Determination of the endocannabinoid anandamide in human plasma by high-performance liquid chromatography.
Anandamide (N-arachidonylethanolamine) is an endogenous cannabinoid receptor ligand that has been implicated in various physiological and pathophysiological functions. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with fluorometric detection was validated and applied for the analysis of anandamide in human plasma. Following derivatization with the fluorogenic reagent 4-(N,N-dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methyl-amino)-2,1,3-benzoxadiazole (DBD-COCl), the analyte was separated using an acetonitrile-water gradient at a flow rate of 0.8 mL/min, and spectrophotometric detection at 560 nm with an excitation wavelength of 450 nm. The retention times for anandamide and R+-methanandamide (internal standard) were 27.1 and 30.7 min, respectively. The validated quantification range was 1-15 ng/mL. The developed procedure was applied to determine anandamide levels in human plasma following a 24 h incubation of human whole blood at 37 degrees C in the presence or absence of phenylmethylsulfonyl fluoride, an inhibitor of the anandamide-degrading enzyme fatty acid amide hydrolase. Anandamide levels determined under both conditions were within the validated concentration range with anandamide levels being 2.3-fold higher in plasma from PMSF-treated blood.
Schmidt
Andreas
A
Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany.
Brune
Kay
K
Hinz
Burkhard
B
D001095:Arachidonic Acids / Q000097:blood*; D063385:Cannabinoid Receptor Modulators / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides
D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; C078814:anandamide