A computer program for automated data evaluation to support in vitro higher-throughput screening for drug metabolism and pharmacokinetics attributes

With the advances in analytical techniques, higher-throughput screening for drug metabolism and pharmacokinetics (DMPK) attributes has become an integral part of drug discovery. However, as the number of compounds increases, the volume of data that needs to be processed and evaluated increases exponentially. As a result, a major challenge for the analytical chemist is how to quickly process the vast amount of data so as to keep up with the throughput of the screening assay. We have developed a customized computer program for automated evaluation of the liquid chromatography/tandem mass spectrometric (LC/MS/MS) data generated from the in vitro DMPK screening assays. This program performs automatic data processing and quality control. It identifies analytical anomalies, such as low internal standard intensity and poor reproducibility of replicates. All analytical anomalies for individual compounds are summarized into an 'E-Log' in a color-coded format for reviewing. With the use of this program and other supporting software, data processing and evaluation for up to 100 compounds are accomplished in several minutes.     

An integrated bioanalytical platform for supporting high-throughput serum protein binding screening

Quantification of small molecules using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a triple quadrupole mass spectrometer has become a common practice in bioanalytical support of in vitro adsorption, distribution, metabolism and excretion (ADME) screening. The bioanalysis process involves primarily three indispensable steps: MS/MS optimization for a large number of new chemical compounds undergoing various screening assays in early drug discovery, high-throughput sample analysis with LC/MS/MS for those chemically diverse compounds using the optimized MS/MS conditions, and post-acquisition data review and reporting. To improve overall efficiency of ADME bioanalysis, an integrated system was proposed featuring an automated and unattended MS/MS optimization, a staggered parallel LC/MS/MS for high-throughput sample analysis, and a sophisticated software tool for LC/MS/MS raw data review as well as biological data calculation and reporting. The integrated platform has been used in bioanalytical support of a serum protein binding screening assay with high speed, high capacity, and good robustness. In this new platform, a unique sample dilution scheme was also introduced. With this dilution design, the total number of analytical samples was reduced; therefore, the total operation time was reduced and the overall throughput was further improved. The performance of the protein binding screening assay was monitored with two controls representing high and low binding properties and an acceptable inter-assay consistency was achieved. This platform has been successfully used for the determination of serum protein binding in multiple species for more than 4000 compounds.     

Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.     

Automatic mass spectrometry method development for drug discovery: application in metabolic stability assays

High-throughput metabolic screening has been requested routinely to keep pace with high-throughput organic synthesis. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a fast gradient has become the method of choice for the task due to its sensitivity and selectivity. We have developed an automated system that consists of a robotic system for in vitro incubation and a commercially available software package for automatic MS/MS method development. A short, generic LC gradient and MS conditions that are applicable to most compounds have been developed to minimize the method development time and data analysis. This system has been used to support a number of in vitro screening assays in early drug discovery phase including microsomal stability and protein binding.     

A generic fast solid-phase extraction high-performance liquid chromatography/mass spectrometry method for high-throughput drug discovery

High-performance liquid chromatography/mass spectrometry (HPLC/MS) is increasingly perceived to be an essential tool in drug discovery at many key steps, like drug screening, lead identification, ADME profiling, and drug metabolism and pharmacology studies. High-throughput screenings in the early phase for metabolic stability, protein binding, permeability (ADME) and bioavailability are widely used to weed out compounds that do not exhibit the necessary characteristics. For such high-throughput LC/MS studies, a generic LC/MS method that can be used for a variety of compounds is desired. In this study, we used a small set of compounds with a wide range of properties to guide method development, and achieved a sample throughput of 1.7 min/sample. Here, we present a generic fast method that achieves good peak separation and peak shape on conventional HPLC systems using a column-switching mechanism for on-line solid-phase extraction (SPE)-HPLC/MS analysis. The method has a linear response range from 1 to 500 nM for the tested compounds. When a larger set of 658 randomly picked small molecules were analyzed using this method, 612 were observed with good signal intensity and HPLC peak shapes. This generic fast SPE-LC/MS method has been used to screen more than 1.5 million compounds repetitively against over 200 protein targets for hit confirmation and semi-quantitation of binding constants from biological assays. Over 7000 different compounds for a variety of protein-binding assays have been studied using this method for quantitative analysis as well.     

