Estradiol Valerate Injection

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 Valerate Injection is a sterile solution of Estradiol Valerate in a suitable vegetable oil. It contains NLT 90.0% and NMT 115.0% of the labeled amount of estradiol valerate (C₂₃H₃₂O₃).

2 IDENTIFICATION

A.

Phenol reagent (Folin-Ciocalteu reagent): In a 1500-mL flask connected by a standard taper joint to a reflux condenser, dissolve 100 g of sodium tungstate and 25 g of sodium molybdate in 700 mL of water. Add 50 mL of phosphoric acid and 100 mL of hydrochloric acid, and reflux gently for 10 h. Cool, and add 150 g of lithium sulfate, 50 mL of water, and 4–6 drops of bromine. Boil the mixture without the condenser for 15 min to remove the excess bromine. Cool, transfer to a 1-L volumetric flask, dilute with water to volume, and filter. The filtrate is golden yellow in color, and has no greenish tint. Store the filtrate in a tight container in a refrigerator. Mix the filtrate with water (1:2) before use as the Phenol reagent.

Analysis: Transfer 0.5 mL of Injection to a separator containing 10 mL of solvent hexane and 10 mL of 80% methanol. Shake the contents for 2 min, and allow the phases to separate. Add 1 mL of Phenol reagent and 3 mL of sodium carbonate solution (1 in 5) to 1 mL of the bottom layer, and mix.

Acceptance criteria: A blue color develops.

3 ASSAY

Procedure

Mobile phase: Dissolve 0.8 g of ammonium nitrate in 300 mL of water, add 700 mL of acetonitrile, and mix.

Internal standard solution: 8.0 mg/mL of Testosterone benzoate in tetrahydrofuran

Standard solution: 0.8 mg/mL of USP Estradiol Valerate RS, prepared as follows. Transfer 20 mg of USP Estradiol Valerate RS to a 25-mL volumetric flask. Add 5.0 mL of the Internal standard solution, and dilute with tetrahydrofuran to volume.

Sample solution: Nominally 0.8 mg/mL of estradiol valerate, prepared as follows. Using a “to contain” pipet, transfer an accurately measured volume of Injection, equivalent to 20 mg of estradiol valerate, to a 25-mL volumetric flask. Rinse the pipet with small portions of tetrahydrofuran, collecting the washings in the volumetric flask. Add 5.0 mL of Internal standard solution, and dilute with tetrahydrofuran to volume.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4-mm × 30-cm; packing L1

Flow rate: 2 mL/min

Injection volume: 10 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for estradiol valerate and testosterone benzoate are 1.0 and 1.2, respectively.]

Suitability requirements

Resolution: NLT 3.0 between estradiol valerate and the internal standard

Column efficiency: NLT 1100 theoretical plates for estradiol valerate

Relative standard deviation: NMT 1.5%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of estradiol valerate (C₂₃H₃₂O₃) in the portion of Injection taken:

Result = (R_U/R_S) × (C_S/C_U) × 100

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

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

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

C_U = nominal concentration of the Sample solution (mg/mL)

Acceptance criteria: 90.0%–115.0%

4 IMPURITIES

Limit of Estradiol

Standard solution: A solution of estradiol in acetone containing 30.0% of the labeled concentration of the Injection. Dilute 1.0 mL of this solution with oil labeled as vehicle for Injection to 10.0 mL.

Sample solution: Injection

Chromatographic system

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

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel

Application volume: 5 μL

Developing solvent system: Cyclohexane and ethyl acetate (7:3)

Spray reagent: A 3-in-10 solution of methanol in sulfuric acid, prepared as follows. To 30 mL of methanol in a 100-mL volumetric flask in an ice bath, cautiously add sulfuric acid to volume.

Analysis

Samples: Standard solution and Sample solution

Apply the samples at spots 2.5 cm from the bottom edge of the plate. Allow the applications to be absorbed by the layer without air-drying. Develop the chromatogram in an unlined chamber until the solvent front has moved about 15 cm above the point of application.

Remove the plate, dry at 90° for 30 min, and spray the plate lightly with Spray reagent. Heat the plate at 90° for 30 min.

Acceptance criteria: NMT 3.0%; any spot from the Sample solution close to the origin and corresponding to the estradiol spot is not larger or more intense than that from the Standard solution.

5 SPECIFIC TESTS

Other Requirements: Meets the requirements in Injections and Implanted Drug Products 〈1〉

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose or multiple-dose, light-resistant containers, preferably of Type I or Type III glass.

USP Reference Standards 〈11〉

USP Estradiol Valerate RS