Structural elucidation of succinimide-derived hydrolytic isomers of atosiban using a conventional CID-based Asp/IsoAsp discrimination strategy.

The succinimide-mediated degradation system of atosiban generates four hydrolytic impurities that share identical mass shifts and modification sites, posing a significant analytical challenge in peptide drug development. In this study, a discrimination strategy for Asp/isoAsp isomers was developed under conventional collision-induced dissociation (CID) conditions by exploiting subtle differences in MS/MS fragmentation patterns, in combination with the oxazolone-mediated b-ion formation mechanism. The observed fragmentation behavior is mechanistically rationalized by the conformational constraint of isoAsp residues in the gas phase. This study demonstrates, for the first time, that Asp and isoAsp impurities in singly charged peptide ions can be differentiated using only widely accessible CID techniques, without reliance on high charge-state fragmentation methods such as electron transfer dissociation (ETD) or electron capture dissociation (ECD), or specialized instrumentation such as electron activated dissociation (EAD). This significantly enhances the practical feasibility of the method in industrial quality control settings. The D/L configurations of the hydrolytic products were assigned based on an α-carbon carbanion-mediated racemization mechanism, supported by hydrolysis experiments under acidic and basic conditions. As a result, complete structural elucidation of four hydrolytic impurities with identical mass and modification sites was achieved. This work provides a practical and mechanistically grounded analytical strategy for the structural characterization of succinimide-derived impurities in peptide drugs, with potential applicability to other peptide systems sharing similar structural features.