Jin D, Nagakura K, Murofushi S, Miyahara T, Toyo'oka T
Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
J Chromatogr A. 1998 Oct 2;822(2):215-24. doi: 10.1016/s0021-9673(98)00617-7.
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.
采用反相液相色谱法研究了用荧光手性标记试剂4-(3-异硫氰酸根合吡咯烷-1-基)-7-(N,N-二甲基氨基磺酰基)-2,1,3-苯并恶二唑[R(-)-DBD-PyNCS]衍生化后17种DL-氨基酸的完全分离情况。该试剂与氨基酸的反应在55℃、1%三乙胺存在下有效进行20分钟，生成相应的荧光非对映异构体（激发波长460nm，发射波长550nm）。以含1%乙酸的水-乙腈为流动相，可有效分离每对生成的衍生物。峰分辨率在0.92（DL-精氨酸）至9.8（DL-半胱氨酸）范围内。虽然使用洗脱溶剂可以实现一些DL-氨基酸的相互分离，但即使采用一些梯度洗脱，单次色谱运行也难以同时分离17种DL-氨基酸。因此，采用梯度洗脱和等度洗脱系统对DL-氨基酸进行完全分离。通过梯度洗脱和等度洗脱系统，17种DL-氨基酸得到了很好的分离。所提出的衍生化和洗脱方法应用于酸奶中DL-氨基酸的测定。结果表明，在酸奶的甲醇提取物中发现了一些L-氨基酸，即谷氨酸、天冬氨酸、丝氨酸、甘氨酸、丙氨酸、苏氨酸、脯氨酸、赖氨酸、苯丙氨酸和蛋氨酸。另一方面，提取物中鉴定出的D-氨基酸为D-谷氨酸、D-天冬氨酸和D-丙氨酸，它们相对于各自L-氨基酸的平均百分比分别为11.9%（D-谷氨酸）！27.6%（D-天冬氨酸）和56.7%（D-丙氨酸）。
