Test solution— Evaporate 1 mL of the Assay preparation, prepared as directed in the Assay, on a steam bath just to dryness, and dissolve the residue in 0.5 mL of alcohol.

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

Procedure— Proceed as directed in the chapter. Locate the spots by lightly spraying with dilute sulfuric acid (1 in 2) and heating on a hot plate or under a lamp until spots appear.

Developing solvent— Prepare a mixture of chloroform, methanol, and ammonium hydroxide (175:20:1).

Tetramethylammonium hydroxide reagent— Dilute 20 mL of tetramethylammonium hydroxide TS with alcohol to make 100 mL.

Standard preparation— Dissolve a suitable quantity of USP Betamethasone RS, accurately weighed, in a mixture of chloroform and methanol (1:1) to obtain a solution having a known concentration of about 0.6 mg per mL.

Assay preparation— Use a pipet calibrated to contain a suitable volume, and transfer to a 50-mL centrifuge tube an accurately measured volume of Oral Solution, equivalent to about 1.2 mg of Betamethasone. Rinse the pipet with 15 mL of 0.1 N hydrochloric acid, then with 20 mL of ethyl acetate, and add the rinsings to the centrifuge tube. Rotate for about 10 minutes, or shake manually for about 1 minute. [note—Do not use a mechanical shaker.] Centrifuge to separate the phases. Transfer the upper phase (ethyl acetate) to a small, pear-shaped flask. Extract the aqueous phase twice more with 20-mL portions of ethyl acetate, and add the extracts to the pear-shaped flask. Evaporate the combined extracts on a steam bath under a gentle stream of nitrogen to dryness. Allow to cool to room temperature. Dissolve the residue in about 0.5 mL of chloroform and methanol (1:1), using a vortex mixer. Transfer the solution to a 2-mL volumetric flask with small portions of a mixture of chloroform and methanol (1:1), dilute with the mixture of chloroform and methanol (1:1) to volume, and mix.

Procedure— Apply 200-µL portions of the Assay preparation and the Standard preparation to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel. Allow the spots to dry, and develop the chromatogram, using the Developing solvent, until the solvent front has moved about 15 cm. Remove the plates from the developing chamber, and allow them to dry for about 15 minutes. Mark the betamethasone bands, using short-wavelength UV light, to include similar zones of silica gel for the Assay preparation, the Standard preparation, and a zone containing no betamethasone for the blank. Scrape off these zones, and transfer them to separate 50-mL centrifuge tubes. Add 15.0 mL of alcohol to each, and rotate for 20 minutes. Centrifuge to clarify. Transfer 10.0-mL portions of the supernatant to separate, stoppered tubes. To each tube add 1.0 mL of blue tetrazolium TS, followed by 1.0 mL of Tetramethylammonium hydroxide reagent, and mix. Heat in a 35 water bath for about 1 hour. Remove from the water bath, add 1.0 mL of glacial acetic acid to each tube, and mix. Cool to room temperature. Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 525 nm, with a suitable spectrophotometer. Calculate the quantity, in mg, of betamethasone (C22H29FO5) in each mL of the Oral Solution taken by the formula: in which C is the concentration, in mg per mL, of USP Betamethasone RS in the Standard preparation; V is the volume, in mL, of Oral Solution taken; and AU, AS, and AB are the absorbances of the solutions from the Assay preparation, the Standard preparation, and the blank, respectively.