Increasing the throughput and productivity of Caco-2 cell permeability assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism

The Caco-2 cell monolayer permeability assay has become a standard model of human intestinal absorption and transport. This paper reviews recent progress in increasing the throughput of Caco-2 cell monolayer assays and in expanding the scope of this assay to include modeling intestinal drug metabolism. The state-of-the-art in Caco-2 cell monolayer permeability assays combines multi-well plates fitted with semi-permeable inserts on which Caco-2 cells have been cultured with liquid chromatography-mass spectrometry (LC-MS) or LC-tandem mass spectrometry (LC-MS-MS) for the quantitative analysis of test compounds and the identification of their intestinal metabolites. After reviewing the progress in increasing the throughput of Caco-2 cell monolayer assays for both modeling human intestinal permeability or transport and the metabolism of xenobiotic compounds, we demonstrate the application of LC-MS and LC-MS-MS to the measurement of resveratrol permeability and metabolism in the Caco-2 model. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenolic compound occurring in grapes, peanuts and other food sources, that is under investigation as a cancer chemoprevention agent. The apparent permeability coefficient for apical (AP) to basolateral (BL) movement of resveratrol was 2.0 x 10(-5)cm/sec. Resveratrol was not a substrate for P-glycoprotein or the multi-drug resistance associated proteins (MRP). No phase I metabolites were observed, but the phase II conjugates resveratrol-3-glucuronide and resveratrol-3-sulfate was identified based on LC-MS and LC-MS-MS analysis and comparison with synthetic standards. Although these data indicate that resveratrol diffuses rapidly across the intestinal epithelium, extensive phase II metabolism during absorption might reduce resveratrol bioavailability.     

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.     

A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites

An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8 min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation.     

Demonstration of the capabilities of a parallel high performance liquid chromatography tandem mass spectrometry system for use in the analysis of drug discovery plasma samples.

There is a continuing need for increased throughput in the evaluation of new drug entities in terms of their pharmacokinetic (PK) parameters. This report describes an alternative procedure for increasing the throughput of plasma samples assayed in one overnight analysis: the use of parallel high performance liquid chromatography (HPLC) combined with tandem mass spectrometry (parallel LC/MS/MS). For this work, two HPLC systems were linked so that their combined effluent flowed into one tandem MS system. The parallel HPLC/APCI-MS/MS system consisted of two Waters 2690 Alliance systems (each one included an HPLC pump and an autosampler) and one Finnigan TSQ 7000 triple quadrupole mass spectrometer. Therefore, the simultaneous chromatographic separation of the plasma samples was carried out in parallel on two HPLC systems. The MS data system was able to deconvolute the data to calculate the results for the samples. Using this system, 20 compounds were tested in one overnight assay using the rapid rat PK screening model which includes a total of 10 standards plus samples and two solvent blanks per compound tested. This application provides an additional means of increasing throughput in the drug discovery PK assay arena; using this approach a two-fold increase in throughput can be achieved in the assay part of the drug discovery rat PK screening step.     

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.     

High-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry determination of cholesterol uptake by Caco-2 cells

A simple, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric method (LC/APCI-MS/MS) was developed and applied to quantitative determination of uptake of cholesterol by Caco-2 human intestine cells. Caco-2 cells were cultured in medium containing cholesterol-3,4-13C2 and phytosterols from nutritional supplements after in vitro digestion. Cellular cholesterol (cholesterol-3,4-13C2) and endogenous cholesterol were extracted using methanol/chloroform (1:2, v/v) and directly analyzed using LC/APCI-MS/MS with selected reaction monitoring (SRM), using cholesterol-2,2,3,4,4,6-d6 as an internal standard. Detection and quantification limits were 2.2 and 7.2 pmol, respectively. This method provides an effective tool for rapid determination of cholesterol uptake by cells with increased selectivity and sensitivity in comparison to previously reported LC/APCI-MS analysis using selected ion monitoring (SIM).     

Simultaneous LC-MS/MS Determination of Danshensu and Paeoniflorin for Permeability Studies in Caco-2 Intestinal Absorption Model

