Premium
Sensitive determination of anandamide in rat brain utilizing a coupled‐column HPLC with fluorimetric detection
Author(s) -
Arai Yoshiko,
Fukushima Takeshi,
Shirao Mika,
Yang Xiangjing,
Imai Kazuhiro
Publication year - 2000
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/(sici)1099-0801(200004)14:2<118::aid-bmc936>3.0.co;2-o
Subject(s) - anandamide , chemistry , chromatography , high performance liquid chromatography , pmsf , phenylmethylsulfonyl fluoride , biochemistry , cannabinoid receptor , receptor , enzyme , agonist
A fluorimetric determination method for N ‐arachidonoylethanolamine (anandamide) was developed using a precolumn fluorescence derivatization followed by coupled‐column high‐performance liquid chromatography (HPLC). Anandamide extracted from the rat brain tissue was derivatized with 4‐ N ‐chloroformylmethyl‐ N ‐methylamino‐7‐ N,N ‐dimethylaminosulfonyl‐2,1,3‐benzoxadiazole (DBD‐COCl), purified by a solid‐phase extraction (Empore™), and assayed by the coupled‐column HPLC. The HPLC consisted of phenyl (100 × 4.6 mm i.d.) and octadecylsilica columns (250 × 4.6 mm i.d.), both connected by a six‐port valve. The concentration of anandamide in rat brain was 3.37 ± 0.73 pmol/g with 6.47 and 3.57% of intra‐ and inter‐day precisions, respectively. Using this method, we investigated the alteration of anandamide concentration in rat brain 30 min after administration of anandamide (2 mg/kg, i.p.) to rats pretreated with or without phenylmethylsulfonyl fluoride (PMSF; 30 mg/kg, i.p.), an inhibitor of amidohydrolase. In rats pretreated with PMSF, the brain concentration of anandamide was approx. 16‐fold higher than that of rats without PMSF ( p < 0.01). Copyright © 2000 John Wiley & Sons, Ltd.