Ethinyl Estradiol Tablets
DEFINITION
Ethinyl Estradiol Tablets contain NLT 90.0% and NMT 115.0% of the labeled amount of C20H24O2.
IDENTIFICATION
• Thin-Layer Chromatographic Identification Test 
201
201
Standard solution:
0.03 mg/mL of USP Ethinyl Estradiol RS in methanol
Sample solution:
Transfer 25 Tablets to a suitable container, add 50 mL of water, and sonicate until the Tablets disintegrate (if needed, remove any coating with water before sonication). Place the sample in a separatory funnel, add 25 mL of ether, and shake well to extract the actives. Using a glass pipet, transfer the ether layer to a clean beaker, and evaporate to 10 mL.
Spray reagent:
Methanol and sulfuric acid (1:1)
Application volume:
30 µL
Developing solvent system:
Chloroform and alcohol (24:1)
Analysis
Samples:
Standard solution and Sample solution
Proceed as directed under Chromatography 621, Thin-Layer Chromatography. Spray the plate with Spray reagent, place in an oven at 105
for about 5 min, and examine the plate.
621, Thin-Layer Chromatography. Spray the plate with Spray reagent, place in an oven at 105 for about 5 min, and examine the plate.
Acceptance criteria:
Meet the requirements
ASSAY
• Procedure
Mobile phase:
Acetonitrile and 20 mM potassium phosphate buffer, pH 6.0 (1:1)
Diluent:
Acetonitrile and water (1:1)
Standard stock solution:
0.3 mg/mL of USP Ethinyl Estradiol RS in methanol
Standard solution:
0.12 µg/mL of USP Ethinyl Estradiol RS from Standard stock solution in Diluent
Sample solution:
0.12 µg/mL of ethinyl estradiol from NLT 20 Tablets in Diluent. [Note—Shake for about 30 min before makeup to volume. Centrifuge a portion of the solution, and makeup with Diluent. ]
Chromatographic system
Mode:
LC
Detector:
Spectrofluorometric, with an excitation wavelength of 285 nm and an emission wavelength of 310 nm
Column:
4.6-mm × 15-cm; packing L11
Guard column:
4.6-mm × 12.5-mm; packing L11
Flow rate:
2 mL/min
Injection size:
200 µL
System suitability
Sample:
Standard solution
Suitability requirements
Tailing factor:
NMT 2.0 for ethinyl estradiol
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of C20H24O2 in the portion of Tablets taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response of ethinyl estradiol from the Sample solution |
| rS | = | = peak response of ethinyl estradiol from the Standard solution |
| CS | = | = concentration of USP Ethinyl Estradiol RS in the Standard solution (µg/mL) |
| CU | = | = nominal concentration of the Sample solution (µg/mL) |
Acceptance criteria:
90.0%–115.0%
PERFORMANCE TESTS
• Dissolution
[Note—Care must be taken not to expose any of the solutions to plastic or rubber. Fluorescent material will leach into the solutions and interfere with the quantitation of ethinyl estradiol. Also, adsorption may occur. ]
Test 1
Medium:
0.3% sodium lauryl sulfate in water; 500 mL, degassed
Apparatus 2:
100 rpm
Time:
30 min
pH 6.0 phosphate buffer:
Transfer 2.7 g of monobasic potassium phosphate to a 1-L volumetric flask. Dissolve in 900 mL of water. Adjust with 1 N sodium hydroxide to a pH of 6.0, and dilute with water to volume.
Mobile phase:
pH 6.0 phosphate buffer and acetonitrile (1:1)
Standard stock solution:
0.25 mg/mL of USP Ethinyl Estradiol RS in methanol. This solution is stable for 14 days.
Standard solution:
Dilute the Standard stock solution with Medium to a final concentration of 0.06 µg/mL. Add 1 or 2 drops of methanol to dissipate the bubbles, if necessary. This solution is stable for 24 h.
Sample solution:
Centrifuge the solution under test for 10 min at 2000 rpm. Use the supernatant.
Chromatographic system
Mode:
LC
Detector:
Fluorescence, excitation at 285 nm, emission at 310 nm
Column:
4.6-mm x 15-cm, 5-µm packing L11
Guard column:
4.6-mm × 1.25-cm, 5-µm packing L11
Flow rate:
2.0 mL/min
Injection size:
200 µL
System suitability
Sample:
Standard solution
Suitability requirements
Relative standard deviation:
NMT 3.0%
Analysis:
Calculate the percentage of ethinyl estradiol dissolved:
Result = (rU/rS) × (CS/L) × V × 100
| rU | = | = peak response from the Sample solution |
| rS | = | = peak response from the Standard solution |
| CS | = | = concentration of the Standard solution (mg/mL) |
| L | = | = label claim (mg/Tablet) |
| V | = | = volume of Medium, 500 mL |
Tolerances:
NLT 80% (Q) of the labeled amount of ethinyl estradiol is dissolved.
Test 2:
If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 2.
Medium:
5 ppm of polysorbate 80 in water; 500 mL, deaerated with helium
Apparatus 2:
75 rpm
Time:
45 min
Standard stock solution:
Transfer 10 mg of USP Ethinyl Estradiol RS and 50 mg of USP Norgestrel RS to a 500-mL volumetric flask. Add 250 mL of acetonitrile, and sonicate until dissolved. Cool to room temperature, and dilute with water to volume. The final concentration is about 20 µg/mL of ethinyl estradiol and 100 µg/mL of norgestrel. This solution is stable for 15 days.
