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目的建立一种反相高效液相色谱荧光检测法,测定大鼠血清和脑组织中茶氨酸含量。方法用ODS-SP C18色谱柱(4.6 mm×250 mm,粒径5μm),以磷酸二氢钾缓冲液(0.1 mol/L,pH6.0):四氢呋喃:甲醇体积比=62:3:35为流动相;高丝氨酸为内标;邻苯二甲醛柱前衍生;流速0.85 ml/min;荧光检测器激发波长340 nm、发射波长455 nm检测大鼠血清、脑组织中茶氨酸含量。结果茶氨酸浓度在1～20μmol/L范围内线性关系良好,相关系数为0.9997;浓度为1、10、20μmol/L时日内RSD(%)分别为3.51、1.58、0.75,日间RSD(%)分别为4.54,3.25,2.87;茶氨酸平均回收率范围为91.8%～99.1%;最低检测限为0.02μmol/L。SD大鼠给予200 mg/kg.bw茶氨酸灌胃后,血清中峰值出现在0.5～1.0 h之间,脑组织中浓度5 h达到峰值。结论该反相高效液相色谱荧光法灵敏度高,重复性好,能有效分离检测大鼠血清、脑组织中茶氨酸含量。
Objective To establish a reverse-phase high-performance liquid chromatography (HPLC) fluorescence detection method for the determination of theanine content in rat serum and brain tissue. Methods ODS-SP C18 column (4.6 mm × 250 mm, particle size 5 μm) was used with potassium dihydrogen phosphate buffer (0.1 mol / L, pH 6.0): tetrahydrofuran: methanol volume ratio = 62: 3: 35 Mobile phase; homoserine as internal standard; orthophthaldehyde pre-column derivatization; flow rate of 0.85 ml / min; fluorescence detector excitation wavelength of 340 nm, emission wavelength of 455 nm in rat serum and brain tissue theanine content. Results The linearity of theanine concentration in the range of 1 ~ 20μmol / L was good with a correlation coefficient of 0.9997. The intraday RSD (%) were 3.51, 1.58 and 0.75 respectively at 1,10 and 20μmol / ) Were 4.54,3.25,2.87 respectively. The average recoveries of theanine ranged from 91.8% to 99.1%. The lowest detection limit was 0.02μmol / L. SD rats given 200 mg / kg.bw theanine gavage, the serum peak appeared in 0.5 to 1.0 h, brain tissue concentration reached its peak 5 h. Conclusion The reversed-phase high performance liquid chromatography with high sensitivity and repeatability can effectively separate and detect the content of theanine in the serum and brain tissue of rats.