Showing papers on "Bioanalysis published in 2011"

It is time for a paradigm shift in drug discovery bioanalysis: from SRM to HRMS.

Abstract: It can be argued that the last true paradigm shift in the bioanalytical (BA) arena was the shift from high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection to HPLC with tandem mass spectrometry (MS/MS) detection after the commercialization of the triple quadrupole mass spectrometer in the 1990s. HPLC-MS/MS analysis based on selected reaction monitoring (SRM) has become the gold standard for BA assays and is used by all the major pharmaceutical companies for the quantitative analysis of new drug entities (NCEs) as part of the new drug discovery and development process. While LC-MS/MS continues to be the best tool for drug discovery bioanalysis, a new paradigm involving high-resolution mass spectrometry (HRMS) and ultrahigh-pressure liquid chromatography (uHPLC) is starting to make inroads into the pharmaceutical industry. The ability to collect full scan spectra, with excellent mass accuracy, mass resolution, 10-250 ms scan speeds and no NCE-related MS parameter optimization, makes the uHPLC-HRMS techniques suitable for quantitative analysis of NCEs while preserving maximum qualitative information about other drug-related and endogenous components such as metabolites, degradants, biomarkers and formulation materials. In this perspective article, we provide some insight into the evolution of the hybrid quadrupole-time-of-flight (Qq-TOF) mass spectrometer and propose some of the desirable specifications that such HRMS systems should have to be integrated into the drug discovery bioanalytical workflow for performing integrated qualitative and quantitative bioanalysis of drugs and related components.

Bioanalytical approaches to analyzing peptides and proteins by LC--MS/MS.

Abstract: Peptides and proteins have been utilized as therapeutic agents for over 40 years. Traditional approaches to quantify these molecules in biological matrices have utilized immunoassay approaches that can be time inefficient, lack assay specificity and have limited analytical ranges. The advances in sample preparation technologies, chromatographic systems and their chemistries, mass spectrometers and their software over the last decade have meant that LC-MS/MS approaches to peptide and protein quantification are feasible and can overcome the problems associated with quantification by immunoassay. In this article we present an overview of the challenges and approaches to overcome them when performing quantitative bioanalysis of peptides and proteins by LC-MS/MS.

Sample treatment based on extraction techniques in biological matrices

Abstract: The importance of sample preparation methods as the first stage in bioanalysis is described. In this article, the sample preparation concept and strategies will be discussed, along with the require ...

Recommendations on: internal standard criteria, stability, incurred sample reanalysis and recent 483s by the Global CRO Council for Bioanalysis

Abstract: “The Global CRO Council (GCC) for Bioanalysis was formed in an effort to bring together many CRO leaders to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry”

Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein

Abstract: Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charg...

Quality requirements for critical assay reagents used in bioanalysis of therapeutic proteins: what bioanalysts should know about their reagents

Abstract: Ligand-binding assays are the standard technology used for bioanalysis of therapeutic proteins, for example, for drug quantification (pharmacokinetics assays) and immunogenicity testing (antidrug antibody assays). Besides the selection of the most suitable technology platform (e.g., ELISA, electrochemiluminescence assays and surface plasmon resonance assays) and assay procedure, a pivotal prerequisite for good assay performance on any technology platform is the design, production and characterization of high quality reagents. To enable bioanalytical project support over the complete product life cycle, an appropriate long-term reagent supply is needed. This perspective describes our opinion on the requirements for generation and QC of critical reagents used in ligand-binding assays for drug quantification and antidrug antibody detection to enable high-quality assays and long-term supply, including reagent batch switches. The critical parameters during reagent design, production and long-term supply, along with the appropriate analytical methods for QC testing and appropriate certification, are discussed.

