MS bioanalysis.

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Current advances and strategies towards fully automated sample preparation for regulated LC–MS/MS bioanalysis

Robotic liquid handlers (RLHs) have been widely used in automated sample preparation for liquid chromatography–tandem mass spectrometry (LC–MS/MS) bioanalysis. Automated sample preparation for regulated bioanalysis offers significantly higher assay efficiency, better data quality and potential bioanalytical cost-savings. For RLHs that are used for regulated bioanalysis, there are additional requirements, including 21 CFR Part 11 compliance, software validation, system qualification, calibration verification and proper maintenance. This article reviews recent advances in automated sample preparation for regulated bioanalysis in the last 5 years. Specifically, it covers the following aspects: regulated bioanalysis requirements, recent advances in automation hardware and software development, sample extraction workflow simplification, strategies towards fully automated sample extraction, and best practices in automated sample preparation for regulated bioanalysis.

Background Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has become the primary technique used for the quantitation of drugs and metabolites in various biological specimens in support of toxicokinetic (TK) and pharmcokinetic (PK) studies [1,2] . Sample preparation, including sample aliquoting, dilution and extraction, for LC–MS/MS bioanalysis is still the most time-consuming task in modern bioanalytical laboratories. In fact, the sample preparation procedures have a dramatic impact on assay throughput, assay performance and overall cost [3,4] . Automated sample extraction has demonstrated several advantages, including standardizing the sample extraction procedure, increasing processing efficiency and assay throughput, minimizing human exposure to biohazards, and improving the assay reproducibility and data consistency [5–8] . The extent in which robotic liquid handlers (RHLs) provide better data quality and the extent of bioanalytical cost savings usually depend on several factors, including the automated sample preparation strategies, application of specific RLH platforms and the cost of a

10.4155/BIO.14.161 © 2014 Future Science Ltd

Naiyu Zheng*,1, Hao Jiang1 & Jianing Zeng1 Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Route 206 & Province Line Road, Princeton, NJ 08540, USA *Author for correspondence: Tel.: +1 609 252 5494 Fax: +1 609 252 3845 [email protected]

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‘full time equivalent’ (FTE) in a particular laboratory [3,4] . Biological matrices, such as plasma or urine, contain significant amounts of endogenous compounds (e.g., proteins and phospholipids), which can interfere with analyte detection such as affecting the LC separation or causing ion suppression [1,2,9–13] . Traditionally, these unwanted matrix components are removed using extraction techniques such as protein precipitation (PPT), liquid–liquid extraction (LLE) and solidphase extraction (SPE) [1,2,9–11] . Without automation, these sample extraction methods can be very tedious and time-consuming for large batch sample analysis. The introduction of RLHs for automated sample-preparation with 96-well plate format has revolutionized bioanalysis from pharmaceutical discovery to development [14–17] . Today, more and more sample extraction methods for LC–MS/MS assays are performed in an automated or semiautomated manner using 96-well plates with at least one step being performed by a RLH. Several review papers were published on recent developments in automation of LC–MS/MS analysis for drug discovery and development

Bioanalysis (2014) 6(18), 2441–2459

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ISSN 1757-6180

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Review Zheng, Jiang & Zeng or clinical bioanalysis [9,18–24] . Most reviews have emphasized the application of high-throughput bioanalysis in drug discovery. The focus of this article is the automated sample preparation for small-molecule regulated bioanalysis in biofluids (such as plasma or urine) using LC–MS/MS. Recently, LC–MS/MS methodology has been increasingly used for the quantitation of proteins or peptides. This application requires additional sample treatment steps such as protein denaturation and trypsin digestion [25] . However, many sample clean-up steps for protein or peptide analysis by LC–MS/MS can be handled by a RLH in similar way to those for small molecules even though the procedures may be different. In general, most of the automation tools and methods used for biofluid analysis can also be applied to solid samples such as tissue samples or dried blood spots after the analytes are eluted from solid phases into the liquid phases with pretreatment using homogenization or sonication [26–28] . In this article, we review recent progress in hardware and software development, automated workflow improvement, automated system integration, and new strategies towards fully automated sample extraction for regulated LC–MS/MS bioanalysis. Key terms Robotic liquid handler: A robotic liquid handler (RLH) is used in automation of chemical or biochemical procedures. It is used to automatically dispense a selected quantity of reagent, samples or other liquid to a designated container. More complex RLHs can perform complex operations, such as on-deck sample transport, sample mixing, weighing, manipulation and incubation. Protein precipitation: Sample extraction technique by addition of miscible organic solvents to a sample resulting in the precipitation of proteins in the matrix. Liquid–liquid extraction: Sample extraction technique involving two immiscible solvents (usually water and an organic solvent) used to partition target analyte(s) from one liquid phase to another liquid phase based on differential solubility and partitioning equilibrium of analyte(s) between aqueous and organic phases. Solid-phase extraction: Separation process using packed solid sorbent and appropriate elution solvent(s). The target analyte(s) are retained onto the sorbent and the interferences are washed off. The analyte(s) are selectively eluted and separated from other compounds in the same mixture based on their physical and chemical properties. Incurred sample reanalysis: Reanalysis of selected study samples from a clinical or nonclinical study to determine the assay reproducibility by comparison of the concentration values between the repeat and initial results. 21 CFR Part 11: Known as Title 21 CFR Part 11 of the Code of Federal Regulations. Refers to the US FDA guidelines on electronic records and electronic signatures. Specifically, it defines the criteria under which electronic records and electronic signatures are considered to be trustworthy, reliable and equivalent to paper records.

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Bioanalysis (2014) 6(18)

