Enantiomeric separation of d,l‐tryptophan and d,l‐kynurenine by HPLC using pre‐column fluorescence derivatization with R(−)‐DBD‐PyNCS

Abstract

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).