Determination of taurine in biological samples by high-performance liquid chromatography using 4-fluoro-7-nitrobenzofurazan as a derivatizing agent.

OBJECTIVE: A highly sensitive and rapid high-performance liquid chromatography method with pre-column derivatization with 4-fluoro-7-nitrobenzofurazan was developed for determination of taurine in biological samples, including plasma, brain, and liver. METHODS: The optimum derivatization reaction temperature was 70 °C, and at this temperature the reaction was complete within 3 min. The derivatized taurine was separated using phosphate buffer (0.02 mol/L, pH 6.0):acetonitrile (84:16, v/v) as the mobile phase at a flow rate of 1.0 mL/min, and a column temperature of 25 °C. The taurine derivatives were separated within 20 min (tR:14.5 min) and fluorometrically detected at 530 nm with excitation at 470 nm. RESULTS: The intra- and the inter-day coefficients of variation for the method were 5.3% and 7.7%, respectively. The calibration curve was linear from 0.1 µmol/L to 30.0 µmol/L with a correlation coefficient of 0.9995. CONCLUSION: This method can be used to determine the taurine contents in plasma, brain, and liver from normal rats and human plasma.

Role of oxidative/nitrative stress in hepatic encephalopathy induced by thioacetamide.

The study was designed to reveal the pathogenic mechanism of peroxynitrite in hepatic encephalopathy (HE), assess oxidative/nitrative stress in relation to HE induced by thiacetamide (TAA) and provide new ideas and scientific basis for the etiology and treatment of HE. Male Wistar rats were divided into four groups randomly: A (control), B (model), C (ebselen) and D (solvent). All the groups were treated with TAA by intraperitoneal (i.p.) except group A (treated with saline i.p.) to manufacture the model of HE. When rats treated with TAA came to the second stage of HE the four groups were administered intragastrically (i.g.) with saline (A, B), ebselen (C) and dimethyl sulfoxide (DMSO) (D), respectively. Plasma was collected to detect the levels of 3-nitrotyrosine (3-NT), NO, T-SOD and MDA. The results showed that the levels of 3-NT, NO, MDA significantly increased and T-SOD decreased obviously in rats suffering from HE. With the development and progression of HE the extent of oxidative/nitrative stress increased. When treated with ebselen the symptoms of HE mitigated and the levels of biochemical indicators ameliorated significantly. This indicates that oxidative/nitrative stress is involved in the mechanisms of hepatic encephalopathy.

[Effects of garlic oil, age and sex on n-hexane metabolism in rats].

OBJECTIVE: To investigate effects of garlic oil (GO), age and sex on n-hexane metabolism in rats. METHODS: The Wistar rats were used as experimental animals. (1) Intragastric administration: n-hexane group (3000 mg/kg n-hexane), GO treated group (80 mg/kg GO ig. an hour earlier than 3000 mg/kg n-hexane), then blood was taken from tails of rats at 8, 12, 16, 20, 24, 28, 32 h points after n-hexane administration. (2) Intraperitoneal injection: n-hexane group (1000 mg/kg n-hexane), GO treated group (80 mg/kg GO ig. an hour earlier than 1000 mg/kg n-hexane), then took blood was taken from tails of rats at 8, 12, 16, 20, 24, 28 h points after n-hexane injection. (3) 7 rats each group of 6, 8, 10 weeks age were administrated by 3000 mg/kg n-hexane intragastrically, then were taken blood from tails at 16, 20, 24 h points after administration. (4) 7 male and 7 female rats of 8 weeks age were administrated by 3000 mg/kg n-hexane intragastrically, then were taken blood from tails at 16, 20, 24, 28 h points after administration. The gas chromatography was used to determine the metabolite 2, 5-hexanedione concentration of n-hexane in serum and 2, 5-hexanedione concentration was compared between GO and no GO treated rats, different ages and different sexes. RESULTS: (1) Intragastric administration: 2, 5-hexanedione concentrations in serum of n-hexane group and GO treated group had the peak 19.2 and 12.3 µg/ml at 20h and 24 h points. Compared with n-hexane group, the serum 2, 5-hexanedione concentration of GO treated group was lower at time points prior to peak and 2, 5-hexanedione eliminating process was slower after peak. (2) Intraperitoneal injection: effects of GO on the serum 2, 5-hexanedione concentrations was very similar to intragastric administration, 2, 5-hexanedione concentrations in serum of n-hexane group and GO treated group had the peak 15.0 and 6.7 µg/ml at 12 h and 16 h points. (3) Comparison of the serum 2, 5-hexanedione concentrations of different weeks age rats: The serum 2, 5-hexanedione concentrations of 6, 8, 10 weeks age rats were 25.5, 15.0, 12.8 µg/ml each (8, 10 weeks age significantly lower than 6 weeks age) at 16 h point; at 20 h point, they were 24.7, 18.3, 15.0 µg/ml each (10 weeks age significantly lower than 6 weeks age); at 24 h point, they were 11.0, 14.7, 8.1 µg/ml each (10 weeks age significantly lower than 8 weeks age). (4) Comparisons of the serum 2, 5-hexanedione concentrations of different sex rats: the serum 2, 5-hexanedione concentrations of male and female rats were 22.5, 17.2 µg/ml each at 16 h point (different significantly); at 20, 24, 28 h points, they were 27.6, 22.9 µg/ml, 24.6, 19.1 µg/ml, 19.1, 13.8 µg/ml each (different non-significantly). CONCLUSION: GO reduces production of 2, 5-hexanedione in serum generated by n-hexane in rats; the metabolic capacity of low age rats on n-hexane is stronger than high age ones.

Quantification of glutathione in plasma samples by HPLC using 4-fluoro-7-nitrobenzofurazan as a fluorescent labeling reagent.

A rapid and highly sensitive high-performance liquid chromatograpy method with fluorescence detection has been developed for determination of glutathione (GSH) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. 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 2°C. The eluted derivatives were fluorometrically detected at an excitation wavelength 470 nm and an emission wavelength 530 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1 µmol/L to 10.0 µmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra- and inter-day coefficient of variation being 6.3%, 6.9%, respectively. This method has been used to determine glutathione concentrations in plasma samples from healthy individuals.

Improved determination of D-glucosamine hydrochloride in health foods by HPLC using 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole as a derivative.

This article presents an improved method to detect D-glucosamine hydrochloride in health foods. A simple precolumn derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized D-glucosamine hydrochloride (NBD-D-glucosamine hydrochloride) was performed using a mobile phase consisting of acetonitrile, potassium dihydrogen phosphate (0.01 mol/L), and trifluoroacetic acid (350:649.74:0.26, volume ratio) at a flow rate of 1.0 mL/min with the column temperature 35 °C. Under the optimum chromatographic conditions, the peak area of NBD-D-glucosamine hydrochloride compared with its absolute value of the peak area of NBD-D-glucosamine hydrochloride in a standard solution concentration range from 1.0 to 500.0 mg/L showed a good linear calibration (R = 0.9999). Recoveries, at spiked concentrations of 10.0, 40.0, and 500.0 mg/L, varied between 97.2% and 102.6% with relative standard deviations ranging from 0.4% to 1.5%. The present method provides sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ). LOD was determined from the signal-to-noise ratios (S/N) of NBD-D-glucosamine hydrochloride peak of at least 3 in the recovery test at 0.02 mg/L, and the estimated LOQ was 0.06 mg/L (S/N = 10). The proposed method was successfully applicable to detect D-glucosamine hydrochloride in health foods and drugs containing a variety of complex materials.