A Comprehensive, Curated, High‑Throughput Method for the Detailed Analysis of the Plasma Lipidome

Significance of the Topic

The detailed analysis of the human plasma lipidome offers critical insight into the molecular underpinnings of health and disease. Lipids regulate cell structure, energy storage, and signaling pathways. High-throughput lipid profiling at the population level reveals metabolic alterations linked to cardiometabolic disorders, diabetes, and inflammatory diseases. A robust, reproducible workflow capable of quantifying hundreds of lipid species from minimal plasma volumes accelerates large-cohort studies, biomarker discovery, and personalized medicine.

Objectives and Study Overview

This study presents a targeted liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of 763 lipid species across 44 classes from just 10 µL of human plasma. A balance between comprehensive coverage and sample throughput was achieved via dynamic multiple reaction monitoring (dMRM) on a triple quadrupole instrument. Key goals included optimizing sample preparation, lipid extraction, chromatographic separation, instrument parameters, and data processing to support high-throughput clinical and population research.

Methodology and Instrumentation

Sample Preparation and Extraction

• Internal standard mixture composed of stable isotope-labeled and nonphysiological lipids prepared in chloroform:methanol (1:1) and stored at –80 °C.
• Plasma lipids extracted by mixing 10 µL plasma with 100 µL butanol:methanol (1:1) containing 10 mM ammonium formate and ISTDs, followed by vortexing, bath sonication, centrifugation, and transfer to autosampler vials.
• Pooled QC samples (PQC, TQC, NIST 1950) and blanks inserted at regular intervals to monitor extraction and instrumental performance.

Chromatography and MS Conditions

• Agilent 1290 Infinity/Infinity II LC system with single or dual ZORBAX Eclipse Plus C18 column at 45 °C.
• Gradient elution over 16 min (single-column) or 13 min (dual-column) with mobile phases containing ammonium formate, deactivator additive, water, acetonitrile, and 2-propanol.
• Agilent 6495C triple quadrupole MS with Jet Stream ionization, positive/negative switching, dynamic MRM, unit resolution, and optimized source parameters.
• dMRM windows tailored to each lipid’s retention time, allowing up to 115 concurrent transitions with dwell times >5 ms.

Main Results and Discussion

Instrument Reproducibility

• 65 TQC injections: median CVs of 4.1 % for peak area and 4.0 % for calculated concentration across 763 lipids.
• Background contributions < 1 % for 585 lipids; 28 low-abundance species showed > 20 % blanks, highlighting targets requiring caution.

Batch Alignment Across 2,500 Samples

• Six batches of 417 plasma samples with QCs achieved alignment via time reference and batch-wise correction.
• PQC and NIST1950 QC median CVs: 6.6 % and 7.7 % (single batch); batch alignment reduced interbatch variability.
• PCA distinguished biological variance from QC tight clustering, demonstrating data quality for large-scale studies.

Structural Annotation and Isomer Distinction

• Offline standards and isotopic overlap assessments enabled discrimination of isomers (e.g., PC(36:2) positional and plasmalogen forms).
• Acid-hydrolysis confirmed vinyl-ether-containing lipids.

Benefits and Practical Applications of the Method

This workflow supports high-throughput lipid profiling with minimal sample input, rapid turnaround, and stringent quality controls. It enables:

• Population-level lipidomics to identify disease-associated lipid signatures.
• Comparability across laboratories through standardized NIST1950 QC alignment.
• Flexible adaptation to alternate matrices (e.g., tissues, cell extracts) by rerouting chromatographic separation and annotation strategies.

Future Trends and Applications

Emerging directions include:

• Integration with ion mobility and high-resolution MS to resolve additional isomeric species.
• Expansion of targeted panels to include oxidized lipids and rare subclasses.
• Automation on 96-well platforms to further increase throughput.
• Machine-learning-driven data processing for rapid interpretation of large lipidomic datasets.

Conclusion

The described targeted LC/MS/MS protocol provides fast, reproducible quantitation of a comprehensive plasma lipidome. Coupled with rigorous quality control and batch alignment strategies, it empowers large-cohort studies and cross-lab comparisons for biomedical research and clinical applications.

Reference

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