Showing papers in "Bioanalysis in 2013"
TL;DR: An overview is given of the different aspects of the 'hematocrit problem' in quantitative DBS analysis, and the different strategies that try to cope with this problem are discussed, along with their potential and limitations.
Abstract: Dried blood spot (DBS) sampling for quantitative determination of drugs in blood has entered the bioanalytical arena at a fast pace during the last decade, primarily owing to progress in analytical instrumentation. Despite the many advantages associated with this new sampling strategy, several issues remain, of which the hematocrit issue is undoubtedly the most widely discussed challenge, since strongly deviating hematocrit values may significantly impact DBS-based quantitation. In this review, an overview is given of the different aspects of the 'hematocrit problem' in quantitative DBS analysis. The different strategies that try to cope with this problem are discussed, along with their potential and limitations. Implementation of some of these strategies in practice may help to overcome this important hurdle in DBS assays, further allowing DBS to become an established part of routine quantitative bioanalysis.
212 citations
TL;DR: The strategy has involved novel protein structural characterization tools to help understand ADC biotransformations in vivo and use of the analyte knowledge gained to guide the development of quantitative bioanalytical assays.
Abstract: Antibody-drug conjugates (ADCs) are monoclonal antibodies with covalently bound cytotoxic drugs. They are designed to target tumor antigens selectively and offer the hope of cancer treatment without the debilitating side-effects of conventional therapies. The concept of ADCs is not new; however, development of these therapeutics is challenging and only recently are promising clinical data emerging. These challenges include ADC bioanalysis, such as quantifying in serum/plasma for PK studies and strategies for assessing immunogenicity. ADCs have complex molecular structures incorporating large- and small-molecule characteristics and require diverse analytical methods, including ligand-binding assays and MS-based methods. ADCs are typically mixtures with a range of drug-to-antibody ratios. Biotransformations in vivo can lead to additional changes in drug-to-antibody ratios resulting in dynamically changing mixtures. Thus, a standard calibration curve consisting of the reference standard may not be appropriate for quantification of analytes in vivo and represents a unique challenge. This paper will share our perspective on why ADC bioanalysis is so complex and describe the strategies and rationale that we have used for ADCs, with highlights of original data from a variety of nonclinical and clinical case studies. Our strategy has involved novel protein structural characterization tools to help understand ADC biotransformations in vivo and use of the analyte knowledge gained to guide the development of quantitative bioanalytical assays.
210 citations
TL;DR: Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.
Abstract: Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.
140 citations
TL;DR: Dried blood spots are widely applied in numerous bioanalytical assays and have gained a significant role in the screening of inherited metabolic diseases, in PK and PD modeling; in the treatment and diagnosis of infectious diseases; and in therapeutic drug monitoring.
Abstract: Dried blood spots have become a popular method in a variety of micro blood-sampling techniques in the life sciences sector, consequently competing with the field of conventional, invasive blood sampling by venepuncture Dried blood spots are widely applied in numerous bioanalytical assays and have gained a significant role in the screening of inherited metabolic diseases, in PK and PD modeling; in the treatment and diagnosis of infectious diseases; and in therapeutic drug monitoring Recent technological developments such as automation, online extraction, mass spectrometric direct analysis and also conventional dried blood spot bioanalysis, as well as future developments in dried blood spot bioanalysis are highlighted and presented in this article
115 citations
TL;DR: A critical overview of the literatures on SALLE is provided and perspectives of the future bioanalytical application of this often overlooked extraction technique are discussed.
