Identification of fluoroquinolone protonation isomers in mass spectrometry using ion mobility spectrometry and infrared ion spectroscopy

Fluoroquinolone antibiotics are compounds containing multiple basic sites, which allows for the formation of multiple protonation isomers (protomers) in acid-base reactions. In mass spectrometric analyses using electrospray ionization (ESI), the actual site of protonation affects the gas-phase structure and reactivity of the ions, which can lead to differences in tandem mass spectrometry (MS/MS) spectra and collision cross section (CCS) values. This structural variability may depend on subtle differences in ESI conditions, which may therefore complicate compound identification based on MS/MS and/or CCS reference library data. While previous ion mobility spectrometry (IMS) studies investigated protomer formation in ESI for several fluoroquinolones, this study combines MS, IMS, and infrared ion spectroscopy (IRIS) to establish the protomers actually present in the mass analyzer. As a follow up to our recent investigation of ciprofloxacin, we extend our studies here to include pazufloxacin, gemifloxacin, moxifloxacin and danofloxacin. Differences in ion mobility distributions were observed between the investigated compounds, indicating variations in the number and nature of protomers formed. These differences are related to structural features of the molecules, which influence protonation preferences as well as the likelihood of proton migration in the gas phase. In particular, the structure of the molecule and relative positioning of protonation sites can enable proton migration during ion transfer and storage prior to IRIS interrogation, altering the protomer distribution even after IMS separation. These results highlight the importance of considering protomer formation in analytical workflows and show that combining IMS and IRIS can help to better understand and resolve protomer-related complexity in MS workflows.