ANALYSIS OF PHYTOESTROGENS AND POLYPHENOLS IN PLASMA, TISSUE, AND URINE USING HPLC WITH COULOMETRIC ARRAY DETECTION

The study of phytoestrogens in food sources and their metabolism, effe cts, and mechanism of action in animals requires very selective and of ten sensitive analytical techniques. We have applied coulometric array detection, which uses a series of flow-through electrochemical sensor s each providing 100% electrolytic efficiency, for measurement of a va riety of phytochemicals in complex matrices. Recent work has involved the resolution of coumestrol (COM), daidzein (DE), daidzin (DI), dieth ylstilbestrol (DES), enterodiol (ED), enterolactone (EL), equol (EQ), estradiol (E2), estriol (E3), estrone (E), genistein (GE), and quercet in (QE). Binary gradient reversed-phase (C18) chromatography was used with a sodium acetate buffer (pH 4.8)-methanol-acetonitrile solvent sy stem. Eight coulometric sensors were set at 260, 320, 380, 440, 500, 5 60, 620, and 680 mV (vs Pd reference). Compounds were resolved in 30 m in via both their oxidation/reduction characteristics and chromatograp hic behavior. Respective maximal oxidation potentials (mV) were: COM = 380; DE = 500; DI = 620; DES = 440; ED = 620; EL = 620; EQ = 560; E2 = 560; E3 = 560; El = 560; GE = 500; and QE = 260 with limits of detec tion of 5-50 pg. Uterine tissue homogenates (30 mg/ml in Tris-EDTA) an d plasma from Sprague-Dawley rats sacrificed 1 hr after sc injection w ith either vehicle, dimethylsulfoxide, 10 mu g DES, or 1.0 mg Ea were analyzed before and after enzymatic hydrolysis with beta-glucuronidase /sulfatase. Urine samples from humans receiving a Boston-area diet wit h or without soy protein isolate supplements were also analyzed. Ethan ol extracts were evaporated and reconstituted in 20% methanol before H PLC analysis. DE, ED, EL, Ea, and GE were determined in urine with les s than 5% (R.S.D.) intraassay imprecision and 85%-102% recovery. Level s (ng/ml) of GE (1.8), QE (11.2), and EQ (1.7) were found in control p lasma before hydrolysis and GE (293), QE (183), and EQ (22) after hydr olysis. Higher concentrations, corresponding to sc injection, in free and total EQ were found in both tissue and plasma.