O-Phthalaldehyde—N-acetyl- L-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid ernantiomers and its application to conventional amino acid analysis

Abstract

A useful fluorescence derivatization reagent, o-phthalaldehyde-N-acetyl- l-cysteine, has been developed for the optical resolution of enantiomeric amino acids and for conventional amino acid analysis. Amino acids rapidly reacted with o-phthalaldehyde in the presence of N-acetyl- L-cysteine to give intensely fluorescent products, which were diastereoisomers. When this reaction was used in the precolumn derivatization of amino acid enantiomers, their diastereomeric derivatives were efficiently resolved on a reversed-phase column. Simultaneous analysis of common protein amino acid enantiomers was achieved by gradient elution within 70 min. In addition, the reagent was applied to the post-column derivatization of protein amino acids, combined with hypochlorite oxidation for the detection of amino acids, such as proline. Seventeen protein amino acids were sufficiently separated in 17 min by ion-pair chromatography with sodium dodecyl sulphate as a counter-ion, and they were determined at the same level of sensitivity by the above post-column fluorimetric detection system. Applications of both the precolumn and the post-column derivatization methods with the new reagent to hydrolysed protein samples are also described.