Test solution: 1 mg per mL, prepared by concentrating 10 mL of the Assay preparation on a steam bath to 1 mL.

Standard solution— Prepare a solution of USP Betamethasone RS in dehydrated alcohol having a concentration of 1 mg 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 105 for 10 minutes.

Mobile phase, Internal standard solution, Standard preparation, and Chromatographic system— Prepare as directed in the Assay under Betamethasone.

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

Procedure— Separately inject equal volumes (about 10 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 C22H29FO5 in the portion of Cream taken by the formula: in which C is the concentration, in mg per mL, of USP Betamethasone RS in the Standard preparation; and RU and RS are the peak height ratios of the betamethasone peak and the internal standard peak obtained from the Assay preparation and the Standard preparation, respectively.