Wilson Shou

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%.

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P‐glycoprotein (P‐gp) inhibition screen

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.

Recent development in software and automation tools for high-throughput discovery bioanalysis

Bioanalysis with LC-MS/MS has been established as the method of choice for quantitative determination of drug candidates in biological matrices in drug discovery and development. The LC-MS/MS bioanalytical support for drug discovery, especially for early discovery, often requires high-throughput (HT) analysis of large numbers of samples (hundreds to thousands per day) generated from many structurally diverse compounds (tens to hundreds per day) with a very quick turnaround time, in order to provide important activity and liability data to move discovery projects forward. Another important consideration for discovery bioanalysis is its fit-for-purpose quality requirement depending on the particular experiments being conducted at this stage, and it is usually not as stringent as those required in bioanalysis supporting drug development. These aforementioned attributes of HT discovery bioanalysis made it an ideal candidate for using software and automation tools to eliminate manual steps, remove bottlenecks, improve efficiency and reduce turnaround time while maintaining adequate quality. In this article we will review various recent developments that facilitate automation of individual bioanalytical procedures, such as sample preparation, MS/MS method development, sample analysis and data review, as well as fully integrated software tools that manage the entire bioanalytical workflow in HT discovery bioanalysis. In addition, software tools supporting the emerging high-resolution accurate MS bioanalytical approach are also discussed.

Cassette incubation followed by bioanalysis using high-resolution MS for in vitro ADME screening assays

BACKGROUND High-resolution MS (HRMS) has recently received a considerable interest in quantitative bioanalysis using full-scan acquisition mode. The benefits include complete elimination of compound-specific MS method development, and simultaneous collection of mass spectral data on both targeted and non-targeted components. One additional advantage that has not been widely discussed is its suitability for simultaneous quantitation of, theoretically, an unlimited number of compounds, which is not possible with selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. MATERIALS & METHODS We took advantage of this unique bioanalytical capability of HRMS and developed a novel in vitro ADME workflow of cassette incubation of as many as 32 compounds, followed by quantitative bioanalysis using full-scan acquisition on an Orbitrap HRMS. The workflow was evaluated for a serum protein-binding assay and a parallel artificial membrane permeability (PAMPA) assay. RESULTS The bioanalytical assay displayed acceptable sensitivity, selectivity and linearity for all compounds in the cassettes, and the biological results obtained using this approach were similar to those from discrete incubation and analysis, demonstrating the feasibility of the workflow. Additional benefits of this platform include a saving of analysis time due to the reduced sample numbers from the cassette approach, as well as cost saving due to the reduction in the required assay reagents. CONCLUSION Cassette incubation with bioanalysis using HRMS is a feasible approach for high-throughput in vitro ADME assays evaluated in this study.

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.

Discovery of 3-hydroxy-4-cyano-isoquinolines as novel, potent, and selective inhibitors of human 11β-hydroxydehydrogenase 1 (11β-HSD1).

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11β-HSD1 inhibitors with good cross species activity. Optimization of substituents at the 1 and 4 positions of the isoquinoline group in addition to the core modifications, with a special focus on enhancing metabolic stability and aqueous solubility, resulted in the identification of several compounds as potent advanced leads.

Approach to improve compound recovery in a high-throughput Caco-2 permeability assay supported by liquid chromatography-tandem mass spectrometry.

The Caco-2 cell culture system is widely employed as an in vitro model for prediction of intestinal absorption of test compounds in early drug discovery. Poor recovery is a commonly encountered issue in Caco-2 assay, which can lead to difficulty in data interpretation and underestimation of the apparent permeability of affected compounds. In this study, we systematically investigated the potential sources of compound loss in our automated, high-throughput Caco-2 assay, sample storage, and analysis processes, and as a result found the nonspecific binding to various plastic surfaces to be the major cause of poor compound recovery. To minimize the nonspecific binding, we implemented a simple and practical approach in our assay automation by preloading collection plates with organic solvent containing internal standard prior to transferring incubations samples. The implementation of this new method has been shown to significantly increase recovery in many compounds previously identified as having poor recovery in the Caco-2 permeability assay. With improved recovery, permeability results were obtained for many compounds that were previously not detected in the basolateral samples. In addition to recovery improvement, this new approach also simplified sample preparation for liquid chromatography-tandem mass spectrometric analysis and therefore achieved time and cost savings for the bioanalyst.

Sample reduction strategies in discovery bioanalysis

It is a constant challenge to provide timely bioanalytical support for the evaluation of drug-like properties and PK/PD profiles for the ever-increasing numbers of new chemical entities in a cost-effective manner. While technological advancement in various aspects of LC-MS/MS analysis has significantly improved bioanalytical efficiency, a number of simple sample reduction strategies can be employed to reduce the number of samples requiring analysis, and as a result increase the bioanalytical productivity without deploying additional instruments. In this review, advantages and precautions of common sample reduction strategies, such as sample pooling and cassette dosing, are discussed. In addition, other approaches such as reducing calibration standards and eliminating over-the-curve sample reanalysis will also be discussed. Taken together, these approaches can significantly increase the capacity and throughput of discovery bioanalysis without adding instruments, and are viable means to enhance the overall productivity of the bioanalytical laboratory.

Coupling Laser Diode Thermal Desorption with Acoustic Sample Deposition to Improve Throughput of Mass Spectrometry–Based Screening

The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)–based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)–tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1′-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4′-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless steel 384-LazWell plate, then desorbed by an infrared laser directly from the plate surface into the gas phase, ionized by atmospheric pressure chemical ionization (APCI), and analyzed by MS/MS. Using this method, we achieved a sample analysis speed of 2.14 s/well, with bioanalytical performance comparable to the current online solid-phase extraction (SPE)–based MS method. An even faster readout speed was achieved when postreaction sample multiplexing was applied, where three reaction samples, one for each CYP, were transferred into the same well of the LazWell plate. In summary, LDTD coupled with acoustic sample deposition and multiplexing significantly decreased analysis time to 0.7 s/sample, making this MS-based approach feasible to support high-throughput screening (HTS) assays.

A high‐speed liquid chromatography/tandem mass spectrometry platform using multiplexed multiple‐injection chromatography controlled by single software and its application in discovery ADME screening

RATIONALE Multiplexed liquid chromatography (LC) coupled with multiple-injection-chromatogram acquisition has emerged as the method of choice for high-speed discovery bioanalysis, because it significantly reduces injection-to-injection cycle time while maintaining the chromatography quality. Historically, systems utilizing this approach had been custom built, and therefore relied on custom software tools to communicate with multiple vendor software for system control, which lacked transferability, flexibility and robustness. METHODS In this study, we refined a multiplexed bioanalytical system previously reported, by implementing open-deck auto-sampler manifold and multiple-injection-chromatogram acquisition, all on a commercially available system with single software control. RESULTS As a result of these improvements, the developed LC/tandem mass spectrometry (MS/MS) method on the system was nearly three times faster than the previous method, while demonstrating comparable analytical accuracy, precision and robustness. This system has been evaluated for in vitro ADME screening assays including metabolic stability, CYP inhibition and Caco-2. The biological data generated on the developed system displayed good correlation with those from the previous LC/MS/MS approaches. CONCLUSIONS The developed platform demonstrated applicability to the in vitro screening assays evaluated and has been successfully implemented to support the high-throughput metabolic stability assay, with a significantly improved bioanalytical throughput, capacity and data turnaround.