Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept |
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Authors: | Kenji Hamase Yurika Miyoshi Kyoko Ueno Hai Han Junzo Hirano Akiko Morikawa Masashi Mita Tsuneaki Kaneko Wolfgang Lindner Kiyoshi Zaitsu |
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Institution: | 1. Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan;2. Innovative Science Research and Development Center, Shiseido Co., Ltd., 2-12-1 Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan;3. Institute of Analytical Chemistry and Food Chemistry, University of Vienna, Waehringerstrasse 38, A-1090 Vienna, Austria |
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Abstract: | A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the non-chiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The d-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios. |
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Keywords: | Hydrophilic amino acid Enantiomer separation Two-dimensional HPLC Fluorescence |
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