正交试验设计优化小鼠脑组织中氨基酸类神经递质测定的实验研究

Research on optimization of experiment conditions for determination of amino acid neurotransmitters in mouse brain with orthogonal experiment design

目的用正交试验设计法优化测定小鼠脑组织中氨基酸类神经递质谷氨酸(glutamic acid,Glu)和γ-氨基丁酸(γ-aminobutyric acid,GABA)的实验条件,简化实验方法,提高检测方法的灵敏度。方法以2,4-二硝基氟苯(2,4-dinitrofluorobenzene,DNFB)为衍生化试剂柱前衍生,采用正交试验设计法优化样品的衍生化条件。SymmetryC18色谱柱分离,等度洗脱,流动相为30 mmol/L NaAc缓冲液(pH 5.6)∶乙腈(3∶1,v/v),紫外检测波长360 nm,保留时间定性,外标法定量。结果最佳衍生化条件:1%2,4-二硝基氟苯50μl、0.5 mol/L碳酸氢钠100μl、衍生温度85℃、衍生时间30 min。在优化的实验条件下,Glu和GABA在20μg/ml^400μg/ml浓度范围内线性良好,相关系数均为1.0;加标回收率分别为91.5%~98.7%和94.0%~97.7%;Glu的日内和日间精密度分别为2.83%~7.18%和3.10%~8.16%;GABA的日内和日间精密度分别为2.59%~6.00%和2.46%~5.64%。结论该方法简便、准确、灵敏,适用于小鼠脑组织中氨基酸类神经递质的测定。

Objective To optimize the experiment conditions for determination of amino acid neurotransmitters（ glutamic acid,Glu and γ- aminobutyric acid,GABA） in mouse brain with orthogonal experiment design,so as to improve the sensitivity of the measurement method. Methods DNFB was used as derivation agent for precolumn derivation,and the derivatization conditions were optimized with orthogonal experiment design. The sample solution was separated by Waters Symmetry  C18column using isocratic elution after derivatization with 30 mmol /L sodium acetate buffer solution（ pH 5. 6） ∶ acetonitrile（ 3∶1,v /v） as mobile phase,and UV detection wavelength was 360 nm. Retention time was used for qualitative analysis and external standard method was used for quantitative analysis. Results The optimal conditions of derivatization were 1% DNFB 50 μl and0. 5 mol /L NaHCO3100 μl,the derivatization temperature at 85℃ and the derivatization time of 30 min. Using the optimized experimental conditions,the linearity of Glu and GABA were both well in the range of 20 μg /ml - 400 μg /ml and the linear correlation coefficients were both 1. 0. The recoveries of Glu and GABA were 91. 5% - 98. 7% and 94. 0% - 97. 7%,respectively. The intra- day and inter- day precision of Glu were 2. 83% - 7. 18% and 3. 10% - 8. 16%,respectively. While those of GABA were 2. 59% - 6. 00% and 2. 46% - 5. 64%,respectively. Conclusion The method is simple,accurate,sensitive,and it is suitable for determination of amino acid neurotransmitters in mouse brain tissue.