2000(C/Q)(RU/RS),

Dibasic potassium phosphate solution— Dissolve 4.35g of dibasic potassium phosphate in water to make 1000mL.

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 0.07mg per mL.

Clotrimazole standard stock solution— Transfer about 50mg of USP Clotrimazole RS,accurately weighed,to a 25-mLvolumetric flask,add dehydrated alcohol to volume,and mix.

Clotrimazole related compound Astandard stock solution— Prepare a solution of USP Clotrimazole Related Compound A RSin dehydrated alcohol having a known concentration of about 0.1mg per mL.

Standard preparation— Mix 5.0mLof Clotrimazole standard stock solution,5.0mLof Clotrimazole related compound Astandard stock solution,and 10.0mLof Internal standard solution.

Assay preparation— Accurately weigh a portion of freshly mixed Lotion,equivalent to about 10mg of clotrimazole,and transfer to a screw-capped,50-mLcentrifuge tube.Add 10.0mLof Internal standard solution,place the cap on the tube,and heat at 50in a water bath for 5minutes,with occasional shaking.Remove the tube from the bath,and shake vigorously for 5minutes.Cool in a methanol-ice bath for 15minutes,and promptly centrifuge.Transfer the supernatant to a test tube.Add 10.0mLof 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 á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 2.1-mm ×6-cm guard column that contains 10-µm packing L2and a 3.9-mm ×30-cm analytical column that contains 10-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.9for clotrimazole related compound A,1.0for clotrimazole,and 1.5for testosterone propionate;the resolution,R,between clotrimazole related compound Aand 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 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 Lotion taken by the formula: in which Cis the concentration,in mg per mL,of USP Clotrimazole RSin the Standard preparation;Wis the weight,in g,of Lotion taken;and RUand RSare the peak response ratios of clotrimazole to testosterone propionate obtained from the Assay preparationand the Standard preparation,respectively.