Sensitive Fluorescence Determination of Tryptophan Enantiomers Derivatized with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole Using a Photo-Reactor

For the sensitive fluorescence detection of tryptophan (Trp) enantiomers derivatized with 4-fluoro-7-nitro-2,1,3- benzoxadiazole (NBD-F), a photo-reactor was designed and integrated into a chiral high-performance liquid chromatographic (HPLC) system with tandemly-connected ultraviolet/visible (UV/VIS) and fluorescence detectors. The photo-reactor was inserted between the two detectors. After monitoring the separation of the NBD-Trp enantiomers by the absorbance (470 nm), light-irradiation (200 nm) was performed and the peaks derived from both enantiomers were fluorometrically detected (ex. 470 nm, em. 530 nm). To improve the fluorescence detection sensitivity, the effects of the compositions of the organic solvents in the mobile phase and the flow rate were investigated. As a result, using an acetonitrile solution containing 0.3 % formic acid at the flow rate of 100 µL/min, the highest fluorescence intensity (the ratio of the intensity obtained by a fluorescence detector and that obtained by a UV/VIS detector) derived from NBD-d-Trp was acquired. The obtained ratio was approximately 1600 times higher than that obtained when a mixed solution of acetonitrile/methanol (50/50, v/v) containing 0.1 % formic acid at the flow rate of 200 µL/min was adopted as the commonly-used mobile phase conditions for separating NBD-Trp enantiomers using a Pirkle-type column. These results indicate that the developed photo-reactor is effective for the sensitive detection of Trp enantiomers (lower limit of detection: 5 fmol/injection) and promotes further clarifications, such as tissue distribution and amounts under various physiological conditions in mammals.