Received for publication February 15, 2008.
Revised April 17, 2008.
Accepted for publication April 23, 2008.

Plasma profiling of intact isoflavone metabolites by HPLC and mass spectrometric identification of flavone glycosides, daidzin and genistin in human plasma after administration of kinako

Abstract

The roles of isoflavones in the prevention of several hormone-dependent cancers and osteoporosis are of great interest. Despite many pharmacokinetics studies of the isoflavones, precise information on the actual types of conjugates circulating in the body, and the position(s) of conjugation sites on the flavone skeleton, is still uncertain. This is because, in general, conjugated compounds in biological fluids have been evaluated by measuring the free aglycones obtained after selective enzymatic hydrolysis. Using an HPLC-UV-DAD method combined with solid phase extraction, we have obtained HPLC profiles of isoflavone glycosides (daidzin, Din; genistin, Gin) and of intact isoflavone metabolites in human plasma: daidzein, genistein, daizein-7-glucuronide, daidzein-4'-glucuronide, genistein-7-glucuronide, genistein-4'-glucuronide, daidzein-7-sulfate, daidzein-4'-sulfate, genistein-7-sulfate and genistein-4'-sulfate. We investigated the plasma profile of intact isoflavone metabolites in plasma obtained 1 h - 7 h after orally administrating 50 g of kinako (baked soybean powder) to two healthy volunteers. The results of DAD (diode-array detector) analysis indicated that the main isoflavone metabolite peaks were identified on the HPLC chromatogram. Furthermore, the intact glycosides Din and Gin were detected in 1 h plasma samples by their positive ESI-MS, demonstrating that the glycoside Din and Gin can be absorbed from the gut.

Key words: glucuronidation, HPLC, mass spectrometry, metabolite identification, metabolite kinetics, sulfate conjugation