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‐chloroformylme...
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Veröffentlicht in: | Biomedical chromatography 2000-04, Vol.14 (2), p.118-124 |
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Sprache: | eng |
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Zusammenfassung: | 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 |
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ISSN: | 0269-3879 1099-0801 |
DOI: | 10.1002/(SICI)1099-0801(200004)14:2<118::AID-BMC936>3.0.CO;2-O |