A: Infrared Absorption 197M.

B: Thin-Layer Chromatographic Identification Test 201—

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

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

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

Application volume: 5 µL.

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

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. After air-drying the plate for 5 minutes, place it in a closed container with a dish containing 100 g of iodine in a shallow layer, and allow to remain for 60 minutes. Remove the plate from the container, and observe the chromatogram. Any brown spot obtained from the Test solution at an RF value corresponding to the principal spot from the Standard solution is 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, and Chromatographic system— Proceed as directed in the Assay.

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

Test solution— Use the Assay stock solution used to prepare the Assay preparation in the Assay.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of clotrimazole related compound A in the portion of Clotrimazole taken by the formula: in which C is the concentration, in mg per mL, of USP Clotrimazole Related Compound A RS in the Standard solution; W is the weight, in mg, of Clotrimazole taken; and rU and rS are the responses for clotrimazole related compound A obtained from the Test solution and the Standard solution, respectively: not more than 0.5% is found.

Dibasic potassium phosphate solution— Dissolve 4.35 g of dibasic potassium phosphate in water to make 1000 mL of 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 33 mg of testosterone propionate to a 200-mL volumetric flask, add 125 mL of methanol to dissolve it, add 50 mL of Dibasic potassium phosphate solution, dilute with methanol to volume, and mix.

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

Resolution solution— Transfer 3 mL of the Standard stock solution used to prepare the Standard preparation and 5 mL of the Stock solution used to prepare the Standard solution in the test for Limit of clotrimazole related compound A to a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix.

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

Chromatographic system (see Chromatography 621)— 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 1.5 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for clotrimazole related compound A and 1.0 for clotrimazole; and the resolution, R, between clotrimazole and clotrimazole related compound A is not less than 1.9. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 1.0 for clotrimazole and 1.5 for testosterone propionate; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C22H17ClN2 in the portion of Clotrimazole taken by the formula: in which C is the concentration, in mg per mL, of USP Clotrimazole RS in the Standard preparation; and RU and RS are the peak response ratios of clotrimazole to testosterone propionate obtained from the Assay preparation and the Standard preparation, respectively.