Estradiol Vaginal Cream

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

Estradiol Vaginal Cream contains NLT 90.0% and NMT 110.0% of the labeled amount of estradiol (C₁₈H₂₄O₂) in a suitable cream base.

2 IDENTIFICATION

A. Thin-Layer Chromatography

Standard solution: 0.5 mg/mL of USP Estradiol RS in chloroform

Sample solution: Transfer a portion of Vaginal Cream, equivalent to 1 mg of estradiol, to a 150-mL beaker. Add 25 mL of acetonitrile, and gently heat to boiling. Boil for 45 s, and cool to room temperature. Add 25 mL of water, and swirl. Filter with the aid of suction. Transfer the filtrate to a 125-mL separator, add 50 mL of chloroform, and shake. Allow the layers to separate, drain the chloroform layer into a flask, and evaporate in a rotary evaporator to dryness. Dissolve the residue in 2 mL of chloroform.

Chromatographic system

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

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture

Application volume: 50 μL. Dry the applications with the aid of a stream of nitrogen.

Developing solvent system: Toluene and acetone (4:1)

Spray reagent: Sulfuric acid and methanol (1:1)

Analysis

Samples: Standard solution and Sample solution

Spray the plate with a fine mist of Spray reagent, then heat the plate for 3–5 min at 90°. Observe the plate under visible light.

Acceptance criteria: The R value and color of the principal spot of the Sample solution correspond to those of the Standard solution.

3 ASSAY

Procedure

Mobile phase: Acetonitrile and water (1:1)

Internal standard solution: 60 μg/mL of dydrogesterone in acetonitrile. Use a freshly prepared solution.

Standard solution: 10 μg/mL of USP Estradiol RS and 7.5 μg/mL of USP Estrone RS, prepared as follows. Transfer 10 mg of USP Estradiol RS and 7.5 mg of USP Estrone RS to a 1000-mL volumetric flask. Add 50.0 mL of Internal standard solution and 450 mL of acetonitrile, and mix.

Dilute with water to volume.

Sample solution: Nominally 10 μg/mL of estradiol, prepared as follows. Transfer a portion of Vaginal Cream, equivalent to 0.5 mg of estradiol, to a 150-mL beaker. Add 2.5 mL of Internal standard solution, 22.5 mL of acetonitrile, and a few boiling chips. Cover with a watch glass, and heat gently until the Vaginal Cream melts, swirling occasionally. Heat to boiling for about 45 s. Allow to cool to room temperature, add 25.0mL of water, and mix. Pass first through paper and then through a microdisk filter.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 3.9-mm × 30-cm; packing L1

Flow rate: 1 mL/min

Injection volume: 50 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for estradiol, estrone, and the internal standard are 1.0, 1.25, and 2.0, respectively.]

Suitability requirements

Resolution: NLT 1.9 between estradiol and estrone

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of estradiol (C₁₈H₂₄O₂) in the portion of Vaginal Cream taken:

Result = (R_U/R_S) × (C_S/C_U) × 100

R_U = peak response ratio of estradiol to the internal standard from the Sample solution

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

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

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

Acceptance criteria: 90.0%–110.0%

4 PERFORMANCE TESTS

Minimum Fill 〈755〉: Meets the requirements

5 SPECIFIC TESTS

Microbial Enumeration Tests 〈61〉andTests for Specified Microorganisms 〈62〉: Meets the requirements of the tests for absence of Staphylococcus aureus and Pseudomonas aeruginosa

pH 〈791〉: 3.5–6.5

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in collapsible tubes or in tight containers.

USP Reference Standards 〈11〉

USP Estradiol RS

USP Estrone RS