Structure Elucidation of Macrolide Antibiotics Using MSⁿ Analysis and Deuterium Labelling

The 14- and 16-membered macrolide antibiotics are an important structural class. Ubiquitously produced by a number of bacterial strains, namely actinomycetes, purification and structure elucidation of the wide array of analogs is challenging, both for discovery efforts and methodologies to monitor for byproducts, metabolites, and contaminants. Collision-induced dissociation mass spectrometry offers an attractive solution, enabling characterization of mixtures, and providing a wealth of structural information. However, interpretation of these spectra can be difficult. We present a study of 14- and 16-membered macrolide antibiotics, including MSⁿ analysis for unprecedented depth of coverage, and complimentary analysis with D₂O and H₂¹⁸O labeling to elucidate fragmentation mechanisms. These analyses contrast the behaviors of varying classes of macrolides and highlight how analogues can be identified in relation to similar structures, which will provide utility for future studies of novel macrolides, as well as impurities, metabolites, and degradation products of pharmaceuticals. Graphical Abstract.