MS System for the Bioanalysis of Samples

Anal. Chem. 2001, 73, 1495-1501

A High-Capacity LC/MS System for the Bioanalysis of Samples Generated from Plate-Based Metabolic Screening John S. Janiszewski,*,† Katrina J. Rogers,†,‡ Kevin M. Whalen,† Mark J. Cole,† Theodore E. Liston,† Eva Duchoslav,§ and Hassan G. Fouda†

Groton Labs, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, and MDS Sciex, 71 Four Valley Drive, Concord, Ontario, L4K4V8 Canada

HPLC/MS is a linear technique characterized by serial injection and analysis of individual samples. Parallelformat high-throughput screens for druglike properties present a significant analytical challenge. Analysis speed and system ruggedness are key requirements for bioanalysis of thousands of samples per day. The tasks involved in LC/MS analysis are readily divided into three areas, sample preparation/liquid handling, LC/MS method building/sample analysis, and data processing. Several automation and multitasking strategies were developed and implemented to minimize plating and liquid handling errors, reduce dead times within the analysis cycle, and allow for comprehensive review of data. Delivering multiple samples to multiple injectors allows the autosampler time to complete its wash cycles and aspirate the next set of samples while the previous set is being analyzed. A dual-column chromatography system provides column cycling and peak stacking and allows rapid throughput using conventional LC equipment. Collecting all data for a compound into a single file greatly reduces the number of data files collected, increases the speed of data collection, allows rugged and complete review of all data, and provides facile data management. The described systems have analyzed over 40 000 samples per month for two years and have the capacity for over 2000 samples per instrument per day. Drug metabolism parameters derived from in vitro absorption and hepatic stability studies are integral components of contemporary drug discovery. They permit the assessment of compound structure relative to metabolism and pharmacokinetic (PK) properties. PK-related defects account for nearly 40% of lead compound failures in drug development stages.1-3 Determination of the absorption, distribution, metabolism, and excretion (ADME) * Corresponding author: (e-mail) [email protected]; (phone) 860-441-8445; (fax) 860-715-7846. † Pfizer Global Research and Development. ‡ Current address: MDS Pharma Services, 11804 North Creek Pkwy., Bothell, WA 98011. § MDS Sciex. (1) Kennedy, T. Drug Discovery Today 1997, 2, 436-444. (2) Prentis, R. A.; Lis, Y.; Walker, S. R. Br. J. Clin. Pharmacol. 1988, 25, 387396. (3) Lin, J. H.; Lu, A. Y. Pharmacol Rev. 1997, 49, 403-449. 10.1021/ac0013251 CCC: $20.00 Published on Web 03/03/2001

