Characterization of Gas-Phase Charge-Site Isomerism in Flavonoids and Flavonoid Glycosides by Cyclic Ion Mobility-Mass Spectrometry with Computational Validation

Flavonoids exhibit high structural complexity not only from their diverse cores and substitutions but also from glycosylation with various glycans, leading to numerous isomeric species. This study employed cyclic ion mobility mass spectrometry (cIM-MS) to achieve high-resolution separation of flavonoid and flavonoid glycoside isomers with subtle structural differences. A multipass cIM strategy featuring adjustable drift path lengths significantly enhanced both resolution and operational flexibility. Notably, multiple ion mobility peaks were frequently observed for individual pure compounds, which were identified as charge-site isomers arising primarily from sodium adduction at distinct molecular sites. Computational chemistry and collision cross section (CCS) analysis were integrated to elucidate the three-dimensional configurations corresponding to these different mobility peaks. The results reveal that sodium ions, by coordination with multiple electronegative oxygen atoms within the molecule, stabilize specific gas-phase conformations, leading to distinct CCS values resolvable by ion mobility spectrometry. This work advances the understanding of charge-site isomerism by providing atomic-level insights into metal ion–molecule interactions. It also establishes an integrated cIM-MS and computational modeling framework, which substantially enhances analytical depth for complex isomeric systems.