ANALYTICAL INSTRUMENTATION: Competitiveness of Bioanalytical Laboratories— Technical and Regulatory Perspectives

The discovery and development of a new drug costs around $1 billion, and it may take approximately 10 years for the drug to reach the marketplace. Drug discovery and development are the processes of generating compounds and evaluating all of their properties to determine the feasibility of selecting one new chemical entity (NCE) to become a safe and efficacious drug.
Among many criteria, obtaining experimental pharmacokinetics (PK) data from laboratory animals in the nonclinical stage is critical to evaluating a drug candidate before it can qualify to be tested in the clinical trials for safety and efficacy evaluation.
A key parameter in pharmacokinetics is the plasma or tissue concentration of the new drug after its administration to laboratory animals. Therefore, developing an accurate and fast analytical method for measuring the concentrations of a compound in plasma or tissue is the first step toward yielding the PK of a compound. As the drug candidate moves down in the pipeline, the requirements for PK information differ at the different stages of drug discovery and development, leading to the introduction of “fit for purpose” analytical strategies that provide the appropriate level of bioanalytical support for the purpose at hand, while simultaneously minimizing resource expenditures.
Due to its superior sensitivity and selectivity, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has become the analytical technique primarily used by bioanalytical laboratories performing analyses for preclinical and clinical studies. The increased number of biological agents used as therapeutics—in the form of recombinant proteins, monoclonal antibodies, vaccines, and so on—has prompted the pharmaceutical industry to review and refine aspects of the development and validation of bioanalytical methods for the quantification of these therapeutics in biological matrices in support of preclinical and clinical studies. Most of these methodologies are used in quantitative assays supporting PK and toxicokinetic parameters of the therapeutic agents. To date, an alternative approach for dealing with macromolecules is to utilize ligand-binding assays (LBAs) to generate data.
LC–MS/MS has played an incredible role in drug metabolism and pharmacokinetics studies at various drug discovery and development stages since its introduction to the pharmaceutical and biotechnology industries. This section will elaborate on the most recent advances in sample preparation and separation, along with the mass spectrometric aspects of high-throughput quantitative bioanalysis of drugs and metabolites in biological matrices. Recently introduced techniques such as ultra-performance or ultra-speed liquid chromatography (UPLC or USLC), with small particles (sub-2 mm) and monolithic chromatography, offer improvements in speed, resolution, and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed-phase LC–MS/MS.
Sample preparation formatted to 96-well and/or 384-well plates has allowed for semi-automation of off-line sample preparation techniques, significantly improving throughput. On-line solid phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications to reduce both the time and labor required for generating bioanalytical results. Extraction sorbents for on-line SPE extend to an array of media, including large particles for turbulent-flow chromatography, restricted access materials, monolithic materials, and disposable cartridges utilizing traditional packings such as those used in Spark Holland (Symbiosis) systems. In the latter part of this section, recent studies of matrix effect in LC–MS/MS analysis and ways to reduce/eliminate matrix effect in method development and validation will be also discussed.
Bioanalytical laboratories in the pharmaceutical and life science industries are constantly under pressure to reduce overall time for discovery and development. This pressure is often accompanied by an increase in the number of biological samples requiring PK analysis and a decrease in the desired quantitation levels. Hyphenated techniques are examples of new tools adopted for developing fast and cost-effective analytical methods. LC–MS/MS has led to major breakthroughs in the field of quantitative bioanalysis since the 1990s due to its inherent specificity, sensitivity, and speed.1-2 It is now generally accepted as the preferred technique for quantitating small-molecule drugs, metabolites, and other xenobiotic biomolecules in biological matrices. The use of LC–MS/MS has grown exponentially in the last decade due to its unmatched sensitivity, extraordinary selectivity, and rapid rate of analysis. Typical workflow of bioanalysis from sample generation to data delivery is summarized in Figure 6.1.

Figure 6.1: Typical workflow of bioanalysis from sample generation to data delivery

Samples from biological matrices are usually not directly compatible with LC–MS/MS methodologies. Sample preparation has traditionally used such approaches as protein precipitation (PPT), liquid–liquid extraction (LLE), or SPE. Manual operations associated with these processes—in particular with LLE and SPE—are fairly labor intensive and time consuming. Parallel sample processing in a 96-well format using robotic liquid handlers has significantly shortened the time analysts have to spend in the laboratory for sample preparation. An alternative sample extraction method that has generated a lot of interest in recent years is the direct injection of plasma using an on-line extraction method. A major advantage of on-line SPE over off-line extraction techniques is that the sample preparation step is embedded into the chromatographic separation and thus eliminates most of the sample preparation time traditionally performed at the bench, potentially resulting in more variability from batch to batch and analyst to analyst. Steep gradients and relatively short columns were first utilized in early applications of high-throughput LC–MS/ MS assays to reduce run times.
A better understanding of how matrix effects can compromise the integrity of bioanalytical methods has re-emphasized the need for adequate chromatographic separation of analytes from endogenous biological components in quantitative bioanalysis using the LC–MS/MS technique. New developments from chromatographic techniques such as UPLC and USLC with sub-2 mm particles and monolithic chromatography show promise in delivering higher speed, better resolution, and increased sensitivity for high-throughput analysis while minimizing matrix effects. LC–MS/MS applications for quantitative bioanalysis have been documented in hundreds of articles in just the past several years, and a number of reviews dealing with one or more aspects of quantitative LC–MS/MS bioanalysis have been published.3-6 Within this section, publications related to technology development for throughput improvement, associated applications, and discussions of key developments in quantitative analysis from 2002 to 2009 will be reviewed and elaborated upon.

