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The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “Context of Use – COU”); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 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. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15–29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global 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. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

Aim: Capillary microsampling of 15 μl whole blood from fingersticks or heelsticks was used to collect pharmacokinetic (PK) samples from pediatric subjects in two projects. Results: In a mebendazole multisite study in Ethiopia and Rwanda in subjects between 1 and 16 years old, complete PK profiles (7 timepoints) could be obtained, although some of the fingerstick samples were contaminated by the dosing formulation. In a multisite study with a respiratory syncytial virus drug in children between 1 and 24 months old, sparse PK sampling was done (2 samples). All samples were successfully analyzed even though some capillaries were not properly filled. Conclusion: CMS shows potential for PK sampling in pediatrics but may need further optimization.

The 2018 12th Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9–13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, 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 bioanalysis involving LC–MS, hybrid ligand binding assay (LBA)/LC–MS and LBA/cell-based assays approaches. This 2018 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. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC–MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC–MS for biotherapeutics and regulatory agencies’ inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.

Aim: Quantitative LC–MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. Methodology & results: A set of 20 single stranded antisense oligonucleotides (ASO) and five siRNAs were extracted from plasma and tissue homogenates. Anion Exchange (AEX) SPE was selected as generic extraction approach, resulting in recoveries from plasma >70%. Extraction from tissue homogenates showed often more variation and lower recoveries. A proof of concept of a novel tailored hybridization extraction is demonstrated for two 16-mer reference OGNs. Conclusion: Two methods for extraction of OGNs were investigated and applied for quantitative analysis. The AEX-SPE is considered a more generic approach preferred when multiple compounds are evaluated. Hybridization extraction has great potential but critical reagents per analyte are needed.

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1–5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. 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 bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 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. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations on Innovation in Small Molecules and Oligonucleotides & Mass Spec Method Development Strategies for Large Molecules Bioanalysis. Part 2 (2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) and Part 3 (New Insights in Biomarkers Assays Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in drug discovery & development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and The Gene Therapy Bioanalytical Challenges) are published in volume 11 of Bioanalysis, issues 23 and 24 (2019), respectively.

Aim: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC–MS/MS. Calibration curves were established (0.1–50 μg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC–MS/MS were comparable with a validated ELISA. Conclusion: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC–MS/MS. The hybridization extraction in combination with LC–MS/MS is a novel extraction approach.

In Alzheimer's disease (AD) brain, one of the histopathological hallmarks is the neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. Currently many tau binding antibodies are under development to target the extracellular species responsible for the spreading of the disease in the brain. As such, an in-house developed antibody JNJ-63733657 with picomolar affinity towards tau phosphorylated at both T212 and T217 (further named p217+tau) was recently tested in phase I clinical trial NCT03375697. Following multiple dose administration in healthy subjects and subjects with AD, there were dose dependant reductions in free p217+tau fragments in cerebrospinal fluid (CSF) following antibody administration, as measured with a novel single molecule ELISA assay (Simoa PT3 x PT82 assay), demonstrating epitope engagement of the therapeutic antibody [Galpern, Haeverans, Janssens, Triana-Baltzer, Kolb, Li, Nandy, Mercken, Van Kolen, Sun, Van Nueten, 2020]. Total p217+tau levels also were reduced in CSF as measured with the Simoa PT3 x PT82 assay. In this study we developed an orthogonal immunoprecipitation - liquid chromatography - triple quadrupole mass spectrometry (IP-LC-TQMS) assay to verify the observed reductions in total p217+ tau levels. In this assay, an excess of JNJ-63733657 is added to the clinical CSF to ensure all p217+tau is bound by the antibody instead of having a pool of bound and unbound antigen and to immunoprecipitate all p217+tau, which is followed by on-bead digestion with trypsin to release surrogate peptides. Tryptic peptides with missed cleavages were monitored when phosphorylation occurred close to the cleavage site as this induced miscleavages. Compared with acidified mobile phases typically used for peptide analysis, reversed phase LC with mobile phase at basic pH resulted in sharper peaks and improved selectivity and sensitivity for the target peptides. With this setup a diphospho-tau tryptic peptide SRTPSLPTPPTREPK*2 could be measured with pT217 accounting for at least one of the phospho-sites. This is the first time that the presence of a diphopsho-tau peptide is reported to be present in human CSF. A two-dimensional LC-TQMS method was developed to remove matrix interferences. Selective trapping of diphospho-peptides via a metal oxide chromatography mechanism was achieved in a first dimension with a conventional reversed phase stationary phase and acidified mobile phase. Subsequent elution at basic pH enabled detection of low picomolar p217+tau levels in human CSF (lower limit of quantification: 2 pM), resulting in an approximate 5-fold increase in sensitivity. This enabled the quantification of total p217+tau in CSF leading to the confirmation that in addition to reductions in free p217+tau levels total p217+tau levels were also reduced following administration of the tau mAb JNJ-63733657, correlating with the previous measurement with the PT3 x PT82 Simoa assay. An orthogonal sample clean-up using offline TiO2/ZrO2 combined with 1DLC-TQMS was developed to confirm the presence of mono-ptau (pT217) tryptic peptides in CSF. ispartof: JOURNAL OF CHROMATOGRAPHY A vol:1651 ispartof: location:Netherlands status: published

