Limit of clotrimazole related compound A—
Using the chromatograms of the
Assay preparation and the
Standard preparation obtained as directed in the
Assay, calculate the percentage of clotrimazole related compound A in the portion of Lotion taken by the formula:
2000(C / Q)(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Clotrimazole Related Compound A RS in the
Standard preparation; Q is the quantity, in mg, of clotrimazole (C
22H
17ClN
2) in the portion of Lotion taken; and
RU and
RS are the peak response ratios of clotrimazole related compound A to testosterone propionate obtained from the
Assay preparation and the
Standard preparation, respectively: not more than 5% is found.
Assay—
Dibasic potassium phosphate solution—
Dissolve 4.35 g of dibasic potassium phosphate in water to make 1000 mL.
Mobile phase—
Prepare a mixture of methanol and Dibasic Potassium phosphate solution (3:1), pass through a membrane filter having a 0.5-µm or finer porosity, and degas.
Internal standard solution—
Prepare a solution of testosterone propionate in dehydrated alcohol having a concentration of about 0.07 mg per mL.
Clotrimazole standard stock solution—
Transfer about 50 mg of
USP Clotrimazole RS, accurately weighed, to a 25-mL volumetric flask, add dehydrated alcohol to volume, and mix.
Clotrimazole related compound A standard stock solution—
Prepare a solution of
USP Clotrimazole Related Compound A RS in dehydrated alcohol having a known concentration of about 0.1 mg per mL.
Standard preparation—
Mix 5.0 mL of
Clotrimazole standard stock solution, 5.0 mL of
Clotrimazole related compound A standard stock solution, and 10.0 mL of
Internal standard solution.
Assay preparation—
Accurately weigh a portion of freshly mixed Lotion, equivalent to about 10 mg of clotrimazole, and transfer to a screw-capped, 50-mL centrifuge tube. Add 10.0 mL of
Internal standard solution, place the cap on the tube, and heat at 50
in a water bath for 5 minutes, with occasional shaking. Remove the tube from the bath, and shake vigorously for 5 minutes. Cool in a methanol-ice bath for 15 minutes, and promptly centrifuge. Transfer the supernatant to a test tube. Add 10.0 mL of dehydrated alcohol to the residue in the centrifuge tube, and repeat the extraction as directed above, beginning with “place the cap on the tube.” Transfer the supernatant to the test tube containing the supernatant from the first extraction, mix, and use this solution as the
Assay preparation.
Chromatographic system (see Chromatography 
621
)—
The liquid chromatograph is equipped with a 254-nm detector and a 2.1-mm × 6-cm guard column that contains 10-µm packing L2 and a 3.9-mm × 30-cm analytical column that contains 10-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 0.9 for clotrimazole related compound A, 1.0 for clotrimazole, and 1.5 for testosterone propionate; the resolution,
R, between clotrimazole related compound A and clotrimazole is not less than 1.2; the resolution,
R, between clotrimazole and testosterone propionate is not less than 1.9; 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 clotrimazole (C
22H
17ClN
2) in each g of Lotion taken by the formula:
20(C / W)(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Clotrimazole RS in the
Standard preparation; W is the weight, in g, of Lotion taken; and
RU and
RS are the peak response ratios of clotrimazole to testosterone propionate obtained from the
Assay preparation and the
Standard preparation, respectively.