General requirements for automated sample preparation in regulated bioanalysis In drug development, bioanalytical work is commonly conducted as part of evaluating the safety and/or efficacy of drug candidates in support of nonclinical and clinical studies. These types of studies are regulated by global health authorities. From a regulatory perspective, bioanalytical assays need to be validated to ensure the reliability, accuracy and integrity of the data [29–31] . In order to meet the stringent regulatory requirements for regulated bioanalysis, extensive time and resources are usually required in method development to ensure good assay performance during method validation and sample analysis, including incurred sample reanalysis (ISR) [32–34] . ISR is required to determine the assay reproducibility by comparison of the concentration values between the repeat and initial results. In general, representative incurred samples have to be selected across different dose groups, dosing periods and collection time. The majority of the sample extraction methods for regulated bioanalysis involve PPT, LLT or SPE in a 96-well format or online SPE extraction with different extents of automation [9,11,34,35] . Regardless of what type of automation and to what extent automation is used for regulated bioanalysis, the following are generally required before implementing any automated sample preparation procedures: • Assay validation requirement: a bioanalytical method that includes automation requires a full validation. If a manual method is being validated, those steps being automated may require additional validation; • System qualification, calibration and maintenance. All computer software used for RLHs needs to be validated when they are used for good laboratory practice (GLP) or clinical regulated bioanalysis [36,37] . The regulations and guidance for computerized systems come from the GLP Regulations for Nonclinical Laboratory Studies of the US FDA (21 CFR Part 58) [38] and the 21 CFR Part 11 requirement [39,40] . In addition to 21 CFR Part 11, there are additional requirements for all RLH systems, including installation qualification (IQ), operational qualification (OQ), calibration verification and proper maintenance. In addition to the hardware costs, all of these requirements will add further base costs for any assays performed by automation; • Sample’s chain-of-custody requirement: due to concerns of sample misidentification or contamination during shipping and storage, biofluid samples are usually collected and stored in capped tubes with

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Advances & strategies towards fully automated sample preparation for regulated LC–MS/MS bioanalysis

proper labeling for regulated bioanalysis [41,42] . 1D or 2D barcodes have been very useful to track the samples’ identifications during sample shipment, storage and analysis; • Sample quality and volume requirement: although automated liquid detection is available in most RLHs, extra sample volume collected in automation-friendly tubes (e.g., a conical-shaped or round-bottomed tube) is usually required in order to facilitate accurate liquid transfer by a RLH. For example, a minimum of 75 μl of sample is needed for accurate sample transfer of 50 μl of sample. Samples requiring enzyme stabilizer treatment may not be suitable for automated sample pipeting due to the occasional gelling of the plasma; • Data management requirement: regulated samples are usually managed by using a laboratory information management system (LIMS), such as Watson® LIMS. In early robotic systems, the sample list generated from the Watson database was not compatible with the automation software. In the following section, the importance of the conversion of Watson LIMS into RLHs executable files will be discussed. Recent progress in hardware development for automated sample preparation Most commercially available RLHs are sold as open platforms that allow individual customization based on application needs. They offer the users extensive ability to configure, scale and upgrade. As a result, integrated instrumentation for automated sample extraction in every step is usually custom-made by adding individual commercially available automation components. Based on liquid handling functions and flexibility, the RLHs can be categorized into two types. RLH platforms with fixed 96-channel pipetting arms

These platforms are usually equipped with a fixed 96-channel pipetting arm that is able to simultaneously transfer 96 samples from one 96-well plate to another plate. In general, these platforms cannot be used to transfer samples from tubes in a custom rack into a 96-well plate. They are very useful for rapid liquid transfer for sample extraction using the 96-well format regardless of the actual extraction method. These fixed 96-channel pipetting arm systems are usually very easy to set up and use without extensive training. These systems are also able to transfer liquids with high precision across the wells and are especially suitable for adding buffer solutions, transferring the extracted samples or adding the reconstitution solution. They can also be


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used for accurate transfer of liquids providing the volumes have been calibrated. There are many standalone RLHs with a 96-channel pipetting arm. Representatives of such RLHs include Tomtec Quadra™ 4 from Tomtec, Inc (CT, USA), PerkinElmer JANUS® Mini MDT™ from PerkinElmer (MA, USA) and Hamilton MICROLAB® NIMBUS 96 from Hamilton Company (NV, USA). As discussed below, recently dual arms with one eight-channel arm and one 96-channel arm have become available in one automated workstation and are very popular for more advanced applications. RLH platforms with independent 8- or 16-channel pipetting arms

These platforms are essential to transfer samples from individual tubes in a custom rack to the same or a different format such as a 96-well plate. There are choices of washable or disposable tips for accurate handling of aqueous, organic or biological solutions and suspensions. Disposable tips are commonly used for most of the applications because they can achieve sample transfer with excellent precision and accuracy and obviate carryover concerns. The washable, fixed tips made of stainless steel or ceramic material are designed for pipetting liquid without the need to change the tips. A washing step is needed between each pipetting. The washable fixed tips are suitable for liquid transfer from tubes with piercieable caps without decapping and recapping [43,44] . The advantages of using fixed tips include cost-saving in disposable tips and space-saving on the automated deck since the disposable tip boxes can take up considerable space. The disadvantage of fixed tip system includes potential carryover concerns and potential dilution effects from washing solvent that could impact the accuracy and precision when preparing standard curve and QC samples [45–47] . In general, the carryover when using fixed-tip RLHs is analyte-dependent, which needs to be evaluated by modifications of washing solvents, washing volume and washing times in each pipeting cycle. In most cases the carryover can be overcome by tip washing with acetonitrile/water or methanol/water solvent that contains formic acid [44] . Both platforms with washable or disposable tips can be used for standard curve and QC preparation and sample dilution, but washable tips require additional sample conditioning steps [45–47] . If the RLHs with an eight-channel pipetting arm are used to transfer 96 samples to a 96-well plate, they will take a longer time than using a RLH with a 96-channel arm because the eight-channel pipetting arm needs to aspirate and dispense 12 times to complete the cycle. Representative robotic platforms with independent 8- or 16-channel pipetting arm include TECAN Freedom EVO® from Tecan Group Ltd

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Review Zheng, Jiang & Zeng (Männedorf, Switzerland), Hamilton MICROLAB STAR Line (STARlet, STAR and STAR Plus) from Hamilton Company and PerkinElmer JANUS Varispan Automated Workstation from PerkinElmer. All of these platforms offer the option to add a 96-channel pipetting arm, and labware grippers. In general, these RLHs allow users to add a wide array of other devices, and thereby perform complex methods. Recent hardware development in RLHs

