Simultaneous determination of glucuronic acid and sulfuric acid conjugated metabolites of daidzein and genistein in human plasma by high-performance liquid chromatography

Isoflavone aglycones daidzein (Dein) and genistein (Gein) are mainly present as glucuronides and sulfates in human plasma, and small amounts of the intact aglycones are also detected. In the present study, we have developed a high-performance liquid chromatography (HPLC)-UV-diode-array detector (DAD) method for the determination of intact 16 metabolites of Dein and Gein in plasma, especially focusing on highly polar conjugated metabolites at both 4′ and 7 positions on the isoflavone ring with glucuronic acid and/or sulfuric acid (7-glucuronide-4′-sulfates and 4′,7-diglucuronides). Luteolin-3′,7-di-O-glucoside was used as an internal standard. Solid-phase extraction was performed on an Oasis® HLB cartridge (60 mg, 3 cm3) with a recovery of >ca. 80%. The HPLC assay was performed on a Hydrosphere C18 column (100 mm × 4.6 mm I.D., particle size 3 μm). The mobile phase consisted of a mixture of 10 mM ammonium acetate solution and acetonitrile run under gradient mode at a flow rate of 1.5 ml/min. The UV detection wavelength was set at 250 nm. For UV spectral analysis, the diode-array detection wavelength was set at 220–360 nm. All HPLC analyses were performed at 45 °C. Each calibration for the determination of 16 metabolites gave a linear signal (r > 0.997) over a concentration range of 5–5000 ng/ml. The lower limits of quantification of these metabolites were 21.1–23.4 ng/ml and the lower limits of detection were 7.9–9.4 ng/ml. This method was used in a preliminary experiment to determine the plasma concentration of intact 16 metabolites after oral administration of kinako (baked soybean powder) to a healthy volunteer. The present HPLC-UV-DAD method should be useful for the metabolic and pharmacokinetic investigations of isoflavones in humans.