Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS
Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS
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DOI：
10.1021/ac020361s
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发表时间：
2003-07-01
影响因子：
7.4
通讯作者：
Chavez-Eng, CM
中科院分区：
文献类型：
--
作者：
Matuszewski, BK;Constanzer, ML;Chavez-Eng, CM
近年来，高效液相色谱（HPLC）串联质谱（MS/MS）检测已被证明是一种强大的技术，用于定量测定药物和代谢物在生物体液中。然而，利用HPLC-MS/MS实际上保证选择性的常见和早期认识受到了许多报告的由于样品基质和代谢物干扰引起的离子抑制或增强而缺乏选择性的例子的挑战。鉴于这些严重的方法缺陷，人们提出了如何开发和验证可靠的HPLC-MS/MS方法的问题，特别是用于支持长期人体药代动力学研究。核心问题是，除了通常为传统生物分析方法提供的验证数据外，还需要进行哪些实验来确认HPLC-MS/MS测定的选择性和可靠性。目前的法规要求包括需要评估和消除生物分析中的基质效应。方法，但评估基质效应所需的实验程序没有详细说明。本文介绍了研究、鉴别和消除基质对HPLC-MS/MS定量分析结果影响的实用实验方法。作为一个例子，一组验证实验进行我们的研究新药候选人之一，“绝对”与“相对”矩阵效应的定量评估的概念被引入。此外，使用HPLC-MS/MS测定分析物的“真实”回收率的实验描述了消除基质对测定该常用测量方法参数的影响的不确定性。通过测定基质效应，可以评估现有HPLC-MS/MS方法的可靠性和选择性。如果这些研究的结果不令人满意，则所确定的参数可为需要对方法进行哪些变更以提高测定选择性提供指导。此外，在其他相同的样品制备和色谱条件下，使用两种不同的界面（加热雾化器，HN和离子喷雾，ISP）对基质效应的程度进行了直接比较。结果表明，对于研究中的试验药物，当使用ISP接口时，明显观察到基质效应，但当使用HN接口时，基质效应不存在。
In recent years, high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection has been demonstrated to be a powerful technique for the quantitative determination of drugs and metabolites in biological fluids. However, the common and early perception that utilization of HPLC-MS/MS practically guarantees selectivity is being challenged by a number of reported examples of lack of selectivity due to ion suppression or enhancement caused by the sample matrix and interferences from metabolites. In light of these serious method liabilities, questions about how to develop and validate reliable HPLC-MS/MS methods, especially for supporting long-term human pharmacokinetic studies, are being raised. The central issue is what experiments, in addition to the validation data usually provided for the conventional bioanalytical methods, need to be conducted to confirm HPLC-MS/MS assay selectivity and reliability. The current regulatory requirements include the need for the assessment and elimination of the matrix effect in the bioanalytical. methods, but the experimental procedures necessary to assess the matrix effect are not detailed. Practical, experimental approaches for studying, identifying, and eliminating the effect of matrix on the results of quantitative analyses by HPLC-MS/MS are described in this paper. Using as an example a set of validation experiments performed for one of our investigational new drug candidates, the concepts of the quantitative assessment of the "absolute" versus "relative" matrix effect are introduced. In addition, experiments for the determination of, the "true" recovery of analytes using HPLC-MS/MS are described eliminating the uncertainty about the effect of matrix on the determination of this commonly measured method parameter. Determination of the matrix effect allows the assessment of the reliability and selectivity of an existing HPLC-MS/MS method. If the results of these studies are not satisfactory, the parameters determined may provide a guide to what changes in the method need to be made to improve assay selectivity. In addition, a direct comparison of the extent of the matrix effect using two different interfaces (a heated nebulizer, HN, and ion spray, ISP) under otherwise the same sample preparation and chromatographic conditions was made. It was demonstrated that, for the investigational drug under study, the matrix effect was clearly observed when ISP interface was utilized but it was absent when the HN interface was employed.