Cyclic IMS-MS Reveals Protonation-Site Preservation in Isobaric Fragments of Quizartinib Protomers via Charge-Remote Fragmentation

The protonation site of a molecule can significantly influence its gas-phase behavior and fragmentation, especially when multiple protonation sites are accessible. Here, we characterize two gas-phase protonation site isomers (hereafter referred to as protomers) of quizartinib using cyclic ion mobility-mass spectrometry (cIMS-MS), tandem MS, and molecular modeling. Despite density functional theory (DFT) calculations indicating a gas-phase preference for protonation at the central imidazole nitrogen (hereafter N21), two mobility-separated species were observed, suggesting kinetic trapping of a solution-phase protomer. To probe this hypothesis, solvent-phase molecular modeling using implicit water and acetonitrile models was performed, revealing that the morpholine nitrogen (hereafter N15) is the most favorable protonation site in solution. This supports a dual-phase model: one protomer arises from the liquid-phase favored protonation site, and the other from the gas-phase protonation site. Post-IMS fragmentation of the protomers revealed a common <i>m</i>/<i>z</i> 421 product ion, along with other shared fragments at <i>m</i>/<i>z</i> 395 and <i>m</i>/<i>z</i> 114. Product ion <i>m</i>/<i>z</i> 308 was unique to protomer 1, while <i>m</i>/<i>z</i> 334, <i>m</i>/<i>z</i> 307, <i>m</i>/<i>z</i> 281,276 and <i>m</i>/<i>z</i> 87 were unique to protomer 2. To investigate the origins of <i>m</i>/<i>z</i> 421 and <i>m</i>/<i>z</i> 395, these ions were generated by pre-IMS activation, isolated, and subjected to cIMS separation. Both ions exhibited two distinct arrival time peaks, indicating that they retain the protonation-site memory of their precursors. The two mobility-separated <i>m</i>/<i>z</i> 421 ions further yielded unique as well as some common fragments upon dissociation. We propose a charge remote hydrogen transfer mechanism for formation of <i>m</i>/<i>z</i> 421 and <i>m</i>/<i>z</i> 395, initiated from either the morpholine (N15) or imidazole (N21) protonation. Structural assignments were proposed for the major common and unique product ions of each protomer. These findings highlight a mechanistic link between solution- and gas-phase protonation and demonstrate the utility of cIMS-MS for probing structure-specific fragmentation and isobaric dissociation product ion resolution in small molecules with multiple heteroatoms.