LipidQuan: HILIC-based LC-MS/MS High-Throughput Targeted Free Fatty Acid Screen

Importance of the Topic

Free fatty acids (FFAs) are a key metabolically active lipid class in blood, serving as energy substrates and signaling molecules. Accurate, high-throughput quantification of FFAs supports biomedical research, clinical metabolomics, and lipidomics workflows.

Objectives and Study Overview

This study introduces a HILIC-based LC-MS/MS method using the LipidQuan platform to rapidly and selectively quantify 24 FFAs in human plasma within eight minutes. The approach leverages standardized Quanpedia methods and SOPs to streamline deployment, reduce method development, and lower training costs.

Methodology

• Sample Preparation: Protein precipitation with cold isopropanol (1:5 plasma:IPA), vortexing, incubation at –20 °C and 4 °C, centrifugation at 10 300 g.
• Chromatography: ACQUITY UPLC BEH Amide column (2.1×100 mm, 1.7 µm) with acetonitrile/water (10 mM ammonium acetate); flow 0.6 mL/min; 8-minute gradient (0.1 %–20 % B in 2 min, 20 %–80 % B in 3 min, 3 min re-equilibration).
• Mass Spectrometry: Negative ESI MRM on Xevo TQ-S micro/XS or TQ-S; capillary voltage 1.9 kV; source temp 120 °C; desolvation 500 °C; cone gas 150 L/hr; desolvation gas 1000 L/hr.

Instrumentation

• Waters ACQUITY UPLC I-Class system
• Waters Xevo TQ-S micro, Xevo TQ-XS, or Xevo TQ-S mass spectrometers
• ACQUITY UPLC BEH Amide column
• TargetLynx and Skyline software for data processing

Main Results and Discussion

The HILIC separation produced a distinct FFA elution band (~0.5 min) with minimal co-elution of other lipid classes. Calibration curves spanned four orders of magnitude, achieving R²>0.95 and CV<30 % using SPLASH LIPIDOMIX™ standards. Normal plasma levels of C12–C22 saturated and unsaturated FFAs were reliably detected from 50 µL samples. Compared to GC-MS and RP-LC-MS, this workflow removes the need for derivatization, simplifies sample preparation, and reduces solvent usage.

Benefits and Practical Applications

• High throughput: 24 analytes in an 8-minute run.
• Operational simplicity: streamlined sample prep and preconfigured method files.
• Cost efficiency: fewer isotope standards required; no derivatization reagents.
• Data flexibility: compatible with TargetLynx and third-party informatics (Skyline).

Future Trends and Applications

Ongoing trends include expanding coverage to additional lipid classes within comprehensive lipidomics pipelines, improving isotope-labeled standard availability, and enhancing informatics automation for real-time quantitation and data integration.

Conclusion

The described HILIC-based LC-MS/MS method on the LipidQuan platform offers a rapid, robust, and cost-effective solution for targeted FFA quantification in plasma, addressing limitations of traditional GC-MS and RP-LC-MS workflows.

References

1. Ran-Ressler RR, Bae S, Lawrence P, Wang DH. Branched-chain fatty acid content of food and estimated intake in the USA. Br J Nutr. 2014;112(4):565–572.
2. Ran-Ressler RR, Devapatla S, Lawrence P, Brenna JT. Branched chain fatty acids are constituents of the normal healthy newborn gastrointestinal tract. Pediatr Res. 2008;64(6):605–609.
3. Quehenberger O, Armando AM, Brown AH, et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res. 2010;51:3299–3305.
4. Brevik A, Veierød MB, Drevon CA, Andersen LF. Evaluation of the odd fatty acids 15:0 and 17:0 in serum and adipose tissue as markers of intake of milk and dairy fat. Eur J Clin Nutr. 2005;59(12):1417–1422.
5. Dennis EA, Norris PC. Eicosanoid storm in infection and inflammation. Nat Rev Immunol. 2016;15(8):511–523.
6. Cifkova E, Holcapek M, Lisa M, et al. Nontargeted quantitation of lipid classes using HILIC-ESI-MS with single internal standard and response factor approach. Anal Chem. 2012;84(22):10064–10070.
7. Kopf T, Schmitz G. Analysis of non-esterified fatty acids in human samples by SPE and GC-MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;938:22–26.