 20 mL of water: the solution is clear and colorless. (2) SulfatePerform the test with 1.0 g of Ipratropium Bromide. Prepare the control solution with 0.50 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (3) Heavy metalsProceed with 2.0 g of Ipratropium Bromide according to Method 4 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 10 ppm). (4) ArsenicPrepare the test solution with 2.0 g of Ipratropium Bromide according to Method 4 and perform the test (not more than 1 ppm). (5) Isopropylatropine bromideDissolve 25 mg of Ipratropium Bromide in the mobile phase to make exactly 100 mL and use this solution as the test solution. Perform the test with 25 L of the test solution as directed under the Liquid Chromatography according to the following conditions. Determine the peak area, Aa, of ipratropium and the peak area, Ab, having a ratio of the retention time to ipratropium about 1.3 by the automatic integration method: Ab/(Aa +Ab) is not more than 0.01 and no peak other than the peak of ipratropium and the peak having a ratio of the retention time to ipratropium about 1.3 appears within about 14 minutes of the retention time after the solvent peak.

Each mL of 0.1 mol/L silver nitrate VS = 20.464 mg of C11H9I3N2O4

Packaging and Storage tight containers.

Preserve in light-resistant,

Ipratropium Bromide Hydrate

H O H3CNCH(CH 3)2 H H O C CH CH2OH Br H2 O

C20H30BrNO3H2O: 430.38 Ipratropium Bromide, when dried, contains not less than 99.0% and not more than 101.0% of ipratropium bromide (C20H30BrNO3: 412.36).

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 210 nm).

530 Monographs, Part I Column: A stainless steel column, about 4 mm in inside diameter and 10 cm to 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A room temperature. Mobile phase: A mixture of diluted phosphoric acid (1 in 200), acetonitrile and methanesulfonic acid (1000 : 120 : 1). Flow rate: Adjust the flow rate so that the retention time of ipratropium is about 7 minutes. System suitability System performance: Heat a solution of Ipratropium Bromide in 1 mol/L hydrochloric acid TS (1 in 100) at 100 C for 1 hour and cool. To 2.5 mL of this solution, add the mobile phase to make 100 mL. When the procedure is run with 25 L of this solution according to the above operating conditions, the resolution, between the peak of ipratropium and the peak having a ratio of the retention time to ipratropium about 0.6, is not less than 3.0. Detection sensitivity: Adjust the detection sensitivity so that the peak height of ipratropium obtained from 25 L of the test solution composes 50% to 80% of the full scale. (6) Apo-compoundsDissolve 0.14 g of Ipratropium Bromide in 0.01 mol/L hydrochloric acid TS to make 100 mL. Perform the test with this solution as directed under the Ultraviolet-visible Spectrophotometry and determine the absorbances, A1 and A2, at 246 nm and 263 nm, respectively: A1/A2 is not more than 0.91.
Loss on Drying Between 3.9% and 4.4% (1 g, 105 C, 4 hours). Residue on Ignition Not more than 0.10% (1 g). Assay Weigh accurately about 0.3 g of Ipratropium Bromide, previously dried, dissolve in 40 mL of glacial acetic acid, add 40 mL of dioxane and 2.5 mL of bismuth nitrate TS and titrate with 0.1 mol/L per chloric acid VS (potentiometric titration, Endpoint Detection Method in Tetrimetry). Perform a blank determination and make any necessary correction.

Isoconazole Nitrate
Cl Cl Cl

O N Cl N

HNO3

and enantiomer C18H14Cl4N2OHNO3 : 479.14

Isoconazole Nitrate contains not less than 99.0% and not more than 101.0% of isoconazole nitrate (C18H14Cl4N2OHNO3), calculated on the dried basis.
Description Isoconazole Nitrate is a white powder. Isoconazole Nitrate is soluble in methanol, slightly soluble in ethanol, and very slightly soluble in water. Identification (1) Determine the infrared spectra of Isoconazole Nitrate and Isoconazole Nitrate RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wavenumbers. (2) Dissolve 30 mg of Isoconazole Nitrate in 5 mL of methanol and use as the test solution. Separately, dissolve 30 mg of Isoconazole Nitrate RS in 5 mL of methanol and use this solution as the standard solution (1). Dissolve 30 mg of Isoconazole Nitrate RS and 30 mg of Econazole Nitrate RS in 5 mL of methanol and use this solution as the standard solution (2). Perform the test with 5 L each of the test solution and the standard solutions (1) and (2) as directed under Thin Layer Chromatography. Spot 5 L each of the test solution and the standard solutions (1) and (2) on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of methanol, dioxane and ammonium acetate (40 : 40 : 20) to a distance of about 15 cm and warm air-dry the plate for 15 minutes. Expose this plate to iodine vapor. The principal spots from the test solution and the standard solution show the same color and Rf value. This test is valid only if two spots in the chromatogram obtained with the standard solution (2) separate clearly. (3) Weigh the amount of Isoconazole Nitrate, equivalent to 1 mg of nitric acid, add to a mixture of 0.1 mg of nitrobenzene and 0.2 mL of sulfuric acid and allow to stand for 5 minutes. Cool on ice water and add 5 mL of water slowly with stirring. When 5 mL of 10 mol/L sodium hydroxide TS and 5 mL of acetone were added and allowed to stand, the upper layer turn into dark violet. Melting Point

Each mL of 0.1 mol/L perchloric acid VS = 41.24 mg of C20H30BrNO3

Packaging and Storage Preserve in tight containers.

Between 178 C and 182 C.

KP 9 531
Specific Optical Rotation [ ]20 D : between -0.10 and +0.10 (0.2 g, methanol, 20 mL, 100 mm) Purity (1) Clarity and color of solutionTo 0.2 g of Isoconazole Nitrate add methanol to make 20 mL: it is clear. (2) Related substancesWeigh accurately 0.10 g of Isoconazole Nitrate, dissolve in mobile phase to make exactly 10 mL and use this solution as the test solution. Separately, dissolve 2.5 mg of Isoconazole Nitrate RS and 2.5 mg of Econazole Nitrate RS in mobile phase to make exactly 100 mL and use this solution as the standard solution (1). To 1.0 mL of the test solution add mobile phase to make exactly 100 mL, pipet 5.0 ml of this solution, add mobile phase to make exactly 20 mL and use this solution as the standard solution (2). Perform the test with exactly 10 L each of the test and standard solutions (2) as directed under Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of any peak other than the principal peak in the chromatogram from the test solution is not greater than the area of the principal peak from the standard solution (2) (0.25%); the total area of all peaks other than the principal peak with the test solution is not greater than twice the area of the principal peak in the chromatogram from the standard solution (2) (0.5%). Disregard any peaks due to the solvent. nitrate ion and any peak with an area less than 0.2 times that of the principal peak in the chromatogram from the standard solution (2). Assay Weigh accurately about 0.35 g of Isoconazole Nitrate, add 75 ml of a mixture of methyl ethyl ketone and anhydrous acetic acid (7 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry).

Each mL of 0.1 mol/L perchloric acid VS = 47.91 mg of C18H15Cl4N3O4.

Packaging and Storage well-closed containers.

