Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography

Abstract

A highly reactive amine derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, has been synthesized. In a rapid, one-step procedure, the compound reacts with amino acids to form stable unsymmetric urea derivatives which are readily amenable to analysis by reversed phase HPLC. Studies on derivatization conditions demonstrate excellent derivative yield over the pH range 8.2-10.0. Maximal yields are observed with a molar reagent excess of approximately three or greater. The reaction is extremely tolerant of common buffer salts and detergents, with no discernible decrease in reaction yield with well-buffered samples. Selective fluorescence detection of the derivatives with excitation at 250 nm and emission at 395 nm allows for the direct injection of the reaction mixture with no significant interference from the only major fluorescent reagent by-product, 6-aminoquinoline. Separation of the derivatized amino acids has been optimized on a C18 column with complete resolution in less than 35 min. Excellent response linearity is demonstrated over the concentration range 2.5-200 microM for all hydrolysate amino acids. Detection limits range from 40 fmol for phenylalanine to 800 fmol for cystine. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 30 ng of sample.