Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry

Studying the permeability of compounds across a Caco-2 cell monolayer is an established in vitro model to screen for oral absorption and to evaluate the mechanism of transport. This assay can also be used to evaluate compounds as potential P-glycoprotein substrates and/or inhibitors. The traditional methods of sample analysis (high-performance liquid chromatography (HPLC) with a UV or fluorescence detector) limit the throughput and sensitivity of this assay. Data are presented here describing the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the analysis of samples derived from the Caco-2 cell studies. During the analysis an automatic switching valve was used to divert the flow from the HPLC column to waste for the first minute, preventing the early eluting salts from entering and contaminating the LC/MS interface. This approach allows the rapid and accurate determination of drug transport across the Caco-2 cell monolayer. The high sensitivity and specificity of LC/MS/MS make this technique an ideal candidate for the low concentration and high throughput routine analysis of Caco-2 cell solutions, especially if multiple compounds are administered and analyzed simultaneously. Thus, the use of LC/MS/MS will increase the value of the Caco-2 cell assay as an in vitro screening tool.     

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.     

Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput

Recent years have seen increasing usage of large particle size stationary phases and ultra-high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra-high flow rate liquid chromatography using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow-bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour.     

Rapid bioanalysis of vancomycin in serum and urine by high-performance liquid chromatography tandem mass spectrometry using on-line sample extraction and parallel analytical columns

A novel high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS) method is described for the determination of vancomycin in serum and urine. After the addition of internal standard (teicoplanin), serum and urine samples were directly injected onto an HPLC system consisting of an extraction column and dual analytical columns. The columns are plumbed through two switching valves. A six-port valve directs extraction column effluent either to waste or to an analytical column. A ten-port valve simultaneously permits equilibration of one analytical column while the other is used for sample analysis. Thus, off-line analytical column equilibration time does not require mass spectrometer time, freeing the detector for increased sample throughput. The on-line sample extraction step takes 15 seconds followed by gradient chromatography taking another 90 seconds. Having minimal sample pretreatment the method is both simple and fast. This system has been used to successfully develop a validated positive-ion electrospray bioanalytical method for the quantitation of vancomycin. Detection of vancomycin was accurate and precise, with a limit of detection of 1 ng/mL in serum and urine. The calibration curves for vancomycin in rat, dog and primate were linear in a concentration range of 0.001-10 microg/mL for serum and urine. This method has been successfully applied to determine the concentration of vancomycin in rat, dog and primate serum and urine samples from pharmacokinetic and urinary excretion studies.     

Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

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.     

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.     

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.     

Higher-throughput screening for Caco-2 permeability utilizing a multiple sprayer liquid chromatography/tandem mass spectrometry system

In the current drug discovery environment, higher-throughput analytical assays have become essential to keep pace with the screening demands for drug metabolism and pharmacokinetics (DMPK) attributes. This has been dictated by advances primarily in chemical procedures, notably combinatorial and parallel syntheses, which has resulted in many-fold increases in the number of compounds requiring DMPK evaluation. Because of its speed and specificity, liquid chromatography/tandem mass spectrometry (LC/MS/MS) has become the dominant technology for sample analysis in the DMPK screening assays. For higher-throughput assays, analytical speed as well as other factors such as method development, data processing, quality control, and report generation, must be optimized. The four-way multiplexed electrospray interface (MUX), which allows for the analysis of four LC eluents simultaneously, has been adopted to maximize the rate of sample introduction into the mass spectrometer. Generic fast-gradient HPLC methods that are suitable for approximately 80% of the new chemical entities encountered have been developed. In-house-written software programs have been used to streamline information flow within the system, and for quality control by automatically identifying analytical anomalies. By integrating these components together with automated method development and data processing, a system capable of screening 100 compounds per week for Caco-2 permeability has been established.     

Fast characterization of intact proteins using a high-throughput eight-channel parallel liquid chromatography/mass spectrometry system

The preparation of protein substrates requires that a large number of chromatographic fractions be analyzed for the presence of reactants, products and by-products. Analyses using linear matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or single column liquid chromatography/mass spectrometry (LC/MS) have been inadequate because of mass resolution or throughput. Therefore, a high-throughput method employing an eight-channel parallel reverse-phase LC/MS system was developed. This system is capable of screening fractions from preparative ion-exchange chromatography with the required mass accuracy and throughput so that the protein purification process can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weight of 8.9 kDa is described and the detection of a contaminating by-product that differs in size by less than 20 Da is demonstrated. Using the current instrumentation and approach, it is practical to analyze 50 protein-containing fractions from column chromatography in less than 1 hour using parallel LC/MS.     