Addressing the analytical throughput challenges in ADME screening using rapid ultra-performance liquid chromatography/tandem mass spectrometry methodologies

High-throughput ADME screening for compound drug development properties has become an essential part of the modern drug discovery process, allowing more informed decisions to be made on the best compounds to take forward in the discovery/development process. This however is a time-consuming process requiring multiple tests to be performed, demanding a significant amount of liquid chromatography/mass spectrometry (LC/MS) instrument time. This article focuses on the use of sub-2 microm porous particle LC coupled to tandem quadrupole MS/MS mass spectrometry for the rapid screening of ADME properties. Using this approach analysis times from 30 s to 1 min were achievable allowing analysis times to be cut by 80%. The use of the small particles coupled to high flow rates allowed for sufficient resolution, even with very short analysis time, to resolve the analytes of interest from similar compounds that would interfere with the assay. The use of dedicated, intelligent, software packages allowed for the user-free generation of MS/MS conditions and the processing of the data.     

Dodging matrix effects in liquid chromatography tandem mass spectrometric assays—compilation of key learnings and perspectives

Triple quad liquid chromatography mass spectrometric assays (LC/MS/MS) have revolutionized the analysis of drug(s)/metabolite(s) with exceptional speed, sensitivity and selectivity features. From inception to date, several new and innovative features have been regularly proposed by researchers to further enhance the value in the applicability of this analytical tool. However, owing to such compressed run times and scanty sample preparation procedures, LC/MS/MS assays that are not fully optimized generally have issues of matrix effects, where ionization potential is either suppressed or enhanced due to the presence of other materials (endogenous/exogenous) in the matrix. By definition, even co‐medications, isomeric or isobaric impurities, and drug excipients used in dosing solutions could also potentially contribute to matrix effects. This article captures some of the interesting work carried out by researchers to understand and handle matrix effects. Additionally, it provides perspectives to effectively deal with matrix effects. Copyright © 2008 John Wiley & Sons, Ltd.     

Fast LC/MS in the analysis of small molecules

Liquid chromatography-mass spectrometry (LC/MS) has become one of the most widely used analytical techniques in both qualitative and quantitative analysis of small molecules. Recently, with the increasing demand for ever-higher sample throughput, the use of faster chromatographic separations has become popular, along with other LC/MS methods that decrease analytical cycle-time. The burgeoning use of LC/MS has meant that the primary expertise of many practitioners today is not in the field of LC/MS, which has been facilitated by the ease-of-use of modern LC/MS systems. An examination of the current state of the literature, relating to "fast LC/MS", should serve well to those new to LC/MS, and should help them in the development of fast LC/MS methods that are effective in terms of both the chromatography and the utilization of the mass spectrometer. This review paper focuses on fast LC/MS analyses of small molecules that have been reported in peer-reviewed publications.     

关于中药质量控制与体内代谢研究的思考

质量控制是保证中药安全、有效的重要基础。体内代谢研究可以为阐明中药的治病机制提供依据。本文以蟾酥为例,讨论了当前中药质量控制及体内代谢研究存在的主要问题和解决办法:认为把中药指纹图谱的全面指认与多指标成分定量相结合是有效控制中药质量的可行途径;液相色谱-质谱联用技术(LC/MS)将在中药化学研究、体内代谢研究等方面发挥重要作用;中药的化学指纹图谱应与药理活性相关联,从而建立合理的中药质量评价体系;结构修饰对基于天然产物的新药发现具有重要意义。     

Cutting-edge LC–MS/MS applications in clinical mass spectrometry: Focusing on analysis of drugs and metabolites

