Leveraging Multidimensional Separations to Enhance Traditional LC-MS Lipidomics Workflows

Importance of the Topic

Lipidomics aims to comprehensively characterize lipid species in biological systems, but complexity arises from isomeric forms and overlapping ion signals. Integrating multidimensional separations such as two-dimensional liquid chromatography (2D-LC) and ion mobility mass spectrometry (IM-MS) enhances specificity and confidence in lipid identification.

Objectives and Study Overview

The study explores the combination of 2D-LC with All Ions IM-MS workflows to improve lipidomic analyses through:

• Evaluation of HILIC and reversed-phase LC in a heart-cutting, high-resolution 2D-LC setup
• Application of All Ions IM-MS for drift time alignment of fragments and precursors
• Assessment of data analysis pipelines for untargeted and targeted lipid profiling

Methodology and Instrumentation

2D-LC experiments employed an Agilent 1290 Infinity system with a HILIC column (3.0×100 mm, 1.8 μm) and a Poroshell 120 EC-C18 column (3.0×100 mm, 2.7 μm). Both heart-cutting and comprehensive 2D approaches were performed using tailored solvent gradients. Ion mobility separations and All Ions fragmentation were conducted on the Agilent 6560 IM-QTOF platform, aligning MS/MS fragments with their precursors based on drift time. Data processing utilized Lipid Annotator, Mass Profiler Professional, ID Browser, and Skyline.

Main Results and Discussion

Integrating 2D-LC with IM-MS yielded enhanced separation of lipid classes, reducing spectral congestion and increasing annotation count, especially for phosphatidylcholines in negative mode. Lipid Annotator provided drift-aligned feature views and match details, enabling export of a PCDL containing accurate mass, retention time, collision cross section (CCS), and MS/MS spectra. Two workflows—untargeted alignment via Mass Profiler and ID Browser, and targeted extraction with Skyline—demonstrated robust profiling of MS1 data.

Benefits and Practical Applications of the Method

• Improved lipid identification confidence through drift time alignment
• Enhanced annotation numbers by reducing sample complexity with 2D-LC
• Compatibility with both untargeted and targeted data analysis workflows
• Generation of customizable PCDLs for routine lipid profiling

Future Trends and Potential Applications

Ongoing developments will focus on advanced visualization of multidimensional datasets, refinement of precursor–fragment alignment algorithms in All Ions IM-MS, and optimization of 2D-LC protocols to maximize separation efficiency for diverse lipid classes. These advances will broaden applications in clinical research, biomarker discovery, and quality control.

Conclusion

The coupling of high-resolution 2D-LC with All Ions IM-MS and comprehensive data analysis tools markedly improves the depth and reliability of lipidomic workflows. This multidimensional strategy offers a powerful solution to overcome inherent challenges in lipid identification and quantification.

Used Instrumentation

• Agilent 1290 Infinity 2D-LC system
• Agilent 6560 Ion Mobility Q-TOF LC/MS
• HILIC column (3.0×100 mm, 1.8 μm)
• Poroshell 120 EC-C18 column (3.0×100 mm, 2.7 μm)

References

1. G. Vanhoenacker, R. t’Kindt, F. David, P. Sandra, K. Sandra, Unraveling the Complexity of Lipidomes by Multiple Heart Cutting Q-TOF LC-MS with the Agilent 1290 Infinity 2D-LC Solution, Application Note: Biotherapeutics and Biosimilars, 2015.
2. J. Koelmel, M. Sartain, J. Salcedo, A. Murali, X. Li, S. Stow, Improving Coverage of the Plasma Lipidome Using Iterative MS/MS Data Acquisition Combined with Lipid Annotator Software and 6546 LC/Q-TOF, Application Note: Lipidomics, 2019.
3. S. M. Stow et al., An Interlaboratory Evaluation of Drift Tube Ion Mobility Collision Cross Section Measurements, Anal. Chem., 2017, 89(17), 9048–9055.
4. B. MacLean et al., Skyline: An Open Source Document Editor for Creating and Analyzing Targeted Proteomics Experiments, Bioinformatics, 2010, 26(7), 966–968.