Identification of Glyceollin Metabolites Derived from Conjugation with Glutathione and Glucuronic Acid in Male ZDSD Rats by Online Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

[Image: see text] Glyceollin-related metabolites produced in rats following oral glyceollin administration were screened in plasma, feces, and urine, and these metabolites were identified by precursor and product ion scanning using liquid chromatography coupled online with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Precursor ion scanning in the negative ion (NI) mode was used to identify all glyceollin metabolites based on production of a diagnostic radical product ion (m/z 148) upon decomposition. Using this approach, precursor peaks of interest were found at m/z 474 and 531. Tandem mass spectra of these two peaks allowed us to characterize them as byproducts of glutathione conjugation. The peak at m/z 474 was identified as the deprotonated cysteinyl conjugate of glyceollins with an addition of an oxygen atom, whereas m/z 531 was identified as the deprotonated cysteinylglyceine glyceollin conjugate plus an oxygen. These results were confirmed by positive ion (PI) mode analyses. Mercapturic acid conjugates of glyceollins were also identified in NI mode. In addition, glucuronidation of glyceollins was observed, giving a peak at m/z 513 corresponding to the deprotonated conjugate. Production of glucuronic acid conjugates of glyceollins was confirmed in vitro in rat liver microsomes. Neither glutathione conjugation byproducts nor glucuronic acid conjugates of glyceollins have been previously reported.