Sample Generation, Shipment, and Storage

Figure 6.2: Hamilton STAR

From a bioanalytical standpoint, prior to sample analysis, it is important to maintain the quality and integrity of samples to be generated from discovery in nonclinical and clinical programs regardless of whether the studies are nonregulated or regulated. Laboratory management regarding sample handling/custody is key to the success of a bioanalytical operation. These processes generally include but are not limited to:

• Sample receipt;
• Login (creating unique identification numbers or ID);
• Storage (adequate and acceptable conditions);
• Transfers/relocations from lab to lab and facility to facility; and
• Sample retention and/or disposal.

Zhang and colleagues described an integrated sample collection and handling process for bioanalysis.7 The sample handling process consumes a significant portion of the resources of a bioanalytical laboratory. One of the primary purposes for analytical automation is the possibility of analyzing a huge number of samples, either simultaneously or sequentially, without any human intervention. When large amounts of samples are analyzed, mainly in routine analyses, the importance of the correct identification of each sample in order to obtain the appropriate correlation between the sample analyzed and the results obtained is obvious.
One of the most efficient and reliable ways to identify a sample, from collection to result, is by using bar codes. A bar code can best be defined as an “optical Morse code.” Series of black bars and white spaces of varying widths are printed on labels to identify items uniquely. The bar code labels are read with a scanner, which measures reflected light and interprets the code into numbers and letters that are passed on to a computer. All bar codes have start/stop characters that allow the bar code to be read from both left to right and right to left.
Unique characters placed at both the beginning and end of each bar code, the stop/start characters provide timing references, symbol identification, and direction of reading information to the scanner. By convention, the unique character on the left of the bar code is considered the “start,” while the character on the right of the bar code is considered the “stop.” Besides the linear bar code, today’s new bar codes are two- dimensional, electronic, or small computer chips, which sort data in inconspicuous places like a credit card. This technology has been included in clinical and bioanalytical analyzers, where large amounts of samples are analyzed daily and a correct assignation of the results obtained is mandatory.
Automation for sample handling meets the business needs of improving productivity and reducing the documentation required for compliance. Procedural elements involved in maintaining bioanalytical data integrity for good laboratory practices and regulated studies are discussed by James and Hill.8 The elements can be divided into three areas.
First, there are elements ensuring the integrity of the analyte until analysis, through correct sample collection, handling, shipment, and storage procedures. Incorrect procedures can lead to loss of analyte due toinstability, addition of analyte through contamination or instability of related metabolites, or changes in the matrix composition that may adversely affect the performance of the analytical method.
Second, the integrity of the sample identity must be maintained to ensure that the result that is reported relates to the individual sample that was taken. Possible sources of error include sample mix-up or mislabeling or errors in data handling.
Finally, there is the overall integrity of the documentation that supports the analysis, along with any pre-study validation of the method. This includes a wide range of information, from paper and electronic raw data through standard operating procedures, analytical procedures, and facility records to study plans and final reports. These are critical, allowing an auditor or regulatory body to reconstruct the study. A laboratory information management system (LIMS) is a preferable database and handling system that maintains a detailed pedigree for each sample by capturing processing parameters, protocols, stocks, tests, and analytical results for the sample’s complete life cycle. Project and study data are also maintained to define each sample in the context of the research tasks it supports. LIMS is required for each analytical pipeline to track all aspects of sample handling.

