Identification of Isoflavone Metabolites Dihydrodaidzein, Dihydrogenistein, 6′-OH-O-dma, and cis-4-OH-equol in Human Urine by Gas Chromatography–Mass Spectroscopy Using Authentic Reference Compounds

Abstract

The metabolic products of daidzein and genistein, the principal isoflavones of soy, were examined. Six volunteers included soy into their normal diet for a 2-week period and urine samples were analyzed before and after soy consumption. Isolation and characterization of the urinary metabolites were carried out with absorption chromatography on Sephadex LH-20 and gas chromatography–electron ionization mass spectrometry (GC–EIMS). The structures of the isoflavones isolated were confirmed by using authentic reference compounds. Dihydrogenistein, 6′-OH-O-desmethylangolensin, and cis-4-OH-equol were identified, in addition to known isoflavonoids daidzein, genistein, glycitein, and the known metabolites equol, O-desmethylangolensin, and dihydrodaidzein, by comparing the retention times and the spectra of the urinary compounds with those of the synthesized reference standards. The mammalian lignans enterolactone and enterodiol were also identified. Derivatization of the isoflavones for GC–MS was examined by comparing two silylating reagents, N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and pyridine:hexamethyldisilazan:trimethylchlorosilane (QSM), both used for the derivatization of these compounds. The silylation experiments revealed significant differences in the compositions of the derivatization products. Some corrections were made concerning the earlier published data of dihydrogenistein and 6′-OH-O-dma.