Recent advances in analytical techniques have revealed trace amounts of D-amino acids in higher animals. A variety of their functions, which differ from those of L-forms, have also been clarified (e.g., regulation of neuronal transmission, hormonal synthesis/secretion, antioxidative effects, and barrier function in the skin). As dietary intake is considered one of the sources of D-amino acids in mammals, including humans, finding foodstuffs containing high amounts of D-forms and utilizing them as the functional ingredients are expected. For the analysis of chiral amino acids in real-world samples, high-performance liquid chromatography (HPLC) is widely adopted. However, determining minor amino acid enantiomers in complicated matrices is often affected by the co-elution of countless interfering substances, so highly selective methods are recommended for accurate quantification. To address this issue, we have developed highly selective analytical techniques for proteinogenic and metabolic-related chiral amino acids in complex matrices, either by extending the enantioselective column length or by integrating multiple separation modes (i.e., multi-dimensional HPLC). This article introduces the HPLC methods we have developed for determining chiral amino acids, particularly in food and beverage samples, and presents the results of their application.
