Sensitive determination of anandamide in rat brain utilizing a coupled-column HPLC with fluorimetric detection

✍ Scribed by Yoshiko Arai; Takeshi Fukushima; Mika Shirao; Xiangjing Yang; Kazuhiro Imai

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 80 KB
• Volume: 14
• Category: Article
• ISSN: 0269-3879
• DOI: 10.1002/(sici)1099-0801(200004)14:2<118::aid-bmc936>3.0.co;2-o

✦ Synopsis

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-2,1,3benzoxadiazole (DBD-COCl), purified by a solid-phase extraction (Empore 2 ), and assayed by the coupled-column HPLC. The HPLC consisted of phenyl (100 Â 4.6 mm i.d.) and octadecylsilica columns (250 Â 4.6 mm i.d.), both connected by a six-port valve. The concentration of anandamide in rat brain was 3.37 AE 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).