Methanolic sodium chloride ,Mobile phase,and Chromatographic system—Prepare as directed in the Assayunder Flurandrenolide Cream.

Flurandrenolide standard solution— Dissolve about 7mg of USP Flurandrenolide RS,accurately weighed,in 50mLof methanol in a 100-mLvolumetric flask.Dilute with methanol to volume,and mix.

Internal standard solution— Dissolve about 4mg of Testosterone in 50mLof methanol in a 100-mLvolumetric flask.Dilute with methanol to volume,and mix.

Standard preparation— Pipet 3.0mLof Flurandrenolide standard solutionand 4.0mLof Internal standard solutioninto a 10-mLvolumetric flask.Dilute with water to volume,and mix.

Assay preparation— Accurately measure and cut a portion of Tape,equivalent to about 200µg of flurandrenolide.Remove and discard the paper liner from the portion of Tape.Touch the flattened end of a glass rod to the adhesive side of the Tape,and carefully transfer the tape to the bottom of a 600-mLbeaker containing 15mLof anhydrous methanol,taking care that the adhesive side of the tape does not adhere to the wall of the beaker.Remove the glass rod from the tape,and wash it with 5mLof anhydrous methanol,adding the wash to the beaker.Place the beaker containing the Tape and the methanol in an ultrasonic bath for 3minutes,rotating the beaker in such manner that the methanol is in contact with all portions of the Tape.Transfer the methanol to a 250-mLseparator.Extract the Tape,using sonication,with two additional 20-mLportions of anhydrous methanol,adding each portion to the separator.To the combined methanol extract add 15mLof sodium chloride solution (1in 10)and 50mLof hexane,and shake vigorously.Allow the phases to separate,and drain the lower aqueous phase into a second separator containing 15mLof hexane.Shake vigorously,allow the phases to separate,and drain the lower phase into a third 250-mLseparator containing 100mLof water.Serially extract the hexane phases remaining in the two separators with one 25-mLportion of Methanolic sodium chloride,adding the extract to the third separator.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.Evaporate the solution on a steam bath under a stream of nitrogen to near dryness.Remove the beaker from the steam bath and evaporate the remaining solution with the aid of nitrogen to dryness.Add 10mLof Mobile solventto the beaker,and place it in an ultrasonic bath to dissolve the residue.Pass the solution through a suitable porosity filter having a 0.5-µm filter with a prefilter above the membrane filter to prevent clogging.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 2for testosterone and 1.0for flurandrenolide.Calculate the quantity,in µg,of C24H33FO6in the portion of Tape taken by the formula: in which Cis the concentration,in µg 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.