Standard solution:
Dilute the Standard stock solution with Medium to obtain a final concentration of 0.02 µg/mL of ethinyl estradiol. This solution is stable for 6 days.
Sample solution:
Centrifuge the solution under test at about 3000 rpm for 20 min. Use the supernatant. This solution is stable for 12 h.
Mobile phase:
Water, acetonitrile, and methanol (55:40:5)
Chromatographic system
Mode:
LC
Detector:
UV 200 nm
Column:
4.6-mm × 10-cm, 3-µm packing L1
Column temperature:
30
Flow rate:
1.2 mL/min
Injection size:
200 µL
System suitability
Sample:
Standard solution
Suitability requirements
Resolution:
NLT 6.0 between the ethinyl estradiol and norgestrel peaks
Relative standard deviation:
NMT 3.0%, for ethinyl estradiol
Analysis:
Calculate the percentage of ethinyl estradiol dissolved:
Result = (rU/rS) × (CS/L) × V × 100
| rU | = | = peak response from the Sample solution |
| rS | = | = peak response from the Standard solution |
| CS | = | = concentration of the Standard solution (mg/mL) |
| L | = | = label claim (mg/Tablet) |
| V | = | = volume of Medium, 500 mL |
Tolerances:
NLT 80% (Q) of the labeled amount of ethinyl estradiol is dissolved.
• Uniformity of Dosage Units:
Meet the requirements
IMPURITIES
Organic Impurities
• Procedure
Solution A:
Acetonitrile and 20 mM potassium phosphate buffer, pH 6.0 (1:1)
Solution B:
Acetonitrile and 20 mM potassium phosphate buffer, pH 6.0 (4:1)
Mobile phase:
See the gradient table below.
| Time (min) |
Solution A (%) |
Solution B (%) |
Flow Rate (mL/min) |
|---|---|---|---|
| 0 | 100 | 0 | 2 |
| 20 | 100 | 0 | 2 |
| 20.1 | 100 | 0 | 2.5 |
| 25.0 | 0 | 100 | 2.5 |
| 25.1 | 0 | 100 | 3 |
| 30.0 | 0 | 100 | 3 |
| 30.1 | 0 | 100 | 2 |
| 32.0 | 100 | 0 | 2 |
| 35.0 | 100 | 0 | 2 |
Diluent:
Acetonitrile and water (1:1)
Standard stock solution:
0.3 mg/mL of USP Ethinyl Estradiol RS in methanol
Standard solution:
0.12 µg/mL of USP Ethinyl Estradiol RS from Standard stock solution in Diluent
Sample solution A:
Transfer 20 Tablets into a 200-mL volumetric flask. Add 120 mL of Diluent, and shake for about 30 min. Dilute with Diluent to volume. Centrifuge a portion of the dissolution sample, and use the clear supernatant.
Sample solution B:
0.6 µg/mL of ethinyl estradiol from Sample solution A in Diluent
Chromatographic system
Mode:
LC
Detectors:
UV detector at 210 nm; and a spectrofluorometric detector with an excitation wavelength of 285 nm and an emission wavelength of 310 nm
Column:
4.6-mm × 15-cm; packing L11
Guard column:
4.6-mm × 12.5-mm; packing L11
Flow rate:
See the gradient table above.
Injection size:
200 µL
System suitability
Sample:
Standard solution
Suitability requirements
Tailing factor:
NMT 2.0 for ethinyl estradiol
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Sample solution A and Sample solution B
[Note—Measure the peak heights for the major peaks obtained within 20 min. Use the response from Sample solution A for estrone and all other impurities. Use Sample solution B for 17
-ethinyl estradiol. ]
-ethinyl estradiol. ]
Calculate the percentage of 17-ethinyl estradiol in the portion of Tablets taken:
-ethinyl estradiol in the portion of Tablets taken: Result = (rU/rS) × 100
| rU | = | = height of any peak at the relative retention time of 1.16, using the spectrofluorometric detector |
| rS | = | = peak height of ethinyl estradiol, using the spectrofluorometric detector |
Calculate the percentage of estrone in the portion of Tablets taken:
Result = [(rU/rS) × 100] 
E
E| rU | = | = height of any peak at the relative retention time of 1.2, using the UV detector at 210 nm |
| rS | = | = peak height of ethinyl estradiol, using the UV detector at 210 nm |
| E | = | = percentage of 17-ethinyl estradiol |
Calculate the percentage of any other impurity:
Result = (rU/rS) × 100
| rU | = | = height of any peak other than those mentioned above |
| rS | = | = peak height of ethinyl estradiol, using the UV detector at 210 nm |
Acceptance criteria
17-Ethinyl estradiol:
NMT 0.5%
-Ethinyl estradiol:
NMT 0.5%
Estrone:
NMT 0.5%
Any other impurity:
NMT 0.5%
Total impurities:
NMT 2.0 %
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers.
• Labeling:
When more than one Dissolution test is given, the labeling states the Dissolution test used only if Test 1 is not used.
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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 3127
Pharmacopeial Forum: Volume No. 31(4) Page 1067