Conjugated Estrogens
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 as concomitant components as sodium sulfate conjugates NLT 13.5% and NMT 19.5% of 17
-dihydroequilin, NLT 2.5% and NMT 9.5% of 17-estradiol, and NLT 0.5% and NMT 4.0% of 17
-dihydroequilin of the labeled content of Conjugated Estrogens.
-dihydroequilin, NLT 2.5% and NMT 9.5% of 17-estradiol, and NLT 0.5% and NMT 4.0% of 17-dihydroequilin of the labeled content of Conjugated Estrogens.IDENTIFICATION
• A.
The relative retention times of the 17-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.
-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 17-estradiol and 17-dihydroequilin at retention times of about 0.24 and 0.35, relative to that of 3-O-methylestrone.
-estradiol and 17-dihydroequilin at retention times of about 0.24 and 0.35, relative to that of 3-O-methylestrone.
ASSAY
• Procedure
Solution A:
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-O-methylestrone in methanol
Stock solution:
160 µg/mL, 70 µg/mL, and 50 µg/mL each of USP Estrone RS, USP Equilin RS, and USP 17-Dihydroequilin RS, respectively, in alcohol
-Dihydroequilin RS, respectively, in alcohol
Standard solution:
Pipet 1.0 mL of the Internal standard solution and 1.0 mL of the 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.
. 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 (17-estradiol) in alcohol, 1.0 mL of Internal standard solution, and 1.0 mL of 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…”.
-estradiol) in alcohol, 1.0 mL of Internal standard solution, and 1.0 mL of 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 Solution A 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. Place in a sonic bath 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…”.
. 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…”.
Chromatographic system
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 and a split injection system
Temperature
Column:
208
Injector port:
260
Detector:
260
Carrier gas:
Hydrogen
Flow rate:
2 mL/min
Split flow rate:
40–60 mL/min
Injection size:
1 µL
System suitability
Samples:
Standard solution and System suitability solution
[Note—Adjust the operating conditions as necessary to maintain the elution time of the 3-O-methylestrone peak between 17 and 25 min. ]
[Note—The relative retention times for 17-estradiol, 17-dihydroequilin, estrone, equilin, and 3-O-methylestrone are 0.29, 0.30, 0.80, 0.87, and 1.00, respectively. ]
-estradiol, 17-dihydroequilin, estrone, equilin, and 3-O-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 = (RU/RS) × (CS/CU) × F × 100
| RU | = | = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution |
| RS | = | = ratio of the peak response of the appropriate analyte to that of the internal standard from the Standard solution |
| CS | = | = concentration of USP Estrone RS or USP Equilin RS in the Standard solution (µg/mL) |
| CU | = | = 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
OTHER COMPONENTS
• Content of 17-Dihydroequilin, 17-Dihydroequilin, and 17-Estradiol (concomitant components)
-Dihydroequilin, 17-Dihydroequilin, and 17-Estradiol (concomitant components)
Solution A, Internal standard solution, Stock solution, Standard solution, System suitability solution, Sample solution, and Chromatographic system:
Proceed as directed in the Assay.
[Note—The relative retention times for 17-estradiol, 17-dihydroequilin, and 17-dihydroequilin are about 0.82, 1.00, and 1.11, respectively. ]
-estradiol, 17-dihydroequilin, and 17-dihydroequilin are about 0.82, 1.00, and 1.11, respectively. ]
Analysis
Samples:
Standard solution and Sample solution
Identify the peaks for 17-estradiol, 17-dihydroequilin, and 17-dihydroequilin from the Sample solution. Calculate the percentages of 17-estradiol, 17-dihydroequilin, and 17-dihydroequilin as their sodium sulfate salts in the portion of Conjugated Estrogens taken:
-estradiol, 17-dihydroequilin, and 17-dihydroequilin from the Sample solution. Calculate the percentages of 17-estradiol, 17-dihydroequilin, and 17-dihydroequilin as their sodium sulfate salts in the portion of Conjugated Estrogens taken: Result = (RU/RS) × (CS/CU) × F × 100
| RU | = | = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution |
| RS | = | = ratio of the peak response of 17-dihydroequilin to that of the internal standard from the Standard solution |
| CS | = | = concentration of USP 17-Dihydroequilin RS in the Standard solution (µg/mL) |
| CU | = | = concentration of the Sample solution (µg/mL) |
| F | = | = conversion factor (ratio of molecular weight of sodium salts to free estrogen), 1.381 |
IMPURITIES
Organic Impurities
• Procedure 1: Limits of 17-Dihydroequilenin, 17-Dihydroequilenin, and Equilenin (signal impurities)
-Dihydroequilenin, 17-Dihydroequilenin, and Equilenin (signal impurities)
Solution A, Internal standard solution, Stock solution, Standard solution, System suitability solution, Sample solution, and Chromatographic system:
Proceed as directed in the Assay.
