Mobile phase— Prepare a suitable degassed solution of methanol (about 3in 5)such that the retention time of dienestrol is about 8to 10minutes and that of methyltestosterone is about 11to 14minutes.Make adjustments,if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Dissolve methyltestosterone in methanol to obtain a solution having a concentration of about 125µg per mL.

Standard preparation— Accurately weigh a suitable quantity of USP Dienestrol RS,dissolve in methanol,and dilute quantitatively and stepwise with methanol to obtain a concentration of about 50µg per mL.Pipet 10mLof this solution,5mLof Internal standard solution,and 5mLof water to a 50-mLvolumetric flask,dilute with methanol to volume,and mix.The concentration of dienestrol in the Standard preparationis about 10µg per mL.

Assay preparation— Pipet 20mLof methanol and 5mLof Internal standard solutioninto a 50-mLscrew-capped tube.Using a 5-mLplastic syringe,transfer an accurately weighed quantity of Cream,equivalent to about 0.5mg of dienestrol,to the tube.Cap the tube,and disperse the mixture with a suitable vibrating mixer at high speed for 3minutes,then in an ultrasonic bath for 5minutes.Add 20mLof methanol,continue shaking by mechanical means for another 10minutes,then chill in an ice bath for 5minutes.Filter the mixture through paper,discarding the first 5mLof the filtrate.

Procedure— Introduce equal volumes (about 50µL)of the Assay preparationand the Standard preparationinto a high-pressure liquid chromatograph operated at room temperature,by means of a suitable microsyringe or sampling valve.Typically,the apparatus is fitted with a 25-cm ×4.6-mm column containing packing L1and equipped with an UVdetector capable of monitoring absorption at 254nm and a suitable recorder.The Mobile phaseis maintained at a flow rate of about 2mLper minute.In a suitable chromatographic system,six replicate injections of the Standard preparationshow a relative standard deviation of not more than 2.0%and a resolution factor of not less than 2.0between the peaks for dienestrol and the internal standard.Calculate the quantity,in mg,of dienestrol (C18H18O2)in the portion of Cream taken by the formula: in which Cis the concentration,in µg per mL,of USP Dienestrol RSin the Standard preparation,and RUand RSare the peak area ratios of dienestrol to methyltestosterone obtained from the Assay preparationand the Standard preparation,respectively.