How does your HILIC Method Stack Up? Optimization and Comparison of Common HILIC Columns, Mobile Phases, and Additives for Metabolomics

Significance of the Topic

Hydrophilic interaction liquid chromatography (HILIC) has become a cornerstone technique for both targeted and untargeted metabolomics, enabling broad coverage of polar metabolites. Achieving reproducible retention times and high sensitivity is critical for quantitative workflows, data alignment, and discovery studies in biological and clinical research.

Objectives and Study Overview

This work systematically evaluates common HILIC columns, mobile phase buffers, flow parameters, and additives to optimize metabolomics performance. The study compares column chemistries and dimensions, aqueous buffer compositions and pH, and the impact of a medronic acid-based deactivator on metal-sensitive analytes. Method robustness is assessed using equimolar metabolite standards and plasma extracts.

Instrumentation Used

• Agilent 1290 Infinity II Bio LC system equipped with Poroshell 120 HILIC-Z columns (1.9 µm and 2.7 µm, 2.1 × 100 mm or 150 mm).
• Agilent Bravo Sample Prep Platform with Captiva EMR-Lipid SPE for plasma extraction.
• Agilent 6495D LC/TQ mass spectrometer for targeted analyses (dMRM).
• Agilent 6546 LC/Q-TOF mass spectrometer for untargeted profiling.

Methodology and Instrumentation

Standards were prepared at varying concentrations in 70:20:10 acetonitrile:water:methanol and analyzed alongside bovine plasma extracts. Chromatography employed 20 mM ammonium acetate buffer at pH 9.3 with 5 µM medronic acid deactivator (mobile phase A) against pure acetonitrile (mobile phase B). A 24-minute gradient at 0.4 or 0.6 mL/min on 2.1 × 150 mm or 2.1 × 100 mm columns was used. Source conditions optimized for dual AJS ESI ensured high sensitivity and minimal background.

Main Results and Discussion

• Buffer comparison revealed that 20 mM ammonium acetate at pH 9.3 reduced background ions by 15-fold and improved peak shape for organic acids.
• Medronic acid deactivator significantly enhanced sensitivity and peak sharpness for metal-sensitive metabolites without causing ion suppression or carryover.
• Column dimensions and particle size studies showed 1.9 µm, 2.1 × 150 mm columns at 0.4 mL/min provided optimal separation of isobaric pairs (e.g., leucine/isoleucine, glucose-6-phosphate/fructose-6-phosphate).
• Inclusion of a 2.1 × 5 mm guard column extended column lifetime with negligible impact on retention times and peak areas.

Benefits and Practical Applications

This optimized HILIC workflow delivers reproducible retention times for targeted quantitation and narrow alignment windows for untargeted profiling. The medronic acid additive ensures consistent performance of metal-sensitive analytes, while small-particle columns enhance resolution of critical isomers. The protocol supports high-throughput metabolomics in clinical, pharmaceutical, and biotechnological settings.

Future Trends and Opportunities

Emerging directions include integration of microflow HILIC for reduced solvent consumption, expansion of spectral libraries for automated identification, and coupling with ion mobility for enhanced isomer differentiation. Advances in additive chemistries and surface passivation are expected to further improve metal-sensitive analyses.

Conclusion

The combination of an ammonium acetate buffered mobile phase at pH 9.3, medronic acid deactivator, and high-efficiency HILIC-Z columns delivers a robust, sensitive, and reproducible metabolomics platform. This method supports both targeted and untargeted workflows and can be readily adopted for diverse biological applications.

Reference

1. Yannell KE et al. An End-to-End Targeted Metabolomics Workflow. Agilent Application Note 5994-5628EN (2023).
2. Yannell KE et al. A Comprehensive Untargeted Metabolomics LC/Q-TOF Workflow with an Unknowns Identification Strategy. ASMS (2022).
3. Yannell KE et al. Mastering HILIC-Z Separation for Polar Analytes. Agilent Application Note 5994-5949EN (2023).
4. Van de Bittner GC et al. An Automated Dual Metabolite + Lipid Sample Preparation Workflow. Agilent Application Note 5994-5065EN (2022).
5. Sartain M et al. Enabling Automated, Low-Volume Plasma Metabolite Extraction. Agilent Application Note 5994-2156EN (2020).