Showing papers in "Bioanalysis in 2016"
TL;DR: An LC-MS/MS method is established to measure 11 molecules, namely tryptophan, KYN, 3-OH-KYN,3-OH -anthranilic acid, quinolinic acid, picolinic Acid, kynurenic acid, xanthurenic Acid, serotonin, dopamine and neopterin within 5.5 min, with sufficient sensitivity to quantify these molecules in small sample volumes of plasma, cerebrospinal fluid and brain tissue.
Abstract: Aim: The kynurenine (KYN) pathway is implicated in diseases such as cancer, psychiatric, neurodegenerative and autoimmune disorders. Measurement of KYN metabolite levels will help elucidating the involvement of the KYN pathway in the disease pathology and inform drug development. Methodology: Samples of plasma, cerebrospinal fluid or brain tissue were spiked with deuterated internal standards, processed and analyzed by LC–MS/MS; analytes were chromatographically separated by gradient elution on a C18 reversed phase analytical column without derivatization. Conclusion: We established an LC–MS/MS method to measure 11 molecules, namely tryptophan, KYN, 3-OH-KYN, 3-OH-anthranilic acid, quinolinic acid, picolinic acid, kynurenic acid, xanthurenic acid, serotonin, dopamine and neopterin within 5.5 min, with sufficient sensitivity to quantify these molecules in small sample volumes of plasma, cerebrospinal fluid and brain tissue.
105 citations
TL;DR: The methodology of paper spray MS, which is capable of detecting drugs directly from dried blood, plasma and urine spots at the low ng/ml to pg/ml levels without sample preparation, is discussed.
Abstract: Paper spray MS is part of a cohort of ambient ionization or direct analysis methods that seek to analyze complex samples without prior sample preparation. Extraction and electrospray ionization occur directly from the paper substrate upon which a dried matrix spot is stored. Paper spray MS is capable of detecting drugs directly from dried blood, plasma and urine spots at the low ng/ml to pg/ml levels without sample preparation. No front end separation is performed, so MS/MS or high-resolution MS is required. Here, we discuss paper spray methodology, give a comprehensive literature review of the use of paper spray MS for bioanalysis, discuss technological advancements and variations on this technique and discuss some of its limitations.
79 citations
TL;DR: The challenges underlying metabolomics-based experiments are described, discussing step by step the potential pitfalls of the analytical process, including study design, sample collection, storage, as well as preparation, chromatographic and electrophoretic separation, detection and data analysis.
Abstract: Metabolomics-based strategies have become an integral part of modern clinical research, allowing for a better understanding of pathophysiological conditions and disease mechanisms, as well as providing innovative tools for more adequate diagnostic and prognosis approaches. Metabolomics is considered an essential tool in precision medicine, which aims for personalized prevention and tailor-made treatments. Nevertheless, multiple pitfalls may be encountered in clinical metabolomics during the entire workflow, hampering the quality of the data and, thus, the biological interpretation. This review describes the challenges underlying metabolomics-based experiments, discussing step by step the potential pitfalls of the analytical process, including study design, sample collection, storage, as well as preparation, chromatographic and electrophoretic separation, detection and data analysis. Moreover, it offers practical solutions and strategies to tackle these challenges, ensuring the generation of high-quality data.
77 citations
TL;DR: This review discusses strategies for the identification of metabolites in complex biological mixtures, as encountered in metabolomics, which have emerged in the recent past, including NMR database-assisted approaches and novel combinations of NMR and MS analysis methods.
Abstract: This review discusses strategies for the identification of metabolites in complex biological mixtures, as encountered in metabolomics, which have emerged in the recent past. These include NMR database-assisted approaches for the identification of commonly known metabolites as well as novel combinations of NMR and MS analysis methods for the identification of unknown metabolites. The use of certain chemical additives to the NMR tube can permit identification of metabolites with specific physical chemical properties.
75 citations
TL;DR: A proof of concept study demonstrating the feasibility of bioanalysis of large therapeutic proteins at intact level using LC-HRMS based on deconvoluted high-resolution MS (HRMS) peaks.
