Determination of 17 alpha-dihydroequilenin in rat, rabbit and monkey plasma by high-performance liquid chromatography with fluorimetric detection.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of total (unconjugated and conjugated) 17 alpha-dihydroequilenin in male and female rat, female rabbit and male and female rhesus monkey plasma is described here. Plasma sample preparation involved hydrolysis with enzyme (Glusulase), addition of internal standard (14 beta-equilenin) and solvent extraction. The extracts were chromatographed on a C6, 5-microns reversed-phase HPLC column and detection was accomplished with a fluorescence detector operated at an excitation wavelength of 210 nm and an emission wavelength of 370 nm. The assay was linear over a range of 2.5 to 100 ng/ml in male and female rat plasma, and 5 to 500 ng/ml in female rabbit and male and female monkey plasma. The method was specific, accurate and reproducible (percent differences < 14.5; coefficients of variation < 9.5%) in all matrices examined. The applicability of this method was successfully tested by quantifying total plasma concentrations of 17 alpha-dihydroequilenin in ovariectomized female rats, ovariectomized female rabbits and a normal female rhesus monkey receiving 2.0, 8.3 and 0.1 mg/kg, respectively, of 17 alpha-dihydroequilenin sulfate intragastrically.