Off-line two-dimensional LC-MS/MS determination of tryptophan enantiomers in mammalian urine and alteration of their amounts in D-amino acid oxidase deficient mice

Chiharu Ishii, Noburo Takizawa, Takeyuki Akita, Masashi Mita, Tomomi Ide, Ryuichi Konno, Kenji Hamase

Research output: Contribution to journalArticlepeer-review

Abstract

D-Tryptophan (D-Trp) is one of the minor D-enantiomers of amino acids discovered in microbes and mollusca. In the present study, a highly-selective 2D chiral LC-MS/MS method has been designed and developed focusing on the determination of Trp enantiomers to investigate the presence and regulation of free D-Trp in mammals. The developed system consisted of a reversed-phase separation for the first dimension, an enantioselective separation for the second dimension and also the detection using a triple quadrupole mass spectrometer for the third/fourth dimensions. Using the present method, urinary D-Trp in mammals, including healthy human volunteers and mice, were successfully determined. Although only L-Trp was observed in a mixed urine sample of healthy volunteers, small amounts of D-Trp were detected in the C57BL/6J mice (n = 5, %D=6.18 ± 0.47). In B6DAO- mice lacking the activity of D-amino acid oxidase (DAO), relatively high levels of D-Trp were observed (n = 6, %D=27.43 ± 3.26). The obtained %D values of Trp in the urine of the C57BL/6J mice and B6DAO- mice were confirmed using various enantioselective columns having different separation properties. These results indicate that the urinary D-Trp level is regulated by DAO in mammals, and further investigations, such as tissue distribution and physiological significance of the intrinsic D-Trp, are expected.

Original languageEnglish
Article number114919
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume219
DOIs
Publication statusPublished - Sep 20 2022

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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