Expanding the Coverage of Metabolome Using Multiple Liquid Chromatography Modes

Importance of the Topic

Liquid chromatography serves as a critical front-end for expanding coverage in global metabolomics workflows. It addresses the wide chemical diversity and concentration range of endogenous small molecules by providing tailored retention profiles for nonpolar, polar, and mixed-mode analytes.

Objectives and Study Overview

This study aims to perform a comprehensive side-by-side comparison of three liquid chromatography modes—reversed-phase (RPLC), hydrophilic interaction (HILIC), and mixed-mode chromatography—for the separation of 300 human endogenous metabolites. The goal is to evaluate retention behavior, peak quality, and detection efficiency across diverse compound classes.

Methodology and Instrumentation

Sample Preparation:

• A panel of 300 metabolite standards validated against the Human Metabolome Database was prepared in “batches” of 24, dissolved at 0.5 mg/mL (HILIC and mixed-mode) and 20%/80% MeOH/H2O (RPLC).
• d5-Hippuric acid internal standard spiked at 0.1 mg/mL.
• Samples filtered through 0.22 µm membranes prior to injection.

Instrumentation:

• Thermo Scientific Dionex UltiMate 3000 HPG LC system.
• Thermo Scientific Q Exactive quadrupole-Orbitrap mass spectrometer with full-scan at 70 000 resolution (m/z 200) and targeted MS/MS at 35 000 resolution.
• Electrospray ionization with positive/negative polarity switching.

Main Results and Discussion

• RPLC Classification:
  • “RP-Green”: Nonpolar metabolites with strong retention, symmetric peaks, and robust MS responses.
  • “RP-Yellow”: Polar species eluting early yet displaying acceptable peak shapes.
  • “RP-Red”: Compounds exhibiting poor chromatographic performance (broadening, splitting, tailing) and low signal.
• HILIC showed enhanced retention for highly polar analytes but limited performance for nonpolar species.
• Mixed-mode chromatography provided intermediate selectivity, capturing compounds that were challenging in single-mode separations.
• Sample solvent composition strongly influenced peak shape; optimal ratios varied by column chemistry (e.g., 10% MeOH/90% H2O improved split peaks on C18 AQ).
• Mass spectrometric ionization varied across metabolites: some analytes favored positive ESI, others negative, highlighting the need for dual-polarity acquisition.

Benefits and Practical Applications

• Multi-mode LC workflows enhance coverage of chemical space in metabolomics studies.
• Classification schemes aid in method development by matching compound classes to appropriate chromatographic modes.
• Guidance on solvent and column selection improves peak shape and detection sensitivity.
• Dual-polarity MS acquisition maximizes the number of detectable metabolites.

Future Trends and Possibilities

• Development of novel column chemistries combining multiple interaction mechanisms.
• Integration of machine-learning tools for automated method optimization and retention prediction.
• Standardization of multi-mode LC-MS workflows across laboratories to support large-scale metabolomics.
• Expansion toward ultra-high-throughput and multiplexed analyses in clinical and environmental applications.

Conclusion

A systematic evaluation of RPLC, HILIC, and mixed-mode chromatography demonstrates that each mode contributes unique retention and separation characteristics that, when combined, substantially improve the analytical coverage of metabolite libraries. Methodological insights into solvent effects and ionization behavior further support optimized workflows for comprehensive metabolomics analyses.