Bioanalytical LC–MS of therapeutic oligonucleotides

Abstract: Therapeutic oligonucleotides (OGNTs) are important biopharmaceutical drugs for the future, due to their ability to selectively reduce or knockout the expression of target genes. For the development of OGNTs, reliable and relatively high-throughput bioanalytical methods are required to perform the quantitative bioanalysis of OGNTs and their metabolites in biological fluids (e.g., plasma, urine and tissue). Although immunoaffinity methods, especially ELISA, are currently widely applied for this purpose, the potential of LC-MS in OGNT analysis is under investigation. Owing to its inherent ability to monitor the individual target OGNTs as well as their metabolites, LC-MS is now evolving into the method-of-choice for the bioanalysis of OGNTs. In this paper, the state-of-the-art of bioanalytical LC-MS of OGNTs and their metabolites in biological fluids is critically reviewed and its advantages and limitations highlighted. Finally, the future perspective of bioanalytical LC-MS, that is, lower detection levels and potential generic LC-MS methodology, is discussed.

Dried matrix spot direct analysis: evaluating the robustness of a direct elution technique for use in quantitative bioanalysis.

Abstract: Background: The surge in interest in switching from traditionally used wet plasma to dried matrix spot (DMS) sampling and analysis to support pharmaceutical drug development is due to the significant ethical, financial and data quality advantages on offer. Unfortunately these advantages do not extend to sample bioanalysis, as DMS extraction is more complex than the protein precipitation method typically used for wet plasma analysis. Direct elution techniques coupled to HPLC–MS/MS have been identified as a potential means to counter this additional complexity. Results: The robustness and reproducibility of DMS HPLC–MS/MS data generated using a CAMAG DBS–MS 16 prototype automated direct elution instrument has been demonstrated to meet or exceed results obtained using a conventional manual extraction methodology. Conclusion: The data generated suggest that a simple and fast direct elution method of DMS samples that does not require additional sample or extract clean-up, offers sufficiently robust performance...

Bioluminescence and Its Impact on Bioanalysis

Abstract: There is an increasing need for versatile yet sensitive labels, posed by the demands for low detection in bioanalysis. Bioluminescent proteins have many desirable characteristics, including the ability to be detected at extremely low concentrations; no background interference from autofluorescent compounds present in samples; and compatibility with many miniaturized platforms, such as lab-on-a-chip and lab-on-a-CD systems. Bioluminescent proteins have found a plethora of analytical applications in intracellular monitoring, genetic regulation and detection, immuno- and binding assays, and whole-cell biosensors, among others. As new bioluminescent organisms are discovered and new bioluminescence proteins are characterized, use of these proteins will continue to dramatically improve our understanding of molecular and cellular events, as well as their applications for detection of environmental and biomedical samples.

Single-drop microextraction in bioanalysis

Abstract: Bioanalysis usually requires a preparation procedure for sample cleanup or preconcentration. Conventional sample preparation techniques are often time consuming and labor intensive. Among recent progress in sample preparation, single drop microextraction (SDME) is one of the most efficient techniques providing both sample cleanup and preconcentration capabilities. In SDME, analytes are extracted from a sample solution into an acceptor drop and the drop is introduced to subsequent analysis. Since the volume of the acceptor drop is 1-10 µl or less, the consumption of solvents can be minimized and the preconcentration effect is enhanced. In this review, the basic principles of two-phase and three-phase SDME are described briefly and then recently developed modes of SDME, coupling with analytical instruments, and methods to enhance the drop stability are discussed. Recent applications of SDME to biological samples, including urine, blood and saliva, for the analysis of drugs, metal ions and biomarkers are reviewed.

Incurred sample accuracy assessment: design of experiments based on standard addition.

Abstract: It is commonly acknowledged that random and systematic analytical errors contribute to poor data quality, and moreover, to imprecise and inaccurate pharmacokinetic parameters. To investigate the random errors in GLP bioanalysis, common ground has been found in today’s bioanalysis to assess the reproducibility of the method by reanalyzing part of the incurred samples. The undesired systematic errors in bioanalysis affecting the trueness of the method and leading to inaccurate data remain relatively unattended so far. In order to obtain both precise and accurate data it is suggested in this paper to apply standard addition experiments to calculate the relative systematic errors as an estimate for the incurred sample accuracy. This approach, which can be seen as an important extension to current guidelines in GLP bioanalysis, is illustrated by assessing the accuracy of the bioanalytical results for a bioequivalence study for alendronate.

