A high‐throughput bioanalytical platform using automated infusion for tandem mass spectrometric method optimization and its application in a metabolic stability screen

Abstract

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the bioanalytical method of choice to support plate-based, in vitro early ADME (Absorption, Distribution, Metabolism and Excretion) screens such as metabolic stability (Metstab) assessment. MS/MS method optimization has historically been the bottleneck in this environment, where samples from thousands of discrete compounds are analyzed on a monthly basis, mainly due to the lack of a high-quality commercially available platform to handle the necessary MS/MS method optimization steps for sample analysis by selected reaction monitoring (SRM) on triple quadrupole mass spectrometers. To address this challenge, we recently developed a highly automated bioanalytical platform by successfully integrating QuickQuan 2.0, a unique high-throughput solution featuring MS/MS method optimization by automated infusion, with a customized in-house software tool in support of a Metstab screen. In this platform, a dual-column setup running parallel chromatography was also implemented to reduce the bioanalytical cycle time for LC/MS/MS sample analysis. A set of 45 validation compounds was used to demonstrate the speed, quality and reproducibility of MS/MS method optimization, sample analysis, and data processing using this automated platform. Metstab results for the validation compounds in microsomes from multiple species (human, rat, mouse) showed good consistency within each batch, and also between batches conducted on different days. We have achieved and maintained a monthly throughput of 1300 compound assays representing 500 discrete compounds per instrument per month on this platform, and it has been used to generate metabolic stability data for more than 25 000 compounds to date with an overall success rate of more than 95%.

In vitro metabolic stability and metabolite profiling of TJ0711 hydrochloride, a newly developed vasodilatory β-blocker, using a liquid chromatography–tandem mass spectrometry method

“…Metabolic stability is a critical property of drug candidates because it plays an important role in oral bioavailability and overall pharmacokinetic performance [1]. Metabolic stability is usually quantified by monitoring the disappearance of the parent compound over time in liver preparations [2][3][4]. Currently, commercially available human liver microsomes (HLM) have been the most widely used in vitro model for metabolic stability studies [4].…”

A review of LC–MS techniques and high-throughput approaches used to investigate drug metabolism by cytochrome P450s

“…Examples of approaches/techniques that have been developed, include but are not limited to on-line sample preparation [19], cassette dosing or analysis of compounds (i.e. where multiple compounds are administered simultaneously to animal or samples pooled and simultaneously analysed) [33], and staggered parallel high-performance liquid chromatography/tandem mass spectrometry [34]. Further efficiency gains can be made through the use of different LC-MS/MS approaches.…”

LC‐MS in Drug Metabolism and Pharmacokinetics: A Pharmaceutical Industry Perspective

“…The aforementioned bioanalytical approach of automated MS/MS optimization followed by either multiplexed LC -MS [49] or online SPE/MS [60] analysis is widely used to support metabolic stability assays. In addition, sample reduction strategies such as cassette incubation or cassette analysis are often used to further reduce the number of injections and achieve even higher throughput [69 -71] .…”

“…Over the years, vendors have developed high -throughput software tools to manage the ADME workfl ow and enable batch mode MS/MS optimization, sample analysis, and data reviewing [54] . Previously mentioned software such as QuickQuan [49] and DiscoveryQuant [51] not only provides automated optimization for SRM MS/MS methods, but also is capable of storing the optimized conditions in a shared database, generating acquisition and processing methods, performing sample analysis, and processing results in an automated fashion.…”