Targeted stable-isotope dilution GC–MS/MS analysis of the endocannabinoid anandamide and other fatty acid ethanol amides in human plasma

Abstract

Anandamide (arachidonoyl ethanol amide, AEA) is an endocannabinoid, acting on CB1 and CB2 receptors. Elevated plasma AEA concentrations in humans have been associated amongst others with obesity, psychological disorders and miscarriage. The occurrence in human plasma of ethanol amides of other unsaturated and saturated fatty acids, including oleic acid and palmitic acid, has also been reported. Most data available on anandamide and other fatty acid ethanol amides (FAEA) until now have been generated by using the LC–MS/MS methodology. Here, we describe a stable-isotope dilution GC–MS/MS method for the quantitative determination of AEA, oleic acid ethanol amide (OEA) and palmitic acid ethanol amide (PEA) in human plasma using their stable-isotope labeled analogs as internal standards. Other FAEA were found in plasma and their concentration was estimated. The present method involves a single solvent extraction of FAEA and their internal standards from plasma (50–1000 μl) with toluene, derivatization to the pentafluorobenzamide pentafluoropropionyl derivatives (FAEA–PFBz–PFP), and simultaneous quantification by selected reaction monitoring of the carboxylate anions produced by collision-induced dissociation of the parent ions [M−PFBz] −. The present method was fully validated for anandamide. Thus, accuracy and imprecision of the method were within the range of 100 ± 20% and less than 20%, respectively, in the range investigated (0–4 nM). Mean overall recovery was 90 ± 3%. The LOQ and LOD values of the method were determined to be 0.25 nM of added AEA in plasma samples and 400 amol of injected AEA–PFBz–PFP derivative, respectively. In plasma of 16 healthy individuals AEA concentration was measured to be 1.35 ± 0.32 nM. This finding is concordant to literature AEA plasma concentrations as measured by LC–MS/MS. The plasma concentrations of OEA, PEA and other FAEA are higher than that of AEA. This GC–MS/MS method is straightforward, accurate, precise, highly specific for FAEA and useful in basic and clinical research.