The following highlights recent advances in the capability of RLHs: • Some platforms, such as Hamilton MICROLAB Star Plus, can have up to 16 independent pipetting channels with optional multi-channels (96, 384 or Nano); however, the 16 independent channels limit the reach to some positions on the deck. Not all positions on the deck are available to all channels. In contrast to a 16-channel arm, an eight-channel arm can reach all positions on the deck. Each liquid channel offers optimized pipetting parameters for different types of liquid and volumes; • Multiple arms are able to work independently in parallel to enhance throughput; • Barcode identification capability to ensure the integrity of the samples; • Fast wash capability to minimize carryover of fixed tips and maintain high throughput; • Total aspiration and dispense monitoring; • Dual liquid level detection; • Plug-and-play accessories available; • Upgradable system design for later addition with any arms and third party labware – making it possible to expand to a completely walk-away system. Recent advances in RLH techniques have significantly improved the accuracy and precision of liquid handling, as indicated by the increasing number of reports using RLHs for the preparation of standard curve, quality controls (QCs) or automated sample dilution [48–51] . Plasma pipetting using a TECAN Freedom EVO has been demonstrated to be very accurate by pipetting 25 μl of plasma with six replicates from sample tubes containing 50, 75, 100 or 200 μl of rat plasma [52] . The % deviation (%Dev) obtained from six replicates of pipetting 25 μl was within ±1.9% of the nominal value (defined as 25.4 mg by weighing of 25 μl of rat plasma) and %CV was ≤1.9% regardless of the sample volume in the tubes; that is, even 50 μl of plasma in the tubes was sufficient for pipetting 25 μl volumes [52] . Most of the built-in softwares are able to control a variety of on-deck devices, including

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those from third parties, such as a plate sealer, vortexer, centrifuge, seal piercer [49] and 2D barcode reader [51] . Of particular interest, the on-deck centrifuge option using a RLH has become commercially available. Two representative robotic centrifuges include a robotic temperature-controlled centrifuge (Hectic Rotanta® 46 RSC, TECAN Group Ltd) and a robotic microplate centrifuge (Agilent, CA, USA). These centrifuges eliminate the need for manual intervention during a variety of sample preparation steps, including LLE. Recently, automation of the evaporation step, the most challenging step in automation, was demonstrated using the new Ultravap™ RC microplate evaporator from Porvair Sciences (Leatherhead, UK). The latest fully robot-compatible version of Ultravap nitrogen blow-down evaporator can be integrated into several leading laboratory liquid handling robots. In general, on-deck centrifugation and on-deck evaporators are less commonly used for automated sample preparation. Integration of these on-deck devices into a RLH will increase on-deck time to finish sample preparation, and can reduce the time the instrument is available for other users. In addition, the robustness of these steps may need to be carefully evaluated before using for regulated sample analysis. For example, centrifugation normally requires balancing the weight of samples in the centrifuge before starting. On-deck evaporators may need to be evaluated for potential inter-well contamination of analytes. Progress in software development for automated sample preparation Due to the diversity and complexity of sample extraction procedures, application software scripting or custom-developed software programming is necessary. Significant progress has been made in recent years in software development for automation by the vendors as well as in custom-developed software programming to create specific method procedures for automation. Traditionally, the RLHs are designed for applications that are not specific to any type of sample extractions or molecule. Before the systems can be used to automate a designated sample preparation procedure, hardware configuration and application software scripting are usually required. For example, bioanalysts may need to develop a detailed and executable script for the preparation of analytical standards (STDs), QCs and sample dilution. For this application, the bioanalyst needs to provide the application software with a lot of detailed information, including the number and concentration levels of analytical STD and QC samples, liquid class and volume of each liquid transfer, the order each liquid is added, sample dilution factors, sample location and final sample sequence to



be injected to LC–MS/MS. The script editing work is assay- or sample batch-specific, and can take several hours or even days. In particular, the script for one analytical run may consist of several hundred lines, so it is not practical to edit the script for every analytical run. Recently, several vendors have recognized the importance of their instrument control software and its impact on the system usability and end-user satisfaction. Several intuitive, graphical user interface (GUI) applications have been developed to simplify the programming environment for basic method creation, making their RLHs accessible to everybody. Examples include the JANUS Application Assistant™ as an integrated part of the WinPREP 4.8® software from PerkinElmer, TouchTools™ software as part of the exiting EVOware™ software from TECAN, and the INSTINCT™ software from Hamilton. These user interfaces have made it easier to execute sample extraction workflows, simplify the interaction and boost productivity. For more sophisticated applications using an integrated RLH system, software application scripting or custom-developed software programming is still required for regulated bioanalysis. For instance, most of the commercially available RLHs do not provide a software interface to link the application software to the laboratory information management system (LIMS). Several bioanalytical laboratories have successfully developed software that converts the sample run list from the Watson™ LIMS system into a TECAN program [47,52] or a Hamilton program [51] for bioanalytical sample preparation, which includes sample transfer, dilution, extraction and reconstitution. As shown in Figure 1, by using TECAN Freedom RLH, our laboratory has successfully developed and validated a Microsoft Excel–based robotic sample preparation program (RSPP) software that automatically transforms Watson worklist information (identification, sequence and dilution factor) to comma-separated value (CSV) files [52] . Based on the CSV file, TECAN EVOware™ software automatically generates a TECAN executable work list (.gwl files), allowing the robot to perform sample dilutions at variable dilution factors [52] . Thus, the extracted sample sequence obtained from TECAN is identical to the intended LC–MS/MS injection sequence obtained from Watson. The RSPP program has been tested for the analysis of asunaprevir (BMS-650032), an HCV NS3 protease inhibitor, in dog plasma [52] . The mean %Dev of the concentrations from corresponding nominal concentrations was demonstrated to be within ±7.7% at different dilution factors and concentration levels [52] . The assay reproducibility using RSPP was demonstrated by incurred sample reanalysis because the %Dev of the repeat value


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Key term Graphical user interface: Software program’s graphical representation of user commands that incorporate icons, pictures, menus and other visual indicators instead of text entry input. It allows users to interact with a robotic liquid handler through graphical icons or visual indicators, instead of using text-based commands.

from the mean of the initial value and the repeat value was within ±7.6% for all 20 samples selected from different animals and collection times [52] . Acceptance criteria for the assay reproducibility testing using the incurred samples was based on the difference of a repeat result from the mean of the two values (initial and repeat) being within 10.0% of the mean for at least two-thirds of the samples tested. Similarly, the preparation of standard curve and QC preparation can be done using a Hamilton MICROLAB robotic system based on application software scripting to reflect the serial dilution scheme [48,50–51] . Several laboratories have customized the Hamilton MICROLAB STAR RLH platform using their custom-developed GUIs in combination with the instrument control software and application software for each type of automated sample preparation, including PPT [50] , supported liquid extraction (SLE) [51] and SPE [53] . A novel approach to combine multiple types of extraction techniques, including PPT, SPE and LLE, into one integrated and automated Watson worklist (.xls) LC–MS/MS data

Export

Watson LIMS™

RSPP macro Excel file (.csv) TECAN software TECAN file (.gwl)

LC–MS/MS analysis Automated sample dilution and preparation

Figure 1. Automated sample dilution and preparation. RSPP: an Excel macro program transforms the Watson Worklists (.xls) to TECAN readable files (.csv). Tecan EVOware™ software transfers the information of sample sequences and dilution factors in these CSV files to TECAN executable files (.gwl), which enables automated sample dilution. The dashed line represents the automated workflow using the RSPP program. LIMS: Laboratory information management system; RSPP: Robotic sample preparation program.


