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.
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 degrees 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.
Jin
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D
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan.
Miyahara
T
T
Oe
T
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Toyo'oka
T
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D000596:Amino Acids / Q000032:analysis* / Q000652:urine; D001628:Beverages / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003611:Dairy Products / Q000032:analysis; D005456:Fluorescent Dyes; D005502:Food; D006801:Humans; D017879:Isothiocyanates; D018551:Solanum lycopersicum / Q000737:chemistry; D010069:Oxadiazoles; D013237:Stereoisomerism
C107817:4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole; D000596:Amino Acids; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D010069:Oxadiazoles