Sensitivity is often a critical parameter in quantitative bioanalyses in drug development. For liquid-chromatography-based methods, sensitivity can be improved by reducing the column diameter, but practical sensitivity gains are limited by the reduced sample loading capacity on small internal diameter (I.D.) columns. We developed a set-up that has overcome these limitations in sample loading capacity. The set-up uses 4 columns with gradually decreasing column diameters along the flow-path (2.1 → 1 → 0.5 → 0.15 mm). Samples are pre-concentrated on-line on a 2.1 mm I.D. trapping column and back flushed to a 1 mm I.D. UHPLC analytical column and separated. The peak(s) of interest are transferred using heartcutting to a second trapping column (0.5 mm I.D.), which is back-flushed to a 0.15 mm I.D. micro-UHPLC analytical column for orthogonal separation. The proof of concept of the set-up was demonstrated by the simultaneous analysis of midazolam and 1'-hydroxy midazolam in plasma by injection of 80 μL of protein precipitated plasma. The 4-column funnel set-up proved to be robust and resulted in a 10-50 times better sensitivity compared to a trap-elute approach and 250-500 fold better compared to direct micro-UHPLC analysis. A lower limit of quantification of 100 fg/mL in plasma was obtained for both probe compounds.

The strategy of outsourcing bioanalytical services at Janssen has been evolving over the last years and an update will be given on the recent changes in our processes. In 2016, all internal GLP-related activities were phased out and this decision lead to the re-orientation of the in-house bioanalytical activities. As a consequence, in-depth experience with the validated bioanalytical assays for new drug candidates is currently gained together with the external partner, since development and validation of the assay and execution of GLP preclinical studies are now transferred to the CRO. The evolution to externalize more bioanalytical support has created opportunities to build even stronger partnerships with the CROs and to refocus internal resources. Case studies are presented illustrating challenges encountered during method development and validation at preferred partners when limited internal experience is obtained or with introduction of new technology.

Aim: To evaluate alternative analytical strategies to extend the dynamic range in quantitative LC–MS/MS. Methods & results: Two approaches based on prior or no prior knowledge of expected exposure levels were evaluated. These approaches make use of two analytical strategies, which include the use of more than one injection volume or dilution of sample extract with solvents or solvent mixtures. A total of 16 compounds with varying logP values were classified into polar and nonpolar groups and used in this evaluation. From the two analytical strategies, three workflows were derived. Conclusion: All three workflows were successfully evaluated and resulted in good accuracy (80–120%) for all the compound groups.

Aim: Microsampling in preclinical pharmacokinetics (PK) studies is currently widely adopted across the pharmaceutical industry. Materials & methods: The European Bioanalysis Forum liquid microsampling consortium member companies assessed the accuracy and precision of handheld pipettes and microcapillaries at volumes of less than 10 μl. The following key factors on pipetting performance were also evaluated: Pipette type (positive displacement, air displacement and microcapillary), experience of user and the liquid type. Water was selected as a best-case scenario for accuracy and precision determination and blood plasma as a ‘real world’ bioanalysis sample type. Conclusion: Accuracy and precision on the pipetted volume decreased at lower volumes and experienced laboratory technicians performed better compared with the infrequent users. With respect to the pipetting devices used, microcapillaries showed better or equivalent accuracy and precision compared with handheld pipettes across the volume range 1–8 μl independent of the matrix used.

Following the completion of a detailed experimental protocol into the potential inhomogeneity of capillary liquid microsamples, which was performed at seven European Bioanalysis Forum member companies, the summary and conclusion on the data are reported here. It has been demonstrated that it is possible to generate homogeneous samples using these microsampling techniques; that the resultant microsamples can be accurate and precise and that capillary liquid microsampling data can be consistent with conventional larger volume plasma samples. However, the data contain some variability which is contributed to by the different range of experiences that each investigating site had with these techniques. Therefore, knowledge of the compounds, well-designed experiments and experience with these techniques are essential for the delivery of high quality data.

