Evaluating the Effect of Chromatographic Chelator Modifiers on Metabolite Levels in Untargeted Liquid Chromatography–Mass Spectrometry

Abstract Metal-sensitive metabolites frequently exhibit poor chromatographic performance during liquid chromatography–mass spectrometry (LC-MS) due to analyte interactions with stainless-steel components of the chromatographic system. Although mobile phase chelators are widely used to improve targeted analyses, their broader effects on untargeted metabolomics remain unclear. We systematically evaluated the impact of chromatographic chelator modifiers on untargeted LC-MS detection of Pseudomonas aeruginosa secondary metabolites using the metal-chelating quorum-sensing molecule Pseudomonas Quinolone Signal (PQS) as a model analyte. Analysis of publicly available metabolomics data sets revealed substantial variability in PQS chromatographic performance across laboratories. From this observation, we hypothesized that metal-mediated adsorption may contribute to inconsistent detection of PQS. We compared the effects of EDTA and citrate as mobile phase additives on P. aeruginosa metabolite detection across multiple culture media. Both chelators markedly improved PQS peak shape and enhanced detection of PQS and the siderophore pyochelin. However, high concentrations of EDTA caused ion suppression, altered adduct formation, and reduced detection of several metabolite classes, including phenazines and rhamnolipids, while citrate eliminated detection of phenazine-1-carboxylic acid (PCA). Reducing EDTA concentration to low micromolar levels largely preserved chromatographic improvements while minimizing adverse effects on metabolite coverage, although suppression of PCA persisted. These trends were consistent across chemically distinct growth media. Collectively, our findings demonstrate that mobile phase chelators substantially influence untargeted metabolomic profiles beyond improving chromatography. Careful selection of chelator identity and concentration is therefore essential to balance chromatographic performance with comprehensive metabolite detection in untargeted LC-MS analyses of metal-sensitive metabolites.