Protomer Formation Can Aid the Structural Identification of Caffeine Metabolites

The structural annotation of isomeric metabolites remains a key challenge in untargeted electrospray ionization/high-resolution mass spectrometry (ESI/HRMS) metabolomic analysis. Many metabolites are polyfunctional compounds that may form protomers in electrospray ionization sources and therefore yield multiple peaks in ion mobility spectra. Protomer formation is strongly structure-specific. Here, we explore the possibility of using protomer formation for structural elucidation in metabolomics on the example of caffeine, its eight metabolites, and structurally related compounds. It is observed that twothirds of the studied compounds formed high- and low-mobility species in highresolution ion mobility. Structures in which proton hopping was hindered by a methyl group at the purine ring nitrogen (position 3) yielded structure-indicative fragments with collision-induced dissociation (CID) for high- and low-mobility ions. For compounds where such a methyl group was not present, a gas-phase equilibrium could be observed for tautomeric species with two-dimensional ion mobility. We show that the protomer formation and the gas-phase properties of the protomers can be related to the structure of caffeine metabolites and facilitate the identification of the structural isomers.

Automated summary

The structural annotation of isomeric metabolites is a significant challenge in untargeted metabolomic analysis. This study explores the use of protomer formation for structural elucidation in metabolomics, using caffeine and its metabolites as examples. The results show that the formation of protomers is structure-dependent and can facilitate the identification of structural isomers.