© 2001 American Chemical Society

properties of new chemical entities (NCE) in early drug discovery should allow defects to be corrected prior to time-consuming and expensive nonclinical and clinical stage development. An appreciation of the relationship between candidate survival and its ADME profile, coupled with the ever-increasing productivity of combinatorial and high-speed synthesis approaches, has led to the rapid evolution of high-throughput drug metabolism screening.4-7 These in vitro drug metabolism screens are plate based and processed in parallel format. In order for ADME screening to integrate seamlessly into the drug discovery process, data turnaround must keep pace with synthesis cycles. This requires coordination between the compound management, biology, and bioanalytical groups as plates and data move through the system. Due to the structural diversity of compound sets, and relatively low detection levels needed for quantitative bioanalysis, LC/MS is the current method of choice to support high-throughput metabolic screening.8-12 Its main limitation has been that it is essentially a serial technique characterized by the injection and analysis of individual samples. This is a disadvantage in support of parallel-format high-throughput screens that produce multiple samples simultaneously (i.e., “96 or 384 at a time”). We have approached this challenge by developing a standardized LC/MSbased, bioanalysis protocol that provides rapid turnaround of (4) Sahakian, D. C.; Sisk, R. L.; Polzer, R. J. The Evaluation of Rat Primary Hepatocyte Models for Predicting In Vivo Metabolic Clearance; American Association of Pharmaceutical Sciences Annual Meeting, San Francisco, CA, November 1998. (5) Sweetland, R. L.; Polzer, R. J. Comparison of Traditional 21-day CACO-2 Cultures to Biocoat Intestinal Epithelium Differentiation Environment -Cultured CACO-2 Cells for the Ability to Predict Active and Passive Transport; American Association of Pharmaceutical Sciences Annual Meeting, San Francisco, CA, November 1998. (6) Johnson, D.; Janiszewski, J.; Cohen, L.; Mankowski, D.; Whalen, R.; Tweedie, D. In Vitro Inhibition Studies in 96 Well Plates: Higher Throughput Methods to Assess Drug Interaction Potential; Society for Biomolecular Screening, San Diego, CA, September 1997. (7) Yee, S. Pharm. Res. 1997, 14 (6), 763-766. (8) Bu, H.-Z.; Poglod, M.; Micetich, R. G.; Khan, J. K. Rapid Commun. Mass Spectrom. 2000, 14, 523-528. (9) Korfmacher, W. A.; Palmer, C. A.; Nardo, C.; Dunn-Meynell, K.; Grotz, D.; Cox, K.; Lin, C.-C.; Elicone, C.; Liu, C.; Duchoslav, E. Rapid Commun. Mass Spectrom. 1999, 13, 901-907. (10) Chu, I.; Favreau, L.; Soares, T.; Lin, C.-C.; Nomeir, A. A. Rapid Commun. Mass Spectrom. 2000, 14, 207-214. (11) Wang, Z.; Hop, C., E. C. A.; Leung, K. H.; Pang, J. M. J. Mass Spectrom. 2000, 35, 71-76. (12) Yin, H.; Racha, J.; Li, S.-Y.; Olejnik, N.; Satoh, H.; Moore, D. Xenobiotica 2000, 30, 141-154.

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Figure 1. Overall system layout showing fluid paths and electronic communication signals. In the upper left portion of the figure is the 215 Multiprobe autosampler setup in dual-needle mode with 17-plate deck. A multiply injected run begins after a ready out signal is received from the MS. Unipoint (PC) controls the sequence of injection and coordinates scheduling within the system.

quantitative data in support of ADME/PK screening. This generic approach integrates system setup, bioanalysis, and data handling to maximize sample throughput and data delivery. Each instrumental setup routinely analyzes between 300 and 400 NCEs per screen per week. Each compound entering a given screen (e.g., membrane permeability (caco-2), metabolic stability, etc.) generates between 10 and 20 samples; thus, the sample load averages ∼5000 samples per instrument per week. The described approach organizes the components of the bioanalysis process, such as injection sequence, chromatography, mass spectrometry, and data handling, in an automated way to minimize idle time and streamline information flow through the system. The resulting systems allow serial LC/MS analysis to keep pace with the parallel-format ADME screens. EXPERIMENTAL SECTION Sample Preparation. Compounds submitted for screening are received in deep-well 96-well plate (DW-plates, 1.2-mL Marsh Tubes, Marsh Biomedical, Rochester, NY) format from Pfizer compound management services. The plates contain 90 compounds each. Each plate is associated with a MS-Excel template file that includes the individual analyte structure, putative molecular weight, and well position for every compound in that plate. The template file coordinates sample tracking through the biological and analytical parts of the assay. Caco-2 and hepatocyte samples are received in Deep-Well (DW, 1.2 mL) polypropylene plates with scored lids (Marsh Biomedical). 1496

