Development and Application of Analytical Methods for Chiral Amino Acids and Related Metabolites

Three different types of analytical methods for D,L-amino acid were developed. First, a method for simultaneous analysis of D,L-amino acids in food was constructed by combining pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), UHPLC separation, and UV and circular dichroism (CD) detection. Using this method, D,L-amino acids in food samples was determined at a very short time of 5.5 minutes. Second, a pre-column derivatization LC/MS method (direct method) for D-alanine, D-serine, and D-aspartic acid and L-isomers in biological samples was developed. In this method, D,L-amino acids were derivatized with an achiral reagent, p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide (TAHS), separated with a chiral column, and detected using mass spectrometry (MS). Third, a pre-column derivatization LC/MS method (indirect method) for highly sensitive and selective analysis of 20 D,L-amino acids using a chiral derivatization reagent, (R)-4-nitrophenyl-N-[2-(diethylamino)-6,6-dimethyl-[1,1-biphenyl]-2-yl] carbamate hydrochloride ((R)-BiAC), was developed. The unique structure of the (R)-BiAC reagent, which has a biaryl axially chiral structure, allowed for higher resolution and a high sensitive analysis of the 20 D,L-amino acids within 12 minutes. This method has been applied to various D-amino acid research studies, and it can be applied to a wide range of analyses from food to biological samples. Lastly, an LC/MS method was established that simultaneously analyzes tryptophan and 15 of its metabolites, which is involved in neurotransmission and immunity in vivo.