A multiplex quantitation method for eicosanoids and platelet-activating factor using column-switching reversed-phase...

Area of Science:

• Lipidomics
• Analytical Chemistry
• Biochemistry

Background:

• Eicosanoids and platelet-activating factor (PAF) are critical phospholipid-derived lipid mediators involved in various physiological processes.
• Accurate and simultaneous quantification of these mediators is essential for comprehensive biological analysis.
• Existing methods often face challenges with sensitivity and reliability due to the adsorptive nature of lipids.

Purpose of the Study:

• To develop a sensitive and reliable column-switching reversed-phase liquid chromatography-tandem mass spectrometry method for multiplex quantitation of eicosanoids and PAF.
• To overcome signal loss issues associated with lipid adsorption in conventional systems.
• To establish a high-throughput method suitable for metabolomics and screening applications.

Main Methods:

• Development of a novel column-switching LC-MS/MS system incorporating an online-dilution technique.
• Optimization of sample solvent (100% methanol) and addition of formic acid to prevent adsorption and ensure successful analyte introduction.
• Validation of the method for rapid analysis of 14 lipid mediators with high throughput and low limits of quantification.

Main Results:

• The optimized method effectively prevented sample adsorption by utilizing 100% methanol as the sample solvent.
• Addition of 0.2% formic acid facilitated LTC4 introduction and minimized carryover.
• Achieved a throughput of 96 samples/24 h, with lower limits of quantification of 5 pg on column and linear calibration ranges up to 5000 pg.
• Demonstrated compatibility with solid-phase-extracted samples, allowing direct injection of methanol-eluted fractions.

Conclusions:

• The developed column-switching LC-MS/MS method offers a sensitive, reliable, and high-throughput solution for simultaneous quantitation of eicosanoids and PAF.
• The online-dilution approach effectively addresses lipid adsorption challenges, enhancing method robustness.
• The method is suitable for cell-based assays in a 96-well format, supporting metabolomics and screening strategies for lipid mediator analysis.