A high sensitive method based on liquid chromatography tandem mass spectrometry（LC-MS/MS） was developed and validated for the study of permeability of danshensu（DS） and paeoniflorin（PF） in Caco-2 intestinal absorption model. The DS and PF were extracted from cell culture by vacuum-lyophilizing and then separated on a Zorbax Stable Bond C18 column with 0.1% acetic acid aqueous solution and methanol as mobile phase. Detection was carried out by negative electrospray ionization（ESI ） with selected reaction monitoring（SRM） mode. The apparent permeability coefficients（Papp） of DS and PF in Caco-2 cell medium were calculated and the effects of verapamil on the coefficients Papp of the two test compounds were also illustrated. The permeability of PF was much better than that of DS when the two compounds were administrated individually. Co-administration of DS and PF led to the decrease of the transport from apical side to basolateral side for both the compounds. However, the transport in the contrary direction were accelerated. It was also observed that verapamil could accelerate the transport of the test compounds from apical side to basolateral side. However, the absorption-enhanced effect of verapamil was attenuated when DS and PF were co-administrated. These observations suggest that both passive diffusion and active efflux involved in P-gp would effect the passage of DS and PF across Caco-2 cell monolayer. At the same time, the co-administration of DS and PF to an alteration of transport behavior, which suggests that the interaction must be taken into account when ‘n-in-one＇ samples were used in Caco-2 intestinal model.     

Description and validation of a staggered parallel high performance liquid chromatography system for good laboratory practice level quantitative analysis by liquid chromatography/tandem mass spectrometry.

The Aria LX4 staggered parallel high performance liquid chromatography (HPLC) system is evaluated for application to good laboratory practice (GLP) level quantitative analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This system consists of four fully independent binary HPLC pumps, a modified autosampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality. Validation results for four different compounds, each analyzed on a separate channel of the Aria system, show precision and accuracy equivalent to that required of a single-channel system. The results show that sample throughput can be increased nearly four-fold without requiring significant changes in current analytical procedures. The flexibility and ease of use of the Aria system suggest that it should be possible to quickly implement it in any analytical LC/MS/MS environment.     

LC–MS/MS in the routine clinical laboratory: has its time come?

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been increasingly used in routine clinical laboratories during the last two decades. The high specificity, sensitivity, and multi-analyte potential make it an ideal alternative to immunoassays or conventional high-performance liquid chromatography (HPLC). It also provides higher throughput than gas chromatography–mass spectrometry (GC–MS). LC–MS/MS also offers higher flexibility than immunoassays because LC–MS/MS assays are typically developed in-house. In addition, abundant information can be obtained from a single LC–MS/MS run which can produce a large amount of quantitative or qualitative data. In this review, typical LC–MS/MS clinical applications are presented, personal experiences are shared, and strengths and weakness are discussed. It is foreseeable that LC–MS/MS will become a key instrument in routine clinical laboratories.     

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.     

Permeability of the Perindopril Arginine under In Vitro Conditions across Caco-2 Monolayer and Biomimetic Phospholipid Membrane

Perindopril arginine (PA) as an angiotensin-converting enzyme (ACE) inhibitor is widely used in cardiovascular diseases, especially in systemic hypertension and heart failure. Although the pharmacokinetics of PA are well documented, there is no available detailed data on its permeation in in vitro conditions. The present study aimed to assess the transport of PA across both biological membranes and artificial biomimetic ones. For the determination of PA transport, the Caco-2 cell line was selected as a reliable in vitro model of gastrointestinal biological barriers. Additionally, a novel 96-well plate with phospholipid membrane PermeaPad was used to evaluate the transport of PA by passive diffusion. We confirmed that PA is relatively poorly permeable across the Caco-2 monolayer. The permeability results obtained from the non-cell-based model demonstrated higher transport of PA as compared to that of Caco-2. Thus, PA transport across the biological membranes might be suggested to be regulated by the membrane transporters.     

On-line sample preparation for high throughput reversed-phase LC/MS analysis of combinatorial chemistry libraries

An on-line sample preparation method utilizing a time-programmed autosampler is described for high throughput liquid chromatography/mass spectrometry (LC/MS). This approach is particularly helpful for the LC/MS analysis of samples which require solvents incompatible with HPLC in the sample preparation process. The on-line sample preparation approach minimizes a bottleneck in throughput and improves sample recovery under some circumstances.     

Trace-level quantitation of iralukast in human plasma by microbore liquid chromatography/tandem mass spectrometry

Iralukast (CGP 45715A) is a potent peptido-leukotriene antagonist that is active in various in vitro and animal models for the treatment of asthma. An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ) of 10 pg/mL for the analysis of iralukast when administered at low doses during clinical trials. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compound. First, iralukast appeared to be light sensitive and unstable at room temperature under acidic conditions. Second, a LLOQ of 10 pg/mL was needed to support several clinical trials. Third, positive electrospray ionization of iralukast did not yield the necessary sensitivity required for studies in humans. Consequently, LC/MS/MS conditions were optimized for the negative ion mode of detection. Fourth, sample preparation steps proved to be critical to reduce the possibility of microbore HPLC column (50 mm x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix-mediated electrospray ion suppression. While our validated method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, plasma concentration-time profiles for patients with moderate asthma after oral administration of 200, 500, 1000, and 5000 microgram/kg/day of iralukast were successfully obtained.     

Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice)

Gan-Cao, or licorice, is a popular Chinese herbal medicine derived from the dried roots and rhizomes of Glycyrrhiza uralensis, G. glabra, and G. inflata. The main bioactive constituents of licorice are triterpene saponins and various types of flavonoids. The contents of these compounds may vary in different licorice batches and thus affect the therapeutic effects. In order to ensure its efficacy and safety, sensitive and accurate methods for the qualitative and quantitative analyses of saponins and flavonoids are of significance for the comprehensive quality control of licorice. This review describes the progress in chemical analysis of licorice and its preparations since 2000. Newly established methods are summarized, including spectroscopy, thin-layer chromatography, gas chromatography, high-performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry (LC/MS), capillary electrophoresis, high-speed counter-current chromatography (HSCCC), electrochemistry, and immunoassay. The sensitivity, selectivity and powerful separation capability of HPLC and CE allows the simultaneous detection of multiple compounds in licorice. LC/MS provides characteristic fragmentations for the rapid structural identification of licorice saponins and flavonoids. The combination of HPLC and LC/MS is currently the most powerful technique for the quality control of licorice.     

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.     

Ultra-performance liquid chromatography/tandem mass spectrometric quantification of structurally diverse drug mixtures using an ESI-APCI multimode ionization source

We report in this paper an ultra-performance liquid chromatography/tandem mass spectrometric (UPLC(R)/MS/MS) method utilizing an ESI-APCI multimode ionization source to quantify structurally diverse analytes. Eight commercial drugs were used as test compounds. Each LC injection was completed in 1 min using a UPLC system coupled with MS/MS multiple reaction monitoring (MRM) detection. Results from three separate sets of experiments are reported. In the first set of experiments, the eight test compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes (ESI+, ESI-, APCI-, and APCI+) during an LC run. Approximately 8-10 data points were collected across each LC peak. This was insufficient for a quantitative analysis. In the second set of experiments, four compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes during an LC run. Approximately 15 data points were obtained for each LC peak. Quantification results were obtained with a limit of detection (LOD) as low as 0.01 ng/mL. For the third set of experiments, the eight test compounds were analyzed as a batch. During each LC injection, a single compound was analyzed. The mass spectrometer was detecting at a particular ionization mode during each LC injection. More than 20 data points were obtained for each LC peak. Quantification results were also obtained. This single-compound analytical method was applied to a microsomal stability test. Compared with a typical HPLC method currently used for the microsomal stability test, the injection-to-injection cycle time was reduced to 1.5 min (UPLC method) from 3.5 min (HPLC method). The microsome stability results were comparable with those obtained by traditional HPLC/MS/MS.     

A mass spectrometry based functional assay for the quantitative assessment of ABC transporter activity

ATP‐Binding Cassette (ABC) transporters are highly expressed in pharmacological barriers limiting the access of drugs to their targets. Since characterization of a compound as a transporter substrate or inhibitor bears significant consequences in drug development, there is a great need for reliable tools that enable the rapid analysis of the transport susceptibility of drugs. Here we describe a simple but very efficient high‐performance liquid chromatography/mass spectrometry (HPLC/MS) assay for measuring the ABC transporter‐dependent vesicular transport of compounds. In addition, we provide evidence that the requirement for sample preparation can be minimized using desorption electrospray ionization (DESI)‐MS, paving the way for a direct, high‐throughput investigation of drug‐transporter interactions. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of on-line nano-liquid chromatography/mass spectrometry in metabolite identification studies

Metabolite identification is an important part of the drug discovery and development process. High sensitivity is necessary to identify metabolic products in vitro and in vivo. The most common method utilizes standard high-performance liquid chromatography (4.6 mm i.d. column and 1 mL/min flow rate) coupled to tandem mass spectrometry (HPLC/MS/MS). We have developed a method that utilizes a nano-LC system coupled to a high-resolution tandem mass spectrometer to identify metabolites from in vitro and in vivo samples. Using this approach, we were able to increase the sensitivity of analysis by approximately 1000-fold over HPLC/MS. In vitro samples were analyzed after simple acetonitrile precipitation, centrifugation, and dilution. The significant improvement in sensitivity enabled us to conduct experiments at very low substrate concentrations (0.01 μM), and very low incubation volumes (20 μL). In vivo samples were injected after simple dilution without any pre-purification. All the metabolites identified by conventional HPLC/MS/MS were also identified using the nano-LC method. This study demonstrates a very sensitive approach to identifying phase I and II metabolites with throughput and separation equivalent to the standard HPLC/MS/MS method.     

A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.     

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.