Regioselectivity of Human UDP-Glucuronsyltransferase 1A1 in the Synthesis of Flavonoid Glucuronides Determined by Metal Complexation and Tandem Mass Spectrometry

• Published in: Journal of the American Society for Mass Spectrometry 2007-11, Vol.19 (2), p.246-256
• Main Authors: Davis, Barry D, Brodbelt, Jennifer S
• Format: Article
• Language: English
• ISSN: 1044-0305
• DOI: 10.1016/j.jasms.2007.11.004

A three-part tandem mass spectrometric strategy that entails MS n analysis and a post-column LC-MS cobalt complexation method is developed to identify flavonoid monoglucuronide metabolites synthesized using the 1A1 isozyme of human UDP-glucuronosyltransferase (UGT). Ten flavonoid aglycons were used as substrates, spanning the subclasses of flavones, flavonols and flavanones. The products were characterized by LC-MS and LC-MS n , with post-column cobalt complexation employed to pinpoint the specific sites of conjugation. The dissociation of complexes of the form [Co(II) (flavonoid glucuronide – H) (4,7-diphenyl-1,10-phenanthroline) 2 ] + allowed identification of the products and differentiation of isomers. The correlation between glycosylation site and elution order is used to provide additional structural confirmation. Flavonoids lacking a 3′ hydroxyl group were glucuronidated only at position 7, while those containing this functionality also formed 3′-O-glucuronides and sometimes 4′-O-glucuronides, thus supporting the conclusion that the presence or absence of the 3′-OH group is the major determinant of the regioselectivity of glucuronidation. Moreover, the specific distribution of multiple glucuronide products (7-O, 3′-O, 4′-0) is governed by the subclass of flavonoid.