5.Monograph_Part I(J~Z)

8/10/2019 5.Monograph_Part I(J~Z)

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KP 9 539

Josamycin Propionate

O

O

CH 3

H

OHH3C

H

HOHC

O

CH 3

HH

O

CH 3O

OHO

H

CH 3H

H

OH

NCH 3CH 3

HOO

H

H

CH 3OH

CH 3

O

H

O

H3C

CH 3

CH 3

O

C45H73 NO 16: 884.06

Josamycin Propionate is a derivative of josamycin.Josamycin Propionate contains not less than 843 g(potency) per mg of josamycin (C 42H69 NO 15: 828.00),calculated on the dried basis.

Description Josamycin Propionate is a white to lightyellowish white crystalline powder, and is odorless andtasteless.Josamycin Propionate is very soluble in methanol or inethanol, soluble in ether, and practically insoluble inwater.

Identification (1) Dissolve 2 mg of Josamycin Pro- pionate in 5 mL of sulfuric acid: the solution turns tored-brown.

(2) Determine the absorption spectrum of a solutionof Josamycin Propionate in methanol (1 in 100000) asdirected under Ultraviolet-visible Spectrophotometry, itexhibits maximum between 230 nm and 233 nm.

Purity Heavy metals Proceed with 1.0 g of Josamy-cin Propionate according to Method 2 and perform thetest. Prepare the control solution with 3.0 mL of stan-dard lead solution (not more than 30 ppm).

Loss on Drying Not more than 2.0 % (0.5 g, in va-cuum, 60 C, 3 hours).

Residue on ignition Not more than 0.2 % (1 g).

Assay The Cylinder-plate method method (1) Agarmedia for seed and base layer- Use the medium in I 21) (1) 1 under Microbial Assay for Antibiotics.

(2) Test organism- Bacillus subtilis ATCC 6633.(3) Weigh accurately about 20 mg (potency) of Jo-

samycin Propionate, dissolve in 10 mL of methanol,and add 0.06 mol/L phosphate buffer solution, pH 5.6to make solution so that each mL contains 400.0 g(potency). Take exactly a suitable amount of this solu-tion, add 0.06 mol/L phosphate buffer solution, pH 5.6to make solutions so that each mL contains 80.0 g(potency) and 20.0 g (potency), and use these solu-

tions as the high concentration test solution and the lowconcentration test solution, respectively. Separately,weigh accurately about 20 mg of Josamycin Propionate

RS, dissolve in 10 mL of methanol, and add 0.06 mol/L phosphate buffer solution, pH 5.6 to make solution sothat each mL contains 400.0 g (potency), and use thissolution as the standard stock solution. Keep the stan-dard stock solution at not exceeding 5 C and use with-in 3 days. Take exactly a suitable amount of the stan-dard stock solution before use, add 0.06 mol/L phos-

phate buffer solution, pH 5.6 to make solutions so thateach mL contains 80.0 g (potency) and 20.0 g (po-tency), and use these solutions as the high concentra-tion standard solution and the low concentration stan-dard solution, respectively. Perform the test with thesesolutions according to the Cylinder-plate method (I 8)as directed under Microbial Assay for Antibiotics.

Packaging and Storage Preserve in tight containers.

Kanamycin Monosulfate

O

O

OH

O

NH2

NH2

HO

O

OH

H2SO 4

H2N

NH2

OH

HO

OH

OH

C18H36 N4O11H2SO 4: 582.58

Kanamycin Monosulfate is the sulfate of an aminogly-coside substance having antibacterial activity produced

by the growth of Streptomyces kanamyceticus .Kanamycin Monosulfate contains not less than 750 g(potency) per mg of kanamycin (C 18H36 N4O11: 484.50),calculated on the dried basis.

Description Kanamycin Monosulfate is a white toyellowish white powder, is odorless or has a little bit ofodor, and has a little bit of bitter taste.Kanamycin Monosulfate is freely soluble in water, and

practically insoluble in ethanol or in ether.

Identification (1) To 50 mg of Kanamycin Monosul-fate add 3 mL of water and dissolve, and add 2 mL ofanthrone TS: a blue-purple color develops.

(2) To 20 mg of Kanamycin Monosulfate, add 2 mLof 0.06 mol/L phosphate buffer solution, pH 5.6, dis-solve, and add 1 mL of ninhydrin TS and boil: a blue-

purple color develops.(3) To 10 mg of Kanamycin Monosulfate add 1 mL

of water and dissolve, and add 1 mL of ninhydrin solu-tion in n-butanol (1 in 500) and 0.5 mL of pyridine, andheat in water bath for 5 minutes and add 1 mL of water:a dark purple color develops.

(4) A solution of Kanamycin Monosulfate responds


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540 Monographs, Part I

to Qualitative Tests for sulfate salt.

pH The pH of a solution obtained by dissolving 0.1 gof Kanamycin Monosulfate in 10 mL of water is be-tween 6.5 and 8.5.

Sterility Test It meets the requirement, when Kana-mycin Monosulfate is used in a sterile preparation.

Bacterial Endotoxins Less than 0.50 EU per mg ofkanamycin, when Kanamycin Monosulfate is used in asterile preparation.


Residue on Ignition Not more than 1.0 % (1 g).

Assay The Cylinder-plate method method (1) Agarmedia for seed and base layer- Use the medium in I 21) (1) 1 under Microbial Assay for Antibiotics.

(2) Test organism- Bacillus subtilis ATCC 6633.(3) Weigh accurately about 20 mg (potency) of Ka-

namycin Monosulfate, dissolve in sterile purified water,to make a solution so that each mL contains 400.0 g(potency), and use this solution as the test stock solu-tion. Take exactly a suitable amount of this solution,add 0.1 mol/L phosphate buffer solution, pH 8.0 tomake solutions so that each mL contains 20.0 g (po-tency) and 5.0 g (potency), and use these solutions asthe high concentration test solution and the low concen-

tration test solution, respectively. Separately, weigh ac-curately about 20 mg of Kanamycin Sulfate RS, pre-viously dried, dissolve in 0.05 mol/L phosphate buffersolution, pH 6.0 to make solution so that each mL con-tains 400.0 g (potency), and use this solution as thestandard stock solution. Keep the standard stock solu-tion between 5 C and 15 C, and use within 30 days.Take exactly a suitable amount of the standard stocksolution before use, add 0.1 mol/L phosphate buffer so-lution, pH 8.0 to make solutions so that each mL con-tains 20.0 g (potency) and 5.0 g (potency), and usethese solutions as the high concentration standard solu-tion and the low concentration standard solution, re-

spectively. Perform the test with these solutions accord-ing to the Cylinder-plate method (I 8) as directed underMicrobial Assay for Antibiotics.


Kanamycin Sulfate

O

O

OH

O

NH2

NH2

HO

O

OH

xH2SO 4

H2N

NH2

OH

HO

OH

OH

C18H36 N4O11 xH 2SO 4

Kanamycin Sulfate is the sulfate of an aminoglycosidesubstance having antibacterial activity produced by the

growth of Streptomyces kanamyceticus .Kanamycin Sulfate is prepared by adding not more than1 mol of sulfuric acid to 1 mol of kanamycin monosul-fate.Kanamycin Sulfate contains not less than 600 g (po-tency) per mg of kanamycin (C 18H36 N4O11: 484.50),calculated on the dried basis.

Description Kanamycin Sulfate is a white to yello-wish white powder, is odorless or has a little bit of odor,and has a little bit of bitter taste.Kanamycin Sulfate is very soluble in water, and practi-cally insoluble in ethanol, in acetone, or in ether.

Identification Perform the test according to Identifi-cation in Kanamycin Monosulfate.

pH The pH of a solution obtained by dissolving 0.5 gof Kanamycin Monosulfate in 10 mL of water is be-tween 6.0 and 8.5.

Sterility Test It meets the requirement, when Kana-mycin Sulfate is used in a sterile preparation.

Pyrogen Test It meets the requirement, when Kana-mycin Sulfate is used in a sterile preparation,. Weigh anappropriate amount of Kanamycin Sulfate, dissolve inwater, make the solution so that each mL contains 10.0mg, and use the solution as the test solution. Theamount of injection is 1.0 mL of the test solution per kgof body weight of rabbit.


Assay Perform the test according to Assay in Kana-mycin Monosulfate. Weigh accurately about 20 mg(potency) of Kanamycin Sulfate, dissolve in sterile pu-rified water to make a solution so that each mL con-tains 400.0 g (potency), and use this solution as thetest stock solution. Take exactly a suitable amount of


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KP 9 541

this solution, add 0.1 mol/L phosphate buffer solution, pH 8.0 to make solutions so that each mL contains 20.0g (potency) and 5.0 g (potency), and use these solu-tions as the high concentration test solution and the lowconcentration test solution, respectively.


Kainic Acid

HN

H

C

CH 2CO 2H

H

H

CO 2H

CH 3

H2C

H2O

C10H15 NO 4H2O: 231.25

Kainic Acid, previously dried, contains not less than99.0% and not more than 101.0% of kainic acid(C10H15 NO 4: 213.23).

Description Kainic Acid is white crystals or crystal-line powder, is odorless and has an acid taste.Kainic Acid is sparingly soluble in water or in warmwater, very slightly soluble in glacial acetic acid or inethanol and practically insoluble in ether.Kainic acid dissolves in dilute hydrochloric acid or insodium hydroxide TS.

Melting point About 252 C (with decomposition). pH The pH of a solution of Kainic Acid (1 in 100)

is between 2.8 and 3.5.

Identification (1) To 5 mL of a solution of KainicAcid (1 in 5000), add 1 mL of ninhydrin TS and warmin a water-bath at a temperature between 60 C and 70C for 5 minutes: a yellow color is produced.

(2) Dissolve 50 mg of Kainic Acid in 5 mL of gla-cial acetic acid and add 0.5 mL of bromine TS: the col-or of bromine disappears immediately.

Specific Optical Rotation 20D][ : Between -13 and -17 (0.5 g, water, 50 mL, 200 mm).

Purity (1) Clarity and color of solution Dissolve0.10 g of Kainic Acid in 10 mL of water: the solution isclear and colorless.

(2) Chloride Take 0.5 g of Kainic Acid in a plati-num crucible, dissolve in 5 mL of sodium carbonate TSand evaporate In a water-bath to dryness. Heat the cruc-ible slowly at first and then ignite until the sample isalmost incinerated. After cooling, add 12 mL of dilutenitric acid to the residue, dissolve by warming and filter.

