Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography Mass Spectrometry (Unified-HILIC/AEX/MS): A Single-Run Method for Comprehensive and Simultaneous Analysis of Polar Metabolome

One of the technical challenges in the field of metabolomics
is
the development of a single-run method to detect the full complement
of polar metabolites in biological samples. However, an ideal method
to meet this demand has not yet been developed. Herein, we proposed
a simple methodology that enables the comprehensive and simultaneous
analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange
liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with
a polymer-based mixed amines column composed of methacrylate-based
polymer particles with primary, secondary, tertiary, and quaternary
amines as functional groups. The optimized unified-HILIC/AEX/MS method
is composed of two consecutive chromatographic separations, HILIC-dominant
separation for cationic, uncharged, and zwitterionic polar metabolites
[retention times (RTs) = 0–12.8 min] and AEX-dominant separation
for polar anionic metabolites (RTs = 12.8–26.5 min), by varying
the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH
9.8). A total of 400 polar metabolites were analyzed simultaneously
through a combination of highly efficient separation using unified-HILIC/AEX
and remarkably sensitive detection using multiple reaction monitoring-based
triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted
metabolomic approach using unified-HILIC/AEX high-resolution mass
spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive
information on polar metabolites (3242 metabolic features) in HeLa
cell extracts than the conventional HILIC/HRMS method (2068 metabolic
features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS
methods have several advantages over conventional techniques, including
polar metabolome coverage, throughput, and accurate quantitative performance,
and represent potentially useful tools for in-depth studies on metabolism
and biomarker discovery.