Lysine is the principal amino acid that participates in the Maillard reaction between polypeptides and reducing sugars in foods and in vivo. For mechanistic studies of the early Maillard reaction, diagnostic MS/MS fragmentations have been recently employed to discriminate between Amadori rearrangement products (ARP) and Schiff bases of glycated aliphatic amino acids. However, the utility of this method to identify ARP and Schiff bases of glycated dibasic amino acids, such as lysine, has not been explored yet. In this study, we report the potential of MS/MS fragmentations in the negative ionization mode to distinguish Schiff bases of Nα,Nε-diglycated lysines from their Amadori isomers, as well as utilization of 15 N-labeled lysines and the MS/MS fragmentations under positive ionization mode to distinguish between the Nα- and Nε-regioisomers of mono-glycated lysine. The applicability of this approach is exemplified in a mass-spectrometric analysis of a solid-state reaction between lysine and glucose in a ball mill. The analysis of the MS/MS fragmentation pattern of the glycated adduct has revealed the simultaneous formation of mono-glycated adducts, mainly Nε-Schiff bases, and di-glycated adducts, predominantly a mixture of Nα,Nε-Schiff–Schiff and Nα,Nε-Schiff–Amadori isomers. [ABSTRACT FROM AUTHOR]