Structural elucidation of 14‐membered ring macrolide antibiotics using electrospray ionization tandem mass spectrometry and density functional theory calculations

Rationale Macrolides are critical antibiotics featuring a macrocyclic lactone core with deoxy sugars. Understanding their gas‐phase fragmentation is challenging but essential for improving structural elucidation in mass spectrometry, which has implications for drug discovery and development. Methods We used electrospray ionization collision‐induced dissociation tandem mass spectrometry (ESI‐CID‐MS) combined with quantum chemical calculations to investigate the fragmentation pathways of erythromycin A and roxithromycin. This approach helps elucidate the preferred fragmentation routes influenced by protonation sites. Results Macrolides showed similar fragmentation patterns, including sequential losses of saccharide or amino sugar units and dehydration from the macrocycle core. Multiple competitive pathways were observed, influenced by protonation sites. Computational studies confirmed the most favorable protonation sites and their impact on fragmentation, providing insights into key diagnostic product ions. Subsequent fragments involved rearrangement pathways such as alkene formation and cleavages via remote hydrogen transfers and pericyclic reactions. Conclusions Our integrated approach offers a comprehensive understanding of macrolide fragmentation, enhancing structural elucidation and potential applications in drug development. This study advances mass spectrometry analysis of macrolides, contributing to pharmaceutical research by integrating orthogonal annotation methods and fragmentation studies.