Three Candidates, Two Peaks: Addressing Conflicting Assignments of Fentanyl Protomers with DMS-UVPD

Fentanyl and its analogs, many of which share the piperidine-4-carboxamide core, consistently exhibit two distinct peaks when detected as the protonated ion on the various low-field IMS platforms (one major, one minor). These peaks, widely presumed to arise from prototropic isomers (i.e., protomers, which are isomers differing by the site of protonation), are generally agreed to originate from protonation at the piperidine nitrogen for the major feature, the most basic site in both the solution and gas phases. However, the origin of the minor feature remains unresolved, with prior studies attributing it to protonation either at the amide nitrogen or at the carbonyl oxygen, despite theoretical predictions placing the amide-N protomer roughly 35 kJ/mol higher in energy than its O-protonated counterpart. To resolve this, we combine differential mobility spectrometry (DMS), ultraviolet photodissociation (UVPD), tandem mass spectrometry (DMS-MS²/MS³), and quantum chemical calculations to interrogate the protomer landscape of ten fentanyl analogs. Mapping of fragmentation pathways using DMS-MS²/MS³, along with DMS-MS-UVPD supported by in silico vertical-gradient Franck-Condon (VG|FC) simulations of UV absorption spectra, indicates that the major feature arises from piperidine-N protonation while the minor feature corresponds to amide carbonyl-O protonation. Collision cross section (CCS) measurements further corroborate these assignments, with calculated CCSs matching experimental values within 1% (on average) for both the N- and O-protomers, while calculated CCSs for the amide-N protomers deviate by an average of 7%. Microsolvation studies reveal that serial solvation progressively narrows the energy gap between the piperidine-N and O-protomers via the formation of a solvent bridge between the two sites, suggesting that rapid desolvation during electrospray ionization can kinetically trap a fraction of the ensemble in the higher-energy O-protonated form.