Low-Temperature HILIC Provides Enhanced Separations and Stability for LC-MS-Based Metabolomics

Liquid chromatography-mass spectrometry is a potent and robust tool for studying metabolism. However, conventional workflows can suffer from poor peak shapes, limited pressure tolerance, coelution of polar metabolites, and unstable retention times. Here, we describe the development of a more stable HILIC method for LC-MS metabolomics of human plasma and cell extracts, optimizing a zwitterionic HILIC (Z-HILIC) column for improved untargeted performance. We found that using high-pH ammonium bicarbonate with 90% acetonitrile in mobile phase B (ABC B) can greatly improve peak shapes of select metabolites when compared to 100% acetonitrile (ACN B), but at the cost of poor retention time stability. We therefore focused on optimizing chromatography for the ACN B method and observed that cooling the column to 5 °C substantially enhanced peak shape for the BEH-bound Z-HILIC and amide columns but had little effect on the polymeric ZIC-pHILIC column. The low-temperature method with the Z-HILIC column (LT-ZHILIC) enables high-resolution separation of 471 metabolite library standards and from both cellular extracts and human plasma and demonstrates robust stability over 100 consecutive injections and multiple days. Application of the untargeted LT-ZHILIC method to characterize the metabolic consequences of glutamine and pyruvate deficiency in human cells revealed a striking change in nucleotide phosphates─a perturbation that was not observed in the ZIC-pHILIC analysis of the same samples likely due to inadequate elution profiles. In sum, the LT-ZHILIC workflow offers a robust platform to advance untargeted metabolomics by improving metabolite coverage, resolution, and retention time stability, making it a promising technique for providing novel insights into cellular metabolic rewiring and the human plasma metabolome.