Sensitive determination of anandamide in rat brain utilizing a coupled-column HPLC with fluorimetric detection.
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 (Emporetrade mark), and assayed by the coupled-column HPLC. The HPLC consisted of phenyl (100 x 4.6 mm i.d. ) and octadecylsilica columns (250 x 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).
Arai
Y
Y
Department of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Fukushima
T
T
Shirao
M
M
Yang
X
X
Imai
K
K
D000818:Animals; D001095:Arachidonic Acids / Q000032:analysis*; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D063388:Endocannabinoids; D008297:Male; D010664:Phenylmethylsulfonyl Fluoride / Q000008:administration & dosage; D053284:Polyunsaturated Alkamides; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence
D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D010664:Phenylmethylsulfonyl Fluoride; C078814:anandamide