title: 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).
authors.affiliation: Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Iizuka
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hirasa
authors.forename: Kazumi
authors.initials: K
authors.lastname: Kubo
authors.forename: Kana
authors.initials: K
authors.lastname: Ishii
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fukushima
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D017879:Isothiocyanates / Q000737:chemistry*; D007737:Kynurenine / Q000737:chemistry / Q000302:isolation & purification*; D010069:Oxadiazoles / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D014364:Tryptophan / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: C107817:4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D010069:Oxadiazoles; D007737:Kynurenine; D014364:Tryptophan