High-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies. This study aimed to investigate the possibilities and limitations of high-throughput Quan/Qual analysis by ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS), to develop a widely applicable Quan/Qual UHPLC-HRMS method and to provide recommendations for Quan/Qual method development. A widely applicable 4.25-min UHPLC method for small-molecules was used to investigate and optimize mass spectrometric parameters of a Synapt G2S for Quan/Qual analysis. The method was applied on a rat metabolomics study investigating the effect of the fasting state and administration of a dosing vehicle on the rat plasma metabolic profile. scan mode with a short scan time of 30 ms. This 4.25-min Quan/Qual analysis method enabled quantification with accuracy and precision values ≤ 20% at the lowest quality control (QC) level and ≤15% at higher QC levels for 16 out of 19 tested analytes. It provided both parent m/z values and fragmentation spectra for compound identification with limited loss of chromatographic resolution and it revealed biologically relevant metabolites in its application to the metabolomics study. Quan/Qual method development requires balancing between the amount of qualitative data, the quality of the quantitative data and the analysis time. Recommendations are provided for MS resolution, scan mode, scan rate, smoothing and peak integration in Quan/Qual method development and analysis. BACKGROUND METHODS RESULTS CONCLUSION

Coproporphyrins are proposed as endogenous biomarkers of hepatic Organic Anion Transporting Polypeptide (OATP)1B functional activity. In this study, a new sample extraction method based on a mixed-mode anion exchange sorbent (SPE clean-up using Oasis 30mg Max 96 well plates) was developed for absolute quantification of coproporphyrin I and III (CP-I and CP-III) in human plasma. Chromatographic separation was performed with an Ace Excel 2 C18 PFP, 3μm, 2.1×150mm, maintained at 60°C. A 10mM ammonium formate containing 0.1% HCOOH and acetonitrile (100%) was used as mobile phase A and B, respectively. Mass transition, m/z 655.3→596.3 was selected to monitor CP-I and CP-III, while m/z 659.3→600.3 transition was used for the stable isotope labelled internal standard. Optimization of the liquid chromatography tandem mass spectrometry method ensured a lower limit of quantification (LLOQ) of 20pg/mL. Both CP-I and CP-III had extraction recoveries of 70%. The calibration range was 0.02-100ng/mL for both CP-I and CP-III, yielding calibration curves with correlation coefficients greater than 0.988. Inter day precision (CV9%) and accuracy (84.3-103.9%) complied with the recommendation of the European Bioanalytical Forum. The optimized method was used to analyse plasma samples originating from three independent clinical studies. Obtained CP-I and CP-III plasma baseline levels in healthy volunteers were in good agreement with previously published data. Moreover, CP-I and CP-III plasma levels in human subjects dosed with a clinically confirmed OATP inhibitor were significantly increased compared to their baseline levels. These data demonstrate the potential of CP-I and CP-III as endogenous biomarkers to predict the drug-drug interaction (DDI) related to hepatic OATP1B inhibition. Stability of CP-I and CP-III in plasma and solvents under different processing and storage conditions was also evaluated.

Aim: Capillary microsampling (CMS) to collect microplasma volumes is gradually replacing traditional, larger volume sampling from rats in GLP toxicology studies. Methodology: About 32 µl of blood is collected with a capillary, processed to plasma and stored in a 10- or 4-µl capillary which is washed out further downstream in the laboratory. CMS has been standardized with respect to materials, assay validation experiments and application for sample analysis. Conclusion: The implementation of CMS has resulted in blood volume reductions in the rat from 300 to 32 µl per time point and the elimination of toxicokinetic satellite groups in the majority of the rat GLP toxicology studies. The technique has been successfully applied in 26 GLP studies for 12 different projects thus far.

Janssen Research & Development, a Division of Janssen Pharmaceutica N.V., Belgium The role of MS in peptide quantification For quantitative analysis of small-molecule drugs, LC–MS is considered the gold standard. However, focusing on peptide drugs, the majority of peptide development programs still rely on immunoassays for evaluation of exposure of the peptide drug in study samples. The advantages of these assays are the unrivaled sensitivity and the high throughput. Selectivity issues and method development times are, on the other hand, often rate-limiting. LC–MS technology can offer an alternative to improve selectivity and reduce method development time (no reagent preparation needed), but at the expense of lower sensitivity and throughput. Therefore, in our portfolio, for early peptide drug programs, quantitative workflows with LC–MS are preferred to address the short cycle times requested at this stage. In development and also with the aim to evaluate anti-drug antibody response, immunological assays are primarily preferred. An LC–MS assay can then be requested as an orthogonal assay to confirm the selectivity of the immunoassay or to address specific issues. In rare cases, the immunological assay is confronted with considerable challenges and LC–MS(/MS) can be developed faster and with more confidence to support the program.

