L-Ascorbic acid and two distinct anomers, namely the α-D-glucopyranosyl and β-D-glucopyranosyl-(1→2)-L-ascorbic acid (stereoisomers), were studied within the scope of collision-induced dissociation (CID) experiments, performed by linear ion acceleration and collision with argon atoms inside a hexapole quadrupole hexapole ion beam guide, which is coupled to an ion cyclotron resonance (ICR) cell with a 12 Tesla magnet for high-resolution measurements. Loss of C2H4O2 neutral from the [M–H]− anion of L-ascorbic acid was observed. Density functional theory (DFT) calculations on the 6-311+G(2d,p)//6-31+G(d) level of theory reveal a new concerted mechanism for an intramolecular gas-phase rearrangement, through which the observed ejected neutral C2H4O2 can take place. A similar rearrangement also occurs in the case of α- and β-D-glucopyranosyl-(1→2)-L-ascorbic acid. For the α isomer, only homolytic glycoside fragmentation was observed. For the β isomer, both homolytic and heterolytic glycoside cleavages were possible. The mechanisms behind all observed fragmentation pathways were fully understood by the implementation of accurate DFT calculations. Stereoisomeric differentiation between α and β isomers of the L-ascorbic acid-2-O-glucoside could be revealed by tandem mass spectrometry (MS/MS) experiments and were explained theoretically. Copyright © 2011 John Wiley & Sons, Ltd.