Levonorgestrel 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

Levonorgestrel and Ethinyl Estradiol Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of levonorgestrel (C₂₁H₂₈O₂) and NLT 90.0% and NMT 110.0% of the labeled amount of ethinyl estradiol (C₂₀H₂₄O₂).

2 IDENTIFICATION

A. The retention times of the two major peaks of the Sample solution correspond to those of levonorgestrel and ethinyl estradiol in the Standard solution, as obtained in the Assay.

B. Finely powder 20 Tablets and transfer a portion of the powder, equivalent to 4 mg of levonorgestrel, to a suitable container. Add 250 mL of a solvent mixture consisting of isooctane and chloroform (3:1). Sonicate the mixture for 3 min, and then stir it by mechanical means for 30 min.

Filter the mixture and evaporate the filtrate to dryness in a rotating vacuum evaporator. Dissolve the residue in 3 mL of chloroform, and transfer with a pipet to a 60-mL separator containing 18 mL of isooctane. Rinse the evaporator flask with an additional 3-mL portion of chloroform, and add the rinsing to the separator. Add 10 mL of 1 N sodium hydroxide, shake vigorously, and allow the layers to separate. Discard the lower aqueous phase, and filter the organic phase through 3 g of anhydrous sodium sulfate on filter paper into a 50-mL beaker. Rinse the filter with several small portions of the mixture of isooctane and chloroform (3:1), adding the filtered rinsings to the filtrate, and evaporate under nitrogen on a steam bath to dryness. Dissolve the residue in 1–2 mL of hot toluene, and transfer with a pipet to a small glass vial. Reduce the volume of the solution to 0.1 mL under nitrogen with warming. [Note—During this step, any crystals that deposit on the vial wall should be transferred to the bottom, and allowed to redissolve.]

Store the vial containing the clear toluene solution at 4° overnight to allow crystallization to occur. Remove and discard the mother liquor with a pipet, rinse the crystals with two 0.5-mL portions of anhydrous ether, and discard the rinsings. Dry the vial containing the rinsed crystals in a vacuum desiccator at 60° for 4 h.

Acceptance criteria: The melting point of the dried crystals of levonorgestrel so obtained is not lower than 220°, using the procedure described under Melting Range or Temperature 〈741〉, Class I.

3 ASSAY

Procedure

Mobile phase: Acetonitrile, methanol, and water (35:15:45)

Standard solution: 15 μg/mL of USP Levonorgestrel RS and 3 μg/mL of USP Ethinyl Estradiol RS in Mobile phase

Sample solution: Transfer a number of Tablets, equivalent to 3 mg of levonorgestrel, to a 200-mL volumetric flask. Dilute with Mobile phase to volume, sonicate to disintegrate the Tablets, then shake by mechanical means for 20 min. Centrifuge, and use the clear supernatant.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm × 15-cm; 5- to 7-μm packing L7

Flow rate: 1 mL/min

Injection size: 50 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for ethinyl estradiol and levonorgestrel are about 0.7 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 2.5 between the two major peaks

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of C₂₁H₂₈O₂ and C₂₀H₂₄O₂ in the portion of Tablets taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of the corresponding analyte from the Sample solution

r_S = peak response of the corresponding analyte from the Standard solution

C_S = concentration of the appropriate USP Reference Standard in the Standard solution (μg/mL)

C_U = nominal concentration of the corresponding analyte in the Sample solution (μg/mL)

Acceptance criteria: 90.0%–110.0% of the labeled amount of C₂₁H₂₈O₂, 90.0%–110.0% of the labeled amount of C₂₀H₂₄O₂

4 PERFORMANCE TESTS

Dissolution 〈711〉: Determine the amount of C₂₁H₂₈O₂ and C₂₀H₂₄O₂ dissolved by employing the following method.

Medium: Polysorbate 80 (5 μg/g) in water; 500 mL

Apparatus 2: 75 rpm

Time: 60 min

Mobile phase: Acetonitrile and water (6:4)

Standard solution: Prepare a solution of USP Levonorgestrel RS and USP Ethinyl Estradiol RS in Medium having known concentrations corresponding approximately to the concentrations that would be obtained by dissolving 1 Tablet in 500 mL of Medium.

[Note—A volume of alcohol not exceeding 2% of the final total volume of solution may be used to aid in dissolving the Reference Standards.]

Sample solution: Withdraw 15-mL portions of liquid from each vessel, and pass through a polyvinylidene filter, discarding the first 10 mL of the filtrate.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 247 nm (for levonorgestrel analysis); a spectro fluorometric detector (for ethinyl estradiol analysis), with an excitation wavelength of 285 nm, and an emission wavelength of 310 nm

Column: 4-mm × 15-cm; packing L7

Flow rate: 1 mL/min

Injection size: 100 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for ethinyl estradiol and levonorgestrel are about 0.7 and 1.0, respectively.]

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of C₂₁H₂₈O₂ and C₂₀H₂₄O₂ dissolved:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of the corresponding analyte from the Sample solution

r_S = peak response of the corresponding analyte from the Standard solution

C_S = concentration of the appropriate USP Reference Standard in the Standard solution (μg/mL)

C_U = nominal concentration of the corresponding analyte in the Sample solution (μg/mL)

Tolerances

Uncoated Tablets: NLT 80% (Q) of the labeled amount of C₂₁H₂₈O₂, and 75% (Q) of the labeled amount of C₂₀H₂₄O₂ is dissolved.

Coated Tablets: NLT 60% (Q) of the labeled amount of C₂₁H₂₈O₂ and C₂₀H₂₄O₂ is dissolved.

Uniformity of Dosage Units 〈905〉: Meet the requirements

Sample solution: Place 1 Tablet in a 40-mL centrifuge tube, add 10.0 mL of Mobile phase, and proceed as directed in the Assay.

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers.

USP Reference Standards 〈11〉

USP Ethinyl Estradiol RS

USP Levonorgestrel RS