A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

已经开发出了一种二维（2D）HPLC系统，其重点是确定哺乳动物生理液中的苯丙氨酸（Phe）对映异构体。有人指出，β-Phe在某些神经元和代谢性疾病中具有作为疾病生物标志物和治疗分子的潜在价值，因此对哺乳动物中γ-Phe的调控是一个令人关注的问题。然而，在复杂的生物样品中氨基酸对映体的精确测定是困难的，并且期望开发一种具有实际可接受的灵敏度，选择性和通量的分析方法。在本研究中，设计了二维液相色谱系统，该系统在第一维上配备了反相色谱柱，在第二维上配备了对映选择性色谱柱，随后将目标氨基酸对映体用4-氟-7-荧光进行了荧光衍生化。硝基-2,1，3-苯并恶二唑（NBD-F）。该分析方法已通过血浆和尿液样品验证，并成功应用于人，大鼠和小鼠的体液。测定血浆中的痕量ᴅ-Phe，所有物种的％ᴅ值约为0.1％。在尿液中，观察到相对大量的ᴅ-Phe，人，大鼠和小鼠的％the值分别为3.99、1.76和5.25％。还阐明了-氨基酸氧化酶（DAO）的酶活性与固有ᴅ-Phe量之间的关系，并观察到了高ᴅ-Phe量（血浆中约0.3％，尿中约50％ ）在DAO缺陷的大鼠和小鼠中。测定血浆中的痕量ᴅ-Phe，所有物种的％ᴅ值约为0.1％。在尿液中，观察到相对大量的ᴅ-Phe，人，大鼠和小鼠的％the值分别为3.99、1.76和5.25％。还阐明了-氨基酸氧化酶（DAO）的酶活性与固有ᴅ-Phe量之间的关系，并观察到了高ᴅ-Phe量（血浆中约0.3％，尿中约50％ ）在DAO缺乏的大鼠和小鼠中。测定血浆中的痕量ᴅ-Phe，所有物种的％ᴅ值约为0.1％。在尿液中，观察到相对大量的ᴅ-Phe，人，大鼠和小鼠的％the值分别为3.99、1.76和5.25％。还阐明了-氨基酸氧化酶（DAO）的酶活性与固有ᴅ-Phe量之间的关系，并观察到了高ᴅ-Phe量（血浆中约0.3％，尿中约50％ ）在DAO缺陷的大鼠和小鼠中。