Preserve in light-resistant

Isoflurane
F F Cl H F H O F F

and enantiomer C3H2ClF5O: 184.49

Isoflurane contains not less than 99.0% and not more than 101.0% of isoflurane (C3H2ClF5O), calculated on the anhydrous basis.
Description Isoflurane is a clear, colorless fluidal liquid. Isoflurane is slightly soluble in water. Isoflurane is miscible with ethanol, methanol or o-xylene. Isoflurane is volatile, and has no inflammability. Isoflurane shows no optical rotation. 20 : about 1.30 Refractive Index nD Boiling PointBetween 47 C and 50 C Identification (1) The test solution obtained by the Oxygen Flask Combustion Method with 50 L of Isoflurane, using 40 mL of water as the absorbing liquid, responds to the Qualitative Tests for chloride and fluoride. (2) Determine the infrared spectra of Isoflurane and Isoflurane RS as directed in the liquid film method under Infrared Spectrophotometry, respectively: both spectra exhibit similar intensities of absorption at the same wave numbers. Specific Gravity
20 : Between 1.500 and 1.520 d 20

Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 235 nm). Column: A stainless steel column 4.6 mm in inside diameter and 10 cm in length, packed with octadecylsilanized silica gel for Liquid Chromatography (3 m in particle diameter). Mobile phase: Dissolve 6 g of ammonium acetate in a mixture of water, methanol and acetonitrile (380 : 320 : 300). Flow rate: 2.0 mL/minute System suitability System performance: When the procedure is run with 10 L of the standard solution (1) under the above operating condition, econazole and isoconazole are eluted in this order with the retention times being about 10 minutes and 14 minutes, respectively and resolution between the peaks of econazole and isoconazole is nol less than 5.0. Sensitivity of the system: Adjust so that the height of the principal peak in the chromatogram obtained with 10 L of the standard solution (2) is at least 50% of the full scale of the recorder.
Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g).

Purity (1) Acidity or alkalinityTo 10 mL of Isoflurane add 5 mL of freshly boiled and cooled water, and shake for 1 minute: the water layer is neutral. (2) Soluble chlorideTo 60 g of Isoflurane add 40 mL of water, shake thoroughly, and separate the water layer. To 20 mL of the water layer add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test with this solution as the test solution directed under Chloride Limit Test. Prepare the control solution with

532 Monographs, Part I 0.25 mL of 0.01 mol/L hydrochloric acid VS (not more than 3 ppm). (3) Soluble fluorideTo 6 g of Isoflurane add 12 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20), and shake for 10 minutes. Transfer 4.0 mL of the 0.01 mol/L sodium hydroxide TS (1 in 20) layer into a Nessler tube, add 30 mL of a mixture of alizarin complexone TS, acetic acidpotassium acetate buffer solution (pH 4.3) and cerium (III) nitrate TS (1 : 1 : 1), add water to make 50 mL, allow to stand for 60 minutes, and use this solution as the test solution. Separately, to 0.4 mL of the fluorine standard solution and 4.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20) in a Nessler tube add 30 mL of the mixture of alizarin complexone TS, acetic acid-potassium acetate buffer solution (pH 4.3) and cerium (III) nitrate TS (1 : 1 : 1), then proceed in the same manner as for the preparation of the test solution, and use the solution so obtained as the standard solution. Determine the absorbances of the test solution and the standard solution at 600 nm, respectively, as directed under Ultravioletvisible Spectrophotometry, using a solution, obtained by proceeding in the same manner as above with 4.0 mL of diluted 0.01 mol/L sodium hydroxide TS (1 in 20), as the blank: the absorbance of the test solution is not more than that of the standard solution (not more than 2 ppm). (4) Related substancesUse Isoflurane as the test solution. To exactly 1 mL of the test solution add oxylene to make exactly 100 mL. Pipet exactly 1 mL of this solution, add o-xylene to make exactly 100 mL, and use this solution as the standard solution. Perform the test with exactly 5 L each of the test solution and the standard solution as directed under Gas Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of the peak other than isoflurane is not more than the peak area of isoflurane from the standard solution, and the total area of all peaks other than isoflurane is not more than 3 times the peak area of isoflurane from the standard solution. Operating conditions Detector, column, column temperature, carrier gas, and flow rate: proceed as directed in the operating conditions in the Assay. System suitability System performance and System repeatability : proceed as directed in System suitability under the Assay. Test for required detectability: To exactly 1 mL of the standard solution add o-xylene to make exactly 2 mL. Confirm that the peak area of isoflurane obtained with 5 L of this solution is equivalent to 35 to 65% of that with 5 L of the standard solution. Time span of measurement: About 5 times as long as the retention time of isoflurane after injection of the test solution. (5) PeroxideTo 10 mL of Isoflurane add 1 mL of a solution of potassium iodide (1 in 10), freshly prepared, shake vigorously, and allow to stand in a dark place for 1 hour: the water layer is not yellow. (6) Residue on evaporationPipet exactly 65 mL of Isoflurane, evaporate on a water bath, and dry the residue at 105 C for 1 hour: not more than 1 mg. Fluorine standard solution: Dissolve exactly 2.21 g of sodium fluoride in water to make exactly 1000 mL. Pipet exactly 10 mL of this solution, and add water to make exactly 1000 mL. Each mL of this solution contains 0.01 mg of fluorine (F).
Water Not more than 0.1% (2 g, Coulometric titration). Assay Pipet exactly 5 mL each of Isoflurane and Isoflurane RS (separately determined water content in the same manner as Isoflurane), add exactly 3 mL of ethyl acetate as the internal standard, then add o-xylene to make exactly 50 mL each. To 5 mL each of these solutions add o-xylene to make 50 mL, and use these solutions as the test solution and the standard solution, respectively. Perform the test with 2 L each of the test solution and standard solution as directed under Gas Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of isoflurane to that of the internal standard.

Amount (mg) of isoflurane (C3H2ClF5O) in 5 mL of Isoflurane = VS

QT 1000 1.506 QS

VS : Amount of Isoflurane RS, calculated on the anhydrous basis (mL) 20 ) of isoflurane 1.506: Specific gravity ( d 20

Operating conditions Detector: A hydrogen flame-ionization detector. Column: A stainless steel column about 3 mm in inside diameter and about 3.5 m in length, packed with siliceous earth for gas chromatography (between 125 and 149 m in particle diameter), coated in 10% with nonylphenoxypoly(ethyleneoxy)-ethanol for gas chromatography and 15% with polyalkylene glycol for gas chromatography. Column temperature: A constant temperature of about 80 o C . Carrier gas: Nitrogen Flow rate: Adjust the flow rate so that the retention time of isolfurane is about 7 minutes. System suitability System performance: When the procedure is run with 2 L of the standard solution under the above operating conditions, isoflurane and the internal standard are eluted in this order with the resolution between these peaks being not less than 3.

KP 9 533 System repeatability: When the test is repeated 6 times with 2 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of isoflurane is not more than 1.0%.
Packaging and Storage Preserve in tight containers and store at not more than 30 C.

L-Isoleucine
H H 5C 2 C H C COOH

CH 3 NH 2

C6H13NO2: 131.17
L-Isoleucine, when dried, contains not less than 98.5% and not more than 101.0% of L-isoleucine (C6H13NO2).

