A:Ultraviolet Absorption á197Uñ—

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.

Solvent— Use dimethyl sulfoxide.

Diluent— Prepare a mixture of alcohol and water (1:1).

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (3:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Diethylstilbestrol RSin Diluent,and dilute quantitatively,and stepwise if necessary,with Diluentto obtain a solution having a known concentration of about 20µg per mL.

System suitability solution— Dissolve 10mg of USP Diethylstilbestrol RSin 50mLof chloroform,and allow the solution to stand in the dark for not less than 5hours.Pipet 5.0mLof this solution into a 50-mLvolumetric flask,and evaporate to dryness under a current of air.Dissolve the residue (the cis-and trans-isomers of diethylstilbestrol)in Diluent,sonicating if necessary.Dilute with Diluentto volume,and mix.

Assay preparation— Dissolve an accurately weighed quantity of Diethylstilbestrol in Diluent,and dilute quantitatively,and stepwise if necessary,with Diluentto obtain a solution having a concentration of about 20µg per mL.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 1.00for trans-diethylstilbestrol and 1.33for cis-diethylstilbestrol;and the resolution,R,between trans-diethylstilbestrol and cis-diethylstilbestrol is not less than 4.0.Chromatograph the Standard preparation,and record the peak responses for the trans-isomer as directed for Procedure:the column efficiency is not less than 3000theoretical plates;the tailing factor is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas of the peak responses for the cis-and trans-isomers of diethylstilbestrol.Calculate the quantity,in µg,of C18H20O2in the portion of Diethylstilbestrol taken by the formula: in which Cis the concentration,in µg per mL,of USP Diethylstilbestrol RSin the Standard preparation;and rt,Uand rt,Sare the peak responses for the trans-isomer obtained from the Assay preparationand the Standard preparation,respectively;and rc,Uand rc,Sare the peak responses for the cis-isomer obtained from the Assay preparationand the Standard preparation,respectively.