Wash the residue with 15 mL of water, combine thewashings and the filtrate and add water to make 50 mL.

Perform the test (not more than 0.021%).

Control solution Add 5 mL of sodium carbonateTS to 0.30 mL of 0.01 mol/L hydrochloric acid VS and

proceed as directed above.

(3) Sulfate Dissolve 0.5 g of Kainic Acid in 40mL of water by warming. Cool, add 1 mL of dilute hy-drochloric acid and water to make 50 mL and performthe test. Prepare the control solution with 0.30 mL of0.005 mol/L sulfuric acid VS (not more than 0.028%).

(4) Ammonium Take 0.25 g of Kainic Acid and perform the test. Prepare the control solution with 5.0mL of standard ammonium solution (not more than0.02%).

(5) Heavy metals Proceed with 1.0 g of KainicAcid according to Method 2 and perform the test. Pre-

pare the control solution with 2.0 mL of standard leadsolution (not more than 20 ppm).

(6) Arsenic Dissolve 1.0 g of Kainic Acid in 5 mLof dilute hydrochloric acid and perform the test (notmore than 2 ppm).

(7) Related substances Dissolve 0.10 g of KainicAcid in 10 mL of water and use this solution as the testsolution. Pipet 2.0 mL of this solution and add water tomake exactly 100 mL. Pipet 1.0 mL of this solution,add water to make exactly 20 mL and use this solutionas the standard solution. Perform the test as directedunder the Thin-layer Chromatography with these solu-tions. Spot 10 L each of the test solution and the stan-dard solution on a plate of silica gel for thin-layerchromatography. Develop the plate with the superna-tant liquid of a mixture of water, n-butanol and glacialacetic acid (5 : 4 : 1) to a distance of about 10 cm andair-dry the plate. Spray evenly a solution of ninhydrinin acetone (1 in 50) on the plate and dry the plate at 80C for 5 minutes: the spots other than the principal spotfrom the test solution are not more intense than the spotobtained form the standard solution.

Loss on Drying Between 6.5% and 8.5% (1 g, 105C , 4 hours).

Residue on Ignition Not more than 0.1% (0.5 g).

Assay Weigh accurately about 0.4 g of Kainic Acid, previously dried and dissolve in 50 mL of warm water,cool and titrate with 0.1 mol/L sodium hydroxide VS(indicator: 10 drops of bromothymol blue TS).

Each mL of 0.1 mol/L sodium hydroxide VS= 21.323 mg of C 10H15 NO 4



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Kallidinogenase

Kallidinogenase is an enzyme obtained from healthy porcine pancreas, and has kinin-releasing activity basedon vleavage of kininogen. Kallidinogenase contains not less than 25 Kallidinoge-nase Units per mg.Usually, Kallidinogenase is diluted with Lactose Hy-drate or the like. Kallidinogenase contains not less than 90.0% and notmore than 110.0% of the labeled Units.

Description Kallidinogenase is a white to light brown powder. Kallidinogenase is odorless or has afaint, characteristic odor. Kallidinogenase is freely soluble in water, and practi-

cally insoluble in ethanol and in ether. pH The pH of a solution of Kallidinogenase (1 in300) is between 5.5 and 7.5.

Identification (1) Weigh accurately an appropriateamount of Kallidinogenase according to the labeledUnits, and dissolve in 0.05 mol/L phosphate buffer so-lution, pH 7.0 to prepare a solution containing 10 Kal-lidinogenase Units per mL. Pipet 5 mL of this solution,and add exactly 1 mL of trypsin inhibitor TS and 0.05mol/L phosphate buffer solution, pH 7.0 to make exact-ly 10 mL. Pipet 4 mL each of this solution into twoseparate test tubes, add exactly 1 mL each of aprotinin

TS and 0.05 mol/L phosphate buffer solution, pH 7.0separately to each test tube, allow them to stand atroom temperature for 20 minutes, and use these solu-tions as the test solutions (1) and (2). Separately, pipet1 mL of trypsininhibitor TS, and 0.05 mol/L phosphate

buffer solution, pH 7.0 to mke exactly 10 mL. Pipet 4mL each of this solution into two separate test tubes,add exactly 1 mL each of aprotinin TS and 0.05 mol/L

phosphate buffer solution, pH 7.0 separately to eachtube, allow them to stand at room temperature for 20minutes, and use these solutions as the test solutions (3)and (4). Then, pipet 2.5 mL of substrate TS for Kallidi-nogenase assay (1), previously warmed at 30.0 0.5 for 5 minutes, place in a 10-mm cell, add exactly 0.5mL of the test solution (1) warmed at 30.0 0.5 for5 minutes, and start simultaneously a chronograph. Per-form the test at 30.0 0.5 as directed under Ultra-violet-visible Spectrophotometry using water as the

blank, and determine the absorbances at 405 nm, 21 A and 61 A , of this solution, after having allowed it tostand for exactly 2 and 6 minutes. Perform the sametest with the test solutions (2), (3) and (4), and deter-mine the absorbances, 22 A , 62 A , 23 A , 63 A ,

24 A and 64 A , of these solutions. Calculate I by us-

ing the following equation: the value of I does not ex-ceed 0.2.

)()()()(

I24642262

23632161

=

A A A A A A A A

(2) Pipet 2.9 mL of substrate TS for Kallidinoge-nase assay (2), previously warmed at 30.0 0.5 C for5 minutes, place in a 10-mm cell, add exactly 0.1 mL ofthe test solution obtained in the Assay, and start simul-taneously a chromatograph. Perform the test at 30.0 0.5 C as directed under Ultraviolet-visible Spectropho-tometry, and determine the change of the absorbance at253nm for to inhibitor TS, and add 0.05 mol/L phos-

phate buffer solution, pH 7.0 to make exactly 10 mL.Add exactly 0.1 mL of this solution to exactly 2.9 mLof substrate TS for Kallidinogenase assay (2), previous-ly warmed at 30.0 0.5 C for 5 minutes, and use thissolution as the blank. If the rate of change in the absor-

bance remains constant, determine the change of absorbance per 1 minute, A , and calculate R by using thefollowing equation: the value of R is between 0.12 and0.16.

ba A

R

= 10.0383

a: Amount (mg) of Kallidinogenase in 1 mL of thetest solution.

b: Amount (unit) of Kallidinogenase in 1 mg of Kal-lidinogenase obtained in the Assay.

Specific Activity Perform the test with Kallidinoge-nase as directed under Nitrogen Determination to de-termine the nitrogen content, convert 1 mg of nitrogen(N:14.01) into 6.25 mg of protein, and calculate thespecific activity using the amount (Units) of Kallidino-genase obtained in the Assay: it is not less than 100Kallidinogenase Units per 1 mg of protein.

Purity (1) Fat To 1.0 g of Kallidinogenase add 20mL of ether, extract with occasional shaking for 30 mi-nutes, and filter. Wash the residue with 10 mL of ether,combine the washing with the filtrate, evaporate theether, and dry the residue at 105 C for 2 hours: the

mass of the residue is not more than 1 mg. (2) Kininase (i) Bradykinin solution: Weighan appropriate amount of bradykinin, and dissolve ingelatin-phosphate buffer solution, pH 7.4 to prepare asolution containing 0.200 g of bradykinin per mL. (ii) Kallidinogenase solution: Weigh accurately asuitable amount of Kallidinogenase according to the la-

beled unit, dissolve in gelatin-phosphate buffer solution, pH 7.4 to make a solution containing 1 unit of Kallidi-nogenase per mL. (iii) Test solution: Pipet 0.5 mL of bradykinin solu-tion, warm at 30.0 0.5 C for 5 minutes, then add ex-actly 0.5 mL of Kallidinogenase solution previously

warmed at 30.0 0.5 C for 5 minutes, and mix imme-diately. After allow this solution to stand at 30.0 0.5


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KP 9 543

C for exactly 150 seconds, add exactly 0.2 mL of a so-lution of a solution of trichloroacetic acid (1 in 5), andshake. Boil for 3 minutes, then cool in ice immediately,centrifuge, and allow to stand at a room temperature for15 minutes. Pipet 0.5 mL of the supernatant liquid, addexactly 0.5 mL of gelatin-tris buffer solution, pH 8.0,and mix. Pipet 0.1 mL of this solution, add exactly 0.9mL of trichloroacetic acid gelatin-tris buffer solution,and mix. Pipet 0.2 mL of this solution, add exactly 0.6mL of trichloroacetic acid- gelatin-tris buffer solution,shake, and use this solution as the test solution. (iv) Control solution: Proceed with 0.5 mL of gelatin-

phosphate buffer solution, pH 7.4 as described in (iii),and use the solution so obtained as the control solution. (v) Procedure: Add 0.1 mL of anti-bradykinin antibo-dy TS to anti-rabbit antibody-coated wells of a 96-wellmicroplate, shake, and allow to stand at a constant tem-

perature of about 25 C for 1 hour. Remove the anti- bradykinin antibody TS, add 0.3 mL of phosphate buf-fer solution for microplate washing to the wells, thenremove. Repeat this procedure 3 times, take off thewashings thoroughly, then add 100 L each of the testsolution and the control solution, and 50 L of gelatin-

phosphate buffer solution, pH 7.0, shake, and allow tostand at a constant temperature of about 25 C for 1hour. Then add 50 L of peroxidase-labeled bradykininTS, shake, and allow to stand in a cold place for a night.Take off the solution, add 0.3 mL of phosphate buffersolution for microplate washing, and remove. Repeatthis procedure more 4 times, take off the washings tho-roughly, add 100 L of substrate solution for perox-

idase determination, and allow to stand as a constanttemperature of about 25 C for exactly 30 minuteswhile protecting from light. Then add 100 L of dilutedsurfuric acid (23 in 500), shake, and determine the ab-sorbance at 490 ~ 492 nm. Separately, dissolve a suita-

ble amount of bradykinin in gelatin-phosphate buffersolution, pH 7.0 to make solution containing exactly100 ng, 25 ng, 6.25 ng, 1.56 ng, 0.39 ng and 0.098 ngof bradykinin per mL, and use these solutions as thestandard solution (1), the standard solution (2), thestandard solution (3), the standard solution (4), thestandard solution (5), the standard solution (6), respec-tively. Use 1 mL of gelatin-phosphate buffer solution,

pH 7.0 as the standard solution (7). To each of the welladd 50 L each of the standard solutions and 100 L oftrichloroacetic acid-gelatin-tris buffer solution, and

proceed in the same manner as for the test solution andfor the control solution. Prepare the standard curvefrom the amounts of bradykinin in the standard solu-tions and their absorbances, and determine the amountof bradykinin, T B (pg) and S B (pg), of the test solu-tion and the control solution. The absorbance is usuallydetermined by using a spectrophotometer for micro-

plate. Since the wells are used as the cell for absorbance determination, take care for dirt and scratch ofthe well. Light pass length of the well is changeable bythe amount of the liquid, exact addition of the liquid isnecessary.