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Review Zheng, Jiang & Zeng

Key term Supported liquid extraction: Separation process using a plate packed with modified diatomaceous earth. When biological samples are applied to the plate, the aqueous portion is absorbed by the hydrophilic surface. The organic extraction solvent is then used as the elution solvent to elute analytes. SLE is used as an alternative format of liquid–liquid extraction.

sample-preparation suite was also reported using a Hamilton MICROLAB STAR robotic liquid handler [48] . In these methods, a custom GUI was developed to centralize the user’s input, apply built-in business logic rules, calculate a liquid-transfer scheme and generate an output file for a Hamilton MICROLAB STAR RLH platform [48,50,51,53,54] . The Hamilton software executes the scripts to perform the actual liquid-transfer steps on the deck based on the liquid-transfer scheme it received. Similarly, by using the custom GUI program, the real-time 2D barcode processing has be successfully incorporated into a Hamilton RLH for categorizing and organizing study samples according to Watson LIMS sequence [51] . Briefly, the process starts with the generation of a Watson run list, followed by converting it to a Hamilton assay template and saving as a Microsoft Excel file (*.xls). As soon as the unknown samples with 2D barcodes (containing Custom ID and positional information) are scanned offline into the RLH, a 2D barcode text file (*.txt) is generated. Both Watson run list and barcode text file are entered via the GUI, leading to the generation of Hamilton run files (*.mdb) [51] . Similarly, real-time 2D barcode processing can be integrated to TECAN Freedom EVO or other RLHs by scanning of samples and generating a rack barcode and Minitube 2D code file using a standalone Ziath 2D code scanner. Typical samples tubes and racks that can be handled by RLHs include Thermo Scientific’s Matrix (NH, USA), Micronic’s (PA, USA), FluidX’s (Cheshire, UK) or REMP® (Brooks Automation, Inc., MA, USA) 2D barcoded sample tube racks with a 1D barcode on the side. Recent progresses in automated sample extraction & strategies towards fully automated sample extraction Presample extraction steps

Many presample extraction steps can be easily accomplished using a RLH. The most automation-friendly steps include aliquoting of standards, QCs and study samples to a 96-well plate, and the addition of solutions including internal standard, buffers and extraction solvents. As shown in Figure 2, several presample extraction steps are still performed manually in many regulated bioanalytical laboratories. These manual intervention steps include STD preparation from ana-

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lyte stock solutions, sample dilution, sample sorting/ rearrangement and uncapping/recapping of the sample tubes. Current strategies towards fully automated sample extraction include the replacement of these manual steps with automated procedures or a simplification of the workflow to facilitate the automation. For example, bubbles or clots in the samples may cause inaccuracy during pipetting. With brief centrifugation using an on-deck centrifugation, the issues related to bubbles or clots in the samples can be significantly reduced. As shown in Figure 2, other manual steps can be automated using more sophisticated labware or software scripting or programming: • STD preparation and sample dilution can be performed by using application software scripting or custom software development [47,48,50–52] ; in particular, software scripting or development has enabled conversion of Watson sample lists to the executable worklists with necessary sample identification and dilution factors, allowing the robot to perform sample dilutions at variable dilution factors [48,50–52,54] ; • Sample sorting/rearrangement could be automated by incorporating 2D barcode processing into an automation workflow, which has significantly improved the extraction workflow efficiency and increased quality by eliminating accidental human errors [48,51] ; • Decappers/recappers have become commercially available. Representative systems include on-deck Hamilton LabElite DeCapper™ from Hamilton Company, and screw cap decapping/recapping units from Fluidx or Thermo Scientific. For manual decapping/recapping of 96 samples, it can take 30–45 min depending on the skills of the bioanalysts. By using a decapper machine, a complete rack of 48 or 96 tubes can be decapped within 1 min. In order to use a decapper/recapper, all samples need to be collected in sample tubes that are compatible with these decappers. Another strategy to automate the manual steps is to simplify the sample extraction workflow before integrating more hardware or software that is needed. A simplified automated workflow was developed in our laboratory to overcome these challenges that used piercing caps for direct sampling and evaporation-free SPE (Figure 3) [44] . By using pierceable cap sample tubes, a RLH was able to aliquot the samples without uncapping or recapping [43,44] . Considerable time was saved by eliminating uncapping and recapping steps. Since the samples were pipetted by a TECAN RLH from tubes with closed caps, it also prevented



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Thawed study samples Strategies towards fully automated sample preparation Centrifugation to avoid clots (optional)

Labeling of the second tubes for samples (optional)

STD preparation

Application software scripting

Sample dilution

Custom software development

Sorting/sample rearrangement

2D barcode processing

Vortex mixing

On-deck vortex-mixing

Uncapping/recapping

Using pierceable caps or decapper/recapper

Aliquoting all samples to 96-well plate

Adding IS/buffer/extraction solvent

On-deck vortex-mixing

Extraction (PPT/LLE/Offline SPE/Online SPE)

Manual steps

Automated steps

Figure 2. Current presample extraction workflow and strategies towards fully automated sample preparation. IS: Internal standard; LLE: Liquid–liquid extraction; PPT: Protein precipitation; SPE: Solid-phase extraction; STD: Analytical standard.



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A

B Blank/STD samples

Manual intervention

Study/QC samples in capped tubes

Uncapping and recapping

Blank/STD/QC/study samples in tubes with pierceable caps

1

Aliquot using an eight-channel RLH

Aliquot using an eight-channel RLH

Samples in 96-well plate

Samples in 96-well plate Add IS/buffer solvent

Add IS/buffer solvent Conditioning

96-well SPE plate

96-well SPE plate

Washing the plate

Conditioning Washing the plate

Elute the plate using pure or high organic solvent

Elute the plate using 3 lower organic solvent Eluent in 96-well plate

Eluent in 96-well plate Manual intervention Evaporation

2

Dilute with aqueous solution

Reconstitution using mobile phase solvents Eluent in 96-well plate

4

Eluent in 96-well plate

Figure 3. Illustration of two solid-phase extraction sample extraction processes for bioanalysis using LC–MS/MS. (A) Conventional SPE sample extraction with a RLH, in which steps 1 and 2 require manual interventions; (B) direct biofluid transfer with evaporation-free SPE sample extraction, in which no manual interventions are required for steps 1 and 2. The evaporation step was eliminated by modification of elution solvent followed by dilution (steps 3 and 4). IS: Internal standard; QC: Quality control; RLH: Robotic liquid handler; SPE: Solid-phase extraction; STD: Analytical standard. Reproduced with permission from [44] © 2013 Elsevier BV (2013).

the bioanalysts’ exposure to potentially biohazardous fluids. In this particular application, washable fixed tips with independent eight-channel pipetting arm on a TECAN platform were used. As mentioned earlier, there are potential carryover concerns and potential dilution effects from washing solvent that need to be addressed when using fixed tips in a RLH for STD and QC preparation [45–47] .