In recent years, liquid chromatography with tandem mass spectrometry (LC–MS/MS) has become a fundamental technology in clinical practice. In Japan, the LC–MS/MS system is used in many large hospitals. It has become popular among pharmacists and laboratory technicians. LC–MS/MS has some advantages in terms of accuracy, speed, and comprehensiveness compared to conventional automated chemical testing equipment. However, LC–MS/MS is by no means a universal method, and it is necessary to understand its characteristics before using it. In the field of therapeutic drug monitoring (TDM), there is an issue with linearity in comprehensive measurement; however, ion-abundance adjustment methods, such as in-source collision-induced dissociation, have been proposed as a solution to this problem. The development of a biomarker analysis includes search, identification, and quantification, and it is necessary to select an appropriate mass spectrometric method for each step. In this paper, we review cutting-edge technologies that can expand the performance of LC–MS/MS in the clinical field and consider current issues and future prospects.     

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

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%. Copyright © 2009 John Wiley & Sons, Ltd.     

FastTrack to supercritical fluid chromatographic purification: Implementation of a walk-up analytical supercritical fluid chromatography/mass spectrometry screening system in the medicinal chemistry laboratory

Medicinal chemists often depend on analytical instrumentation for reaction monitoring and product confirmation at all stages of pharmaceutical discovery and development. To obtain pure compounds for biological assays, the removal of side products and final compounds through purification is often necessary. Prior to purification, chemists often utilize open-access analytical LC/MS instruments because mass confirmation is fast and reliable, and the chromatographic separation of most sample constituents is sufficient. Supercritical fluid chromatography (SFC) is often used as an orthogonal technique to HPLC or when isolation of the free base of a compound is desired. In laboratories where SFC is the predominant technique for analysis and purification of compounds, a reasonable approach for quickly determining suitable purification conditions is to screen the sample against different columns. This can be a bottleneck to the purification process. To commission SFC for open-access use, a walk-up analytical SFC/MS screening system was implemented in the medicinal chemistry laboratory. Each sample is automatically screened through six column/method conditions, and on-demand data processing occurs for the chromatographers after each screening method is complete. This paper highlights the “FastTrack” approach to expediting samples through purification.     

Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies

The study of the metabolic fate of drugs is an essential and important part of the drug development process. The analysis of metabolites is a challenging task and several different analytical methods have been used in these studies. However, after the introduction of the atmospheric pressure ionization (API) technique, electrospray and atmospheric pressure chemical ionization, liquid chromatography/mass spectrometry (LC/MS) has become an important and widely used method in the analysis of metabolites owing to its superior specificity, sensitivity and efficiency. In this paper the feasibility of LC/API-MS techniques in the identification, structure characterization and quantitation of drug metabolites is reviewed. Sample preparation, LC techniques, isotope labeling, suitability of different MS techniques, such as tandem mass spectrometry, and high-resolution MS in drug metabolite analysis, are summarized and discussed. Automation of data acquisition and interpretation, special techniques and possible future trends are also the topics of the review.     

Improvement of "hit-to-lead" optimization by integration of in vitro HTS experimental models for early determination of pharmacokinetic properties

Development of predictive in vitro surrogate methods for traditional approaches assessing bioavailability and pharmacokinetics of lead compounds must be made to both keep pace with high-throughput (HT) lead identification and to mitigate the high costs associated with progression of compounds with poor chances of developmental success. Indeed opportunities for improvement still exist in the lead optimization phase versus the lead identification phase, where HT methodologies have been nearly optimized. Review of examples, limitations, and development of high-throughput microtiterplate-based assays for evaluating metabolic liabilities, such as in vitro radiometric and fluorometric assays for inhibition of cytochrome p450 (CYP) activity, determination of stability of a compound in liver microsomes, or cloned CYPs coupled to reconstituting systems are described. Parallel approaches to improve speed, resolution, sample preparation, as well as data analysis using LC/MS and LC/MS/MS approaches and technologies to assess compound integrity and biotransformation by automation and multiplexing are also discussed. Realization of the benefits in automation of cell-based models for determining drug permeability to predict drug absorption are still hampered by bottlenecks in analytical analysis of compounds. The implementation and limitations of surrogate physiochemical methods for passive adsorption such as immobilized artificial membranes (IAM) and parallel artificial membrane permeation assays (PAMPA), and compound solubility by laser nephelometry are reviewed as well. Additionally, data from a high-throughput 96-well equilibrium dialysis device, showing good correlation to classical methods, is presented. Finally, the impact of improvements in these downstream bottlenecks in lead optimization and preclinical drug discovery are discussed in this review.     

