Enantioselective Determination of Phenylalanine, Tyrosine and 3,4-Dihydroxyphenylalanine in the Urine of D-Amino Acid Oxidase Deficient Mice Using Two-Dimensional High-Performance Liquid Chromatography

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 1 st 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 2 nd 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.

Multi-Dimensional High-Performance Liquid Chromatographic Determination of Chiral Amino Acids and Related Compounds in Real World Samples

“…The amounts of several D-amino acids are measured in rodents lacking these enzymes. DAO is an enzyme degrading neutral D-amino acids [33], and the changes in the amounts of intrinsic D-amino acids including Ala [22,34], Leu [15], Phe [35], Pro [15,36] and Ser [22,34] are demonstrated. The amounts of D-Ala significantly increase in all the brain regions, peripheral tissues, plasma and urine of both mice and rats lacking DAO activity.…”

“…On the other hand, the amounts of D-Pro are mostly the same between the two strains (DAO −/− mice and control DAO +/+ mice) in all the brain and peripheral tissues [15,36], although extremely high levels of D-Pro are observed in the urine of the DAO −/− mice (20 times higher than those in the DAO +/+ mice). In the urine of DAO −/− mice, D-forms of several other proteinogenic amino acids, such as Phe and Tyr [35], and also non-proteinogenic amino acids, such as citrulline and ornithine, are likely to increase [38].…”

Multi-Dimensional HPLC Analysis of Metabolic Related Chiral Amino Acids -Method Development and Biological/Clinical Applications-

“…For the accurate, precise and simultaneous determination of Phe, Tyr and DOPA enantiomers, a 2D-HPLC system was developed consisting of a monolithic ODS column and an enantioselective column [61].…”

Development of a Three-Dimensional HPLC System for the Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mammalian Urine

“…In the past 10-20 years, various researchers have developed 2D-HPLC systems for the chiral analyses of trace levels of intrinsic compounds in order to obtain high sensitivity and high selectivity. Actually, a large number of reports using 2D-HPLC was published for the trace analysis of enantiomers including amino acids [40][41][42][43][44], dipeptides [45] and hydroxy acids [32,34,35,38,39]. Concerning the hydroxy acids, 2D-HPLC methods for the LA and 3HB enantiomers have been reported including heart-cutting 2D-HPLC [32,34,35] and whole-fraction transfer 2D-HPLC [38,39].…”