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Offline sample extraction

In regulated LC–MS/MS bioanalysis, the development of a robust assay is very desirable. In general, a robust assay is highly reproducible and will provide high-quality data and fewer failed analytical runs. The method development usually starts from systematic method screening and optimization [1,2] . As mentioned earlier, several laboratories have utilized Hamilton



MICROLAB STAR RLH platform with custom software scripting for automated method development for different extraction methods, including PPT [50] , SLE [51] , SPE [53] and integrated multiple assay extraction platform [48] . The following sections focus on recent advances and strategies towards fully automated sample extraction using offline PPT, LLE and SPE. Automated PPT

PPT is the most convenient sample preparation technique among three offline sample extraction methods [9,11] , and is still used in regulated bioanalysis. Most reported PPT methods have been semi-automated assays, and RLH was used to add samples, IS and extraction solvent in a precipitation plate. Then, the plate was manually placed on a vortexer for vortexmixing, followed by centrifugation. The preparation plate was returned to the deck and the supernatant was transferred by a liquid-handling workstation to a clean 96-deep-well plate. The samples were usually required to be evaporated and reconstituted before injection into an LC–MS/MS system. Today, PPT is still used by many bioanalysts due to its simplicity and consistent recovery for most of the analytes with minimal method development time. As shown in Figure 4, for automated sample extraction using PPT, several steps including sealing the plate, extraction, centrifugation and evaporation usually require manual intervention. A strategy towards fully automated sample extraction was reported to incorporate all necessary accessories, including ondeck plate sealer, plate shaker, centrifuge and robotic arms for plate handling on a TECAN [49] . However, the majority of the automated PPT methods in a 96-well format still require off-deck centrifugation before transferring the supernatant into another plate (Figure 4) . The most popular strategy to automate PPT methods is to simplify the PPT workflow. For example, the centrifugation step can be eliminated by using PPT filtration plates. The samples are filtered through single-use filtration plates via vacuum instead of centrifugation. The proteins and unwanted components are retained on the filtration membrane, while clean filtrate is collected [55,56] . The evaporation step can be performed using an Ultravap RC microplate evaporator or simply eliminated by using aqueous diluents to reduce the percentage of organic solvent in extracted samples. PPT methods are generally considered ‘dirty’ methods, even using the PPT filtration plate, since it cannot efficiently remove the matrix effect-causing phospholipids [2] . To maintain the simplicity of automated sample analysis using a PPT method while improving the effectiveness in removing the phospholipids,


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HybridSPE™ plates (from Sigma–Aldrich/Supelco, PA, USA) packed with a proprietary zirconia-coated silica have been introduced. These 96-well plates are able to dramatically remove the residual phospholipids in biological samples, leading to a significant reduction in matrix effects [57–61] . The HybridSPE precipitation plate can be used without prior conditioning, and all procedures can be automated and performed on a Hamilton Star Plus RLH [56,57] . There are other commercially available protein precipitation plates, such as Waters Ostro® (Waters Corporation, MA, USA) and Phree® (Phenomenex, Cheshire, UK), which are capable of removing phospholipids, similar to the HybridSPE™ plate. Automated LLE & SLE

LLE methods provide cleaner extracts than PPT because they more effectively remove phospholipids [2,62–64] . LLE is also better than SPE in terms of assay reproducibility since SPE-based extraction in a 96-well format has been occasionally observed to have SPE plate lot-to-lot variation, which can result in potential run-to-run variability in assay performance [2] . Today, LLE is one of the most popular sample extraction methods for regulated bioanalysis in the pharmaceutical industry due to its clean sample extract, excellent assay reproducibility and potential in cost saving. However, without automation, LLE method can be very tedious and time-consuming. Most reported LLEbased assays are performed using a semi-automated procedure on a RLH with one 96-channel pipetting arm, such as JANUS Mini or Tomtec RLH [62,63] . Specifically, after the addition of the biological samples into each well of a 96-well plate or 1 ml microtubes in a microrack (96-well format), an IS working solution and a buffer solution were added using a JANUS Mini liquid handler. After manually vortex-mixing, a water immiscible solvent (extraction solvent), was added to each well using the RLH. After that, the samples were manually capped and shaken on a reciprocating shaker for 10–20 min, followed by being manually placed in a centrifuge for a few minutes. After uncapping, the samples were placed on the deck of the RLH, and the supernatant (upper layer) was transferred into a 96-well collection plate using the RLH. To maximize the organic layer being transferred and minimize the accidental aspiration of the unwanted aqueous layers, manual measurements of the plate height are usually required during method development and the organic phase volume transfer is set at a fixed height for the aspirate steps. The collected sample plate is manually transferred to an evaporator and dried under heated nitrogen stream. The reconstitution solvent is added to the sample plates using a RLH and the plate is


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Strategies towards fully automated sample preparation

Samples with IS/buffer/solvent

Sealing plate

On-deck sealer

Extraction (vortexing/shaking)

On-deck mixing/ vortexer

Centrifugation

On-deck centrifuge or PPT filtration/SLE

Transferring supernatant to another plate

Skipping, on-deck evaporator or SLE with HILIC

Evaporation

Reconstitution

On-deck sealer/ vortexer

Sealing/vortexing

Centrifugation (optional)


Manual steps

Automated steps

Figure 4. Current sample preparation workflows for protein precipitation and liquid–liquid extraction and strategies towards fully automated sample extraction. HILIC: Hydrophilic interaction chromatography; IS: Internal standard; PPT: Protein precipitation; SLE: Supported liquid extraction.

