反相高效液相色谱荧光法测定大鼠血清和脑组织中茶氨酸含量

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。

DETERMINATION OF THEANINE IN RAT SERUM AND BRAIN BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND FLUORESCENCE DETECTION

Objective To establish a HPLC method of determining theanine in rat serum and brain tissue.Method Using ODS-SP C18 chromatography column(4.6 mm×250 mm,5μm),the mobile-phase was 0.1 mol/L monopotassium phosphate buffer solution(pH 6.0):THF: methanol =63:2:35,the HPLC system with fluorescence detection(FL) was used to examine the theanine in rat serum and brain tissues with pre-column derivatization with o-phthalaldehyde(OPA) in the presence of the internal standard homoserine.Flow rate was set at 0.85 ml/min.The excitation wavelength was 340 nm while the emission wavelength was 455 nm.Results The method had good linearity with the concentration ranges of 1-20 μmol/L for theanine.The correlation coefficient(r) was 0.9997.The intraday precisions(RSD,%) of theanine with the concentration of 1 μmol/L,10 μmol/L,20 μmol/L were 3.51,1.58,0.75,while the interday precisions(RSD,%) were 4.54,4.25,2.87.The recoveries were between 91.8%-99.1%.The detection limits was 0.02 μmol/L.When 200 mg/kg·bw of theanine was intragastrically administered to rats,the serum concentration of theanine reached its highest level during 0.5-1.0 h after i.g.,while the highest level of brain appeared after 5 h.Conclusion This improved high performance liquid chromatography(HPLC) with fluorescence method is sensitive,and reproducible.It can separate and detect theanine of serum and brain tissue in rat effectively.