High-throughput comprehensive analysis of d- and l-amino acids using ultra-high performance liquid chromatography with a circular dichroism (CD) detector and its application to food samples

Abstract

A rapid and comprehensive analytical method for d- and l-enantiomers of proteinogenic amino acids was developed using ultra-high performance liquid chromatography (UHPLC) equipped with a circular dichroism (CD) detector. Pre-column derivatization reagents were examined for enhanced sensitivity and selectivity for UV and CD detection: 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was selected. The method, using a CD detector, does not require separation of optical isomers on a column to calculate the enantio ratio (% D) using the g-factor value and produces a simple chromatogram in comparison to other reported methods. Using this advantage, combined with UHPLC technology, analysis time for the derivatized proteinogenic amino acids was within 5.5 min. The UV detection limit was 4.9–23 pmol/injection and the CD detection limit was 11–64 pmol/injection. The method was applied to the analysis of d- and l-amino acids in food samples. d-Ala, d-Asp, d-Glu and d-Ser were detected at high concentrations in some Japanese black vinegars, fermented milks and yogurts. The results were identical to the results determined by the OPA method. We suggest the UHPLC–CD method would be useful in screening the d-amino acid content of foods and in helping to clarify the importance and reason for the presence of d-amino acids in foods.