An Interlaboratory Evaluation of a Targeted Lipidomics Method in Plasma

Importance of the Topic

Targeted lipidomics enables accurate quantification of hundreds of lipid species in plasma, critical for understanding metabolic dysregulation, disease biomarkers, and therapeutic monitoring. Robust, transferrable methods are essential for large cohort studies and cross-laboratory comparisons.

Objectives and Study Overview

• Evaluate interlaboratory transferability of a 16-minute targeted LC/TQ lipidomics method.
• Assess precision of retention times and peak areas across four laboratories in the US.
• Demonstrate robustness of method implementation and troubleshooting processes.

Methodology and Instrumentation

Plasma samples included NIST SRM 1950 and mixed-gender normal human plasma. Lipids were extracted using a single-phase butanol–methanol protocol with SPLASH II internal standards. A 16-minute reversed-phase LC/TQ method covered 44 lipid classes (~763 lipids in 0.1 µL plasma). Data were acquired on independent Agilent LC/MS systems using MassHunter software and processed with dynamic MRM and retention time referencing.

Used Instrumentation

• Agilent 1290 Infinity II LC with ZORBAX Eclipse Plus C18 column (100 × 2.1 mm, 1.8 µm).
• Agilent 6495 triple quadrupole mass spectrometer.
• Agilent MassHunter Acquisition and Quantitative Analysis software.

Key Results and Discussion

Retention time RSDs for 12 internal standards were below 0.25% intraday across sites. Slight RT shifts observed with new mobile phase preparation were addressed by an automated RT adjustment procedure. Peak abundance RSDs for 377–516 lipids were under 10% intraday. PCA separated NIST and BIO samples consistently, indicating high reproducibility. A QC lapse at one site on day 2 underscored the importance of standardized quality control protocols.

Benefits and Practical Applications

• High-throughput profiling with broad lipid coverage suitable for large population cohorts.
• Demonstrated interlaboratory precision enabling cross-site data integration.
• Robust chromatography enhances isomer and isobar separation critical for accurate annotation.

Future Trends and Applications

Automation of retention time adjustments and QC checks will further streamline transferability. Expanding targeted panels to disease-specific lipid biomarkers and integrating with untargeted discovery workflows can enhance biological insights. Standardized interlaboratory exercises will support method harmonization in clinical metabolomics.

Conclusion

The curated LC/TQ lipidomics method delivers consistent, high-quality lipid quantification across different laboratories. Low retention time and peak area variability, combined with effective QC procedures, make it a reliable tool for large-scale clinical and translational studies.

References

• Sartain M. et al. A Comprehensive, Curated, High-Throughput Method for the Detailed Analysis of the Plasma Lipidome. Agilent Technologies Application Note 5994-3747EN, 2021.
• Agilent Technologies. Lipidomics Analysis with Lipid Annotator and Mass Profiler Professional. Technical Overview 5994-1111EN, 2020.
• Agilent Technologies. Lipid Profiling Workflow Demonstrates Disrupted Lipogenesis Induced with Drug Treatment in Leukemia Cells. Application Note 5994-1356EN, 2020.
• Huynh K. et al. High-Throughput Plasma Lipidomics: Detailed Mapping of the Associations with Cardiometabolic Risk Factors. Cell Chem. Biol. 26(1):71–84.e4, 2019.
• Quehenberger O. et al. Lipidomics Reveals a Remarkable Diversity of Lipids in Human Plasma. Journal of Lipid Research 51(11):3299–3305, 2010.
• Bowden J. A. et al. Harmonizing Lipidomics: NIST Interlaboratory Comparison Exercise for Lipidomics Using SRM 1950-Metabolites in Frozen Human Plasma. Journal of Lipid Research 58(12):2275–2288, 2017.