Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS
Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS
复制标题
DOI：
10.1021/ac802391b
复制
发表时间：
2009-03-15
影响因子：
7.4
通讯作者：
Kennedy, Robert T.
中科院分区：
文献类型：
--
作者：
Li, Qiang;Zubieta, Jon-Kar;Kennedy, Robert T.
采用毛细管液相色谱-三级质谱法（LC-MS 3），以确定内源性阿片肽微透析样品中收集在体内的方法开发，验证，并应用于测量大鼠纹状体。当在室温或-80 ℃下储存时，透析液中的肽迅速降解。添加乙酸至5%的最终浓度使肽稳定5天，允许级分的储存和离线测量，这证明比先前使用的在线方法更方便和可靠。研究了透析流速从0.2 μ L/min到2 μ L/min和柱内径（i.d.）从25到75 μ m对肽获得的相对信号的分析表明，最低流速和最小柱i. d.给出了最高的相对信号该方法进行了测试，为10种不同的神经肽和检测限（LOD）为0.5至60 pM（4 μ L样品）的最。当直接检测时，β-内啡肽的LOD为5 nM，但可通过检测胰蛋白酶消化产生的特征肽（LOD为3 pM）来定量测定。这种方法可能对其他巨大的神经肽也很有用。该方法用于测定体内甲硫氨酸脑啡肽、亮脑啡肽、强啡肽A（1-8）和β-内啡肽。内吗啡肽I和2低于体内方法的检测限。通过标准加入实验验证了外标法定量测定亮脑啡肽的准确性。与以前的方法相比，使用毛细管LC-MSn的改进使得体内神经肽监测对于各种神经肽更加实用和可行。
A method using capillary liquid chromatography-triple-stage mass spectrometry (LC-MS3) to determine endogenous opioid peptides in microdialysis samples collected in vivo was developed, validated, and applied to measurements in the rat striatum. Peptides in dialysate rapidly degraded when stored at room temperature or -80 degrees C. Adding acetic acid to a final concentration of 5% stabilized the peptides for 5 days allowing storage of fractions and off line measurements which proved more convenient and reliable than previously used on-fine methods. Study of the effect of dialysis flow rate from 0.2 to 2 mu L/min and column inner diameter (i.d.) from 25 to 75 pm on the relative signal obtained for peptides revealed that lowest flow rates and smallest column i.d. gave the highest relative signal. The method was tested for 10 different neuropeptides and limits of detection (LODs) were from 0.5 to 60 pM (4 mu L samples) for most. beta-Endorphin had an LOD of 5 nM when detected directly, but it could be quantitatively determined by detecting a characteristic peptide produced by tryptic digestion with an LOD of 3 pM. This approach may prove useful for other huge neuropeptides as well. The method was used to determine met-enkephalin, leu-enkephalin, dynorphin A(1-8), and beta-endorphin in vivo. Endomorphin I and 2 were below the detection limit of the method in vivo. Quantitative determination of leuenkephalin using external calibration was verified by standard addition experiments. The improvements over previous approaches using capillary LC-MSn make in vivo neuropeptide monitoring more practical and feasible for a variety of neuropeptides.