Determination of the glycosylation site in flavonoid mono-O-glycosides by collision-induced dissociation of electrospray-generated deprotonated and sodiated molecules.

The influence of the glycosylation site on the fragmentation behavior of 18 flavonoid glycoside standards was studied using positive and negative electrospray ionization mass spectrometry in combination with collision-induced dissociation and tandem mass spectrometry. The glycosylation position is shown to affect the relative abundance of the radical aglycone ions that can be observed in the [M-H]- collision-induced dissociation spectra. In particular, the radical aglycone ions are very abundant for deprotonated flavonol 3-O-glycosides. Collisional activation of the radical aglycone ions produced from positional isomers revealed minor differences: m,nB0- product ions are pronounced for 7-O-glycosides, whereas m,nA0- product ions are relatively more abundant for 4'-O-glycosides. In addition, the ratio between the radical aglycone and the regular aglycone ions in the [M+Na]+ high-energy collision-induced dissociation spectra gives an indication about the glycosylation site. This ion ratio allows the differentiation between flavonoid 3-O- and 7-O-glycosides or can be useful in the comparison of unknown compounds with standards. Unambiguous differentiation between O-glycosylation at the common positions of flavonoid O-glycosides, i.e. the 3-, 4'- and 7-positions, is achieved by collisional activation of sodiated molecules at high collision energy. The presence of a B-ring product ion containing the sugar residue indicates 4'-O-glycosylation, whereas the loss of the B-ring part from the aglycone product ion is characteristic of 3-O-glycosylation and the loss of the B-ring part from both the [M+Na]+ precursor ion and the aglycone product ion points to 7-O-glycosylation. Copyright (c) 2005 John Wiley & Sons, Ltd.