Employing ballistic gradients, vacuum jacketed columns and prototype benchtop multi reflecting time-of-flight (MRT) to increase lipidomic throughput whilst maintaining highly confident identifications

Significance of the Topic

Lipidomics plays a pivotal role in biomarker discovery and disease mechanism research, yet the rapidly expanding scale of patient cohorts challenges traditional chromatographic workflows. High-throughput lipid profiling that preserves identification confidence and sensitivity is essential for large-scale clinical studies and quality control in pharmaceutical and biotechnological applications.

Objectives and Study Overview

This study compares conventional UPLC-based lipid analysis with methods employing vacuum-jacketed columns (VJC) and a prototype multi-reflection time-of-flight mass spectrometer (Xevo MRT MS). It aims to assess improvements in throughput, peak capacity, sensitivity, and feature detection across healthy control and cancer patient serum samples.

Methodology and Instrumentation

Ultrasonic extraction of serum lipids using isopropanol included an EquiSPLASH internal standard. Conventional UPLC separations used a 2.1×50 mm CSH column at 0.8 mL/min over a four-minute gradient. VJC configurations used the same stationary phase scaled to 0.5 mL/min and gradients of four, two, or one minute. Eluates were analyzed on the Xevo MRT MS in MS mode with alternating low and high collision energies and scan rates up to 30 Hz. Data processing involved conversion to mzML, feature extraction with Lipostar2 against the LIPID MAPS database, and multivariate analysis (PLS-DA) via MetaboAnalyst 6.

Main Results and Discussion

Use of VJC reduced chromatographic band broadening and frictional heating, yielding narrower peaks, higher peak heights, and improved signal-to-noise ratios. Transitioning from conventional columns to VJC increased total detected lipid features by 3.5-fold while cutting solvent use by ~37%. A two-minute VJC method doubled throughput and cut solvent consumption by ~70% compared to the standard four-minute run, with no loss in mass resolution at fast scan rates. Even a one-minute VJC protocol delivered robust PLS-DA separation between healthy and cancer cohorts, achieving a 300% throughput increase and ~85% solvent reduction.

Benefits and Practical Applications of the Method

• High-throughput capacity for large cohort lipidomics
• Enhanced sensitivity and peak capacity for low-abundance lipid species
• Substantial reduction in solvent consumption and operational costs
• Compatibility with data-independent MS workflows for comprehensive profiling
• Potentially greener analytical practices supporting sustainable laboratory operations

Future Trends and Applications

Further integration of VJC and high-resolution, fast-scan mass spectrometers can support ultra-large cohort studies, real-time clinical diagnostics, and environmental lipidomics. Ongoing developments may include automated column heating control, advanced AI-driven data processing pipelines, and coupling with ion mobility for enhanced isomer discrimination.

Conclusion

The combination of vacuum-jacketed columns and the Xevo MRT MS delivers unprecedented lipidomic throughput without sacrificing resolution or sensitivity. Method transfers from conventional UPLC to VJC yield multi-fold gains in feature detection and significant solvent savings. Shortened gradients down to one minute maintain high data quality, making this approach ideal for large-scale lipidomics in research and industrial settings.

References

1. Gray N. et al. Analytical Chemistry 2016;88(11):5742-5751. DOI:10.1021/acs.analchem.6b00038.
2. Plumb R.S. et al. J. Proteome Res. 2022;21(3):691-701. DOI:10.1021/acs.jproteome.1c00836.
3. Conroy M.J. et al. Nucleic Acids Res. 2023. DOI:10.1093/nar/gkad896.
4. Pang Z. et al. Nucleic Acids Res. 2024. DOI:10.1093/nar/gkae253.