Matuszewski B K, Constanzer M L, Chavez-Eng C M
Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Anal Chem. 2003 Jul 1;75(13):3019-30. doi: 10.1021/ac020361s.
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.
近年来，高效液相色谱法（HPLC）结合串联质谱检测（MS/MS）已被证明是一种用于定量测定生物流体中药物和代谢物的强大技术。然而，由于样品基质引起的离子抑制或增强以及代谢物的干扰导致缺乏选择性的一些报道实例，正在挑战人们普遍且早期的观念，即使用HPLC-MS/MS实际上可保证选择性。鉴于这些严重的方法缺陷，人们提出了关于如何开发和验证可靠的HPLC-MS/MS方法的问题，尤其是用于支持长期人体药代动力学研究的方法。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之外，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。当前的监管要求包括需要评估和消除生物分析方法中的基质效应，但评估基质效应所需的实验程序并未详细说明。本文描述了用于研究、识别和消除基质对HPLC-MS/MS定量分析结果影响的实用实验方法。以我们的一种研究性新药候选物进行的一组验证实验为例，介绍了“绝对”与“相对”基质效应定量评估的概念。此外，还描述了使用HPLC-MS/MS测定分析物“真实”回收率的实验，消除了基质对这一常用测定方法参数测定影响的不确定性。基质效应的测定可以评估现有HPLC-MS/MS方法的可靠性和选择性。如果这些研究结果不令人满意，所确定的参数可能为改进测定选择性所需的方法变化提供指导。此外，在相同的样品制备和色谱条件下，对两种不同接口（加热雾化器，HN，和离子喷雾，ISP）的基质效应程度进行了直接比较。结果表明，对于所研究的研究性药物，使用ISP接口时明显观察到基质效应，而使用HN接口时则不存在。
