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

Abstract

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.

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

“…Without using chiral reagents, a UPLC method equipped with a circular dichroism detector [30] and a chiral HPLC-MS/MS system [31] have been used for the analysis of all the proteinogenic amino acids in the vinegar samples. To perform a higher selective analysis, a two-dimensional (2D) HPLC system combining reversed-phase and enantioselective columns [32,33] and a 2D-HPLC-MS/MS system [34] have been developed and clarified the developmental changes of the D-amino acid concentrations during the fermentation processes. By using these analytical methods, the %D values of Asp, Ser and Ala in the rice vinegar samples were reported to be 1.3-35.3, 0.9-21.6 and 1.1-38.3, respectively [28][29][30]32,34].…”

Multi-Dimensional High-Performance Liquid Chromatographic Determination of Chiral Amino Acids and Related Compounds in Real World Samples

“…In some complex matrices, however, co-eluted compounds are fractionated together with the target amino acids in the first dimension, and disturbed the precise determination of the amino acid enantiomers in the second dimension. Therefore, an additional column is placed between the reversed-phase and enantioselective columns for further separation of the target amino acids from interfering compounds [58,59].…”

Development of a Three-Dimensional HPLC System for the Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mammalian Urine

“…In the past 10-20 years, various researchers have developed 2D-HPLC systems for the chiral analyses of trace levels of intrinsic compounds in order to obtain high sensitivity and high selectivity. Actually, a large number of reports using 2D-HPLC was published for the trace analysis of enantiomers including amino acids [40][41][42][43][44], dipeptides [45] and hydroxy acids [32,34,35,38,39]. Concerning the hydroxy acids, 2D-HPLC methods for the LA and 3HB enantiomers have been reported including heart-cutting 2D-HPLC [32,34,35] and whole-fraction transfer 2D-HPLC [38,39].…”