Determination of D-amino acids labeled with fluorescent chiral reagents, R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7 (N,N- dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in biological and food samples by liquid chromatography

Dongri Jin, Taketsune Miyahara, Tomoyuki Oe, Toshimasa Toyo'Oka

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66 Citations (Scopus)

Abstract

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)- 2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55°C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water: 30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5.2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPL-C-electrospray ionization-MS.

Original languageEnglish
Pages (from-to)124-132
Number of pages9
JournalAnalytical Biochemistry
Volume269
Issue number1
DOIs
Publication statusPublished - 1999 Apr 10
Externally publishedYes

Keywords

  • D- amino acids in biological and food samples
  • Diastereomer formation
  • Fluorescence detection
  • Indirect resolution
  • Precolumn derivatization
  • Reversed-phase liquid chromatography

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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