[Note—The relative retention times for dihydroequilenin, 17-dihydroequilenin, 3-O-methylestrone, and equilenin are 0.56, 0.64, 1.0, and 1.3, respectively. ]
-dihydroequilenin, 3-O-methylestrone, and equilenin are 0.56, 0.64, 1.0, and 1.3, respectively. ]
Analysis
Samples:
Standard solution and Sample solution
Identify any peaks for dihydroequilenin, 17-dihydroequilenin, 3-O-methylestrone, and equilenin from the Sample solution. Calculate the percentages of 17-dihydroequilenin, 17-dihydroequilenin, and equilenin as their sodium sulfate salts in the portion of Conjugated Estrogens taken:
-dihydroequilenin, 3-O-methylestrone, and equilenin from the Sample solution. Calculate the percentages of 17-dihydroequilenin, 17-dihydroequilenin, and equilenin as their sodium sulfate salts in the portion of Conjugated Estrogens taken: Result = (RU/RS) × (CS/CU) × F × 100
| RU | = | = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution |
| RS | = | = ratio of the peak response of estrone to that of the internal standard from the Standard solution |
| CS | = | = concentration of USP Estrone RS in the Standard solution (µg/mL) |
| CU | = | = 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 17-dihydroequilenin, 17-dihydroequilenin, and equilenin, respectively, as their sodium sulfate salts.
-dihydroequilenin, 17-dihydroequilenin, and equilenin, respectively, as their sodium sulfate salts.
• Procedure 2: Limits of 17-Estradiol and D8,9-Dehydroestrone
-Estradiol and D8,9-Dehydroestrone
Solution A, Internal standard solution, Stock solution, Standard solution, System suitability solution, Sample solution, and Chromatographic system:
Proceed as directed in the Assay.
[Note—The relative retention times of 17-estradiol, 3-O-methylestrone, and D8,9-dehydroestrone are about 0.29, 1.0, and 0.9, respectively. ]
-estradiol, 3-O-methylestrone, and D8,9-dehydroestrone are about 0.29, 1.0, and 0.9, respectively. ]
Analysis
Samples:
Standard solution and Sample solution
Identify any peaks for 17-estradiol, 3-O-methylestrone, and D8,9-dehydroestrone from the Sample solution. Calculate the percentages of 17-estradiol and D8,9-dehydroestrone as their sodium sulfate salts in the portion of Conjugated Estrogens taken:
-estradiol, 3-O-methylestrone, and D8,9-dehydroestrone from the Sample solution. Calculate the percentages of 17-estradiol and D8,9-dehydroestrone as their sodium sulfate salts in the portion of Conjugated Estrogens taken: Result = (RU/RS) × (CS/CU) × F × 100
| RU | = | = ratio of the peak response of the appropriate analyte to that of the internal standard from the Sample solution |
| RS | = | = ratio of the peak response of estrone to that of the internal standard from the Standard solution |
| CS | = | = concentration of USP Estrone RS in the Standard solution (µg/mL) |
| CU | = | = 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 17-estradiol and D8,9-dehydroestrone, respectively, as their sodium sulfate salts.
-estradiol and D8,9-dehydroestrone, respectively, as their sodium sulfate salts.
• Procedure 3: Limit of Estrone, Equilin, and 17-Dihydroequilin (free steroids)
-Dihydroequilin (free steroids)
Solution A, Internal standard solution, Stock solution, System suitability solution, and Chromatographic system:
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 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. Prepare a reagent blank in the same manner.
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.
Analysis
Samples:
Free steroids standard solution and Sample solution
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 Sample solution, correcting for any reagent blank peaks.
-dihydroequilin relative to the area of the internal standard in the Sample solution, correcting for any reagent blank peaks.
Acceptance criteria:
NMT 0.65 (1.3% of free steroids), for the ratio RU/RS, where RS is the peak response ratio of estrone to that of the internal standard from the Free steroids standard solution
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers. Store at 25; excursions permitted between 15 and 30.
; excursions permitted between 15 and 30.
• Labeling:
Label it to state the content of Conjugated Estrogens on a weight-to-weight basis.
• USP Reference Standards 
11
11
USP 17-Dihydroequilin RS
C18H22O2 270.37
-Dihydroequilin RS
C18H22O2 270.37
USP Equilin RS 
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USP Estradiol RS
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USP Estrone RS
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Domenick Vicchio, Ph.D.
Senior Scientific Liaison 1-301-998-6828 |
(SM42010) Monographs - Small Molecules 4 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 3112
Pharmacopeial Forum: Volume No. 37(3)