Analysis of Omega-3 Fatty Acid-Derived N-Acylethanolamines in Biological Matrices

The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.