High-performance liquid chromatographic method for the quantification of anthranilic and 3-hydroxyanthranilic acid in rat brain dialysate

Anthranilic acid (ANA) and 3-hydroxyanthranilic acid (3-HANA) have attracted considerable attention as two of the L-tryptophan kynurenine pathway metabolites in the central nervous system. In this study, a highly sensitive and accurate method for the quantification of ANA and 3-HANA has been developed using reversed-phase high performance liquid chromatography (HPLC) with fluorimetric detection. The HPLC assay was carried out using a C-18 column (5 mum, 250 x 4.6 mm W.). The mobile phase consisted of a mixture of 25 mM sodium/acetic acid buffer (pH 5.5) and methanol (90:10 v/v). Fluorimetric detection at lambda(ex) = 316 nm and lambda(em) = 420 nm was used. The assay was applied to the measurement of ANA and 3-HANA acid in rat brain dialysate following administration Of L-tryptophan or L-kynurenine. 3-HANA and ANA levels were progressively increased during 90 min following administration Of L-tryptophan, then decreased progressively to basal levels. 3-HANA levels were significantly higher than ANA levels after L-kynurenine administration. These findings suggest that the assay developed should provide an improved means for investigation of neurobiology of kynurenine pathway. (C) 2003 Elsevier Science B.V. All rights reserved.