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Flavonoids are a class of secondary plant metabolites existing in great variety in nature. Due to this variety, identification can be difficult, especially as overlapping compounds in both chromatographic separations and mass spectrometric detection are common. Methods for distinguishing isobaric flavonoids using MS2 and MS3 have been developed. Chromatographic separation of various plant extracts was done with RP-HPLC and detected with positive ESI-MS operated in information-dependent acquisition (IDA) mode. Two methods for the determination of flavonoid identity and substitution pattern, both featuring IDA criteria, were used together with the HPLC equipment. A third method where the collision energy was ramped utilized direct infusion. With the developed strategies, it is possible to differentiate between many isobaric flavonoids. Various classes of flavonoids were found in all of the plant extracts, in the red onion extract 45 components were detected and for 29 of them the aglycone was characterized, while the substituents were tentatively identified for 31 of them. For the strawberry extract, those numbers were 66, 30 and 60, and for the cherry extract 99, 56 and 71. The great variety of flavonoids, several of them isobaric, found in each of the extracts highlights the need for reliable methods for flavonoid characterization. Methods capable of differentiating between most of the isobars analyzed have been developed. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]