Test solution— Extract a quantity of weighed Cream, equivalent to about 500 µg of flurandrenolide, as directed for the Assay preparation. Omit the addition of the internal standard, and evaporate the chloroform extracts on a steam bath under a stream of nitrogen to about 3 mL. Transfer the chloroform solution to a 10-mL flask, and evaporate with the aid of a stream of nitrogen to dryness. Dissolve the residue in 2 mL of chloroform.

Application volume: 4.0 µL.

Developing solvent system: a mixture of ethyl acetate and ethyl ether (70:30).

Methanolic sodium chloride— Transfer 100 mL of sodium chloride solution (1 in 10) to a 500-mL volumetric flask. Dilute with methanol to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (70:30). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Transfer about 10 mg of testosterone to a 100-mL volumetric flask, add methanol to volume, and mix.

Standard preparation— Transfer about 16 mg of USP Flurandrenolide RS, accurately weighed, to a 100-mL volumetric flask, add methanol to volume, and mix. Transfer 3.0 mL of this solution to a 10-mL volumetric flask, add 4.0 mL of Internal standard solution, dilute with water to volume, and mix to obtain a solution having a known concentration of about 48 µg of USP Flurandrenolide RS per mL.

Assay preparation— Transfer an accurately weighed quantity of Cream, equivalent to about 500 µg of flurandrenolide, to a 125-mL separator. Add 50 mL of hexanes and 25 mL of Methanolic sodium chloride, and shake until the Cream is thoroughly dispersed. Allow the phases to separate, and drain the lower aqueous phase into a second 125-mL separator containing 15 mL of hexanes. Shake vigorously, allow the phases to separate, and drain the lower aqueous phase into a 250-mL separator containing 75 mL of water. Serially extract the hexane phases remaining in the two 125-mL separators with two additional 25-mL portions of Methanolic sodium chloride, adding each aqueous phase to the 250-mL separator. Discard the hexane phases. Extract the combined aqueous phases with four 25-mL portions of chloroform. Filter each chloroform extract through 10 g of anhydrous sodium sulfate into a 125-mL conical beaker. Rinse the sodium sulfate with water-washed chloroform, and add the wash to the beaker. Add 4.0 mL of Internal standard solution to the beaker containing the chloroform extract. Evaporate the solution on a steam bath under a stream of nitrogen nearly to dryness. Remove the beaker from the steam bath, and evaporate the remaining solution with the aid of nitrogen to dryness. Add 10 mL of Mobile phase to the beaker, and place it in an ultrasonic bath to dissolve the residue. Pass the solution through a suitable filter having a 0.5-µm porosity and a prefilter above the membrane filter to prevent clogging.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 240-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 2 for testosterone and 1.0 for flurandrenolide; the resolution, R, between the analyte and internal standard is not less than 2.0; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 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 flurandrenolide (C24H33 FO6) in the portion of Cream taken by the formula: in which C is the concentration, in mg per mL, of USP Flurandrenolide RS in the Standard preparation; and RU and RS are the peak response ratios obtained from the Assay preparation and the Standard preparation, respectively.