Test solution: 1mg per mL,prepared by concentrating 10mLof the Assay preparationon a steam bath to 1mL.

Standard solution— Prepare a solution of USP Betamethasone RSin dehydrated alcohol having a concentration of 1mg per mL.

Developing solvent system: a mixture of chloroform and diethylamine (2:1).

Spray reagent: a mixture of sulfuric acid,methanol,and nitric acid (10:10:1).

Procedure— Proceed as directed in the chapter,except to spray the plate with the Spray reagent,and heat at 105for 10minutes.

Mobile phase,Internal standard solution,Standard preparation,andChromatographic system— Prepare as directed in the Assayunder Betamethasone.

Assay preparation— Transfer an accurately weighed quantity of Cream,equivalent to about 2mg of betamethasone,into a capped 50-mLcentrifuge tube.Add 10.0mLof Internal standard solutionand 10.0mLof alcohol.Mix by rotation for about 20minutes.Centrifuge at 2500rpm for about 10minutes.Transfer a portion of the supernatant to a suitable vial.

Procedure— Separately inject equal volumes (about 10µL)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 C22H29FO5in the portion of Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Betamethasone RSin the Standard preparation;and RUand RSare the peak height ratios of the betamethasone peak and the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.