Description L-Isoleucine is a white crystal or crystalline powder, is odorless or has a slightly bitter taste. L-Isoleucine is freely soluble in formic acid, sparingly soluble in water and practically insoluble in ethanol or in ether. L-Isoleucine dissolves in dilute hydrochloric acid. Identification Determine the infrared spectra of LIsoleucine and L-Isoleucine RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Specific Optical Rotation [ ]20 D : Between +39.5 and +41.5 (after drying, 1 g, 6 mol/L hydrochloric acid TS. 25 mL, 100 mm). pH Dissolve 1.0 g of L-Isoleucine in 100 mL of water: the pH of this solution is 5.5 - 6.5. Purity (1) Clarity and color of solutionDissolve 0.5 g of L-Isoleucine in 20 mL of 1mol/L hydrochloric acid TS: the solution is clear and colorless. (2) ChloridePerform the test with 0.5 g of LIsoleucine. Prepare the control solution with 0.30 mL of 0.01 mol/L hydrochloric acid VS (not more than 0.021%). (3) SulfatePerform the test with 0.6 g of LIsoleucine. Prepare the control solution with 0.35 mL of 0.005 mol/L sulfuric acid VS (not more than 0.028%). (4) AmmoniumPerform the test with 0.25 g of LIsoleucine. Prepare the control solution with 5.0 mL of standard ammonium solution (not more than 0.02%). (5) Heavy metalsDissolve 1.0 g of L-Isoleucine in 40 mL of water and 2 mL of dilute acetic acid by warming, cool and add water to make 50 mL. Perform the test. Prepare the control solution as follows: to 2.0

mL of standard lead solution, add 2 mL of dilute acetic acid and water to make 50 mL (not more than 20 ppm). (6) ArsenicPrepare the test solution with 1.0 g of L-Isoleucine according to Method 2 and perform the test. (not more than 2 ppm). (7) Other amino acidsDissolve 0.10 g of LIsoleucine in 25 mL of water and use this solution as the test solution. Pipet 1.0 mL of the test solution and add water to make exactly 50 mL. Pipet 5.0 mL of this solution, add water to make exactly 20 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 5 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of n-butanol, water and glacial acetic acid (3 : 1 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly the plate with a solution of ninhydrin in acetone (1 in 50) and heat at 80 C for 5 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Loss on Drying Not more than 0.3% (1 g, 105 C, 3 hours). Residue on Ignition

Not more than 0.1% (1 g).

Assay Weigh accurately about 0.25 g of L-Isoleucine, previously dried and dissolve in 3 mL of formic acid, add 50 mL of glacial acetic acid and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from orange-yellow through yellowish green to green (indicator: 0.5 mL of -naphtholbenzeine TS). Perform a blank determination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS = 13.117 mg of C6H13NO2

Packaging and Storage Preserve in tight containers.

Isoniazid
N

CONHNH 2

Isonicotinic Acid Hydrazide

C6H7N3O: 137.14

Isoniazid, when dried, contains not less than 98.5% and not more than 101.0% of isoniazid (C6H7N3O).
Description Isoniazid is a colorless crystal or a white, crystalline powder and is odorless. Isoniazid is freely soluble in water, in glacial acetic acid, sparingly soluble in ethanol, slightly soluble in acet-

534 Monographs, Part I ic anhydride and very slightly soluble in ether.

Identification (1) Dissolve about 20 mg each of Isoniazid and Isoniazid RS in water to make 200 mL and to 5 mL of these solutions, add 1 mL of 0.1 mol/L hydrochloric acid TS and water to make 50 mL. Determine the absorption spectra of these solutions as directed under the Ultraviolet-visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths.. (2) Determine the infrared spectra of Isoniazid and Isoniazid RS, previously dried, as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point

Isoniazide Injection
Isonicotinic acid hydrazid Injection Isoniazid Injection is an aqueous solution for injection. Isoniazid Injection contains not less than 95.0% and not more than 105.0% of the labeled amount of isoniazid (C6H7N3O: 137.14).
Method of Preparation Prepare as directed under Injections, with Isoniazide. Description Isoniazide Injection is a clear, colorless liquid. pHBetween 6.5 and 7.5. Identification To a volume of Isoniazid Injection, equivalent to 20 mg of Isoniazid according to the labeled amount, and add water to make 200 mL. To 5 mL of the solution add 1 mL of 0.1 mol/L hydrochloric acid TS and water to make 50 mL. Determine the absorption spectrum of this solution as directed under Ultraviolet-visible Spectrophotometry: it exhibits a maximum absorption between 264 - 268 nm. Sterility Test It meets the requirement. Foregin Insoluble Matter Test ment

Between 170 C and 173 C.

pH Dissolve 1.0 g of Isoniazid in 10 mL of freshly boiled and cooled water: the pH of this solution is between 6.5 and 7.5. Purity (1) Clarity and color of solutionDissolve 1.0 g of Isoniazid in 20 mL of water: the solution is clear and colorless. (2) Heavy metalsProceed with 1.0 g of Isoniazid according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (3) ArsenicPrepare the test solution with 0.40 g of Isoniazid according to Method 3 and perform the test. In this case, add 10 mL of a solution of magnesium nitrate in ethanol (1 in 50), then add 1.5 mL of strong hydrogen peroxide water and ignite the ethanol to burn (not more than 5 ppm). (4) HydrazineDissolve 0.10 g of Isoniazid in 5 mL of water, add 0.1 mL of a solution of salicylaldehyde in ethanol (1 in 20), shake immediately and allow to stand for 5 minutes: no turbidity is produced. Loss on Drying Not more than 0.5% (1 g, 105 C, 2 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.3 g of Isoniazid, previously dried, Dissolve in 50 mL of glacial acetic acid and 10 mL of acetic anhydride and titrate with 0.1 mol/L perchloric acid VS until the color of the solution changes from yellow to seen (indicator: 0.5 mL of naphtholbenzeine TS). Perform a blank determination and make any necessary correction.

It meets the require-

Insoluble Particulate Matter Test for Injections It meets the requirement Determination of Volume of Injection in Containers It meets the requirement. Assay To an exactly measured volume of Isoniazid Injection, equivalent to about 50 mg of isoniazid (C6H7N3O), add water to make exactly 100 mL. Pipet 5 mL of the solution, add exactly 5 mL of the internal standard and the mobile phase to make 50 mL, and use this solution as athe sample solution. Separately, weigh accurately about 50 mg of isoniazid for assay, previously drid at 105 C for 2 hours, and dissolve in water to make exactly 100 mL. Pipet 5 mL of this solution. Add exactly 5 mL of the internal standard and the mobile phase to make 50 mL, and use this solution as the standard solution. Perform the test with 5 L each of the sample solution and standard solution as directed under Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS , of the peak area of isoniazid to that of the internal standard.

Each mL of 0.1 mol/L perchloric acid VS = 13.714 mg of C6H7N3O

Packaging and Storage tight containers.

Preserve in light-resistant,

Amount (mg) of isoniazid (C6H7N3O) = amount (mg) of Isoniazide RS

QT QS

KP 9 535 Operating conditions Detector: An ultraviolet absorption photometer (wavelength : 265 nm). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about . Mobile phase: Disssolve 6.80 g of potassium dihydrogen phosphate in water to make 1000 mL. Separately, to 5.76 g of phosphoric acid add water to make 1000 mL. Mix these solutions to make a solution having pH 2.5. To 500 mL of this solution add 500 mL of methanol, and add 2.86 g of sodium tridecanesulfonate to dissolve. Flow rate: Adjust the flow rate so that the retention time of isoniazid is about 5 minutes. System suitability System performance: When the procedure is run with 5 L of the standard solution under the above operating conditions, isoniazid and the internal standard are eluted in this order with the resolution between these peaks being not less than 10. System repeatability: When the test is repeated 6 times with 5 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of the peak areas of isoniazid to that of the internal standard is not more than 1.3%.
Packaging and Storage containers.

lution 20 minutes after starting the test and filter through a membrane filter with pore size of not more than 0.45 m. Discard the first 10 mL of the filtrate, pipet 5.0 mL of the subsequent, add water to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 0.10 g of Isoniazid RS, previously dried at 105 C, for 2 hours, dissolve in water to make exactly 100 mL, then pipet 5.0 mL of this solution, add water to make exactly 50 mL and then pipet 5.0 mL of this solution, add water to make exactly 50 mL and use this solution as the standard solution. Determine the absorbances, AT and AS , of the test solution and the standard solution, respectively, at 267 nm as directed under the Ultraviolet-visible Spectrophotometry. The dissolution rate of Isoniazid Tablets in 20 minutes is not less than 75%. Dissolution rate (%) with respect to the labeled amount of isoniazid (C6H7N3O) = W S
AT 90 AS C

WS : Amount (mg) of Isoniazid RS. C: Labeled amount (mg) of isoniazid (C6H7N3O) in 1 tablet.

