LipidQuan for Comprehensive and High-Throughput HILIC-based LC-MS/MS Targeted Lipid Quantitation

Importance of the Topic

Lipid molecules play critical roles in energy storage, membrane structure, and signaling pathways. Recent advances have linked lipid dysregulation to diseases such as cancer, inflammation, and cardiovascular disorders. Comprehensive, high-throughput lipid analysis is essential for biomarker discovery and understanding lipid metabolism, but the structural diversity and varied physicochemical properties of lipids pose analytical challenges.

Objectives and Study Overview

This work aims to establish a rapid, robust HILIC-based LC-MS/MS platform (LipidQuan) for simultaneous quantitation of both polar and non-polar lipid classes in human plasma. The method targets 16 lipid classes and over 500 lipid species, enabling large-scale lipidomics studies with minimal development overhead.

Methodology and Instrumentation

• Sample Preparation: Protein precipitation using chilled isopropanol (1:5 plasma:IPA), followed by centrifugation to extract lipid-rich supernatant.
• Chromatography: ACQUITY UPLC H-Class PLUS or I-Class system with BEH Amide column (2.1×100 mm, 1.7 μm), 45 °C, flow rate 0.6 mL/min. Gradient from 0.1% to 20% B in 2 min, 20% to 80% B in 3 min, 3 min re-equilibration.
• Mass Spectrometry: Xevo TQ-XS or TQ-S with ESI in positive/negative modes; MRM acquisition; capillary voltage 2.8 kV (+)/1.9 kV (–); desolvation at 500 °C; source at 120 °C.
• Data Processing: Predefined LipidQuan Quanpedia method file and TargetLynx or Skyline for peak integration and quantitation.

Main Results and Discussion

The HILIC method achieved complete class-based separation of 16 lipid classes within an 8-minute run, reducing isobaric interferences and ion suppression. Using stable isotope-labeled internal standards (SPLASH LIPIDOMIX), calibration curves spanned four orders of magnitude with correlation coefficients >0.992 (positive) and >0.980 (negative). A total of 508 endogenous lipid species (261 positive mode, 247 negative mode) were successfully quantified in NIST SRM 1950 plasma.

Benefits and Practical Applications

• High throughput: 8-minute analysis supports large cohort studies.
• Comprehensive coverage: 16 lipid classes, 2041 MRM transitions.
• Cost efficiency: Class-based separation reduces the number of stable isotope standards required.
• Ease of deployment: Downloadable Quanpedia method eliminates manual method setup.
• Flexible informatics: Compatibility with TargetLynx and Skyline accelerates data processing.

Future Trends and Applications

Integration with advanced informatics and machine learning will enhance lipid identification and pathway analysis. Expanding the method to tissue extracts and other biofluids will broaden its applicability. Emerging techniques such as ion mobility or high-resolution MS may be combined with HILIC to resolve isomeric lipids and deepen structural insights.

Conclusion

• A robust HILIC LC-MS/MS workflow was developed for rapid, quantitative analysis of polar and non-polar plasma lipids.
• Sixteen lipid classes separated in 8 minutes, enabling high-throughput lipidomics.
• Quantification of 508 lipid species with excellent linearity and sensitivity.
• Reduced method development time and cost via preconfigured Quanpedia protocols.
• Platform suited for large-scale studies in clinical and research settings.

References

1. Quehenberger O., Dennis E.A. The Human Plasma Lipidome. N Engl J Med. 2011;365(19):1812–1823.
2. Lisa M., Holčapek M. High-throughput and comprehensive lipidomic analysis using ultrahigh-performance supercritical fluid chromatography mass spectrometry. Anal Chem. 2015;87(13):7187–7195.
3. Grumbach E.S., Fountain K.J. Comprehensive Guide to HILIC. Hydrophilic Interaction Chromatography. 2010.
4. Cifkova E., Holcapek M., Lisa M., Ovcacikova M., Lycka A., Lynen F., Sandra P. Nontargeted quantitation of lipid classes using HILIC-ESI-MS with single internal standard and response factor approach. Anal Chem. 2012;84(22):10064–10070.
5. Sarafian M.H., Gaudin M., Lewis M.R., Martin F.P., Holmes E., Nicholson J.K., Dumas M.E. Optimization of sample preparation for ultra-high throughput untargeted blood plasma lipid profiling by UPLC-MS. Anal Chem. 2014;86(11):5766–5774.