Profiling of grape monoterpene glycosides (aroma precursors) by ultra‐high performance‐liquid chromatography‐high resolution mass spectrometry (UHPLC/QTOF)

A ‘suspect screening analysis’ method for grape metabolomics by ultra‐high performance‐liquid chromatography (UHPLC) and high‐resolution quadrupole‐time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)‐based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Generic fast gradient liquid chromatography/tandem mass spectrometry techniques for the assessment of the in vitro permeability across the blood-brain barrier in drug discovery

Rapid, generic gradient liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays, designed to accelerate sample analyses, have been developed to keep pace with the productivity of advanced synthetic procedures. In this study, LC/MS/MS was combined with an in vitro, cell-based, blood-brain barrier (BBB) model to evaluate the potential of new chemical entities (NCEs) to cross the BBB. This in vitro assay provides the permeability of discovery compounds across a monolayer of a primary culture of bovine brain microvessel endothelial cells in a fraction of the time that is required for in vivo studies (brain/plasma concentrations), using only 2 mg of the compound. The results are consistent with in vivo brain/plasma concentration ratio data.     

Examples of doping control analysis by liquid chromatography-tandem mass spectrometry: ephedrines, beta-receptor blocking agents, diuretics, sympathomimetics, and cross-linked hemoglobins

The application of modern and powerful analytical instruments consisting of liquid chromatographs (LCs), sophisticated atmospheric pressure ion sources, and sensitive mass analyzers has improved quality as well as speed of doping control analyses markedly during the last 5 years. Numerous compounds such as beta-receptor blocking agents or diuretics require derivatization prior to gas chromatographic (GC) and mass spectrometric (MS) measurement, which is the reason for extended sample preparation periods. In addition, several substances demonstrate poor GC-MS properties even after chemical modification, and peptide hormones such as cross-linked hemoglobins cannot be analyzed at all by means of GC-MS. With the availability of electrospray ionization and robust tandem MSs (e.g., triple-stage quadrupole or ion trap instruments) many new or complementary screening and confirmation assays have been developed, providing detailed qualitative and quantitative information on prohibited drugs. With selected categories of compounds (ephedrines, beta-blockers, b2-agonists, diuretics, and bovine hemoglobin-based oxygen therapeutics) that are banned according to the rules of the World Anti-Doping Agency and International Olympic Committee, the advantages of LC-MS-MS procedures over conventional GC-MS assays are demonstrated, such as enhanced separation of analytes, shorter sample pretreatment, and identification of substances that are not identified by GC-MS.     

Evolution of an open-access quantitative bioanalytical mass spectrometry service in a drug discovery environment

Increased demand for assays for compounds at the early stages of drug discovery within the pharmaceutical industry has led to the need for open-access mass spectrometry systems for performing quantitative analysis in a variety of biological matrices. The open-access mass spectrometers described here are LC/MS/MS systems operated in 'multiple reaction monitoring' (MRM) mode to obtain the sensitivity and specificity required to quantitate low levels of pharmaceutical compounds in an excess of biological matrix. Instigation of these open-access systems has resulted in mass spectrometers becoming the detectors of choice for non-expert users, drastically reducing analytical method development time and allowing drug discovery scientists to concentrate on their core expertise of pharmacokinetics and drug metabolism. Setting up an open-access facility that effectively allows a user with minimal mass spectral knowledge to exploit the MS/MS capability of triple quadrupole mass spectrometers presents a significantly different challenge from setting up qualitative single stage mass spectrometry systems. Evolution of quantitative open access mass spectrometry within a pharmaceutical drug metabolism and pharmacokinetics group, from its beginnings as a single generic system to a series of specialist fully integrated walk-up facilities, is described.