Investigation of several chromatographic approaches for untargeted profiling of central carbon metabolism.

Monitoring the central carbon metabolism (CCM) network using liquid chromatography/mass spectrometry (LC-MS) analysis is hampered by the diverse chemical nature of its analytes, which are extremely difficult to analyze using single chromatographic conditions. Furthermore, CCM-related compounds present non-specific adsorption on metal surfaces, causing detrimental chromatographic effects and sensitivity loss. In this study, polar reversed-phase, mixed-mode (MMC), and zwitterionic hydrophilic interaction chromatography (HILIC) featuring low-adsorption hardware were investigated towards untargeted analysis of biological samples with a focus on energy metabolism-related analytes. Best results were achieved with sulfoalkylbetaine HILIC with different supports, where polymeric option featured the highest coverage and inert hybrid silica facilitated best throughput and kinetic performance at a cost of less selectivity for small carboxylic acids. MMC demonstrated excellent performance for strongly anionic analytes such as multiresidue phosphates. The obtained experimental data also suggested that an additional hydrophilic modulation might be necessary to facilitate better resolution of carboxylic acids in zHILIC mode, as found during the application of the developed method to study the effect of two different mutations on the energy metabolism of S. aureus.