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

Kenji Hamase, Akiko Morikawa, Tomohiro Ohgusu, Wolfgang Lindner, Kiyoshi Zaitsu

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71 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)105-111
Number of pages7
JournalJournal of Chromatography A
Volume1143
Issue number1-2
DOIs
Publication statusPublished - Mar 2 2007

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Mammals
Amino Acids
Liquids
Enantiomers
Tissue
Urine
Fluids
Metabolism
Phase separation
Isomers
Rats
Fatty Acids
High Pressure Liquid Chromatography
Pharmacology
Dogs
Derivatives
Equipment and Supplies
Chemical analysis

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

Cite this

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title = "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",
abstract = "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.",
author = "Kenji Hamase and Akiko Morikawa and Tomohiro Ohgusu and Wolfgang Lindner and Kiyoshi Zaitsu",
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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

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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