Fluorometric assay of amino acids and amines by use of 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole(NBD-F) in high-performance liquid chromatography
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The reaction of a newly developed fluoregenic reagent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole(NBD-F), with amino acids and biogenic amines was investigated. NBD-F was reactive to both primary and secondary amines including amino acids and biogenic amines such as catecholamines. The amino acids were reacted with the reagent, separated by high-performance liquid chromatography on μ-Bondapak C18 and detected at 10 to 100 fmol level. A few μg of protein hydrolysates, rabbit pyruvate kinase M1, rabbit aldolase A and papain, were adequate for the amino acids quantitation. An automatic amino acid analyzer with fluorometric detection by the post-column derivatization with NBD-F enabled the amino acid profile analysis in blood samples present in a paper disc of 3 mm diameter.