液相色谱-串联质谱在生物样品定量测定中存在的不准确因素分析和对策

Discussions on the Inaccuracy Problems and Preventing Strategies Associated with the Use of Liquid Chromatography-Tandem Mass Spectrometry in Quantitative Assay of Biosamples

液相色谱-串联质谱(LC-MS/MS)技术是目前生物样本中小分子成分及部分蛋白和多肽定量分析中最广泛使用的技术。与LC相比,LC-MS/MS具有分析速度快、分辨率和灵敏度高、方法开发简单、可多组分同时测定等优点。在方法学开发过程中,分析者的主要操作一般是遵循法规的要求逐项进行方法学验证。但是,基于LC-MS/MS定量分析的原理,即使所建立的分析方法完全按照法规的要求进行了验证,在一些方面如不加以注意,也可能带来定量不准确的问题,即所谓的陷阱(生物分析风险)。笔者归纳了使用LC-MS/MS进行生物样本分析中常见的与不准确有关的问题,尤其是一些不能通过常规方法学验证发现的问题,包括:生物样本处理过程的逆转化、基质效应、源内裂解以及质荷比相近离子的干扰等。为了解决以上问题,除加强质量体系建设和进行严格的分析方法验证外,还可以采取去除基质效率高的样本处理方法、加强色谱分离、选择合适的离子反应、使用给药后样本进行方法学验证等措施。

Liquid chromatography coupled with tandem mass spectrometry （LC-MS/MS） is a widely used technique in the quantitative analysis of small molecules, part of peptides and proteins in biological matrix. When compared with LC, the LC-MS/MS posesses many advantages, such as high sample throughput, high sensitivity and resolutions, simple method development and simultaneous quantification of multiple components. At the stage of method development, the main practices for analysts are to validate analytical methods according to the regulated requirements. However, due to the quantitative mechanisms of LC-MS/MS, even though the used method is fully validated, analytical inaccuracy problem, which is the so called bioanalytical risk （pitfalls）, may still exist, if we do not pay attentions to some details. In the present paper, the factors which may cause inaccuracy in quantitative analysis are summarized, especially those could not be found during routine method validation. Those factors include back transformation of unstable drug metabolites to analyte in the handling of biosamples, matrix effects, in-source conversion, and ion interference caused by ions with similar or same mass-to-charge ratio, etc. In addition to QA/QC oversight and strict method validation, the preventing strategies may contain the follows： selecting biosample processing method with high proficiency in eliminating matrix interferences, enhancing chromatographic separation, selection of suitable precursor-product ions and validation with incurred samples, etc.