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The caco-2 samples are diluted with 75-125 µL of acetonitrile containing an internal standard using an AutoChem liquid handler (Cardinal Instruments, Princeton, NJ) prior to injection. The internal standard is a proprietary compound (MW 649) that has favorable chromatographic behavior and forms both positive and negative ions. Hepatocyte samples are diluted 2.5-3-fold with acetonitrile containing internal standard to induce protein precipitation. The samples are clarified by centrifugation, and the resulting supernatants are transferred to fresh DW plates. A 100-µL volume of deionized water is added to each well, and the plates are mixed prior to LC/MS analysis. Liquid handling during these steps was completed using a Personal Pipettor 96-well pipettor (Apricot Designs, Sunnyvale, CA) and a CCS Packard MiniTrak system (Packard Instruments, Meridan, CT). HPLC Instrumentation. A dual-injection column-switching HPLC system was designed, built, and configured as depicted in Figure 1. The autosampler is a Gilson 215 Multiprobe (Gilson Instruments, Middleton, WS) equipped with a custom-made dualinjection manifold. Custom plate racks built for the autosampler have plate capacities of 16 or 17 96-well plates. A total of three six-port, two-position valves are used in this setup. One of the valves is used to direct the aqueous mobile-phase flow, and the other two are injection port valves. A 10-port two-position valve was used for column switching. All valves were from Valco Instruments (Houston, TX). Gilson Unipoint software controlled the injection sequence and injection valves through a Gilson 506A

Figure 2. Dual-column 10-port valve plumbing diagram. The columns toggle between load and elution modes. Note that the fluid direction valve, and aqueous loading flow, are always directed to waste, and likewise, elution flow is always into the detector.

system interface. Two HPLC pumps were used (model PU-980, Jasco Inc., Easton, MA). Pump I delivered primarily aqueous mobile phase, 10:90 acetonitrile/2 mM ammonium acetate (v/v). Pump II delivered primarily organic mobile phase consisting of 90%, acetonitrile/methanol (1:1) and 10% 2 mM ammonium acetate (v/v). The flow rate for each pump was 1.5 mL/min. The timing for the aqueous flow and column switch valves was controlled by timed event signals sent from pump I or through Unipoint. The dual-column-switching 10-port valve layout is diagramed in Figure 2. The columns were 1 × 15 mm, 40-µm pellicular C18 (Optimize Technologies, Oregon City, OR). The mobile phase was plumbed to direct the aqueous “loading” flow through the autosampler, through the first column, and out to waste. The organic “eluent” flow was simultaneously directed through the second column and into the MS. At 18 s after injection, the 10port valve was switched and the analyte was eluted from column I into the MS and column II received aqueous flow. At 24 s after injection, aqueous flow was directed through the autosampler injection manifold and the second sample was loaded onto column II. The column-switching valve was modified in-house by addition of an electronic relay that allowed “pulse logic” control. That is, a single 0.005-s pulse from pump I turned the valve. Mass Spectrometry. The mass spectrometers were from PE Sciex (Concord, ON, Canada). An API 150 single-quadrupole instrument was used for caco-2 sample analysis, and an API 2000 tandem mass spectrometer was used for analysis of hepatocyte samples. A Turbo IonSpray (TISP) interface was used on both systems, and the eluent flow was split 5:1 such that flow at the sprayer was ∼300 µL/min. The auxiliary, nebulizing, and collision gases were nitrogen obtained from an in-house nitrogen-generating system.13 The TISP interface was maintained at 350 °C on all

