Conjugated Estrogens

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Conjugated Estrogens is a mixture of sodium estrone sulfate and sodium equilin sulfate, derived wholly or in part from equine urine or synthetically from estrone and equilin. It contains other conjugated estrogenic substances of the type excreted by pregnant mares. It is a dispersion of the estrogenic substances on a suitable powdered diluent.

Conjugated Estrogens contains NLT 52.5% and NMT 61.5% of sodium estrone sulfate and NLT 22.5% and NMT 30.5% of sodium equilin sulfate, and the total of sodium estrone sulfate and sodium equilin sulfate is NLT 79.5% and NMT 88.0% of the labeled content of Conjugated Estrogens. Conjugated Estrogens contains concomitant components as sodium sulfate conjugates NLT 13.5% and NMT 19.5% of 17α-dihydroequilin, NLT 2.5% and NMT 9.5% of 17α-estradiol, and NLT 0.5% and NMT 4.0% of 17ẞ-dihydroequilin of the labeled content of Conjugated Estrogens.

2 IDENTIFICATION

A. The relative retention times of the 17α-dihydroequilin peak, the estrone peak, and the equilin peak from the Sample solution correspond to those from the Standard solution, as obtained in the Assay.

B. The chromatogram of Conjugated Estrogens from the Sample solution in the Assay exhibits additional peaks or shoulders, corresponding to 17α-estradiol and 17ẞ-dihydroequilin at retention times of about 0.24 and 0.35, relative to that of 3-0-methylestrone.

3 ASSAY

3.1 PROCEDURE

Buffer: Sodium acetate TS, 1 N acetic acid, and water (79:21:400). Adjust with 1 N acetic acid or sodium acetate TS to a pH of 5.2 ± 0.1, if necessary.

Internal standard solution: 150 µg/mL of 3-0-methylestrone in methanol

Standard stock solution: 160 µg/mL, 70 µg/mL, and 50 µg/mL each of USP Estrone RS, USP Equilin RS, and USP 17α-Dihydroequilin RS, respectively, in alcohol

Standard solution: Pipet 1.0 mL of the Internal standard solution and 1.0 mL of the Standard stock 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 min. Add 0.5 mL of toluene, and mix.

System suitability solution: Pipet 1.0 mL of a 2.0-µg/mL solution of USP Estradiol RS (17ẞ-estradiol) in alcohol, 1.0 mL of Internal standard solution, and 1.0 mL of Standard stock solution into a centrifuge tube fitted with a tight screw cap or stopper. Proceed as directed for Standard solution, beginning with "Evaporate the mixture...".

Sample solution: Transfer a quantity of Conjugated Estrogens, equivalent to 2 mg of total conjugated estrogens, to a 50-ml centrifuge tube,

fitted with a polytef-lined screw cap, containing 15 mL of Buffer and 1 g of barium chloride. Cap the tube tightly, and shake for 30 min. If necessary, adjust the solution with 1 N acetic acid or sodium acetate to a pH of 5.0 ± 0.5. Sonicate for 30 s, then shake for an additional 30 min. Add a suitable sulfatase enzyme solution equivalent to 2500 Units, and shake for 20 min 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 min. Centrifuge for 10 min or until the lower layer is clear. Transfer as much of the organic phase as possible, and dry by rapidly passing through a filter consisting of a pledget of dry glass wool and 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 for Standard solution, beginning with "Evaporate the mixture...".

3.1.1 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 0.25 - mm * 15 - m fused silica capillary column, bonded with a 0.25-µm layer of phase G19

3.1.2 Temperature

Column: 208°

Injector port: 260"

Detector: 260*

Carrier gas: Hydrogen

Flow rate: 2 mL/min

Injection mode: Split

Split flow rate: 40-60 mL/min

Injection size: 1 µL

3.1.3 System suitability

Samples: Standard solution and System suitability solution

[NOTE-Adjust the operating conditions as necessary to maintain the elution time of the 3-0-methylestrone peak between 17 and 25 min.]

[NOTE-The relative retention times for 17ẞ-estradiol, 17α-dihydroequilin, estrone, equilin, and 3-0-methylestrone are 0.29, 0.30, 0.80, 0.87, and 1.00, respectively.]

Suitability requirements

Resolution: NLT 1.2 between estrone and equilin, System suitability solution

Tailing factor: NMT 1.3 for the estrone peak, System suitability solution

Relative standard deviation: NMT 2.0% for the estrone peak ratios for NLT four injections of the Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of sodium estrone sulfate and sodium equilin sulfate in the portion of Conjugated Estrogens taken:

                Result = (R_U/R_S) x (C_S/C_U) x F x 100

R_U = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution Ru

R_S = ratio of the peak response of the appropriate analyte to that of the internal standard from the Standard solution

C_S = concentration of USP Estrone RS or USP Equilin RS in the Standard solution (µg/mL)

C_U = concentration of the Sample solution (µg/mL)

F = conversion factor (ratio of molecular weight of sodium salts to free estrogen), 1.381

Acceptance criteria: 52.5%-61.5% of sodium estrone sulfate and 22.5%-30.5% of sodium equilin sulfate

4 OTHER COMPONENTS

4.1 CONTENT OF 17-DIHYDROEQUILIN, 17B-DIHYDROEQUILIN, AND 17-ESTRADIOL (concomitant components)

Buffer, Internal standard solution, Standard stock solution, Standard solution, System suitability solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

4.1.1 Analysis

Samples: Standard solution and Sample solution

[NOTE-The relative retention times for 17a-estradiol, 17a-dihydroequilin, and 178-dihydroequilin are about 0.82, 1.00, and 1.11, respectively.]

Identify the peaks for 17a-estradiol, 17a-dihydroequilin, and 178-dihydroequilin from the Sample solution.

