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首页> 外文期刊>CHROMATOGRAPHY >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
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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

机译：二维高效液相色谱法对D-氨基酸氧化酶缺陷小鼠尿液中苯丙氨酸，酪氨酸和3,4-二羟基苯丙氨酸的对映选择性测定

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摘要

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.
机译：为了同时测定苯丙氨酸（Phe），酪氨酸（Tyr）和3,4-二羟基苯丙氨酸（DOPA）对映异构体，采用了采用4柱前衍生的全自动二维高效液相色谱（2D-HPLC）系统-氟-7-硝基-2,1,3-苯并恶二唑（NBD-F）已开发并验证。这三种氨基酸与芳香族氨基酸的代谢途径有关，因此需要一种灵敏且对映选择性的分析方法来评估其固有量和代谢。为了在1〜（st）尺寸上进行反相分离，使用整体式ODS色谱柱（0.53 mm x 1000 mm），使用Pirkle型色谱柱KSAACSP-001S（1.5 mm x 250 mm）作为色谱柱。 2〜（nd）维2D-HPLC系统用于测定D-氨基酸氧化酶（DAO）缺陷小鼠和对照C57BL小鼠尿液中的Phe，Tyr和DOPA对映异构体。至于Phe和Tyr，在两组小鼠的尿液中都发现了全部4种对映体，DAO缺陷小鼠的％D-Phe（D-Phe / Phe x 100）值显着高于Dhe对照小鼠。关于DOPA对映异构体，在C57BL小鼠和DAO缺陷小鼠的尿液中未观察到峰。另一方面，在腹膜内给予消旋DOPA后，尿中发现大量的D-DOPA。这些结果表明，本二维液相色谱系统是测定生物基质中芳香族氨基酸对映体的有力工具，尿液中Phe对映体的比例可能是DAO活性的非侵入性指标之一。

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