Strategies for the Identification of Cyclic Peptide Drugs and Their Impurities

Cyclic peptide drugs pose significant analytical challenges due to their constrained architectures and multiple intramolecular linkages, which complicate fragmentation behavior and hinder structural identification. In this study, electron-transfer/higher-energy collision dissociation (EThcD) was systematically optimized to analyze three clinically relevant classes of cyclic peptides: disulfide-bridged, ester- and disulfide-bridged, and macrolactam-bridged peptides. Compared with higher-energy collisional dissociation (HCD) and electron transfer dissociation without supplemental activation (ETnoD), EThcD significantly increased the abundance of both c/z and b/y fragment ions, providing an improved sequence coverage. Distinct diagnostic fragmentation behaviors were observed. Disulfide-bridged cyclic peptides exhibited dominant M33 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. Macrolactam-bridged peptides showed characteristic M-28 neutral losses consistent with CO elimination. These neutral losses serve as diagnostic markers for rapid classification and impurity identification. Targeted MS3 experiments further supported the proposed formation mechanisms of these product ions. An EThcD MS2-CID MS3 strategy was also evaluated to investigate fragmentation mechanisms. However, for sequence determination, EThcD MS2 spectra provided more regular c/z ion series and reduced spectral complexity, enabling straightforward analysis using commercial software. Applying optimized EThcD conditions and established fragmentation rules, impurities in atosiban and carbetocin were characterized, leading to the identification of three impurities in atosiban and two in carbetocin. This approach demonstrates the effectiveness of EThcD for structural elucidation and impurity profiling of cyclic peptide therapeutics.