A: The chromatogram exhibits peaks for 17a-dihydroequilin,estrone,and equilin at relative retention times corresponding to those exhibited in the chromatogram of the Standard preparation.

B: The chromatogram of Conjugated Estrogens exhibits additional peaks or shoulders,corresponding to 17a-estradiol and 17b-dihydroequilin at retention times of about 0.24and 0.35,respectively,relative to that of 3-O-methylestrone.

Internal standard solution,Stock solution,pH5.2Acetate buffer,System suitability solution,Standard preparation,and Chromatographic system— Proceed as directed in the Assay.

Test preparation— Prepare as directed for Assay preparationin the Assay.

Procedure— Separately inject equal volumes (about 1µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and identify the peaks due to 17a-estradiol,17a-dihydroequilin,and 17b-dihydroequilin in the chromatogram of the Test preparation.The relative retention times relative to 17a-dihydroequilin are about 0.82,1.00,and 1.11for 17a-estradiol,17a-dihydroequilin,and 17b-dihydroequilin,respectively.Separately calculate the quantities,in mg,of 17a-estradiol,17a-dihydroequilin,and 17b-dihydroequilin as their sodium sulfate salts in the portion of Conjugated Estrogens taken by the formula: in which CSis the concentration,in µg per mL,of USP17a-Dihydroequilin RSin the Stock solution;RUis the ratio of the peak response of the appropriate analyte to that of the internal standard obtained from the Test preparation;and RSis the ratio of the peak response of 17a-dihydroequilin to that of the internal standard obtained from the Standard preparation.

Internal standard solution,Stock solution,pH5.2Acetate buffer,System suitability solution,Standard preparation,and Chromatographic system— Proceed as directed in the Assay.

Test preparation— Prepare as directed for Assay preparationin the Assay.

Procedure— Separately inject equal volumes (about 1µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and identify any peaks due to dihydroequilenin,17b-dihydroequilenin,3-O-methylestrone,and equilenin in the chromatogram of the Assay preparation.The relative retention times for these peaks are about 0.56,0.64,1.0,and 1.3,respectively.Separately calculate the quantities,in mg,of 17a-dihydroequilenin,17b-dihydroequilenin,and equilenin as their sodium sulfate salts in the portion of Conjugated Estrogens taken by the formula: in which CSis the concentration,in µg per mL,of USP Estrone RSin the Stock solution;RUis the ratio of the peak response of the appropriate analyte to that of the internal standard obtained from the Test preparation;and RSis the ratio of the peak response of estrone to that of the internal standard obtained from the Standard preparation.The limits of 17a-dihydroequilenin,17b-dihydroequilenin,and equilenin as their sodium sulfate salts are not more than 3.25%,2.75%,and 5.5%,respectively,of the labeled content of Conjugated Estrogens.

Internal standard solution,Stock solution,pH5.2Acetate buffer,System suitability solution,Standard preparation,and Chromatographic system— Proceed as directed in the Assay.

Test preparation— Prepare as directed for Assay preparationin the Assay.

Procedure— Separately inject equal volumes (about 1µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and identify any peaks due to 17b-estradiol,3-O-methylestrone,and D8,9-dehydroestrone in the chromatogram of the Test preparation.The relative retention times of these peaks are about 0.29,1.0,and 0.9,respectively,relative to the internal standard.Separately calculate the quantities,in mg,of 17b-estradiol and D8,9-dehydroestrone as their sodium sulfate salts in the portion of Conjugated Estrogens taken by the formula: in which CSis the concentration,in µg per mL,of USP Estrone RSin the Stock solution;RUis the ratio of the peak response of the appropriate analyte to that of the internal standard obtained from the Test preparation;and RSis the ratio of the peak response of estrone to that of the internal standard obtained from the Standard preparation.The limits of 17b-estradiol and D8,9-dehydroestrone as their sodium sulfate salts are not more than 2.25%and 6.25%,respectively,of the labeled content of Conjugated Estrogens.

Internal standard solution,pH5.2Acetate buffer,Stock solution,and System suitability solution— Proceed as directed in the Assay.

Free steroids standard solution— Dilute the Stock solutiontenfold.Pipet 1.0mLof the resulting solution and 1.0mLof the Internal standard solutioninto a suitable centrifuge tube fitted with a tight screw cap or stopper.Proceed as directed for Standard preparationin the Assay,beginning with “Evaporate the mixture.”

Test solution— Proceed as directed for Assay preparationin the Assaywith the following exceptions:do not add the sulfatase enzyme preparation,and transfer 6.0mLof the filtrate instead of 3.0mLin the preparation of the test specimen.Prepare a reagent blank in the same manner.

Chromatographic system— Proceed as directed in the Assaywith the additional requirement that the relative standard deviation for the ratio of the peak response of estrone to that of the internal standard in the Free steroids standard solutionis not greater than 5.5%,on the basis of not less than two replicate injections.

