Exploring Fluoroquinolone Protomer Populations Formed with MALDI and Plasma Post-Ionization Mass Spectrometry

Molecules that possess more than one protonation site can form protonation site isomers (protomers) in mass spectrometry. During the ionization process, a change in protonation site can alter the resulting collision-induced dissociation mass spectrum, which can hamper the accurate confirmation of unknown analytes in mass spectrometry workflows. In this study, we report the presence of protomers for ten fluoroquinolone antibiotics in a conventional matrix-assisted laser desorption ionization (MALDI) ion source and compare this against an atmospheric pressure MALDI plasma postionization (AP-MALDI-PPI) source. Irrespective of matrix composition, only the most stable protomer forms in a conventional MALDI ion source, which is consistent with a thermodynamically driven MALDI ionization process. In contrast, between 1-3 protomers form with an AP-MALDI-PPI source and this demonstrates that a different mechanism is responsible for analyte protonation. Protomer populations can be biased to favor the most stable protomer with high proton affinity MALDI matrices. Protomer populations can also be biased by doping the plasma with methanol or acetonitrile solvent vapor. Fluoroquinolone protomers are separated by trapped-ion mobility spectrometry (TIMS) and subjected to collision-induced dissociation. The protonation sites are assigned by the presence of unique fragment ions and comparing experimentally derived collisional cross sections ^TIMSCCS_N2 against trajectory method CCS_N2 calculations at the ωB97X-D/aug-cc-pVDZ//ωB97X-D/cc-pVDZ level of theory. This composite study shows that protomer population ratios change between MALDI and AP-MALDI-PPI ion sources, sample preparation method, and analyte structure, which can affect their ion mobility distributions and potentially impact their faithful confirmation.