A: Ultraviolet Absorption 197U—

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

Solvent— Use dimethyl sulfoxide.

(Official January 1, 2007)

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 Suitability under Chromatography 621).

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

System suitability solution— Dissolve 10 mg of USP Diethylstilbestrol RS in 50 mL of chloroform, and allow the solution to stand in the dark for not less than 5 hours. Pipet 5.0 mL of this solution into a 50-mL volumetric 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 Diluent to volume, and mix.

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

Chromatographic system (see Chromatography 621)— 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 1 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 1.00 for trans-diethylstilbestrol and 1.33 for 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 3000 theoretical 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 50 µL) of the Standard preparation and the Assay preparation into 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 C18H20O2 in the portion of Diethylstilbestrol taken by the formula: in which C is the concentration, in µg per mL, of USP Diethylstilbestrol RS in the Standard preparation; and rt, U and rt, S are the peak responses for the trans-isomer obtained from the Assay preparation and the Standard preparation, respectively; and rc, U and rc,S are the peak responses for the cis-isomer obtained from the Assay preparation and the Standard preparation, respectively.