Identification of isomeric flavonoid glucuronides in urine and plasma by metal complexation and LC‐ESI‐MS/MS

Noncovalent complexes were used for structural determination and isomer differentiation of flavonoid glucuronides. Several flavonoid glucuronides including naringenin‐7‐ O ‐glucuronide, synthesized here for the first time, were used as test compounds. Electrospray ionization quadrupole ion trap mass spectrometry with collision‐induced dissociation (CID) was used to analyze complexes of the form [Co(II) (L‐H) (Aux)] + and [Co(II) (L‐H) (Aux) 2 ] + , in which L is the flavonoid glucuronide and Aux is a phenanthroline‐based ligand. These complexes yielded characteristic fragmentation patterns that facilitated assignment of the substitution position of the glucuronides. The methods were adapted to liquid chromatography/tandem mass spectrometry (LC‐MS/MS) with postcolumn cobalt complexation and were tested on extracts from biological fluids. The metabolites naringenin‐7‐ O ‐glucuronide and naringenin‐4′‐ O ‐glucuronide were detected in human urine following the consumption of grapefruit juice. Isomeric quercetin glucuronides were identified and differentiated after spiking rat plasma at the 1 µ M level, proving that the new methods are effective at biologically relevant concentrations. Copyright © 2006 John Wiley & Sons, Ltd.