(vi) Judgement: The value R calculated by the follow-ing equation is not less than 0.8.

R = ( S T B B / )

(3) Trypsin-like substances Pipet 4 mL of the teststock solution prepared for the Assay, add exactly 1 mLof trypsin inhibitor TS and 0.05 mol/L phosphate buffersolution , pH 7.0 to make exactly 10 mL, and use thissolution as the test solution. Pipet 2.5 mL of substrateTS for kallidinogenase assay (1), previously warmed at30.0 0.5 for 5 minutes, place in a 10-mm cell, addexactly 0.5 mL of the test solution, warmed at 30.0 0.5 C for 5 minutes, and start simultaneously a chro-nograph. Perform the test at 30.0 0.5 C as directedunder Ultraviolet-visible Spectrophotometry using wa-ter as the blank, and determine the absorbances at 405nm, 2 A and 6 A , of this solution after having al-

lowed it to stand for exactly 2 and 6 minutes. Seperate-ly, pipet 4 mL of the test stock solution prepared for theAssay, add 0.05 mol/L phosphate buffer solution, pH7.0 to make exactly 10 mL, and use this solution as thecontrol solution. Perform the same test with the controlsolution, and determine the absorbances, 2' A and

6' A . Calculate T by using the following equation: thevalue of T does not exceed 0.05.

)}''/()()''{( 262626 A A A A A AT =

(4) Protease Weigh accurately an appropriateamount of kallidinogenase according to the labeledUnits, dissolve in 0.05 mol/L phosphate buffer solution,

pH 7.0 to prepare a solution containing 1 Kallidinoge-nase Unit per mL, and use this solution as the test solu-tion. Pipet 1 mL of the test solution, place in a test tube,and allow to stand at 35 0.5 C for 5 minutes. Then,

pipet 5 mL of substrate TS for kallidinogenase assay(3), previously warmed to 35 0.5 C add quickly tothe test solution in the test tube, and allow to stand at35 0.5 C for exactly 20 minutes. Then add exactly 5mL of trichloroacetic acid TS, shake well, allow tostand at room temperature for 1 hour, and filter through

a membrane filter (5 m in pore size). Discard the first3 mL of the filtrate, and determine the absorbance, A .

of the subsequent filtrate at 289 nm within 2 hours asdirected under Ultraviolet-visible Spetrophotometry,using water as the blank. Separately, pipet 1 mL of thetest solution, add exactly 5 mL of trichloroacetic acidTS, shake well, and add exactly 5 mL of the substrateTS for kallidinogenase assay (3). Proceed in the samemanner as described for the test solution, and determinethe absorbance, 0 A , of this solution. Calculate thevalue of ( 0 A A ): it is not more than 0.2.

Loss on Drying Not more than 2.0% (0.5g, in va-cuum, phosphorus (V) oxide, 4 hours).


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Residue on Ignition Not more than 3.0% (0.5g,650~750 C).

Kinin-Releasing Activity (i) Kallidinogenase solu-tion: Weigh accurately a suitable amount of Kallidino-genase, according to the labeled unit, dissolve in 0.02mol/L phosphate buffer solution, pH 8.0 to make a so-lution containing 0.1 unit of Kallidinogenase per mL.Perform this procedure by using glassware. (ii) Test solution: Pipet 0.5 mL of kininogen TS, warmat 30 0.5 C for 5 minutes, then add exactly 0.5 mLof Kallidinogenase solution previously warmed at 30 0.5 C for 5 minutes, and mix immediately. After allowthis solution to stand at 30 0.5 C for exactly 2 mi-nutes, add exactly 0.2 mL of a solution of trichloroacet-ic acid (1 in 5), and shake. Boil for 3 minutes, then coolin ice immediately, centrifuge, and allow to stand at a

temperature for 15 minutes. Pipet 0.5 mL of the super-nant liquid, addd exactly 0.5 mL of gelatin-tris buffersolution, pH 8.0, and shake. Pipet 0.1 mL of this solu-tion, add exactly 1.9 mL of trichloroacetic acid-gelatin-tris buffer solution, shake, and use this solution as thetest solution. (iii) Procedure: Perform the test with the test solutionas directed in the Purity (2), and determine the amount,

B (pg), of kinin per well. The kinin-releasing activity per 1 unit of Kallidinogenase calculated by the follow-ing equation is not less than 500 ng bradykinin equiva-lent/min/unit.

Kinin-releasing activity (ng bradykinin equiva-lent/min/unit) per 1 unit of Kallidinogenase

= B 4.8

Assay Weigh accurately an appropriate amount of Kallidinogenase according to the labeled units, dissolvein 0.05 mol/L phosphate buffer solution, pH 7.0 to pre-

pare a solution containing about 10 KallidinogenaseUnits per mL, and use this solution as the test stock so-lution. Pipet 4 mL of the test stock solution, add exact-ly 1 mL of trypsin inhibitor TS and 0.05 mol/L phos-

phate buffer solution, pH 7.0 to make exactly 10 mL,and use this solution as the test solution. Pipet 2.5 mLof substrate TS for Kallidinogenase assay (1), previous-ly warmed at 30 0.5 C for 5 minutes, place in a 1-cmcell, add exactly 0.5 mL of the test solution, warmed at30 0.5 C for 5 minutes, and start simultaneously achronograph. Perform the test at 30 0.5 C as directedunder the Ultraviolet-visible Spetrophotometry usingwater as the blank, and determine the absorbances at405 nm, 2T A and 6T A , of this solution after allowingto stand for exactly 2 and 6 minutes. Separately, dis-solve Kallidinogenase reference standard in 0.05 mol/L

phosphate buffer solution, pH 7.0 to make a solution sothat each mL contains exactly 10 Units, and use this so-

lution as the standard stock solution. Pipet 4 mL of thestock solution, add exactly 1 mL of trypsin inhibitor TS

and 0.05 mol/L phosphate buffer solution, pH 7.0 tomake exactly 10 mL, and use this solution as the stan-dard solution. Take exactly 0.5 mL of the standard so-lution, perform the test in the same manner as describedfor the test solution, and determine the absorbances,

2S A and 6S A , of the solution after allowing to standfor exactly 2 and 6 minutes. Separately, take exactly 1mL of the trypsin inhibitor TS, and add 0.05 mol/L

phosphate buffer solution, pH 7.0 to make exactly 10mL. Pipet 0.5 mL of this solution, perform the test inthe same manner as described for the test solution, anddetermine the absorbances, 02 A and 06 A , of the solu-tion after allowing to stand for exactly 2 and 6 minutes.

Units per 1 mg of Kallidinogenase =

ba

W

A A A A

A A A A 1)()(

)()( S

0206S2S6

0206T2T6

=

S W : Amount (Units) of Kallidinogenase Refer-ence Standard

a: Volume (mL) of the standard stock solution b: Amount (mg) of Kallidinogenase in 1 mL of the

test stock solution

Packaging and Storage Preserve in tight containers

Ketamine Hydrochloride

HCl

NHCH 3

OCl

and enantiomer C13H16ClNOHCl: 274.19

Ketamine Hydrochloride, when dried, contains not lessthan 99.0% and not more than 101.0% of ketamine hy-drochloride (C 13H16ClNOHCl).

Description Ketamine Hydrochloride is a white crys-tal or crystalline powder.Ketamine Hydrochloride is very soluble in formic acid,freely soluble in water or in methanol, sparingly so-luble in ethanol or in glacial acetic acid and practicallyinsoluble in acetic anhydride or in ether.A solution of Ketamine Hydrochloride (1 in 10) showsno optical rotation.

Melting point 258 C (with decomposition).

Identification (1) Determine the absorption spectraof solutions of Ketamine Hydrochloride and Ketamine

Hydrochloride RS in 0.1 mol/L hydrochloric acid TS (1in 3000) as directed under the Ultraviolet-visible Spec-trophotometry: both spectra exhibit similar intensities


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KP 9 545

of absorption at the same waveleng.(2) Determine the infrared spectra of Ketamine Hy-

drochloride and Ketamine Hydrochloride RS as di-rected in the potassium bromide disk method under theInfrared Spectrophotometry: both spectra exhibit simi-lar intensities of absorption at the same wavenumbers.

(3) A solution of Ketamine Hydrochloride (1 in 10)responds to the Qualitative Tests (2) for chloride.

Absorbance %1cm1 E (269 nm): Between 22.0 and 24.5(after drying, 30 mg, 0.1 mol/L hydrochloric acid TS,100 mL).

pH Dissolve 1.0 g of Ketamine Hydrochloride in 10mL of freshly boiled and cool water: the pH of this so-lution is between 3.5 and 4.5.

Purity (1) Clarity and color of solution Dissolve

1.0 g of Ketamine Hydrochloride in 5 mL of water: thesolution is clear and colorless.(2) Heavy metals Proceed with 1.0 g of Ketamine

Hydrochloride according to Method 1 and perform thetest. Prepare the control solution with 2.0 mL of stan-dard lead solution (not more than 20 ppm).

(3) Arsenic Prepare the test solution with 1.0 g ofKetamine Hydrochloride, according to Method 1 and

perform the test (not more than 2 ppm).(4) Related substances Dissolve 0.5 g of Keta-

mine Hydrochloride in 10 mL of methanol and use thissolution as the test solution. Pipet 1.0 mL of the test so-lution, add methanol to make exactly 200 mL and use

this solution as the standard solution. Perform the testwith the test solution and the standard solution as di-rected under the Thin-layer Chromatography. Spot 2 Leach of the test solution and the standard solution on a

plate of silica gel for thin-layer chromatography. De-velop the plate with a mixture of cyclohexane and iso-

propylamine (49 : 1) to a distance of about 10 cm andair-dry the plate. Spray evenly Dragendorffs TS forspraying on the plate, dry the plate and then sprayevenly hydrogen peroxide TS: the spots other than the

principal spot from the test solution are not more in-tense than the spot from the standard solution.