Aim: A procedure was developed for the assessment of photochemical stability of drugs in blood and plasma under standardized conditions. The procedure avoids a variable outcome of photochemical stability experiments and tests relevant worst case conditions so that unnecessary light protection is avoided. Results/methodology: Blood and plasma were spiked with a mixture of drugs and incubated in a Suntest CPS+, in the laboratory on the bench and near the window on a sunny summer day. The results were compared. Discussion/conclusion: No protection from light, limited protection from light and full protection from light are advised for drugs that are stable in plasma in the Suntest CPS+ at 250 W/m2 for at least 30 min, for 5–30 min and for

Background: Peptides represent a growing class of potential drugs. Information on metabolic clearance can be valuable for peptide drug development but different challenges are encountered with the identification of peptide metabolites in comparison to the process used for small-molecule therapeutics. Results: Enfuvirtide was selected as a test compound and dosed intravenously at 2 mg/kg to rats. Plasma samples were collected and analyzed on two different quadrupole-TOF instruments in positive and negative ion modes. Different post-acquisition processing tools were evaluated to identify the metabolites of a peptide drug in the presence of an in vivo matrix. Charge state filtering and ion mobility extraction were applied to reduce the matrix background and combined with more comprehensive software tools generally used for large molecule analyses as well as tools designed for small-molecule metabolite identification work. Conclusion: Both ion mobility spectrometry and charge state filtration proved to be successful in extracting peptide ions and significantly reducing background signals. Both small- and large-molecule software tools contain specific capabilities that could be usefully combined in a single package for peptide metabolite identification.

Further to the discussions on the bioanalysis of samples generated using microsampling techniques [1–6] and more specifically the ongoing work of the European Bioanalysis Forum (EBF) Liquid Microsampling Consortium [7], this article seeks to highlight some of the ‘philosophical’ aspects around liquid microsampling and to introduce some of the experimental elements that will form part of future efforts by the Consortium. Discussion will be focused on three major areas: sample manipulation, homogeneity of samples and validation of assays. The scope of this manuscript will be liquid microsampling and will not focus on adsorption techniques such as dried blood spots (DBS) and solid phase microextraction. Different microsampling techniques have created great interest from toxicokinetic (TK) and pharmacokinetic (PK) scientists, since they offer the potential to reduce sample volumes for exposure assessment in biofluids. For preclinical studies microsampling can facilitate the generation of serial profiles in rodent exposure evaluation studies, rather than working with composite designs. Microsampling has facilitated the removal of satellite animal groups leading to substantial reductions in the number of animals required and the reduction or elimination of rodent warming. Benefits in the clinical environment include the ability to take reduced sample volumes from pediatric, elderly and critically ill patient populations. In a recent publication the NC3R group proposes the following definition of a blood microsample: the sample should ideally not contain more than 50 μl whole blood [8]. As a consequence the subsequent plasma volumes are 20 μl or lower. Another, more philosophical consideration would be the impact of sampling volume on the animal or subject; for example, 50 μl of blood may not be considered as a ‘microsample’ in the context of juvenile animals or newborn babies. Two major liquid microsampling approaches can be envisaged

The present study investigated the practical use of modern ultra-high performance liquid chromatography (UHPLC) separation techniques for drug metabolite profiling, aiming to develop a widely applicable, high-throughput, easy-to-use chromatographic method, with a high chromatographic resolution to accommodate simultaneous qualitative and quantitative analysis of small-molecule drugs and metabolites in biological matrices. To this end, first the UHPLC system volume and variance were evaluated. Then, a mixture of 17 drugs and various metabolites (molecular mass of 151-749Da, logP of -1.04 to 6.7), was injected on six sub-2μm particle columns. Five newest generation core shell technology columns were compared and tested against one column packed with porous particles. Two aqueous (pH 2.7 and 6.8) and two organic mobile phases were evaluated, first with the same flow and temperature and subsequently at each column's individual limit of temperature and pressure. The results demonstrated that pre-column dead volume had negligible influence on the peak capacity and shape. In contrast, a decrease in post-column volume of 57% resulted in a substantial (47%) increase in median peak capacity and significantly improved peak shape. When the various combinations of stationary and mobile phases were used at the same flow rate (0.5mL/min) and temperature (45°C), limited differences were observed between the median peak capacities, with a maximum of 26%. At higher flow though (up to 0.9mL/min), a maximum difference of almost 40% in median peak capacity was found between columns. The finally selected combination of solid-core particle column and mobile phase composition was chosen for its selectivity, peak capacity, wide applicability and peak shape. The developed method was applied to rat hepatocyte samples incubated with the drug buspirone and demonstrated to provide a similar chromatographic resolution, but a 6 times higher signal-to-noise ratio than a more traditional UHPLC metabolite profiling method using a fully porous particle packed column, within one third of the analysis time. In conclusion, a widely applicable, selective and fast chromatographic method was developed that can be applied to perform drug metabolite profiling in the timeframe of a quantitative analysis. It is envisioned that this method will in future be used for simultaneous qualitative and quantitative analysis and can therefore be considered a first important step in the Quan/Qual workflow.

