Analysis of Alanine and Serine Enantiomers in Vinegars Using a Meter-long High-resolution Enantioselective Column

Over the past few decades, D-amino acids, the minor enantiomers of widely present L-amino acids, have been found in mammals, and several D-forms including D-serine (D-Ser) and D-alanine (D-Ala) are expected as new physiologically-active molecules. Since orally ingested D-amino acids are transferred to mammalian tissues, foodstuffs and beverages naturally containing D-amino acids would be useful for human health. Therefore, the determination of chiral amino acids in various foods/beverages, especially in fermented products, is expected for the discovery of functional materials. However, the precise determination of trace levels of D-amino acids is a challenging task, and continuous improvement of both the enantiomer separation and the chemo-selectivity of the analytical methods is required. Regarding these issues, extension of the column length is one of the effective approaches. In the present study, the enantioselective separations of Ala and Ser using long enantioselective columns (the total lengths were 250, 500, 750 and 1000 mm) were evaluated. As a result, using a column of 1000-mm length, the resolution values for the Ala and Ser enantiomers were improved by 1.5– 1.6 times compared to the results obtained using a 250-mm column. The effective theoretical plate numbers for L-Ala and L-Ser were also improved by 2.2–2.3 times. By using the chiral HPLC method equipped with the meter-long enantioselective column, the Ala and Ser enantiomers in various vinegar samples were successfully determined. In the Japanese black vinegar samples produced by traditional manufacturing processes, relatively high amounts of D-enantiomers were found. Especially, in the black vinegars made in Kagoshima prefecture, the concentrations of D-Ala and D-Ser were 1.79–5.58 μmol mL^–1 and 0.12–0.89 μmol mL^–1, respectively. Additionally, the significant increase (p < 0.05) in the D-Ser amounts with the extension of the fermentation/aging period was demonstrated, indicating that the long-term fermented black vinegars are useful as functional materials.