High-Performance Liquid Chromatographic Determination of Chiral Amino Acids Using Pre-Column Derivatization with o-Phthalaldehyde and N-tert-Butyloxycarbonyl-D-cysteine and Application to Vinegar Samples

Abstract

A reversed-phase high-performance liquid chromatographic (HPLC) method using pre-column derivatization with o-phthalaldehyde (OPA) plus N-tert-butyloxycarbonyl-D-cysteine (Boc-D-Cys) has been developed for the determination of aspartic acid (Asp), serine (Ser) and alanine (Ala) enantiomers. D-Amino acids in the real world samples are trace in most cases, and their small peaks should be eluted faster than the huge peaks of the L-forms in order to avoid overlapping. Amino acids were rapidly derivatized at room temperature with OPA plus Boc-D-Cys under simple conditions and were detected by their fluorescence. The target amino acid enantiomers were separated within 60 min on a reversed-phase column, CAPCELL PAK C18 MG II (4.6 x 200 mm), and their resolution values were higher than 2.14. The developed system was successfully validated using standard amino acids, and sufficient calibration lines (r² > 0.9983) and precision (RSD < 5.29%) results were obtained. In a Japanese traditionally fermented amber rice vinegar, all of the target D-amino acids were observed, and their %D values were 21.5 for Asp, 6.8 for Ser and 22.9 for Ala.