利用联用柱高效液相色谱-荧光检测法灵敏测定大鼠脑中的花生四烯酸乙醇胺。

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

作者信息

Arai Y, Fukushima T, Shirao M, Yang X, Imai K

机构信息

Department of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

出版信息

Biomed Chromatogr. 2000 Apr;14(2):118-24. doi: 10.1002/(SICI)1099-0801(200004)14:2<118::AID-BMC936>3.0.CO;2-O.

DOI:10.1002/(SICI)1099-0801(200004)14:2<118::AID-BMC936>3.0.CO;2-O

PMID:10694706

Abstract

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).

摘要

建立了一种N-花生四烯酸乙醇胺（花生四烯酸酰胺）的荧光测定方法，该方法采用柱前荧光衍生化，随后进行双柱高效液相色谱（HPLC）分析。从大鼠脑组织中提取的花生四烯酸酰胺用4-N-氯甲酰甲基-N-甲基氨基-7-N，N-二甲基氨基磺酰基-2,1,3-苯并恶二唑（DBD-COCl）进行衍生化，通过固相萃取（Empore商标）进行纯化，并通过双柱HPLC进行测定。HPLC由苯基柱（100×4.6 mm内径）和十八烷基硅柱（250×4.6 mm内径）组成，两者通过六通阀连接。大鼠脑中花生四烯酸酰胺的浓度为3.37±0.73 pmol/g，日内和日间精密度分别为6.47%和3.57%。使用该方法，我们研究了在给用或未用酰胺水解酶抑制剂苯甲基磺酰氟（PMSF；30 mg/kg，腹腔注射）预处理的大鼠腹腔注射花生四烯酸酰胺（2 mg/kg，腹腔注射）30分钟后，大鼠脑中花生四烯酸酰胺浓度的变化。在用PMSF预处理的大鼠中，花生四烯酸酰胺的脑浓度比未用PMSF的大鼠高约16倍（p<0.01）。