Packaging and storage—
Preserve in tight, light-resistant containers.
Specific rotation 
781S
:
between +41
and +47
.
Test solution:
25 mg, uncorrected for moisture, per mL, in dioxane.
Limit of estradiol—
Apply 5 µL of a solution of Estradiol Valerate in acetone, containing 5 mg per mL, and 5 µL of a solution of estradiol in acetone, containing 50 µg per mL, about 2.5 cm from the lower edge of a thin-layer chromatographic plate (see
Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel. Develop the chromatogram in a solvent system consisting of a mixture of cyclohexane and ethyl acetate (7:3) 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 minutes, and spray the plate lightly with a 3 in 10 solution of methanol in sulfuric acid, prepared by cautiously adding sulfuric acid to 30 mL of methanol in a 100-mL volumetric flask, in an ice bath, to volume. Heat the plate at 90
for 30 minutes: any spot in the chromatogram of Estradiol Valerate close to the origin and corresponding to the estradiol spot is not larger nor more intense than that produced by the standard. (The limit is 1.0% of estradiol.)
Free acid—
Neutralize 25 mL of alcohol, in a conical flask, with 0.01 N sodium hydroxide VS to a faint blue color, using
bromothymol blue TS. Accurately weigh 500 mg of Estradiol Valerate, and dissolve it in the neutralized alcohol. Titrate rapidly with 0.01 N sodium hydroxide VS to a faint blue color. Each mL of 0.01 N sodium hydroxide is equivalent to 1.021 mg of valeric acid. The free acid content, expressed as valeric acid, does not exceed 0.5%.
Ordinary impurities 466—
Test solution:
acetone.
Standard solution:
acetone.
Eluant:
a mixture of cyclohexane and ether (4:1).
Visualization:
5 followed by 1.
Assay—
Mobile phase—
Dissolve 0.8 g of ammonium nitrate in 300 mL of water, add 700 mL of acetonitrile, and mix. Filter, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Internal standard solution—
Prepare a solution of testosterone benzoate in tetrahydrofuran having a concentration of about 2.0 mg per mL.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Estradiol Valerate RS in
Internal standard solution, and dilute quantitatively with
Internal standard solution to obtain a solution having a known concentration of about 1 mg of
USP Estradiol Valerate RS per mL.
Assay preparation—
Transfer about 25 mg of Estradiol Valerate, accurately weighed, to a 25-mL volumetric flask, add Internal standard solution to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 1.2 for testosterone benzoate and 1.0 for estradiol valerate; the column efficiency determined from the analyte peak is not less than 1100 theoretical plates; the resolution,
R, between the analyte and internal standard peaks is not less than 3.0; and the relative standard deviation for replicate injections is not more than 1.5%.
Procedure—
Separately inject equal volumes (about 10 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 C
23H
32O
3 in the portion of Estradiol Valerate taken by the formula:
25C(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Estradiol Valerate RS in the
Standard preparation; and
RU and
RS are the peak response ratios obtained from the
Assay preparation and the
Standard preparation, respectively.
)-, 17-pentanoate.