Quantification of Epoxyeicosatrienoic acids Enantiomers: The development of reliable and practical liquid chromatography mass Spectrometry assay

IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Journal of Chromatography B Pub Date : 2024-10-15 DOI:10.1016/j.jchromb.2024.124346
Fadumo A. Isse , Sara Helal , Ahmed A. El-Sherbeni , Dion R Brocks , Ayman O.S. El-Kadi
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Abstract

Epoxyeicosatrienoic acids (EETs) are increasingly recognized as key metabolites in the arachidonic acid (AA) metabolic pathway. EETs are epoxy derivatives of AA with two chiral centers formed by cytochrome P450 (CYP) enzymes. EETs have reported biological activities as racemates; however, knowledge on specific optical isomers of EET is lacking. A main reason is the absence of practical assay to quantify EETs isomers associated with specific pathological conditions and enzymes. The reported underivatized chiral LC-MS/MS assays utilize different mobile phases and flow rates or required long run times to achieve separation of EET stereoisomers. Others incorporated a derivatization step before the separation of EETs in their assays. Therefore, the objective of this study was to develop and validate a stereoselective assay for the simultaneous quantitation of underivatized EET enantiomers using Liquid Chromatography Mass Spectrometry (LC-MS/MS) with an optimum baseline separation using binary mobile phase and gradient elution. Herein, we report the development and validation of an LC-MS/MS assay, and its application to quantify the formation of EET enantiomers mediated by human liver microsomes. Assay linearity extends over 10–600 ng/mL with r2 > 0.99 for all EETs enantiomers. The inter-run accuracy was within ± 15 %, and precision was ≤ 15 %, and < 20 % for the LLOQ. The matrix effect for the current assay was within ≤ ±20 %, and the mean recovery for quantitative methods was 70–125 %. The assay proved to be reliable and practical for chiral analysis.
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环二十碳三烯酸对映体的定量:开发可靠实用的液相色谱质谱分析法。
环二十碳三烯酸(EETs)越来越被认为是花生四烯酸(AA)代谢途径中的关键代谢物。EETs 是 AA 的环氧衍生物,由细胞色素 P450(CYP)酶形成两个手性中心。据报道,EET 具有外消旋体的生物活性;然而,目前还缺乏有关 EET 特定光学异构体的知识。其中一个主要原因是缺乏实用的检测方法来量化与特定病理条件和酶相关的 EETs 异构体。已报道的手性液相色谱-质谱/质谱检测方法使用不同的流动相和流速,或需要较长的运行时间才能实现 EET 立体异构体的分离。还有一些检测方法在分离 EET 之前加入了衍生步骤。因此,本研究的目的是开发并验证一种立体选择性检测方法,利用液相色谱质谱法(LC-MS/MS)同时定量未充分活化的 EET 对映体,并使用二元流动相和梯度洗脱法进行最佳基线分离。在此,我们报告了一种 LC-MS/MS 检测方法的开发和验证情况,并将其应用于量化由人类肝脏微粒体介导的 EET 对映体的形成。测定线性范围为 10-600 ng/mL,所有 EET 对映体的 r2 > 0.99。运行间准确度在± 15 %以内,精密度≤ 15 %,并且
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来源期刊
Journal of Chromatography B
Journal of Chromatography B 医学-分析化学
CiteScore
5.60
自引率
3.30%
发文量
306
审稿时长
44 days
期刊介绍: The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis. Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches. Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.
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