A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,both relative to the internal standard,as obtained in the Assay.

B: Place a quantity of Ointment,equivalent to about 1.25mg of alclometasone dipropionate,in a 50-mLcentrifuge tube,add 10mLof 2,2,4-trimethylpentane,insert a stopper securely into the tube,and disperse the specimen using a vortexing mixer.Add 5.0mLof a solution of methanol in water (45in 50),insert the stopper securely,shake vigorously for 2minutes,and centrifuge at 2500rpm for 3minutes.Remove the lower,aqueous alcohol phase,and transfer this test solution to a stoppered vial.Apply separately 20µLof the test solution and 20µLof a Standard solution of USP Alclometasone Dipropionate RSin methanol containing about 0.25mg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture,and dry the applications with the aid of a stream of nitrogen.Position the plate in a saturated,unlined chromatographic chamber,and develop the chromatograms in a solvent system consisting of a mixture of chloroform and acetone (7:1)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.Observe the plate under short-wavelength UVlight:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

Methanol solution— Dilute 450mLof methanol with water to 500mL,and mix.

0.05M Monobasic potassium phosphate— Transfer 3.40g of monobasic potassium phosphate to a 500-mLvolumetric flask,add water to volume,and mix.

Mobile phase— Prepare a filtered and degassed mixture of methanol and 0.05M Monobasic potassium phosphate(2:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Transfer about 30mg of betamethasone dipropionate to a 200-mLvolumetric flask,add Methanol solutionto volume,and mix.

Standard preparation— Transfer about 20mg of USP Alclometasone Dipropionate RS,accurately weighed,to a 200-mLvolumetric flask,add Methanol solutionto volume,and mix.Transfer 5.0mLof this solution to a small stoppered flask,add 5.0mLof Internal standard solution,and mix to obtain a Standard preparationhaving a known concentration of about 0.05mg of USP Alclometasone Dipropionate RSper mL.

Assay preparation— Transfer an accurately weighed quantity of Ointment,equivalent to about 0.5mg of alclometasone dipropionate,to a 50-mLcentrifuge tube,add 10mLof 2,2,4-trimethylpentane,insert a stopper securely into the tube,and disperse the specimen using a vortexing mixer.Add 5.0mLof Internal standard solutionand 5.0mLof Methanol solution,insert the stopper securely,shake vigorously for 2minutes,and centrifuge at 2500rpm for 3minutes.Remove the lower,aqueous alcohol phase,and transfer this Assay preparationto a stoppered vial.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 1.2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the analyte and internal standard peaks is not less than 3.0;and the relative standard deviation for replicate injections is not more than 2%.

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.The relative retention times are about 0.7for alclometasone dipropionate and 1.0for betamethasone dipropionate.Calculate the quantity,in mg,of alclometasone dipropionate (C28H37ClO7)in the portion of Ointment taken by the formula: in which Cis the concentration,in mg per mL,of USP Alclometasone Dipropionate RSin the Standard preparation;and RUand RSare the peak height ratios obtained from the Assay Preparation and the Standard preparation,respectively.