高级检索

    超高效液相色谱-串联质谱法测定小鼠纹状体中多巴胺及其代谢物的含量

    Determination of Dopamine and Its Metabolites in Mice Striatum by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry

    • 摘要: 提出了超高效液相色谱-串联质谱法同时测定小鼠纹状体中多巴胺(DA)及其代谢物高原儿茶酸(DOPAC)与高香草酸(HVA)含量的方法。取小鼠纹状体约10 mg,用200 μL 2%(体积分数)甲酸溶液提取、离心。取上清液80 μL,加入40 μg·L-1异丙肾上腺素(内标)溶液10 μL和含0.1%(体积分数,下同)乙酸的甲醇溶液10 μL,涡旋混匀,再加入100 μL甲醇,涡旋、离心。上清液中DA及其代谢物在Gemini NX-C18色谱柱上分离,以不同体积比的0.1%乙酸溶液和含0.1%乙酸的甲醇溶液混合液为流动相进行梯度洗脱,质谱分析采用电喷雾离子源正、负离子模式,选择反应监测(SRM)模式扫描。结果表明,3种目标物在7.00 min内能完成测定。DA、DOPAC和HVA标准曲线的线性范围分别为0.05~100,0.5~1 000,0.25~500 μg·L-1,检出限分别为0.015,0.150,0.075 μg·L-1。按照标准加入法进行回收试验,回收率为84.4%~117%,日内精密度和日间精密度试验所得测定值的相对标准偏差均小于15%。质控样品在自动进样器中保存8 h、4℃保存24 h以及-80℃保存30 d前后3种目标物测定值的相对误差绝对值不大于20%,说明样品稳定性良好。

       

      Abstract: A method was proposed for the simultaneous determination of dopamine (DA) and its metabolites of high protocatechuic acid (DOPAC) and high vanillic acid (HVA) in mice striatum by ultra-high performance liquid chromatography tandem mass spectrometry. About 10 mg of mice striatum was extracted with 200 μL of 2% (volume fraction) formic acid solution and centrifuged. The supernatant (80 μL) was taken and 10 μL of 40 μg·L-1 isoproterenol (internal standard) solution and 10 μL of methanol solution containing 0.1% (volume fraction, the same below) acetic acid were added. After vortex mixing, 100 μL of methanol was added, followed by vortex mixing and centrifugation. The DA and its metabolites in the supernatant were separated on a Gemini NX-C18 column using a mixture of 0.1% acetic acid solution and methanol solution containing 0.1% acetic acid at different volume ratios as the mobile phase for gradient elution. And the mass spectrometry analysis was performed by electrospray ion source with positive and negative ion modes, and scanned by selective reaction monitoring (SRM) mode. As shown by the results, the three targets could be determined within 7.00 min. The linear ranges of the standard curves for DA, DOPAC and HVA were 0.05-100, 0.5-1 000, 0.25-500 μg·L-1, and the detection limits were 0.015, 0.150, 0.075 μg·L-1, respectively. According to the standard addition method, the recoveries of the three targets ranged from 84.4%-117%. RSDs of the determined values of tests for intra-day precision and inter-day precision were less than 15%. The determined values of the relative error for the three targets in quality control samples stored in an autosampler for 8 h, at 4℃ for 24 h and at -80℃ for 30 d were not more than 20%, indicating good sample stability.

       

    /

    返回文章
    返回