Standard preparation— Dissolve an accurately weighed quantity of USP Diethylstilbestrol RSin alcohol,and dilute quantitatively and stepwise with alcohol to obtain a solution having a known concentration of about 20µg per mL.Mix 25.0mLof this solution with an equal volume of dibasic potassium phosphate solution (1in 55).

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to not less than 1mg and not more than 50mg of diethylstilbestrol,to a 125-mLseparator containing 75mLof isooctane.Extract the solution with one 20-mLand two 10-mLportions of 1Nsodium hydroxide.Wash the combined alkaline extracts with two 10-mLportions of chloroform.Transfer the alkaline solution to a 150-mLbeaker,and with the careful addition of 2.5Mphosphoric acid adjust the pHof the solution to 9.5.Transfer the adjusted solution,with the aid of a small volume of water,to a 125-mLseparator,and extract with four 20-mLportions of chloroform.Filter the extracts into a 100-mLvolumetric flask through a chloroform-wetted pledget of cotton,washing the filter with several small portions of chloroform to adjust the solution to volume.If necessary,dilute the solution quantitatively and stepwise with chloroform so that it contains about 10µg of diethylstilbestrol per mL.Transfer 20mLof this solution to a 50-mLconical flask,and evaporate with the aid of gentle heating in a current of air to about 5mL.Complete the evaporation of the solvent in the air current without further application of heat.Dissolve the residue in 10.0mLof alcohol,add 10.0mLof dibasic potassium phosphate solution (1in 55),and mix.

Procedure— [Caution—Protect the eyes from direct rays of UVlight throughout this procedure. ] Transfer 4mLof the Standard preparationto a stoppered,1-cm quartz cell,place about 5cm from a low-pressure,short-wave mercury lamp rated at from 2to 20watts,and irradiate for about 5minutes.Place the cell in the sample compartment of a spectrophotometer,and measure the absorbance at the wavelength of maximum absorbance at about 418nm,using water as the blank.Continue irradiation for successive 1-to 3-minute intervals,measuring at 418nm until the maximum absorbance (about 0.7)has been obtained.If necessary,adjust the geometry of the irradiation apparatus so as to obtain maximum,reproducible absorbance at 418nm.Similarly irradiate a 4-mLportion of the Assay preparation,recording the absorbance at 418nm,at successive short intervals until maximum absorbance is obtained.Concomitantly determine the absorbances of the Assay preparationand the Standard preparationin 1-cm cells at 418nm,using water as the blank,and subtract these values from those for the respective irradiated solutions to obtain the corrected maximum absorbances.Calculate the quantity,in µg,of diethylstilbestrol (C18H20O2)in each mLof the Assay preparationtaken by the formula: in which Cis the concentration,in µg per mL,of USP Diethylstilbestrol RSin the Standard preparation,and AUand ASare the corrected maximum absorbances of the irradiated Assay preparationand Standard preparation,respectively.