Mobile phase— Prepare a filtered and degassed mixture of methanol,water,and acetonitrile (23:18:9).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Dissolve suitable quantities of USP Methyltestosterone RSin methanol to obtain a solution containing about 250µg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Betamethasone Benzoate RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.5mg per mL.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,add 10.0mLof Internal standard solution,dilute with methanol to volume,and mix.

Assay preparation— Transfer an accurately weighed portion of Gel,equivalent to about 0.5mg of betamethasone benzoate,to a 125-mLseparatory funnel,add 20mLof water and 2mLof saturated sodium acetate solution,shake to disperse the Gel,add 2.0mLof Internal standard solution,and mix.Extract this solution with one 50-mLportion of chloroform followed by three 40-mLportions of chloroform.Discard the aqueous layer.Wash the chloroform extract with 10mLof water,allow to stand for 10minutes,then pass through chloroform-wetted glass fiber filter paper and anhydrous sodium sulfate into a suitable container.Evaporate to dryness under vacuum at 30.Dissolve the residue in 10mLof methanol.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 236-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.The column temperature is maintained at 30.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 1.33for betamethasone benzoate and 1.0for methyltestosterone;the resolution,R,between methyltestosterone and betamethasone benzoate is not less than 3.0;and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 10µL)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 betamethasone benzoate (C29H33FO6)in the portion of Gel taken by the formula: in which Cis the concentration,in µg per mL,of USP Betamethasone Benzoate RSin the Standard preparation;and RUand RSare the ratios of the peak responses obtained for betamethasone benzoate and methyltestosterone from the Assay preparationand the Standard preparation,respectively.