When do you need a validated assay

Abstract: As science advances, the questions we need to address in drug-development burgeons. However, resources are limited, and it is necessary to use these resources strategically. Clearly, it is impossible to do all things. Bioanalytical science is no exception to this phenomenon. In the recent past, some areas of scientific endeavor have expanded extensively, such as better characterization of metabolites and the exploitation of biomarkers in drug-development. As measuring these entities in vivo plays a critical role, many questions have arisen about how much method validation is needed, and when. The issue of metabolites in safety testing has evolved over a number of years and culminated with the publication of the US FDA Metabolites in Safety Testing Guidance in 2008 [101]. This guidance dealt with the evaluation of novel or unique metabolites observed in early human clinical trials, which were not observed in preclinical safety studies. The concern that is addressed is the unknown safety risks of these metabolites and the document provides several strategies to assess the toxicology of these entities. Some of the difficulty with this issue arises in the timing of these events: these metabolites are seen in early clinical trials (hopefully), when drug-development plans and timelines have already been implemented. Hence, backtracking is difficult. A requisite corollary to the toxicological assessment of these metabolites is their measurement in vivo. In fact, the guidance stipulates that studies need to be conducted for a metabolite (to assess its toxicity) if its exposure is greater than 10% compared with the parent. This prerequisite raises a number of questions that are difficult to answer. How do you assess metabolite AUC without a validated assay (for a metabolite you haven’t seen previously)? How do you validate an assay for this metabolite when no authentic standards are available? Do you need to go back and repeat the trial with a validated assay? Assessing the activity of the metabolite and possibly measuring it in vivo are necessary steps. However, most of these questions boil down to: how much validation is needed and by what stage of development? The European Bioanalytical Forum has developed a paradigm for addressing this issue [1]. Essentially, the scheme describes the development and use of an analytical method with varying degrees of validation. In cases where the metabolite is known or expected to be toxicologically active, the notion is to use a validated method for the metabolite beginning with preclinical studies and continuing with clinical development. In the case where the activity of the metabolite is unknown, less validated screening and qualified methods may be more appropriate until a determination is made regarding the toxicology of the metabolite. The European Bioanalytical Forum scheme makes very reasonable sense and may be a very valuable tool for industry. However, from the regulatory perspective, there are two key issues. First is the determination of metabolite activity. Unexpectedly high concentrations of a metabolite or an altogether new metabolite are not especially problematic if it is inactive, because no further characterization is needed. So the decision to develop a fully validated analytical method for the metabolite hinges on the determination of its toxicology (although, you could argue that measuring the metabolite may contribute to the understanding of its metabolism and the fate of its parent molecule). The trigger is the appearance of 10% or more of the metabolite compared with parent. Although this is a useful rule, care should be paid to the activity of the molecule. For example, SN-38 is the metabolite of irinotecan. Overall, it accounts for less than 10% of the parent. Yet its activity is 1000-times that of the parent [2]. If one simply followed the guidance rule, SN-38 may not have been measured, although it contributes significantly to the activity (efficacy and toxicity) of this drug. The second key issue is the overall purpose of the study. In early studies, the extent of method When do you need a validated assay?

Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid–liquid extraction LC–MS/MS analytical approach

Abstract: Background: A significant challenge of oligonucleotide bioanalysis is the selective extraction from complex tissue samples, where the molecules that distribute into the intracellular space are extensively protein bound and sit amongst a high concentration of endogenous nucleic acid material. Published analytical methodology currently purports extensive sample preparation requirements that include cell lysis steps, homogenization and dual cleanup with liquid–liquid extraction and solid-phase extraction, prior to injection. Results: We have developed a simple liquid–liquid extraction approach to rapidly isolate antisense oligonucleotides from biological tissues with high recovery and combined these preparative steps with a robust monolithic column LC–MS/MS setup. The platform showed improved chromatographic resolution and detection sensitivity over standard reversed-phase columns and required a low sample volume. Conclusion: The high-throughput method was sufficient to accurately quantify multiple antisense...

Minimizing matrix effects while preserving throughput in LC-MS/MS bioanalysis.

