General workflow for untargeted plasma lipidomics on Orbitrap mass spectrometers

Importance of the Topic

Lipids are fundamental mediators of cell structure, energy storage, and signaling. Comprehensive profiling of the plasma lipidome enables the discovery of disease markers in conditions such as cancer and diabetes. Untargeted lipidomics expands understanding of complex lipid networks, supporting precision medicine and early disease detection.

Objectives and Study Overview

This technical note outlines an optimized, untargeted plasma lipidomics workflow on Orbitrap mass spectrometers. It aims to maximize lipid coverage through advanced UHPLC separation, high resolution accurate mass spectrometry, and intelligent data acquisition. The study demonstrates methods for sample preparation, data-dependent acquisition, and multi-stage fragmentation to enhance lipid identification in complex matrices.

Methodology and Instrumentation

The workflow begins with plasma extraction using methanol/chloroform precipitation and stable isotope–labeled internal standards for QC. Samples are randomized and pooled QC injections monitor instrument performance. Chromatographic separation is achieved on a C30 column with a gradient of acetonitrile, isopropanol, water, and ammonium formate additives. High resolution full-scan MS (120,000 resolution) is conducted in both polarities with EASY-IC calibration. AcquireX intelligent acquisition automates iterative MS2 or MSn targeting, excluding background and redundant ions to ensure in-depth fragmentation of low-abundance species.

Used Instrumentation

• Vanquish Horizon UHPLC system with Accucore C30 HPLC column (2.1×150 mm, 2.6 μm)
• Orbitrap Exploris 120 and 240 mass spectrometers with fast polarity switching and sub-ppm mass accuracy
• Orbitrap IQ-X Tribrid mass spectrometer enabling HCD, CID, and MSn experiments
• UltimateSPLASH ONE Mass Spec Standard for lipid internal standardization

Main Results and Discussion

Implementing the AcquireX workflow increased lipid annotations by over 50% compared to conventional DDA, revealing more than 1,300 unique lipid species in plasma. High resolution HCD MS2 spectra provided confident molecular composition assignments. Complementary positive and negative ion fragmentation enhanced annotation specificity, particularly for phosphatidylcholines. The IQ-X Tribrid platform resolved co-eluting triglyceride isomers via targeted MS3, enabling unambiguous identification of fatty acyl combinations.

Benefits and Practical Applications

• Expanded lipidome coverage and deeper structural characterization
• Improved specificity in distinguishing isobaric and isomeric species
• Robust quality control with pooled QC and stable-isotope standards
• High-throughput analysis suitable for large cohort studies
• Support for biomarker discovery and metabolic phenotyping

Future Trends and Potential Applications

Integration of AI-driven data analysis and machine learning will streamline lipid annotation and quantitation. Advances in chromatography and ion mobility may further resolve complex isomers. Expanding workflows to diverse sample types such as tissues or single cells will broaden applications in clinical diagnostics, nutrition research, and environmental lipidomics.

Conclusion

The combination of UHPLC C30 separation, high-resolution Orbitrap instrumentation, and the AcquireX intelligent acquisition workflow delivers unparalleled depth and confidence in untargeted plasma lipidomics. These methods enhance detection of low-abundance species and enable precise structural annotation, positioning Orbitrap-based platforms as a leading choice for advanced lipid research.

References

• 1. Bashar A et al. Quality assurance and quality control in metabolomics: achieving high-quality data for high-quality results. Thermo Scientific Application Note 001771.
• 2. Hackbusch S et al. Reproducible measurement and confident identification of the plasma lipidome from obese mice using the Orbitrap Exploris 240 mass spectrometer. Thermo Scientific Application Note 000696.
• 3. Narváez-Rivas M, Zhang Q. Comprehensive untargeted lipidomic analysis using core-shell C30 particle column and high field Orbitrap mass spectrometer. J Chromatogr A. 2016;1440:123–134.