Abstract
For the simultaneous determination of phenylalanine (Phe), tyrosine (Tyr) and 3,4-dihydroxyphenylalanine (DOPA) enantiomers, a fully-automated two-dimensional high-performance liquid chromatographic (2D-HPLC) system using pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been developed and validated. These three amino acids are related to the aromatic amino acid metabolic pathways, and a sensitive and enantioselective analytical method is required to evaluate their intrinsic amounts and metabolism. For the reversed-phase separation in the 1st dimension, a monolithic ODS column (0.53 mm x 1000 mm) was used and a Pirkle-type column, KSAACSP-001S (1.5 mm x 250 mm), was used for the 2nd dimension. The 2D-HPLC system was applied to the determination of the Phe, Tyr and DOPA enantiomers in the urine of D-amino acid oxidase (DAO) deficient mice and control C57BL mice. As for Phe and Tyr, all of the 4 enantiomers were found in the urine of both groups of mice, and the values of %D-Phe (D-Phe/Phe x 100) were significantly higher in the DAO deficient mice than those in the control mice. Concerning the DOPA enantiomers, no peaks were observed in the urine of the C57BL mice and DAO deficient mice. On the other hand, a significant amount of D-DOPA was found in their urine after the intraperitoneal administration of racemic DOPA. These results indicate that the present 2D-HPLC system is a powerful tool for the determination of aromatic amino acid enantiomers in biological matrices, and the ratio of Phe enantiomers in the urine could be one of the non-invasive indicators of DAO activity.
