TY - JOUR
T1 - Comprehensive analysis of branched aliphatic d-amino acids in mammals using an integrated multi-loop two-dimensional column-switching high-performance liquid chromatographic system combining reversed-phase and enantioselective columns
AU - Hamase, Kenji
AU - Morikawa, Akiko
AU - Ohgusu, Tomohiro
AU - Lindner, Wolfgang
AU - Zaitsu, Kiyoshi
N1 - Funding Information:
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Takeda Science Foundation, and Industrial Technology Research Grant Program in ’05 from New Energy and Industrial Technology Development Organization (NEDO) of Japan. The authors appreciate Shiseido Co. Ltd. (Tokyo, Japan) for providing the HPLC system (NANOSPACE series) and the technical support. The authors express their gratitude to Dr. Kevin Schug for skilled proofreading of the manuscript.
PY - 2007/3/2
Y1 - 2007/3/2
N2 - A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic d-amino acids in the presence of large amounts of their l-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the d-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60 min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5 min. In the rat, significant amounts of d-Leu were found in all tissues and physiological fluids tested (trace - 1.3 nmol/g tissue), and in the urine, the presence of high amounts of d-allo-Ile (d-isomer of a non-proteinogenic amino acid, 22.2 nmol/ml) was demonstrated. d-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual d-amino acid and the potential need to clarify its unique metabolism in mammals.
AB - A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic d-amino acids in the presence of large amounts of their l-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the d-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60 min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5 min. In the rat, significant amounts of d-Leu were found in all tissues and physiological fluids tested (trace - 1.3 nmol/g tissue), and in the urine, the presence of high amounts of d-allo-Ile (d-isomer of a non-proteinogenic amino acid, 22.2 nmol/ml) was demonstrated. d-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual d-amino acid and the potential need to clarify its unique metabolism in mammals.
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U2 - 10.1016/j.chroma.2006.12.078
DO - 10.1016/j.chroma.2006.12.078
M3 - Article
C2 - 17223114
AN - SCOPUS:33846794212
SN - 0021-9673
VL - 1143
SP - 105
EP - 111
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 1-2
ER -