Evaluation of ultra-performance liquid chromatography in the bioanalysis of small molecule drug candidates in plasma

Ultra-performance liquid chromatography (UPLC) combined with mass spectrometric detection (MS) is used successfully in the bioanalysis of small molecule drug candidates in plasma. UPLC-MS is shown to increase sample throughput by reducing run times over 3-fold, without compromising analytical sensitivity or analyte resolution. The technique is demonstrated to be practical and robust on a commercially available ultra-high pressure system when injecting extracts of plasma and has also shown to be a technique that can be used effectively on a conventional high-performance liquid chromatography system fitted with short columns (相似文献   

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.     

Direct injection versus liquid-liquid extraction for plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry.

Direct injection versus liquid-liquid extraction for post-dose human plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) have been studied using a drug candidate compound. For the direct-injection method, an Oasis(R) HLB column (1 x 50 mm, 30 micrometer) was used as the on-line extraction column and a conventional Waters symmetry C18 column (3.9 x 50 mm, 5 micrometer) was used as the analytical column. Each plasma sample (100 microL) was mixed with 100 microL of a working solution of the internal standard in aqueous 0.05 M ammonium acetate (pH 6.9), and portions (10 microL) of these samples were then injected into the LC/MS/MS system. For the liquid-liquid extraction method, a YMC Basic C18 column (2.0 x 50 mm, 5 micrometer) was used as the analytical column. Each sample (0.5 mL) was extracted with methyl tert-butyl ether and the extract was reconstituted and injected into the LC/MS/MS system. The total analysis time for both methods was 2.0 min per sample. The accuracy, inter-day precision and intra-day precision obtained from the quality control samples were within 8% for both methods. The analysis results of post-dose human plasma samples showed that the deviations of 91% of the concentrations obtained using the direct-injection method were within +/-20% from the concentrations obtained using the liquid-liquid extraction method, and the overall average percentage deviation was -1.5%. The results showed that the two methods were equivalent in terms of total chromatographic run time, accuracy and precision. However, for a batch of 100 samples, the sample preparation time for the direct-injection method was only about 25% of the time required for liquid-liquid extraction. This decrease in sample preparation time resulted in the doubling of the overall sample analysis throughput.     

High-speed solubility screening assay using ultra-performance liquid chromatography/mass spectrometry in drug discovery

Ultra-performance liquid chromatography (UPLC) was investigated as an alternative to high-performance liquid chromatography (HPLC) for analyzing pharmaceutical drug candidates. We previously developed a 96-well-based high-speed solubility assay system (HSSOL) using HPLC/UV and a LogD assay system (HSLogD) using HPLC/MS [Y. Dohta, T. Yamashita, S. Horiike, T. Nakamura, T. Fukami, Anal. Chem. 79 (2007) 8312]. We have introduced the UPLC/MS system into this previously developed HSSOL system to increase throughput. Results obtained by the UPLC/MS and HPLC/UV systems showed good agreement, validating the usefulness of the UPLC/MS system. A high-speed solubility assay system was developed employing the UPLC/MS system, thereby tripling the throughput.     

Quantitative liquid chromatography/tandem mass spectrometry determination of chloramphenicol residues in food using sub-2 microm particulate high-performance liquid chromatography columns for sensitivity and speed

The use of chloramphenicol (CAP)--a highly effective broad-spectrum antibiotic used in animal husbandry--is banned in many countries. Therefore, a very low minimum required performance limit (MRPL) of 0.3 microg/kg CAP in meat for human consumption has been defined. Analytical methods capable of quantifying and confirming such low residue levels require sophisticated instrumentation. Preferably sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) or gas chromatography/mass spectrometry (GC/MS) methods have been used. This paper suggests the use of sub-2 microm particulate high-performance liquid chromatography (HPLC) columns to gain additional sensitivity and improve resolution as well as speed. Depending on the operating conditions, higher chromatographic resolution and speed can be obtained at the price of a significantly increased operating pressure, requiring dedicated LC equipment. A 3-4-fold overall improvement of the signal-to-noise ratio for CAP was obtained compared to more classical 5 microm particulate HPLC columns. The proposed analytical methodology includes an enzymatic digestion, which liberates glucuronide-bound CAP from kidney tissue. The extracts obtained after an Extrelut clean-up are sufficiently pure to permit routine injection of biological samples into the sub-2 microm particulate HPLC column, without observing rapid deterioration of peak shape or column clogging problems. The time for one chromatographic run was 4.2 min. The described method was validated for two particularly difficult matrices (kidney and honey). Decision limits (CC alpha) were 0.007 microg/kg (honey) und 0.011 microg/kg (kidney), which are significantly below the current MRPL.     

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.     

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.     

Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry

The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two.     

Structure elucidation of four related substances in gramicidin with liquid chromatography/mass spectrometry

A selective reversed phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of related substances in commercial gramicidin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and the negative ion mode. The LCQ is ideally suited for identification of related substances because it provides on-line LC/MS(n) capability. Compared with UV detection the main advantage of this hyphenated LC/MS(n) technique is the efficient identification of novel related substances without time-consuming isolation and purification procedures. Using this method four novel related substances were separated and identified in a commercial sample.     

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.