Plasma protein binding (PPB) is an important parameter for a drug’s efficacy and safety that needs to be investigated during each drug-development program. Even though regulatory guidance exists to study the extent of PPB before initiating clinical studies, there are no detailed instructions on how to perform and validate such studies. To explore how PPB studies involving bioanalysis are currently executed in the industry, the European Bioanalysis Forum (EBF) has conducted three surveys among their member companies: PPB studies in drug discovery (Part I); in vitro PPB studies in drug development (Part II); and in vivo PPB studies in drug development. This paper reflects the outcome of the three surveys, which, together with the team discussions, formed the basis of the EBF recommendation. The EBF recommends a tiered approach to the design of PPB studies and the bioanalysis of PPB samples: ‘PPB screening’ experiments in (early) drug discovery versus qualified/validated procedures in drug development.

Background: With an increased interest in peptides and proteins as potential new drug candidates, new approaches for sensitive and selective quantitative analysis are required. LC–MS/MS analysis provides a good alternative to immunoassays with reduced method development times and increased specificity. Results: We have evaluated two state-of-the-art triple quadrupole and high-resolution TOF mass spectrometers with respect to their performance for quantification of six peptides (glufibrinopeptide B, somatostatin, enfuvirtide, TRI1144, C34 and exenatide). The peptides were spiked into protein-precipitated plasma supernatant. Triple quadrupole quantification was performed in SRM mode, and in high-resolution, MS narrow-width extracted chromatograms were generated for quantification. Specificity, accuracy, reproducibility and robustness were found to be comparable between the two instruments. The triple quadrupole instrument is still the most sensitive instrument for quantification of peptides with a median factor of about four-times higher sensitivity (based on LLOQ evaluation). Conclusion: Based on sensitivity, the newest generation triple quadrupole MS systems are still the preferred technology for quantification of peptides. Since the sensitivity difference between triple quadrupole instruments and the new-generation high-resolution TOF-MS instruments is minor, the latter offer a useful alternative whenever additional selectivity is preferred or the use of a generic approach not requiring method optimization is advantageous.

Background: Amyloid peptides are established biomarkers for Alzheimer’s disease but their analysis presents major challenges. Results: In this article, we describe the use of ultra high-performance LC and API4000 triple quadrupole instrumentation for the quantification of amyloid peptides (Aβ1-38 and Aβ1-42) in cerebrospinal fluid (CSF), using electrospray ionization with negative ion multiple reaction monitoring transitions. Sample preparation was simplified by the addition of acetonitrile and ammonium hydroxide. Although excellent sensitivity and precision was demonstrated, we observed differences in matrix suppression effects when using artificial CSF versus canine CSF for calibration curves and quality control samples. Conclusion: A case study shows that the method can be used to determine the relative levels of two key peptides (Aβ1-38 and Aβ1-42) compared with their predose values (a screening assay) in support of preclinical studies.

An LC-MS/MS method for the simultaneous quantification of azide and azidoalanine in bacterial and mammalian cellular systems was developed to support mutagenicity evaluation of azido containing nucleoside drugs. With the aim to enhance ionization efficiency and chromatographic retention, derivatisation with dansyl chloride was optimized; study samples (diluted 1/10) were derivatised for 2 h at room temperature and subsequently analysed with reversed phase liquid chromatography in combination with electrospray mass spectrometry in the positive ion mode. Good linearity and accuracy were obtained with a lower limit of quantification of 10.0 ng mL−1 for both analytes. The method was applied in mutagenicity experiments and showed time-dependent release of azide, but no formation of azidoalanine could be detected.