Abstract: Salting-out assisted liquid-liquid extraction (SALLE) applies the salting-out effect to separate water-miscible organic solvent such as acetonitrile from plasma or other aqueous biofluids, and can extract a wide range of drug and metabolites, including many hydrophilic compounds. In most cases, the separated organic phase can be directly injected for bioanalysis, or with a simple dilution. SALLE provides similar simplicity to protein precipitation, but cleaner extracts due to a true phase separation. SALLE is also faster, more environmentally friendly and more cost-efficient than conventional liquid-liquid extraction and SPE. Through 96-well automation, SALLE can be easily integrated into the overall high-throughput LC-MS/MS bioanalysis strategy to increase productivity. This article provides a critical overview of the literatures on SALLE and perspectives of the future bioanalytical application of this often overlooked extraction technique. Important parameters impacting SALLE-LC-MS/MS assays are also discussed.
114 citations
TL;DR: The results from the conducted experiments show that the issues of DBS inregulated bioanalysis are real and that the technology will need improvements to be ready for use as a general tool for regulated bioanalysis.
Abstract: Background: The European Bioanalysis Forum dried blood spots (DBS)/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. This paper is focused on the impact of hematocrit changes on DBS analyses. Results: The hematocrit can have an effect on the size of the blood spot, on spot homogeneity and on extraction recovery in a compound-dependent manner. The extraction recovery can change upon aging in an hematocrit-dependent way. Different card materials can give different outcomes. Conclusions: The results from the conducted experiments show that the issues of DBS in regulated bioanalysis are real and that the technology will need improvements to be ready for use as a general tool for regulated bioanalysis. © 2013 Future Science Ltd.
110 citations
TL;DR: The characterization data generated by affinity capture LC-MS or hydrophobic interaction chromatography-UV provided critical mechanistic insights into understanding the stability and bioactivity of ADCs in vivo, and also helped the development of appropriate quantitative ELISAs.
Abstract: Background: Antibody–drug conjugates (ADCs) are a new class of cancer therapeutics that deliver potent cytotoxins specifically to tumors to minimize systemic toxicity. However, undesirable release of covalently linked drugs in circulation can affect safety and efficacy. The objective of this manuscript was to propose and assess the assays that allow for the characterization of the drug deconjugation in plasma/serum. Results: ADCs of three main drug conjugation platforms, linked via lysine, site-specific engineered cysteine or reduced interchain disulfide cysteine residues, were analyzed using affinity capture for sample enrichment coupled with LC–MS or hydrophobic interaction chromatography–UV for detection. These novel approaches enabled measurement of the relative abundance of individual ADC species with different drug-to-antibody ratios, while maintaining their structural integrity. Conclusion: The characterization data generated by affinity capture LC–MS or hydrophobic interaction chromatography–UV pr...
105 citations
TL;DR: The European Medicines Agency's 2011 guideline on bioanalytical method validation (BMV) was evaluated and subsequently intensely discussed by the European Bioanalysis Forum (EBF) during a 2-day workshop to help facilitate a smooth implementation at laboratories.
Abstract: The European Medicines Agency's (EMA) 2011 guideline on bioanalytical method validation (BMV) was evaluated and subsequently intensely discussed by the European Bioanalysis Forum (EBF) during a 2-day workshop (EBF Workshop on the implementation of the EMA guideline on BMV, Château de Limelette, Limelette, Belgium, 15-16 March 2012). The goal of the evaluation and discussions was to come to a uniform interpretation of the guideline and thus to help facilitate a smooth implementation at our laboratories. Up front preparations for the workshop by dedicated teams concentrated on challenges on implementation: ambiguities, technical or operational challenges and issues in general. In addition, common understandings were identified as well as main differences to the 2011 US FDA guideline. The guideline was perceived as being well written with a clear structure, separating method validation from sample analysis and treating all relevant aspects one-by-one in a logical order. It is the first BMV guideline clearly addressing the specifics for ligand binding assays and it shows a good match with current scientific thinking. The EBF community considers the EMA BMV guideline an excellent basis for countries that are in the process of developing or updating their own BMV guideline.
104 citations
TL;DR: Results from experiments performed by the European Bioanalysis Forum dried blood spots/microsampling consortium demonstrate that the issues of DBS in regulated bioanalysis are real and that the technology will need additional improvements to be ready for use as a general tool forregulated bioanalysis.
