用于從基于板的代謝篩選產(chǎn)生的樣品的生物分析的高容量LC/MS系統(tǒng)-PP5+1

Personal Pipettor是Apricot Designs研發(fā)生產(chǎn)的集多種移液功能于一身的緊湊型全自動(dòng)移液工作站，具有高效、便捷、精準(zhǔn)的特性，能同時(shí)兼容1/8/12/16/24/96/384通道的液體處理，開放式的設(shè)計(jì)可以便于整合疊板機(jī)和機(jī)械臂等。

以下為有關(guān)Personal Pipettor的Application，請(qǐng)參考：

A High-Capacity LC/MS System for the Bioan alysisof 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) 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 * Corresponding author: (e-mail) john_s_janiszewski@groton.pfizer.com; (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, 387-396.
(3) Lin, J. H.; Lu, A. Y. Pharmacol Rev. 1997, 49, 403-449.
(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.

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.

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).
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 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.
1496 Analytical Chemistry, Vol. 73, No. 7, April 1, 2001 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 diagramedin 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 10-port 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.