Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples.

Over the past decade, the number of known eicosanoids has expanded immensely and we have now developed an ultra-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometric (UPLC-QTRAP/MS/MS) method to monitor and quantify numerous eicosanoids. The UPLC-QTRAP/MS/MS approach utilizes scheduled multiple reaction monitoring (MRM) to optimize sensitivity, number of metabolites that can be analyzed and the time requirement of the analysis. A total of 184 eicosanoids including 26 deuterated internal standards can be separated and monitored in a single 5min UPLC run. To demonstrate a practical application, human plasma samples were analyzed following solid-phase extraction (SPE) and the recovery rate and matrix effects were determined for the 26 deuterated internal standards added to the plasma. The method was validated and shown to be sensitive with the limit of quantitation at pg levels for most compounds, accurate with recovery rates of 70-120%, and precise with a CV<30 for all compounds. Also, the method showed a linear response over a range spanning several orders of magnitude. In a QC human plasma sample, we identified and rigorously quantified over 120 eicosanoids.