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vortexed. The detailed steps for a semi-automated LLE method are shown in Figure 4. Several reports indicated that semi-automated LLE method provided robust assay performance and have been used for studies that required good assay specificity and sensitivity [62–64] . In a recent example, a semi-automated LLE method was successfully used in an important assay to evaluate oral absolute bioavailability of daclatasvir, an HCV NS5A inhibitor, with an intravenous microdose of a stable isotopically labeled drug [65] . As shown in Figure 4, LLE methods are usually performed manually or in a semi-automated fashion due to the need for centrifugation and evaporation steps. The strategy to fully automate LLE sample extraction includes the incorporation of more labwares to facilitate on-deck sample sealing, on-deck sample mixing similar to PPT [49] . The offline phase mixing was reported to be eliminated by performing in-tip mixing of samples on the deck of an RLH [66] . On-deck centrifugation and evaporation have not been reported for regulated bioanalysis. This could be applied in the future since the robotic version of the centrifuge and evaporator is now commercially available. An alternative strategy for fully automated LLE is the application of supported liquid extraction (SLE), which replaces the centrifugation steps with vacuum filtration and was found to provide clean extracts with high analyte recovery [51,67–70] . The SLE plate is usually packed with a modified form of diatomaceous earth. When biological samples are applied to the SLE plates, the aqueous portion is absorbed by the hydrophilic surface. The organic extraction solvent is then used as the elution solvent to elute analytes. As shown in Figure 4, SLE is particularly suitable for the 96-well format operation with fully automated potential since the centrifugation step is optional. By using HILIC chromatography separation, the SLE extract can be directly injected into the HILIC–MS/MS system without the need for solvent evaporation and reconstitution [67] . Automated SPE

As shown in Figure 5, SPE in a 96-well format is more automation-friendly than other methods and has been widely used in drug discovery and development [1–2,9,11,21] . Automated sample extraction using SPE is very popular in regulated bioanalysis due to its ability to achieve a satisfactory extraction recovery for analytes with a variety of chemotypes by using different SPE sorbent chemistries [2,9,11] . In some cases, a SPE-based extraction method has been the unique solution to achieve high extraction recoveries for multiple analytes within an assay with minimal matrix effect. In a recent example, an LC−MS/MS method, using automated SPE extraction strategy, followed by automated LLE,


Review

was developed to quantify five HIV-1 integrase inhibitors at a LLOQ of 1–2 pg/ml in plasma in support of a microdose clinical trial study [71] . Despite the advantages of automated SPE method mentioned above, SPE method development is usually very time-consuming because it requires substantial efforts in selecting appropriate SPE sorbents, pH range and washing and elution conditions. There are a variety of SPE sorbents to choose from, such as C2, C8, C18, phenyl, polar endcapped and polar-embedded stationary phases, just to cite those available for reversedphase extraction. To eliminate the need to dramatically change the automated sample extraction protocols for different analytes, several very generic SPE plates including Strata-X® (Phenomenex), EVOLUTE® ABN (Biotage, NC, USA) and Oasis® HLB (Waters) have been developed. These SPE plates can be used for simultaneous quantification of multiple analytes with satisfactory recoveries without the need to significantly modify the automated sample extraction procedure [44] . Recently, an automated SPE method development strategy was implemented using a Hamilton MicroLab STAR and was performed by different bioanalytical laboratories, which significantly reduced the time and errors during SPE method development [48,53] . As shown in Figures 3 & 5, SPE methods usually require manual intervention for sample evaporation and reconstitution after elution. Current strategy towards fully automated SPE was to simplify the SPE workflow by using an evaporation-free SPE method [44] . Traditionally, the purpose of sample evaporation is to concentrate the samples to achieve a LLOQ, or to enable the injection sample to be compatible with the HPLC mobile phase. Due to the availability of highly sensitive LC–MS/MS instrumentation, assay sensitivity is no longer the biggest challenge in LC–MS/MS bioanalysis. In addition, the analyte recovery variation can be compensated for by the use of the stable isotopelabeled IS. As a result, the evaporation step for SPE can be removed if a mobile phase-compatible elution solvent is used to dilute the extracts prior to LC–MS/MS analysis (Figure 3) [44] . This strategy has led to the successful development and validation of three GLP assays for three CNS drug candidates and metabolites [44] : BMS-763534 and its metabolite, BMS-790318, in dog plasma; BMS-694153 in monkey plasma; and, pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. Recently, μElution SPE plates, such as Waters Oasis® μElution plate, have become very popular because the analyte(s) in the plate can be eluted out using as little as 25 μl of solvent, allowing target analyte(s) to be preconcentrated without the need for an evaporation step. In general, evaporation may be required to con-


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Samples with IS/buffer/solvent Manual steps Automated steps

Conditioning the SPE plate

Loading samples to SPE plate

Applying vacuum

Washing samples

Applying vacuum

Setting up sample collection plate Strategies towards fully automated sample preparation Eluting samples

Applying vacuum

Evaporation

Evaporation-free SPE or on-deck evaporator

Reconstitution

Sealing/vortexing

On-deck sealer


Figure 5. Current sample preparation workflow for solid-phase extraction and strategies towards fully automated sample extraction. IS: Internal standard; SPE: Solid-phase extraction.

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centrate analytes to improve the analyte signal-to-noise ratio (S/N) or sensitivity detected by LC–MS/MS. However, during evaporation, some evaporative analytes or adsorptive analytes can be lost; for example, analytes with strong nonspecific binding bind to the sample collection tube (96-well plate) during evaporation to dryness, and may not completely dissolve in the reconstitution solvent since the reconstitution solvent usually contains a much lower percentage of organic solvent. We do not have data that directly support the concerns of nonspecific binding, however, data published by Tan et al. indicated that the S/N for the analysis of sumatriptan using the evaporation-free SPE method was even higher than that of the evaporationbased method despite the dilution factor of 5 in the evaporation-free method [72] . In this case, a better signal achieved in an evaporation-free SPE method than that of an evaporation-based method could be due to many factors, including a higher extraction recovery with less analyte loss without evaporation, a cleaner extract with less matrix effect, and less interference from co-extracted matrix components [73] . Automated online sample extraction method Automated online sample preparation methods have also been used for regulated bioanalysis, although it may be more commonly used in discovery bioanalysis [23,35,74–76] . In general, automated online sample preparation platform consists of extraction and chromatographic separation in one operation [74–76] . A simple and custom-made automated online sample extraction can be accomplished by using one six- or ten-port switching valve for column-switching connected to an LC pumping system [76] . It requires two columns, including one primary column (or SPE cartridge) used as an extraction column, and one secondary column used as an analytical column. First, the biofluid sample is injected into the primary column and the fractions containing analytes are trapped inside the column. The interfering components from the sample matrix are eluted from the primary column to waste. After column switching, the analyte fractions of interest are eluted to the analytical column for mass spectrometric detection. There are two basic column switching modes including straight-flush column switching and back-flush column switching [76] . Back-flushing techniques are more efficient than other modes because they minimize peak broadening of analytes while maximizing the washing of strongly retained matrix components. The advantages of online SPE include the ability to readily automate the analyses without substantial capital investment of expensive RLH systems. In addition, substantial time can be saved dur-


