Distinguishing Isomeric Caffeine Metabolites through Protomers and Tautomers Using Cryogenic Gas-Phase Infrared Spectroscopy

作者：Niklas Geue, Gurpur Rakesh D. Prabhu, Eleonora Renzi, Caitlin Walton‐Doyle, Gerard Meijer, Gert von Helden, Kevin Pagel · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.5c05164 · 被引用次数：4 · 研究领域：Analytical Chemistry and Chromatography、Coffee research and impacts、Mass Spectrometry Techniques and Applications

Caffeine is metabolized through various pathways in the human body, with the primary two steps yielding isomeric products. Distinguishing these metabolites is crucial for mass spectrometry-based metabolomics, for example, to assess specific drug interactions. Here, we investigate the gas-phase structures of caffeine and its metabolites─theophylline, theobromine, paraxanthine, 1-methylxanthine, 3-methylxanthine, and 7-methylxanthine─in their respective protonated ions using cryogenic gas-phase infrared spectroscopy, supported by density functional theory. The analytes exhibit varying preferences for protonation and tautomerism, particularly N9 protonation and, where applicable, a tendency for N3O2 and N1O2 amide-imidic acid and N7N9 imine-imine tautomerism. We further demonstrate that the two isomeric sets of caffeine metabolites can easily be distinguished with gas-phase IR spectroscopy, paving the way for robust identification of such molecules in metabolomics using hyphenated gas-phase techniques.