Uniformity of Dosage Units ment.

It meets the require-

Light-resistant, hermetic

Isoniazid Tablets
Isonicotinic Acid Hydrazide Tablets Isoniazid Tablets contain not less than 95.0% and not more than 105.0% of the labeled amount of isoniazid (C6H7N3O: 137.14).
Method of Preparation Tablets, with Isoniazid.

Prepare as directed under

Identificatlon Take a portion of powdered Isoniazid Tablets, equivalent to 20 mg of Isoniazid according to the labeled amount, add 200 mL of water, shake well and filter. To 5 mL of the filtrate, add 1 mL of 0.1 mol/L hydrochloric acid TS and water to make 50 mL and determine the absorption spectrum of this solution as directed under the Ultraviolet-visible Spectrophotometry: it exhibits a maximum between 264 nm and 268 nm. Dissolution Test Perform the test with 1 tablet of Isoniazid Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of water. Take 20 mL or more of the dissolved so-

Assay Weigh accurately and powder not less than 20 Isoniazid Tablets. Weigh accurately a portion of the powder, equivalent to about 0.10 g of isoniazid (C6H7N3O), add 150 mL of water, shake for 30 minutes, then add water to make exactly 200 mL and filter. Discard the first 10 mL of the filtrate, pipet 5.0 mL of the subsequent filtrate, add the mobile phase to make exactly 50 mL and use this solution as the test solution. Separately, weigh accurately about 50 mg of Isoniazid RS, previously dried at 105 o C for 2 hours, dissolve in water to make exactly 100 mL. Pipet 5.0 mL of this solution, add the mobile phase to make exactly 50 mL and use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine the peak areas, AT and AS , of Isoniazid of the test solution and the standard solution, respectively.

Amount (mg) of isoniazid (C6H7N3O) = amount (mg) of Isoniazid RS

AT AS

Operoting condition Detector: An ultraviolet absorption photometer (wavelength: 265 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with

536 Monographs, Part I octadecysilanized silica gel for liquid chromatography (5 m in particle diameter). Column temperature: A constant temperature of about 40 C. Mobile phase: Dissolve 6.80 g of monobasic potassium phosphate in water to make 1000 mL. Separately, dissolve 5.76 g of phosphoric acid in water to make 1000 mL. Mix these solutions, to adjust the pH to 2.5. To 400 mL of this solution, add 600 mL of methanol and dissolve 2.86 g of sodium tridodecane sulfonate. Flow rate: Adjust the flow rate so that the retention time of Isoniazid is about 5 minutes. System suitability System performance: Dissolve 5 mg of Isoniazid and 5 mg of isonicotinic acid in 100 mL of the mobile phase. When the procedure is run with 10 L of this solution under the above operating conditions, isonicotinic acid and Isoniazid are eluted in this order with the resolution between these peaks being not less than 1.5. System repeatability : When the test is repeated 6 times with 5 L of the standard solution under the above operatig conditions, the relative standard deviation of the ratios of the peak area of isoniazid to that of the internal standard is not more than 1.0%.
Packaging and Storage tight containers.

sists of fine, oblong crystals of 5 to 30 m in major axis and does not contain amorphous substances or large aggregates.
Identification Adjust the pH of Isophan Insulin Injection (Aqueous suspension to Between 2.5 and 3.5 with dilute hydrochloric acid; Particles dissolve and the solution is clear and colorless.. pH

Between 7.0 and 7.4

Preserve in light-resistant,

Isophane Iusulin Injection (Aqueous Suspension)

Isophane Insulin Injection (Aqueous Suspension) is an aqueous suspension for injection. Isophane Insulin Injection contains not less than 90.0% and not more than 110.0% of the labeled insulin units and not less than 0.01 mg and not more than 0.04 mg of zinc (Zn: 65.41) for each labeled 100 units. When sodium chloride is used in the preparation of Isophane Insulin Injection (Aqueous Suspension), this should be stated on the label.
Method of Preparation Prepare as directed under Injections, with Insulin and Protamine Sulfate. To each 100 mL of Isophane Insulin Injection (Aqueous Suspension), add either 0.38 to 0.63 g of dibasic sodium phosphate, 1.4 to 1.8 g of concentrated glycerin, 0.15 to 0.17 g of cresol and 0.06 to 0.07 g of phenol, or 0.38 to 0.63 g of dibasic sodium phosphate, 0.42 to 0.45 g of sodium chloride, 0.7 to 0.9g of concentrated glycerin and 0.18 to 0.22 g of cresol. Description Isophane Insulin Injection (Aqueous Suspension) is a white aqueous suspension. When allowed to stand, it separates into a white precipitate and colorless supernatant liquid and the precipitate returns easily to the suspension state on gentle shaking. When examined microscopically, the precipitate mostly con-

Purity (1) ProteinPerform the test as directed under the Nitrogen Determination: not exceeding 0.85 mg of nitrogen is found for each labeled 100 units. (2) Isophane ratio (i) Buffer solution A: Dissolve 2.0 g of anhydrous dibasic sodium phosphate, 16 g of glycerin, 1.6 g of m-cresol and 0.65 g of phenol in water to make exactly 200 mL. (ii) Buffer solution B: Dissolve 2.0 g of anhydrous dibasic sodium phosphate, 4.35 g of sodium chloride, 8.0 g of glycerin and 2.0 g of n-cresol in water to make exactly 200 mL. (iii) Insulin solution: Weigh accurately 1000 units of insulin RS, dissolve in 1.5 mL of diluted hydrochloric acid (1 in 360) and add 5.0 mL of buffer solution A and water to make 20 mL. Adjust the pH to 7.2 with dilute hydrochloric acid or sodium hydroxide TS. The solution is clear. Dilute with water to make exactly 25 mL, The solution is clear and the pH is between 7.1 and 7.4. When it is stated on the label that sodium chloride is used in the preparation, use 5.0 mL of buffer solution B instead of buffer solution A in the above procedure. (iv) Protamine solution: Weigh accurately 50 mg of protamine sulfate RS and dissolve in 2 mL of buffer solution A and water to make 8 mL. Adjust the pH to 7.2 with dilute hydrochloric acid or sodium hydroxide TS and dilute with water to exactly 10 mL. The solution is clear and the pH is between 7.1 and 7.4. When it is stated on the label that sodium chloride is used in the preparation, use 2 mL of buffer solution B instead of buffer solution A in the above procedure. (v) Procedure: When Isophane Insulin Injection (Aqueous Suspension) contains 40 units per ml, centrifuge a portion of the suspension, measure exactly two 10 mL volumes of the supernatant liquid in two tubes A and B, respectively, add exactly 1 mL of the Insulin solution to tube, A and 1 mL of the protamine solution to tube B, mix the contents of each tube, allow to stand for 10 minutes and determine the turbidity of each mixture by using a photometer or a nephelometer: the turbidity of the mixture in tube B is not greater than that in tube A. When Isophane Insulin Injection (Aqueous Suspension) contains 80 units per ml, measure exactly 5 mL of the supernatant liquid and proceed in the same manner. Sterility Test It meets the requirement. Determination of Volume of Injection in Containers It meets the requirement.

