|
Objective benzofurazan (benzofurazan) class is a class derived agents has been widely used in the determination of biological samples derivatized with amino acids, peptides and proteins derived from partial reaction of the derivatives may be UV or fluorescence detector for detection. By studying benzofurazan class derived agent 4 - fluoro-7 - nitro-2 ,1,3 - benzoxadiazole (4-fluoro-7-nitrobenzofurazan, NBD-F) of spectroscopy and quantum yield characteristics rate, optimizing the NBD-F with the analyte derivative reaction conditions, to establish a simple, rapid and sensitive HPLC health food or biological samples taurine, glutathione, glucosamine hydrochloride Methods. Methods and results were measured using a spectrophotometer NBD-F and its derived products UV-visible absorption spectra, fluorescence spectra and quantum yield was found NBD-F in alkaline conditions, can be an excitation wavelength of 370nm, 475nm, emission wavelength to generate a strong fluorescence at 540nm. In the 0.1-100μmol / L concentration range, the fluorescence intensity and the concentration of linear relationship. Rhodamine B as a reference, NBD-F measured at an excitation wavelength of 475nm at the fluorescence quantum yield of 0.86. NBD-F precolumn derivatization HPLC UV detector measuring health food taurine, fluorescence detector human, rat plasma and rat liver, brain taurine content. Chromatographic conditions: Column Diamonsil C18 column (5μm, 250 x4.6 mm); mobile phase: acetonitrile - phosphate buffer (0.02mol / L, pH 6.0) (16:84, v / v); temperature was 25 ℃; flow rate of 1.0 ml / min; detection wavelength λ = 470 nm; fluorescence detector λex = 475nm, λem = 536nm; injection volume was 10μL. Results The linear range of fluorescence detection taurine 0.1μmol/L-30.0μmol/L, r = 0.9995, the lowest detection limits (S / N = 3) 0.010μmol / L, intra-and inter-assay CVs were 5.3%, 7.7% . This method for the determination of taurine in biological samples Determination of normal men and women in plasma taurine levels were 37.8 ± 5.1,38.1 ± 5.5μmol / L, normal rat plasma taurine content was 263 ± 24μmol / L normal rat liver, brain taurine levels were 1.3 ± 0.2,1.9 ± 0.1μmol / g. Column derivatization HPLC glutathione content in biological samples using acetonitrile-0.02mol / L phosphate solution (23:77, v / v) as the mobile phase, column temperature 25 ℃; flow rate 1.0 mL / min ; fluorescence detection: λex = 475 nm, λem = 542 nm; injection volume was 10μL. Glutathione linear range of fluorescence detection 0.1μmol/L-10.0μmol/L, r = 0.9988 intra-and inter-assay CVs were 6.3%, 6.9%. This method for the determination of glutathione in biological samples, determination of the normal male and female bovine plasma glutathione levels were 4.70 ± 0.92,4.72 ± 0.94μmol / L, determination of the normal group, and hepatic encephalopathy rats plasma glutathione glutathione values ??were 5.70 ± 1.01,3.42 ± 0.54μmol / L, normal rat liver, brain glutathione levels were 5.46 ± 0.62,0.63 ± 0.10μmol / g. Pre-column Derivatization High Performance Liquid Chromatography health foods glucosamine hydrochloride content, chromatographic conditions were: DiamonsilC18 250x4.6mm, 5μm; mobile phase: acetonitrile-0.02mol / L KH2PO4 (0.05% TFA, pH 4.0) = 32:68 (v / v), column temperature: 35 ℃, flow rate: 1.0 ml / min, detection wavelength: 466nm, injection volume was 10μL. Linear range 1.0-500 mg / L, the correlation coefficient of 0.9995, the lowest detection limits (S / N = 3) was 0.20mg / L, sample recovery was 95.6% -103.7% and the relative standard deviation was 2.5% -7.7 %. Conclusions In this paper benzofurazan class derivatizing agent NBD-F spectroscopy features optimized NBD-F and taurine, glutathione, glucosamine hydrochloride derived reaction conditions, and the absorption spectrum of derivative products and stability characteristics are examined. The results show that NBD-F is a strong fluorescence markers, with the amine group containing compound under alkaline conditions derivatization reaction occurs, the reaction conditions are simple derivatives, short reaction time, good stability of derived products, available liquid chromatography UV detector or fluorescence detector, less interference peaks, high sensitivity. This method can be used to determine the food and biological samples taurine, glutathione, glucosamine hydrochloride content.
|