Medium: 0.05MpH6.8phosphate buffer;900mL.

Apparatus 2: 75rpm.

Time: 30minutes.

Diluentand Mobile phase— Proceed as directed in theAssay.

System suitability solution— Transfer about 100mg of 4¢-nitroacetophenone,accurately weighed,to a 100-mLvolumetric flask,and dissolve in methanol,sonicating if necessary.Dilute with methanol to volume,and mix.Pipet 2mLof this solution into a 250-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution— Dissolve an accurately weighed quantity of USP Estropipate RSinDiluent,sonicating if necessary,and dilute quantitatively withDiluentto obtain a solution having a known concentration of about 1mg per mL.Pipet 2mLof this solution into a 100-mLvolumetric flask,dilute with water to volume,and mix.Pipet 4mLof the solution so obtained into a second 100-mLvolumetric flask,add 8.0mLof theSystem suitability solution,dilute with water to volume,and mix.

Test solution— Transfer an accurately measured volume of a filtered portion of the solution under test,equivalent to about 20µg of estropipate,to a 25-mLvolumetric flask.Dilute with water to volume if the contents are less than the nominal volume of the flask,and mix.

Chromatographic system(see Chromatography á621ñ)— Prepare as directed in theAssay.To evaluate the system suitability requirements,use theStandard solution.

Procedure— Separately inject equal volumes (about 300µL)of theStandard solutionand theTest solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity of C18H22O5S·C4H10N2dissolved.

Tolerances— Not less than 80%(Q)of the labeled amount of C18H22O5S·C4H10N2is dissolved in 30minutes.

Diluent,Mobile phase,System suitability solution,Standard preparation,and Chromatographic system— Prepare as directed in theAssay.

Test preparation— Transfer 1Tablet to a 50-mLvolumetric flask,add 20mLof water,insert the stopper,and shake by mechanical means for about 30minutes or until the Tablet disintegrates.Add 20mLof methanol,insert the stopper,and shake by mechanical means for about 60minutes.Dilute withDiluentto volume,and mix.Transfer an accurately measured volume of this stock solution,equivalent to about 0.25mg of estropipate,to a 25-mLvolumetric flask,dilute withDiluentto volume,and mix.Pass this solution through a solvent-resistant membrane filter having a 1-µm or finer porosity,discarding the first portion of the filtrate.

Procedure— Proceed as directed in theAssay,except to use theTest preparationinstead of theAssay preparation.

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

Mobile phase— Dissolve 13.6g of monobasic potassium phosphate in 1000mLof water,and mix.Mix 600mLof the resulting solution with 200mLof methanol,add 200mLof acetonitrile with stirring,and mix.Filter,and degas.Make adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Estropipate RSinDiluent,sonicating if necessary,and dilute quantitatively withDiluentto obtain a solution having a known concentration of about 1mg per mL.Pipet 1.0mLof this solution into a 100-mLvolumetric flask,dilute withDiluentto volume,and mix.

System suitability solution— Transfer about 100mg of 4¢-nitroacetophenone,accurately weighed,to a 100-mLvolumetric flask,and dissolve in methanol,sonicating if necessary.Dilute with methanol to volume,and mix.Pipet 5mLof this solution into a 200-mLvolumetric flask,dilute withDiluentto volume,and mix.Pipet 5mLof the solution so obtained into a 25-mLvolumetric flask,dilute withStandard preparationto volume,and mix.

Assay preparation— Transfer 20Tablets to a 1000-mLvolumetric flask,add 200mLof water,insert the stopper,and shake by mechanical means for about 30minutes or until the Tablets disintegrate completely.Add 200mLof methanol,insert the stopper,and shake by mechanical means for about 60minutes.Dilute withDiluentto volume,and mix.Transfer an accurately measured volume of this stock solution,equivalent to about 0.25mg of estropipate,to a 25-mLvolumetric flask,dilute withDiluentto volume,and mix.Pass this solution through a solvent-resistant membrane filter having a 1-µm or finer porosity,discarding the first portion of the filtrate.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 213-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph theSystem suitability solution,and record the peak responses as directed forProcedure:the relative retention times are about 0.85for 4¢-nitroacetophenone and 1.0for estropipate;the resolution,R,between estropipate and 4¢-nitroacetophenone is not less than 3.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of theStandard preparationand theAssay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of estropipate (C18H22O5S·C4H10N2)in each Tablet taken by the formula: in whichCis the concentration,in mg per mL,of USP Estropipate RSin theStandard preparation;Vis the volume,in mL,of the stock solution taken to prepare theAssay preparation;andrUandrSare the peak responses for estropipate obtained from theAssay preparationand theStandard preparation,respectively.