Thakare Rhishikesh, Chhonker Yashpal S, Gautam Nagsen, Alamoudi Jawaher Abdullah, Alnouti Yazen
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States.
J Pharm Biomed Anal. 2016 Sep 5;128:426-437. doi: 10.1016/j.jpba.2016.06.017. Epub 2016 Jun 11.
Accurate quantitative analysis of endogenous analytes is essential for several clinical and non-clinical applications. LC-MS/MS is the technique of choice for quantitative analyses. Absolute quantification by LC/MS requires preparing standard curves in the same matrix as the study samples so that the matrix effect and the extraction efficiency for analytes are the same in both the standard and study samples. However, by definition, analyte-free biological matrices do not exist for endogenous compounds. To address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS, four approaches are used including the standard addition, the background subtraction, the surrogate matrix, and the surrogate analyte methods. This review article presents an overview these approaches, cite and summarize their applications, and compare their advantages and disadvantages. In addition, we discuss in details, validation requirements and compatibility with FDA guidelines to ensure method reliability in quantifying endogenous compounds. The standard addition, background subtraction, and the surrogate analyte approaches allow the use of the same matrix for the calibration curve as the one to be analyzed in the test samples. However, in the surrogate matrix approach, various matrices such as artificial, stripped, and neat matrices are used as surrogate matrices for the actual matrix of study samples. For the surrogate analyte approach, it is required to demonstrate similarity in matrix effect and recovery between surrogate and authentic endogenous analytes. Similarly, for the surrogate matrix approach, it is required to demonstrate similar matrix effect and extraction recovery in both the surrogate and original matrices. All these methods represent indirect approaches to quantify endogenous compounds and regardless of what approach is followed, it has to be shown that none of the validation criteria have been compromised due to the indirect analyses.
对内源性分析物进行准确的定量分析对于多种临床和非临床应用至关重要。液相色谱-串联质谱法（LC-MS/MS）是定量分析的首选技术。通过LC/MS进行绝对定量需要在与研究样品相同的基质中制备标准曲线，以便标准样品和研究样品中分析物的基质效应和提取效率相同。然而，根据定义，不存在用于内源性化合物的无分析物生物基质。为了解决通过LC-MS/MS定量内源性化合物时缺乏空白基质的问题，使用了四种方法，包括标准加入法、背景扣除法、替代基质法和替代分析物法。这篇综述文章概述了这些方法，引用并总结了它们的应用，并比较了它们的优缺点。此外，我们详细讨论了验证要求以及与FDA指南的兼容性，以确保在定量内源性化合物时方法的可靠性。标准加入法、背景扣除法和替代分析物法允许在校准曲线中使用与测试样品中待分析的基质相同的基质。然而，在替代基质法中，各种基质，如人工基质、去除基质和纯基质，被用作研究样品实际基质的替代基质。对于替代分析物法，需要证明替代内源性分析物和真实内源性分析物在基质效应和回收率方面的相似性。同样，对于替代基质法，需要证明替代基质和原始基质在基质效应和提取回收率方面相似。所有这些方法都是用于定量内源性化合物的间接方法，无论采用何种方法，都必须表明由于间接分析，没有任何验证标准受到损害。
