Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry
Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry
复制标题
DOI：
10.1016/s1570-0232(02)00914-5
复制
发表时间：
2003-03-05
影响因子：
3
通讯作者：
Grant, R
中科院分区：
文献类型：
--
作者：
Polson, C;Sarkar, P;Grant, R
使用分光光度法在血浆中检测了四类蛋白质沉淀技术（有机溶剂、酸、盐和金属离子），以评估不同体积、种属和批次的蛋白质去除效率。发现乙腈、三氯乙酸（TCA）和硫酸锌在去除其类别中的蛋白质方面是最佳的（在沉淀剂与血浆的2：1比率下，蛋白质沉淀效率分别> 96%、92%和91%）。柱后输注LC-MS/MS系统用于评估由使用各种蛋白沉淀剂后剩余的内源性组分引起的蛋白结合药物的电离效应。电离效应的程度随移动的相（~ 20 - 93%）、蛋白质沉淀剂（0.3-86%）而变化，但仅随种类（86-93%）而轻微变化。在阳离子涡轮离子喷雾LC-MS/MS中，用于去除血浆蛋白和探针分子最小电离效应的最佳生物分析方法包括使用TCA进行沉淀，其中移动的流动相由纯有机溶剂（甲醇：水或乙腈：水）组成，或使用所有质谱仪兼容沉淀剂进行沉淀，其中使用甲醇：0.1%甲酸水溶液移动的流动相进行评价。(C)2002年爱思唯尔科学B。V.保留所有权利。
Four categories of protein precipitation techniques (organic solvent; acid, salt and metal ion) were tested in plasma using spectrophotometry to assess protein removal efficiency across a range of volumes, species and lots. Acetonitrile, trichloroacetic acid (TCA) and zinc sulfate were found to be optimal at removing protein in their categories (>96, 92 and 91% protein precipitation efficiency at a 2:1 ratio of precipitant to plasma, respectively). A post-column infusion LC-MS/MS system was used to assess ionization effect of a protein-bound drug caused by the endogenous components remaining after using various protein precipitants. The extent of ionization effect varied with,mobile phase (-20 to 93%), protein precipitant (0.3-86%), but only slightly with species (86-93%). The optimal bioanalytical methodologies for removal of plasma proteins and minimal ionization effect for the probe molecule in positive ion turboionspray LC-MS/MS involve the use of TCA for precipitation with mobile phases consisting of either pure organic solvents (methanol:water or acetonitrile:water) or precipitation with all of,the mass spectrometer compatible precipitants evaluated with a methanol:aqueous 0.1% formic acid mobile phase. (C) 2002 Elsevier Science B. V. All rights reserved.