毛细管电泳-激光诱发荧光法检测大鼠PAG脑片灌流液中谷氨酸和天门冬氨酸的含量

Determination of Glutamate and Aspartate Concentrations in Brain Slices of Pag by Cze-Lifd

目的 建立毛细管电泳 激光诱发荧光法 (CZE LIFD)检测大鼠中脑导水管周围灰质 (PAG)脑片灌流液中谷氨酸 (L Glu)和天门冬氨酸 (L Asp)含量的方法。方法 灌流液中Glu和Asp用NDA衍生标记 ,电泳条件为 :BECKMAN毛细管 ,长度 6 7cm ,内径 75 μm ,激激发波长 4 88λ/nm ,发射波长 5 2 0λ/nm ,分离电压 2 5kV ,分离时间 10min。结果 L Glu的回归方程为Y =2 0 0 9+4.0e-9X ,r =0 .9996 ,平均回收率为 99.8% ;L Asp的回归方程为Y =3384 6 +4.4e-9X .,r=0 .9970 ,平均回收率为 99.2 % ,Y为峰面积 ,X为浓度 (mol·L-1)。该方法能够检测大鼠PAG脑片灌流液在高钾刺激前后的L Glu和L Asp含量。结论 毛细管电泳 激光诱发荧光法可用于脑片灌流液内微量L Glu和L Asp的测定。

OBJECTIVE To detect the concentrations of glutamate( L Glu) and aspartate( L Asp) in brain slices of periaqueductal gray (PAG)through developing the method of Capillary zone eletrophoresis with laser induced fluorescence detection(CZE LIFD). METHOD Glu and Asp in the infusion fluid were labeled by naphthalene 2,3 dicarbox aldehyde(NDA). Electrophresis was consisted of BECKMAN capillary(67cm×75μm) with automated sample injecting system.The laser excited wavelength was 488λ/nm.The emission intensity was measured at a wavelength of 520λ/nm.The running voltage was 25kV.The separation time was 10min.RESULTS The standard curve was linear .The equation about L Glu was Y=2009+4.0e+9X with the correlation coefficient of 0.999 6. The average recovery of L Glu was 99.8%. The equation about L Asp was Y=33846+4.4e+9 X with the correlation coefficient of 0.997 0. The average recovery of L Glu was 99.2%. This method could measure the concentrations of L Glu and L Asp in brain slices of PAG pre and after high K + stimulation. CONCLUSION CZE LIFD can detect the concentrations of L Glu and L Asp in infusion fluid of brain slices.