Mass spectrometric identification and quantification of glycosyl flavonoids, including dihydrochalcones with neutral loss scan mode

We developed a strategy for determination and quantification of glycosyl flavonoids using liquid chromatography–triple quadrupole mass spectrometry with neutral loss scan at 15 and 30 eV collision energy in the positive ion mode. The fragmentation patterns of glycosyl flavonoids at 15 and 30 eV showed that fragmentation of sugar moiety depended on the type of glycosidic bond to aglycone, the site of C-glycosylation, and the type of aglycone. C-Glycosyl dihydrochalcones especially stood out because they produced [M+H−162] + even at 15 eV such as O-glycoside in spite of C-glycoside. C-Glycosides were classified according to (i) the intensity ratio A of [M+H−150] + to [M+H−120] + at 30 eV and (ii) the intensity ratio B of [M+H−120] + at 15 eV to one at 30 eV. The 8- C-glycosides were A < 1 and B < 1, the 6- C-glycosides were A > 1 and B < 1, and the C-glycosyl dihydrochalcones were A > 1 and B ≫ 1. Therefore, the intensity ratios of the neutral loss scan of 120 and 150 Da at 30 eV and those of 120, 162, and 308 Da at 15 eV allowed sequential distinction among these three types of C-glycosides as well as between O- and C-glycosides. Our method was applied for analysis of Rooibos tea, and the identified glycosides could be quantified specifically by the selected reaction monitoring method.