KP 9 537
Assay (1) InsulinTake Isophane Insulin Injection (Aqueous Suspension), add diluted hydrochloric acid (1 in 100) to adjust pH to about 2.5 and proceed with the clear solution as directed in the Assay under Insulin Injection. (2) ZincPipet a volume of Isophane Insulin Injection (Aqueous Suspension), equivalent to about 400 units according to the labeled units, add 1 mL of 0.1 mol/L hydrochloric acid TS and water to make exactly 100 mL, dilute, if necessary, with water to contain 0.6 to 1.0 g of zinc pet mL and use this solution as the test solution. Separately, pipet a volume of standard zinc solution for the Atomic Absorption Spectrophotometry, dilute with water to contain 0.4 to 1.2 g of zinc per mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution according to the Atomic Absorption Spectrophotometry under the following conditions and determine the amount of zinc in the test solution using the analytical curve obtained from the absorbance of the standard solution. Purity (1) Clarity and color of solutionTo 2.0 mL of Isopropanol, add 8 mL of water and shake: the solution is clear. (2) AcidTo 15.0 mL of Isopropanol, add 50 mL of freshly boiled and cooled water and 2 drops of phenolphthalein TS and add 0.40 mL of 0.01 mol/L sodium hydroxide VS: a red color develops. (3) Residue on evaporationEvaporate 20.0 mL of Isopropanol on a water-bath to dryness and dry at 105 C for 1 hour: the weight of the residue is not more than 1.0 mg. Water Not more than 0.75% (2 mL, volumetric titration, direct titration). Distilling Range than 94 vol%.

Between 81 C and 83 C, not less

Packaging and Storage Preserve in remoting from fire, tight containers.

Gas: Combustible gas-Acetylene gas. Supporting gas-Air. Lamp: Zinc hollow-cathode lamp. Wavelength: 213.9 nm.
Packaging and Storage Preserve in hermetic containers in a cold place and avoid freezing. Expiration Date 24 months after preparation.

Isopropylantipyrine
CH3 H3C N N H3C CH O CH3

Propyphenazone

C14H18N2O: 230.31

Isopropanol
OH CH3CHCH3

Isopropylantipyrine, when dried, contains not less than 98.0% and not more than 101.0% of isopropylantipyrine (C14H18N2O).
Description Isopropylantipyrine is a white crystal or crystalline powder, is odorless and has a slightly bitter taste. Isopropylantipyrine is very soluble in glacial acetic acid, freely soluble in ethanol or in acetone, soluble in ether and slightly soluble in water. Identification (1) To 2 mL of a solution of Isopropylantipyrine (1 in 500), add 1 drop of ferric chloride TS: a pale red color develops. Further add 3 drops of sulfuric acid to this solution: the color changes to pale yellow. (2) Add 5 mL of a solution of Isopropylantipyrine (1 in 500) to a mixture of 5 mL of potassium ferricyanide TS and 1 to 2 drops of ferric chloride TS: A dark green color gradually develops. (3) To 2 mL of a solution of Isopropylantipyrine (1 in 500), add 2 to 3 drops of tannic acid TS: a white precipitate is produced. Melting Point

Isopropyl Alcohol

C3H8O: 60.10

Description Isopropanol is a clear, colorless liquid and has a characteristic odor. Isopropanol is miscible with water, with methanol, with ethanol,or with ether. Isopropanol is flammable and volatile. Identification (1) To 1 mL of Isopropanol, add 2 mL of iodine TS and 2 mL of sodium hydroxide TS and shake: pale yellow precipitate is formed. (2) To 5 mL of Isopropanol, add 20 mL of potassium dichromate TS and 5 mL of sulfuric acid with caution and warm gently on a water-bath: the produced gas has the odor of acetone and the gas turns the filter paper, previously wetted with a solution of salicylaldehyde in ethanol (1 in 10) and with a solution of sodium hydroxide (3 in 10), to red brown. Specific Gravity
20 : Between 0.785 and 0.788 d 20

Between 103 C and 105 C.

Purity (1) ChlorideDissolve 1.0 g of Isopropylan-

538 Monographs, Part I tipyrine in 30 mL of dilute ethanol and add 6 mL of dilute nitric acid and water to make 50 mL. Perform the test. Prepare the control solution as follows: to 0.40 mL of 0.01 mol/L hydrochloric acid VS, add 6 mL of dilute nitric acid, 30 mL of dilute ethanol and water to make 50 mL (not more than 0.014%). (2) SulfateDissolve 1.0 g of Isopropylantipyrine in 30 mL of dilute ethanol and add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test. Prepare the control solution as follows: to 0.40 mL of 0.005 mol/L sulfuric acid VS, add 1 mL of dilute hydrochloric acid and 30 mL of dilute ethanol and dilute with water to make 50 mL (not more than 0.019%). (3) Heavy metalsDissolve 1.0 g of Isopropylantipyrine in 25 mL of acetone, add 2 mL of dilute acetic acid and water to make 50 mL and perform the test. Prepare the control solution as follows: to 2.0 mL of standard lead solution, add 2 mL of dilute acetic acid, 25 mL of acetone and dilute with water to make 50 mL (not more than 20 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Isopropylantipyrine according to Method 3 and perform the test (not more than 2 ppm). (5) AntipyrineDissolve 1.0 g of Isopropylantipyrine in 10 mL of dilute ethanol and add 1 mL of sodium nitrite TS and 1 mL of dilute sulfuric acid: no green color develops.
Loss on Drying Not more than 0.5% (1 g, in vacuum, silica gel, 5 hours). Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 0.4 g of Isopropylantipyrine, previously dried, dissolve in 60 mL of a mixture of glacial acetic acid and acetic anhydride (2 : 1) and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration, Endpoint Detection Method in Titrimetry), Perform a blank determination and make any necessary correction.

less than 97.0% and not more than 101.5% of isoproterenol hydrochloride (C11H17NO3HCl).
Description Isoproterenol Hydrochloride is a white, crystalline powder and is odorless. Isoproterenol Hydrochloride is freely soluble in water, sparingly soluble in ethanol and practically insoluble in glacial acetic acid, in acetic anhydride, in ether or in chloroform. Isoproterenol Hydrochloride is gradually colored by air and by light. Identification (1) Determine the absorption spectra of solutions of Isoproterenol Hydrochloride and Isoproterenol Hydrochloride RS in 0.1 mol/L hydrochloric acid TS(1 in 20000) as directed under the Ultravioletvisible Spectrophotometry: both spectra exhibit the similar intensities of absorption at the same wavelengths. (2) Determine the infrared spectra of Isoproterenol Hydrochloride and Isoproterenol Hydrochloride RS, previously dried, as directed in the potassium disk method under the Infrared Spectrophotometry: both spectra exhibit the similar intensities of absorption at the same wavenumbers. Melting Point Between 165 o C and 170 o C . Purity (1) Clarity and color of solutionDissolve 1.0 g of Isoproterenol Hydrochloride in 20 mL of 0.1 mol/L hydrochloric acid TS: the solution is clear and colorless. (2) SulfatePerform the test with 0.10 g of Isoproterenol Hydrochloride. Prepare the control solution with 0.40 mL of 0.005mol/L sulfuric acid VS (not more than 0.2%). (3) IsoproterenoneDissolve 50 mg of Isoproterenol Hydrochloride in 0.01 mol/L hydrochloric acid TS to make exactly 25 mL and determine the absorbance of the solution at 310 nm as directed under the Ultraviolet-visible Spectrophotometry: not more than 0.2. Loss on Drying Not more than 1.0% (1 g, in vacuum, silica gel, 4 hours). Residue on Ignition Not more than 0.2% (1 g).

