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

Abstract

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 μm, 250×4.6 mm i.d.). 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 λ ex=316 nm and λ 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.