Iizuka Hideaki, Hirasa Yasushi, Kubo Kazumi, Ishii Kana, Toyo'oka Toshimasa, Fukushima Takeshi
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510, Japan.
Biomed Chromatogr. 2011 Jul;25(7):743-7. doi: 10.1002/bmc.1525. Epub 2010 Oct 29.
The enantiomeric separation of D,L-tryptophan (Trp) and D,L-kynurenine (KYN) was investigated by high-performance liquid chromatography using pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7- (N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS]. Using an octadecylsilica column, namely, an Inertsil ODS-3 column (250 x 2.0 mm; i.d., 3 μm), four fluorescence peaks of D- and L-Trp as well as D- and L-KYN derivatized with R(-)-DBD-PyNCS were clearly observed, and their chemical structures were confirmed by HPLC-time-of-flight-mass spectrometry. Simultaneous separation was achieved under the mobile phase condition of 1.5% acetic acid in H₂O-CH₃CN (60:40), and the separation factors of D,L-Trp and D,L-KYN derivatized with R(-)-DBD-PyNCS were 1.22 and 1.19, respectively. Fluorescence detection was carried out by setting the emission wavelength at 565 nm, and the excitation wavelength at 440 nm, and the detection limits were approximately 0.3-0.5 pmol (signal-to-noise ratio of 3).
采用高效液相色谱法，使用手性荧光标记试剂R(-)-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]进行柱前荧光衍生化，研究了D,L-色氨酸（Trp）和D,L-犬尿氨酸（KYN）的对映体分离。使用十八烷基硅胶柱，即Inertsil ODS-3柱（250×2.0 mm；内径，3μm），清晰观察到用R(-)-DBD-PyNCS衍生化的D-和L-Trp以及D-和L-KYN的四个荧光峰，并通过HPLC-飞行时间质谱法确认了它们的化学结构。在H₂O-CH₃CN（60:40）中1.5%乙酸的流动相条件下实现了同时分离，用R(-)-DBD-PyNCS衍生化的D,L-Trp和D,L-KYN的分离因子分别为1.22和1.19。通过将发射波长设置为565 nm，激发波长设置为440 nm进行荧光检测，检测限约为0.3 - 0.5 pmol（信噪比为3）。