Abstract: The European Bioanalysis Forum dried blood spots/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. Their experiments focused on the impact of hematocrit changes, IS addition, spot homogeneity, aging of spots and stability of fresh blood and cards. Results from these experiments demonstrate that the issues of DBS in regulated bioanalysis are real and that the technology will need additional improvements to be ready for use as a general tool for regulated bioanalysis. In addition, results on fresh blood and card stability were shared at international meetings and will be reported at a later date.
101 citations
TL;DR: Issues concerning hair structure, collection, storage and analysis are reviewed, and representative examples of drug quantification using hair are summarized, emphasizing its potentialities and limitations as an alternative biological matrix for toxicological analyses.
Abstract: Alternative matrices are steadily gaining recognition as biological samples for toxicological analyses. Hair presents many advantages over traditional matrices, such as urine and blood, since it provides retrospective information regarding drug exposure, can distinguish between chronic and acute or recent drug use by segmental analysis, is easy to obtain, and has considerable stability for long periods of time. For this reason, it has been employed in a wide variety of contexts, namely to evaluate workplace drug exposure, drug-facilitated sexual assault, pre-natal drug exposure, anti-doping control, pharmacological monitoring and alcohol abuse. In this article, issues concerning hair structure, collection, storage and analysis are reviewed. The mechanisms of drug incorporation into hair are briefly discussed. Analytical techniques for simultaneous drug quantification in hair are addressed. Finally, representative examples of drug quantification using hair are summarized, emphasizing its potentialities and limitations as an alternative biological matrix for toxicological analyses.
74 citations
TL;DR: Efforts were made to fully and accurately describe traditional and modern techniques used to determine the components of breath in terms of design, function and also detection limit of different volatile organic compounds.
Abstract: Breath is a rich mixture containing numerous volatile organic compounds at trace amounts (ppbv–pptv level) such as: hydrocarbons, alcohols, ketones, aldehydes, esters or heterocycles. The presence of some of them depends on health status. Therefore, breath analysis might be useful for clinical diagnostics, therapy monitoring and control of metabolic or biochemical cell cycle products. This Review presents an update on the latest developments in breath analysis applied to diagnosing different diseases with the help of high-quality equipment. Efforts were made to fully and accurately describe traditional and modern techniques used to determine the components of breath. The techniques were compared in terms of design, function and also detection limit of different volatile organic compounds. GC with different detectors, MS, optical sensor and laser spectroscopic detection techniques are also discussed.
TL;DR: This Review focuses on recent work in the field of paper microfluidics that specifically addresses the goal of translating the multistep processes that are characteristic of gold-standard laboratory tests to low-resource point-of-care settings.
Abstract: This Review focuses on recent work in the field of paper microfluidics that specifically addresses the goal of translating the multistep processes that are characteristic of gold-standard laboratory tests to low-resource point-of-care settings. A major challenge is to implement multistep processes with the robust fluid control required to achieve the necessary sensitivity and specificity of a given application in a user-friendly package that minimizes equipment. We review key work in the areas of fluidic controls for automation in paper-based devices, readout methods that minimize dedicated equipment, and power and heating methods that are compatible with low-resource point-of-care settings. We also highlight a focused set of recent applications and discuss future challenges.
TL;DR: A simple, specific and reproducible LC-MS/MS method has been developed and validated for measuring Kyn and Trp in human plasma samples and was validated for precision, accuracy, matrix effect, extraction efficiency and stability.
