Mobile phase and Chromatographic system—Proceed as directed in the Assay under Betamethasone Valerate.

Internal standard solution— Transfer about 20 mg of beclomethasone dipropionate to a 50-mL volumetric flask, add a 1 in 1000 solution of glacial acetic acid in alcohol to volume, and mix.

Standard preparation— Transfer about 30 mg of USP Betamethasone Valerate RS, accurately weighed, to a 50-mL volumetric flask, add a 1 in 1000 solution of glacial acetic acid in alcohol to volume, and mix. Transfer 5.0 mL of this solution to a suitable stoppered vial, add 10.0 mL of Internal standard solution, and mix to obtain a solution having a known concentration of about 0.2 mg of USP Betamethasone Valerate RS per mL.

Assay preparation— Transfer an accurately weighed portion of Ointment, equivalent to about 2.5 mg of betamethasone, to a 50-mL centrifuge tube. Add 10.0 mL of the Internal standard solution and 5.0 mL of a 1 in 1000 solution of glacial acetic acid in alcohol. Insert the stopper into the tube, and place in a water bath held at 70 until the specimen melts. Remove from the bath, and shake vigorously until the specimen resolidifies. Repeat the heating and shaking two more times. Place the tube in an ice-methanol bath for 20 minutes, then centrifuge to separate the phases. Decant the clear supernatant into a suitable stoppered flask, and allow to warm to room temperature.

Procedure— Proceed as directed for Procedure in the Assay under Betamethasone Valerate. Calculate the quantity, in mg, of C22H29FO5 in the portion of Ointment taken by the formula: in which 392.46 and 476.59 are the molecular weights of betamethasone and betamethasone valerate, respectively; C is the concentration, in mg per mL, of USP Betamethasone Valerate RS in the Standard preparation; and RU and RS are the peak response ratios obtained from the Assay preparation and the Standard preparation, respectively.