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 m/z 421 product ion, along with other shared fragments at m/z 395 and m/z 114. Product ion m/z 308 was unique
to protomer 1, while m/z 334, m/z 307, m/z 281,276 and m/z 87 were unique
to protomer 2. To investigate the origins of m/z 421 and m/z 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 m/z 421 ions further yielded unique as well as some common
fragments upon dissociation. We propose a charge remote hydrogen transfer
mechanism for formation of m/z 421
and m/z 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.