Each mL of 0.1 mol/L perchloric acid VS = 23.031 mg of C14H18N2O

Packaging and Storage Preserve in tight containers.

Isoproterenol Hydrochloride
OH H N H CH3 HO OH CH3 HCl

and enantiomer C11H17NO3HCl: 247.72

Isoprenaline hydrochloride

Isoproterenol Hydrochloride, when dried, contains not

Assay Weigh accurately about 0.125 g of Isoproterenol Hydrochloride, dissolve in a solution of sodium bisulfite (3 in 1000), dilute with the solution of sodium bisulfite to make 25 mL and mix. Transfer 5.0 mL of this solution, dilute with 0.17 mol/L acetic acid to make exactly 100 mL and mix. Use this solution as the test solution. Separately, weigh accurately a portion of Isoproterenol Hydrochloride RS to make a solution containing about 2.5 mg per mL and dissolve in mobile phase. Pipet 5.0 mL of this solution and dilute with 0.17 mol/L acetic acid to make exactly 50 mL. Use this solution as the standard solution. Perform the test with 10 L each of the test solution and the standard solu-

KP 9 539 tion as directed under the Liquid Chromatography. Determine, AT and AS , the peak areas of isoproterenol for the test solution and the standard solution, respectively. Amount (mg) of isoproterenol hydrochloride (C11H17NO3.HCl) = 0.5 C
AT AS

C: Concentration (g/mL) of Isoproterenol Hydrochloride RS in the standard solution. Operating conditions Detector: An ultraviolet absorption photometer (wavelength: 278 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 30 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 m to 10 m in particle diameter). Mobile phase: 0.17 mol/L of acetic acid. Flow rate: 1.5 mL/minute. System suitability System repeatabily: When the test is repeated 6 times with 10 L of the standard solution, the relative standard deviation of the peak area of isoproterenol is not more than 3.0%.
Packaging and Storage resistant containers.

bath: an orange preciptate is formed. (2) To 2 g of Isosorbide, add 30 mL of pyridine and 4 mL of benzoyl chloride, boil under a reflux condenser for 50 minutes, cool and pour gradually the solution into 100 mL of cold water. Filter the formed precipitate by suction through a glass filter (G3), wash with water, recrystallize twice from ethanol and dry in a desiccator (in vacuum, silica gel) for 4 hours: it melts between 102 o C and 103 o C . (3) Determine the infrared spectrum of Isosorbide and Isosorbide TS as directed in the potassium bromide disk method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavelengths.
Specific Optical Rotation [ ]20 D : Between +45.0 and +46.0 (5 g, calculated on the anhydrous basis, water, 50 mL, 100 mm). Purity (1) Clarity and color of solutionTake 25 g of Isosorbide in a Nessier tube and dissolve in 50 mL of water: the solution is clear and has no more color than the following control solution.

Preserve in tight, light-

Control solutionTo a mixture of 1.0 mL of cobaltous chloride stock CS, 3.0 mL of ferric chloride stock CS and 2.0 mL of cupric sulfate stock CS, add water to make 10.0 mL. To 3.0 mL of this solution, add water to make 50 mL. (2) SulfatePerform the test with 2.0 g of Isosorbide. Prepare the control solution with 1.0 mL of 0.005 mol/L sulfuric acid VS (not more than 0.024%). (3) Heavy metalsProceed with 5.0 g of Isosorbide according to Method 1 and perform the test. Prepare the control solution with 2.5 mL of standard lead solution (not more than 5 ppm). (4) ArsenicPrepare the test solution with 1.0 g of Isosorbide according to Method 1 and perform the test (not more than 2 ppm). (5) Related substancesDissolve 0.10 g of Isosorbide in 10 mL of methanol and use this solution as the test solution. Pipet 2.0 mL of the test solution, add methanol to make exactly 100 mL and use this solution as the standard solution. Perform the test with the test solution and the standard solution as directed under the Thin-layer Chromatography. Spot 10 L each of the test solution and the standard solution on a plate of silica gel for thin-layer chromatography. Develop the plate with a mixture of ethanol and cyclohexane (1 : 1) to a distance of about 10 cm and air-dry the plate. Spray evenly a mixture of ethanol and sulfuric acid (9 : 1) on the plate and heat at 105 C for 30 minutes: the spots other than the principal spot from the test solution are not more intense than the spot from the standard solution.
Water Not more than 1.5% (2 g, volumetric titration, direct titration).

Isosorbide
H HO H O

O H H OH

C6H10O4: 146.14 Isosorbide, contains not less than 98.5% and not more than 101.0% of isosorbide (C6H10O4), calculated on the anhydrous basis.
Description Isosorbide is a white crystal or mass, is odorless or has a faint, characteristic odor and has a bitter taste. Isosorbide is very soluble in water or in methanol, freely soluble in ethanol and slightly soluble in ether. Isosorbide is hygroscopic. Identification (1) To 0.1 g of Isosorbide, add 6 mL of diluted sulfuric acid (1 in 2) and dissolve by heating in a water-bath. After cooling, shake well with 1 mL of a solution of potassium permanganate (1 in 30) and heat in a water-bath until the color of potassium permanganate disappears. To this solution, add 10 mL of 2,4-dinitrophenylhydrazine TS and heat in a water-

540 Monographs, Part I

Residue on Ignition Not more than 0.1% (1 g). Assay Weigh accurately about 10 g of Isosorbide and dissolve in water to make exactly 100 mL. Determine the optical rotation, D , of this solution at 20 1 C in a 100 mm cell.

Amount (g) of isosorbide (C6H10O4) = D 2.1978

Packaging and Storage Preserve in tight containers.

Isosorbide Dinitrate
ONO 2 H H O O H H ONO 2

C6H8N2O8: 236.14 Isosorbide Dinitrate, contains not less than 95.0% and not more than 101.0% of isosorbide dinitrate (C6H8N2O8), calculated on the anhydrous basis.
Description Isosorbide Dinitrate is a white crystal or crystalline powder, is odorless or has a faint odor like that of nitric acid. Isosorbide Dinitrate is very soluble in dimethylformamide or in acetone, freely soluble in chloroform or in toluene, soluble in methanol, in ethanol or in ether and practically insoluble in water. Isosorbide Dinitrate explodes if heated quickly or subjected to percussion. Identification (1) Dissolve 10 mg of Isosorbide Dinitrate in 1 mL of water and dissolve by adding 2 mL of sulfuric acid cautiously. After cooling, superimpose 3 mL of ferrous sulfate TS and allow to stand for 5 minute to 10 minutes: a brown ring is produced at the zone of contact. (2) Dissolve 0.1 g of Isosorbide Dinitrate in 6 mL of diluted sulfuric acid (1 in 2) by heating in a waterbath. After cooling, add 1 mL of a solution of potassium permanganate (1 in 30), stir well and heat in a water-bath until the color of potassium permanganate disappears. Add 10 mL of 2,4-dinitrophenylhydrazine TS and heat in a water-bath: an orange precipitate is produced. Specific Optical Rotation [ ]20 D : Between + 134 and + 139 (1 g, calculated on the anhydrous basis, ethanol, 100 mL, 100 mm). Purity (1) Clarity and color of solutionDissolve 1.0 g of Isosorbide Dinitrate in 10 mL of acetone: the solution is clear and colorless.

