Norethindrone and Ethinyl Estradiol Tablets

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

Norethindrone and Ethinyl Estradiol Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of norethindrone (C₂₀H₂₆O₂), and NLT 90.0% and NMT 110.0% of the labeled amount of ethinyl estradiol (C₂₀H₂₄O₂).

2 IDENTIFICATION

A. THIN-LAYER CHROMATOGRAPHIC TEST

Standard solution: 1 mg/mL of USP Norethindrone RS and 50 µg/mL of USP Ethinyl Estradiol RS in alcohol

Sample solution: Crush 1 Tablet in 1 mL of alcohol in a 15-mL conical centrifuge tube, and warm to 50° for 10 min with gentle swirling. Cool, and centrifuge to obtain a clear solution.

Chromatographic system

(See Chromatography (621), Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel and previously activated by heating at 105° for 30 min

Application volume: 20 µL

Developing solvent system: Toluene and ethyl acetate (4:1)

Spray reagent: Sulfuric acid-methanol, prepared by cautiously adding 70 mL of sulfuric acid in small increments to 30 mL of methanol chilled in an ice-bath, and mixing

Analysis

Samples: Standard solution and Sample solution

Apply both Standard solution and Sample solution at a line 2.5 cm from the bottom of a thin-layer chromatographic plate. Develop the chromatogram in Developing solvent system until the solvent front has moved 10 cm. Remove the plate, and air-dry. Spray the plate with Spray reagent.

Acceptance criteria: The spots from the Sample solution have the same R_F values as the spots from USP Ethinyl Estradiol RS and from USP Norethindrone RS.

3 ASSAY

PROCEDURE

Mobile phase: Acetonitrile and water (60:40)

Diluent: Acetonitrile and water (45:55)

Internal standard solution: Transfer 15 mg of valerophenone into a 250-mL volumetric flask, add 125 mL of acetonitrile, and dilute with water to volume.

Ethinyl estradiol standard stock solution: 0.09 mg/mL of USP Ethinyl Estradiol RS in acetonitrile

Norethindrone standard stock solution: 1.25 mg/mL of USP Norethindrone RS in acetonitrile

Standard solution: Transfer 5.0 mL of Internal standard solution into a 100-mL volumetric flask. Add volumes of Ethinyl estradiol standard stock solution and Norethindrone standard stock solution so that the final known concentrations, in mg/mL, of the Reference Standards correspond numerically to one-twentieth of the labeled amounts of the corresponding ingredients in the Tablets. Add (26-X) mL of acetonitrile. X being the total volume of the standard stock solution taken. Dilute with Diluent to volume.

Sample stock solution: Transfer 10 Tablets to a 100-mL volumetric flask, add 20 mL of water, and shake by mechanical means until the Tablets are completely disintegrated. Add 10.0 mL of Internal standard solution and 60 mL of acetonitrile, and mix. Sonicate for 2 min. Dilute with acetonitrile to volume, and mix. Allow solid particles to settle, or centrifuge if necessary to obtain a slightly turbid solution.

Sample solution: Dilute 5.0 mL of the Sample stock solution with Diluent to 10.0 mL.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 200 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection volume: 25 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for ethinyl estradiol and norethindrone are 0.9 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 2.0 between norethindrone and ethinyl estradiol

Column eciency: NLT 8000 theoretical plates from the internal standard peak

Relative standard deviation: NMT 2.0% for six replicate injections for norethindrone and ethinyl estradiol

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of norethindrone (C₂₀H₂₆O₂) and ethinyl estradiol (C₂₀H₂₄O₂) in each Tablet:

Result = (R_u /R_s ) × (C_s /C_u ) × 100

R_u = peak response ratio, at corresponding retention times, of the Sample solution

R_s = peak response ratio, at corresponding retention times, of the Standard solution

C_s = concentration of the corresponding USP Reference Standard in the Standard solution (mg/mL)

C_u = nominal concentration of the corresponding sample in the Sample solution (mg/mL)

Acceptance criteria: 90.0%~110.0% of the labeled amount of norethindrone (C₂₀H₂₆O₂), and 90.0%~110.0% of the labeled amount of ethinyl estradiol (C₂₀H₂₄O₂)

4 PERFORMANCE TESTS

DISSOLUTION (711)

[NOTE-Exercise care in filtering and handling solutions containing ethinyl estradiol to prevent adsorptive loss of the drug. Centrifugation may be used instead of filtration with nonadsorptive membrane filters. Withdraw dissolution aliquots with glass or polytef pipets or syringes that have been checked for adsorptive loss. Use glass dissolution vessels and polytef-coated or solid polytef paddles.]

Test 1

Medium: 0.09% sodium dodecyl sulfate in 0.1 N hydrochloric acid: 500 mL

Apparatus 2: 75 rpm

Time: 60 min

Buffer: 0.02 M pH 6.0 phosphate buffer

Mobile phase: Acetonitrile and Buffer (35:65)

Standard solution: USP Norethindrone RS and USP Ethinyl Estradiol RS in Medium with concentrations similar to those expected in the solution under test. [NOTE-A volume of methanol not exceeding 4% of the total final volume of the Standard solution may be used in preparing the Standard solution.]

Sample solution: A filtered portion of the solution under test.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 200 nm

Column: 5-mm x 8.3-cm; 3-µm packing L1

Flow rate: 1 mL/min.

Injection volume: 100 µL

System suitability

Sample: Standard solution

[NOTE-The relative retention times for norethindrone and ethinyl estradiol are 0.9 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 1.5 between norethindrone and ethinyl estradiol

Column efficiency: NLT 7000 theoretical plates for the ethinyl estradiol peak

Tailing factor: NMT 2.0 for either peak

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of norethindrone (C₂₀H₂₆O₂) and ethinyl estradiol (C₂₀H₂₄O₂) dissolved by comparison of the corresponding peak responses of the Standard solution and the solution under test.

