Monolithic Column for Improved 4D-Lipidomics Analysis

Importance of the Topic

Large-scale lipidomics requires sensitive and efficient separation strategies to capture structurally diverse and low-abundance lipids. Conventional high-flow LC methods suffer from dilution and high backpressure with viscous mobile phases. Monolithic columns, with their high permeability and broad porosity, enable faster microflow and nanoflow separations, improved throughput, and enhanced detection in 4D-lipidomics workflows.

Study Objectives and Overview

This study compares monolithic and conventional particle-based LC columns for both analytical and nanoLC lipidomics using a timsTOF HT MS system. It evaluates two extraction methods, various mobile phase compositions, and column technologies to maximize lipid annotation, reproducibility, and run efficiency.

Methodology and Instrumentation

Sample preparation involved MTBE and a simplified isopropanol (IPA) extraction. Mobile phases were water/IPA/acetonitrile with acetic and phosphoric acids plus ammonium acetate. Tested columns included YMC C18, Bruker Bio-LP, Waters Acquity BEH C18, and Kyoto Monotech monolithic C8 (analytical) and C18 (nano). Analytical separations used an Agilent 1290 Infinity II UHPLC with VIP-HESI; nanoLC used a Bruker nanoElute 2 with CaptiveSpray. Both systems were coupled to a Bruker timsTOF HT MS operating in DDA-PASEF mode with ion mobility. Data were processed in DataAnalysis 6.2 and MetaboScape 2025b.

Key Results and Discussion

The IPA extraction protocol streamlined preparation and increased lipid annotations by 6.75% versus MTBE. Monolithic analytical columns operated at 150–400 µL/min with reduced backpressure; monolithic nanoLC columns ran at 1.85 µL/min and ~550 bar, enabling 15-minute gradients. Modified mobile phases with YMC columns yielded ~16.9% more annotations, a gain confirmed on monolithic columns. NanoLC monolithics delivered the highest lipid IDs and reproducible profiles. Kendrick Mass Defect plots facilitated rapid lipid class visualization across methods.

Benefits and Practical Applications

• High throughput: 15-minute lipidomic runs at elevated flow rates.
• Improved sensitivity: enhanced detection of low-abundance species.
• Streamlined workflow: fast, easy IPA-based extraction.
• Robust operation: monolithic columns resist clogging and maintain reproducibility.

Future Trends and Opportunities

Custom-designed monolithic phases for specific lipid classes could further improve selectivity. Combining these columns with advanced ion mobility, data-independent acquisition, and automated IPA extraction promises deeper lipidome coverage and fully integrated high-throughput platforms for clinical and industrial lipidomics.

Conclusion

Monolithic LC columns, combined with an IPA extraction protocol, optimized mobile phases, and 4D-timsTOF MS, significantly enhance lipid annotation, throughput, and reproducibility. This integrated approach advances comprehensive lipid profiling of complex biological samples.

Reference

Peng X., Wang B., Krawitzky M., Nakabayashi R., Forsberg E. Monolithic Column for Improved 4D-Lipidomics Analysis. ASMS 2025 WP420.