Fragmentation patterns and structural diagnostic characteristics of 15 flavonoids based on high-resolution mass spectrometry

The structural diversity of flavonoids poses a significant challenge for their rapid identification in biological matrices such as plasma, tissues, and food extracts. Using UHPLC-Orbitrap HRMS in positive electrospray ionization mode with stepped collision energies (60%, 100%, 150%), we systematically investigated the fragmentation behavior of 15 flavonoid reference standards covering flavones, flavonols, isoflavones, flavanones, dihydroflavonols, methoxylated flavonoids, and glycosides. A near-universal strong fragment at m/z 68.997 (C₃HO₂⁺) arising from C-ring 0,4-cleavage was observed for aglycones and O-glycosides but was absent in C-glycosides. A prominent ¹,³A⁺ ion at m/z 153.018 signals a 5,7-dihydroxy A-ring, while ions at m/z 123.008 and 95.013 indicate a 5,6,7-trihydroxy pattern (baicalein type). On the B-ring, the ion at m/z 65.038 (C₅H₅⁺) marks a 4'-OR group, and the relative intensities of m/z 121.028 (⁰,²B⁺) and m/z 135.044 (¹,³B⁺) distinguish 4'-monohydroxy from 3',4'-dihydroxy substitution patterns. Rhamnose-containing O-glycosides yield characteristic ions at m/z 71.049 and 85.028, whereas C-glycosides give a triple-ion series from successive losses of 120, 150, and 178 Da. Dihydroflavone O-glycosides show ¹,³A⁺ as the base peak, while dihydroflavonols show ¹,²B⁺. Flavonols exhibit a diagnostic [M-CO]⁺/[M+H-CO]⁺ doublet. These empirically derived fragmentation rules, based on 15 reference standards, provide a practical framework for flavonoid identification. To our knowledge, this is the first systematic compilation of manually validated positive-ion diagnostic fragment-ion rules covering multiple flavonoid subclasses under unified high-resolution mass spectrometry conditions, with explicit exceptions and cross-interferences listed for each diagnostic ion. The rules are preliminary and require validation on a larger and more diverse set of flavonoids.