Abstract: Background: Phospholipids are known to cause matrix effects in LC–MS analysis and are not effectively removed by one of the most common method of sample preparation: organic solvent protein precipitation. The objective of this research is to minimize phospholipid interferences chromatographically. Results: In this article we examine several chromatographic approaches and highlight the method we developed that allows for the rapid gradient separation of model drug molecules from phospholipids. Conclusion: The new approach (which utilizes a mixture of methanol and acetonitrile as the organic mobile phase on a 2.1 × 20 mm C18 column) minimized phospholipids-related matrix effects in the analysis of plasma samples prepared by protein precipitation and is suitable for high-throughput bioanalysis in drug discovery.

Turbulent flow bioanalysis in drug metabolism and pharmacokinetics.

Abstract: Turbulent flow chromatography (TFC) as an analytical technique was introduced in the mid-1990s for online sample processing in bioanalysis. Turbulent flow columns are packed with large particles permitting the use of high mobile phase linear velocities. Solute molecules travel in a uniform concentrated band in which large matrix biomolecules are effectively separated from lower molecular weight analytes by differential mass transfer effects. In addition, the use of various bonded stationary phases enhances selectivity in TFC separations. Originally, the turbulent flow column was used for both online sample processing and the analytical separation. This mode of operation has largely been replaced with a dual column arrangement, in which sample processing is done online with the turbulent flow column and a second analytical column is employed for separation. Within the pharmaceutical industry, certain laboratories now use TFC routinely for bioanalytical support of in vivo drug discovery pharmacokinetic stud...

Application and interpretation of free and total drug measurements in the development of biologics

Abstract: Ligand-binding assays are used to determine concentration levels of biopharmaceuticals in biological matrices. The whole molecule does not serve as a basis for quantification, but subregions are captured and detected by specific binding critical reagents that have been produced for the sole purpose of bioanalysis. An assay can be designed to measure the free or the total analyte. Depending on the format of the assay, different observations and interpretations could be deemed. In the case studies presented in this article, the same serum samples were subjected to analysis in parallel by two different assay formats. In three out of the four cases presented, the results and the data interpretation were remarkably different. Therefore, it is essential for the bioanalyst to communicate to other stake-holders, such as toxicologists and pharmacokineticists, what the assay detects and measures for plausible data interpretation and implication.

Anticoagulant counter ion impact on bioanalytical LC–MS/MS assays: results from discussions and experiments within the European Bioanalysis Forum

Abstract: Background: In regulated bioanalysis, the need for partial validation when changing the counter ion of the anticoagulant is currently being debated within the bioanalytical community. To date, industry and the health authorities have not yet reached a consensus on this issue. The aim of the present study was to evaluate the impact of a change in counter ion when using the same anticoagulant on LC–MS/MS assay performance for a broad array of new chemical entities, compiling data generated at companies within the European Bioanalysis Forum (EBF). Results: In all, 15 EBF member companies provided experimental data on partial validation. In total, data from 42 LC–MS/MS assays were evaluated. The results show that a change in counter ion when using the same anticoagulant had no impact on assay performance. Conclusion: Based on these results and on conclusions from previous studies, the EBF recommends that in regulated bioanalysis, plasma samples containing different counter ions, but the same anticoagulant, sh...

Large-volume injection of sample diluents not miscible with the mobile phase as an alternative approach in sample preparation for bioanalysis: an application for fenspiride bioequivalence

