Revealing misannotated electrospray ionization artifacts: A systematic pipeline for heterodimer detection in untargeted metabolomics

Heterodimers are non-covalent adducts formed between two co-eluting molecules during electrospray ionization (ESI). Despite their frequent occurrence in MS-based untargeted metabolomics and lipidomics analysis, these artifacts are often misinterpreted as genuine metabolites or entirely overlooked, leading to inflated feature counts, compromised annotation accuracy, and contributing to the so-called dark metabolome. Here, we present a systematic MS1-based pipeline to detect and characterize heterodimers integrating coelution analysis, mass difference evaluation, and validation using binary standard mixtures. We applied this workflow to detect and annotate heterodimers present in untargeted datasets from different separation platforms: (i) polar heterodimers present in a human plasma analyzed by CE-ESI(+)-TOF-MS. (ii) lipid heterodimers from the reference human plasma NIST® SRM® 1950 and rat heart tissue extracts, both analyzed by RP-UHPLC-ESI(+/−)-QTOF-MS. Considering both separation platforms, a total of 124 heterodimers were identified: 101 in ESI(+) and 23 in ESI(−). These predominantly involved lipid species, such as glycerophosphatidylcholines, glycerophosphatidylethanolamines and sphingomyelins in LC-MS, along with polar metabolites, including basic amino acids, in CE-MS. Experiments with binary mixtures confirmed that heterodimer formation is favoured under equimolar conditions, highlighting the influence of stoichiometry and monomer abundance. In CE-MS, we also observed heterodimers formed between genuine metabolites and lock-mass reference compounds, further illustrating the complexity of intermolecular associations during ESI. This practical and systematic pipeline enhances annotation reliability, reduces feature redundancy, and improves interpretability in MS-untargeted datasets. It is broadly applicable across separation platforms and sample types, offering a cost-effective solution for routine studies in metabolomic and lipidomic analyses.