instruments. Prior to quantitative analysis, MS and MS/MS ion monitoring conditions are automatically determined for all compounds. The details of this procedure have been described.14 Data Handling and Review. After selection of SIM and SRM conditions, a custom software application14 is used to produce a text file for import into Sciex Sample Editor to build the sequence of injections. Each compound to be analyzed is assigned a unique data file name identified in the injection sequence by the compound name (in-house label) and plate position (well ID). All samples associated with a given compound are injected into this single file. For caco-2 screening, a total of 20 injections are made per compound per file. For hepatocyte screening, a total of 16 injections are made per compound per file. Therefore, the Sample Editor injection sequence contains a total of 96 files, corresponding exactly to the original electronic file created when the compound plate entered the ADME screening process. At the conclusion of an LC/MS run a software application, EvaLution15 is used to process the chromatograms and produce a text file of peak areas and retention times for each file. The text file is read directly into an MS-Excel spreadsheet. During data review, each chromatographic file is reviewed such that numerical data in MS-Excel and the corresponding chromatograms are viewed simultaneously. For caco-2, if the chromatogram meets acceptance criteria, an apparent permeability (Papp value) is calculated immediately. RESULTS AND DISCUSSION Parallel-format compound synthesis and biological assays stretch serial analysis techniques such as LC/MS to their limit. For LC/MS to impact and contribute to an ADME screening effort in drug discovery, sample analysis must be rapid enough to keep pace with plate-based parallel-format screening protocols. Each component of the analysis process, such as chromatography and data review, needs to be as rapid as possible in context of the overall bioanalysis system. The throughput limitations of various system components must be identified and understood before the analytical system can be designed to integrate components for optimal overall throughput speed. Although the MS is a serial detector, it is not necessarily the limiting factor in terms of throughput speed. As an example, quantification of two analytes using a dwell time of 200 ms, and a general requirement that 10 data points are needed to define a peak with adequate precision, requires chromatographic baseline peak widths of 4 s (2 s fwhh). Therefore, until samples can be delivered to the MS, including the chromatography step, in a time frame of less than 4 s the MS is not rate limiting. These values are illustrative. Actual dwell times and precision tolerances are determined by assay criteria. The point is that the conventional quadrupole MS can make fast measurements, and very high-throughputs can be achieved, dependent upon how fast samples can be delivered to the detector. The LC/MS system described herein was designed to speed sample delivery to the MS detector using a conventional approach. The focus was on the components peripheral to the MS, such as (13) Luther, E. Am. Lab. (Shelton, Conn.) 1999, 31, 62-64. (14) Whalen, K. M.; Rogers, K. J.; Cole, J. M.; Janiszewski, J. S. Rapid Commun. Mass Spectrom. 2000, 14, 2074-2079. (15) Whalen, K. M.; Rogers, K. J.; Janiszewski, J. S.; Luther, E. W.; Fouda, H. G.; Cole, M. J.; Duchoslav, E. 48th ASMS Conference on Mass Spectrometry, Long Beach, CA, June 12-16, 2000; No. 303.


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Figure 3. Aqueous flow-directing valve. Used to flush the dual sample loops, in sequence, into the dual-column 10-port valve.

the autosampler, chromatography system, and data handling. These system components are described in the following sections. Autosampler. The autosampler cycle time has the greatest impact on throughput speed. The cycle time is defined as the time the autosampler takes from injection to injection and includes washing the needles, retrieving the next sample(s), aspirating, and returning to the injection port. The Gilson Multiprobe autosampler can aspirate or dispense one to eight samples, in 9-mm spacing, simultaneously. A modified dual-needle setup was used to inject replicate samples arranged in contiguous wells. At the start of each cycle, two samples are aspirated and transferred to the injection ports, the sample loops are loaded and the samples are flushed in sequence into the dual column system. A flowdirecting valve (see Figure 3) directs the aqueous pump flow through each injection valve in a specific timed sequence. Time is saved as the autosampler is completing its wash routine and aspirating the next set of samples while the previous set is being processed through the injection valve sample loops and chromatography system. The cycle time in the described system is limited to ∼50 s. This means that, given a total needle rinse volume of 1000 µL, it takes 50 s between sample injections no matter how many samples are aspirated at once. The Gilson 215 multiprobe has a requirement that samples must be in contiguous wells for simultaneous aspiration. Therefore, the plate layout has an impact on throughput. At present we are limited to dual injection due to the format of the biological assays. The chromatography system flushes the sample loops within 30 s, leaving ∼20 s of dead time. This dead time can be filled to improve throughput by changing the plate format to accommodate quadruple injections. Chromatography and Column Switching. There were several requirements in development of the chromatography system. Due to the structural diversity of the compound sets, and the sheer numbers of samples, the chromatography methods needed to be 1498


