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 (0.53 mm i.d. x 1000 mm) 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–105 S (1.5 mm i.d. x 250 mm), 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.
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Organic Chemistry