Abstract: Aim: The commonly used LC–MS workflow to quantify protein therapeutics in biological samples is ‘bottom-up’ approach. In this study, the aim is to establish ‘top-down’ approach for absolute quantitation of therapeutic antibodies or proteins of similar sizes in biological samples at intact level. Materials & methods: Using a recombinant human monoclonal antibody as the model molecule, we present a workflow to measure large therapeutic proteins in plasma at intact level based on deconvoluted high-resolution MS (HRMS) peaks. A novel MultiQuant™ software function was developed to automatically deconvolute the peaks and process the data. Results & conclusion: The workflow showed satisfying performance. This is a proof of concept study demonstrating the feasibility of bioanalysis of large therapeutic proteins at intact level using LC-HRMS.
54 citations
TL;DR: The advantages of microfluidic devices in drug screening are introduced, and the critical factors which influence device design are outlined, highlighting recent innovations and advances in the field including a summary of commercialization efforts on micro fluidic cell chips.
Abstract: The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell–drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers.
53 citations
TL;DR: Seven α-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity and had the most intense in vivo signals.
Abstract: Background: Little or no pharmacological or toxicological data are available for novel psychoactive substances when they first emerge, making their identification and interpretation in biological matrices challenging. Materials & methods: A new synthetic cathinone, α-pyrrolidinopentiothiophenone (α-PVT), was incubated with hepatocytes and samples were analyzed using liquid chromatography coupled to a Q ExactiveTM Orbitrap mass spectrometer. Authentic urine specimens from suspected α-PVT cases were also analyzed. Scans were data mined with Compound Discoverer™ for identification and structural elucidation of metabolites. Results/conclusion: Seven α-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity. α-PVT dihydroxypyrrolidinyl, α-PVT 2-ketopyrrolidinyl, α-PVT hydroxythiophenyl and α-PVT thiophenol had the most intense in vivo signals.
46 citations
TL;DR: The application of ruthenium complex, luminol and nanomaterial-based ECL biosensors to making measurements in biological matrices over the past 4 years is reviewed.
Abstract: Electrogenerated chemiluminescence (ECL) is the production of light via electron transfer reactions between electrochemically produced reagents. ECL-based biosensors use specific biological interactions to recognize an analyte and produce a luminescent signal. Biosensors fabricated with novel biorecognition species have increased the number of analytes detected. Some of these analytes include peptides, cells, enzymes and nucleic acids. ECL biosensors are selective, simple, sensitive and have low detection limits. Traditional methods use ruthenium complexes or luminol to generate ECL. Nanomaterials can be incorporated into ECL biosensors to improve efficiency, but also represent a new class of ECL emitters. This article reviews the application of ruthenium complex, luminol and nanomaterial-based ECL biosensors to making measurements in biological matrices over the past 4 years.
44 citations
TL;DR: After normalizing the spot-to-spot hematocrit variations, peptide surrogates of protein biomarkers could be accurately quantitated in DBS and enabled detection of changes over time, thus offering an effective solution for longitudinal personal monitoring of biomarkers relevant in health and disease.
Abstract: Background: The use of DBS for quantitative protein biomarker measurement has been hindered by issues associated with blood hematocrit variations and lack of detection sensitivity, particularly when multiple biomarkers are measured. Materials & methods: An automated, multiplexed SISCAPA analysis was used to normalize blood volume variations in DBS and quantify proteins of varying abundance in longitudinal specimens. Conclusion: The results showed that after normalizing the spot-to-spot hematocrit variations, peptide surrogates of protein biomarkers could be accurately quantitated in DBS. This allowed the establishment of baselines for a variety of biomarkers in multiple individuals and enabled detection of changes over time, thus offering an effective solution for longitudinal personal monitoring of biomarkers relevant in health and disease.
