Fluorimetric assay of dopamine, nerepinephrine and their 3-o-methyl metabolites by using fluorescamine

Abstract

Fluorescamine was subjected to reaction with dopamine and norepinephrine (catecholamines) and with 3-methoxytyramine and normetanephrine (3-methyl metabolites of catecholamines) in phosphate or borate buffer. Catecholamines gave the highest fluorescent intensity at pH 8.0 in phosphate buffer but lower fluorescence in borate buffer. The fluorophores produced in phosphate or borate buffer were the same but the fluorescence intensities were suppressed in borate buffer. The dopamine and norepinephrine fluorophores were separated by high-pressure liquid chromatography on Hitachi 3011 gel with methanol-0.10 M Tris buffer of pH 8.0 (7:3). They were measurable at the 100-pmole level. The metabolites were also measurable by the same chromatography. By using methanol-0.15 M borate buffer of pH 8.0 catechol-O-methyltransferase activity might be assayed.