Review

ing sample extraction because an online SPE system serves as a highly integrated ‘walkaway’ system capable of handling all SPE loading, washing and elution steps without any manual interventions. The most convenient strategy using online SPE as related to fully automated sample extraction is the application of integrated online sample preparation platforms, such as Spark Holland’s Symbiosis™ online SPE system (Spark Holland, The Netherlands) and Thermo Scientific’s LC–MS OnLine Sample Prep and Multiplexing System using TurboFlow™ technology. Integrated online systems offer several advantages, including simplification of sample analysis to reduce potential analyte loss due to nonspecific binding as compared with offline SPE methods. In particular, online methodology is preferable for volatile or unstable samples during sample processing, which has led to the successful determination of 18 perfluorinated organic acids and amides in human serum using online SPE [77] . However, online sample extraction requires more time in method development and poses potential carryover concerns. Recently, a number of review articles have been published describing the application of various methodologies for the direct analysis of biofluid samples [35,76–80] . Best practices in automated sample extraction for regulated bioanalysis GLP compliance considerations

In general, RLHs offer software products with 21 CFR Part 11-compliant E-signatures, audit trails, configurable user roles, secure data storage, and data archival capability. However, RLH systems normally require software validation, system qualification and calibration verification, as well as proper maintenance and documentation, in order to be compliant with GLP regulations [38] . Depending on the protocol, a typical software validation may take two months or longer. For routine applications, a standard operation procedure (SOP) is usually required to govern the operation, maintenance and calibration of the RLH used. The SOP may include several sections such as installation qualification (IQ) and operational qualification (OQ), operation and data management, administration and maintenance, calibration verification, documentation and archiving. The procedure in each section will need to be developed based on the requirements established by regulated agencies. For example, the calibration verification procedure and the acceptance criteria for the RLH are usually specified in the SOP. Typical calibration verification procedures for a RLH used in regulated bioanalysis can be found in recent publications [24,44,52] . In our laboratory, the calibration of each RLH system is verified with at least two transfer volumes that are appro-


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Review Zheng, Jiang & Zeng priate for the scale range of the system and the tip type [44,52] . The volumes used for the calibration verification should bracket the actual volumes used in production as closely as possible. Accuracy and precision is measured gravimetrically using liquid transfers to a container on a balance or measured colorimetrically using transfers of colored solutions for spectrophotometric measurements using an Artel system [24,44,52] . For RLHs with an eighttip format, a minimum of six replicate liquid transfers for each tip is required for the calibration verification. If the percent deviation of the average (n = 6; per tip) measured weight or absorbance was more than ±5% of the theoretical weight or absorbance, the RLH would be considered inaccurate. As a result, the system would require repair service and re-calibration before being used in a regulated bioanalytical assay. Scope of automation in regulated bioanalysis

The scope of automation used in regulated bioanalysis depends on specific assay needs, availability of the RLHs and the bioanalysts’ comfort with using automation. As described before, the extent of automation within an automated sample extraction method used for regulated bioanalysis can vary from a few steps to a fully automated process. For example, liquid transfer from extraction plate to injection plate in 96-well format can be easily accomplished by using a Tomtec Quadra™ or PerkinElmer JANUS Mini™ robotic liquid handler. For other steps, manual intervention is usually required because automating these steps requires additional hardware or accessories that may not be commercially available, are not compatible with the existing automation platform, or are not cost effective. In addition, any new hardware addition may also require additional time and capital investment in system qualification, software validation and substantial training [36] . Therefore, semi-automated sample extraction methods may still best fit the needs of many regulated bioanalytical laboratories. In addition, the RLHs or automated sample preparation workstations are usually shared by several bioanalysts in open access. Due to the complexity of the hardware or software in the integrated RLHs, it is not always feasible to have many duplicate systems in a bioanalytical laboratory. If one bioanalyst runs all steps on one workstation, other bioanalysts will need to wait their turn. Alternatively, a method divided into several automated procedures with fewer steps will give more flexibility for a group of people to share the platform. Some steps, such as centrifugation and evaporation, are usually done off the deck of a RLH, allowing others access. Lastly, some critical liquid transfer steps are best performed manually because the RLHs may not be able to achieve the accuracy that is needed. For

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example, it is still challenging for a RLH to deal with low sample volume or poor sample quality due to partially gelled plasma, or samples with bubbles or clots. In such cases, if the built-in configuration functions in a RLH including proper liquid class, liquid level detection and bubble detection functions cannot resolve the issues, manual sample transfer is required to visually inspect the sample volume and quality. When using a RLH with fixed tips for preparation of standard and QC samples from stock solution, there were potential risks of inaccurate results due to dilution effects caused by the washing solvent [45–47] . Therefore, individual standard or QC samples are usually prepared manually before they are aliquoted for the assay when using a RLH with fixed tips. Considering the significant capital investment and the complexity of integrating multiple components into one system, full automation is usually adopted only if it adds significant value in terms of efficiency, safety and quality improvement. The bioanalyst will judge whether the benefits gained from full automation will outweigh the disadvantages and the time spent on automating the procedure. Nevertheless, with the recent development in hardware and software related to RLHs, it is clear that all the sample preparation steps can be automated. It is expected that more and more fully automated sample extraction methods will not only become possible, but also add significant values as compared with semi-automated methods in regulated bioanalysis. Sample extraction strategy considerations

