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

Abstract

Total resolution of 17 dl-amino acids after derivatization with a fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-( N, N-dimethylaminosulfonyl)-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°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.