Origins of Fluoroquinolone Protomers: Tuning and Separation in MALDI and Plasma Post Ionization.

Matrix-assisted laser desorption ionization (MALDI) is a powerful technique to liberate and ionize analyte molecules for mass spectrometry but the underlying mechanisms of ionization are not fully understood. Protons are one of the most common charge carriers in MALDI. For molecules with multiple protonation sites, this can give rise to analyte ion populations composed of multiple protonation-site isomers (protomers), which only differ by the site of proton attachment. This seeming nuance of proton location can result in profound changes in analyte ion stability, affect fragmentation efficiencies, alter tandem mass spectrometry spectra and ion mobility—all while maintaining the m/z ratio. Investigating protonation site isomers provides insight into the ionization mechanism as analytes undergo the phase transition from condensed to gas-phase. In this study, protonation isomers for ten fluoroquinolone antibiotics are investigated using MALDI and only the most stable protomers form, irrespective of matrix composition, and this is consistent with a MALDI gas-phase equilibrium model. In contrast, multiple protomers are formed when using a modified atmospheric pressure MALDI plasma post-ionization source and this demonstrates a different protonation mechanism. Relative protomer populations are influenced by the choice of matrix and this can be controlled by externally doping the plasma source with solvent vapor. Experimental trapped-ion mobility spectrometry collisional cross sections are matched against protomer simulations from trajectory method calculations at the ωB97X-D/aug-cc-pVDZ//ωB97X-D/cc-pVDZ level. This composite study provides insight into two fundamentally different ionization processes and demonstrates how protomer systems provide insight into the underlying mechanisms of protonation.