A:Infrared Absorption á197Mñ.

B:Ultraviolet Absorption á197Uñ—

Test solution: 10mg per mL,in dioxane.

Mobile phase— Prepare a filtered and degassed mixture of 2,2,4-trimethylpentane,n-butyl chloride,and methanol (45:4:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluting solution— Prepare a filtered and degassed mixture of n-butyl chloride and methanol (5:1).

Test solution— Transfer about 70mg of Estradiol,accurately weighed,to a 10-mLvolumetric flask,dissolve in Diluting solution,shake vigorously to aid dissolution,dilute with Diluting solutionto volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains packing L3.The flow rate is about 2mLper minute.Chromatograph the Test solution,and record the peak responses as directed for Procedure:the resolution,R,between estradiol and any impurity is not less than 1.0;the column efficiency is not less than 800theoretical plates;the tailing factor is not more than 1.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Inject a volume (about 10µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the peak responses.Calculate the percentage of each impurity in the portion of Estradiol taken by the formula: in which riis the peak response for each impurity;and rsis the sum of the responses of all the peaks:not more than 0.5%of any individual impurity is found;and not more than 1.0%of total impurities is found.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (55:45).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Transfer about 300mg of ethylparaben to a 500-mLvolumetric flask,add methanol to volume,and mix.

Standard preparation— Dissolve accurately weighed quantities of USP Estradiol RSand USP Estrone RSin methanol to obtain a solution containing 0.40mg and 0.24mg,respectively,in each mL.Pipet 10mLof this solution and 5mLof the Internal standard solutioninto a 200-mLvolumetric flask.Add 100mLof methanol,dilute with water to volume,and mix to obtain a solution having a known concentration of about 20µg of USP Estradiol RSper mL.

Assay preparation— Transfer about 100mg of Estradiol,accurately weighed,to a 250-mLvolumetric flask,add methanol to volume,and mix.Transfer 10.0mLof this solution to a 200-mLvolumetric flask,add 5.0mLof Internal standard solutionand 100mLof methanol,dilute with water to volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 205-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 for Procedure:the relative retention times are about 0.7for the internal standard,about 1.3for estrone,and 1.0for estradiol;the resolution,R,between the analyte and estrone is not less than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C18H24O2in the portion of Estradiol taken by the formula: in which Cis the concentration,in µg per mL,of USP Estradiol RSin the Standard preparation;and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.