Tolerances: NLT 75% (Q) of each of the labeled amounts of norethindrone (C₂₀H₂₆O₂) and ethinyl estradiol (C₂₀H₂₄O₂), respectively, are dissolved in 60 min.

Test 2: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 2.

Medium: 0.09% (w/v) sodium dodecyl sulfate in 0.1 N hydrochloric acid; 500 mL.

Apparatus 2: 75 rpm

Time: 30 min

Buffer: 0.02 M pH 6.0 phosphate buffer prepared as follows. Dissolve 2.72 g of potassium phosphate, monobasic in 1 L of water. Adjust with 10% potassium hydroxide solution in water to a pH of 6.0.

Mobile phase: Acetonitrile and Buffer (42:58)

Standard stock solution 1: 100 µg/mL of USP Norethindrone RS in acetonitrile. Sonicate to dissolve as needed.

Standard stock solution 2: 3.5 µg/mL of USP Ethinyl Estradiol RS in acetonitrile. Sonicate to dissolve as needed.

Standard solution: 0.8 µg/mL of USP Norethindrone RS and 0.07 µg/mL of USP Ethinyl Estradiol RS in Medium, from Standard stock solution 1 and Standard stock solution 2.

Sample solutions: Centrifuge a portion of the solution under test at about 3000 rpm for 3 min, and use the clear supernatant.

Chromatographic system

(See Chromatography (621), System Suitability)

Mode: LC

Detector: UV 200 nm

Column: 4.6-mm x 10-cm; 3-µm packing 11

Flow rate: 0.8 mL/min

Injection volume: 100 µL

Run time: NLT 2 times the retention time of ethinyl estradiol

System suitability

Sample: Standard solution

[NOTE-The relative retention times for norethindrone and ethinyl estradiol are 0.9 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 1.5 between norethindrone and ethinyl estradiol

Tailing factor: NMT 2.0 for norethindrone and ethinyl estradiol

Relative standard deviation: NMT 3.0% for norethindrone and ethinyl estradiol

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of norethindrone (C₂₀H₂₆O₂) and ethinyl estradiol (C₂₀H₂₄O₂) dissolved:

Result = (r_u /r_s ) × C_s × V × (1/L) × 100

r_u = peak response of norethindrone or ethinyl estradiol from the Sample solution

r_s = peak response of corresponding USP Reference Standard from the Standard solution

C_s = concentration of the corresponding USP Reference Standard in the Standard solution (µg/mL)

V = volume of Medium, 500 mL

L = label claim of norethindrone or ethinyl estradiol (µg/Tablet)

26 Tolerances: NLT 80% (Q) of each of the labeled amounts of norethindrone (C₂₀H₂₆O₂) and ethinyl estradiol (C₂₀H₂₄O₂), respectively, are dissolved in 30 min.

Test 3: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 3.

Medium: 0.09% (w/v) sodium dodecyl sulfate in 0.1 N hydrochloric acid: 500 mL

Apparatus 2: 75 rpm

Time: 30 min

Buffer: 0.02 M pH 6.0 phosphate buffer prepared as follows. Dissolve 2.72 g of potassium phosphate, monobasic in 1 L of water. Adjust with 0.1 N sodium hydroxide solution in water to a pH of 6.0.

Mobile phase: Acetonitrile and Buffer (38:62)

Norethindrone standard stock solution: 0.8 mg/mL of USP Norethindrone RS in methanol. Sonicate to dissolve as needed.

Ethinyl estradiol standard stock solution: 0.135 mg/ml of USP Ethinyl Estradiol RS in methanol. Sonicate to dissolve as needed.

Standard solution: USP Norethindrone RS and USP Ethinyl Estradiol RS in Medium with concentrations similar to those expected in the solution under test, from Norethindrone standard stock solution and Ethinyl estradiol standard stock solution. Add intermediate diluting steps as needed.

Sample solution: Pass a portion of the solution under test through a filter of 0.45-µm pore size and use the clear filtrate.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 200 nm

Column: 4.6-mm × 10-cm; 3-µm packing L1

Column temperature: 30°

Flow rate: 1 mL/min

Injection volume: 100 µL

Run time: NLT 1.4 times the retention time of ethinyl estradiol

System suitability

Sample: Standard solution

[Note—The relative retention times for norethindrone and ethinyl estradiol are 0.9 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 1.5 between norethindrone and ethinyl estradiol

Tailing factor: NMT 2.0 for norethindrone and ethinyl estradiol

Relative standard deviation: NMT 3.0% for norethindrone and ethinyl estradiol

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of norethindrone (C₂₀H₂₆O₂) and ethinyl estradiol (C₂₀H₂₄O₂) dissolved:

Result = (r_u /r_s ) × C_s × V × (1/L) × 100

r_u = peak response of norethindrone or ethinyl estradiol from the Sample solution

r_s = peak response of corresponding USP Reference Standard from the Standard solution

C_s = concentration of the corresponding USP Reference Standard in the Standard solution (µg/mL)

V = volume of Medium, 500 mL

L = label claim of norethindrone or ethinyl estradiol (µg/Tablet)

Tolerances: NLT 80% (Q) of each of the labeled amounts of norethindrone (C₂₀H₂₆O₂) and ethinyl estradiol (C₂₀H₂₄O₂), respectively, are dissolved in 30 min.

Uniformity of Dosage Units 〈905〉, Content Uniformity: Meet the requirements with respect to norethindrone and to ethinyl estradiol

5 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.

USP Reference Standards 〈11〉

USP Ethinyl Estradiol RS

USP Norethindrone RS