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 microM level, proving that the new methods are effective at biologically relevant concentrations.