DETERMINATION OF 17-ALPHA-DIHYDROEQUILENIN IN RAT, RABBIT AND MONKEY PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUOROMETRIC DETECTION

A high-performance liquid chromatographic (HPLC) method with fluoresce nce detection for the determination of total (unconjugated and conjuga ted) 17 alpha-dihydroequilenin in male and female rat, female rabbit a nd male and female rhesus monkey plasma is described here. Plasma samp le preparation involved hydrolysis with enzyme (Glusulase), addition o f internal standard ((14)beta-equilenin) and solvent extraction. The e xtracts were chromatographed on a C-6, 5-mu m reversed-phase HPLC colu mn and detection was accomplished with a fluorescence detector operate d 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 an d female monkey plasma. The method was specific, accurate and reproduc ible (percent differences <14.5; coefficients of variation <9.5%) in a ll matrices examined. The applicability of this method was successfull y tested by quantifying total plasma concentrations of 17 alpha-dihydr oequilenin in ovariectomized female rats, ovariectomized female rabbit s and a normal female rhesus monkey receiving 2.0, 8.3 and 0.1 mg/kg, respectively, of 17 alpha-dihydroequilenin sulfate intragastrically.