Loss on Drying Not more than 0.5% (1 g, 105 C, 3hours).

Residue on Ignition Not more than 0.1% (1 g).

Assay Weigh accurately about 0.5 g of Ketamine Hy-drochloride, previously dried, dissolve in 1 mL of for-mic acid, add 70 mL of a mixture of acetic anhydrideand glacial acetic acid (6 : 1) and titrate with 0.1 mol/L

perchloric acid VS (potentiometric titration, EndpointDetection Method in Titrimetry). Perform a blank de-termination and make any necessary correction.

Each mL of 0.1 mol/L perchloric acid VS= 27.419 mg of C 13H16ClNOHCl


Ketoconazole

NNH3C

O

O

N

N

ClCl

O

O

H and enantiomer

C26H28Cl2 N4O4: 531.43

Ketoconazole contains not less than 98.0% and not

more than 102.0% of ketoconazole (C 26H28Cl2 N4O4),calculated on the dried basis.

Description Ketoconazole is a white powder. Ketoconazole is soluble in methanol, sparingly solublein ethanol, and practically insoluble in water.

Identification Determine the infrared spectra of Ke-toconazole and Ketoconazole RS, as directed in the po-tassium bromide disk method under the Infrared spec-trophotometry: both spectra exhibit similar intensitiesof absorption at the same wavenumbers.

Melting Point Between 148 C and 152 C .

Specific Optical Rotation 20D][ : Between -1 and +1(0.4 g, methanol, 10 mL, 100 mm).

Purity (1) Heavy metals Proceed with 1.0 g of Ke-toconazole according to Method 2, and perform the test.Prepare the control solution with 2.0 mL of standardlead solution (not more than 20 ppm).

(2) Related substances Dissolve 30 mg of Keto-conazole in 3.0 mL of chloroform and use this solutionas the test solution. Separately, weigh accurately a suit-

able quantity of Ketoconazole RS, dissolve it in chloro-form to make a solution containing 10 mg per mL, anduse this solution as the standard solution. Pipet a suita-

ble volume of this solution, add chloroform to make asolution containing 1.0 mg per mL and use this solutionas a diluted standard solution. Perform the test with thetest solution, standard solution and the diluted standardsolution as directed under the Thin-layer Chromatogra-

phy. Spot 10 L each of the test solution and the stan-dard solution, and spot 2 L of the diluted standard so-lution on a plate of silica gel for thin-layer chromato-graphy. Develop the plate with a mixture of n-hexane,ethyl acetate, methanol, water and glacial acetic acid

(42 : 40 : 15 : 2 : 1) to a distance of about 15 cm andair-dry. Allow the plate to stand in iodine vapor: the


8/470


f R value and the size of the principal spot from thetest solution corresponds to the f R value and size ofthe principal spot from the standard solution. The sumof the spots other than the principal spot from the testsolution is not more intense than the principal spot

from the diluted standard solution.Loss on Drying Not more than 0.5% (1 g, in vacuum,80 C , 4 hours).


Assay Weigh accurately about 0.2 g of Ketoconazole,dissolve in 40 mL of glacial acetic acid, and titrate with0.1 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= 26.572 mg of C 26H28Cl2 N4O4


Ketoconazole Tablets

Ketoconazole Tablets contain not less than 90.0% andnot more than 110.0% of the labeled amount of ketoco-nazole (C 26H28Cl2 N4O4: 531.43).

Method of Preparation Prepare as directed underTablets, with Ketoconazole.

Identification Weigh accurately and powder a quan-tity of Ketoconazole Tablets equivalent to about 50 mgof Ketoconazole, add 50 mL of chloroform, shake for 2minutes, filter, and use the filtrate as the test solution.Separately, dissolve a suitable quantity of KetoconazoleRS in chloroform to make a solution containing 1 mg

per mL and use this solution as the standard solution.Perform the test with the test solution and the standardsolution as directed under the Thin-layer Chromatogra-

phy. Spot 10 L each of the test solution and the stan-dard solution on a plate of silica gel for thin-layerchromatography. Develop the plate with a mixture of n-hexane, ethyl acetate, methanol, water and glacial acet-ic acid (42 : 40 : 15 : 2 : 1) to a distance of about 15 cm,and air-dry. And examine the plate under ultravioletlight (main wavelength: 254 nm): the f R value of the

principal spot obtained from the test solution corresponds to that obtained from the standard solution.

Disintegration Test It meets the requirement pro-vided that the time limit of the test is 10 minutes.

Uniformity of Dosage Units It meets the require-ment.

Assay Weigh accurately and powder not less than 20Ketoconazole Tablets. Weigh accurately a portion ofthe powder, equivalent to about 0.2 g of Ketoconazole,transfer to a glass-stoppered centrifuge tube and addexactly 50 mL of a mixture of methanol and dichloro-methane (1 : 1). Shake for 30 minutes with a shaker and

centrifuge. Pipet 5.0 mL of the supernatant, add 5.0 mLof the internal standard solution and add a mixture ofmethanol and dichloromethane (1 : 1) to make exactly50 mL. Use this solution as the test solution. Separately,weigh accurately about 20 mg of Ketoconazole RS, add5.0 mL of the internal standard solution and add a mix-ture of methanol and dichloromethane (1 : 1) to makeexactly 50 mL. Use this solution as the standard solu-tion. Perform the test with 20 L each of the test solu-tion and the standard solution as directed under theLiquid Chromatography according to the followingconditions and calculate the ratios, TQ and SQ , ofthe peak area of Ketoconazole to that of the internalstandard for the test solution and the standard solution,respectively.

Amount (mg) of Ketoconazole (C 26H28Cl2 N4O4)

= amount (mg) of Ketoconazole RS 10S

T QQ

Internal standard solution Weigh a suitable quan-tity of Terconazole RS and dissolve in a mixture of me-thanol and dichloromethane (1 : 1) to make a solutioncontaining 5 mg per mL.

Operating conditionsDetector: 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 withoctadecylsilanized silica gel for liquid chromatography(3 m to 10 m in particle diameter).

Column temperature: A room temperature.Mobile phase: A mixture of diisopropylamine in

methanol (1 in 500) and ammonium acetate in water (1in 200) (7 : 3).

Flow rate: 3 mL/minute.System suitability

System performance: When the procedure is runwith 20 L of the standard solution under the aboveoperating conditions, ketoconazole and the internalstandard are eluted in this order with a resolution be-tween their peaks being not less than 2.0.

System repeatability: When the test is repeated 6times with 20 L of the standard solution under theabove operating conditions, the relative standard devia-tion of the ratios of the peak area of Ketoconazole tothat of the internal standard is not more than 2.0%.



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KP 9 547

Ketoprofen

CCO 2H

CO

H3C

H

and enantiomer Cl6H14O3: 254.28

Ketoprofen, when dried, contains not less than 99.0%and not more than 100.5% of ketoprofen (C l6H14O3).

Description Ketoprofen occurs as a white, crystalline powder.Ketoprofen is very soluble in methanol, freely solublein ethanol or in acetone and practically insoluble in wa-ter.A solution of Ketoprofen in dehydrated ethanol(1 in100) shows no optical rotation.Ketoprofen is colored to pale yellow by light.

Identification (1) Determine the absorption spectraof solutions of Ketoprofen and Ketoprofen RS in me-thanol (1 in 200000) as directed under the Ultraviolet-visible Spectrophotomtry: both spectra exhibit similarintensities of absorption at the same wavelength.

(2) Determine the infrared spectra of Ketoprofenand Ketoprofen RS, previously dried, as directed in the

potassium bromide disk method under the InfraredSpectrophotometry: both spectra exhibit similar intensi-ties of absorption at the wavenumbers.

Melting Point Between 94 C and 97 C

Purity (1) Clarity and color of solution Dissolve1.0 g of Ketoprofen in 10 mL of acetone: the solution isclear and has no more color than the following controlsolution.

Control solution To a mixture of 0.6 mL ofcobalt( ) chloride colorimetric stock solution and 2.4mL of Iron( ) chloride colorimetric stock solution addthe diluted hydrochloric acid (1 in 10) to make 10 mL.To 5.0 mL of this solution add diluted hydrochloric ac-id(1 in 10) to make 100 mL.

(2) Heavy metals Proceed with about 2.0 g of Ke-toprofen according to Method 2 and perform the test.Prepare the control solution with 2.0 mL of standardlead solution (not more than 10 ppm).

(3) Related substances Perform this procedurewith a minimum of exposure to light, using light-resistant vessels.. Dissolve 20 mg of Ketoprofen in 20mL of the mobile phase and use this solution as the test

solution. Pipet exactly 1 mL of the test solution and addthe mobile phase to make exactly 50 mL. Pipet exactly1 mL of this solution, add the mobile phase to makeexactly 10 mL and use this solution as the standard so-lution. Perform the test with exactly 20 L each of thetest solution and the standard solution as directed under

the Liquid Chromatography according to the followingconditions. Determine each peak area by the automaticintegration method: the areas of the peaks from the testsolution, having the relative retention time of about 1.5and about 0.3 with respect to Ketoprofen are not greaterthan 2 times and 4.5 times peak area of ketoprofenfrom the standard solution, respectively, the area of the

peak other than Ketoprofen and the peaks mentionedabove is not larger than the peak area of Ketoprorenfrom the standard solution, and total area of these peaksis not larger than 2 times peak area of Ketoprofen fromthe standard solution.

Operating conditinsDetector: An ultraviolet absorption photometrer

(wavelength : 233 nm)Column: A stainlenn steel column, about 4.6 mm in

inside diameter and about 15 cm in length, packed withoctadecylsilanized silica gel for Liquid Chromatogra-

phy (5 m in particle diameter).Column temperature: A constant temperature of

about 25 CMobile phase: Dissolve 68 g of potassium dihydro

phosphate in water to make 1000mL, and adjust the pHto 3.5 with phosphoric acid, To 20 mL of this solutionadd 430 mL of acetonitrile and 550 mL of water.

Flow rate: Adjust the flow rate so that the retentiontime of ketoprofen is about 7minutes.

System suitabilityTest for required detectability: To exactly 1 mL of

the standard solution, add the mobile phase to make ex-actly 10 mL. Confirm that the peak area of Ketoprofenobtained with 20 L of this solution is equivalent to 9to 11% of that with 20 L of the standard solution.

