Determination ofd-Amino Acids Labeled with Fluorescent Chiral Reagents,R(−)- andS(+)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7- (N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in Biological and Food Samples by Liquid Chromatography

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 HPLC–electrospray ionization–MS.