As discussed earlier, for regulated LC–MS/MS bioanalysis, any of several automated sample extraction techniques including PPT, LLE, SPE or online SPE can be chosen. In general, method development starts from a systematic method screening (or comprehensive) strategy that includes PPT, LLE and SPE methods, followed by sample extraction optimization to maximize the analyte recovery while minimizing the matrix effectcausing components, such as phospholipids [1,2,10] , dosing vehicles [81] and enzyme inhibitors [82,83] . It was previously reported that systematic method development that included PPT, LLE and SPE could be done automatically using a Hamilton MICROLAB STAR RLH platform with custom software scripting [48] . The extraction method selected could have a dramatic impact on the applicability and scope of automation, as well as the assay robustness [1,2,34,62,63,84] . For bioanalysts who are not experts in application software scripting, an automated method development system can be intimidating. Therefore, we developed a simple generic LLE methodology for rapid assay development for the analysis of BMS-927711, an



antimigraine drug candidate, in plasma using a RLH [63] . This LLE strategy was based on previously published results that LLE using N-butyl chloride, MTBE and solvent combination of hexane/ethyl acetate or hexane/2-methyl-1-butanol as the extraction solvents could achieve maximal phospholipid removal and decent extraction recoveries for most of the smallmolecule drugs and metabolites [2,62–64] . Therefore, it is possible to re-use the generic sample preparation procedure on a RLH with changes in extraction solvents or pH obtained from a simplified screening method that includes only two or three extraction solvents with three pH conditions (acidic, neutral and basic) [64] . That way, substantial time in hardware configuration or extensive application software scripting can be saved. However, these LLE methods still require manual intervention for some steps. To eliminate the manual steps, including aqueous/organic phase mixing and centrifugation, SLE plates can be used to replace the traditional LLE method while achieving similar assay reproducibility. If the LLE strategy is not working well for certain analytes, the next choice will be SPE method. To save time in application software scripting or method development, which can take several hours or even days, it is desirable to develop a one-size-fix-all strategy for automated SPE. The evaporation-free SPE methodology, in combination with the generic applicability of EVOLUTE® or Strata-X SPE plates, was able to reduce the time that is needed for software scripting since the procedure is very generic and can be directly used with no or minimal modification [44] . Similarly, PPT or phospholipid-removal PPT plates can be used for generic application if the assay sensitivity is adequate [57–61] . Finally, due to the recent development of integrated online SPE systems, online SPE has become an alternative to the more time-consuming offline sample extraction method, particularly when large numbers of samples are involved. Best practices in the application of RLH in regulated bioanalysis

Labware configuration is an important factor contributing to the failure of a procedure, especially when a group of people share one RLH. Each labware name can only represent one labware configuration. Accidental overwriting of a labware name with configuration changes can lead to the disabling of the labware or cause a collision of the robotic arms during assay implementation. To minimize the number of labware names designated to each individual labware, the same naming convention should be used for all labware within a group. If configuration modifications for shared labware are necessary, it is recommended to use a new name or new extension for the modified labware


Review

to avoid unnecessary errors in labware configurations for other assays. In order to better organize all folders within a RLH to facilitate future archiving or retrieve data, it is recommended not to use the same procedure with same name for more than one assay. Different names should be given to the same procedure if it is used for different assays, so that they can be archived and retrieved separately according to the studies without impacting other ongoing studies. In regulated bioanalysis, assay carryover is an important parameter to evaluate the assay robustness. When fixed tips in a RLH are used for liquid transfer, the carryover contribution to the assay from the RLH should be evaluated. In addition, if the sample tubes with pierceable caps are used for sample collection, the same sample tubes and caps will need to be used during method validation. The total carryover can be evaluated using all tips to transfer an ULOQ followed by washing and then transfer of the blank sample, in triplicate. All the blank samples are injected first followed by the ULOQ. Carryover of the liquid handler is usually calculated as the percentage response in the blank compared with the response in the ULQC, and the largest mean of the three measurements will be reported. In general, carryover contribution from an ULOQ to a blank sample less than 20.0% of the LLOQ is considered as having no impact on the assay performance. For sample dilutions that are done using RLHs, it may be also necessary to determine the accuracy of the dilutions performed by the RLHs during method validation, as well as sample analysis. In addition to the demonstration of dilution accuracy by using a RLH during method validation, in our laboratory, if diluted study specimens are included in an analytical run using RLHs, a minimum of two dilution QC replicates are required to be included in each analytical run during sample analysis. When diluted QC samples are to be used, if the dilution QC samples fail, the data for diluted study specimens will be rejected within the run; although nondiluted samples can be accepted depending on the performance of the analytical run. Conclusion & future perspective Automated sample extraction is widely used in regulated LC–MS/MS bioanalysis to help standardize the sample extraction procedure, increase assay throughput, improve the assay reproducibility and reduce the overall cost. However, automated sample preparation still poses a significant challenge in regulated bioanalysis because it requires additional time and expense for instrument qualification and software validation for RLHs before implementation of any automated sample


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Review Zheng, Jiang & Zeng preparation. A majority of the published methods required some manual intervention, generally in either the centrifugation or evaporation steps. Recent software and hardware development have made it possible to convert the Watson data files into RLH executable files, and automate most or all sample preparation steps in offline PPT, LLE and SPE using an integrated robotic workstation. In addition, a number of new strategies towards fully automated sample preparation have been developed to simplify the automated sample extraction workflow, such as the application of PPT filter plates, SLE plates, phospholipid-removing SPE plates, piercable caps for sample storage, evaporation-free SPE and fully automated online SPE systems. These strategies have significantly improved the automation workflow, increasing efficiency and robustness and lowering costs. Future developments will focus on the identification of bottlenecks in sample preparation workflows, simplification of the vendor’s software and standardization of platforms to make assay transfer easier. Since most of the integrated systems are custom made, it is

very desirable to see future commercialization of these highly integrated ‘walk away’ systems. Acknowledgements The authors would like to thank Mark E Arnold and AnneFrancoise Aubry (Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08540, USA) for their review of this manuscript.

Financial & competing interests disclosure The authors of this article are current employees of BristolMyers Squibb Company (BMS). All financial support for the studies reported herein was provided by BMS. The authors have no further relevant affiliations or financial involvement with any other organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Advantages of automated sample preparation • Automated sample preparation has become predominant in regulated bioanalysis, and has significantly improved the workflow efficiency, assay throughput and reproducibility, as well as data consistency.

Automated sample preparation in regulated bioanalysis • Automated sample preparation still poses a significant challenge in terms of regulatory requirements, such as 21 CFR Part 11 compliance, software validation, system qualification, calibration verification and proper maintenance.

Current advances & strategies towards fully automated sample preparation in regulated bioanalysis • Recent development of some highly integrated automated systems demonstrated that it is possible to automate all sample preparation steps within one software control. • Newly developed automated strategies towards fully automated sample preparation, such as protein precipitation filter plates, phospholipid-removing plates, supported liquid extraction plates, an evaporationfree solid-phase extraction method and online solid-phase extraction, provide the automation workflow with increased efficiency and robustness, and lower costs. • Future development will focus on simplification of application software and commercialization of highly integrated ‘walkaway’ systems.

effects from plasma phospholipids and eliminate potential metabolite interferences.

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Review

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