Troubleshooting in LC-MS/MS method for determining endocannabinoid and endocannabinoid-like molecules in rat brain structures applied to assessing the brain endocannabinoid/endovanilloid system significance

Abstract

Introduction: In recent years, a potential participation of endocannabinoids (eCBs) and related endocannabinoid-like molecules, including N-acylethanolamines (NAEs), in the physiological and pathophysiological processes has been highlighted, whereas measurement of their levels still remains difficult. The aim of this study was to develop a bioanalytical method that would enable researchers to simultaneously determine quantitatively eCBs (anandamide – AEA and 2-arachidonoylglycerol – 2-AG) and NAEs (oleoylethanolamide or oleoylethanolamine – OEA, palmitoylethanolamide or palmitoylethanolamine – PEA and linoleoylethanolamide or linoleoylethanolamine – LEA) in the rat brain. The analytical problems with analysis and possible solutions have been also shown.Methods: The methodology for quantifying eCBs/NAEs by means of a sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray positive ionization and multiple reaction monitoring (MRM) mode was developed and validated. Analytical problems with analyzed compounds were estimated.Results: Reasonably high precision and accuracy of the method were demonstrated in the validation process. The method is linear up to 200 ng/g for AEA, OEA, PEA and LEA and up to 100 μg/g for 2-AG, while the quantification limit reaches 0.2 ng/g and 0.8 μg/g, respectively.Discussion: Simplicity and rapidity of the assay allows analyzing many samples on a routine basis. This article presents the new procedure applied to the analysis of brain tissues.