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

Eicosanoids and platelet-activating factor (PAF) are phospholipid-derived lipid mediators produced by various tissues and cells through a cascade pathway. For a comprehensive analysis of these lipid mediators, a simultaneous quantitation method with sensitivity and reliability is necessary. This article details a development of column-switching reversed-phase liquid chromatography-tandem mass spectrometry for multiplex quantitation of eicosanoids and PAF. The adsorptive nature of lipids caused significant loss of signal in a conventional column-switching configuration. The use of an online-dilution method allowed use of 100% methanol as a sample solvent, which prevented sample adsorption to contacting surfaces. Addition of 0.2% formic acid to the sample solvent was required for the successful introduction of LTC4 to the trapping column and minimizing its carryover. The optimized method provided rapid analysis of 14 lipid mediators with a throughput of 96 samples/24 h, lower limits of quantitation of 5 pg on column, and linear calibration ranges up to 2000-5000 pg. The system was highly compatible with solid-phase-extracted samples, as methanol-eluted fractions were directly injected without reconstitution. The analysis of lipid mediator production of macrophage-like RAW264.7 cells demonstrated that the cell-based assay can be performed in a 96-well format, suitable for metabolomics analyses and/or screening strategies.