Nanoflow ion-pairing LC-MS for ultra-low-input polar metabolomics and isotope tracing

Low-input and single-cell metabolomics remain constrained by the poor retention of polar metabolites in conventional reversed-phase nanoflow LC-MS workflows. Here, we establish nanoflow tributylamine (TBA) ion-pairing LC-MS as a platform for ultra-low-input polar metabolomics and stable isotope tracing. By adapting an analytical-flow TBA ion-pairing method to the nanoflow scale, this workflow extends the sensitivity and inline concentration advantages of nanoflow chromatography to charged metabolites involved in central carbon metabolism. Using mouse liver metabolite extracts, we show that the nanoflow method preserves chromatographic retention and separation of chemically diverse metabolite classes, including adenine nucleotides, nucleotide cofactors, TCA cycle intermediates, acyl-CoAs, and bile acid isomers. Despite loading 20-fold less tissue-equivalent material on column, nanoflow LC-MS produced higher signal intensity than the analytical-flow method for many metabolites. Across representative compounds, the nanoflow workflow reduced the biomass required for detection by approximately 20- to >600-fold, with pronounced gains for low-abundance metabolites such as NADPH and acetyl-CoA. TBA ion-pairing also enabled trap-and-elute nanoflow analysis of retained polar metabolites from single-cell-equivalent inputs. ATP was detected from one cell equivalent using both full-scan and targeted parallel reaction monitoring acquisition, with targeted acquisition further increasing signal over blank. Finally, we applied the workflow to stable isotope tracing in uniformly labeled 13C-glucose-treated cells. 13C-labeled ATP isotopologues were detectable from single-cell-equivalent input, and targeted acquisition improved isotopologue measurement near the detection limit. Together, these results demonstrate that nanoflow TBA ion-pairing LC-MS enables retained, high-sensitivity analysis of polar metabolites from ultra-low inputs and provides a foundation for extending central carbon metabolite analysis and isotope tracing toward single-cell-scale applications.