Profiling primaquine metabolites in primary human hepatocytes using UHPLC-QTOF-MS with 13C stable isotope labeling
Profiling primaquine metabolites in primary human hepatocytes using UHPLC-QTOF-MS with 13C stable isotope labeling
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DOI：
10.1002/jms.3122
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发表时间：
2013-02-01
影响因子：
2.3
通讯作者：
Walker, Larry A.
中科院分区：
文献类型：
--
作者：
Avula, Bharathi;Tekwani, Babu L.;Walker, Larry A.
伯氨喹（PQ）是目前唯一能根治复发间日疟的药物，其疗效和溶血毒性是由其代谢产物介导的。然而，这些代谢物的鉴定仍然是一个重大挑战，显然是由于数量少和它们的反应性质。用稳定同位素标记的候选药物提供了方便的工具，用于通过液相色谱-串联质谱法（LC-MS-MS）跟踪复杂基质中的药物衍生代谢物，并过滤具有可归因于标记的双峰的质量。本研究旨在从13 C6-PQ/PQ 1：1 w/w混合物与原代人肝细胞的体外孵育中鉴别PQ代谢物。Acquity超高效液相色谱（UHPLC）与QTOF-MS集成，将分离效率与高灵敏度、检测选择性和准确的质量测定相结合。采用UHPLC保留时间、差异为6的双质量峰（源自13 C6-PQ/PQ）和MS-MS裂解模式进行表型分析。除了通过PQ氧化脱氨基为醛并随后氧化形成的羧基-PQ（cPQ）外，还鉴别了几种其他代谢产物：包括PQ醇（可预测通过PQ氧化脱氨基为醛并随后还原产生）、其乙酸盐和醇的葡糖苷酸结合物。还检测到PQ和cPQ的痕量醌亚胺代谢物，其可能通过PQ/cPQ喹啉环在5位的羟基化和随后的氧化产生。这些发现进一步阐明了PQ的人体肝脏代谢，该方法可用于鉴定临床前和临床研究中体内产生的反应性PQ代谢物。版权所有（c）2013约翰威利父子有限公司
Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug-derived metabolites in complex matrices by liquid chromatography-tandem mass spectrometry (LC-MS-MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of 13C6-PQ/PQ with primary human hepatocytes. Acquity ultra-performance LC (UHPLC) was integrated with QTOF-MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from 13C6-PQ/PQ), and MS-MS fragmentation pattern were used for phenotyping. Besides carboxy-PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohol's glucuronide conjugate. Trace amounts of quinone-imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5-position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies. Copyright (c) 2013 John Wiley & Sons, Ltd.