universal and rugged. There is not enough time for HPLC method development on each individual analyte or groups of structurally related analytes. The goal of the chromatography system was to deliver samples in the MS time frame, that is, the minimum time frame as dictated by the number of analytes, the MS dwell time, and the number of data points necessary to determine peak area with high precision. Rapid chromatographic methods coupled with multiple autosamplers and/or a multiplexed electrospray source have been described to increase throughput speed.16,17 These approaches can be complex in terms of instrumentation, plumbing, and overall system orchestration. In the system described, samples are desalted and concentrated on-line using a dual-column switching valve (Figure 2). Samples are loaded on the first column in high-aqueous mobile phase (90% aqueous) at a flow rate of 1.5 mL/min. The aqueous column eluent is directed to waste. At the same time high-organic mobile phase (90% organic solvent) is directed through the second column, also at 1.5 mL/min, and into the MS. A typical chromatogram resulting from a caco-2 experiment is shown in Figure 4. The two columns alternate between the loading and eluting modes. This process continually repeats, such that the load step on one column and the elution step on the other are occurring nearly simultaneously. This is possible due to the negligible reequilibration time of the 1 × 15 mm columns. In this format, the column volume is less than 10 µL. At a flow rate of 1.5 mL/min, 0.05 min (3 s) provides aqueous volume (75 µL) sufficient for reequilibration. Chromatographic resolution is minimal in this system, and both the analyte and internal standard elute in an approximate 0.3-min window. There is little or no variability in retention time throughout a run; the internal standard trace for sample 1 and 96 overlay exactly. In fact, a retention time shift would be indicative of a problem. Likewise, a diminished area count or change in peak shape of the internal standard peak would also indicate a problem. Using this setup, the precision of injection is routinely within 6-8%, measured by peak area. The measured precision is equivalent to that obtained using a single-column, single-injection system, all other variables held constant. The interday variability of the system was assessed by preparing 6 point standard curves from 0.125 to 2 µM for 16 compounds on 3 separate days. The standard curves were prepared in replicates of eight such that a total of eight plates were analyzed on each experimental day. Correlation coefficients were better than 0.990 for all analytical runs. Precision was within 10% for slope and R2 across the entire experiment. A variety of column configurations and packings were tested during the development of the system. The main requirements were ease of use, peak shape consistency across structural series, and ruggedness. The column dimensions tested varied from 2 × 30 to 1 × 10 mm. These were mostly cartridge-style columns of either C8 or C18 porous silica. The porous silica columns had a lifetime of about 800-1000 injections per column using the dualcolumn switching setup described. After 800 injections, there was a decrease in performance with these columns in terms of loss of peak shape. There were increases in both peak broadening and asymmetry with all porous silica materials tested. These problems were attributed to perturbation of the packing material at the head (16) Zweigenbaum, J.; Henion, Jack. Anal. Chem. 2000, 72, 2446-2454. (17) Bayliss, M. K.; Little, D.; Mallett, D. N.; Plumb, R. S. Rapid Commun. Mass Spectrom. 2000, 14, 2039-2045.

Figure 4. Typical chromatogram for caco-2 MS-SIM assay. All the samples pertaining to a given analyte are collected in a single file. Note that samples for the apical to basolateral (A f B) transport experiment are on the left side and basolateral to apical (B f A) are to the right side in the chromatogram.

of the column due to fouling and/or the high-flow rate, rapid step gradient conditions used. After testing many options to improve column lifetime, we finally settled on Opti-Lynx 1-mm-i.d. columns from Optimize technologies. These columns are dry-packed with a superficially porous pellicular material. The main differences are a larger particle size (∼40 µm) and a difference in the bead. Rather than using completely porous silica particles, the pellicular packing uses a porous silica surface coated on a glasslike substrate. The columns are cartridge format and have a unique quick-connect cartridge holder that greatly facilitates replacement. The back pressure observed with these columns ranged between 15 and 70 bar over the course of a run. The pressure cycles between the low end of the range for the elution side and high end for aqueous, load side. The columns are routinely used for ∼5000 injections with no loss of peak shape. Due to the small column dimensions and high flow rates used in this approach, it is expected that negligible resolution would be obtained with either porous or pellicular packing material. Of utmost importance is peak shape and reproducibility across very diverse molecular structures. It is presumed that solute masstransfer kinetics at the stationary-phase interface is the key process at work under these conditions and that pellicular packings, due to their superficially porous surface, may be an ideal match for this application.

