UPLC/QExactiveMS检测肉苁蓉多糖促PC12细胞释放神经递质的方法研究

目的：建立UPLC/Q Exactive MS法测定PC12细胞释放的神经递质多巴胺、去甲肾上腺素和谷氨酸含量。方法：采用Ultimate 3000系列高效液相色谱仪和Q Exactive四极杆-静电场轨道阱高分辨质谱系统检测。标准品用超纯水溶解后，以乙腈-水（0.1%甲酸，10 mmol·L^-1醋酸铵）（4：96）为流动相，Hypersil GOLD色谱柱分离，通过高分辨质谱的全扫描/选择离子监测（Full MS/SIM）模式进行定性筛查和定量检测。应用Sigma Plot12.0统计软件包进行数据分析。结果：多巴胺和谷氨酸在3~2000ng·mL^-1浓度范围内线性关系良好，最低定量限为3 ng·mL^-1；去甲肾上腺素在100~2000 ng·mL^-1浓度范围内线性关系良好，最低定量限为100 ng·mL^-1；3种神经递质的加标回收率高，精密度RSD均小于5%；常温放置8 h，4℃冷藏7 d后以及-20℃冷冻1个月后稳定性良好。本实验同时在药物肉苁蓉多糖（CDPS）改善学习记忆功能已有研究的基础上，测定PC12细胞给药后3种神经递质不同时间点的释放浓度，结果显示CDPS对3种神经递质释放量有明显促增趋势。结论：UPLC/Q Exactive MS法适合同时测定PC12细胞分泌的多巴胺、去甲肾上腺素和谷氨酸的含量。

Objective: To establish a method for the determination of dopamine, norepinephrine and glutamate in PC12 cells by UPLC/Q exactive MS(quadrupole electrostatic field orbitrap high resolution mass spectrometry). Methods: the Ultimate 3 000 series high performance liquid chromatography(HPLC)and Q exactive system were used. Standard reference was dissolved in ultrapure water. Acetonitrile-water(0.1% formic acid, 10 mmol·L^-1 ammonium acetate)(4:96)was used as a mobile phase with an Hypersil GOLD column. Full scan/selected ion monitoring high resolution mass spectrometry(Full MS/SIM)model was applied for qualitative screening and quantitative detection. Sigma Plot12.0 statistical software package was used for data analysis. Results: The concentration of dopamine and glutamate in the range of 3-2 000 ng·mL^-1 revealed a good linear relationship, and the limit of quantitation was 3 ng·mL^-1. Norepinephrine concentration in 100-2 000 ng·mL^-1 showed a good linear relationship;the recovery of three kinds of neurotransmitter rate was high, the RSD of precision was less than 5%. The stability of 8 h at room temperature, 7 d in 4℃ refrigerator and a month of frozen in 20℃ was good. Based on the study of Cistanche deserticola polysaccharides (CDPS)to improve the function of learning and memory, the concentration of three neurotransmitters released at different time points after administration was measured. Results showed that CDPS could obviously promote the trend of the three kinds of neurotransmitter release. Conclusion: UPLC/Q Exactive MS method is suitable for simultaneous determination of dopamine, norepinephrine and glutamate in PC12 cells.