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

Area of Science:

• Metabolomics
• Analytical Chemistry
• Biochemistry

Background:

• Liquid chromatography-mass spectrometry (LC-MS) is crucial for metabolomics but faces challenges with peak shape, metabolite coelution, and retention time stability.
• Conventional hydrophilic interaction liquid chromatography (HILIC) methods often struggle with polar metabolites and reproducibility in complex biological samples like human plasma and cell extracts.

Purpose of the Study:

• To develop and optimize a stable and high-resolution HILIC method for untargeted LC-MS metabolomics.
• To improve the separation and detection of polar metabolites in human plasma and cell extracts.
• To enhance the robustness and reproducibility of HILIC-based metabolomics workflows.

Main Methods:

• Optimization of a zwitterionic HILIC (Z-HILIC) column using a high-pH ammonium bicarbonate/acetonitrile mobile phase.
• Investigated the effect of column temperature, specifically cooling to 5 °C (LT-ZHILIC), on peak shape and retention time stability.
• Validated the optimized method using a 471 metabolite library and applied it to analyze cellular extracts and human plasma.

Main Results:

• Cooling the Z-HILIC column to 5 °C significantly improved peak shape and retention time stability compared to ambient temperatures, especially with an acetonitrile-based mobile phase.
• The low-temperature Z-HILIC (LT-ZHILIC) method achieved high-resolution separation of 471 metabolite standards and demonstrated robust performance over 100 injections.
• Application to cellular extracts under nutrient deficiency revealed significant changes in nucleotide phosphates, a perturbation missed by a standard ZIC-pHILIC method.

Conclusions:

• The LT-ZHILIC workflow provides a robust and stable platform for untargeted metabolomics, enhancing metabolite coverage and resolution.
• This optimized method offers improved insights into cellular metabolic rewiring and the human plasma metabolome.
• LT-ZHILIC represents a promising advancement for LC-MS-based metabolomics research.