Determination of endocannabinoids and their conjugated congeners in the brain by means of μSPE combined with UHPLC-MS/MS

The present study encompasses the development of a fast and reliable analytical method to quantify the main endocannabinoids and some of their conjugated congeners, particularly N-arachidonoyl amino acids, in brain tissue. Samples were homogenized and a micro solid phase extraction (μSPE) procedure was developed for brain homogenate clean-up. Miniaturized SPE was selected as it allowed to work with reduced sample amounts, while maintaining high sensitivity; this last feature was mandatory due to the low concentration of endocannabinoids in biological matrices that makes their determination a challenging analytical task. UHPLC-MS/MS was used for the analysis as it provided a great sensitivity, especially for conjugated forms that were detected by negative ionization. Polarity switching was applied during the run; low limits of quantification were between 0.003 ng g−1 and 0.5 ng g−1. This method provided also low matrix effect (lower than 30%) and good extraction recoveries in the brain. To the best of our knowledge, this is the first time that μSPE is applied on this matrix for this class of compounds. The method was validated according to international guidelines, and then tested on real cerebellum samples from mice, which were sub-chronically treated with URB597, a well-known inhibitor of the fatty acid amide hydrolase. [Display omitted] •Novel analytical method for endocannabinoids analysis in brain by UHPLC-MS/MS.•μSPE clean-up provided good recoveries and low matrix effect.•Improvement of sensitivity through polarity switching.•Validation of the method according to European Medical Agency.•Testing on real samples to prove method performance.