| ObjectivesDerivatization reagents having a benzofurazan structure are extremely sensitive fluorescent reagents for determination of peptides, amino acids and some drugs. We have study the absorption and fluorescence characteristics of 4-fluoro-7-nitr-obenzofurazan (NBD-F). To develop a very simple and rapid HPLC method for quantification of taurine, glutathione and D-glucosamine hydrochloride in biological samples and health foods after pre-column derivatization with NBD-F.Methods and ResultsFluorescence spectra and fluorescence quantum yield of NBD-F acetonitrile solution are reported. When pH>7.0, NBD-F gives a strong and steady fluorescence with the maximum excitation wavelengths at 370 and 475 nm and the maximum emission wavelength at 540 nm, respectively. A linear relationship between the fluorescence intensity and NBD-F concentration was found in the range of 0.1-100.0μmol/L. By using Rhodamine B as a reference, the fluorescence quantum yield of NBD-F at maximum excitation wavelength 475 nm was measured to be 0.86.The separation of the taurine in plasma and tissues was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (84:16, v/v) at a flow rate of 1.0 ml/min with the column temperature 25℃. The derivatives were separated within 20 min and fluorometrically detected at 536 nm with excitation at 475 nm. The RSD of the method was satisfactory with the intra-day and the inter-day coefficient of variation 5.3%,7.7%. The calibration curve was linear over the range of 0.1μmol/L to 30.0μmol/L with the correlation coefficient of 0.9995. The proposed method was successfully applied for determination of the taurine in the healthy huaman and rat samples. The mean plasma concentrations of taurine in male and female were 37.8±5.1 and 38.1±5.5μmol/L, the concentrations of rat plasma was 263±24μmol/L. The amounts of taurine in the rat liver and brain were 1.3±0.2,1.9±0.1μmol/g wet tissue.The separation of the derivatized glutathione was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0):acetonitrile (77:23, v/v) at a flow rate of 1.0 ml/min with the column temperature 25℃. The eluted derivatives were fluorometrically detected at excitation wavelength 475 nm and an emission wavelength 542 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1μmol/L to 10.0μmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra-day and the inter-day coefficient of variation 6.3%,6.9%. This method has been used to determine the glutathione concentrations in plasma samples from humans and tissues in rat samples. The mean plasma concentrations of glutathione in male and female were 4.70±0.93μmol/L and 4.72±0.94μmol/L. The mean concentration of glutathione in normal and hepatic encephalopathy rat plasma was 5.70±1.01, 3.42±0.54μmol/L, respectively. The amounts of glutathione in the rat liver and brain were 5.46±0.62 and 0.63±0.1μmol/g wet tissue.Reverse phase chromatography using pre-column derivatization with NBD-F and ultraviolet detection (466nm), were used to quantify the D-glucosamine hydrochloride. The mobile phase was acetonitrile-potassium dihydrogen phosphate(0.02mol/L)-trifluoroacetic acid (320:679.74:0.26, v/v/v)and pumped at a flow rate of 1.0 ml/min. The column temperature was 35℃.Under the optimum chromatographic conditions, the calibration curve was linear over the range of 1.0-500mg/L with the correlation coefficient of 0.9995. The recoveries were in the range of 95.6%-103.7%. The precision, expressed as the relative standard deviation (RSD.), was 2.5%-7.7% at all concentrations. ConclusionsThe derivatization reagent NBD-F is highly fluorescent as the data of the fluorescence spectra and fluorescence quantum yield of NBD-F. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent for the determination of taurine, glutathione and D-glucosamine hydrochloride was employed. The derivatization reaction conditions were evaluated and optimized. It is sensitive, accurate and reproducible, suitable for analysis of large sample numbers in clinic msearch. This method can be used to determine the taurine, glutathione and D-glucosamine hydrochloride concentrations in plasma, tissue and health food.. |
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