Simultaneous determination of 2‐arachidonoylglycerol, 1‐arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry

Abstract

Endocannabinoids (ECs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, 2-AG. Identification and quantification of 2-AG, 1-AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of 2-AG. We have developed a sensitive and specific quantification method for simultaneous determination of 2-AG, 1-AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparation method. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N = 3) at 50, 25 and 65 fmol for 2-AG, 1-AG and AA, respectively. The methods were selective, precise (%R.S.D. < 10%) and sensitive over a range of 0.02-20, 0.01-10 and 0.05-50 ng/mg tissue for 2-AG, 1-AG and AA, respectively. The quantification method was validated with consideration of the matrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of 2-AG from mouse brain extracts containing membrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of 2-AG, 1-AG and AA in mouse brain and adipose tissues.