(2) SulfateDissolve 1.5 g of Isosorbide Dinitrate in 15 mL of dimethylformamide, add 60 mL of water, cool and filter. Wash the filter paper with three 20 mL volumes of water, combine the washings with the filtrate and add water to make 150 mL. To 40 mL of this solution, add 1 mL of dilute hydrochloric acid and water to make 50 mL. Perform the test. Prepare the control solution with 0.40 mL of 0.005 mol/L sulfuric acid VS (not more than 0.048%). (3) NitrateDissolve 50 mg of Isosorbide Dinitrate in 30 mL of toluene and extract with three 20 mL volumes of water. Combine the aqueous layers and wash with two 20 mL volumes of toluene. To the aqueous layer, add water to make exactly 100 mL and use this solution as the test solution. Pipet 5.0 mL of standard nitric acid solution and 25.0 mL of the test solution in each Nessler tube and add water to make exactly 50 mL, respectively. To each of them, add 60 mg of GrissRomijin's nitric acid reagent, stir well, allow to stand for 30 minutes and observe from the side of the Nessler tube: the test solution has no more color than the standard solution. (4) Heavy metalsDissolve 1.0 g of Isosorbide Dinitrate in 30 mL of acetone and add 2 mL of dilute acetic acid and water to make 50 mL. Perform the test. Prepare the control solution as follows: to 2.0 mL of standard lead solution, add 30 mL of acetone, 2 mL of dilute acetic acid and water to make 50 mL (not more than 20 ppm).
Water Not more than 1.5% (0.3 g, volumetric titration, direct titration). Assay Weigh accurately about 0.1 g of Isosorbide Dinitrate in a Kjedahl flask as described under the Nitrogen Deterimination, dissolve in 10 mL of methanol, add 3 g of Devardas alloy and 50 mL of water and connect the flask with the distillation apparatus as described under the Nitrogen Determination. Measure exactly 25 mL of 0.05 mol/L sulfuric acid VS in an absorption flask, add 5 drops of bromocresol greenmethyl red TS and immerse the lower end of the condenser tube in it. Add 15 mL of a solution sodium hydroxide (1 in 2) through the funnel, cautiously rinse the funnel with 20 mL of water, immediately close the clamp attached to the rubber tubing, then begin the distillation until the distillate measures 100 mL. Remove the absorption flask, rinse the end of the condenser tube with a small quantity of water and titrate the distillate and the rinsings with 0.1 mol/L sodium hydroxide VS until the color of the solution changes from red through pale red-purple to pale bluegreen. Perform a blank determination and make any necessary correction.

Each mL of 0.05 mol/L sulfuric acid VS = 11.807 mg of C6H8N2O8

Packaging and Storage Preserve in light-resistant, tight containers in a cold place.

KP 9 541 C: the amount (%) of Isosorbide Dinitrate in Isosorbide Dinitrate RS Internal standard solutionDissolve 3 g of Nitroglycerin RS (10% diluted with lactose) in 100 mL of mobile Phase. Operating conditions Detector : An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column, about 4 mm in inside diameter and about 15 cm to 30 cm in length, packed with octadeylsilanizd silica gel for liquid chromatography (5 m to 10 m in particle diameter). Column temperature: A room temperature. Mobile phase: To the mixture of methanol and 0.005 mol/L triethylamine solution (50 : 50), add acetic acid to adjust pH to 5.0. Flow rate: 1.0 mL/minute. System suitability System performance: When the procedure is run with 10 L of the standard solution under the above operating conditions, Isosorbide Dinitrate and internal standard are eluted in this order with a resolution between their peaks being not more than 2.0. System repeatability: When the test is repeated 6 times with 10 L of the standard solution under the above operating conditions, the relative standard deviation of the ratios of peak area of Isosorbide Dinitrate to that of the internal standard is not more than 2.0%.
Packaging and Storage Preserve in tight containers. Uniformity of Dosage Units ment.

Isosorbide Dinitrate Tablets

Isosorbide Dinitrate Tablets contain not less than 93.0% and not more than 107.0% of the labeled amount of isosorbide dinitrate (C6H8N2O8: 236.14).
Method of Preparation Prepare as directed under Tablets, with Isosorbide Dinitrate. Identification Weigh a portion of powdered Isosorbide Dinitrate Tablets equivalent to 0.1 g of lsosorbide Dinitrate, according to the labeled amount, add 50 mL of ether, shake well and filter. Measure 5 mL of the filtrate, evaporate to dryness cautiously, add 1 mL of water to the residue and dissolve by adding 2 mL of sulfuric acid cautiously. After cooling, superimpose 3 mL of ferrous sulfate TS and allow to stand for 5 to 10 minutes: a brown ring is produced at the zone of contact. Purity NitrateWeigh accurately a quantity of powdered Isosorbide Dinitrate Tablets, equivalent to 50 mg of Isosorbide Dinitrate according to the labeled amount, transfer to a separator, add 30 mL of toluene, shake thoroughly, extract with three 20 mL volumes of water and proceed as directed in Purity (3) under Isosorbide Dinitrate. Disintegration Test It meets the requirement. For sublingual tablets, the time limit of the test is 2 minutes and omit the use of the auxiliary disk.

It meets the require-

Assay Weigh accurately and powder not less than 20 Isosorbide Dinitrate. Weigh accurately a portion of the powder, equivalent to about 5 mg of isosorbide dinitrate (C6H8N2O8), dissolve in mobile phase add 5.0 mL of interal standard solution and mobile phase to make exactly 20 mL, filtrate and use this filter as the test solution. Separately, 0.2 g of Isosorbide Nitrate RS (25%, diluted with lactose), dissolve in mobile phase and to make exactly 50 mL. Pipet 5.0 mL of this solution, add 5.0 mL of internal standard solution and mobile phase to make exactly 20 mL and use this solution as the standard solution. Perform the test with 10L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following conditions and calculate the ratios, QT and QS ,of the peak area of Isosorbide Dinitrate to that of the internal standard, for the test solution and the standard solution, respectively.