System Setup and Integration. The autosampler and sixport injection valves are controlled through Gilson’s Unipoint software package. The injection ports and valve manifold were custom-made in-house to save space on the deck and add flexibility to the system. The autosampler has a capacity of 17 96-well plates in a custom-made deck. The samples for a given analyte are not in contiguous wells but are distributed across the 12-16 plates, in duplicate, depending on the specific assay. So a minimum deck capacity of 12-16 plates is required to process complete experimental sets. All of the samples for a given analyte are injected into a single data file. This minimizes the size of the Sciex sample control file, as an experiment with 96 analytes (16-20 samples/analyte) consists of 96 rows of identification information and 96 corresponding data files. The Unipoint software allows autosampler actions for the injection of individual or groups of samples to be customized by embedding different subroutines in the appropriate line of the injection sequence. In this application, two Unipoint control files are used. One waits for a ready out signal from the mass spectrometer prior to injection of a sample set. This ensures that no samples are injected until the MS is ready. The other control file simply identifies the sample location on the autosampler deck and injects. The runtime is adjusted so that the autosampler is waiting for the MS ready out signal prior to the Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

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Figure 5. Data review interface. Chromatograms are reviewed interactively by “clicking” in the hepatocyte review window. The spreadsheet raw data toggle along to correspond to the chromatogram in the current review screen. The reviewer can click the radio buttons in the hepatocyte review interface to fail the run for the indicated problem.

end of data acquisition. The autosampler sends a contact closure to the HPLC pump after each injection, and the pump controls the flow-directing and column switch valves. To minimize complexity and component idle time, the autosampler, HPLC pumps, and MS share system control. Since the HPLC pump controls chromatography timing, the autosampler is free to rinse the needles and retrieve the next sample. Unipoint could be used to control the entire scheduling process, as it can approximate a multithreaded application, but we found it to be unwieldy and difficult to troubleshoot when it was applied to the entire system. A simple but important feature of the 10-port column switch valve is its ability to operate in a “pulse logic” mode, such that the valve switches position upon receiving a single pulse. This simplifies control, since the columns on the 10-port valve are identical and it does not matter which is in load or elution mode at any given time. Data Handling and Review. In the iterative process of synthesis followed by screening, ADME data need to be provided in the time frame dictated by synthesis speed in order to build the optimal candidate molecule. A bottleneck in this information flow results when ADME data availability lags behind. Another challenge for the bioanalytical component of the process is data processing and review. Screening processes can produce enormous amounts of raw data needing to be reviewed, reduced, and passed on. To maintain a high degree of accountability in the process it is desirable to review each chromatogram and possibly make the relevant end point calculations (e.g., Papp value) or reduce the data to a concise format for further processing. This was the primary reason all samples for a given compound are injected into a single file. This approach reduces by up to 20-fold the number of data files produced and allows the results for a given compound to be located easily, because the data files are related to the original plate’s index file. In addition, the number of chromatograms to review is reduced to a manageable number. The review process has been automated using a custom software 1500


