Metanephrine and normetanephrine are measured in blood plasma to diagnose different diseases. Simpler sample preparation procedures are preferred but tend to yield less purified extracts. Therefore, thorough investigation of matrix effects is required. In this work, several sample preparation methods and chromatographic modes were compared for liquid chromatography tandem mass spectrometric (with electrospray ionization; LC-ESI-MS/MS) analysis of metanephrine and normetanephrine in blood plasma. Protein precipitation with methanol was found to be sufficient for sample preparation and pentafluorophenyl column provided adequate chromatographic separation. A new cheaper and less labor-intensive approach is proposed where necessary quantitation limits are achieved through a sample preparation containing only protein precipitation and dilution of the sample extract. Matrix effects for different sample preparation methods and the use of isotope-labeled internal standards were evaluated. Unusual interference to D3-labeled internal standard of normetanephrine was discovered – signal of interfering compound increased while the matrix effects were reduced by dilution, e.g. dilution eliminates matrix suppression on interfering compound. The results stress the need to monitor interfering compounds and evaluate matrix effects at every step of method development. Matrix effects and interferences can be different for analytes and their corresponding isotopically labeled internal standards. This means that the use of isotopically labeled internal standards cannot guarantee accuracy of obtained results. New method allows quantification of the low nanomolar concentrations of metanephrine and normetanephrine in plasma samples.

中文翻译：

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蛋白质沉淀血浆样品中变肾上腺素的 LC-ESI-MS/MS 分析中的基质干扰

在血浆中测量变肾上腺素和去甲变肾上腺素以诊断不同的疾病。更简单的样品制备程序是首选，但往往会产生较少纯化的提取物。因此，需要彻底研究基质效应。在这项工作中，对血浆中变肾上腺素和去甲变肾上腺素的液相色谱串联质谱（带电喷雾电离；LC-ESI-MS/MS）分析的几种样品制备方法和色谱模式进行了比较。发现用甲醇沉淀蛋白质足以进行样品制备，而五氟苯基柱可提供足够的色谱分离。提出了一种新的更便宜且劳动强度更低的方法，其中通过仅包含蛋白质沉淀和样品提取物稀释的样品制备来实现必要的定量限制。评估了不同样品制备方法的基质效应和同位素标记内标的使用。发现了对 D3 标记的去甲变肾上腺素内标的异常干扰 - 干扰化合物的信号增加，而基质效应因稀释而降低，例如稀释消除了基质对干扰化合物的抑制。结果强调需要在方法开发的每一步监测干扰化合物和评估基质效应。分析物及其相应的同位素标记内标的基质效应和干扰可能不同。这意味着使用同位素标记的内标不能保证获得的结果的准确性。新方法允许量化血浆样品中低纳摩尔浓度的变肾上腺素和去甲变肾上腺素。