高效毛细管电泳法直接测定红豆杉悬浮培养细胞中游离芳香族氨基酸

Direct Determination of Free Aromatic Amino Acids in Suspension of Taxus chinensis Cell by High Performance Capillary Eletrophoresis

建立了红豆杉悬浮培养细胞中游离芳香族氨基酸的高效毛细管电泳的分离直接紫外吸收测定方法。在优化的实验条件下 ,以间苯二酚为内标 ,未涂层融硅毛细管 (75 μmi.d .,总长 37cm ,有效分离长度 30cm)为分离通道 ,40mmol/L磷酸缓冲液 (pH10 .46 )为电泳介质 ,3sec(0 .5 psi)压力进样 ,15kV恒压电泳 ,2 0 0nm检测 ,在 9min内三种氨基酸得到分离测定。色氨酸、苯丙氨酸和酪氨酸的线性范围分别为 2 .5 0～ 2 5 0 μmol/L(r =0 .996 2 ,RSD =2 .5 2 % )、1.2 5～ 2 5 0 μmol/L(r =0 .996 6 ,RSD =2 .12 % )和 2 .5 0～ 2 5 0 μmol/L(r=0 .9940 ,RSD =2 .93% ) ,苯丙氨酸回收率为 96 .8%± 1.37% ,酪氨酸回收率为 99.2 %± 3.0 4%。

A method for separation and direct determination of free aromatic amino acids in the suspension of Taxus chinensis cell by high performance capillary eletrophoresis with UV-absorbance was established. The optimum experimental conditions ware the following: separation column: bare fused-sillica capillary (75μm i.d.,total length 37cm,30cm from the inlet to UV detector); electrolyte: 40mmol/L phosphate buffer,pH 10.46;detection wavelength:20nm;sampling:5sec(0.5 psi);running voltage:15kV,polarity from anode(inlet) to cathode(outlelt);temperature:20℃;internal standard:resorcin. The linear ranges of determination for Trp,Phe and Try were 2.50～250μmol/L(r=0.9962,RSD=2.52%),1.25～250μmol/L(r=0.9966,RSD=2.12%)and 2.50～250μmol/L(r=0.9940,RSD=2.93%) respectively. The recoveries of Phe and Try were 96.8%±1.37% and 99.2%±3.04%.