Kinetic Control of Protonation in Electrospray Ionization

The site of protonation in a molecule can greatly affect the fragments observed in product ion MS/MS spectra. In electrospray positive ionization mass spectra, protonation usually occurs predominantly on the most basic site on the molecule to produce the thermodynamically favored protonated species. However, the literature is unclear whether liquid phase or gas phase thermodynamics has the greater influence. This paper describes the protonation and fragmentation behavior of crizotinib and two of its impurities. Crizotinib has two possible protonation sites, a pyridine nitrogen and a secondary amine, piperidine nitrogen; the former is the favored site in the gas phase and the latter the more favored site in the liquid phase. The impurities contain alkyl substitution on the piperidine nitrogen, producing tertiary amine species. Literature precedence suggests that in the liquid phase, the piperidine nitrogen is still the most basic site but, in the gas phase, the pyridine nitrogen and the piperidine nitrogen have very similar basicities. Fragmentation data for the three molecules suggest that the secondary and tertiary amines protonate preferentially and almost exclusively on different sites. We propose that the secondary amine protonates on the piperidine nitrogen (influenced by solution thermodynamics) and the two tertiary amine structures protonate on the pyridine nitrogen because of steric hindrance at the most basic site of the molecule, allowing kinetic control of the protonation process.