Characterization of the interglycosidic linkage in di-, tri-, tetra- and pentaglycosylated flavonoids and differentiation of positional isomers by liquid chromatography/electrospray ionization tandem mass spectrometry.

Abstract

The LC/UV-DAD/ESI-MSn negative fragmentation mode of 23 O-glycosylated flavonoids with two, three, four and five hexoses was studied. The results show that it is possible to differentiate the (1-->2) and (1-->6) interglucosidic linkages and also to discern between the flavonoid isomers with two glucoses (sophorosides, gentiobiosides and X,Y-diglucosides), three glucoses (sophorotriosides and X-sophoroside-Y-glucoside) and four glucoses (X-sophorotriosides-Y-glucoside and X-sophoroside-Y-sophoroside). In the characterization of the (1-->2) and (1-->6) interglycosidic linkages, the Y1- (-162 u) and Z1- (-180 u) ions play a relevant role. In the first case ions with high relative abundance (13-79%) are found, whereas in the other cases they are in very low abundance or absent. X,Y-di-O-glucoside flavonoids can be differentiated from the O-diglucoside flavonoids by the presence of Y1- (base peak) and Y0- (approximately 30%) ions and the absence of Z1- ions. Regarding flavonoids glycosylated with three glucoses, X-sophoroside-Y-glucoside flavonoids show the Y7(0-) (-162 u) ion as the only peak in MS2 events whereas in sophorotrioside flavonoids various ions due to intermediate fragmentations are observed. These ions are characteristic of a (1-->2) interglucosidic linkage. In MS2 experiments on flavonoids with four glucoses (X-sophorotrioside-Y-glucoside and X-sophoroside-Y-sophoroside), the base peak indicated the total loss of the sugar moieties in position 7. In addition, the characterization of the type of interglycosidic linkage in flavonoids glycosylated with five sugars can be achieved. On the other hand, in tetra- and pentaglycosylated flavonoids, the ions that characterize the (1-->2) interglucosidic linkage formed by intermediate fragmentation of the oligosacharide residues (sophorosides and sophorotriosides) are found in much higher relative abundance in MS3 than in MS2 experiments, where they are almost not detected.