Isotretinoin
H3C CH3 CH3 CH3

CH3

HO

C20H28O2 : 300.44 Isotretinoin contains not less than 98.0% and not more than 102.0% of isotretinoin (C20H28O2),calculated on the dried basis.
Description Isotretinoin is a yellow crystal. Isotretinoin is soluble in chloroform, slightly soluble in ethanol, in isopropanol or in polyethylene glycol 400, and practically insoluble in water. Identification (1) Determine the absorption spectra of solutions of a solution of 0.01 mol/L hydrochloric acid TS in isopropanol (1 in 1000) of Isotretinoin and Isotretinoin RS (1 in 25000) as directed under the Ultraviolet-Visible Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave-

Amount (mg) of isosorbide dinitrate (C6H8N2O8) = amount (mg) of Isosorbide Dinitrate RS

C Q 1 T 100 QS 10

542 Monographs, Part I lengths. (2) Determine the infrared spectra of Isotretinoin and Isotretinoin RS as directed under the paste method of Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wave numbers.
Purity (1) Heavy metalsProceed with 1.0 g of Isotretinoin according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) TretinoinWeigh accurately about 0.25 g of Isotretinoin dissolve, in a small amount of dichloromethane TS, and dilute with isooctane accurately to 100 mL and use this solution as the test solution. Separately, weigh accurately about 25 mg of tretinoin RS, dissolve in a small amount of dichloromethane TS, and dilute with isooctane acculately to 100 mL and use this solution as the standard stock solution, add isooctane to 1.0 mL of this solution to make 100 mL and use this solution as the standard solution. Perform the test with each 20 L of test solution and standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine each peak area of tretinoin from the test solution , AT , and standard solution , AS , by the automatic integration method. The amount of Tretinoin is not more than 1.0 %

times with 50 L of the standard solution according to the above operating conditions, the relative standard deviation of the peak of Tretinoin is not more than 2.0%.
Loss on Drying Not more than 0.5% (1 g, room temperature, vaccum, 16 hours) Residue on Ignition Not more than 0.1% (1 g) Assay Weigh accurately about 0.24 g of Isotretinoin, dissolve in 50 mL of dimethylformamide and titrate with 0.1 mol/L sodium methoxide VS. [indicator: 3 drops of a solution of thymol blue in dimethyl formamide (1 in 100)] until the color of solution changes to green. Perform the blank determination and make any necessary correction.

Each ml of 0.1 mol/L sodium methoxide VS = 30.04 mg of C20H28O2.

Packaging and Storage Preserve in light-resistant, tight containers filled with an atmosphere of an inert gas.

Isradipine
CH3 H3C O O CH3

Amount of Tretinoin (%) = 10 C AT

W AS

C : Concentration of Tretinoin (g/mL) in the standard solution. W : Mass of Tretinoin (mg) used in the test. Operating conditions Detector: An ultraviolet absorption photometer (wavelength : 352 nm) Column: A stainless steel column, about 4 mm in inside diameter and about 25 cm in length, packed with porous silica gel for liquid chromatography (5 m in particle diameter). Mobile phase: A mixture of isooctane, isopropanol and glacial acetic acid (99.65 : 0.25 : 0.1) Flow rate: 1 mL/min System suitability System performance: Dissolve 25 mg of Isotretinoin RS in a small amount of dichloromethane, dilute with isooctane to make 100 mL and use this solution as the system suitability standard solution. Add the system suitability standard solution to 1 mL of standard stock solution to make 100 mL and use this solution as the system suitability solution. When the procedure is run with 20 L of this solution under the above operating conditions, the relative retention times of Isotretinoin and Tretinoin are 0.84 and 1.0, respectively and the resolution between the peaks of Isotretinoin and Tretinoin is not less than 2.0. System repeatability: When the test is repeated 5
N O N O OCH3

NH

CH3

C19H21N3O5 : 371.39 Isradipine contains not less than 98.0% and not more than 102.0% of isradipine (C19H21N5O5), calculated on the dried basis.
Description Isradipine is a yellow, crystalline powder. Isradipine is freely soluble in acetone, soluble in acetonitrile or in methanol, and practically insoluble in nhexane or in water. Identification Determine the infrared spectra of Isradipine and Isradipine RS as directed in the potassium disk method under Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption at the same wavenumbers. Melting Point Between 166 C and 170 C.

KP 9 543
Purity (1) Heavy metalsProceed with 1.0 g of Isradipine according to Method 2 and perform the test. Prepare the control solution with 2.0 mL of standard lead solution (not more than 20 ppm). (2) Related substancesWeigh exactly 50 mg of Isradipine, dissolve in 5.0 mL of methanol, add the mobile phase to make exactly 25 mL, and use this solution as the test solution. Weigh exactly 6 mg of Isradipine RS, dissolve in 5 mL of methanol, add the mobile phase to make exactly 100 mL, add the mobile phase to 5.0 mL of this solution to make exactly 50 mL, and use this solution as the standard solution. Perform the test with each 25 L of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions. Determine the peak area other than Isradipine peak from the test solution: the total area of each peak other than Isradipine peak obtained from the test solution is not greater than 4 times the peak area of Isradipine obtained from the standard solution (1.2%), the area of the largest peak other than Isradipine peak obtained from the test solution is not greater than 1.6 times the peak area of Isradipine obtained from the standard solution (0.5%), the area of each peak other than the peak area of Isradipine obtained from the test solution is not greater than the peak area of Isradipine obtained from the standard solution (0.3%). Residue on Ignition Not more than 0.1% (1 g). Assay Use the light-resistant containers in this procedure. Weigh accurately about 20 mg of Isradipine, dissolve in 20 mL of methanol, add the mobile phase to make exactly 100 mL, and use this solution as the test solution. Separately, weigh accurately a constant mass of Isradipine RS and Isradipine related substances [isopropylmethyl 4-(4-benzofurzanyl)-2,6-dimethyl-3,5pyridinedicarboxylate] RS, dissolve in methanol, add the mobile phase to make the solutions containing 0.2 mg/mL and 10 g /mL respectively, and use these solutions as the standard solution. Perform the test with 25 L each of the test solution and the standard solution as directed under the Liquid Chromatography according to the following operating conditions: determine the peak area, AT and AS , of Isradipine .

Amount (mg) of isradipine C19H21N3O5) = 100 C AT

AS

C: Concentration (mg/mL) of Isradipine in the standard solution. Operating conditions Detector: An ultraviolet-visible absorption photometer (wavelength : 326 nm) Mobile phase: A misture of water , methanol and tetrahydrofuran (50:40:10) Column: A stainless steel column, about 4.6 mm in inside diameter and 10 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (between 3 and 10 m in particle diameter). Flow rate: 1.7 mL/minute System suitability System performance: When the procedure is run with 25 L of the system suitability under the above operating conditions, the resolution between isradipine peak and Isradipine related substance peak are not more than 1.5. System repeatability: When the test is repeated 5 times with 25 L of the standard solution under the above operating conditions, the relative standard deviation of the peak area of Isradipine is not more than 1.5%.
Packaging and Storage tight containers.

Operating conditions Detector: An ultraviolet-visible spectrophotometry (wavelength: 230 nm). Mobile phase: A mixture of water, ethanol and tetrahydrofuran (50 : 40 : 10). Column: A stainless steel column, about 4.6 mm in inside diameter and 10 cm in length, packed with porous silica gel for liquid chromatography (3 m to 10 m in particle diameter) Flow rate: 1.7 mL/minute System suitability System performance: Weigh 0.2 g of Isradipine RS and 10 mg of Isradipine related substance I RS, dissolve in 5 mL of methanol, add the mobile phase to make 100 mL. Add the mobile phase to 5.0 mL of this solution to make 50 mL, and use this solution as the solution of the system suitability. When the procedure is run test with 25 L of this solution as directed under the Liquid Chromatography according to the following operating conditions, the resolution between the peak of isradipine and the peak of isradipine related substance I is not less than 1.5%. System repeatability: When the test is repeated 5 times with 25 L of the standard solution of the system suitability under the above operating conditions, the relative standard deviation of the peak area of isradipine is not more than 1.5%. Time span of measurement: Not less than 3 times as long as the retention time of Isradipine peak.
Loss on Drying Not more than 0.2% (105 C, 4 hours, constant mass).

Preserve in light-resistant,