检测癫痫大鼠脑组织中Glu、Gly、GABA含量的异硫氰酸苯酯柱前衍生HPLC技术的建立

Establishment of HPLC technology of precolumn derivatization with PITC in determining contents of Glu,Gly and GABA in brain tissues of rats with epilepsy

目的建立检测癫痫大鼠脑组织谷氨酸(Glu)、γ-氨基丁酸(GABA)、甘氨酸(Gly)含量的异硫氰酸苯酯(PITC)柱前衍生高效液相色谱(HPLC)技术,并验证其检测效果。方法将海人藻酸(KA)或等量生理盐水注射入大鼠海马CA3区建立癫痫模型或对照,2周后分别取其海马和皮层组织,采用PITC柱前衍生法处理样品,采用醋酸钠缓冲液和乙腈梯度洗脱,Venusil-AA氨基酸分析柱结合紫外检测器检测,计算Glu、GABA、Gly含量。结果在30 min内3种氨基酸完全出峰。在0.20~12.50 mg/L范围内Glu线性关系良好,相关系数为0.999 3,日内精密度为1.46%,日间精密度为1.84%,加样回收率为97.12%±2.98%;在0.01~6.25 mg/L范围内Gly线性关系良好,相关系数为0.998 4,日内精密度为1.17%,日间精密度为1.62%,加样回收率为96.63%±1.28%;在0.10~6.25mg/L范围内GABA线性关系良好,相关系数为0.999 4,日内精密度为1.00%,日间精密度为1.53%,加样回收率为99.25%±2.98%。模型组海马和皮层中Glu含量均高于对照组相应组织(P均<0.05)。结论成功建立了检测癫痫大鼠脑组织Glu、Gly、GABA含量的PITC柱前衍生HPLC技术检测效果较好。

Objective To establish high-performance liquid chromatography( HPLC) technology of precolumn derivatization with phenyl isothiocyanate( PITC) to determine the contents of glutamic acid( Glu),glycine( Gly) and γ-aminobutyric acid( GABA) in the brain tissues of rats with epilepsy,and to verify the results. Methods Kainic acid( KA) was injected into the hippocampus CA3 region of rats to make the epilepsy models. Two weeks later,we obtained the hippocampus and cortex tissues,respectively. The diluted samples were precolumn derivatizated with PITC. The mobile phase consisted of sodium acetate and acetonitrile gradient elution. We calculated the contents of Glu,Gly and GABA in hippocampus and cortex by venusil-AA column combined with UV detector. Results The amino acids were separated within 30 min. A good linearity was obtained from( 0. 20-12. 50) mg / L of Glu,the correlation coefficient was 0. 999 3,the intraday RSD was 1. 46%,interday RSD was 1. 84%,and the average recovery rate was 97. 12% ± 2. 98%. A good linearity was obtained from( 0. 01-6. 25) mg / L of Gly,the correlation coefficient was 0. 998 4,the intraday RSD was 1. 17%,interday RSD was 1. 62%,and the average recovery rate was 96. 63% ± 1. 28%. A good linearity was obtained from( 0. 10-6. 25) mg / L of GABA,the correlation coefficient was 0. 999 4,the intraday RSD was 1. 00%,interday RSD was 1. 53%,and the average recovery rate was99. 25% ± 2. 98%. Conclusion The HPLC technology of precolumn derivatization with PITC in determining the contents of Glu,Gly and GABA in the brain tissues of rats with epilepsy is successfully established.