Arsenomolybdate spray reagent— Dissolve 25g of ammonium molybdate in 450mLof water,add 21mLof sulfuric acid,and mix.Add 3g of dibasic sodium arsenate heptahydrate dissolved in 25mLof water,mix,and incubate at 37±2for 24to 48hours.Store,protected from light.

Procedure— Transfer a portion of Vaginal Cream,equivalent to about 8mg of estropipate,to a container.Add 20mLof methanol,stir to obtain a homogeneous mixture,and pass through filter paper.Apply 30µLof the filtrate and 30µLof a Standard solution of USP Estropipate RSin methanol containing about 400µg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of a mixture of chloroform and methanol (10:8)until the solvent front has moved about 14cm from the origin.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the spots by lightly spraying with Arsenomolybdate spray reagentand by heating at 105for about 20minutes:the RFvalues of the principal spots obtained from the test solution correspond to those obtained from the Standard solution.

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

Resolution solution— Dissolve a suitable quantity of 4¢-nitroacetophenone in methanol to obtain a solution having a known concentration of about 1.0mg per mL.Transfer 5.0mLof the solution so obtained to a 200-mLvolumetric flask,dilute with Diluentto volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Estropipate RSin Diluent,and dilute quantitatively,and stepwise if necessary,with Diluentto obtain a solution having a known concentration of about 1.0mg per mL.Transfer 5.0mLof the solution so obtained to a 100-mLvolumetric flask,dilute with 0.05Mpiperazine to volume,and mix.Transfer 5.0mLof the solution so obtained to a 25-mLvolumetric flask,add 5.0mLof the Resolution solution,dilute with 0.05Mpiperazine to volume,mix,and filter.

Assay preparation— Transfer an accurately weighed portion of Vaginal Cream,equivalent to about 1.5mg of estropipate,to a 125-mLseparator,add 50mLof chloroform and 20mLof 0.05Mpiperazine,and extract for 3minutes.Collect the organic extract in another separator,and extract the aqueous layer with an additional 20-mLportion of chloroform.Combine the organic phases,wash with a 20-mLportion of 0.05Mpiperazine for 3minutes,and collect the aqueous washings.Discard the organic layer.Rinse the separators with 0.05Mpiperazine,and add the rinse to the aqueous washings.Transfer the aqueous extract and the aqueous washings to a 100-mLvolumetric flask,dilute with 0.05Mpiperazine to volume,and mix.Transfer 15.0mLof the solution so obtained to a 25-mLvolumetric flask,dilute with 0.05Mpiperazine to volume,mix,and filter.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 213-nm detector and 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper minute.[NOTE—The injection of a diluted solution of 4¢-nitroacetophenone can be used to better identify the peak locus.]Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.8for 4¢-nitroacetophenone and 1.0for estropipate;the resolution,R,between 4¢-nitroacetophenone and estropipate is not less 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 for not less than 30minutes for the Standard preparationand 90minutes for the Assay preparation,and measure the responses for the major peaks.Calculate the quantity,in mg,of estropipate (C18H22O5S·C4H10N2)in the portion of Vaginal Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Estropipate RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.