Abstract: Background: Liquid–liquid extraction of target compounds from biological matrices followed by the injection of a large volume from the organic layer into the chromatographic column operated under reversed-phase (RP) conditions would successfully combine the selectivity and the straightforward character of the procedure in order to enhance sensitivity, compared with the usual approach of involving solvent evaporation and residue re-dissolution. Large-volume injection of samples in diluents that are not miscible with the mobile phase was recently introduced in chromatographic practice. The risk of random errors produced during the manipulation of samples is also substantially reduced. Results: A bioanalytical method designed for the bioequivalence of fenspiride containing pharmaceutical formulations was based on a sample preparation procedure involving extraction of the target analyte and the internal standard (trimetazidine) from alkalinized plasma samples in 1-octanol. A volume of 75 µl from the octanol layer was directly injected on a Zorbax SB C18 Rapid Resolution, 50 mm length × 4.6 mm internal diameter × 1.8 µm particle size column, with the RP separation being carried out under gradient elution conditions. Detection was made through positive ESI and MS/MS. Aspects related to method development and validation are discussed. Conclusions: The bioanalytical method was successfully applied to assess bioequivalence of a modified release pharmaceutical formulation containing 80 mg fenspiride hydrochloride during two different studies carried out as single-dose administration under fasting and fed conditions (four arms), and multiple doses administration, respectively. The quality attributes assigned to the bioanalytical method, as resulting from its application to the bioequivalence studies, are highlighted and fully demonstrate that sample preparation based on large-volume injection of immiscible diluents has an increased potential for application in bioana lysis.

LC-MS/MS sensitivity enhancement using 2D-SCX/RPLC and its application in the assessment of pharmacokinetics of clonidine in dried blood spots

Abstract: Background: Dried blood spot (DBS) technology offers distinctive preclinical and clinical advantages primarily ascribed to microscale sampling (e.g., 40–80 µl per time point), and the nature of solid-state samples in filter papers. Logistic benefits in sample collection, storage and shipping also result. However, the effective DBS samples available for bioanalysis are finite, that is, in the order of approximately 1 µl equivalent of plasma (3-mm punch) from a DBS of approximately 15–20 µl whole blood samples. This represents 20- to 100-times fewer samples for bioanalysis compared with a typical plasma assay. It is critical to increase LC–MS/MS sensitivity to accommodate DBS bioanalysis. Results: We developed a 2D strong cation exchange reversed-phase LC–MS/MS (2D-SCX/RPLC–MS/MS) for online enrichment, separation and detection of basic polar compounds, using clonidine hydrochloride as a model compound. Positively charged clonidine was retained and enriched in the first dimensional SCX column even in large ...

Reliable procedures to evaluate and repair crosstalk for bioanalytical MS/MS assays.

Abstract: Louis-Philippe Morin is a senior instrument application specialist at Algorithme Pharma, a CRO located in Laval, Canada. He has been working in the bioanalysis industry for the past 10 years where he became a subject matter expert in analytical instrumentation, especially in the MS field. His responsibilities in his current position are to optimize the workflow of the laboratory and to find new procedures, or approaches, to fix complex analytical problems. Louis-Philippe's expertise acquired over the years has led him to multiple publications regarding instrumentation. LC-MS/MS is the analytical technique of choice for the quantification of drugs in biological fluids. In recent years, MS/MS detection has been impacted by the rapid evolution of bioanalysis industry requirements. The availability of fast chromatographic systems, the demand for wider dynamic ranges and the extensive use of stable isotope-labeled internal standards in bioanalysis has pushed some triple quadrupole detectors to their limits of operation. Consequently, this situation has led to a re-evaluation of the problem of crosstalk as a potential cause of issues in bioanalysis. In this article, the importance of crosstalk verification on the MS/MS instrument will be demonstrated. Additionally, procedures to identify, evaluate and fix possible crosstalk issues during bioanalytical assays on MS/MS instruments are proposed.

Insights in regulated bioanalysis of human insulin and insulin analogs by immunoanalytical methods.

Abstract: Despite the long and illustrious history of insulin and insulin analogs as important biotherapeutics, the regulated bioanalysis (in this article, regulated bioanalysis refers to the formalized process for generating bioanalytical data to support pharmacokinetic and toxicokinetic assessments intended for development of insulin and insulin analogs as biotherapeutics, as opposed to the analytical process used for measuring insulin as a biomarker) of these peptides remains a challenging endeavor for a number of reasons. Paramount is the fact that the therapeutic concentrations are often low in serum/plasma and not too dissimilar from the endogenous level, particularly in patients with insulin resistance, such as Type 2 diabetes mellitus. Accordingly, this perspective was written to provide helpful background information for the design and conduct of immunoassays to support regulated bioanalysis of insulin and insulin analogs. Specifically, it highlights the technical challenges for determination of insulin an...