application, EvaLution, such that chromatographic and numerical data are reviewed simultaneously. EvaLution processes chromatograms and writes peak area or height data to a text file. Analyte peaks are located relative to the retention time of internal standard peaks. The system can also find analyte peaks by setting expected retention times prior to processing if no internal standard is present. During data review, the analyst toggles through chromatograms using mouse clicks in a custom graphical interface (Figure 5). At the same time, corresponding numerical peak area data are viewed alongside the chromatogram. If the chromatographic pattern looks peculiar, the chromatogram and data are reviewed more carefully. The number of expected samples is known, and the system signals if the chromatogram contains more or fewer samples than anticipated. Apparent permeability values (Papp) can be calculated “on-the-fly”, and metabolic stability data are flagged as pass/fail relative to analytical evaluation. For example, the analyst can fail a compound for “poor chromatography” if the peak data cannot be accurately integrated. Likewise a compound would fail, and be flagged as “inconsistent data”, during analytical review if a metabolic stability sample shows late time points having significantly more response than a peak at time ) 0 (time ) 0 being equivalent to 100% recovery). An entire set of chromatograms, 96/study plate, can be reviewed in 5-15 min due to the easily recognizable pattern of the chromatography. The reviewer becomes accustomed to the data pattern expected for each assay and can thereby easily spot inconsistencies. The most common analytical flag (see Figure 5) occurrence is “poor MS sensitivity”. This determination is made when the MS signal is nonexistent or too weak to accurately quantify. In our process, poor MS sensitivity is a somewhat generic description in that it is broadly applied to several problems that are difficult to adequately address in a high-throughput environment. While certain compound classes do not ionize well in electrospray mode, in many cases there are solubility issues or the analytical response is low due to extensive nonspecific binding. We have found many

examples in which a given analyte yields a strong ESI signal during MS conditions building,14 and good data are generated in most ADME screens, but no signal at all is seen in one specific screen. In these instances, the poor MS sensitivity call is made, although any number of reasons might apply. It is easiest just to resubmit the compound for reassay on a subsequent week. Further refinement of the data handling processes to address these issues will provide additional guidance to the ADME screen teams and customers. CONCLUSION Optimizing the delivery of samples to the mass spectrometer enhances throughput. The achievable throughput can be determined by considering the tradeoffs between the dwell time, the number of analytes measured, and the required precision. For measurement of a single analyte and ISTD using a quadrupole mass spectrometer, the absolute time frame for highly precise quantitation is approximately 1-2 s. Considering the measurement process in this light provides a frame of reference for the sample delivery system, including the chromatography step. That is, the focus can be placed on serial sample delivery within the MS time frame. In the system described, sample throughput is increased by delivering multiple samples to the injection manifold and processing these in rapid succession through the chromatography system. The factors limiting throughput are the chromatography/ cleanup step, owing to the need to desalt and reequilibrate the column(s), and the format of the samples organized in 96- or 384well plates. The formatting requirement is a tradeoff, as all samples for a given analyte are injected sequentially into a single file. This requires the sequence of injections to proceed from sample to sample, across several plates, before proceeding to the next compound. This approach is a compromise, inasmuch as single

file data collection greatly simplifies the overall process of setup and data handling, offsetting any time savings that might be gained by sampling from well to well, plate to plate, and thereby collecting data for each well into separate files. Collecting single files per compound, containing multiple injections per file, has the advantage of allowing facile data review of chromatographic files. Since review is now a manageable process, there is a high degree of quality control built in and bad data do not reach customers. At the same time problematic analytes or series are identified. Without some type of raw data review, a larger potential exists for errors to compromise the final end point calculations. The ADME screening group’s overall success rate is better than 85%. This means that data are returned, on average across all screens, for about 85-90 out of every 100 compounds submitted for screening. The failure rate for bioanalytical reasons is ∼8%. This figure overstates the number of true analytical problems as it includes failures due to poor electrospray ionization, solubility issues, compound submission errors, and nonspecific binding problems. The failures are broadly defined as such, due to the difficulty in adequately establishing their causes in a highthroughput environment. ACKNOWLEDGMENT The authors thank Don Smith, Carter Courtney (Pfizer, Scientific Instrumentation and Automation Group), El Luther (Pfizer), and Fred Mannarino (Gilson) for their contributions to this work.

Received for review November 10, 2000. Accepted January 30, 2001. AC0013251


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MS System for the Bioanalysis of Samples

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