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

Mobile phase— Prepare a mixture of methanol and Dibasic ammonium phosphate solution(7:3),and adjust with phosphoric acid to a pHof 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 Suitabilityunder Chromatography á621ñ).

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

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

Betamethasone dipropionate standard stock solution— Prepare a solution of USP Betamethasone Dipropionate RSin alcohol having a known concentration of about 6.4Jmg per mL,Jbeing 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 Astandard stock solution— Prepare a solution of USP Clotrimazole Related Compound A RSin methanol having a known concentration of about 0.5mg per mL.

Standard preparation— Transfer 1.0mLof Clotrimazole related compound Astandard stock solutionto a suitable container,and evaporate to dryness in a water bath at room temperature under a stream of nitrogen.To the residue add 2.0mLeach 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 10mg of clotrimazole,and transfer to a screwcapped,50-mLcentrifuge tube.Add 2.0mLof Internal standard solutionand 4.0mLof alcohol,place the cap on the tube,and heat at 60in a water bath for 10minutes,with occasional shaking.Remove the tube from the bath,cool in an ice bath for 20minutes,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 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 1.7mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 1.0for betamethasone dipropionate,1.2for clotrimazole related compound A,1.4for progesterone,and 1.7for clotrimazole;the resolution,R,between betamethasone dipropionate and clotrimazole related compound Ais not less than 1.0,between clotrimazole related compound Aand 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 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 clotrimazole (C22H17ClN2)in each g of Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Clotrimazole RSin the Clotrimazole standard stock solution;Wis the weight,in g,of Cream taken;and RUand RSare the peak response ratios of clotrimazole to progesterone obtained from the Assay preparationand the Standard preparation,respectively.Calculate the quantity,in mg,of betamethasone (C22H29FO5)in each g of Cream taken by the formula: in which 392.46and 504.60are the molecular weights of betamethasone and betamethasone dipropionate,respectively;Cis the concentration,in mg per mL,of USP Betamethasone Dipropionate RSin the Betamethasone dipropionate standard stock solution;Wis the weight,in g,of Cream taken;and RUand RSare the peak response ratios of betamethasone dipropionate to progesterone obtained from the Assay preparationand the Standard preparation,respectively.Calculate the quantity,in mg,of clotrimazole related compound Ain each g of Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Clotrimazole Related Compound A RSin the Clotrimazole related compound Astandard stock solution;Wis the weight,in g,of Cream taken;and RUand RSare the peak response ratios of clotrimazole related compound Ato progesterone obtained from the Assay preparationand the Standard preparation,respectively:the quantity of clotrimazole related compound Afound is not more than 5.0%of the labeled quantity of clotrimazole in the Cream.