title: Enantioselective determination of citrulline and ornithine in the urine of d-amino acid oxidase deficient mice using a two-dimensional high-performance liquid chromatographic system.
abstract: Two-dimensional high-performance liquid chromatographic (2D-HPLC) and 2D-HPLC-mass spectrometric (2D-HPLC-MS) systems have been designed and developed for the determination of the citrulline (Cit) and ornithine (Orn) enantiomers. Several d-amino acids have already been identified as novel physiologically active molecules and biomarkers, and the enantioselective evaluation of the amounts, distributions and metabolisms of non-proteinogenic amino acids gain as well increasing interest. In the present study, highly selective analytical methods were developed using a capillary monolithic ODS column (0.53mm i.d. x 1000mm) for the reversed-phase separation of the target analytes from the matrix compounds in the first dimension, and a narrowbore-Pirkle type enantioselective column, KSAACSP-105S (1.5mm i.d. x 250mm), was used for the enantiomer separation in the second dimension. The amino acids were analyzed after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by the fluorescence detector and MS. The systems were applied to the urine of d-amino acid oxidase (DAO) deficient B6DAO-mice and control C57BL mice to evaluate the presence and metabolism of the Cit and Orn enantiomers in mammals. As a result, all of the 4 target enantiomers (d-Cit, l-Cit, d-Orn, l-Orn) were found in the urine of both strains. The %D value of Cit (d-Cit/Cit×100) increased about 3-fold in the urine of the DAO deficient mice and that of Orn also tended to increase with the DAO deficiency. These results were definitely confirmed by a 2D-HPLC-MS detection system. Further investigations about the biological significance of these d-isomers are currently ongoing.
authors: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors: Reiko
authors: R
authors: Koga
authors: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors: Yurika
authors: Y
authors: Miyoshi
authors: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors: Yu
authors: Y
authors: Sato
authors: Shiseido Co., Ltd., 1-6-2 Higashi-shimbashi, Minato-ku, Tokyo 105-8310, Japan.
authors: Masashi
authors: M
authors: Mita
authors: Department of Pharmaceutical Sciences, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara 324-8501, Japan.
authors: Ryuichi
authors: R
authors: Konno
authors: Institute of Analytical Chemistry, University of Vienna, Waehringerstrasse 38, A-1090 Vienna, Austria.
authors: Wolfgang
authors: W
authors: Lindner
authors: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: hamase@phar.kyushu-u.ac.jp.
authors: Kenji
authors: K
authors: Hamase
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002956:Citrulline / Q000737:chemistry / Q000652:urine*; D003605:D-Amino-Acid Oxidase / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009952:Ornithine / Q000737:chemistry / Q000652:urine*; D013237:Stereoisomerism
chemical_list: D015415:Biomarkers; D002956:Citrulline; D009952:Ornithine; C485958:Dao1 protein, mouse; D003605:D-Amino-Acid Oxidase
keywords: Citrulline; Enantiomer separation; Ornithine; Two-dimensional HPLC; d-amino acid oxidase