Procedure— Separately inject equal volumes (about 1µL)of the Free steroids standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the ratio,RU,of the combined peak areas of estrone,equilin,and 17a-dihydroequilin relative to the area of the internal standard in the Test solution,correcting for any reagent blank peaks.The ratio,RU/RS,where RSis the peak response ratio of estrone to that of the internal standard obtained from the Free steroids standard solution,is not more than 0.65(1.3%of free steroids).

Solvent— Use dimethyl sulfoxide.

Internal standard solution— Prepare a solution of 3-O-methylestrone in methanol containing about 150µg per mL.

Stock solution— Using accurately weighed quantities of USP Estrone RS,USP Equilin RS,and USP17a-Dihydroequilin RS,prepare,by quantitative and stepwise dilution,a solution in alcohol having known concentrations of about 160,70,and 50µg per mL,respectively.

pH5.2Acetate buffer— Mix 79mLof sodium acetate TSwith 21mLof 1Nacetic acid,dilute with water to 500mL,and mix.Adjust to a pHof 5.2±0.1by the addition of 1Nacetic acid or sodium acetate TS,if necessary.

System suitability solution— Dissolve a quantity of USP Estradiol RS(17b-estradiol)in alcohol to obtain a solution containing about 2µg per mL.Pipet 1.0mLof this solution,1.0mLof Stock solution,and 1.0mLof Internal standard solutioninto a centrifuge tube fitted with a tight screw cap or stopper.Proceed as directed for Standard preparation,beginning with “Evaporate the mixture.”

Standard preparation— Pipet 1.0mLof the Stock solutionand 1.0mLof Internal standard solutioninto a suitable centrifuge tube fitted with a tight screw cap or stopper.Evaporate the mixture with the aid of a stream of nitrogen to dryness,maintaining the temperature below 50.To the dry residue add 15µLof dried pyridine and 65µLof bis(trimethylsilyl)trifluoroacetamide containing 1%trimethylchlorosilane.Immediately cover the tube tightly,mix,and allow to stand for 15minutes.Add 0.5mLof toluene,and mix.

Assay preparation— Transfer an accurately weighed quantity of Conjugated Estrogens,equivalent to about 2mg of total conjugated estrogens,to a 50-mLcentrifuge tube,fitted with a polytef-lined screw cap,containing 15mLof pH5.2Acetate bufferand 1g of barium chloride.Cap the tube tightly,and shake for 30minutes.If necessary,adjust the solution with 1Nacetic acid or sodium acetate to a pHof 5.0±0.5.Place in a sonic bath for 30seconds,then shake for an additional 30minutes.Add a suitable sulfatase enzyme preparation equivalent to 2500Units,and shake for 20minutes in a water bath maintained at 50.Add 15.0mLof ethylene dichloride to the warm mixture,cap the tube again,and shake by mechanical means for 15minutes.Centrifuge for 10minutes or until the lower layer is clear.Transfer as much of the organic phase as possible,and dry by filtering rapidly through a filter consisting of a pledget of dry glass wool and about 5g of anhydrous sodium sulfate in a small funnel.Protect from loss by evaporation.Transfer 3.0mLof the solution to a suitable centrifuge tube fitted with a tight screw cap or stopper.Add 1.0mLof Internal standard solution.Proceed as directed under Standard preparation,beginning with “Evaporate the mixture.”

Chromatographic system(see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector maintained at a temperature of 260,a 0.25-mm ×15-m fused silica capillary column bonded with a 0.25-µm layer of phase G19,and a split injection system.The column temperature is maintained at 220and the injection port at 260.The carrier gas is hydrogen flowing at the rate of 2mLper minute,and the split flow rate is 40to 60mLper minute.Inject about 1µLof the System suitability solutioninto the gas chromatograph.Adjust the operating conditions as necessary to maintain the elution time of the 3-O-methylestrone peak at between 17and 25minutes.The relative retention times are about 0.29,0.30,0.80,0.87,and 1.00for 17b-estradiol,17a-dihydroequilin,estrone,equilin,and 3-O-methylestrone,respectively.The tailing factor for the estrone peak is not more than 1.3;the resolution,R,between estrone and equilin is not less than 1.2;and the relative standard deviation of the estrone peak ratios is not greater than 2.0%for not fewer than four injections of the Standard preparation.

Procedure— Separately inject equal volumes (about 1µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Separately calculate the quantities,in mg,of sodium estrone sulfate and sodium equilin sulfate in the portion of Conjugated Estrogens taken by the formula: in which 1.381is the factor converting free estrogen to the conjugate sodium salt;CSis the concentration,in µg per mL,of USP Estrone RSor USP Equilin RSin the Stock solution;and RUand RSare the ratios of the peak response of the appropriate analyte to that of the internal standard obtained from the Assay preparationand the Standard preparation,respectively.