44 citations
Bristol-Myers Squibb1, Hoffmann-La Roche2, Regeneron3, Genentech4, Charles River Laboratories5, Janssen Pharmaceutica6, Durham University7, Daiichi Sankyo8, Health Canada9, Pfizer10, Silver Spring Networks11, GlaxoSmithKline12, National Health Surveillance Agency13, Shire plc14, Biogen Idec15, Amgen16, Merck Serono17, Novartis18
TL;DR: This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance.
Abstract: The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
43 citations
TL;DR: An overview of the available technologies for producing antigen arrays is provided, some of the technical and methodological considerations are highlighted and their applications as discovery tools are discussed.
Abstract: Autoantibodies are a key component for the diagnosis, prognosis and monitoring of various diseases. In order to discover novel autoantibody targets, highly multiplexed assays based on antigen arrays hold a great potential and provide possibilities to analyze hundreds of body fluid samples for their reactivity pattern against thousands of antigens in parallel. Here, we provide an overview of the available technologies for producing antigen arrays, highlight some of the technical and methodological considerations and discuss their applications as discovery tools. Together with recent studies utilizing antigen arrays, we give an overview on how the different types of antigen arrays have and will continue to deliver novel insights into autoimmune diseases among several others.
TL;DR: The potential and challenges of an integrated strategy based on LC-SWATH/MS for simultaneous drug metabolism and metabolomics studies was investigated and it was possible to characterize 28 vinpocetine metabolites in urine and detect endogenous metabolite expression changes in urine after the administration of a single dose of a model drug to rats.
Abstract: Aim: Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) has recently emerged as a powerful high resolution mass spectrometric data independent acquisition technique. In the present work, the potential and challenges of an integrated strategy based on LC-SWATH/MS for simultaneous drug metabolism and metabolomics studies was investigated. Methodology: The richness of SWATH data allows numerous data analysis approaches, including: detection of metabolites by prediction; metabolite detection by mass defect filtering; quantification from high-resolution MS precursor chromatograms or fragment chromatograms. Multivariate analysis can be applied to the data from the full scan or SWATH windows and allows changes in endogenous metabolites as well as xenobiotic metabolites, to be detected. Principal component variable grouping detects intersample variable correlation and groups variables with similar profiles which simplifies interpretation and highlights related ions and fragments. Princi...
TL;DR: The MS methods presented here measure the whole molecule and provide a platform to better understand the various circulating drug forms by allowing for variant quantitation, as a basic platform to be further developed for Good Practice (GxP) applications.
Abstract: Aim: Large-molecule biotherapeutic quantitation in vivo by LC–MS has traditionally relied on enzymatic digestion followed by quantitation of a ‘surrogate peptide’ to infer whole-molecule concentration. MS methods presented here measure the whole molecule and provide a platform to better understand the various circulating drug forms by allowing for variant quantitation. Results: An immunocapture LC–MS method for quantitation of a biotherapeutic monoclonal antibody from human plasma is presented. Sensitivity, precision and accuracy for each molecular portion are presented along with an example of glycoform variant quantitation. Conclusion: The method is presented as a basic platform to be further developed for Good Practice (GxP) applications, critical quality attribute analysis or general understanding of molecular forms present as required for the wide range of drug development processes.
TL;DR: A general understanding that biomarker methods validation cannot be adequately depicted by current PK-centric guidelines emerged as a consensus from the Crystal City VI meeting.
Abstract: Crystal City VI Workshop on Bioanalytical Method Validation of Biomarkers, Renaissance Baltimore Harborplace Hotel, Baltimore, MD, USA, 28-29 September 2015 The Crystal City VI workshop was organized by the American Association of Pharmaceutical Scientists in association with the US FDA to continue discussion on the bioanalysis of biomarkers. An outcome of the Crystal City V workshop, convened following release of the draft FDA Guidance for Industry on Bioanalytical Methods Validation in 2013 was the need to have further discussion on biomarker methods. Biomarkers ultimately became the sole focal point for Crystal City VI, a meeting attended by approximately 200 people and composed of industry scientists and regulators from around the world. The meeting format included several panel discussions to maximize the opportunity for dialogue among participants. Following an initial session on the general topic of biomarker assays and intended use, more focused sessions were held on chromatographic (LC-MS) and ligand-binding assays. In addition to participation by the drug development community, significant representation was present from clinical testing laboratories. The experience of this latter group, collectively identified as practitioners of CLIA (Clinical Laboratory Improvement Amendments), helped shape the discussion and takeaways from the meeting. While the need to operate within the framework of the current BMV guidance was clearly acknowledged, a general understanding that biomarker methods validation cannot be adequately depicted by current PK-centric guidelines emerged as a consensus from the meeting. This report is not intended to constitute the official proceedings from Crystal City VI, which is expected to be published in early 2016.
