Estradiol Vaginal Cream
»Estradiol Vaginal Cream contains not less than 90.0percent and not more than 110.0percent of the labeled amount of C18H24O2in a suitable cream base.
Packaging and storage— Preserve in collapsible tubes or in tight containers.
Identification— Transfer a portion of Vaginal Cream,equivalent to about 1mg of estradiol,to a 150-mLbeaker.Add 25mLof acetonitrile,and gently heat to boiling.Boil for 45seconds,and cool to room temperature.Add 25mLof water,and swirl.Filter with the aid of suction.Transfer the filtrate to a 125-mLseparator,add 50mLof 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 2mLof chloroform to obtain the test solution.Apply separately 50µLof the test solution and 50µLof a Standard solution of USP Estradiol RSin chloroform containing about 0.5mg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)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 3to 5minutes at 90.Observe the plate under visible light:the RFvalue and color of the principal spot obtained from the test solution correspond to those obtained from the Standard solution.
Microbial limits á61ñ— It meets the requirements of the tests for absence of Staphylococcus aureusand Pseudomonas aeruginosa.
Minimum fill á755ñ: meets the requirements.
pHá791ñ: between 3.5and 6.5.
Assay—
Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (1:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).
Internal standard solution— Dissolve a suitable quantity of dydrogesterone in acetonitrile to obtain a solution containing about 60µg per mL.Use a freshly prepared solution.
Standard preparation— Transfer about 10mg of USP Estradiol RSand about 7.5mg of USP Estrone RS,both accurately weighed,to a 1000-mLvolumetric flask.Add 50.0mLof Internal standard solutionand 450mLof acetonitrile,and mix.Dilute with water to volume,and mix to obtain a solution having a known concentration of about 10µg of USP Estradiol RSper mL.
Assay preparation— Transfer an accurately weighed portion of Cream,equivalent to about 0.5mg of estradiol,to a 150-mLbeaker.Add 2.5mLof Internal standard solution,22.5mLof 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 45seconds.Allow to cool to room temperature,add 25.0mLof water,and mix.Filter first through paper and then through a micro disk filter.
Chromatographic system (see Chromatography á621ñ)—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 1mLper 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 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 2.0for the internal standard,1.0for estradiol,and 1.25for estrone.Calculate the quantity,in mg,of C18H24O2in the portion of Vaginal Cream taken by the formula:
0.05C(RU/RS),
in which Cis the concentration,in µg per mL,of USP Estradiol RSin the Standard preparation,and RUand RSare the peak response ratios of estradiol and the internal standard obtained from the Assay preparationand the Standard preparation,respectively.
Auxiliary Information— Staff Liaison:Clydewyn M.Anthony,Ph.D.,Scientist
Expert Committee:(PA1)Pharmaceutical Analysis 1
USP28–NF23Page 772
Phone Number:1-301-816-8139