Optimized EThcD significantly improves cyclic peptide impurity profiling, identifying specific neutral losses for atosiban and carbetocin.

Cyclic peptide drugs offer desirable therapeutic characteristics, including high selectivity and extended half-lives, but their constrained architectures and multiple intramolecular linkages present significant analytical challenges. Traditional mass spectrometry techniques often struggle with their complex fragmentation behavior, hindering accurate structural identification and impurity profiling. This gap complicates drug discovery and quality control for these promising therapeutics, necessitating advanced analytical strategies to overcome the limitations of conventional fragmentation methods for structural elucidation and impurity characterization.

Researchers systematically optimized electron-transfer/higher-energy collision dissociation (EThcD) for analyzing three classes of cyclic peptides: disulfide-bridged, ester- and disulfide-bridged, and macrolactam-bridged. The optimized EThcD method was compared against higher-energy collisional dissociation (HCD) and electron transfer dissociation without supplemental activation (ETnoD). The strategy was then applied to characterize impurities in the clinically relevant cyclic peptides atosiban and carbetocin. Targeted MS3 experiments were also conducted to investigate product ion formation mechanisms, and an EThcD MS2-CID MS3 strategy was evaluated for fragmentation mechanism studies.

Compared to HCD and ETnoD, optimized EThcD significantly increased the abundance of both c/z and b/y fragment ions, leading to improved sequence coverage for cyclic peptides. Distinct diagnostic fragmentation behaviors were observed for different cyclic peptide classes. Disulfide-bridged cyclic peptides exhibited dominant M-33 neutral losses, associated with HS• elimination. Ester- and disulfide-bridged peptides generated both M-33 and M-46 fragments, corresponding to HS• and CH2S losses, respectively. Macrolactam-bridged peptides showed characteristic M-28 neutral losses, consistent with CO elimination. These specific neutral losses serve as diagnostic markers for rapid classification and impurity identification. For sequence determination, EThcD MS2 spectra provided more regular c/z ion series and reduced spectral complexity, simplifying analysis with commercial software. Applying these optimized EThcD conditions and established fragmentation rules, impurities in atosiban and carbetocin were successfully characterized.

The method led to the identification of three impurities in atosiban and two impurities in carbetocin, demonstrating its effectiveness for structural elucidation and impurity profiling.