TL;DR: Steroid precursors can be measured in DBS and it is suggested to implement the method as a second tier testing for CAH in The Netherlands.
Abstract: Background: The aim of this study was to improve the sensitivity of the congenital adrenal hyperplasia (CAH) neonatal screening by including second-tier steroid profiling on a DBS using LC–MS. Results: We developed a method to measure the steroid profile in DBS and established gestational age-specific reference ranges of cortisol, cortisone, 11-deoxycortisol, 21-deoxycortisol, 17-hydroxyprogesterone, testosterone, Δ4-androstenedione, corticosterone and 11-deoxycorticosterone using 450 heel prick samples of neonates, participating in the Dutch Screening Program. Analyzing 92 cards with a positive CAH screening showed that only 21-deoxycortisol was 100% specific for diagnosed CAH patients. Conclusion: Steroid precursors can be measured in DBS and we suggest to implement the method as a second tier testing for CAH in The Netherlands.
TL;DR: This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance.
Abstract: The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event – A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excelle...
TL;DR: An LC-MS/MS method for the analysis of dabigatran, apixaban and rivaroxaban in human plasma has been successfully developed and validated and is easily applicable in the clinical management of patients on anticoagulation therapy.
Abstract: Aim: Novel oral anticoagulants are characterized by a wide therapeutic window, yet the determination of their plasma–drug concentrations may be useful in some clinical conditions. Results: An LC–MS/MS method for the analysis of dabigatran, apixaban and rivaroxaban in human plasma has been successfully developed and validated. The analysis of plasma samples from patients given other concomitant drugs revealed no significant interference. By reanalysis of samples from patients on anticoagulant therapy, we found the percentage difference in results between the concentration of repeat and the original sample to be within the threshold limit of 20% in 60 of 63 specimens. Conclusion: The developed LC–MS/MS assay is easily applicable in the clinical management of patients on anticoagulation therapy.
TL;DR: This review highlights recent progress in the domain of analytical lipidomics, with main emphasis on non-targeted methodologies for large scale clinical applications, as well as discusses some of the key challenges and opportunities in this field.
Abstract: Lipidomic analysis aims at comprehensive characterization of molecular lipids in biological systems. Due to the central role of lipid metabolism in many devastating diseases, lipidomics is being increasingly applied in biomedical research. Over the past years, advances in analytical techniques and bioinformatics enabled increasingly comprehensive and accurate coverage of lipids both in tissues and biofluids, yet many challenges remain. This review highlights recent progress in the domain of analytical lipidomics, with main emphasis on non-targeted methodologies for large scale clinical applications, as well as discusses some of the key challenges and opportunities in this field.
TL;DR: In this article, a series of ligand binding and LC-MS/MS hybrid assays, through different combinations of anti-idiotype (anti-Id), antipayload, or generic capture reagents, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays.
Abstract: Background: Antibody–drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. Methods: A series of ligand binding and LC–MS/MS (LB-LC–MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. Results & conclusion: Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.
TL;DR: The state of the art in metabolomics research is summarized, focusing on efforts to provide a more comprehensive metabolome coverage via improvements in two fundamental processes: sample preparation and MS analysis.
