LC–MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites

• Simultaneous measurement of eicosanoids and docosanoids by HPLC–MS/MS.
• Method for quantification of 24 pro-inflammatory and pro-resolving lipid mediators.
• Rapid and robust method.
• Method validation for application in cellular systems, fluids and tissues.

Lipid autacoids derived from n-3/n-6 polyunsaturated fatty acids (PUFA) are some of the earliest signals triggered by an inflammatory reaction. They are acting also as essential regulators of numerous biological processes in physiological conditions. With regards to their importance, a robust and rapid procedure to quantify a large variety of PUFA metabolites, applicable to diverse biological components needed to be formulated. We have developed a simple methodology using liquid chromatography–tandem mass spectrometry allowing quantification of low-level of PUFA metabolites including bioactive mediators, inactive products and pathway biomarkers. Solid phase extraction was used for samples preparation with an extraction yield of 80% ranging from 65% to 98%. The method was optimized to obtain a rapid (8.5 min) and accurate separation of 26 molecules, with a very high sensitivity of detection and analysis (0.6–155 pg). When applied to biological samples, the method enabled characterization of eicosanoids and docosanoids production in epithelial cells or foam macrophages stimulated with LPS, in biological fluids and tissues from mouse models of peritonitis or infectious colitis. Our results demonstrate that this new method can be used in cultured cells, in fluids and in colonic tissues to quantify pro-inflammatory and pro-resolving PUFA metabolites mediators.