Internal standard solution— Dissolve fluoxymesterone in acetonitrile to obtain a solution containing about 100µg per mL.

Mobile solvent— Prepare a solution containing butyl chloride,water-saturated butyl chloride,tetrahydrofuran,methanol,and glacial acetic acid (95:95:14:7:6).

Standard preparation— Dissolve a suitable quantity of USP Fluorometholone RS,accurately weighed,in Internal standard solutionto obtain a solution having a known concentration of about 50µg per mL.

Assay preparation— Transfer an accurately weighed quantity of Cream,equivalent to about 1mg of fluorometholone,into a suitable container,add 20.0mLof Internal standard solution,and mix.

Procedure— Treat 20.0mLeach of the Standard preparationand the Assay preparationin the following manner.To each add 10.0mLof hexane,and shake for about 15minutes,then allow the layers to separate,and centrifuge,if necessary.Using a suitable microsyringe or sampling valve,inject equal volumes of lower (acetonitrile)layers obtained from the Standard preparationand the Assay preparationinto a suitable high-pressure liquid chromatograph (see Chromatography á621ñ)of the general type equipped with a detector for monitoring UVlight absorption at about 254nm,equipped with a suitable recorder,capable of providing column pressure up to about 1000psi.The instrument contains a 4-mm ×30-cm stainless steel column that contains packing L3.In a suitable chromatogram,the resolution factor,R(see Chromatography á621ñ),is not less than 2.4between peaks for fluorometholone and the internal standard,and the lowest and highest peak area ratios (RS)of three replicate injections of the Standard preparationagree within 2.0%.Calculate the quantity,in mg,of fluorometholone (C22H29FO4)in the portion of Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Fluorometholone RSin the Standard preparation;and RUand RSare the peak area ratios of the fluorometholone peak and the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.