Abstract: Background: Indoleamine 2,3-dioxygenase, catalyzing tryptophan (Trp) metabolism through the kynurenine (Kyn) metabolic pathway, plays important roles in immune suppression and the CNS In this article, we report a simple, rapid and specific LC–MS/MS method for accurate determination of Kyn and Trp concentrations in human plasma from HIV-infected patients Results: The human plasma sample (100 µl) was mixed with Kyn-d4 and Trp-d5 internal standards and then precipitated with trifluoroacetic acid The supernatant was directly analyzed by LC–MS/MS The assay using surrogate matrix calibrators was validated for precision, accuracy, matrix effect, extraction efficiency and stability Some assay validation issues for endogenous substance bioanalysis using an LC–MS/MS method are discussed Conclusion: A simple, specific and reproducible LC–MS/MS method has been developed and validated for measuring Kyn and Trp in human plasma samples
TL;DR: The breath sampling protocol was found to be acceptable for children, and healthy and asthmatic individuals were distinguished on the basis of eight VOCs at elevated levels in the breath ofAsthmatic children.
Abstract: Background: In-community non-invasive identification of asthma-specific volatile organic compounds (VOCs) in exhaled breath presents opportunities to characterize phenotypes, and monitor disease state and therapies. The feasibility of breath sampling with children and the preliminary identification of childhood asthma markers were studied. Method: End-tidal exhaled breath was sampled (2.5 dm3) from 11 children with asthma and 12 healthy children with an adaptive breath sampler. VOCs were collected onto a Tenax®/Carbotrap hydrophobic adsorbent trap, and analyzed by GC–MS. Classification was by retention-index and mass spectra in a ‘breath matrix‘ followed by multivariate analysis. Results: A panel of eight candidate markers (1-(methylsulfanyl)propane, ethylbenzene, 1,4-dichlorobenzene, 4-isopropenyl-1-methylcyclohexene, 2-octenal, octadecyne, 1-isopropyl-3-methylbenzene and 1,7-dimethylnaphtalene) were found to differentiate between the asthmatic and healthy children in the test cohort with complete separa...
TL;DR: It is demonstrated that the use of a high-resolution MS instrument in a regulated environment is a viable technique for quantification of large molecules.
Abstract: Background: Bioanalysts are continuously looking for innovative ideas or instruments to increase the sensitivity and selectivity of their assays. Research for better mass spectrometers is becoming crucial with the emerging trend of large-molecule quantification. This study lists the different advantages of high-resolution MS (HRMS) over standard triple quadrupole instruments and proposes basic guidelines on how to use HRMS for large-molecule quantification in a regulated environment. Results: A direct comparison between HRMS and triple quadrupole instruments for the quantification of six different model peptides (desmopressin, calcitonin, enfuvirtide, exenatide, glucagon and somatostatin) was completed. The HRMS instrument, when used specifically for targeted quantification (‘quant/quant’), showed equivalent or better sensitivity for all compounds tested. Conclusion: This paper demonstrates that the use of a HRMS instrument in a regulated environment is a viable technique for quantification of large molec...
TL;DR: In this article, the relative abundance of 2H (expressed in δ 2H values) in tissues of plants, wildlife and people has evolved into a powerful forensic tool.
Abstract: Measurement of the relative abundance of 2H (expressed in δ 2H values) in tissues of plants, wildlife and people has evolved into a powerful forensic tool. The approach is based on the strong linkage between spatial patterns of δ 2H values in precipitation at local and continental scales, and the tissues of plants and animals produced on these ‘isoscapes’. Unfortunately, despite this exciting potential, difficulties inherent in the measurement of δ 2H values in complex organic materials such as proteins, as well as the accuracy of such measurements, and a reluctance to adopt strict quality assurance/QC approaches to address challenges associated with these measurements, has clearly limited this potential. These challenges are entirely avoidable and techniques now exist for the routine reliable measurement of δ 2H values in materials of forensic interest that will allow completely comparable data among laboratories.
TL;DR: It is demonstrated that sample inhomogeneity is a significant hurdle to the use of dried blood spots for regulated bioanalysis that should be investigated further in the method establishment phase if the whole spot is not sampled.
