Structural characterization and enhanced detection of flavonoids by electrospray ionization mass spectrometry and molecular modeling
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Structural characterization and isomer differentiation of flavonoids was investigated by gas-phase hydrogen/deuterium (H/D) exchange and various metal complexation approaches using electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS). Gas-phase H/D exchange was used to probe the conformations, gas-phase acidities, and sites of deprotonation of isomeric flavonoids. The structural factors that promote or prevent H/D exchange were identified and correlated with collisionally activated dissociation (CAD) patterns and/or molecular modeling data. The use of a transition metal and an auxiliary ligand to form flavonoid/transition metal/auxiliary ligand complexes was proved to be more effective than deprotonation for differentiating isomeric flavonoids using chalcones as an example (a subclass of flavonoids). The relative threshold dissociation energies of such transition metal complexes were measured by energy-variable CAD, and the structural features that influence the threshold energies were identified. The conformations, point charges, and helium (collision gas) accessible surface areas obtained by various computational means were used to rationalize the differences in threshold energies of isomeric flavonoids. Aluminum(III) was used to form strong complexes with flavonoids for more effective isomer differentiation of flavonoids. The CAD patterns of the aluminum complexes were used to differentiate flavonoids, and the structures and conformations of representative flavonoids were also identified by high level computational means. Silver complexation was used to conquer the limitation of the transition metal and aluminum complexation (flavonoids must have a 4-keto group and at least one adjacent hydroxyl group). Silver complexation could not only be used for isomer differentiation of individual flavonoids but could be adapted to characterize flavonoids in mixtures coupled with high performance liquid chromatography (HPLC). HPLC-ESI-MS was used as a sensitive method with high selectivity to detect flavonoids in kale and grapefruit juice and metabolites in urine. The major flavonoids in kale were detected and confirmed as quercetin and kaempferol by CAD and isotopic labeling. The growing conditions were found to significantly affect the flavonoid levels in kale. LC/MS and LC/MS/MS were used to identify the individual metabolites and to gain insight into the overall metabolite profiles in human urine after consumption of grapefruit juice.