Development of a Fast Eicosanoid Profiling Method Using Plasma Microsamples

Significance of the topic

Eicosanoids and omega-3 fatty acid metabolites serve as critical indicators in inflammation, cardiovascular disease and other pathophysiological conditions. Rapid and sensitive quantification in minimal sample volumes enhances biomarker discovery, drug development and clinical diagnostics.

Objectives and Study Overview

This work aimed to establish a high-throughput LC-MS/MS method for the quantitative profiling of over 100 eicosanoids and related fatty acid metabolites in human plasma micro-samples. By refining prior mouse serum protocols, the study sought to shorten analysis time to 10 minutes while maintaining analytical performance.

Methodology and Instrumentation

The analytical workflow combined precise microsampling with ultra-fast UHPLC and triple quadrupole MS detection.

• Sampling: A 5.6 µL plasma aliquot collected using the MSW2 microsampling device.
• Sample preparation: Protein precipitation with methanol (0.1% formic acid) spiked with 18 deuterated internal standards; solid-phase extraction on STRATA-X cartridges; reconstitution in methanol.
• Chromatography: Nexera X3 system with Shim-pack GIST C18-AQ HP column (100×2.1 mm, 1.9 µm); gradient elution at 0.4 mL/min; total run time 10 minutes.
• Mass spectrometry: LCMS-8060 operating in electrospray ionization (positive/negative), multiple reaction monitoring mode with 200 transitions; rapid polarity switching (5 ms); dwell time 10 ms.

Used Instrumentation

• Shimadzu Nexera X3 UHPLC system
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer
• MSW2TM microsampling device (Shimadzu)
• STRATA-X SPE cartridges (Phenomenex)

Main Results and Discussion

• The 10-minute method monitored 114 target metabolites plus 18 internal standards using 200 MRM transitions.
• Chromatographic resolution (R ≈3.0) for key prostaglandin deuterated standards remained robust compared to the prior 20-minute method (R ≈3.8).
• Analysis of 5.6 µL human plasma detected over 50 eicosanoids and related fatty acids, including major arachidonic acid derivatives (5-HETE, 15-HETE, etc.).
• Reproducibility across three replicate injections showed coefficient of variation below 20% for 36 quantified targets.
• Microsampling repeatability was demonstrated with ~3% CV for the intense Lyso-PAF signal, confirming accurate volume collection.

Benefits and Practical Applications

• High throughput: 10-minute runtime accelerates large-scale screening.
• Minimal sample volume: Suitable for studies with limited material, such as clinical microsamples or small animal models.
• Comprehensive coverage: Simultaneous quantitation of a broad range of lipid mediators in a single run.

Future Trends and Potential Applications

The combination of microsampling and ultra-fast LC-MS/MS is poised to expand in personalized medicine, longitudinal biomarker monitoring and preclinical research. Further miniaturization and integration with automated sample handling will enhance throughput and reproducibility.

Conclusion

A rapid, robust LC-MS/MS protocol has been developed for eicosanoid profiling in 5.6 µL human plasma. The method delivers high sensitivity, reproducibility and analytical depth within a 10-minute analysis, supporting diverse applications in biomarker discovery and pathophysiological research.

References

• M. Yamada, T. Nakamura, A. Murata, T. Hattori, MP-546, 67th ASMS (2019).