Analysis of eicosanoids and related metabolites

Importance of the topic

Eicosanoids and related lipid mediators, including prostaglandins, thromboxanes, leukotrienes and hydroxyeicosatetraenoic acids (HETEs), play critical roles in inflammation, vascular homeostasis and immune signaling. Robust quantification of these compounds in biological matrices is essential for biomedical research and clinical diagnostics but is challenged by their low concentrations and structural complexity.

Objectives and overview

This application note presents a validated reversed-phase LC-MS/MS method for simultaneous analysis of 18 eicosanoids and related metabolites in plasma. The workflow employs a Shim-pack GIST C18-AQ HP column coupled to a Nexera X3 UHPLC system and an LCMS-8060 triple quadrupole mass spectrometer, using multiple reaction monitoring (MRM) for targeted quantification.

Methods and instrumentation

• Chromatography conditions
  • Column: Shim-pack GIST C18-AQ HP (100 mm × 2.1 mm I.D., 1.9 µm)
  • Mobile phase A: 0.1% formic acid in water; B: acetonitrile
  • Flow rate: 0.4 mL/min; Column oven: 40 °C; Injection: 5 µL sample co-injected with 15 µL water
• Mass spectrometry conditions
  • Instrument: Shimadzu LCMS-8060
  • Ionization: ESI in positive/negative mode; Mode: MRM
  • Temperatures: DL 250 °C; Interface 270 °C; Block heater 400 °C
  • Gas flows: Nebulizing 2.5 L/min; Drying 10 L/min; Heating 10 L/min; CID gas pressure: 230 kPa
  • Dwell time: 10 ms; Pause: 1 ms
• Internal standards: 18 deuterated analogs including Tetranor-PGEM-d6, 6-keto-PGF1α-d4, TXB2-d4, PGF2α-d4, PGE2-d4, PGD2-d4, LTC4-d5, LTD4-d5, PGA2-d4, LTB4-d4, 14,15-DHET-d11, 15-HETE-d8, 12-HETE-d8, 5-HETE-d8, PAF-d4, 11,12-EET-d11, OEA-d4, AA-d8

Instrumentation used

• Nexera X3 UHPLC system (Shimadzu)
• LCMS-8060 triple quadrupole mass spectrometer (Shimadzu)

Main results and discussion

The method achieved baseline separation of all 18 analytes within a 15-minute gradient. Quantification limits reached low picogram levels with calibration linearity exceeding R² > 0.99. Intra- and inter-day precision was below 8% RSD, and recoveries ranged from 85% to 110%. Matrix suppression was minimized by optimized gradient conditions.

Benefits and practical applications

This high-throughput, sensitive workflow supports accurate eicosanoid profiling in clinical research, pharmacokinetics, and lipidomics. It facilitates biomarker discovery, disease monitoring and quality control in pharmaceutical and nutraceutical development.

Future trends and applications

Advancements such as automated sample preparation, microflow LC-MS, and integration with high-resolution platforms will improve sensitivity and expand multiplexing capabilities. Extending the panel to emerging lipid mediators will propel comprehensive systems lipidomics analyses.

Conclusion

The described LC-MS/MS method delivers robust, reproducible and sensitive analysis of eicosanoids in plasma, meeting the demands of research and clinical laboratories for comprehensive lipid mediator profiling.

References

• Shimadzu Corporation. Application News 01-00124 (JP, ENG). First Edition: Sep. 2024.