Identification—
Mix an amount of Lotion, equivalent to about 5 mg of betamethasone, with a mixture of methanol and chloroform (2:1) to make 10 mL. Apply 20 µL of this solution and 20 µL of a Standard solution of
USP Betamethasone Valerate RS in a mixture of methanol and chloroform (2:1) containing 0.6 mg per mL to a suitable thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of a mixture of chloroform and ethyl acetate (1:1), 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. View the spots under UV light: the
RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.
Assay—
Internal standard solution—
Transfer about 50 mg of beclomethasone dipropionate to a 25-mL volumetric flask, add chloroform to volume, and mix.
Standard preparation—
Transfer about 40 mg of
USP Betamethasone Valerate RS, accurately weighed, to a 25-mL volumetric flask, add chloroform to volume, and mix. Pipet 2 mL of this solution into a 50-mL centrifuge tube, add 10 mL of 0.1 N hydrochloric acid, then add 2.0 mL of
Internal standard solution. Insert the stopper into the tube, shake vigorously for about 2 minutes, and centrifuge to separate the phases. Using a syringe, transfer the lower, chloroform phase to a small stoppered vial. Evaporate the chloroform on a steam bath, at low heat, with the aid of a stream of nitrogen. Add 4.0 mL of a 1 in 1000 solution of glacial acetic acid in methanol, and swirl to dissolve the residue.
Assay preparation—
Transfer an accurately weighed portion of Lotion, equivalent to about 2.5 mg of betamethasone, to a stoppered, 50-mL centrifuge tube. Add 10.0 mL of 0.1 N hydrochloric acid, insert the stopper, and shake to disperse the specimen. Add 2.0 mL of chloroform and 2.0 mL of
Internal standard solution, insert the stopper, and proceed as directed for
Standard preparation, beginning with “shake vigorously for about 2 minutes.”
Procedure—
Proceed as directed for
Procedure in the
Assay under
Betamethasone Valerate. Calculate the quantity, in mg, of betamethasone (C
22H
29FO
5) in the portion of Lotion taken by the formula:
(392.46 / 476.59)(4C)(RU / RS),
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.
