GC/MS Analysis of Anandamide and Quantification of N‐Arachidonoylphosphatidylethanolamides in Various Brain Regions, Spinal Cord, Testis, and Spleen of the Rat

Abstract

Abstract:Anandamide [N-arachidonoylethanolamide (NAE)] was initially isolated from porcine brain and proposed as an endogenous ligand for cannabinoid receptors in 1992. Accumulating evidence has now suggested that, in the tissue, NAE is generated from N-arachidonoylphosphatidylethanolarnides (N-ArPEs) by phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N-ArPE, including organic solvent extraction, reversephase C-18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GUMS) analysis. NAE is converted by a two-step derivatization procedure to a pentafluorobenzoyl ester followed by pentafluoropropionyl acylation. Quantification was performed by isotope dilution GC/MS using deuteriumlabeled NAE (NAE-*H,) as an internal standard. The same chemical derivatization was applicable to N-ArPE quantification. The separated N-ArPE fractions were converted by a two-step cleavage/derivatization procedure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropropionyl amide. The derivative was quantified by GC/MS using deuterium-labeled 1,2-[2H,]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamide as an internal standard. Using these methods, we have found that endogenous NAE levels in rat brain, spleen, testis, liver, lung, and heart were below the level of quantification achievable (0.1 pmol/mg of protein) but that N-ArPE is readily quantifiable and is widely distributed in the rat CNS with the highest level in the spinal cord. The striaturn, hippocampus, and accumbens contain intermediate concentrations of N-ArPE, whereas the value is lowest in the cerebellum. Key Words: Anandamide-N-Arachidonoylphosphatidylethanolamides-Endogenous cannabinoidGas chromatography/mass spectrometry-Deuterium labeling-Reverse-phase C-18 cartridge.

Identification of prostamides, fatty acyl ethanolamines, and their biosynthetic precursors in rabbit cornea

“…Nevertheless, NAPE species are reported to exert bioactive functions independent of being the precursor of the FA-EA [reviewed in ( 30 )]. Concentration of NAPE species can vary greatly depending on the tissue analyzed (e.g., levels described in the rat kidney are 6-fold higher than in rat brain cortex) ( 57,58 ). In the present study, we have identifi ed several NArPE and NAPE species present in corneal tissue ( Table 2 ), and further studies are required to address the pathway of FA-EA generation from these parent phospholipids.…”

Cannabinoids and the immune system: An overview

“…It is found in brain, spleen, skin, kidney and uterus (Yang et al, 1999;Croxford and Yamamura, 2005). AEA is highly produced by brain areas where CB1R is highly represented (striatum, hippocampus, cerebellum) and is implicated in cannabis-related effects like nociception and catalepsy.…”

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

“…For the GC-MS analysis of anandamide, which possesses two functionalities accessible to derivatization, pentafluorobenzoyl chloride (PFBzCl) and pentafluoropropionic anhydride (PFPA) have been shown to be suitable [24]. We observed that anandamide reacts with these reagents to produce different derivatives.…”

“…Also, targeted lipidomic approaches for endocannabinoids including AEA have recently been performed by LC-MS/MS [22]. Methods using GC-MS have only been used to measure AEA in tissue or animal blood so far [23][24][25]. Table 1 summarizes reported data and applied methodologies for the measurement of AEA in human plasma.…”

“…Derivatization prior to GC-MS/MS analysis is carried out with pentafluorobenzoyl chloride (PFBzCl) and pentafluoropropionic anhydride (PFPA). These derivatization procedures have also been used by Yang et al [24] for the GC-MS analysis of AEA in rat tissue. By using the same method we have identified and quantitated simultaneously additional FAEA including oleic acid ethanol amide (OEA) and palmitic acid ethanol amide (PEA) (Fig.…”