A: Infrared Absorption á197Mñ.

B: Thin-Layer Chromatographic Identification Test á201ñ—

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Test solution— Dissolve 500mg of Clotrimazole,accurately weighed,in 5.0mLof chloroform.

Standard solution— Prepare a solution of USP Imidazole RSin chloroform containing 500µg per mL.

Application volume: 5µL.

Developing solvent system: a mixture of methanol and chloroform (3:2).

Procedure— Proceed as directed for Thin-Layer Chromatographyunder Chromatography á621ñ.After air-drying the plate for 5minutes,place it in a closed container with a dish containing 100g of iodine in a shallow layer,and allow to remain for 60minutes.Remove the plate from the container,and observe the chromatogram.Any brown spot obtained from the Test solutionat an RFvalue corresponding to the principal spot from the Standard solutionis not greater in size or intensity than the principal spot obtained from the Standard solution:not more than 0.5%of imidazole.

Dibasic potassium phosphate solution,Mobile phase,Resolution solution,andChromatographic system— Proceed as directed in the Assay.

Standard solution— Transfer about 12.5mg of USP Clotrimazole Related Compound A RS,accurately weighed,to a 25-mLvolumetric flask,add 10mLof methanol to dissolve,add 6.25mLof Dibasic potassium phosphate solution,dilute with methanol to volume,and mix (Stock solution).Transfer 5.0mLof the Stock solutionto a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Test solution— Use the Assay stock solutionused to prepare the Assay preparationin the Assay.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of clotrimazole related compound Ain the portion of Clotrimazole taken by the formula: in which Cis the concentration,in mg per mL,of USP Clotrimazole Related Compound A RSin the Standard solution;Wis the weight,in mg,of Clotrimazole taken;and rUand rSare the responses for clotrimazole related compound Aobtained from the Test solutionand the Standard solution,respectively:not more than 0.5%is found.

Dibasic potassium phosphate solution— Dissolve 4.35g of dibasic potassium phosphate in water to make 1000mLof solution.

Mobile phase— Prepare a mixture of methanol and Dibasic potassium phosphate solution(3:1),pass through a membrane filter having a 0.2-µm or finer porosity,and degas.The ratio of volumes may be changed to obtain the required resolution.

Internal standard solution— Transfer 33mg of testosterone propionate to a 200-mLvolumetric flask,add 125mLof methanol to dissolve it,add 50mLof Dibasic potassium phosphate solution,dilute with methanol to volume,and mix.

Standard preparation— Transfer about 50mg of USP Clotrimazole RS,accurately weighed,to a 50-mLvolumetric flask.Add 25mLof methanol to dissolve,add 12.5mLof Dibasic potassium phosphate solution,dilute with methanol to volume,and mix (Standard stock solution).Transfer 10.0mLof the Standard stock solutionto a 100-mLvolumetric flask,add 4.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix.

Resolution solution— Transfer 3mLof the Standard stock solutionused to prepare the Standard preparationand 5mLof the Stock solutionused to prepare the Standard solutionin the test for Limit of clotrimazole related compound Ato a 25-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Assay preparation— Transfer about 100mg of Clotrimazole,accurately weighed,to a 10-mLvolumetric flask,add 5mLof methanol to dissolve,add 2.5mLof Dibasic potassium phosphate solution,dilute with methanol to volume,and mix (Assay stock solution).Transfer 1.0mLof the Assay stock solutionto a 100-mLvolumetric flask,add 4.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains 10-µm packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.7for clotrimazole related compound Aand 1.0for clotrimazole;and the resolution,R,between clotrimazole and clotrimazole related compound Ais not less than 1.9.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 1.0for clotrimazole and 1.5for testosterone propionate;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C22H17ClN2in the portion of Clotrimazole taken by the formula: in which Cis the concentration,in mg per mL,of USP Clotrimazole RSin the Standard preparation;and RUand RSare the peak response ratios of clotrimazole to testosterone propionate obtained from the Assay preparationand the Standard preparation,respectively.