N-Alkylpyridinium sulfonates as versatile scout compounds for multi-mode highly targeted metabolomics: Application to...

Although targeted metabolomics using multiple reaction monitoring (MRM) offers high sensitivity, it struggles with multiplexing capacity and retention time drifts. Scout-triggered MRM (stMRM) overcomes these limitations by using reference molecules to dynamically frame the acquisition window. However, establishing comprehensive stMRM assays requires reliable and cost-effective scout compounds that are compatible with multi retention mode of liquid chromatography-mass spectrometry (LC-MS). This study evaluates N-alkylpyridinium sulfonates (NAPS) as versatile exogenous scouts for highly multiplexed stMRM. The chromatographic and ionization behaviours of NAPS were characterised across three stationary phases (reversed-phase C₁₈, F₅ and hydrophilic interaction liquid chromatography (HILIC)) in positive and negative electrospray ionization. Due to their zwitterionic structure, NAPS ionised efficiently in both polarities, thereby simplifying dual-polarity acquisitions. Using NAPS as retention-time-independent triggers, we developed a comprehensive multi-mode assay monitoring 559 metabolites. To validate this methodology, we investigated the physiological impact of silver (Ag) exposure on the digestive caeca of the sentinel amphipod Gammarus fossarum. Multivariate analysis revealed metabolic shifts. Mapping these alterations onto species-specific networks highlighted a pronounced disruption of the purine metabolism pathway, characterised by consistent downregulation of xanthine and related intermediate precursors. In conclusion, NAPS are highly effective dual-polarity scout compounds that simplify the multiplexing of stMRM assays. This analytical strategy is a reliable tool for capturing complex molecular responses to environmental stressors in ecotoxicology.