Determination of phenylalanine enantiomers in the plasma and urine of mammals and ᴅ-amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography.
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.
['Hsiao|Sui-Wen|SW|', 'Ishii|Chiharu|C|', 'Furusho|Aogu|A|', 'Hsieh|Chin-Ling|CL|', 'Shimizu|Yukiko|Y|', 'Akita|Takeyuki|T|', 'Mita|Masashi|M|', 'Okamura|Tadashi|T|', 'Konno|Ryuichi|R|', 'Ide|Tomomi|T|', 'Lee|Ching-Kuo|CK|', 'Hamase|Kenji|K|']
D000818:Animals D030801:Animals, Genetically Modified D002851:Chromatography, High Pressure Liquid D003605:D-Amino-Acid Oxidase D006801:Humans D007527:Isoenzymes D008297:Male D051379:Mice D008810:Mice, Inbred C57BL D010649:Phenylalanine D051381:Rats D011916:Rats, Inbred F344 D012680:Sensitivity and Specificity D013237:Stereoisomerism D055815:Young Adult
Phenylalanine ᴅ-Amino acid oxidase Enantiomer separation Two-dimensional HPLC