Direct Identification and Quantification of Flavonoids and Their Structural Isomers Using Ambient Ionization Tandem Mass Spectrometry

ABSTRACT Rationale Flavonoids are phenolic compounds with many health‐benefiting properties. However, differentiating different types of flavonoids and their isomers is challenging due to their highly similar structures of various subtypes and different numbers and sites of substituents. Timely quality evaluation of flavonoid‐based products is currently almost impossible. Methods An ambient ionization method of direct analysis in real time (DART) ion source and tandem mass spectrometry (MS) was used to characterize the fine structures of flavonoids. Different flavonoid subtypes and their isomers with varied numbers and sites of substituents were subjected to DART ionization and collision‐induced fragmentation MS analysis. Results Seven classes of flavonoids, including methoxy‐substituted compounds, exhibited distinctive fragmentation pathways such as retro‐Diels‐Alder reactions, cross‐ring cleavages, and neutral losses. Many flavonoid isomers produced diagnostic MS 2 and MS 3 fragments through DART‐tandem MS, enabling direct identification of various isomers within mixtures. An identification workflow was developed, culminating in the creation of a computational tool called FlavoFinder, which automatically determines flavonoid aglycone subtypes and their isomeric structures. Conclusions The method and the structural elucidation program were successfully used for the qualitative and quantitative analysis of different flavonoid isomers from real samples. The analysis procedure is high‐throughput and is capable of characterizing complex flavonoid structures without extensive sample pretreatment and front‐end chromatographic separations.