Infrared Ion Spectroscopy Combined with Ion Mobility Spectrometry for Identification of Caffeine Metabolite Isomers and Protomers.

Chromatography combined with tandem mass spectrometry is a conventional strategy for metabolite differentiation and identification. However, coelution and overlapping fragmentation patterns often limit confident assignment of isomeric and isobaric species. Caffeine metabolites represent a particularly challenging case. Here, we demonstrate a high-field asymmetric waveform ion mobility spectrometry (FAIMS) approach coupled with infrared multiple photon dissociation (IRMPD) ion spectroscopy for the identification of the major caffeine metabolites paraxanthine (PX), theobromine (TB), and theophylline (TP). FAIMS provided baseline separation of these isomers into four distinct populations, including two distinct protomers of protonated TB. The nature of each FAIMS population was probed by IRMPD, which enabled structural assignment and revealed unique protomeric signatures for PX and TP. This combined FAIMS-IRMPD workflow not only resolves isomeric metabolites but also distinguishes protomeric and tautomeric forms, expanding the scope of ion mobility-spectroscopy approaches in metabolite analysis.