System performance: When the procedure is runwith 20 L of the standard solution under the aboveoperation conditions, the number of the theoretical

plates and symmertrical factor the peak of Ketoprofenis not less than 8000 and not more than 1.5 respectively.

System repeatability: When the test is repeated 6times with 20 L of the standard solution under theabove operating conditions, the relative standard devia-tion of the peak area of Ketoprofen is not more than2.0%.

Time spar of measurement: About 7 times as long asthe retention time of ketoprofen.

Loss on Drying Not more than 0.5% (0.5 g, in va-cuum, 60 C, 24 hours).


Assay Weigh accurately about 0.3 g of Ketoprofen, previously dried, dissolve in 25 mL of ethanol, add 25


10/470


mL of water and titrate with 0.1 mol/L sodium hydrox-ide VS (indicator: 3 drops of phenolphthalein TS). Per-form a blank determination and make any necessarycorrection.

Each mL of 0.1 mol/L sodium hydroxide VS

= 25.428 mg of C l6H14O3

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

Ketorolac Tromethamine

O

NOH

O

HO OH

OH

NH2

C15H13 NO 3C4H11 NO 3: 376.40

Ketorolac Tromethamine contains not less than 98.5%and not more than 101.5% of ketorolac tromethamine

(C15H13 NO 3C4H11 NO 3), calculated on the dried basis.

Description Ketorolac Tromethamine is a white,crystalline powder.

Ketorolac Tromethamine is freely soluble in water or inmethanol, sparingly soluble in ethanol, in dehydratedethanol or in tetrahydrofuran, and practically insolublein acetone, in dichloromethane, in toluene, in ethylace-tate, in dioxane, in hexane, in butanol or in acetonitrile.

Melting point Between 165 C and 170 C (de-composition).

Identification (1) Determine the absorption spectraof solutions of Ketorolac Tromethamine and KetorolacTromethamine RS in methanol (1 in 100000) as di-rected under Ultraviolet-visible Spectrophotometry:

both spectra exhibit similar intensities of absorption atthe same wavelength.

(2) Determine the infrared spectra of KetorolacTromethamine and Ketorolac Tromethamine RS as di-rected in the potassium bromide disk method under theInfrared Spectrophotometry: both spectra exhibit simi-lar intensities of absorption at the same wavenumber.

(3) Dissolve each 50 mg each of Ketorolac Trome-thamine and Ketorolac Tromethamine RS in a mixtureof dichloromethane and methanol (2:1) to make 10 mL,and use these solutions as the test solution and thestandard solution. Perform the test with the test solutionand the standard solution as directed under the Thin-layer Chromatography. Spots each 40 L of the test so-lution and the standard solution on a plate of silica gelfor the thinlayer chromatography. Develop the plate

with a mixture of dichloromethane, acetone and glacialacetic acid (95:5:2) to a distance of about 15 cm, andair-dry the plate. Sprlay evenly a solution of ninhydrinin ethanol (0.3 in 10) on the plate and heat the plate atabout 150 C for 2 to 5 minutes: yellow spots with pinkto purple borders is shown.

pH Dissolve 1 g of Ketorolac Tromethamine in 100mL of water: the pH of this solution is between 5.7 and6.7.

Purity (1) Heavy metals Proceed with 1.0 g of Ke-torolac Tromethamine according to the method 2 and

perform the test. Prepare the control solution with 2.0mL of standard lead solution (not more than 20 ppm).

(2) Related substances Perform the test with 10L of the test solution as directed under the LiquidChromatography according to following operating con-ditions. Determine each peak area by the automatic in-tegration method, and calculate the amount of each re-lated substances: the ketorolac 1-keto analog or the ke-torolac 1-hydroxy analog is not more than 0.1% andany other impurity is not more than 0.5% and the sumof all impurities is not more than 1.0%.

Amount(%) of related substances =S

1 A A

rf i100

1rf : Response factor of each individual related sub-stance peak relative to that of ketorolac; the rf 1

values are 0.52 for the ketorolac 1- to analog, 0.67for the ketorolac 1-hydroxy analog,

2.2 for the related substance peak having a retentiontime of 0.54 relative to that of ketorolac, and

0.91 for the related substance peak at a relative re-tention time of 0.66.

i A : Peak response for each related substance.

S A : Sum of all the peak responses of the relatedsubstance peaks and the principal ketorolac peak.

Operating conditionsTest solution, mobile phase, detector, column, injec-

tion volume are the same in the Assay.

Time span of measurement: About three times as longas the retention time of ketorolac.

Loss on Drying not more than 0.5%.

Residue on Ignition not more than 0.1%.

Assay Weigh accurately about 20 mg each of KetorolacTromethamine and Ketorolac Tromethamine RS, dis-solve in a mixture of water and tetrahydrofuran (70:30)to make exactly 50 mL, and use these solutions as thetest solution and the standard solution. The test solutionand the standard solution are protected from light. Per-

form the test with 10 L each of the test solution andthe standard solution as directed under the LiquidChromatography according to the following operating


11/470

KP 9 549

conditions. Calculate the area of the principal peak ofeach solutions, T A and S A , by the automatic integra-tion method.

Amount(mg) of ketorolac tromethamine

(C15H13 NO 3C4H11 NO 3)= amount (mg) of Ketorolac Tromethamine RS

S

T

A A

Operating conditionsDtector: An ultraviolet absortion photometer (wa-

velength: 313 nm).Column: A stainless steel column, about 4.6 mm in

inside diameter and 25 cm in length, packed with octyl-silanized silica gel for liquid chromatography (5 m in

particle diameter).Column temperature: A constant temperature of

about 40 C.Mobile phase: A mixture of ammonium phosphate buffer and tetrahydrofuran (70:30).

Flow rate : 1.5 mL/minute.System suitability

System performance: In a 250-mL separator, mix100 mL of water, 100 mL of dichloromethane, 30 mgof Ketorolac Tromethamine RS and 1 mL of 1 mol/Lhydrochloric acid, shake, and allow the layers to sepa-rate. Tasfer the lower dichloromethane to a flask, anddiscard the upper layer. Expose the dichloromethanesolution to direct sunlight 10 to 15 minutes. Transfer1.0 mL of this solution to a vial, evaporate in a current

of air or in a stream of nitrogen to dryness, add 1.0 mLof a mixture of water and tetrahydrofuran (70 : 30), anddissolve. This solution may be stored under refrigera-tion, and used as long as the chromatogram obtained asdirected in Assay is suitable for identifying the peaksdue to the ketorolac 1-keto analog and ketorolac 1-hydroxy analog, and for the measurement of the resolu-

tion. When the procedure is run with 10 L of this so-lution according to the above operating conditions, therelative retention time of ketorolac 1-hydroxy analogand ketorolac 1-keto analog to the ketorolac peak areabout 0.63 and 0.89, respectively, with the resolutionsof these peaks being not less than 1.5. Perform the test

with 10 L of standard solution: the theoretical platenumbers are not less than 5500.

System repeatability: When the test is repeated 5times with 10 L of the standard solution under theabove operating conditions, the relative standard devia-tion of the peak area of Ketorolac is not more than1.5%.

Ammonium phosphate buffer Dissolve 5.75 g ofammonium dihydrogen phosphoric acid in water tomake 1000 mL, add phosphoric acid, and adjust to pH3.0. Make adjustments, if necessary, to achieve a reten-

tion time for ketorolac of about 8 to 12 minutes.

Packaging and Storage Preserve in light-resistant,

tight containers.

Lacidipine

HN CH 3H3C

COOC 2H5C2H5OOC

COOC 4H9

C26H33 NO 6: 455.54

Lacidipine contains not less 97.5% and not more than102.0% of lacidipine (C 26H33 NO 6), calculated on theanhydrous and solvent-free basis.

Description Lacidipine is a white to pale yellowcrystalline powder.Lacidipine is freely soluble in dichloromethane or inacetone, sparingly soluble in dehydrated ethanol, and

practically insoluble in water. Melting point About 178 C.

Identification (1) Determine the infrared spectra ofLacidipine and Lacidipine RS as directed in the potas-sium bromide disk method under the Infrared Spectro-

photometry: both spectra exhibit similar intensities ofabsorption at the same wavenumbers.

(2) When proceed as directed in the Assay, the re-tention time of the principal peak in the test solutioncorresponds to that in the standard solution.

Purity (1) Isopropanol Weigh accurately 1.0 g ofLacidipine, add the internal standard solution to makeexactly 50 mL and use this solution as the test solution.Pipet accurately 2 L of isopropanol, add the internalstandard solution to make exactly 100 mL and use thissolution as the standard solution. Perform the test with1 L each of the test solution and the standard solutionas directed under the Gas Chromatography according tothe following operating conditions and calculate thecontent (%) of isopropanol in the test solution (notmore than 0.5%).

Internal standard solution Pipet accurately 2 Lof dioxane and add dimethylacetamide to make exactly100 mL.

Operating conditionsDetector: A hydrogen flame-ionization detector.Column: A glass column, about 0.32 mm in inside

diameter and about 60 cm in length, coated inside sur-

face with polymethylsiloxane for gas chromatography 5m in thickness.Column temperature: Maintain the initial tempera-


12/470


ture of 60 C for 1 minute, up to 110 C at the rate of 3 C per minute, then up to 200 C at the rate of 50 C

per minute and hold at 200 C for 7 minutes.Carrier gas: Nitrogen.Flow rate: 1.7 mL/minute.System suitability

System performance: When the procedure is runwith 1 L of the standard solution as directed under theabove operating conditions, two peaks are clearly sepa-rated with each other and the retention time for isopro-

panol and dioxin is about 6.2 minutes and about 15 mi-nutes, respectively.

(2) Related substances Weigh accurately 10 mgof Lacidipine, add dehydrated ethanol to make exactly10 mL, pipet 1.0 mL of this solution, add the mobile

phase to make exactly 5 mL and use this solution as thetest solution. Pipet 1.0 mL of the test solution, add mo-

bile phase to make exactly 500 mL and use this solu-tion as the standard solution. Perform the test with 20L of the test solution and the standard solution as di-rected under the Liquid Chromatography according tothe following operating conditions and determine thearea of the peaks in the test solution and the standardsolution the area of the peak corresponding to lacidi-

pine related substance I obtained from the test solutionis not more than twice the area of the principal peak ob-tained from the standard solution (0.2%, the relative re-sponse factor: 2), the area of any other related sub-stance from the test solution is not more than the areaof the principal peak from the standard solution (0.2%).Calculate the content of lacidipine related substance Iusing the response factor and the content of any otherrelated substance using the standard solution: totalamount of impurities is not greater than 0.5%.