Sample Preparation

One strategy for high-throughput bioanalytical analysis is to use well-established instrumentation; rigorous, standardized techniques; and automation wherever possible to minimize or replace manual tasks. Automation results in greater performance consistency over time and more reliable methods transfer from site to site. Automated 96-well plate technology is well established and accepted and has been shown to effectively replace manual operations. The 96-well instruments can execute automated off-line extraction and sample cleanups. Automated SPE, LLE, and PPT all can be performed in 96-well format.
Adequate sample preparation is a key aspect of quantitative bioanalysis; without it, bottlenecks can occur during high-throughput analysis. Sample preparation techniques in 96-well format have been well adopted in high-throughput quantitative bioanalysis with a number of applications. Techniques that can use the format include LLE, SPE, and PPT.
Typically, liquid transfer steps, including preparation of calibration standards and quality control samples as well as the addition of the internal standard, have been performed automatically using robotic liquid handling workstations for parallel sample processing (Hamilton STAR, refer to robot liquid handler system in Figure 6.2). The increasing demand for high throughput causes a unique situation of balancing cost versus analysis speed as each sample preparation technique offers unique advantages. Dilute-and-shoot and PPT are among the most popular and effective techniques due to their simplicity.
Sample preparation with PPT is widely used in the bioanalysis of plasma samples. The method has been extended to the quantitation of drug and metabolites from whole blood. Koseki and colleagues developed a sensitive and specific LC–MS/MS method for the simultaneous determination of cyclosporine A (CsA) and its three main metabolites (AM1, AM4N, and AM9) in human blood.9
Mixed-mode polymer-based sorbents (e.g., Waters Oasis MCX cartridge) were introduced in the late 1990’s for the isolation of drugs with ionizable functional groups from biological fluids. The extraction procedures can be a generic protocol or can be optimized if better sample cleanup is desired. The use of SPE often gives superior results to those with a PPT approach but may not be as cost effective as PPT due to the labor and material costs associated with the procedures. Mallet and colleagues described a novel 96-well SPE plate that was designed to minimize the elution volume required for quantitative elution of analytes.10
The plate was packed with 2 mg of a high-capacity SPE sorbent that allows loading of up to 750 mL of plasma. The novel design permitted elution with as little as 25 mL solvent, enabling the plate to offer up to a 30-fold increase in sample concentration. The evaporation and reconstitution step that is typically required in SPE is unnecessary due to the concentrating ability of the sorbent. Yang and colleagues developed a sensitive mElution SPE–LC–MS/MS method for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma.11 In the method, analytes were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well mElution SPE plate with 70 mL methanol as the elution solvent. The lower limit of quantitation was 0.1 ng/mL and the linear calibration range was from 0.1 to 100 ng/mL for both analytes. Several related formats have recently appeared using miniaturized packed-bed formats as well as an adaptation of disk technology for the 96-well format.

Figure 6.3: Spark Holland Symbiosis system

The ultimate goal for the 96-well approach is to add a higher level of parallel sample preparation using essentially a further miniaturized SPE format. By extracting multiple 96-well format SPE plates, up to four 96-well plates or 384 samples may be prepared and then analyzed within 24 hours by one person with one LC–MS/MS system, a significant improvement in sample throughput using commercially available equipment and supplies. Future developments should further increase throughput significantly.
LLE gives excellent sample cleanup but poses engineering challenges for use in an automated high-throughput fashion. Several groups have developed different approaches to solve the mixing and phase separation problems typically observed in a 96-well LLE method. By using vigorous vortexing after well-controlled heat-sealing, or repeated aspiration and dispensing by robotic liquid handler, common extraction solvents such as methyl-t-butyl ether (MTBE) or ethyl acetate (EA) may be used in the routine extraction of plasma, blood, or tissue samples.
Another approach is on-line SPE for the automated preparation of samples prior to LC–MS/MS analysis. This approach uses a commercial device that combines an auto-sampler and a solvent delivery unit to aliquot multiple liquid samples into a flowing stream of solvent. The solvent has preconditioned an in-line SPE cartridge. After conditioning, the SPE cartridge retains the targeted analytes, while the relatively weak solvent elutes unretained salts and polar matrix components to waste. An empirically optimized sequence of increasingly stronger solvents is then used to further elute weakly retained unwanted sample components. A final elution with high performance liquid chromatography (HPLC) mobile phase elutes the targeted analytes off the SPE cartridge and onto an analytical HPLC column for LC–MS/MS analysis. The on-line SPE technique offers speed, high sensitivity by the preconcentration factor, and low extraction cost per sample, but typically requires the use of program-controlled switch valves and column reconfigurations. The on-line technique can be fully automated, however, offering real-time high-throughput sample analysis.
One commercial automated on-line SPE system is the Symbiosis system, manufactured by Spark Holland (Figure 6.3). It includes an auto-sampler (Reliance), two binary HPLC pumps, an on-line SPE unit with two high-pressure solvent delivery pumps, and a combined valve system to direct fluid for different steps of SPE. At the beginning of each run, an on-line SPE cartridge is loaded into the unit. After a conditioning step with high organic solvent and an equilibrium step with low organic aqueous solution, a sample is injected onto the cartridge and washed with aqueous solution. Proteins and other matrix materials from the sample are removed during the washing step. Analyte of interest is then eluted onto the analytical column and detected by mass spectrometry or tandem mass spectrometries. During the sample elution step, a second sample is loaded to a new on-line SPE cartridge for the next analysis. In this parallel mode, the sample analysis cycle time approximates the LC run time without the time required for the SPE procedures. Because the on-line SPE cartridge is disposable and each sample uses a new cartridge, the carry-over problem from the extraction cartridge is eliminated.

References

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8. James CA, Hill HM. Procedural elements involved in maintaining bioanalytical data integrity for good