Multi-Dimensional HPLC Analysis of Serine Containing Chiral Dipeptides in Japanese Traditional Amber Rice Vinegar

Several D-amino acids have been identified in higher animals including humans, and are now increasingly recognized as biologically active substances and/or biomarkers. Therefore, the presence of related compounds, such as dipeptides, containing D-amino acids in biological matrices is one of the recent topics of increasing interest. In the present study, on-line multi-dimensional HPLC systems combining a narrowbore reversed-phase column (KSAARP, 1.5 mm i.d. x 500 mm), a weak anion-exchange column (KSAAAX, 1.5 mm i.d. x 250 mm) and a Pirkle-type enantioselective column (KSAACSP-001S or R, 1.5 mm i.d. x 250 mm) have been designed for the chiral analysis of Ser-containing dipeptides in fermented products. As target analytes, Ser-Gly and Gly-Ser were selected. For the sensitive analysis, the dipeptides were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the NBD-dipeptides, which were detected upon their fluorescence. During the selective multi-dimensional analysis, the target dipeptides were separated from other substances by the reversed-phase and anion-exchange columns as their D/L scalemic mixtures and automatically introduced to the enantioselective column, where the D,L-forms were separated with selectivity / separation factor (α) and resolution (Rs) values higher than 1.07 and 0.71, respectively. The present 3D-HPLC system was well applicable to the analysis of Japanese traditional amber rice vinegar where relatively large amounts of the L-dipeptides and trace amount of D-Ser-Gly were observed.