(Official January 1, 2007)

Dibasic ammonium phosphate solution— Dissolve 6.6 g of dibasic ammonium phosphate in water to make 1000 mL of solution.

Mobile phase— Prepare a mixture of methanol and Dibasic ammonium phosphate solution (7:3), and adjust with phosphoric acid to a pH of 7.0 ± 0.2. Pass through a membrane filter having a 0.45-µm or finer porosity, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Prepare a solution of progesterone in alcohol having a concentration of about 0.15 mg per mL.

Clotrimazole standard stock solution— Prepare a solution of USP Clotrimazole RS in alcohol having a known concentration of about 5 mg per mL.

Betamethasone dipropionate standard stock solution— Prepare a solution of USP Betamethasone Dipropionate RS in alcohol having a known concentration of about 6.4J mg per mL, J being the ratio of the labeled amount, in mg, of betamethasone to the labeled amount, in mg, of clotrimazole in each g of Cream.

Clotrimazole related compound A standard stock solution— Prepare a solution of USP Clotrimazole Related Compound A RS in methanol having a known concentration of about 0.5 mg per mL.

Standard preparation— Transfer 1.0 mL of Clotrimazole related compound A standard stock solution to a suitable container, and evaporate to dryness in a water bath at room temperature under a stream of nitrogen. To the residue add 2.0 mL each of the Clotrimazole standard stock solution, Betamethasone dipropionate standard stock solution, and Internal standard solution, and mix.

Assay preparation— Accurately weigh a portion of Cream, equivalent to about 10 mg of clotrimazole, and transfer to a screwcapped, 50-mL centrifuge tube. Add 2.0 mL of Internal standard solution and 4.0 mL of alcohol, place the cap on the tube, and heat at 60 in a water bath for 10 minutes, with occasional shaking. Remove the tube from the bath, cool in an ice bath for 20 minutes, and promptly centrifuge. Transfer a portion of the supernatant to a test tube, 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 4.6-mm × 25-cm column that contains 10-µm packing L1. The flow rate is about 1.7 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 1.0 for betamethasone dipropionate, 1.2 for clotrimazole related compound A, 1.4 for progesterone, and 1.7 for clotrimazole; the resolution, R, between betamethasone dipropionate and clotrimazole related compound A is not less than 1.0, between clotrimazole related compound A and progesterone is not less than 1.5, and between progesterone and clotrimazole is not less than 1.8; and the relative standard deviation for replicate injections is not more than 2.0% determined from clotrimazole and betamethasone dipropionate, and not more than 4.0% determined from clotrimazole related compound A.

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: in which C is the concentration, in mg per mL, of USP Clotrimazole RS in the Clotrimazole standard stock solution; W is the weight, in g, of Cream taken; and RU and RS are the peak response ratios of clotrimazole to progesterone obtained from the Assay preparation and the Standard preparation, respectively. Calculate the quantity, in mg, of betamethasone (C22H29FO5) in each g of Cream taken by the formula: in which 392.46 and 504.60 are the molecular weights of betamethasone and betamethasone dipropionate, respectively; C is the concentration, in mg per mL, of USP Betamethasone Dipropionate RS in the Betamethasone dipropionate standard stock solution; W is the weight, in g, of Cream taken; and RU and RS are the peak response ratios of betamethasone dipropionate to progesterone obtained from the Assay preparation and the Standard preparation, respectively. Calculate the quantity, in mg, of clotrimazole related compound A in each g of Cream taken by the formula: in which C is the concentration, in mg per mL, of USP Clotrimazole Related Compound A RS in the Clotrimazole related compound A standard stock solution; W is the weight, in g, of Cream taken; and RU and RS are the peak response ratios of clotrimazole related compound A to progesterone obtained from the Assay preparation and the Standard preparation, respectively: the quantity of clotrimazole related compound A found is not more than 5.0% of the labeled quantity of clotrimazole in the Cream.