Abstract
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°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. Copyright © 2005 John Wiley & Sons, Ltd.