An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates
An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates
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DOI：
10.1089/adt.2006.038
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发表时间：
2007-04-01
影响因子：
1.8
通讯作者：
Sanders, Mark
中科院分区：
文献类型：
--
作者：
Drexler, Dieter M.;Belcastro, James V.;Sanders, Mark
描述了一种自动化高通量方法（称为MetFast测定），用于体外评估潜在候选药物的一般微粒体细胞色素P450 β-烟酰胺腺嘌呤二核苷酸磷酸介导的首过代谢稳定性，作为药物分析的实用程序。利用具有多探针液体处理器的机器人方案，将化合物与来自不同物种的肝微粒体一起孵育。然后通过在线液相色谱（LC）-质谱（MS）分析样品，以确定一定时间后剩余的化合物量，从而计算代谢速率。为了通过LC-MS定量评估大量结构不同的化合物，设计了基于迭代两步法的策略。首先通过LC-紫外（UV）/MS（步骤1）对化合物进行定性分析，以确定纯度（UV检测）和结构完整性（MS检测）。该步骤确保仅对具有足够纯度的正确且经验证的化合物进行分析，以获得可重现的高数据质量。此外，收集所有必要信息，以自动生成特定定量方法，用于随后通过LC-U-V/MS对代谢稳定性样品进行生物分析（步骤2）。内部开发的高度灵活和复杂的数据管理软件，称为SmartReport，用于自动定性和定量LC-MS分析设置，数据处理和结果报告。关键方面的集成，用于化合物特异性和灵敏定量MS方法的串联质谱扫描功能的离子阱质谱仪的固有“通用”碰撞诱导解离设置，通用快速LC条件。通用MS仪器设置和SmartReport软件的功能使得分析过程能够常规地提供关于结构不同化合物的可再现的高质量代谢稳定性数据。本文描述了MetFast测定的设置，并给出了测定验证化合物的代谢稳定性数据。
An automated high throughput process, termed the MetFast assay, is described to assess in vilro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphatemediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-U-V/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions., generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.