Complementary Single-Cell Microflow HILIC and Ion Pair LC−MS Reveal Bystander Metabolic Effects in a Macrophage Model of Tuberculosis

Single-cell metabolomics remains analytically challenging due to the low abundance and chemical diversity of metabolites in individual cells. We have developed complementary microflow hydrophilic interaction liquid chromatography (HILIC) and ion pair (IP) liquid chromatography-mass spectrometry (LC−MS) methods to expand metabolite coverage in single macrophages. Ion pair LC−MS was applied to single cells for the first time, enabling retention of highly polar and ionic metabolites that elute early under conventional reversed-phase conditions. Across Mycobacterium bovis Bacillus Calmette−Guérin (BCG)-infected, uninfected bystander, and control-unexposed THP-1 macrophages, both microflow methods detected significantly more features than a previously reported analytical-flow HILIC method. The two microflow methods provided complementary chemical space, together yielding 633 unique name-annotated metabolites with MS2 spectra. This depth enabled pathway-level interpretation at single-cell resolution, revealing infection-associated changes in purine-, arginine-, glutathione-, and one-carbon folate-associated metabolism. Metabolite-level interrogation indicated shared purine and amino acid changes in both infected and neighboring macrophages, while a potential bystander phenotype arose, characterized by elevated glycine and heterogeneous adenosine triphosphate (ATP) levels. Finally, we demonstrate sequential IP and HILIC analysis of the same single cell, establishing a route toward maximal coverage from individual cells. These results position microflow HILIC and IP LC−MS as powerful, orthogonal strategies for advancing single-cell metabolomics and unveiling heterogeneity within complex biological microenvironments.