Structural characterization of isomeric flavonoid glycosides and metabolites by metal complexation and electrospray ionization tandem mass spectrometry
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Flavonoids form a vast group of natural products that occur ubiquitously throughout the plant kingdom. These compounds play a significant role in the field of phytochemistry and are of nutritional interest due to numerous reports of their benefits to human health. Structural characterization of individual flavonoid derivatives is challenging because of widespread isomerism and a lack of sensitive and specific analytical techniques. The goal of this work is to present practical tandem mass spectrometry methods for systematic isomer differentiation of flavonoid glycosides and flavonoid glucuronides. Metal complexation is used extensively as a strategy to achieve this aim. In this approach, flavonoid derivatives and metal ions are mixed in solution, resulting in the rapid self-assembly of complexes which are subsequently infused into a quadrupole ion trap mass spectrometer. Collision-induced dissociation of these flavonoid/metal complexes results in fragment ions that are highly characteristic of specific structural features of the flavonoid derivatives. These methods are adaptable to LC-MS analysis via post-column addition of the complexation reagents. Methods to differentiate the five most common glycosylation sites of monoglucosyl flavonoids are described. Based on the fragment ions yielded from magnesium or manganese complexes, specific indicators of 3-O-glucosylation, 7-O-glucosylation, 4'-O-glucosylation, 6-C-glucosylation and 8-C-glucosylation are observed. The manganese complexation method also differentiates isomeric glucose and galactose sugars at the 3 position, as well as arabinose and xylose sugars. Differentiation of isomeric flavonoid glucuronide metabolites is achieved by cobalt complexation with auxiliary ligands. The effectiveness of these methods is proven in numerous practical applications. Flavonoid glycosides are identified in extracts from apples, onions, and Silphium albiflorum. Flavonoid glucuronide metabolites are identified in urine samples and a cell culture extract. The identifications are achieved without the use of standards or additional analytical techniques. Finally, an enzymatic synthesis of flavonoid glucuronides is used to establish the regioselectivity of UGT1A1, an enzyme involved in flavonoid metabolism. The cobalt complexation method successfully identifies many hitherto uncharacterized metabolites. The significance of this work lies in its potential application to problems in botanical, agricultural, nutritional and disease-prevention studies in which precise flavonoid identification is required.