EMPLOYING BALLISTIC GRADIENTS, VACUUM JACKETED COLUMNS AND A NOVEL MRT TO INCREASE LIPIDOMIC THROUGHPUT WHILST MAINTAINING HIGHLY CONFIDENT IDENTIFICATIONS

Significance of the Topic

Lipidomics plays a pivotal role in biomarker discovery for diseases such as colorectal cancer. Growing patient cohorts demand analytical methods that deliver high throughput without sacrificing data quality or environmental sustainability. Integrating vacuum jacketed columns with advanced mass spectrometry addresses the need for rapid, sensitive, and high confidence lipid identifications.

Objectives and Study Overview

This study aimed to evaluate whether combining ballistic gradients, vacuum jacketed column chromatography, and a prototype Xevo multi reflecting time of flight mass spectrometer could increase lipidomic throughput while preserving chromatographic resolution and identification confidence. Comparisons were made between conventional UPLC columns and vacuum jacketed columns (VJC) under gradient times of four, two and one minute.

Methodology and Instrumentation

Sample Preparation

• Serum lipids extracted from healthy controls and colorectal cancer patients using isopropanol
• EquiSPLASH internal standard spiked at 100 ng/ml

Chromatographic Conditions

• Column dimensions 2.1 mm x 50 mm packed with CSH stationary phase
• Gradients assessed at 4, 2 and 1 minute durations
• Solvent A comprised acetonitrile water and ammonium formate, solvent B comprised isopropanol acetonitrile and ammonium formate

Instrument Configuration

• Xevo multi reflecting time of flight mass spectrometer operating in MSE mode
• Scan speeds of 20 Hz for four and two minute gradients and 30 Hz for the one minute gradient
• Mass resolution maintained above 100 000 FWHM with alternating low and high collision energies

Results and Discussion

Key Findings

• Vacuum jacketed columns maintained narrow chromatographic peak widths comparable to conventional columns while enhancing ionisation efficiency
• Switching to VJC increased peak height and signal to noise ratio, boosting sensitivity
• Four minute VJC method identified 3.5 times more features than the equivalent conventional method
• Reducing gradient time to two minutes yielded a twofold increase in throughput with a 70 reduction in solvent use and further feature gains
• One minute VJC method delivered a 300 increase in sample throughput and 85 reduction in solvent consumption without compromising separation or resolution
• Partial least squares discriminant analysis demonstrated clear grouping of healthy, QC and cancer samples across all VJC methods

Discussion Points

• Shorter gradients combined with high scan speed mass spectrometry maintain data quality and depth of coverage
• Environmental impact is reduced through substantial solvent savings and lower flow rates
• High resolution and fast scanning capabilities of the Xevo MRT MS are critical for profiling ultra narrow peaks

Contributions and Practical Applications

The integration of vibrational chromatographic strategies and advanced mass spectrometry offers analytical laboratories a validated approach for large cohort lipidomic studies. Benefits include significantly accelerated workflows, reliable biomarker detection for clinical research, and reduced solvent costs and environmental footprint. Applications span disease phenotyping, quality control in biopharma and industrial lipid analysis.

Future Trends and Potential Applications

Emerging directions include further miniaturisation of column formats, integration with automation and artificial intelligence driven data processing, exploration of green solvents, and expansion into multiomics platforms. Adoption of even faster scanning mass spectrometers will push throughput limits for real time clinical and bioprocess monitoring.

Conclusion

This work demonstrates that vacuum jacketed columns paired with a fast scanning multi reflecting time of flight instrument can dramatically boost lipidomic throughput by up to fourfold while maintaining high chromatographic resolution and confident identifications. The approach reduces solvent use by up to 85 and supports robust separation of disease and control samples, offering a powerful solution for high throughput lipidomics in research and clinical environments.

References

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