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SECOND INTERNATIONAL SYMPOSIUM
ON THE ROLE OF SOY
IN PREVENTING AND TREATING CHRONIC DISEASESeptember 15-18, 1996
Brussells, Belgium
POSTER ABSTRACTSSensitive Analysis of Phytoestrogens in Animal Tissue by HPLC with Coulometric Array Detection.
P.H. Gamache1, T.J. Maher*2, K.D.R. Setchell3, T.H. Wu2, and I.N. Acworth1,2, ESA Inc.1, Chelmsford, MA 01824, Div. of Pharm. Sciences, Mass. College of Pharmacy & AHS2, Boston, MA 02115 and Children s Hospital Medical Center3, Cincinnati, OH 45229.
Interest in potential health benefits of phytoestrogens has created the need for simple and reliable techniques for their measurement. The objective of this study was to apply coulometric array detection with HPLC for analysis of isoflavones, lignans and metabolites in animal tissue. Eight serial electrochemical sensors, each providing 100% electrolysis, were used at incrementally increasing potentials to generate voltammetric response relationships for each analyte. Optimal settings for sensitivity, peak identification, and voltammetric resolution of potential co-eluents were obtained using potentials of 340, 520, 540, 560, 580, 620, 680 and 760 millivolts (mV vs. Pd reference). Analytes were separated in 15 minutes with isocratic elution using an ODS column (15cm x 3mm; 3 m) and mobile phase (50mM sodium acetate, pH 4.8 - methanol-acetonitrile; 60:30:10; v/v/v) at a flow rate of 0.6mL/min.
Daidzein (Dz) and genistein (Gs) both showed dual oxidation waves with dominant response at 540 and 680mV. Single oxidative response waves were exhibited for equol (Eq, 560mV), 4-ethylphenol (4eP, 560mV), enterodiol (620mV) and enterolactone (El, 620mV). Dz, El, 4eP, Eq and Gs were routinely determined in acetone/ethanol extracts of plasma, tissue homogenates and methanol diluents of tissue perfusates. Voltammetric profiles, measured as response ratios across the array, demonstrated high peak purity for sample peaks. For all analytes, lower limits of detection were 3-5 pg (signal to noise ratio of 3) with linear response of > 3 orders of magnitude and <5% R.S.D. intra-assay imprecision. This methodology has been evaluated by comparison with a GC-MS technique. This approach provides a practical, affordable means of phytoestrogen determination suitable for metabolic, pharmacokinetic and tissue distribution studies.
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