Mobile phase— Prepare a solution of acetonitrile in water containing approximately 30% (v/v) of acetonitrile.

Internal standard solution— Dissolve fluoxymesterone in isopropyl alcohol to obtain a solution having a concentration of about 50 µg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Triamcinolone Acetonide RS in Internal standard solution to obtain a solution having a known concentration of about 75 µg per mL. Mix an accurately measured volume of the resulting solution with an equal volume of Mobile phase to obtain a Standard preparation containing about 37.5 µg of USP Triamcinolone Acetonide RS per mL.

Assay preparation— Transfer an accurately weighed quantity of Cream, equivalent to about 1.5 mg of triamcinolone acetonide, to a screw-cap tube. Add 20.0 mL of Internal standard solution, and cap securely. Heat for 5 minutes at 60, then swirl vigorously for not less than 30 seconds. Repeat the heating and swirling sequence three times. Cool in a methanol-ice bath for 15 to 20 minutes, then centrifuge for 15 minutes at 5. Dilute an accurately measured volume of the supernatant with an equal volume of Mobile phase. Cool in a methanol-ice bath for 10 to 15 minutes, with occasional agitation. Filter first through a pledget of glass wool or a prefilter disk and then through a 0.45-µm porosity membrane to obtain a clear solution.

Procedure— Introduce equal volumes (between 15 and 25 µL) of the Assay preparation and the Standard preparation into a high-pressure liquid chromatograph (see Chromatography 621), operated at room temperature, by means of a suitable microsyringe or sampling valve. Adjust the operating parameters with Mobile phase on the column, such that the separation of triamcinolone acetonide and internal standard is optimized, with a retention time of about 14.5 minutes for triamcinolone acetonide. Typically, the apparatus is fitted with a 30-cm × 4-mm column containing packing L1, and is equipped with a UV detector capable of monitoring absorbance at 254 nm, and a suitable recorder. In a suitable chromatogram, the coefficient of variation for five replicate injections of a single specimen is not more than 3.0%, and the resolution factor, R (see Chromatography 621), between the peaks for triamcinolone acetonide and fluoxymesterone is not less than 2.0. Measure the heights of the internal standard and triamcinolone acetonide peaks, at the same retention times obtained from the Assay preparation and the Standard preparation. Calculate the quantity, in mg, of C24H31FO6 in the portion of Cream taken by the formula: in which C is the concentration, in mg per mL, of USP Triamcinolone Acetonide RS in the Standard preparation, and RU and RS are the ratios of the peak heights of triamcinolone acetonide to the internal standard obtained from the Assay preparation and the Standard preparation, respectively.