Protomer Formation Can Aid the Structural Identification of Caffeine Metabolites

Abstract

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 two-thirds of the studied compounds formed high- and low-mobility species in high-resolution 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.

Figure 1

Base structure for caffeine and its metabolites (R₁, R₂, R₃ = −CH₃/–H).

Figure 2

Acquired arrival time distributions (ATDs) in water (light blue) and acetonitrile (dark blue) for (a) caffeine, (b) theophylline, (c) theobromine, and (d) paraxanthine. The structural differences, presence or absence of a methyl group, are highlighted with blue or green, respectively.

Figure 3

MS/MS spectra of high- and low-mobility species of theobromine (a and b, respectively) in centroid mode. The formation of main observed fragments for caffeine metabolites is shown based on the work of Bianco et al. for (c) loss of (methyl-)isocyanate and (d) loss of water.

Figure 4

Two tautomers of paraxanthine (a) and theobromine (b) cations were separated with three cycles; one species was selected (blue rectangle) to reinject for the second ion mobility measurement. Using the same separation parameters for the second IM separation, two species were separated for paraxanthine. Although the first and second IM separation last the same length of time, the time scale is shifted for second separation as an additional time for reinjection is counted in when acquiring arrival time distributions. The rapid conversion was observed for paraxanthine, 1-methylxanthine, and 7-methylxanthine, while separation of one species was successful for theobromine and 3-methylxanthine. Calculated CCS values for respective structures are presented in Table 2.

Figure 5

Structural fragments of methyl-substituted pyrimidine (without an imidazole ring), indicating similar behavior in protomer and tautomer formation.