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

Abstract

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.

Figure 1

Effect of additive type and its concentration in the aqueous mobile phase (pH 9.8) on separation and elution of (i) cationic, (ii) uncharged, (iii) zwitterionic, and (iv) anionic polar metabolites by a mixed amines polymer column.

Figure 2

LC/MS/MS chromatograms of 52 representative polar metabolite standards in groups (i), (ii), (iii), and (iv) measured on a mixed amines polymer column. Mobile phase A, 40 mM ABC solution at pH 9.8; mobile phase B, acetonitrile. Other LC/MS/MS parameters are detailed in the Experimental Section.

Figure 3

Comparison of RTs (a) and peak widths at 10% peak height (W_0.1) (b) of 52 representative polar metabolites using a glycerol dimethacrylate-based bare polymer column (P column), a spacer-modified (i.e., glycerol-modified) methacrylate polymer column (P–S column), and a mixed amines polymer column (P–S–A column) under identical LC/MS/MS conditions. Values are presented as the mean ± standard deviation (n = 3). (c) Representative LC/MRM chromatograms of polar metabolites (His and ATP) under three different column conditions.

Figure 4

Relationship between RT and log P_ow for 52 polar metabolites in HILIC mode (a) and AEX mode (b) under optimized gradient elution conditions using a mixed amines polymer column. Mobile phase A, 40 mM ABC solution at pH 9.8; mobile phase B, acetonitrile. Other LC/MS/MS parameters are detailed in the Experimental Section.