Sub-3 μm porous graphitic carbon for highly polar metabolomics: orthogonal selectivity and comparison with HILIC and reversed-phase chromatography.

The analysis of the polar metabolome remains a major challenge in LC-MS. Beyond established hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) workflows, alternative and orthogonal chromatographic approaches are required to improve the detection and quantification of compounds with low logP values and structurally related isomers. In this study, we evaluated the performance of sub-3 µm porous graphitic carbon (PGC) stationary phase and benchmarked it against state-of-the-art HILIC and RP approaches. Flow rate, column temperature, organic modifier, mobile-phase composition, and pH were optimized using a representative panel of highly polar endogenous metabolites. The three chromatographic modes exhibited markedly complementary selectivity profiles. HILIC provided the broadest coverage of nucleotides and related metabolites, whereas PGC showed enhanced performance for sugar phosphates, polyamines, and, particularly, tricarboxylic acid (TCA) cycle intermediates. Focused optimization for TCA cycle metabolites improved peak shape and signal-to-noise ratio and enabled the separation of citrate and isocitrate, outperforming both HILIC and reversed-phase chromatography for the detection of this metabolite class under the investigated conditions. In mouse liver extracts, PGC detected a complementary subset of metabolites not observed using HILIC or reversed-phase chromatography, confirming its distinctive selectivity in a complex biological matrix. As a proof of concept, the optimized PGC method was applied to lipopolysaccharide-stimulated macrophages using a hybrid workflow combining multiplexed parallel reaction monitoring for the absolute quantification of TCA cycle intermediates with data-dependent acquisition for global metabolic profiling. The workflow captured the characteristic metabolic reprogramming associated with macrophage inflammatory activation. Overall, sub-3 µm PGC represents a valuable orthogonal chromatographic platform for extending analytical coverage and improving the characterization of highly polar metabolites.