Test solution— Extract a quantity of weighed Cream,equivalent to about 500µg 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 3mL.Transfer the chloroform solution to a 10-mLflask,and evaporate with the aid of a stream of nitrogen to dryness.Dissolve the residue in 2mLof chloroform.

Application volume: 4.0µL.

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

Methanolic sodium chloride— Transfer 100mLof sodium chloride solution (1in 10)to a 500-mLvolumetric 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 Suitabilityunder Chromatography á621ñ).

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

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

Assay preparation— Transfer an accurately weighed quantity of Cream,equivalent to about 500µg of flurandrenolide,to a 125-mLseparator.Add 50mLof hexanes and 25mLof 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-mLseparator containing 15mLof hexanes.Shake vigorously,allow the phases to separate,and drain the lower aqueous phase into a 250-mLseparator containing 75mLof water.Serially extract the hexane phases remaining in the two 125-mLseparators with two additional 25-mLportions of Methanolic sodium chloride,adding each aqueous phase to the 250-mLseparator.Discard the hexane phases.Extract the combined aqueous phases with four 25-mLportions of chloroform.Filter each chloroform extract through 10g of anhydrous sodium sulfate into a 125-mLconical beaker.Rinse the sodium sulfate with water-washed chloroform,and add the wash to the beaker.Add 4.0mLof Internal standard solutionto 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 10mLof Mobile phaseto 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 á621ñ)— 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 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 2for testosterone and 1.0for 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 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of flurandrenolide (C24H33FO6)in the portion of Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Flurandrenolide RSin the Standard preparation;and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.