Determination of Chiral Amino Acids in Various Fermented Products Using a Two-Dimensional HPLC-MS/MS System

Abstract

Chiral amino acids in fermented products including Japanese traditional black vinegar, a processed cheese and nam pla were determined using an on-line two-dimensional (2D) HPLC-MS/MS system. As the target amino acids, Ala, Asp, Glu, Leu, Pro and Ser were selected. Prior to the HPLC separation, the fermented products were appropriately homogenized and deproteinized, then amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Using the 2D-HPLC system, the target NBD-amino acids were individually purified by a microbore reversed-phase column in the first dimension and further separated by a narrowbore enantioselective column in the second dimension. The detection was performed by a fluorescence detector and also by a tandem mass spectrometer. Compared to the 2D-HPLC with fluorescence detection, the target chiral amino acids in complicated real world matrices were successfully determined using the 2D HPLC-MS/MS system without interference by the co-elution of unknown intrinsic compounds. In all the tested fermented products, various D-amino acids were observed, and the obtained values were 0.02-6.21 mmol/L (%D=0.7-29.2%) in the black vinegar, 0.02-0.73 µmol/g (0.2-24.8%) in the processed cheese and 0.07-2.30 mmol/L (0.5-23.5%) in the nam pla.

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].…”

“…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]. The D-amino acid amounts in the traditionally fermented rice vinegars seem to be higher than those in the other rice vinegars.…”

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

“…In foodstuffs and beverages, relatively high levels of D-amino acids are considered to be contained particularly in the fermented products due to the generation by microorganisms. Accordingly, the commercially available fermented foods/beverages including processed cheese, Japanese traditional black vinegar (amber rice vinegar) and nam pla (fish source) are selected and the chiral amino acids in these foodstuffs are analyzed by the 2D HPLC-MS/MS system [53]. The chromatograms obtained using the black vinegar are shown in Fig.…”

Molecular Shape Selectivity for Polycyclic Aromatic Compoundson a Poly(4-vinylpyridine) Stationary Phase in Liquid Chromatography

“…Liquid chromatography (LC) is one of the most powerful separation techniques for various types of complex mixtures and it has been employed in a wide range of scientific fields such as pharmaceutical, analytical and environmental chemistry [1][2][3][4][5]. In order to make sure an effective LC separation of real samples commonly encountered, a suitable setting of the separation conditions, especially, the choice of an appropriate stationary phase is quite important parameter [6][7][8][9][10].…”