Packaging and storage—
Preserve in tight, light-resistant containers. Store at 25
, excursions permitted between 15
and 30
.
Specific rotation 
781S
:
between +158
and +165
.
Test solution:
10 mg, previously dried, per mL, in dioxane.
Limit of equilenin and equilin—
Dissolve 10 mg in sufficient alcohol to make 50 mL. Transfer 5 mL of the solution to a small beaker. Add 5 mL of a buffer solution prepared by dissolving 2 mL of glacial acetic acid and 13.3 g of anhydrous sodium acetate in water to make 100 mL, warm to about 50
, and add 1 mL of a freshly prepared 1 in 200 solution of 2,6-dibromoquinone-chlorimide in alcohol. Mix, and allow to stand for 30 minutes. Transfer the solution to a small separator, add 10 mL of chloroform and 20 mL of 1 N sodium hydroxide, and shake vigorously for 2 minutes. Separate the chloroform layer, and filter rapidly through a dry filter paper into a dry test tube, discarding the first 2 mL of the filtrate. Viewed transversely against a white background, the chloroform filtrate shows no more red color than that produced by similarly treating 5 mL of an alcohol solution containing 20 µg of equilenin.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile and 0.05
M monobasic potassium phosphate (1:1). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Transfer about 20 mg of
USP Estrone RS, accurately weighed, to a 100-mL volumetric flask, add methanol to volume, and mix. If necessary, sonicate to aid solution. Transfer 5 mL of this solution to a 25-mL volumetric flask, dilute with
Mobile phase to volume, and mix to obtain a
Standard preparation having a known concentration of about 40 µg of
USP Estrone RS per mL.
Assay preparation—
Transfer about 20 mg of Estrone, accurately weighed, to a 100-mL volumetric flask, add methanol to volume, and mix. If necessary, sonicate to aid solution. Transfer 5.0 mL of this solution to a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 280-nm detector and a 4-mm × 15-cm column that contains 5-µm 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 column efficiency determined from the analyte peak is not less than 1500 theoretical plates, the tailing factor for the analyte peak is not more than 2.0, and the relative standard deviation for replicate injections is not more than 2.0%.
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. Calculate the quantity, in mg, of C
18H
22O
2 in the portion of Estrone taken by the formula:
0.5C(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Estrone RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
