Dark Reactions in Microdroplets Explain Widespread Artifacts in Metabolomic Profiling

Area of Science:

• Analytical Chemistry
• Biochemistry
• Metabolomics

Background:

• Mass-spectrometry-based untargeted metabolomics faces significant challenges in reliable ion annotation and identification.
• A large number of unexplained ions are frequently observed, complicating data interpretation.

Purpose of the Study:

• To elucidate the identities, sources, and formation mechanisms of previously unexplained ions in untargeted metabolomics.
• To demonstrate how microdroplets formed during electrospray ionization contribute to artifactual ion generation.

Main Methods:

• Investigated chemical transformations occurring within microdroplets during electrospray ionization.
• Analyzed ion formation mechanisms including redox, addition, condensation, reductive amination, decarboxylative coupling, and radical reactions.
• Examined the role of reactive oxygen and nitrogen species at the gas-aqueous interface.

Main Results:

• Microdroplets promote diverse chemical reactions, generating artifactual ions (false positives) from metabolite activation.
• These artifacts are often misassigned as endogenous metabolites, contributing to numerous unidentified features.
• In one case, artifact identification improved annotation rates from 9% to over 50% in untargeted metabolomics data.

Conclusions:

• Microdroplet-induced chemical transformations are a major source of artifactual ions in untargeted metabolomics.
• Addressing these artifacts is crucial for improving the accuracy and reliability of metabolomic analyses.
• Practical strategies can be implemented to minimize artifact formation and enhance metabolite identification.