Abstract: Background: At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consortium did not form a dedicated team to investigate the spot homogeneity. However, two teams performed experiments that produced results relating to sample homogeneity. Results: The data, which were produced via two different approaches (a radiolabeled and a nonradiolabeled approach), are highly complementary and demonstrate clear effects on sample inhomogeneity due to the substrate type, compound and hematocrit levels. Conclusion: The results demonstrate that sample inhomogeneity is a significant hurdle to the use of dried blood spots for regulated bioanalysis that should be investigated further in the method establishment phase if the whole spot is not sampled. © 2013 Future Science Ltd.
Biogen Idec1, Algorithme Pharma2, Bristol-Myers Squibb3, Bayer HealthCare Pharmaceuticals4, Silver Spring Networks5, Intertek6, Hoffmann-La Roche7, Pfizer8, Boehringer Ingelheim9, GlaxoSmithKline10, Fordham University11, Genentech12, Ferring Pharmaceuticals13, Amgen14, Quintiles15, Health Canada16, Janssen Pharmaceutica17, Merck & Co.18, National Health Surveillance Agency19, Medicines and Healthcare Products Regulatory Agency20, United States Military Academy21
TL;DR: This 2013 White Paper addresses important bioAnalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.
Abstract: The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.
TL;DR: Merck's thoughts on why, when and how to use dried blood spot (DBS) technology in a clinical setting are communicated, and a strategic approach, emphasizing the necessary steps, are provided, for successful clinical implementation of this microsampling technique.
Abstract: This paper communicates Merck’s thoughts on why, when and how to use dried blood spot (DBS) technology in a clinical setting, and provides a strategic approach, emphasizing the necessary steps, for successful clinical implementation of this microsampling technique. PK consideration based on relevant in vitro data, that is, blood-to-plasma ratio, hematocrit, plasma unbound fraction and/or blood cell partition, is suggested to be part of the decision tree on when to choose DBS as a surrogate matrix for PK analysis. A quick feasibility assessment addressing analytical challenges, including sensitivity, hematocrit impact and storage stability, needs to be evaluated before initiating DBS studies. Special attention should be paid to the clinical sample collection procedures to ensure data quality. Bridging studies are required to establish the correlation between plasma and DBS data to ensure that pooling of data from the various clinical studies can be used in population PK or PK/PD assessment. Seeking regulat...
TL;DR: A simple SPE LC-MS analysis was developed for direct, simultaneous quantification of insulin glargine, detemir, aspart and glulisine, and it was found that mass spectral fragments m/z >700 provided greater specificity, significantly reducing endogenous background.
Abstract: Background: Intact insulins are difficult to analyze by LC–MS/MS due to nonspecific binding and poor sensitivity, solubility and fragmentation. This work aims to provide a simpler, faster LC–MS method and focuses on solving the above issues. Results: A novel charged-surface chromatographic column produced peak widths for insulin that were significantly narrower than traditional C18 columns when using formic acid as mobile phase. Mass spectral fragments m/z >700 provided greater specificity, significantly reducing endogenous background. Detection limits in human plasma were 0.2 ng/ml for insulin glargine, glulisine and detemir, and 0.5 ng/ml for insulin aspart. Average accuracy for standard curve and QC samples was 93.4%. Conclusion: A simple SPE LC–MS analysis was developed for direct, simultaneous quantification of insulin glargine, detemir, aspart and glulisine.
TL;DR: Liquid-liquid-liquid membrane extraction was successfully performed in a slightly modified commercially available 96-well plate format in a modification of hollow-fiber liquid-phase microextraction.
Abstract: Background: This paper reports development of a new approach towards analytical liquid–liquid–liquid membrane extraction termed parallel artificial liquid membrane extraction. A donor plate and acceptor plate create a sandwich, in which each sample (human plasma) and acceptor solution is separated by an artificial liquid membrane. Parallel artificial liquid membrane extraction is a modification of hollow-fiber liquid-phase microextraction, where the hollow fibers are replaced by flat membranes in a 96-well plate format. Results: Four basic drugs (pethidine, nortriptyline, methadone and haloperidol) were extracted from human plasma in 30 min, followed by analysis with LC–MS/MS. Extraction recoveries for the model analytes were in the range of 34–74% from human plasma. LOQs were in the range of 0.01–0.35 ng/ml, linearity above 0.9955 for all drugs and with RSD values below 12%. Conclusion: Liquid–liquid–liquid membrane extraction was successfully performed in a slightly modified commercially available 96-we...
