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

TL;DR

This paper introduces a new method combining ion mobility and infrared spectroscopy to identify caffeine metabolites that are difficult to distinguish using traditional techniques.

Contribution

The novel workflow using FAIMS and IRMPD enables the separation and identification of caffeine metabolite isomers and protomers.

Findings

FAIMS separated caffeine metabolites into four distinct populations, including two protomers of theobromine.

IRMPD spectroscopy revealed unique structural signatures for paraxanthine and theophylline.

The combined workflow distinguishes isomeric metabolites and their protomeric forms.

Abstract

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.

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