Abstract: Metabolomics, focusing on comprehensive analysis of all the metabolites in a biological system, provides a direct signature of biochemical activity. Using emerging technologies in MS, it is possible to simultaneously and rapidly analyze thousands of metabolites. However, due to the chemical and physical diversity of metabolites, it is difficult to acquire a comprehensive and reliable profiling of the whole metabolome. Here, we summarize the state of the art in metabolomics research, focusing on efforts to provide a more comprehensive metabolome coverage via improvements in two fundamental processes: sample preparation and MS analysis. Additionally, the reliable analysis is also highlighted via the combinations of multiple methods (e.g., targeted and untargeted approaches), and analytical quality control and calibration methods.
TL;DR: An overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation is presented.
Abstract: LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.
TL;DR: A new version of the antibody-conjugated drug assay for valine-citrulline-linked monomethylauristatin E (vcMMAE) ADCs was presented to assess the difference in ex vivo drug-linker stability of native-cysteine versus engineered cysteine ADCs and to determine the number of drugs per antibody of a native- Cysteine ADC in vivo.
Abstract: Background: Antibody–drug conjugates (ADCs) require multiple assays to characterize their PK. These assays can separately evaluate the ADC by quantifying the antibody or the conjugated drug and may give different answers due to assay measurement differences, heterogeneous nature of ADCs and potential biotransformations that occur in vivo. Results: We present a new version of the antibody-conjugated drug assay for valine-citrulline-linked monomethylauristatin E (vcMMAE) ADCs. A stable isotope-labeled internal standard, protein A affinity capture and solid-phase cleavage of MMAE using papain was used prior to LC–MS/MS analysis. Conclusion: The assay was used to assess the difference in ex vivo drug-linker stability of native-cysteine versus engineered cysteine ADCs and to determine the number of drugs per antibody of a native-cysteine ADC in vivo.
TL;DR: In this article, the authors evaluate changes to extraction recovery across time for one analyte, the glycopeptide antibiotic vancomycin, in plasma using two dried micro sampling formats, dried plasma spots and volumetric absorptive microsampling.
Abstract: The reliability of extraction recovery of an analyte in bioanalysis is fundamentally important for downstream analytical testing. For dried format microsamples, if the recovery changes with time the concentration in clinical samples, derived from calibration standards and alongside quality control samples prepared following different drying protocols, may not reflect the true result. The purpose of this paper was therefore to evaluate changes to extraction recovery across time for one analyte, the glycopeptide antibiotic vancomycin, in plasma using two dried microsampling formats, dried plasma spots and volumetric absorptive microsampling.
TL;DR: The immunoaffinity-LC-MS/MS assay validation results across seven antibody therapeutics, using comparable conditions, illustrate the 'plug-and-play' nature of the IA- LC- MS/MS mAb framework peptide assay format.
Abstract: Background: Biotherapeutics development requires validated assays in biological matrices for pharmacokinetic assessment. Historically, ligand-binding assays have been the predominant platform available. Recently, alternative hybrid methods, combining ligand-binding analyte enrichment with LC–MS detection have emerged. Methodology & results: The validation of an immunoaffinity (IA)-LC–MS/MS method to quantify a monoclonal antibody biotherapeutic in cynomolgus monkey serum is described. This method includes immunoaffinity capture of the antibody in serum, followed by enzymatic digestion and detection of a framework peptide. Using similar method conditions, six additional biotherapeutic assays were readily validated in different nonhuman mammalian species, including mouse, rat and monkey. Conclusion: The immunoaffinity-LC–MS/MS assay validation results across seven antibody therapeutics, using comparable conditions, illustrate the ‘plug-and-play’ nature of the IA-LC–MS/MS mAb framework peptide assay format.
TL;DR: This review will highlight the phenomenon of new psychoactive substances and review methods for oral fluid drug testing analysis using on-site tests, immunoassays and chromatographic methods.
Abstract: Oral fluid has become an important matrix for drugs of abuse analysis. These days the applicability is challenged by the fact that an increasing number of new psychoactive drugs are coming on the market. Synthetic cannabinoids and synthetic cathinones have been the main drug classes, but the diversity is increasing and other drugs like piperazines, phenethylamines, tryptamines, designer opioids and designer benzodiazepines are becoming more prevalent. Many of the substances are very potent, and low doses ingested will lead to low concentrations in biological media, including oral fluid. This review will highlight the phenomenon of new psychoactive substances and review methods for oral fluid drug testing analysis using on-site tests, immunoassays and chromatographic methods.
