A: The chromatogram exhibits peaks for 17-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 17-estradiol and 17-dihydroequilin at retention times of about 0.24 and 0.35, respectively, relative to that of 3-O-methylestrone.

Internal standard solution, Stock solution, pH 5.2 Acetate buffer, System suitability solution, Standard preparation, and Chromatographic system— Proceed as directed in the Assay.

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

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

Internal standard solution, Stock solution, pH 5.2 Acetate buffer, System suitability solution, Standard preparation, and Chromatographic system— Proceed as directed in the Assay.

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

Procedure— Separately inject equal volumes (about 1 µL) of the Standard preparation and the Test preparation into the chromatograph, record the chromatograms, and identify any peaks due to dihydroequilenin, 17-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 17-dihydroequilenin, 17-dihydroequilenin, and equilenin as their sodium sulfate salts in the portion of Conjugated Estrogens taken by the formula: in which CS is the concentration, in µg per mL, of USP Estrone RS in the Stock solution; RU is the ratio of the peak response of the appropriate analyte to that of the internal standard obtained from the Test preparation; and RS is the ratio of the peak response of estrone to that of the internal standard obtained from the Standard preparation. The limits of 17-dihydroequilenin, 17-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, pH 5.2 Acetate buffer, System suitability solution, Standard preparation, and Chromatographic system— Proceed as directed in the Assay.

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

Procedure— Separately inject equal volumes (about 1 µL) of the Standard preparation and the Test preparation into the chromatograph, record the chromatograms, and identify any peaks due to 17-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 17-estradiol and D8,9-dehydroestrone as their sodium sulfate salts in the portion of Conjugated Estrogens taken by the formula: in which CS is the concentration, in µg per mL, of USP Estrone RS in the Stock solution; RU is the ratio of the peak response of the appropriate analyte to that of the internal standard obtained from the Test preparation; and RS is the ratio of the peak response of estrone to that of the internal standard obtained from the Standard preparation. The limits of 17-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, pH 5.2 Acetate buffer, Stock solution, and System suitability solution— Proceed as directed in the Assay.

Free steroids standard solution— Dilute the Stock solution tenfold. Pipet 1.0 mL of the resulting solution and 1.0 mL of the Internal standard solution into a suitable centrifuge tube fitted with a tight screw cap or stopper. Proceed as directed for Standard preparation in the Assay, beginning with “Evaporate the mixture.”

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

Chromatographic system— Proceed as directed in the Assay with 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 solution is not greater than 5.5%, on the basis of not less than two replicate injections.

Procedure— Separately inject equal volumes (about 1 µL) of the Free steroids standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the ratio, RU, of the combined peak areas of estrone, equilin, and 17-dihydroequilin relative to the area of the internal standard in the Test solution, correcting for any reagent blank peaks. The ratio, RU / RS, where RS is 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).

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

Stock solution— Using accurately weighed quantities of USP Estrone RS, USP Equilin RS, and USP 17-Dihydroequilin RS, prepare, by quantitative and stepwise dilution, a solution in alcohol having known concentrations of about 160, 70, and 50 µg per mL, respectively.

pH 5.2 Acetate buffer— Mix 79 mL of sodium acetate TS with 21 mL of 1 N acetic acid, dilute with water to 500 mL, and mix. Adjust to a pH of 5.2 ± 0.1 by the addition of 1 N acetic acid or sodium acetate TS, if necessary.

System suitability solution— Dissolve a quantity of USP Estradiol RS (17-estradiol) in alcohol to obtain a solution containing about 2 µg per mL. Pipet 1.0 mL of this solution, 1.0 mL of Stock solution, and 1.0 mL of Internal standard solution into 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 1.0 mL of the Stock solution and 1.0 mL of Internal standard solution into 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 15 µL of dried pyridine and 65 µL of bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. Immediately cover the tube tightly, mix, and allow to stand for 15 minutes. Add 0.5 mL of toluene, and mix.

Assay preparation— Transfer an accurately weighed quantity of Conjugated Estrogens, equivalent to about 2 mg of total conjugated estrogens, to a 50-mL centrifuge tube, fitted with a polytef-lined screw cap, containing 15 mL of pH 5.2 Acetate buffer and 1 g of barium chloride. Cap the tube tightly, and shake for 30 minutes. If necessary, adjust the solution with 1 N acetic acid or sodium acetate to a pH of 5.0 ± 0.5. Place in a sonic bath for 30 seconds, then shake for an additional 30 minutes. Add a suitable sulfatase enzyme preparation equivalent to 2500 Units, and shake for 20 minutes in a water bath maintained at 50. Add 15.0 mL of ethylene dichloride to the warm mixture, cap the tube again, and shake by mechanical means for 15 minutes. Centrifuge for 10 minutes 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 5 g of anhydrous sodium sulfate in a small funnel. Protect from loss by evaporation. Transfer 3.0 mL of the solution to a suitable centrifuge tube fitted with a tight screw cap or stopper. Add 1.0 mL of Internal standard solution. Proceed as directed under Standard preparation, beginning with “Evaporate the mixture.”

Chromatographic system (see Chromatography 621)— 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 220 and the injection port at 260. The carrier gas is hydrogen flowing at the rate of 2 mL per minute, and the split flow rate is 40 to 60 mL per minute. Inject about 1 µL of the System suitability solution into the gas chromatograph. Adjust the operating conditions as necessary to maintain the elution time of the 3-O-methylestrone peak at between 17 and 25 minutes. The relative retention times are about 0.29, 0.30, 0.80, 0.87, and 1.00 for 17-estradiol, 17-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 1 µL) of the Standard preparation and the Assay preparation into 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 1.381 is the factor converting free estrogen to the conjugate sodium salt; CS is the concentration, in µg per mL, of USP Estrone RS or USP Equilin RS in the Stock solution; and RU and RS are the ratios of the peak response of the appropriate analyte to that of the internal standard obtained from the Assay preparation and the Standard preparation, respectively.