TL;DR: Various data-dependent and -independent acquisition methods in combination with accurate mass-based data mining tools for metabolite identification in drug discovery and development are discussed.
Abstract: Metabolite identification plays a pivotal role through all stages of drug discovery and development. The task of detecting and characterizing drug metabolites in complex biological matrices is very challenging, due in part to the co-existence of drug-related material with a large excess of endogenous material. Deciphering information on drug metabolites in these complex biological systems requires not only sophisticated LC-MS systems, but also software that can help differentiate drug-related compounds from endogenous material in the MS data. Fortunately, there have been considerable advances in high-resolution MS technologies with improved mass accuracy. The high resolution and mass accuracy capabilities have necessitated and augmented the development of integrated data acquisition methods, which have significantly facilitated metabolite detection and identification. In this review, we discuss various data-dependent and -independent acquisition methods in combination with accurate mass-based data mining tools for metabolite identification in drug discovery and development.
TL;DR: The bioanalytical strategy was successfully applied to the drug development of T-DM1 and ensured that key analytes were accurately measured in support of nonclinical and clinical development.
Abstract: Background: Antibody–drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs and are heterogeneous mixtures that can biotransform in vivo, resulting in additional complexity. ADC bioanalytical strategies require novel analytical methods, as well as existing large- and small-molecule methods. Because ADCs in late-stage clinical development are relatively new, regulatory guidelines and standard industry best practices for developing strategies for bioanalytical PK assays are still being established. Results: A PK assay strategy was developed that included comprehensive novel reagent and assay characterization approaches for the ADC ado-trastuzumab emtansine (T-DM1). Conclusion: The bioanalytical strategy was successfully applied to the drug development of T-DM1 and ensured that key analytes were accurately measured in support of nonclinical and clinical development.
TL;DR: Different parameters affecting the equilibrium and analysis are discussed, together with suggestions on how to control these parameters in order to produce as trustworthy results for unbound concentrations/fractions as possible.
Abstract: Knowledge regarding unbound concentrations is of vital importance when exploring the PK and PD of a drug. The accurate and reproducible determination of plasma protein binding and unbound concentrations for a compound/drug is a serious challenge for the bioanalytical laboratory. When the drug is in equilibrium with the binding protein(s), this equilibrium will shift when physiological conditions are not met. Furthermore, the true unbound fraction/concentration is unknown, and there are numerous publications in the scientific literature reporting and discussing data that have been produced without sufficient control of the parameters influencing the equilibrium. In this Review, different parameters affecting the equilibrium and analysis are discussed, together with suggestions on how to control these parameters in order to produce as trustworthy results for unbound concentrations/fractions as possible.
TL;DR: The basis for why DBS techniques are likely to be part of the future is discussed, as well as offering insights into where these benefits may be realized.
Abstract: The use of DBS is an appealing approach to employing microsampling techniques for the bioanalysis of samples, as has been demonstrated for the past 50 years in the metabolic screening of metabolites and diseases. In addition to its minimally invasive sample collection procedures and its economical merits, DBS microsampling benefits from the very high sensitivity, selectivity and multianalyte capabilities of LC-MS, which has been especially well demonstrated in newborn screening applications. Only a few microliters of a biological fluid are required for analysis, which also translates to significantly reduced demands on clinical samples from patients or from animals. Recently, the pharmaceutical industry and other arenas have begun to explore the utility and practicality of DBS microsampling. This review discusses the basis for why DBS techniques are likely to be part of the future, as well as offering insights into where these benefits may be realized.