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 withcyanosilyl silica gel for liquid chromatography (5 min particle diameter).

Mobile phase: A mixture of n-hexane and dehy-

drated ethanol (97:3). Adjust the composition so thatthe retention time of lacidipine is about 10 min.Flow rate: About 2.0 mL/minute.

Water Not more than 0.2% (0.5 g, coulometric titra-tion).

Assay Weigh accurately 10 mg each of Lacidipineand Lacidipine RS, add dehydrated ethanol to make ex-actly 10 mL each, pipet 5.0 mL each, add the mobile

phase to make exactly 100 mL each and use these solu-tions as the test solution and the standard solution, re-spectively. Perform the test with 20 L of the test solu-

tion and the standard solution as directed under theLiquid Chromatography according to the following op-erating conditions and determine the area of lacidipine

peak in the test solution, T A , and the standard solu-

tion, S A .

Amount (mg) of lacidipine (C 26H33 NO 6)

= amount (mg) of Lacidipine RS S

T

A

A

Operating conditionsProceed as directed in the operating conditions of

the Related Substances under the Purity.

Packaging and Storage Preserve in well-closed con-tainers.

Lactulose

OOH

HH OH

H

CH 2OH

H

OH

O

H HO

HOH 2C OH

O H

CH 2OH

H

H

C12H22O11: 342.30

Lactulose is a solution of Lactulose prepared by isome-rizing lactose under the existing of alkaline and purified

by ion-exchange resin.Lactulose contains not less than 50.0% and not morethan 56.0% of lactulose (C 12H22O11).

Description Lactulose is a clear, colorless of paleyellow, viscous liquid, is odorless and has a sweet taste.Lactulose is miscible with water and with formamide .

Identification (1) To 0.7 g of Lactulose, add 10 mLof water, 10 mL of ammonium molybdate (1 in 25) and

0.2 mL of glacial acetic acid and heat in a water-bathfor 5 minutes to 10 minutes: a blue color is observed.

(2) Mix 0.3 g of Lactulose and 30 mL of water, add16 mL of 0.5 mol/L iodine TS, then immediately add2.5 mL of 8 mol/L sodium hydroxide TS, allow to standfor 7 minutes and add 2.5 mL of diluted sulfuric acid (3in 20). To this solution, add a saturated solution of so-dium sulfite until the solution turns pale yellow, thenadd 3 drops of methyl orange TS, neutralize with a so-lution of sodium hydroxide (4 in 25) and add water tomake 100 mL. To 10 mL of this solution, add 5 mL ofFehlings TS and boil for 5 minutes: a red precipitate is

produced.

Specific gravity 2020d : Between 1.320 and 1.360.


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KP 9 551

pH Dissolve 2.0g of Lactulose in 15 mL of water: the pH of this solution is between 3.5 and 5.5.

Purity (1) Heavy metals Proceed with 5.0 g ofLactulose according to Method 4 and perform the test.Prepare the control solution with 2.5 mL of standardlead solution (not more than 5 ppm).

(2) Arsenic Prepare the test solution with 1.0 g ofLactulose according to Method 1 and perform the test(not more than 2 ppm).

(3) Other saccharides Determine the heights ofthe peaks corresponding to D-galactose and Lactose,respectively, on the chromatogram obtained in Assayfrom the test solution and the standard solution and cal-culate the ratios of the peak heights of D-galactose andLactose to that of the internal standard from the test so-lution, TaQ and TbQ and then from the standard so-

lution, SaQ and SbQ : it contains D-galactose of notmore than 11.0% and lactose monohydrate of not morethan 6.0%.

Amount (mg) of galactose (C 6H12O6)

= amount (mg) of D-galactose Sa

Ta

QQ

Amount (mg) of lactose monohydrate (C 12H22O11H2O)

= amount (mg) of lactose monohydrate Sb

Tb

QQ

Loss on Drying Not more than 35.0% (0.5 g, in va-cuum, 80 C, 5 hours).


Assay Weigh accurately about 1 g of Lactulose, add10.0 mL of the internal standard solution and water tomake exactly 50 mL and use this solution as the test so-lution. Separately, weigh accurately about 0.5 g of Lac-tulose RS, accurately about 80 mg of D-galactose andaccurately about 40 mg of lactose monosaccharide, add10.0 mL of the internal standard solution and water to

make exactly 50 mL and use this solution as the stan-dard solution. Perform the test with 20 L each of thetest solution and the standard solution as directed underthe Liquid Chromatography according to the followingconditions and calculate the ratios, TQ and SQ , ofthe peak height of lactulose to that of the internal stan-dard, for the test solution and the standard solution re-spectively.

Amount (mg) of lactulose (C 12H22O11)

= amount (mg) of Lactulose RS S

T

QQ

Internal standard solution A solution of D-mannitol (1 in 20).

Operating conditionsDetector: A differential refractometer.Column: A stainless steel column, about 8 mm in

inside diameter and about 50 cm in length, packed withgel type strong acid ion-exchange resin for liquid

chromatography (degree of crosslinkage: 6%) (11 min particle diameter).

Column temperature: A constant temperature ofabout 75 C.

Mobile phase: Water.Flow rate: Adjust the flow rate so that the retention

time of lactulose is about 18 minutes.System suitability

System performance: When the procedure is runwith 20 L of the standard solution under the aboveoperating conditions, lactulose and internal standard areeluted 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 20 L of the standard solution under theabove operating conditions, the relative standard devia-tion of the ratios of the peak heights of lactulose, galac-tose and lactose to that of the internal standard are notmore than 2.0% respectively.


Lamivudine

S

ON

HON

NH2

O

C8H11 N3O3S: 229.26

Lamivudine contains not less than 98.0% and not morethan 102.0% of lamivudine (C 8H11 N3O3S), calculatedon the anhydrous and solvent-free basis.

Description Lamivudine is a white solid.

Lamivudine is soluble in water. Melting point About 176 C.

Identification (1) Determine the infrared spectra ofLamivudine and Lamivudine RS as directed in the

paste method under the Infrared Spectrophotometry: both spectra exhibit similar intensities of absorption atthe same wavenumbers.

(2) When proceed as directed in the Lamivudineenantiomer under the Purity, the retention time of the

principal peak from the test solution corresponds to thatfrom the standard solution.

Purity (1) Clarity and color of solution Proceedwith a solution of Lamivudine (1 in 20) as directed un-der the Infrared Spectrophotometry and measure the


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absorbance of this solution at 440 nm in 4 cm: not morethan 0.0015.

(2) Lamivudine enantiomer Weigh accuratelyabout 25 mg of Lamivudine, add water to make 100 mLand use this solution as the test solution. Perform thetest with 10 L of the test solution as directed under theLiquid Chromatography according to the following op-erating conditions, determine the areas of the principal

peaks and calculate the content of lamivudine enantio-mer: not more than 0.3%.

Content (%) of lamivudine enantiomer

=S i

i

A A A+

100

i A : Peak area of lamivudine enantiomer

S A : Peak area of lamivudine



inside diameter and about 25 cm in length, packed with beta-cyclodextrin bonded to silica gel for liquid chro-matography (5-10 m in particle diameter).

Mobile phase: A mixture of 0.1 mol/L ammoniumacetate solution and methanol (95:5).

Flow rate: About 1.0 mL/minute.System suitability

System performance: Dissolve the content of one

vial of lamivudine resolution mixture I RS in 5 mL ofwater, wash the inner side of the vial with 2 mL each ofwater, combine the washings and the dissolved content,and add water to make exactly 10 mL. Perform the testwith 10 L of this solution as directed under the LiquidChromatography according to the above operating con-ditions, the relative retention times for lamivudine peakand lamivudine enantiomer peak are about 1.0 and 1.2,respectively, with the resolution between lamivudine

peak and lamivudine enantiomer peak being not lessthan 1.5.

0.1 mol/L Ammonium acetate solution Dissolveabout 7.7 g of ammonium acetate in water and dilutewith water to make 1000 mL.

(3) Residual solvents Weigh accurately 5 g ofLamivudine, add 10 mL of the internal standard solu-tion, add a mixture of dimethylsulfoxide and water(1:1) to make exactly 100 mL and use this solution asthe test solution. To 10 mL of the internal standard so-lution, add accurately 100 L each of dehydrate ethanol,isopropyl acetate, methanol and triethylamine, add amixture of dimethyl sulfoxide and water (1:1) to makeexactly 100 mL and use this solution as the standard so-lution. Perform the test with 0.5 L each of the test so-

lution and the standard solution as directed under theGas Chromatography according to the following oper-ating conditions, determine the areas of peaks in these

solutions and calculate the content of residual solventsin Lamivudine: not more than 0.2% of ethanol, notmore than 0.2% of isopropyl acetate, not more than0.1% of triethylamine and not more than 0.3% of totalresidual solvents.

Content (%) of each residual solvent=

S

i

Q

Q

W C 10

C: Concentration (mg/mL) of the respective analytein the standard solution

W: Weight (g) of Lamivudine takeniQ : Peak area ratio of the respective analyte to the

internal standard in the test solutionSQ : Peak area ratio of the respective analyte to the

internal standard in the standard solution

Internal standard solution Pipet 1 mL of 2- pentanone and add a mixture of dimethylsulfoxide andwater (1:1) to make exactly 100 mL.

Operating conditionsDetector: A hydrogen flame-ionization detector.Column: A fused silica tube, about 0.53 mm in in-

side diameter and about 50 m in length, coated insidesurface with dimethylpolysiloxane for gas chromato-graphy 5 m in thickness.

Column temperature: Maintain the initial tempera-ture of 70 C for 3 minute, up to 200 C at the rate of30

C per minute and hold at 200

C for 6.5 minutes.

Carrier gas: Helium.Injection port temperature: 150 C.Flow rate: 320 mL/minute.Detector temperature: 250 C.

