Amino acid analysis by derivatization with o-phthaldialdehyde and chiral thiols

Abstract

The chiral resolution of standard mixtures composed of a large number of DL-amino acids was investigated by reversed-phase high-performance liquid chromatography (HPLC) of the corresponding isoindolyl derivatives, formed by automated precolumn derivatization with o-phthaldialdehyde (OPA) and a series of chiral N-acyl-L-cysteines (acyl = n-octanoyl, n-butyryl, isobutyryl) which had been synthesized in our laboratory. The resolutions were compared with those obtained when N-acetyl-D-penicillamine and the thiol drug ‘Captopril’ (1-[(2S)-3-mercapto-2-methylpropanoyl]-L-proline) were used as chiral reagents. All reagents made the more or less complete enantioseparation of amino acid mixtures possible; however, n-butyryl-L-cysteine and isobutyryl-L-cysteine (i-But-Cys) in particular gave high resolution coefficients for most of the DL-amino acids. For HPLC the liquid Chromatograph was equipped with a device for automated precolumn derivatization, a fluorescence detector and a workstation for data processing. Chromatography was performed by employing octadecylsilyl silica (3 μm) as the stationary phase and gradients of sodium acetate buffers with acetonitrile as the eluents. The utility of the method for the determination of minor amounts of enantiomers is demonstrated by the detection of 0.2% D-Trp and 0.5% D-Ser in the presence of an excess of the respective L-enantiomer by use of the OPA/i-But-Cys reagent.