Faculty of Medicine and Health, School of Pharmacy, University of Sydney, Sydney, Australia; Centre for Education and Research on Ageing, Concord Repatriation General Hospital, Concord, Australia; Bosch Mass Spectrometry Facility, University of Sydney, Sydney, Australia.
Bosch Mass Spectrometry Facility, University of Sydney, Sydney, Australia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 1;1140:122013. doi: 10.1016/j.jchromb.2020.122013. Epub 2020 Feb 1.
Measuring in vivo changes in the drug metabolizing activity of cytochrome P450 (CYP) enzymes is critical to understanding and assessing drug-drug, drug-diet and drug-disease interactions. The sensitivity and specificity of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) makes it an ideal tool for analyzing drugs and their metabolites in biological matrices, and has demonstrated utility in CYP phenotyping across varied applications. Published CYP phenotyping cocktail assays often require large plasma sample volumes (0.5-1 mL), have relatively low sensitivity and multi-step complex sample preparation and extraction procedures. Further, variability exists in the way that recovery and matrix effects are investigated and reported, and some studies fail to report these data altogether. Therefore, the aim of this study was to develop, validate and optimize a simplified assay for the probe drugs caffeine (metabolized by CYP1A2), omeprazole (CYP2C19), losartan (CYP2C9), dextromethorphan (CYP2D6), midazolam (CYP3A4) and their respective enzyme-specific metabolites in small volumes (100 μL) of human plasma, that addresses the issues noted. Analyte extraction involved protein precipitation with acetonitrile and solid-phase extraction (SPE). Samples were analyzed using an Agilent 1290 infinity LC system in tandem with 6460A triple quadrupole mass spectrometers. The assay met FDA guideline-recommended requirements for specificity, sensitivity (analyte LLOQs 0.78-23.4 ng/mL), accuracy (intra-day RE% nominal concentration 90.7-110.2%; inter-day RE% 87.0-110.5%) and precision (intra-day analyte RSD% 0.46-11.4%; inter-day RSD% 1.36-11.2%). Recovery and matrix effects were thoroughly investigated and excluded as potential interferers with assay performance. This assay has been used successfully to phenotype CYP activity in a human clinical trial participant. Importantly, the authors provide a contemporary commentary on commonly found issues in the CYP phenotyping cocktail assay literature, and make recommendations concerning best-practice approaches and the standardization of data reporting in this area.
测量细胞色素 P450（CYP）酶在体内的药物代谢活性变化对于理解和评估药物-药物、药物-饮食和药物-疾病相互作用至关重要。超高效液相色谱串联质谱（UHPLC-MS/MS）的灵敏度和特异性使其成为分析生物基质中药物及其代谢物的理想工具，并在各种应用中证明了用于 CYP 表型分析的效用。已发表的 CYP 表型分析鸡尾酒检测法通常需要大量的血浆样本量（0.5-1 毫升），具有相对较低的灵敏度和多步骤复杂的样本制备和提取程序。此外，在调查和报告回收率和基质效应的方式上存在差异，有些研究根本没有报告这些数据。因此，本研究的目的是开发、验证和优化一种简化的探针药物咖啡因（由 CYP1A2 代谢）、奥美拉唑（CYP2C19）、洛沙坦（CYP2C9）、右美沙芬（CYP2D6）、咪达唑仑（CYP3A4）及其各自的酶特异性代谢物的分析方法，该方法解决了上述问题。分析物提取涉及用乙腈沉淀蛋白质和固相萃取（SPE）。样品采用安捷伦 1290 无穷液相色谱系统与 6460A 三重四极杆质谱联用仪进行分析。该方法符合 FDA 指南推荐的特异性、灵敏度（分析物LLOQ 0.78-23.4ng/mL）、准确度（日内 RE% 标称浓度 90.7-110.2%；日间 RE% 87.0-110.5%）和精密度（日内分析物 RSD% 0.46-11.4%；日间 RSD% 1.36-11.2%）的要求。回收率和基质效应得到了彻底的研究，并被排除为影响分析性能的潜在干扰因素。该方法已成功用于人类临床试验参与者的 CYP 活性表型分析。重要的是，作者对 CYP 表型分析鸡尾酒检测法文献中常见问题进行了当代评论，并就该领域的最佳实践方法和数据报告标准化提出了建议。
