Total resolution of 17 DL-amino acids labelled with a fluorescent chiral reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-y1)-7-(N,N-dimethylaminosulfonyl)- 2,1,3-benzoxadiazole, by high-performance liquid chromatography.

Total resolution of 17 DL-amino acids after derivatization with a fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfony l)-2,1,3- benzoxadiazole [R(-)-DBD-PyNCS], was studied by reversed-phase liquid chromatography. The reaction of the reagent with amino acids proceeds effectively at 55 degrees C for 20 min in the presence of 1% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). Each pair of the resulting derivatives was efficiently separated with water-acetonitrile containing 1% acetic acid as the mobile phase. Peak resolution was in the range of 0.92 (DL-Arg)-9.8 (DL-Cys). Although mutual separation of some DL-amino acids was possible using the elution solvent, simultaneous resolution of 17 DL-amino acids was difficult with a single chromatographic run, even if some gradient elutions were adopted. Therefore, both gradient and isocratic elution systems were used for total resolution of the DL-amino acids. Thus, 17 DL-amino acids were well resolved by a gradient and an isocratic elution systems. The proposed derivatization and elution methods were applied to the determination of DL-amino acids in yogurt. The results showed that some of the L-amino acids, i.e., Glu, Asp, Ser, Gly, Ala, Thr, Pro, Lys, Phe and Met, were found in the methanol extracts of yogurt. On the other hand, the D-amino acids that were identified in the extracts were D-Glu, D-Asp and D-Ala, and the mean % to each L-amino acid were 11.9% (D-Glu), 27.6% (D-Asp) and 56.7% (D-Ala), respectively.