» Clotrimazole Cream contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of clotrimazole (C22H17ClN2).
Packaging and storage Preserve in collapsible tubes or tight containers, at a temperature between 2 and 30.
Labeling Cream that is packaged and labeled for use as a vaginal preparation shall be labeled Clotrimazole Vaginal Cream.
Identification In a suitable chromatographic chamber, arranged for thin-layer chromatography (see Chromatography 621) containing 200 mL of ether, place a beaker containing 25 mL of ammonium hydroxide. Cover the chamber, and allow to equilibrate for 2 hours. Place a portion of Cream, equivalent to about 5 mg of clotrimazole, in a 50-mL centrifuge tube, add 5 mL of chloroform, mix, and centrifuge to obtain a clear test solution. Apply 20 µL of the lower, chloroform phase and 20 µL of a solution of USP Clotrimazole RS in chloroform containing 1 mg per mL to a suitable thin-layer chromatographic plate (see Chromatography 621), coated with a 0.25-mm layer of chromatographic silica gel mixture. Develop the chromatogram until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Locate the spots on the plate by examination under short-wavelength UV light: the RF value of the principal spot from the test solution corresponds to that obtained from the Standard solution. Dissolve 3 g of bismuth subnitrate and 30 g of potassium iodide in 10 mL of dilute hydrochloric acid (1 in 4), dilute with water to 100 mL, mix, and prepare a spray reagent by diluting 10 mL of this solution and 5 mL of dilute hydrochloric acid (1 in 4) with water to 200 mL, and mixing. Spray the plate evenly with this spray reagent: the principal spots from the test solution and the Standard solution are orange.
Dibasic potassium phosphate solution and Mobile phase Prepare as directed in the Assay under Clotrimazole.
Internal standard solution Prepare a solution of testosterone propionate in dehydrated alcohol having a concentration of about 0.07 mg per mL.
Standard stock solution Transfer about 50 mg of USP Clotrimazole RS, accurately weighed, to a 50-mL volumetric flask, dilute with dehydrated alcohol to volume, and mix.
Standard preparation Mix 10.0 mL of Standard stock solution and 10.0 mL of Internal standard solution.
Resolution solution Prepare a solution of USP Clotrimazole Related Compound A RS in dehydrated alcohol having a concentration of about 0.12 mg per mL. Mix 7 mL of this solution and 1 mL of Standard stock solution.
Assay preparation Accurately weigh a portion of Cream, equivalent to about 10 mg of clotrimazole, and transfer to a 50-mL screw-capped centrifuge tube. Add 10.0 mL of Internal standard solution, 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 starting with heat at 50 in a water bath. Transfer the supernatant to the test tube containing the supernatant from the first extraction, and mix. 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 L7 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 Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.9 for clotrimazole related compound A and 1.0 for clotrimazole; and the resolution, R, between clotrimazole related compound A and clotrimazole is not less than 1.2. 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 clotrimazole (C22H17ClN2) in each g of Cream 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 Cream 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.
Auxiliary Information Please check for your question in the FAQs before contacting USP.Chromatographic Column
USP32NF27 Page 1998Pharmacopeial Forum: Volume No. 27(2) Page 2131
Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.