Comprehensive evaluation of dual-mode LC-MS conditions for enhanced metabolomic profiling: Application to microplastic exposure studies.

Polystyrene microplastics (PS-MPs) are emerging contaminants of concern due to their potential health impacts and widespread presence in the environment. Metabolomics offers a powerful approach to investigate biological responses to such exposures. However, current LC-MS methods are often limited by the suitability of chromatographic conditions for metabolites with diverse physicochemical properties, leading to suboptimal coverage and analytical redundancy. This study addresses these limitations by establishing a robust, broadly applicable dual-mode LC-MS strategy to improve coverage and analytical efficiency in microplastic exposure studies. This study evaluated 18 chromatographic conditions using six commercial columns including amide, silica, Obelisc N, C18, pentafluoophenyl (F5), and cyanopropyl (CN), to optimize metabolite separation in both positive and negative electrospray ionization (ESI) modes. Mouse large intestine extracts exposed to PS-MPs showed broad metabolome coverage under optimized conditions. In positive mode, the amide column with ammonium acetate/acetic acid (AmAc/AcA) effectively captured diverse polar metabolites. In negative mode, the F5 column with ammonium formate/formic acid (AmF/FA) excelled in phospholipid detection and lipid separation. Combining these conditions enabled complementary profiling with minimal overlap. Additionally, 42 differential metabolites affected by PS-MPs were associated with key metabolic pathways, including amino acid, taurine, hypotaurine, and glutathione metabolism. This optimized, high-coverage LC-MS strategy provides a novel analytical framework that maximizes metabolome profiling efficiency and minimizes sample input. It improves detection of diverse metabolite classes and supports robust biological interpretation, offering broad applicability for future studies on environmental exposures and complex biological challenges.