(4) Related substances Weigh accurately about25 mg of Lamivudine, dissolve in the mobile phase asdirected in the Assay to make exactly 100 mL and usethis solution as the test solution. Weigh accurately a

portion of salicylic acid, add the mobile phase as di-rected in the Assay to render the concentration of 0.625g per mL and use this solution as the salicylic acid so-

lution. Perform the test with 10 L each of the test so-lution and the salicylic acid solution as directed underthe Liquid Chromatography according to the operatingconditions directed in the Assay, determine the area ofsalicylic acid peak in these solutions and calculate thecontent of salicylic acid according to the equation (1):not more than 0.1%. Calculate the content of other re-lated substances according to the equation (2): not morethan 0.3% for the related substance having the relativeretention time of about 0.4, not more than 0.2% for therelated substance having the relative retention time ofabout 0.9 and not more than 0.6% for total related sub-stances.


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KP 9 553

Content (%) of salicylic acid =S

T

A A

W C 10 (1)

C: Concentration (g/mL) of salicylic acid in thesalicylic acid solution

W: Weight (mg) of Lamivudine taken

T A : Peak area of salicylic acid obtained from thetest solution

S A : Peak area of salicylic acid obtained from thesalicylic acid solution

Content (%) of other related substance =S

i

A A100 (2)

i A : Peak area of related substance other than sali-cylic acid obtained from the test solution

S A : Total area of peaks obtained from the test solu-tion

Water Not more than 0.2% (1 g, coulometric titra-tion).

Assay Weigh accurately about 25 mg each of Lami-vudine and Lamivudine RS, add a mixture of 0.025mol/L ammonium acetate buffer and methanol (95:5) tomake exactly 100 mL each and use these solutions asthe test solution and the standard solution, respectively.Perform the test with 10 L each of the test solutionand the standard solution as directed under the LiquidChromatography according to the following operatingconditions and determine the area of the principal peakin the test solution, T A , and in the standard solution,

S A .

Amount (mg) of lamivudine (C 8H11 N3O3S)

= amount (mg) of Lamivudine RS S

T

A A


(wavelength: 277 nm).

Column: A stainless steel column about 4.6 mm ininside diameter and about 25 cm in length, packed withoctadecylsilanized silica gel for liquid chromatography(3-10 m in particle diameter).

Mobile phase: A mixture of 0.025 mol/L ammo-nium acetate buffer and methanol (95:5).

Column temperature: A constant temperature ofabout 35 C.

Flow rate: About 1.0 mL/minute.System suitability

System performance: Dissolve the content of onevial of lamivudine resolution mixture II RS in 5 mL ofthe mobile phase. Perform the test with 10 L of this

solution as directed under the above operating condi-tions, the relative retention times for lamivudine peakand lamivudine enantiomer peak are about 1.0 and 0.9,

respectively, with the resolution between lamivudine peak and lamivudine enantiomer peak being not lessthan 1.5.

System repeatability: When the test is repeated 5times with 10 L of the standard solution under theabove operating conditions, the relative standard devia-tion of the areas of lamivudine peak is not more than2.0%.

0.025 mol/L Ammonium acetate buffer Weighabout 1.9 g of ammonium acetate, dissolve in 900 mLof water and adjust the pH to 3.8 0.2 with acetic acidand add water to make 1000 mL.

Packaging and Storage Preserve in light-resistant,well-closed containers.

Lanatoside C

O

O

O

CH 3

HH

H

OH

H

OH H

CH 3

H

H

OH

OHCH 3 H

H

O

CH 3

HH

H

OOH

H

O

O

CH 3

HH

H

OO

H

O

CH 2OH

HOH

H

HOH

H

C

O

CH 3

OH

H

C49H76O20: 985.12

Lanatoside C, when dried, contains not less than 90.0%and not more than 102.0% of lanatoside C (C 49H76O20).

Description Lanatoside C is a colorless or whitecrystal or a white, crystalline powder and is odorless.Lanatoside C is soluble in methanol, slightly soluble inethanol and practically insoluble in water or in ether.Lanatoside C is hygroscopic.

Identification Place 1 mg of Lanatoside C to a smalltest tube having an internal diameter of about 10 mm,dissolve in 1 mL of a solution of ferric chloride in gla-cial acetic acid (1 in 10000) and underlay gently with 1mL of sulfuric acid: at the zone of contact of the twoliquids, a brown ring is produced and the color of theupper layer near the contact zone gradually changes to


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blue through purple. Finally the color of the entire acet-ic acid layer changes to blue-green through deep blue.

Specific Optical Rotation 20D][ : Between + 32 and+ 35 (after drying, 0.5 g, methanol, 25 mL, 100 mm).

Purity Related substances Dissolve 10 mg of La-natoside C in exactly 5 mL of methanol and use this so-lution as the test solution. Separately, dissolve 1.0 mgof Lanatoside C RS in exactly 5 mL of methanol anduse this solution as the standard solution. Perform thetest with the test solution and the standard solution, asdirected under Thin-layer Chromatography. Spot 20 Leach of the test solution and the standard solution on a

plate of silica gel for thin-layer chromatography. De-velop the plate with a mixture of dichloromethane, me-thanol and water (84 : 15 : 1) to a distance of about 13cm and air-dry the plate. Spray evenly dilute sulfuric

acid on the plate and heat the plate at 110 C for 10 mi-nutes: no spot other than the principal spot from the testsolution is larger nor darker than the standard solution.

Loss on Drying Not more than 7.5% (0.5 g, in va-cuum, P 2O5, 60 C, 4 hours).

Residue on Ignition Not more than 0.5% (0.1 g).

Assay Weigh accurately about 50 mg each of Lanato-side C and Lanatoside C RS, previously dried, dissolveseparately in methanol to make exactly 25 mL each, pi-

pet 5.0 mL of each solution, add methanol to make 100

mL each and use these solution as the test solution andthe standard solution, respectively. Pipet 5.0 mL eachof the test solution and the standard solution into light-resistant, volumetric flasks and add 5 mL each of picricacid TS and 0.5 mL each of a solution of sodium hy-droxide (1 in 10), shake well and add methanol to make25 mL each. Allow these solutions to stand between 18 C and 22 C for 25 minutes. Separately pipet 5.0 mLof methanol, proceed in the same manner and use thissolution as the blank solution. Determine the absor-

bances, T A and S A , of the solutions at 485 nm as di-rected under the Ultraviolet-visible spectrophotometryfor the test solution and the standard solution, respec-tively.

Amount (mg) of lanatoside C (C 49H76O20)

= amount (mg) of Lanatoside C RS S

T

A

A


Lanatoside C Tablets

Lanatoside C Tablets contain not less than 90.0% andnot more than 110.0% of the labeled amount of lanato-

side C (C 49H76O20: 985.12).

Method of Preparation Prepare as directed underTablets, with Lanatoside C.

Identification (1) Shake a portion of powdered Lana-

toside C Tablets, equivalent to 1 mg of Lanatoside Caccording to the labeled amount with 3 mL of ether andfilter. Wash the residue with two 3 mL volumes of etherand air-dry. To the remaining residue, add 10 mL of amixture of chloroform and methanol (9 : 1), shake andfilter. Wash the residue with two 5 mL volumes of amixture of chloroform and methanol (9 : 1), combinethe filtrate and washings and evaporate on a water-bathto a smaller volume. Transfer the solution to a smalltest tube having an internal diameter of about 10 mm,further evaporate on a water-bath to dryness and pro-ceed as directed in the Identification under LanatosideC.

(2) Perform the test with the test solution and thestandard solution obtained in the Assay as directed un-der the Thin-layer Chromatography. Spot 25 L each ofthe test solution and the standard solution on a plate ofsilica gel for thin-layer chromatography. Develop the

plate with a mixture of dichloromethane, methanol andwater (84 : 15 : 1) of 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: thespots obtained from the test solution and the standardsolution show a black color and have the same Rf val-ues.

Dissolution Test Take 1 tablet of Lanatoside C Tab-lets and perform the test with 500 mL of diluted hy-drochloric acid (3 in 500), degassed by a suitable me-thod, at 100 revolutions per minute as directed in Me-thod 2 under the Dissolution Test. Take 20.0 mL of thedissolved solution at 60 minutes after starting the testand filter through a membrane filter (not more than 0.8m). Discard the first 10 mL of the filtrate and use thesubsequent filtrate as the test solution. Separately,weigh accurately a portion of Lanatoside C RS, dried invacuum over P2O5 at 60 C for 4 hours, equivalent to100 times an amount of the labeled amount of Lanato-

side C (C 49H76O20), dissolve in methanol to make ex-actly 100 mL. Pipet 1 mL of this solution, add the testsolution to make exactly 500 mL, warm at 37 C for 60minutes and use this solution as the standard solution.Pipet 3.0 mL each of the test solution, the standard so-lution and the dissolution solution and transfer to glass-stoppered brown test tubes, T, S and B, respectively. Tothese solutions, add exactly 10 mL each of 12 mg/dLascorbic acid-hydrochloric acid TS and shake. Imme-diately, add exactly 0.2 mL each of diluted hydrogen

peroxide TS (1 in 100), shake well and allow to stand ata constant temperature between 30 C and 37 C for 45minutes. Determine immediately the fluorescence in-

tensities, T F , S F and B F , of the test solution, thestandard solution and the dissolution solution, respec-


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KP 9 555

tively, at 355 nm of the excitation wavelength and at490 nm of the fluorescence wavelength as directed un-der the Fluorometry.Individual dissolution rate of Lanatoside C Tablets forthe six tablets after 60 minutes is not less than 65%.Requirement of retest is not applied to Lanatoside C

Tablets.

Dissolution rate (%) to labeled amount of lanatoside C

(C49H76O20) =C F F

F F W

BS

BT S

1

W S: Amount (mg) of Lanatoside C RS,C : Labeled amount (mg) of lanatoside C

(C49H76O20) in each tablet.

Uniformity of Dosage Units It meets the require-ment when the content uniformity test is performed ac-

cording to the following procedure. Warm 1 tablet ofLanatoside C Tablets with 5 mL of water until the tabletis disintegrated, add 30 mL of ethanol, disperse finelythe particles with the aid of ultrasonic wave, add etha-nol to make exactly V mL of a solution containingabout 5 g of lanatoside C (C 49H76O20) per mL and fil-ter. Discard the first 10 mL of the filtrate and use thesubsequent filtrate as the test solution. Separately,weigh accurately about 25 mg of Lanatoside C RS,

previously dried in vacuum over P 2O5 at 60 C for 4hours and dissolve in ethanol to make exactly 100 mL.Pipet 2.0 mL of this solution, add 10 mL of water, addethanol to make exactly 100 mL and use this solution

as the standard solution. Pipet 2.0 mL each of the testsolution, the standard solution and diluted ethanol (17in 20) into three brown glass-stoppered test tubes, T, Sand B, previously containing exactly 10 ml of 12mg/dL ascorbic acid-hydrochloric acid TS, add exactly1 mL each of dilute hydrogen peroxide TS immediately,shake vigorously and allow to stand at a constant tem-

perature between 25 C and 30 C for 40 minutes. De-termine the fluorescence intensities, T F , S F and B F ,of the test solution, the standard solution and the di-luted ethanol (17 in 20), respectively, at 355 nm of theexcitation wavelength and at 490 nm of the fluores-cence wavelength as directed under the Fluorometry.

