Quantification of endocannabinoids in human plasma

Abstract

The objective is to quantify 4 endocannabinoids in human plasma. The compounds of interest are anandamide (AEA), 2-arachidonylglycerol (2-AG), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). Endocannabinoids are of research interest within different disease areas including multiple sclerosis. The development of this method could be used to measure endogenous plasma levels and compare the levels between a patient group and control group or as part of monitoring the levels in experimental treatment with medical cannabis products. The extraction of endocannabinoids from plasma samples is a liquid-liquid extraction (LLE) based on toluene as the solvent on a fully automated robotic setup. Toluene extracts are evaporated and reconstituted in 50% acetonitrile in water. An LC-MS/MS method was developed to quantify the four compounds of interest and to separate them from isobaric interferences. Deuterium labeled internal standards were used for all four compounds. Calibration curves are measured in water due to unavailability of a blank matrix. The chromatography allowed for separation of analytes of interest and isobaric interferences as well as separation of 2-AG and its isomer 1-arachidonylglycerol (1-AG). All the calibrations curves for the LC-MS/MS were linear with a R2 > 0.98 in the range 0.25 to 10 μg/kg. 2-AG rapidly undergoes isomerization to form a mixture of 1-AG and 2-AG; both isomers must be measured and pooled to give the total plasma concentration. The analyte OEA has a structural isomer, vaccenic acid ethanolamide (VEA), and the two are difficult to separate resulting in prolonged analysis times. VEA originates from dairy products and can vary from individual to individual, making it necessary to separate it from OEA. During the study a contamination of PEA was found in a bottle of toluene and from literature it is a known contaminant in glass pasteur pipettes. This can present itself as a potential problem, and materials used should be checked beforehand to be sure they are free of contamination. Despite issues occurring from the presence of isomers, the presented methodology allows for quantification of selected endocannabinoids in human plasma at sufficiently low limits of quantifications. Other endocannabinoid and endocannabinoid-like compounds could potentially be included in the analysis without impairing the quality of or prolonging the analysis.