(es'' tra dye' ol).
Hemihydrate 281.39 [35380-71-3].
» Estradiol contains not less than 97.0 percent and not more than 103.0 percent of C18H24O2, calculated on the anhydrous basis.
Packaging and storage Preserve in tight, light-resistant containers. Store at 25, excursions permitted between 15 and 30.
Labeling The hemihydrate form is so labeled.
USP Reference standards 11
B: Ultraviolet Absorption 197U
Solution: 50 µg per mL.
Absorptivities at 280 nm, calculated on the anhydrous basis, do not differ by more than 3.0%.
Melting range, Class I 741: between 173 and 179. [noteDry over silica gel for not less than 16 hours prior to testing. ]
Specific rotation 781S: between +76 and +83.
Test solution: 10 mg per mL, in dioxane.
Water, Method I 921: not more than 3.5%.
Chromatographic purity [noteMake all solutions fresh daily. ]
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 Suitability under Chromatography 621).
Diluting solution Prepare a filtered and degassed mixture of n-butyl chloride and methanol (5:1).
Test solution Transfer about 70 mg of Estradiol, accurately weighed, to a 10-mL volumetric flask, dissolve in Diluting solution, shake vigorously to aid dissolution, dilute with Diluting solution to volume, and mix.
Chromatographic system (see Chromatography 621) 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 2 mL per 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 800 theoretical 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 10 µL) of the Test solution into 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:
100(ri / rs)in which ri is the peak response for each impurity; and rs is 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 Suitability under Chromatography 621).
Internal standard solution Transfer about 300 mg of ethylparaben to a 500-mL volumetric flask, add methanol to volume, and mix.
Standard preparation Dissolve accurately weighed quantities of USP Estradiol RS and USP Estrone RS in methanol to obtain a solution containing 0.40 mg and 0.24 mg, respectively, in each mL. Pipet 10 mL of this solution and 5 mL of the Internal standard solution into a 200-mL volumetric flask. Add 100 mL of methanol, dilute with water to volume, and mix to obtain a solution having a known concentration of about 20 µg of USP Estradiol RS per mL.
Assay preparation Transfer about 100 mg of Estradiol, accurately weighed, to a 250-mL volumetric flask, add methanol to volume, and mix. Transfer 10.0 mL of this solution to a 200-mL volumetric flask, add 5.0 mL of Internal standard solution and 100 mL of methanol, dilute with water to volume, and mix.
Chromatographic system (see Chromatography 621) 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 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for the internal standard, about 1.3 for estrone, and 1.0 for 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 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C18H24O2 in the portion of Estradiol taken by the formula:
5C(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 obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 3099Pharmacopeial Forum: Volume No. 33(6) Page 1167