TL;DR: The sensitivity and precision of adduct detection has increased to the point of enabling subtherapeutic dosing for diagnostics applications, termed diagnostic microdosing, prior to the initiation of full-dose therapy.
Abstract: The personalized medicine revolution is occurring for cancer chemotherapy. Biomarkers are increasingly capable of distinguishing genotypic or phenotypic traits of individual tumors, and are being linked to the selection of treatment protocols. This review covers the molecular basis for biomarkers of response to targeted and cytotoxic lung and bladder cancer treatment with an emphasis on platinum-based chemotherapy. Platinum derivatives are a class of drugs commonly employed against solid tumors that kill cells by covalent attachment to DNA. Platinum–DNA adduct levels in patient tissues have been correlated to response and survival. The sensitivity and precision of adduct detection has increased to the point of enabling subtherapeutic dosing for diagnostics applications, termed diagnostic microdosing, prior to the initiation of full-dose therapy. The clinical status of this unique phenotypic marker for lung and bladder cancer applications is detailed along with discussion of future applications.
TL;DR: MFLC-MS/MS can be used to perform bioanalytical method validations with increased MS signal, reduced source contamination and reduced solvent consumption.
Abstract: Background: In support of bioanalysis, there has always been a desire to improve detection limits and reduce scale. Microflow LC (MFLC) coupled with MS accomplishes both of these goals. Results: As such, MFLC coupled with an MS system was used to generate bioanalytical validation data that met US FDA criteria. The MFLC–MS/MS data was compared with the same method with the use of conventional HPLC–MS/MS and a more than 14× S/N improvement was found with the MFLC–MS/MS method. Methotrexate was used as a model molecule to demonstrate the validation of the method from human plasma. The MFLC–MS/MS method was demonstrated to be accurate (±7%) and precise (12.9% at the LLOQ and a maximum of 11.6% at all other concentrations) across the dynamic range of the assay (1–1000 ng/ml) and compared well with the HPLC–MS/MS method. The MFLC bioanalytical validation was performed at a flow rate of 35 µl/min on a 0.5-mm inner diameter (I.D.) column, whereas, for the same linear velocities on the 2.0-mm I.D. column, the conv...
TL;DR: This report is an attempt to clarify how to validate and use capillary microsampling methods in a regulatory environment.
Abstract: Capillary microsampling (CMS) has recently been introduced as a response to the demands for more ethical use of laboratory animals according to the 3R principles. In CMS, an exact volume of the blood, plasma or other biofluid is collected in a capillary from which it is washed out, resulting in a diluted sample that can be handled using the existing equipment in the bioanalytical laboratory. CMS differs from traditional large volume sampling as the microsample is diluted before further handling and analysis, and reanalysis is performed using the diluted sample. This has some implications for the validation and this report is an attempt to clarify how to validate and use CMS methods in a regulatory environment. CMS also shows some distinct new opportunities: labile analytes can be immediately stabilized at sample collection and the addition of the internal standard to the whole sample can improve analytical performance. The experiences from 5 years use of CMS of plasma and blood for determination of drug exposure in animal studies are reviewed.
TL;DR: A simple and robust novel approach for the collection of small plasma volumes from rodent TK studies has been demonstrated.
Abstract: Background: A novel device and procedure for the collection and isolation of microvolumes of plasma have been developed and two pilot rodent PK studies have been completed. Results: This method involves collection of blood into a plastic-wrapped, EDTA-coated capillary tube, containing a small amount of a thixotropic gel and a porous plug. Following blood collection, the capillary is placed into a secondary labeled container suitable for centrifugation and plasma is generated. During centrifugation, the thixotropic gel isolates the plasma from the red blood cells and creates a physical barrier between the two matrices. The plasma is then dispensed from the capillary tube into a separate container for storage or processing. Conclusion: A simple and robust novel approach for the collection of small plasma volumes from rodent TK studies has been demonstrated.