Unveiling Flavonoid Reactivity: A High-Resolution Mass Spectrometry Journey Through the Silylation of Quercetin

Rationale: Quercetin is an example of a pentahydroxylated flavonol compound studied extensively due to its excellent biological activity (e.g., antioxidant, antiviral, and antimicrobial). The antioxidant properties of flavonoids are influenced by the arrangement of the substituents around the molecule, a phenomenon known as the structure–activity relationship (SAR). SAR studies the relationship between compounds' structural characteristics and their biological activity in drug design. Methods: Quercetin was silylated with MTBSTFA using a semiautomated flow system, and the order in which hydroxyl groups were silylated was used to derive the sequential reactivity of quercetin. Results: The collision-induced dissociation MS/MS fragmentation of the precursor ion of quercetin is influenced by the electrospray ionization (+ and − modes) and an increase in collision energy (CE). Structure elucidation with in-depth high-resolution tandem mass spectrometric analysis revealed that silylation primarily occurs at A7 and is sequentially followed by B3′, B4′, C3, and A5. Conclusions: Retro-Dials Alder cleavage of the C-ring plays a significant role in the MS/MS fragmentations of silylated quercetin, maintaining the integrity of the fragment ions and subsequently allowing tracking of the position of the silyl group.