EVALUATION OF AQUEOUS-ACETONITRILE BASED MOBILE PHASE FOR UNTARGETED ANALYSIS OF POLAR METABOLOME AND LIPIDOME

Significance of the Topic

Comprehensive profiling of the polar metabolome and lipidome in biological samples is essential for understanding biochemical pathways, disease biomarkers, and pharmacological effects. Differences in analyte polarity, ionization, and structural diversity often require multiple chromatographic conditions, increasing analysis time and complexity. Developing a single, versatile mobile phase enables streamlined workflows, improved reproducibility, and cost savings.

Objectives and Study Overview

This study aimed to identify and validate a generic aqueous-acetonitrile (ACN) mobile phase capable of separating both polar metabolites and diverse lipid classes in an untargeted workflow. Various reverse-phase and HILIC column chemistries were screened against the conventional isopropanol (IPA)-based lipid mobile phase. The ultimate goal was to use one mobile phase on different columns within the same LC-MS system for comprehensive metabolomics and lipidomics.

Methodology and Instrumentation

A simple protein precipitation using pre-cooled IPA was applied to rat and human plasma, with supernatant diluted in ACN/IPA before analysis. Chromatographic screening involved:

• Reverse-phase columns: CSH Phenyl-Hexyl, CSH C18, CORTECS Phenyl, CORTECS C8
• HILIC column: BEH Amide
• Mobile phase composition: water as MP A; ACN/water (95:5) with 0.1% formic acid and 1 mM ammonium formate as MP B
• Gradient: 70–99% MP B over 6–8 min at flow rates of 0.4–0.6 mL/min; column temperatures between 40 °C and 70 °C

Used Instrumentation

• Waters ACQUITY Premier UPLC system
• Columns: ACQUITY Premier CSH Phenyl-Hexyl (2.1×150 mm), CSH C18 (2.1×100 mm), CORTECS Phenyl (2.1×100 mm), CORTECS Premier C8 (2.1×100 mm), ACQUITY BEH Amide (2.1×100 mm)
• Mass spectrometer: Waters SYNAPT XS TOF in positive and negative electrospray modes

Main Results and Discussion

Column screening demonstrated that the aqueous/ACN mobile phase delivered equivalent or superior separation of lipids compared with the IPA-based standard. The CSH Phenyl-Hexyl column achieved complete elution of neutral lipids such as triacylglycerols and cholesterol esters in under 10 min with excellent peak resolution. Switching the same mobile phase to the HILIC BEH Amide column enabled efficient retention and elution of polar metabolites in positive and negative modes. No significant carryover was observed.

Benefits and Practical Applications

• Single mobile phase for both lipidomics and metabolomics reduces method development time.
• Cost savings through reduced solvent consumption and simpler solvent management.
• Improved reproducibility and robustness across large sample sets.
• Flexibility to analyze diverse sample types by selecting appropriate columns without changing mobile phase.

Future Trends and Opportunities

• Extension to other biofluids, tissues, and environmental samples for holistic omics profiling.
• Integration with high-resolution mass spectrometry platforms and data-independent acquisition workflows.
• Automation of sample preparation and inline workflow integration for high-throughput screening.
• Development of greener solvent systems and novel stationary phases to further enhance coverage and sustainability.
• Advanced data processing and machine learning for automated feature annotation and pathway mapping.

Conclusion

A single aqueous-acetonitrile mobile phase has been successfully validated for untargeted analysis of polar metabolites and lipid species on multiple LC columns using one LC-MS platform. This approach streamlines workflows, enhances reproducibility, and supports comprehensive biochemical profiling with minimal carryover.

Reference

1. Isaac G., Munjoma N., Gethings L.A., Mullin L., Plumb R.S. A Robust and Reproducible Reversed-Phase Lipid Profiling Method for Large Sample Sets. Waters Application Note 720006959en, July 2020.