Estradiol Vaginal Cream
» Estradiol Vaginal Cream contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of C18H24O2 in a suitable cream base.
Packaging and storage— Preserve in collapsible tubes or in tight containers.
USP Reference standards 11
USP Estradiol RS Click to View Structure
USP Estrone RS Click to View Structure
Identification— Transfer a portion of Vaginal Cream, equivalent to about 1 mg of estradiol, to a 150-mL beaker. Add 25 mL of acetonitrile, and gently heat to boiling. Boil for 45 seconds, 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 to obtain the test solution. Apply separately 50 µL of the test solution and 50 µL of a Standard solution of USP Estradiol RS in chloroform containing about 0.5 mg per mL to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture, and dry the applications with the aid of a stream of nitrogen. Position the plate in a chromatographic chamber, and develop the chromatograms in a solvent system consisting of a mixture of toluene and acetone (4:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Spray the plate with a fine mist of a mixture of sulfuric acid and methanol (1:1), then heat the plate for 3 to 5 minutes at 90. Observe the plate under visible light: the RF value and color of the principal spot obtained from the test solution correspond to those obtained from the Standard solution.
Microbial enumeration tests 61 and Tests for specified microorganisms 62 It meets the requirements of the tests for absence of Staphylococcus aureus and Pseudomonas aeruginosa.
Minimum fill 755: meets the requirements.
pH 791: between 3.5 and 6.5.
Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (1:1). Make adjustments if necessary (see System Suitability under Chromatography 621).
Internal standard solution— Dissolve a suitable quantity of dydrogesterone in acetonitrile to obtain a solution containing about 60 µg per mL. Use a freshly prepared solution.
Standard preparation— Transfer about 10 mg of USP Estradiol RS and about 7.5 mg of USP Estrone RS, both accurately weighed, 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, and mix to obtain a solution having a known concentration of about 10 µg of USP Estradiol RS per mL.
Assay preparation— Transfer an accurately weighed portion of Cream, equivalent to about 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 Cream melts, swirling occasionally. Heat to boiling for about 45 seconds. Allow to cool to room temperature, add 25.0 mL of water, and mix. Filter first through paper and then through a micro disk filter.
Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed under Procedure: the resolution, R, between the analyte and estrone peaks is not less than 1.9, and the relative standard deviation for replicate injections is not more than 3.0%.
Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 2.0 for the internal standard, 1.0 for estradiol, and 1.25 for estrone. Calculate the quantity, in mg, of C18H24O2 in the portion of Vaginal Cream taken by the formula:
0.05C(RU / RS)
in which C is the concentration, in µg per mL, of USP Estradiol RS in the Standard preparation, and RU and RS are the peak response ratios of estradiol and the internal standard obtained from the Assay preparation and the Standard preparation, respectively.
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USP35–NF30 Page 3101