Calculate the percentages of 17a-estradiol, 17a-dihydroequilin, and 17ß-dihydroequilin as their sodium sulfate salts in the portion of Conjugated Estrogens taken:

                Result = (R_U/R_S) x (C_S/C_U) x F x 100

R_U = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution Ru

R_S = ratio of the peak response of 17a-dihydroequilin to that of the internal standard from the Standard solution

C_S = concentration of USP 17α-Dihydroequilin RS in the Standard solution (µg/mL)

C_U = concentration of the Sample solution (µg/mL)

F = conversion factor (ratio of molecular weight of sodium salts to free estrogen), 1.381

Acceptance criteria: NLT 13.5% and NMT 19.5% of 17a-dihydroequilin, NLT 2.5% and NMT 9.5% of 17a-estradiol, and NLT 0.5% and NMT 4.0% of 178-dihydroequilin, as their sodium sulfate conjugates

5 IMPURITIES

5.1 LIMITS OF 17-DIHYDROEQUILENIN, 17ẞ-DIHYDROEQUILENIN, AND EQUILENIN (signal impurities)

Buffer, Internal standard solution, Standard stock solution, Standard solution, System suitability solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

5.1.1 Analysis

Samples: Standard solution and Sample solution

[NOTE-The relative retention times for dihydroequilenin, 17ẞ-dihydroequilenin, 3-0-methylestrone, and equilenin are 0.56, 0.64, 1.0, and 1.3, respectively.]

Identify any peaks for dihydroequilenin, 17ẞ-dihydroequilenin, 3-0-methylestrone, and equilenin from the Sample solution. Calculate the percentages of 17α-dihydroequilenin, 17ẞ-dihydroequilenin, and equilenin as their sodium sulfate salts in the portion of Conjugated

Estrogens taken:

                Result = (R_U/R_S) x (C_S/C_U) x F x 100

R_U = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution Ru

R_S = ratio of the peak response of estrone to that of the internal standard from the Standard solution

C_S = concentration of USP Estrone RS in the Standard solution (µg/mL)

C_U = concentration of the Sample solution (µg/mL)

F = conversion factor (ratio of molecular weight of sodium salts to free estrogen), 1.381

Acceptance criteria: NMT 3.25%, NMT 2.75%, and NMT 5.5% of the labeled content of Conjugated Estrogens for 17α-dihydroequilenin, 17ẞ-dihydroequilenin, and equilenin, respectively, as their sodium sulfate salts

5.2 LIMITS OF 17B-ESTRADIOL Δ8,9-DEHYDROESTRONE

Buffer, Internal standard solution, Standard stock solution, Standard solution, System suitability solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

5.2.1 Analysis

Samples: Standard solution and Sample solution

[NOTE-The relative retention times of 17ẞ-estradiol, 3-0-methylestrone, and A8.9-dehydroestrone are about 0.29, 1.0, and 0.9, respectively.]

Identify any peaks for 17ẞ-estradiol, 3-0-methylestrone, and A8.9-dehydroestrone from the Sample solution.

Calculate the percentages of 17ẞ-estradiol and A8.9-dehydroestrone as their sodium sulfate salts in the portion of Conjugated Estrogens taken:

                Result = (R_U/R_S) x (C_S/C_U) x F x 100

R_U = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution Ru

R_S = ratio of the peak response of estrone to that of the internal standard from the Standard solution

C_S = concentration of USP Estrone RS in the Standard solution (µg/mL) 

C_U = concentration of the Sample solution (µg/mL) 

F = conversion factor (ratio of molecular weight of sodium salts to free estrogen), 1.381

Acceptance criteria: NMT 2.25% and NMT 6.25% of the labeled content of Conjugated Estrogens for 178-estradiol and A8.9-dehydroestrone, respectively, as their sodium sulfate salts

5.3 LIMIT OF ESTRONE, EQUILIN, AND 170-DIHYDROEQUILIN (free steroids)

Buffer, Internal standard solution, Standard stock solution, System suitability solution, and Chromatographic system: Proceed as directed in the Assay.

Free steroids standard solution: Dilute the Standard 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 solution in the Assay, beginning with "Evaporate the mixture...".

Sample solution: Proceed as directed for Sample solution in the Assay with the following exceptions: do not add the sulfatase enzyme solution, and transfer 6.0 mL of the solution, instead of 3.0 mL, to the centrifuge tube.

Blank solution: Prepare a reagent blank in the same manner as the Sample solution.

System suitability: 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 NMT 5.5%, from NLT two replicate injections.

5.3.1 Analysis

Samples: Free steroids standard solution and Sample solution

Calculate the total percentage of estrone, equilin and 17α-dihydroequilin (free steroids) in the portion of Conjugated Estrogens taken:

                Result = (R_U/R_S) x (C_S/C_U) x F x 100

R_U = ratio of the sum of the estrone, equilin, and 17a-dihydroequilin peak areas (corrected for any peaks found in the Blank solution) to the internal standard peak area from the Sample solution Ru

R_S = ratio of the estrone peak area to the internal standard peak area from the Standard solution S

C_S = concentration of USP Estrone RS in the Standard solution (µg/mL)

C_U = concentration of the Sample solution (µg/mL)

Acceptance criteria: NMT 1.3% of free steroids

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in well-closed containers. Store at controlled room temperature, or under refrigeration.

LABELING: Label it to state the content of Conjugated Estrogens on a weight-to-weight basis.

USP REFERENCE STANDARDS (11)

USP 17-Dihydroequilin RS

Estra-1,3,5(10),7-tetraene-3,17a-diol.

C₁₈H₂₂O₂         270.37

USP Equilin RS

USP Estradiol RS

USP Estrone RS