Dark Reactions in Microdroplets Explain Widespread Artifacts in Metabolomic Profiling

Reliable ion annotation and identification remain persistent challenges in mass-spectrometry-based untargeted metabolomics. Here, we elucidate the identities, sources, and formation mechanisms of numerous previously unexplained ions by showing that microdroplets formed during electrospray ionization can promote a wide array of chemical transformations. These include redox, addition, condensation, reductive amination, decarboxylative coupling, and radical reactions, many of which are facilitated at the gas-aqueous interface by reactive oxygen and nitrogen species. Activation of metabolite chemical bonds generates cations, anions, and radical intermediates through the loss of protons, electrons, or functional groups, ultimately leading to bond formation and the generation of artifactual ions or false positive ions. These artifacts are frequently misassigned as endogenous metabolites and account for hundreds of thousands of previously unidentified features. As an example, we show that in a recently published untargeted metabolomics analysis of 1969 ions, the annotation rate was substantially improved to over 50% from the previous value of 9%, showing the importance of this new form of identification. Finally, we describe practical strategies to minimize artifactual ion formation, thereby improving the reliability of metabolomic analyses.