Assay—
Mobile phase—
Prepare a suitable and degassed solution containing 35 volumes of acetonitrile and 65 volumes of water.
Internal standard solution—
Dissolve phenol in acetonitrile to obtain a solution having a concentration of about 35 µg per mL.
Standard preparation—
Dissolve a suitable quantity of
USP Equilin RS, accurately weighed, in
Internal standard solution to obtain a solution having a known concentration of about 0.2 mg per mL.
Assay preparation—
Transfer about 10 mg of Equilin, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with Internal standard solution to volume, and mix.
Chromatographic system
(see
Chromatography 
621
)—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 2 mL per minute. Chromatograph five replicate injections of the
Standard preparation, and record the peak responses as directed under
Procedure: the relative standard deviation is not more than 2.0%, and the resolution factor between equilin and phenol is not less than 5. Adjust the operating parameters such that the peak obtained from the
Standard preparation is about 0.7 full-scale.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph by means of a suitable microsyringe or sampling valve, record the chromatograms, and measure the responses for the major peaks: the retention times for equilin and phenol are about 14 and 3 minutes, respectively. Calculate the quantity, in mg, of C
18H
20O
2 in the portion of Equilin taken by the formula:
50C(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Equilin RS in the
Standard preparation, and
RU and
RS are the ratios of the peak responses of the equilin peak to the internal standard peak obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
and +316