Amount (mg) of lanatoside C (C 49H76O20)= amount (mg) of lanatoside C RS

5000V

F F F F

BS

BT

Assay Weigh accurately and powder not less than 20Lanatoside C Tablets. Weigh accurately a portion of the

powder, equivalent to about 5 mg of lanatoside C(C49H76O20), into a light-resistant volumetric flask, add50 mL of ethanol and shake for 15 minutes. Then dilutewith ethanol to make exactly 100 mL. Filter this solu-

tion, discard the first 20 mL of the filtrate and use thesubsequent filtrate as the test solution. Separately,

weigh accurately about 5.0 mg of Lanatoside C RS, previously dried in vacuum over P 2O5 at 60 C for 4hours, dissolve in ethanol to make exactly 100 mL anduse this solution as the standard solution. Pipet 5.0 mLeach of the test solution and the standard solution intolight-resistant, glass-stoppered test tubes, add 3.0 mLeach of alkaline picric acid TS, shake well and allow tostand between 22 C and 28 C for 25 minutes. Deter-mine the absorbances, T A and S A , of the subsequenttest solution and the standard solution, respectively, at490 nm as directed under the Ultraviolet-visible Spec-trophotometry, using a solution, prepared by the samemanner with 5 mL of ethanol, as the blank.

Amount (mg) of lanatoside C (C 49H76O20)

= amount (mg) of Lanatoside C RS S

T

A A


Lansoprazole

N

HN

S

N

CH 3

O

CF 3

O

C16H14F3 N3O2S: 369.36

Lansoprazole contains not less than 99.0% and notmore than 101.1% of lansoprazole (C 16H14F3 N3O2S),calculated on the anhydrous basis.

Description Lansoprazole is a white or brown crys-

talline powder.Lansoprazole is freely soluble in dimethylformamide,soluble in methanol, sparingly soluble in dehydratedethanol, very slightly soluble in ether and practicallyinsoluble in water.

Melting point About 166 C (with decomposition)

Identification (1) Determine the absorption spectraof solutions of Lansoprazole and Lansoprazole RS inmethanol (1 in 100000) as directed under the Ultravio-let-visible Spectrophotometry: both spectra exhibit sim-ilar intensities of absorption at the same wavelengths.

(2) Determine the infrared spectra of Lansoprazole

and Lansoprazole RS as directed in the potassium bro-mide disk method under the Infrared Spectrophotome-try: both spectra exhibit similar intensities of absorp-


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tion at the same wavenumbers.

Purity Related substances Weigh accurately about0.125 g of Lansoprazole, dissolve in methanol to makeexactly 50 mL, pipet 1.0 mL of this solution, add amixture of the mobile phase A and the mobile phase B(9:1) to make exactly 10 mL and use this solution as thetest solution. Prepare the test solution immediately be-fore use. Separately, weigh accurately about 25 mg ofLansoprazole RS, dissolve in methanol to make exactly100 mL, pipet 5.0 mL of this solution, add methanol tomake exactly 50 mL. To 1.0 mL of this solution, add amixture of the mobile phase A and the mobile phase B(9:1) to make exactly 10 mL and use this solution as thestandard solution. To 9 mL of a mixture of the mobile

phase A and the mobile phase B (9:1), add 1 mL of me-thanol and use this solution as the blank solution. Per-form the test with 40 L each of the test solution, thestandard solution and the blank solution as directed inthe peak area percentage method under the LiquidChromatography. Disregard the peaks obtained fromthe test solution corresponding to the peaks obtainedfrom the blank solution and determined the peak areaof each related substance other than the principal peakfrom the test solution, i A , and the peak area of the

principal peak from the standard solution, S A : totalcontent of related substances is not more than 0.1%.

Content (%) of each related substance

=S

i

A A

W C 50

C: Concentration (g/mL) of lansoprazole in thestandard solution

W: Weight (mg) of Lansoprazole takenOperating conditions

Detector: An ultraviolet absorption photometer(wavelength: 285 nm).

Column: A stainless steel column about 4.6 mm ininside diameter and about 15 cm in length, packed withoctadecylsilanized silica gel for liquid chromatography(5 m in particle diameter).

Mobile phase: Control the step or concentrationgradient by mixing the mobile phases A and B as di-rected in the following table.

Mobile phase A: WaterMobile phase B: A mixture of acetonitrile, water

and triethylamine (160:40:1). Adjust the pH of the so-lution to 7.0 by the addition of phosphoric acid.

Time(min)

Mobile phase A(vol%)

Mobile phase B(vol%)

Elutioncondition

0-40 90 20 10 80 Lineargradient40-50 20 80 Isocratic

50-51 20 90 80 10 Lineargradient51-60 90 10 Isocratic

Flow rate: 0.8 mL/min.System suitability

System performance: Weigh 5 mg each of Lan-soprazole RS and lansoprazole related substance I {2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridyl]methyl]

sulfonyl]benzimidazole} RS, dissolve in methanol tomake 200 mL, pipet 1.0 mL of this solution and add amixture of the mobile phase A and the mobile phase B(9:1) to make exactly 10 mL. When the procedure isrun with 40 L of this solution under the above operat-ing conditions, the resolution between lansoprazole

peak and the related substance I peak is not less than 6.System repeatability: Weigh 2.5 mg of lansopra-

zole related substance I RS, dissolve in methanol tomake 100 mL, pipet 1.0 mL of this solution and add amixture of the mobile phase A and the mobile phase B(9:1) to make exactly 10 mL. When the test is repeated6 times with 40 L each of this solution under theabove operating conditions, the relative standard devia-tion of the areas of lansoprazole related substance I

peak is not more than 3%.

Water Not more than 0.1% (1.0 g, volumetric titra-tion, direct titration).Use 50 mL of a mixture of pyridine and ethylene glycol(9:1 or 8:2) as the solvent in this test.


Assay Weigh accurately about 50 mg each of Lanso- prazol and Lansoprazole RS, dissolve in the internalstandard solution to make 10 mL each, pipet 1 mL eachof these solution, add a mixture, prepared by mixingwater, acetonitrile and triethylamine (60:40:1) and byadjusting the pH of the solution to 10 with phosphoricacid, to make exactly 50 mL each and use these solu-tions as the test solution and the standard solution, re-spectively. Perform the test with 10 L each of the testsolution and the standard solution as directed under theLiquid Chromatography according to the following op-erating conditions and determined the ratios of the peakarea of lansoprazole to that of the internal standardfrom the test solution, TQ , and from the standard solu-

tion, SQ .

Amount (mg) of lansoprazole (C 16H14F3 N3O2S)

= amount (mg) of Lansoprazole RS S

TQ

Q

Internal standard solution Weigh about 25 mg of4-ethoxyacetophenone and dissolve in a mixture, pre-

pared by mixing water, acetonitrile and triethylamine(60:40:1) and by adjusting the pH of the solution to 10with phosphoric acid, to make 10 mL.

Operating conditionsDetector: An ultraviolet absorption photometer


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KP 9 557


inside diameter and about 15 cm in length, packed withoctadecylsilanized silica gel for liquid chromatography(5 m in particle diameter).

Mobile phase: A mixture of water, acetonitrile and

triethylamine (60:40:1). Adjust the pH of the solutionto 7.0 by the addition of phosphoric acid.

Flow rate: 1 mL/min.System suitability

System performance: Weigh 5 mg each of Lan-soprazole RS and lansoprazole related substance I RS,and dissolve in a mixture, prepared by mixing water,acetonitrile and triethylamine (60:40:1) and by adjust-ing the pH of the solution to 10 with phosphoric acid,to make exactly 50 mL. When the procedure is run with10 L of this solution under the above operating condi-tions, the resolution between lansoprazole peak and therelated substance I peak is not less than 5.

System repeatability: When the test is repeated 5times with 10 L each of the standard solution underthe above operating conditions, the relative standarddeviation of the areas of lansoprazole peak is not morethan 0.5%.

Packaging and Storage Preserve in light-resistant,well-closed containers.

Letrozole

N

NC CN

N

N

C17H11 N5 : 285.30

Letrozole contains not less than 98.0% and not morethan 102.0% of letrozole (C 17H11 N5), calculated on theanhydrous basis.

Description Letrozole is a white to yellowish-whitecrystal or crystalline powder.Letrozole is freely soluble in dichloromethane, slightlysoluble in ethanol and practically insoluble in water.

Identification (1) Determine the infrared spectra ofLetrozole and Letrozole RS as directed in the paste me-thod under the Infrared Spectrophotometry: both spec-tra exhibit similar intensities of absorption at the samewavenumbers.

(2) When proceed as directed in the Assay, the re-tention time of the principal peak from the test solution

corresponds to that from the standard solution.

Purity (1) Heavy metals Proceed with 1.0 g of Le-trozole according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of standardlead solution (not more than 20 ppm).

(2) Related substances Weigh accurately about 25mg of Letrozole, dissolve in 75 mL of acetonitrile, addwater to make exactly 250 mL and use this solution asthe test solution. Separately, to 5.0 mL of the test solu-tion, add a mixture of water and acetonitrile (7:3) tomake exactly 50 mL, pipet 5.0 mL of this solution, adda mixture of water and acetonitrile (7:3) to make exact-ly 50 mL and use this solution as the standard solution.Perform the test with 15 L each of the test solutionand the standard solution as directed under the LiquidChromatography according to the following operatingconditions and determine the areas of peaks from thesesolutions: not more than 0.3% for letrozole related sub-stance I (4,4-(1 H -1,3,4-triazol-1-ylmethylene) diben-zonitrile), not more than 0.2% for 4,4,4-methylidenetrisbenzonitrile, not more than 0.1% forany other related substance and not more than 0.3% fortotal related substances (the relative retention time for4,4,4-methylidenetrisbenzonitrile is about 1.9).

Content (%) of each related substance=S

5.Monograph_Part I(J~Z)

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