TL;DR: This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance.
Abstract: The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 2) discusses the recommendations for Hybrid LBA/LCMS and regulatory inputs from major global health authorities. Parts 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
TL;DR: The proposed method affords very low LODs and RSD and allowed its successful application to the determination of EDCs in human urine, blood and breast milk.
Abstract: Background: Humans are exposed to hazardous substances including endocrine-disrupting chemicals (EDCs). These compounds have been associated with some diseases such as cancer and ascribed adverse effects on life-essential organs. Results: The method, which allows the determination of both free and conjugated forms of EDCs, involves the liquid–liquid extraction from the sample with ethyl acetate, followed by its preconcentration and clean-up by SPE in a continuous system for the subsequent determination by GC–MS. The proposed method affords very low LODs and RSD. Conclusion: This allowed its successful application to the determination of EDCs in human urine, blood and breast milk. The most frequently founded were methylparaben, ethylparaben, bisphenol A and triclosan.
TL;DR: It is shown that it is possible to gain sensitivity when going to smaller scale LC-MS (UPLC-MS to μLC- MS) and a lower LOQ could be achieved for infliximab compared with a previously developed UPLC-ms method.
Abstract: Background: TNO Triskelion has applied its general workflow for the development of quantitative LC–MS methods for proteins in biological matrices to the quantification of infliximab in rat serum using bottom up μLC–MS/MS. Results/methodology: The general workflow consists of sample purification, analyte processing and LC–MS analysis. In the development of a quantitative μLC–MS/MS method for infliximab in rat serum the analyte processing part and the LC–MS part were optimized, in order to meet the different sample requirements of μLC–MS as compared with UPLC–MS. Using the optimized μLC–MS/MS method the LOQ was 75 ng/ml. Conclusion: The present study showed that it is possible to gain sensitivity when going to smaller scale LC–MS (UPLC–MS to μLC–MS). Due to the combination of a modified sample preparation approach and the application of μLC–MS a lower LOQ could be achieved for infliximab compared with a previously developed UPLC–MS method.
TL;DR: Breath volatiles were readily analyzed on a portable mass spectrometer through a simple inlet modification and induced changes in exhaled profiles were detectable with high sensitivity and measurable in real-time.
Abstract: Aim: Breath analyses have potential to detect early signs of disease onset. Ambient ionization allows direct combination of breath gases with MS for fast, on-line analysis. Portable MS systems would facilitate field/clinic-based breath analyses. Results & methodology: Volunteers ingested peppermint oil capsules and exhaled volatile compounds were monitored over 10 h using a compact mass spectrometer. A rise and fall in exhaled menthone was observed, peaking at 60–120 min. Real-time analysis showed a gradual rise in exhaled menthone postingestion. Sensitivity was comparable to established methods, with detection in the parts per trillion range. Conclusion: Breath volatiles were readily analyzed on a portable mass spectrometer through a simple inlet modification. Induced changes in exhaled profiles were detectable with high sensitivity and measurable in real-time.
TL;DR: This review summarizes the mechanisms and applications of some of the current most commonly used techniques in each category: hybridization-based assays commonly used by molecular biologists and chromatographic assays routinely used by chemists.
Abstract: Technical advances and demands for high-throughput accurate quantification of oligonucleotide therapeutics and biomarkers in pharmaceutical research and clinical diagnosis have aided evolution in quantitative bioanalysis of oligonucleotides. Many bioanalytical methods are available for absolute quantification of oligonucleotides in biological matrices. They can be broadly classified into two categories: hybridization-based assays commonly used by molecular biologists and chromatographic assays routinely used by chemists. Each category has its own advantages and disadvantages for specific applications. This review summarizes the mechanisms and applications of some of the current most commonly used techniques in each category.