Unveiling Ciprofloxacin Protonation Isomers: an Integrated Approach with Mass Spectrometry, Ion Mobility Spectrometry and Infrared Ion Spectroscopy.

Ciprofloxacin is a widely studied fluoroquinolone antibiotic and serves here as a relevant test case for molecules exhibiting multiple protonation isomers (protomers) with abundances that vary depending on the ionization conditions using mass spectrometry (MS). Understanding the protonation behavior of such molecules during positive-ion electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) is interesting from a perspective of fundamental ion chemistry, but also crucial in achieving efficient ionization, detection, and accurate identification in an analytical context. For example, different protomers may exhibit different fragmentation patterns in tandem MS applications. While previous research has utilized MS with ion mobility spectrometry (IMS) or infrared ion spectroscopy (IRIS) to analyze ciprofloxacin, this study integrates all three techniques by performing ion mobility-selected IRIS experiments to definitively assign the protomers of ciprofloxacin and of related piperazinyl building blocks. This combined approach exposes a subtle scenario in which the type of ion source and its mode of operation influence the protomeric structures that are produced and, moreover, where the downstream ion transfer and storage stages may induce proton-migration. These findings provide guidance for the development of general workflows for mass spectrometry-based assessment of analytes with multiple protomers.