High‐performance liquid chromatography and fluorometric detection of arachidonylethanolamide (anandamide) and its analogues, derivatized with 4‐(<i>N</i>‐chloroformylmethyl‐<i>N</i>‐methyl) amino‐7‐<i>N</i>, <i>N</i>‐dimethylaminosulphonyl‐2,1,3‐benzoxadiazole (DBD‐COCl)

Abstract

The endogeneous ligand for the cannabinoid receptor, arachidonylethanolamide (anandamide) and its analogues, oleinylethanolamide, palmitylethanolamide and eicosapentaenoylethanolamide, were derivatized with a fluorogenic reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminsulpho ny1-2,1,3- benzoxadiazole (DBD-COCl). They were separated on a reversed phase HPLC with a mobile phase of acetonitrile:water. The fluorometric detection of the derivatives was made at 560 nm with excitation at 450 nm and the detection limits for anandamide was 20 fmol on column. The structures of DBD-CO-ethanolamides were confirmed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS).