Aim: Investigations have shown that for the antibody-drug conjugate (ADC) belantamab mafodotin, concentrations of the cysteine-conjugated metabolite, Cys-mcMMAF, were overestimated in the presence of the ADC during sample processing when utilizing a historical SPE method. Results: A new assay was developed utilizing an acidic protein precipitation to remove the ADC early in the extraction process, thus eliminating the risk of overestimating Cys-mcMMAF in the presence of belantamab mafodotin. In vitro experiments demonstrated a linear relationship between the concentration of belantamab mafodotin and the release of Cys-mcMMAF. Extensive stability assessments were performed to cover storage of study samples. Conclusion: This work emphasized the critical importance of understanding the performance of a bioanalytical method for free toxic payload in the presence of the ADC.

Inducible T-cell costimulator (ICOS), a homodimeric protein expressed on the surface of activated T-cells, is being investigated as a potential therapeutic target to treat various cancers. Recent studies have reported aberrant increases in the soluble form of ICOS (sICOS) in human serum in disease-state patients, primarily using commercial ELISA kits. However, results from our in-house immunoassay did not show these aberrant increases, leading us to speculate that commercial sICOS ELISAs may be prone to interference. We directly tested that hypothesis and found that one widely used commercial kit yields false-positives and is prone to human anti-mouse antibody interference. We then analyzed a panel of healthy, cancer, chronic hepatitis C virus, systemic lupus erythematosus, and diffuse cutaneous systemic sclerosis human serum using our in-house immunoassay and reported the measured sICOS concentrations in these populations. Since even well characterized immunoassay methods are prone to non-specific interference, we also developed a novel sICOS LC-MS/MS method to confirm the results. Using these orthogonal approaches, we show that sICOS is a low abundance soluble protein that cannot be measured above approximately 20 pg/mL in human serum.

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.

We describe an analytical strategy allowing for the direct quantification of stable isotope label incorporation in newly synthesized proteins following administration of the stable isotope tracer deuterium oxide. We present a demonstration of coupling high-resolution mass spectrometry, metabolic stable isotope labeling, and MS/MS-based isotopologue quantification for the measurement of protein turnover. Stable isotope labeling with deuterium oxide, followed by immonium ion isotopologue quantification, is a more sensitive strategy for determining protein fractional synthesis rates compared to peptide centric mass isotopomer distribution analysis approaches when labeling time and/or stable isotope tracer exposure is limited and, as such, offers a great advantage for human studies.

Quantitative bioanalytical data are crucial in pharmaceutical research and development, allowing project teams to make informed scientific decisions on the progression of candidate molecules to medicines. Many challenges are often encountered during the bioanalysis of drugs in biological matrices which require resolution in a timely manner. In this publication, guidance is provided to bioanalytical scientists on how to identify potential problems before they become an obstacle for the drug development and to share our experiences dealing some of most common problems encountered in the bioanalytical laboratory. Relevant topics in bioanalysis such as stabilization approaches for glucuronides (Acyl and N-); prodrugs (phosphate and esters), amides, amines, N-oxides; bioanalysis of light sensitive molecules, halogenated drugs and lactones are discussed in this publication.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

Aim: GSKA is a compound that was in development in clinical trials. A bioanalysismethod to quantify GSKA using volumetric absorptive microsampling (VAMS) was developed and hematocrit (HCT) related assay bias was investigated. Methodology: After accurate sampling of 10 mu l blood, VAMS tips were air dried approximately 18 h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated. Conclusion: HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

BioanalysisVol. 9, No. 14 EditorialFree AccessPlatforms and techniques used for biomarker assays: where are we now?Charles Hottenstein, Matthew Szapacs, Kerensa Fuller & Christopher EvansCharles Hottenstein*Author for correspondence: E-mail Address: charles.s.hottenstein@gsk.com GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity & Biomarkers; 709 Swedeland Road, King of Prussia, PA 19406 USA, Matthew Szapacs GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity & Biomarkers; 709 Swedeland Road, King of Prussia, PA 19406 USA, Kerensa Fuller GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity and Biomarkers; Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK & Christopher Evans GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity & Biomarkers; 709 Swedeland Road, King of Prussia, PA 19406 USAPublished Online:24 Jul 2017https://doi.org/10.4155/bio-2017-0107AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: biomarkerligand binding assayLC–MSparallelismplatformsurrogate analytesurrogate matrixFirst draft submitted: 28 April 2017; Accepted for publication: 15 May 2017; Published online: 24 July 2017Currently, the term biomarker has many connotations, as such, it is important to properly define this term for the context of this editorial. In the broadest definition, biomarkers are "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention" [1]. However, in the context of biomarkers evaluated during the course of clinical drug trials, we can categorically divide the term into three categories which include biomarkers for determining risk (predisposition), biomarkers used for diagnosis (screening, prognosis and diagnosis) and biomarkers specific to pharmacology (predictive, target engagement, mechanism engagement, clinical engagement, surrogate and clinical end point).As the number of drugs targeting a specific biological mechanism increases, the pharmacodynamic (PD) biomarkers involved in that mechanism can provide a valuable understanding of target engagement, efficacy and/or safety to assist in guiding the drug development process for that mechanism. When measuring PD biomarkers in samples from preclinical and/or clinical studies, it is important that the robustness and limitations of the analytical methods as well as stability of biomarker in the matrix of interest is well understood allowing for minimal analytical variation and lack of biomarker degradation during sample collection.A variety of bioanalytical platforms are routinely applied to measurement of biomarker analytes, including: ligand-binding assays (LBA), LC–MS, flow cytometry and immunohistochemistry. Although each of these platforms can be used to understand the impact of a drug on PD end points, LBA and LC–MS are the two platforms most commonly utilized and will be the focus of this editorial. The most appropriate platform to monitor a given biomarker depends on the matrix, analyte and specific biological question to be answered. However, in some instances either platform could be used to support an analytical measurement, therefore understanding the biological question and/or problem and the benefits of a given platform should guide this selection. This editorial aims to address the challenges and benefits of either of these platforms in relation to the unique issues associated with specific biomarker analyte quantitation.Sensitive versus selective biomarker platformsAs a number of biomarkers of interest are present in matrices at very low concentrations (i.e., cytokines), development of a highly sensitive assay is a critical component to platform selection. However, as the need to develop ever more sensitive assays increases, problems with developing selective assays becomes more challenging. Historically, photometric LBAs were the most commonly used platform for both commercial and bespoke assays, however, the electrochemiluminescent format from Meso Scale Discovery (MSD) has over the past decade become the industry standard, with a large number of commercially available biomarker kits that can be used as is or optimized and validated for regulated use. Although the MSD platform offers improved selectivity and dynamic range over more traditional photometric methods, it represents a single vendor platform with proprietary instrumentation and microtiter plate labware. Where bespoke assays are developed, consistency in microtiter plate lot-to-lot variability of the background and overall signal range have caused issues when changing microtiter plate lots for established methods. Therefore, the benefits and risks should be closely considered when choosing single vendor platforms for biomarker quantitation.In addition, various vendors have been developing proprietary ultrasensitive instrumentation to allow quantitation of very low abundance biomarkers, including: MSD platform (S-Plex), Singulex and Quanterix Simoa with the various platforms providing different sensitivities depending on the analyte of interest [2,3]. Although these platforms can provide high sensitivity assays, they also suffer from additional challenges associated with selectivity from individual samples due to the low levels of analyte that are being detected and often lead to a trade-off between assay sensitivity and overall selectivity. It should be noted that the Singulex and Quanterix Simoa are also single-vendor platforms and the same considerations listed above for the MSD platform should apply when selecting these higher sensitivity instrument for use.One platform that has emerged to address inherent selectivity challenges identified in LBA platforms is LC–MS/MS. Through the use of immunocapture reagents followed by enzymatic digestion and selection of a surrogate peptide which is coupled to LC–MS, we and others [4–6] have shown that this platform can be used to develop highly selective and sensitive assays utilizing immunocapture reagents that may not be suitable for use in a typical LBAs. In addition to mitigating the observed selectivity issues in LBAs, there are also occasions when the LC–MS platform may provide the more sensitive analytical option as the selectivity and overall S/N of the assay is improved; therefore platform selection needs to be made on a case-by-case basis.As well as sensitivity, LBA formats often require less expensive specialized equipment and are often a more economical option than LC–MS platforms. In our laboratories, we employ all of the above described approaches for biomarker quantitation depending on the availability of reagents, while balancing the benefits of sensitivity and selectively when choosing one platform over another allowing one to accurately answer to biological question at hand.Measuring biomarkers in the presence of endogenous analyteAnother common challenge encountered during quantitation of biomarkers is determination of the absolute concentration of a biomarker. Unlike pharmacokinetic methods, where the calibration matrix is typically analyte free, allowing for the detection of absolute drug concentrations; biomarker matrices often contain some level of endogenous analyte and the biomarker bioanalyst often relies on either a depleted matrix approach to provide analyte free matrices for calibration purposes or the use of low- and high-concentration megapools with subsequent admixing of these pools to create a uniform control and potentially use for calibration purposes [7].However, both approaches are not without issue as depleting the matrix may remove other components and the megapools often are not available for novel or less well-studied biomarkers. With this in mind, the use of surrogate matrix or surrogate analyte strategies to generate a standard calibration curve without interference from endogenous biomarkers continues to be standard practice. Surrogate matrix strategies are the most common approaches and use buffer with added proteins like bovine serum albumin, commercially available matrices (SeraSub, UriSub) or other in-house mixtures of proteins in an aqueous buffer all with spiked analyte. The use of surrogate matrix requires that a parallelism assessment is completed by spiking analyte in both surrogate matrix and authentic matrix across the assay range. If the surrogate matrix curve is 'parallel' to the authentic matrix curve then the surrogate matrix is representative of the authentic matrix [8].As LC–MS continues to grow as a platform for biomarker bioanalytical support, the use of a surrogate analyte approach has become more commonplace. Surrogate analyte methodologies utilize an isotopically labeled biomarker that can be used to create a standard calibration curve in the authentic matrix as it has a unique mass that can be differentiated from the authentic, endogenous analyte using LC–MS [9]. This technique has enormous promise for development of biomarker assays as the authentic matrix that is identical to that of the samples can be used for study support and allow for absolute quantitation. In addition, the use of longer gradients or high-resolution LC–MS provides increased confidence and confirmation of the endogenous analyte being measured and demonstrates that it is not a cross-reactive species or a molecule with a similar mass transition in the MS.Measuring the various forms of biomarker targetsOne of the most common challenges for biotherapeutics is the development of a free, total and/or complex target engagement assays. Traditionally, LBA-based platforms have been used for the quantitation of free, total or complex target engagement and have been reviewed previously [10]. However, the ex vivo manipulation of samples required before analysis (i.e., sample dilution, extraction, etc.) can cause an equilibrium bias between free, total and complex forms in solution. Therefore, the development of assays that provide 'true' free, total and/or complex measurements is quite challenging and the assays often do not demonstrate agreement when used in combination.In our laboratories, we prefer the combination of total and complex assay measurements to understand biomarker PDs in relation to the administered drug and feel that free assays are the most challenging and often generate less robust data for a number of reasons including equilibrium shifts from native sample matrix upon dilution, the density of the solid phase capture mechanism concentration compared with analyte concentration in solution and the length of primary incubation times. Typically, we avoid free assays unless the methods are highly characterized and the quality of the data can be assured. Overall, drug target complex and total target assays are less susceptible to these issues and have been shown to demonstrate the best measure of target engagement and target concentration [11].ConclusionThe best-suited quantitation platform for a given biomarker measurement depends on a number of factors including the required sensitivity, selectivity with the ability to use the actual matrix and actual biomarker analyte or will a surrogate or combination approach need to be used. The ability to select from more than one analytical option in many situations is beneficial for the biomarker bioanalyst and allows for the ability to proactively address analytical issues allowing for high-quality reliable data for internal or pivotal decision-making purposes regarding the safety or efficacy of a drug candidate over the course of the drug development process. With the recent bioanalytical advancements and ability to couple platforms (hybrid LBA/LC–MS), the biomarker bioanalyst now has the tools that are critical to address and resolve the unique biological questions and challenges that exist over the course of the drug development process.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1 Atkinson AJ, Colburn WA, Degruttola VG, Demets DL. Surrogate endpoints: preferred definitions and conceptual framework* biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin. Pharmacol. Ther. 69(3), 89–95 (2001).Crossref, Medline, Google Scholar2 Oberoi P, Suschak J, Stenglin M et al. True sensitivity of immunoassays: are concentrations of low abundance analytes real or artifacts? J. Immunol. 196(Suppl. 1), 138–139 (2016).Google Scholar3 Fischer SK, Joyce A, Spengler M et al. Emerging technologies to increase ligand binding assay sensitivity. AAPS J. 17(1), 93–101 (2015).Crossref, Medline, CAS, Google Scholar4 Bronsema KJ, Bischoff R, Pijnappel WP, van der Ploeg AT, van de Merbel NC. Absolute quantification of the total and antidrug antibody-bound concentrations of recombinant human α-glucosidase in human plasma using protein G extraction and LC–MS/MS. Anal. Chem. 87(8), 4394–4401 (2015).Crossref, Medline, CAS, Google Scholar5 Palandra J, Finelli A, Zhu M, Masferrer J, Neubert H. Highly specific and sensitive measurements of human and monkey interleukin 21 using sequential protein and tryptic peptide immunoaffinity LC–MS/MS. Anal. Chem. 85(11), 5522–5529 (2013).Crossref, Medline, CAS, Google Scholar6 Ackermann BL, Berna MJ. Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers. Expert Rev. Proteomics 4(2), 175–186 (2007).Crossref, Medline, CAS, Google Scholar7 Arnold ME, Booth B, King L, Ray C. Workshop Report: Crystal City VI – Bioanalytical Method Validation for Biomarkers. AAPS J. 18(6), 1366–1372 (2016).Crossref, Medline, CAS, Google Scholar8 Jones BR, Schultz GA, Eckstein JA, Ackermann BL. Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules. Bioanalysis 4(19), 2343–2356 (2012).Link, CAS, Google Scholar9 Jenkins RG. Accuracy: a potential quandary in regulated bioanalysis of 'endogenous' analytes. Bioanalysis 8(23), 2393–2397 (2016).Link, CAS, Google Scholar10 Talbot JJ, Calamba D, Pai M, Ma M, Thway TM. Measurement of free versus total therapeutic monoclonal antibody in pharmacokinetic assessment is modulated by affinity, incubation time, and bioanalytical platform. AAPS J. 17(6), 1446–1454 (2015).Crossref, Medline, CAS, Google Scholar11 Lee JW, Kelley M, King LE et al. Bioanalytical approaches to quantify "total" and "free" therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development. AAPS J. 13(1), 99–110 (2011).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByProgress with Metabolomic Blood Tests for Gastrointestinal Cancer Diagnosis—An Assessment of Biomarker Translation10 October 2022 | Cancer Epidemiology, Biomarkers & Prevention, Vol. 31, No. 12An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatricesJournal of Chromatography A, Vol. 1676One-dollar microfluidic paper-based analytical devices: Do-It-Yourself approachesMicrochemical Journal, Vol. 165Development of a Meso Scale Discovery ligand-binding assay for measurement of free (drug-unbound) target in nonhuman primate serumYun Liu, Ronald Robinson, Thao Ung, Chrysanthe Spais, Justin Schreiber, Jacquelyn Lyons, Jean Husten, Hussein Hallak & Thelma Angeles22 March 2021 | Bioanalysis, Vol. 13, No. 7High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths4 February 2019 | Analytical Chemistry, Vol. 91, No. 5A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?Cytokine, Vol. 111 Vol. 9, No. 14 Follow us on social media for the latest updates Metrics History Published online 24 July 2017 Published in print July 2017 Information© 2017 Future Science LtdKeywordsbiomarkerligand binding assayLC–MSparallelismplatformsurrogate analytesurrogate matrixFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Assay multiplicity and complexity generally requires analytical staff to possess a certain amount of scientific nimbleness, in other words, the ability to apply a variety of formats or assay platforms

Supercritical fluid chromatography in combination with chiral stationary phases has proven to be a great tool for chiral resolution, alleviating some of the challenges associated with bioanalysis of stereoisomers.

BACKGROUND:FTIH studies can be challenging due to the varying dosing regimens and rapid data delivery. Chemists are asked to provide ultra-low limits of quantitation to provide an understanding of patient efficacy and safety in order to progress drug development. In a recent dermal study it became necessary to reduce the LLOQ of a small molecule drug from 50 to 1 pg/ml due to reductions in the dose and surface area of drug application.METHODOLOGY:The 50-fold increase in assay sensitivity necessitated the use of a high-resolution mass spectrometer (LC-HRMS) to separate matrix interferences observed when using a unit resolution triple quadrupole MS.CONCLUSION:A sensitive, robust assay was validated to support of a FTIH study using a LC-HRMS.

GSK1278863 is an investigative drug under investigation for treatment of anemia associated with chronic kidney disease. Its metabolism is primarily metabolized by P450 enzymes where 19 unique metabolic species have been identified. These include multiple products of mono-, di-, and tri-oxygenation. Initially, two separate and complex ultra high performance liquid chromatography (UHPLC) reverse phase methodologies were developed, validated and applied to measure parent and various predominant and circulating metabolites in numerous clinical studies. However, 5 of the 6 oxidative metabolites may exist in different stereoisomeric forms, resulting in 14 separate species; therefore a chiral methodology was required to determine which stereoisomeric forms circulated in human. A variety of conventional approaches were explored, where in the end a supercritical fluid chromatography (SFC) method was required to separate this complex mixture of 14 stereoisomeric metabolites; data from these experiments provided important information on which species circulate in human. The details of these methodologies will be discussed herein.

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.

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 excellence, improved quality and better regulatory compliance. This white paper is published in 3 parts due to length. This part (Part 1) discusses the recommendations for small molecules, peptides and small molecule biomarkers by LCMS. Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will be published in the Bioanalysis journal, issue 23.

This paper was developed with the support of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). IQ is a not-for-profit organization of pharmaceutical and biotechnology companies with a mission of advancing science-based and scientifically driven standards and regulations for pharmaceutical and biotechnology products worldwide. Within the IQ, various working groups (WG) have been formed, where the microsampling WG is committed to providing a scientific forum for the advancement of both wet and dry microsampling techniques within the pharmaceutical industry. This first output from the microsampling WG is to summarize and reflect on the current knowledge and opinions on DBS sampling, to stimulate discussion, and to encourage future creative applications of DBS sampling. Dried blood spot (DBS) sampling has established itself as an innovative sampling technique where wet blood is spotted onto absorbent paper or other paper materials and allowed to dry (1–4). DBS offers several potential benefits inherent to the technique, namely a low blood volume, simplified blood sample collection (5), and convenient sample storage and transfer. In certain applications, DBS sampling has been shown to stabilize certain analytes or metabolites without the addition of chemical modifiers (6–9). DBS has been routinely applied for decades in neonatal screening for phenylketonuria and other congenital metabolic disorders (10). The utility of DBS sampling has also been demonstrated for therapeutic drug monitoring (11) and for epidemiological studies (e.g., HIV and HBV detection/monitoring) (12) due to the practical advantages along with simplified sample collection and handling procedures. Finally, DBS can also be used for quantitative biomarker (PD) assessment from blood, where appropriate. However, the technique is relatively new to the pharmaceutical industry and to government regulators overseeing new drug applications. Nevertheless, over the past 5 to 7 years, the technique has been extensively evaluated for quantifying drug exposure in nonclinical and/or clinical studies in various stages of drug discovery and development. The ease to collect, transfer, store, and process small volumes of blood samples has generated considerable interest in providing utility in volume-limited situations (e.g., small rodent, human pediatric studies) for toxicokinetic (TK), pharmacokinetic (PK), or pharmacodynamic (PD) sampling. Discovery and nonclinical studies Rodent animal models are typically employed in these studies. The reduced blood volumes required for DBS can enable serial bleeding and, consequently, elimination of satellite animal groups and reduction of compound use. The ability to eliminate the satellite animal groups enables the assessment of exposure and toxic effects within the same animal. Studies involving expensive animal models (i.e., transgenic mice, knock-out mice, humanized mice, etc.) further highlight a persuasive scientific and economic case for DBS sampling since a complete pharmacokinetic profile can be obtained from a single study animal without the need for extra rodents merely for generating exposure data. These are perfectly in line with the principles of the 3Rs: reduction, refinement, and replacement of humane animal research (13–15). With greater emphasis from the regulatory authorities to study new drugs for infants, neonates, and pediatric populations, the requirement to conduct associated nonclinical juvenile rodent toxicity studies serves as an ideal scenario where the advantage of low blood volume in DBS sampling is undeniable. Although the advantages of DBS heavily favor rodent studies, it can also be used to refine non-rodent studies.

BioanalysisVol. 7, No. 19 Themed Issue: Derivatization in Bioanalysis – ForewordFree AccessChemical derivatization in bioanalysisHermes Licea Perez & Christopher A EvansHermes Licea Perez*Author for correspondence: E-mail Address: hermes.2.licea-perez@gsk.com Bioanalytical Sciences & Toxicokinetics, PTS-DMPK, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA & Christopher A Evans Bioanalytical Sciences & Toxicokinetics, PTS-DMPK, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USAPublished Online:3 Nov 2015https://doi.org/10.4155/bio.15.182AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: chemical derivatizationchiral analysischromatographic separationionization efficiencypolar compoundsselectivityApplicationsChemical derivatization has long proved itself as an analytical technique in bioanalysis to overcome problems associated with low ionization efficiency, compound instability, poor selectivity or unacceptable chromatographic performance (poor retention, bad peak shape and carryover issues) and even poor volatility for GC separation [1,2]. This technique is a powerful tool in many areas of chemistry including medical, forensic, food science, doping control and environmental disciplines. The goal of chemical derivatization is to modify the structure of the analyte (either a nucleophile or electrophile) using a chemical reagent (either an electrophile or nucleophile depending on the nature of the analyte) and, as a result, a new compound (the reaction's derivate) with improved chemical and physical properties for analysis is formed. The reaction conditions (amount of the reagent, reaction time and temperature, etc) are optimized in favor of the formation of the desired derivative with the highest possible reaction yield. Additional sample clean up procedures can be developed to eliminate unwanted byproducts and excess reagents, thereby minimizing analyte inferences upon analysis.Using chemical derivatization, analysis of the impossible becomes possible. Many examples of this have been presented in the literature; impacting GC, LC–MS/MS [1–3] and NMR [4–6] detection. Most notable has been the chromatographic separation of enantiomers through chiral derivatization using specific resolving reagents without the use of specialized chiral columns and separation conditions [7–9].ConsiderationsThe selection of the appropriate chemical reagent is essential for a successful derivatization and is dependent on the specific application. In general, if the target analyte is a nucleophile (compound with an excess of electrons), an electrophile (compounds with overall electron deficiency) is selected as reagent, and vice versa. Reagents need to be selective (target one specific site of molecule), thereby avoiding derivatization at multiple sites in the target molecule, metabolites or endogenous components. As an example, for a molecule containing both hydroxyl and amino functional groups the use of acid chlorides or anhydrides as derivitization reagents should be avoided as these will derivatize both functional groups. On the contrary, the use of dansyl chloride as a derivitization reagent is appropriate for amino and phenol functional groups as it does not react with aliphatic alcohols. Other requisites to be considered in the selection of the reagent include availability (commercially), purity and cost. Typically, the cost of reagents is minimal, and therefore does not represent a barrier for use.Using optimized conditions, chemical derivatization procedures are typically robust enough to be applied for pharmaceutical bioanalysis, and are capable of meeting regulatory expectations. This is usually demonstrated during a rigorous validation process where several parameters, including but not limited to, accuracy, precision, selectivity, matrix effect, etc. The selection of internal standard is essential to correct for any possible analyte loss during the various steps of sample handling and bioanalysis; thus ensuring robustness of the assay. When possible, a deuterium or 13C stable internal standard should be used, otherwise an analog with similar reactivity, recovery, and chromatographic properties could be substituted. Also, it is imperative to consider and evaluate, where possible, metabolic pathways of the analyte of interest; conversion of metabolites back to parent molecule need to be avoided during the derivatization procedure, as these processes often involve harsh conditions (pH, heat, long incubation times, etc.). Unfortunately, this can be complicated by the lack of reference standards for the metabolites, and the lack of metabolic information early in the drug development lifecycle due to differential or accelerated development strategies.Chemical derivatization as an art formThe use of chemical derivatization has declined in recent years as new separation technologies have evolved and become more commonplace. The evolution of supercritical fluid chromatography (SFC), for example, has opened a new avenue for chiral stereoisomeric analysis; thereby reducing the need for chiral derivatization in certain instances [10]. More sensitive generations of triple quadrupole mass spectrometric instrumentation with novel or improved ionization technologies are pushing the detection limits to low picogram levels, and as result the demand for chemical derivatization to improve assay sensitivity (through improved ionization or selectivity) have decreased. Other technologies including UHPLC; micro-/nano-LC (for better ionization efficiency); TOF instruments with ion mobility capability (electronic separation, rather than chemical/physical) have also contributed to the decline of chemical derivitization in the bioanalytical laboratory. With that said however, the technique is still applied for very complex separations where aforementioned technologies cannot make an adequate impact. Sometimes coupling chemical derivatization with one of these technologies has an enhanced/additive impact. In particular, combination of SFC with chiral derivatization has shown to be superior for chiral separation as compared with that SFC analysis (data not shown).Due to this decline of the technique and its complexity as compared with other analytical techniques, chemical derivatization has evolved into a specialty 'art form' in the laboratory, requiring specialized skills combined with a strong chemistry proficiency. As a consequence, fewer scientists in DMPK environments succeed to master the technique and become proficient in its application. The question then becomes how to preserve these skills and pass them on to the future generations of analytical scientists. Special issues such as this, review articles, book chapters, guidance containing experimental protocols will hopefully facilitate and promote the use of chemical derivatization as great analytical tool.OutlineThis themed issue covers advances in existing derivatization techniques used in bioanalytical research, as well as innovative new methods and approaches (e.g., combination of derivatization with microflow LC–MS and the idea of new chemical tagging techniques by Niwa et al. [11]).The issue aims to cover aspects related to: Derivatization methods in LC–MS bioanalysis (including HPLC);Peptide derivatization for analysis of protein therapeutics;Chiral derivatization reagents applied to biological samples (see Vashistha et al. [12]);Derivatization for analysis of endogenous compounds (see 'Beyond Classical Derivatization: Analyte 'derivatives' in the bioanalysis of endogenous and exogenous compounds' by Barnaby et al. [13], or 'Derivatization of steroids in biological samples for GC–MS and LC–MS analyses' by Marcos et al. [14]);Derivatization procedures in human doping control (see interesting review by Athanasiadou et al. [15]).While it is true that chemical derivatization is just another tool in the bioanalytical toolbox, it is a 'must have' for a DMPK laboratory and one that will continue to have an impact addressing many bioanalytical challenges.Therefore, if you don't like your analyte, change it (with chemical derivatization that is)!Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Papers of special note have been highlighted as: •• of considerable interestReferences1 Knapp D. Handbook of Analytical Derivatization Reactions. John Wiley & Sons, NY, USA (1979).•• Highly recommended reference.Google Scholar2 Handbook of Derivatives for Chromatography. Blau K, King GS (Eds). Heyden & Sons, London, UK (1977).Google Scholar3 Gas Chromatography (GC) Derivatization. Regis Chromatography Catalog. www.chromspec.com/pdf/e/rg01.pdf.Google Scholar4 Dale JA, Dull DL, Mosher HS. α-Methoxy-α-trifluoromethylphenylacetic acid, a versatile reagent for the determination of enantiomeric composition of alcohols and amines. J. Org. Chem. 34(9), 2543–2549 (1969).Crossref, CAS, Google Scholar5 Dale JA, Mosher HS. Nuclear magnetic resonance enantiomer regents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and α-methoxy-α-trifluoromethylphenylacetate (MTPA) esters. J. Am. Chem. Soc. 95(2), 512–519 (1973).Crossref, CAS, Google Scholar6 Ward DE, Rhee CK. A simple method for the microscale preparation of Mosher's acid chloride. Tetrahedron Lett. 32(49), 7165–7166 (1991).Crossref, CAS, Google Scholar7 Chandrul KK, Srivastava B. Enantiomeric separation in pharmaceutical analysis: a chromatographic approach. J. Chem. Pharm. Res. 2(4), 923–934 (2010).CAS, Google Scholar8 Porter WH. Resolution of chiral drugs. Pure Appl. Chem. 63(8), 1119–1122 (1991).Crossref, CAS, Google Scholar9 Görög S, Gazdag M. Enantiomeric derivatization for biomedical chromatography. J. Chromatogr. B. 659(1–2), 51–84 (1994).Crossref, Medline, CAS, Google Scholar10 Zhao Y, Woo G, Thomas S, Semin D, Sandra P. Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry. J. Chromatogr. A 1003(1–2), 157–166 (2003).Crossref, Medline, CAS, Google Scholar11 Niwa M, Miyuki Watanabe M, Watanabe N. Chemical derivatization in LC–MS bioanalysis: current and future challenges. Bioanalysis 7(19), 2443–2449 (2015).Link, CAS, Google Scholar12 Vashistha VK, Bhushan R. Bioanalysis and enantioseparation of DL-carnitine in human plasma by derivatization approach. Bioanalysis 7(19), 2477–2488 (2015).Link, CAS, Google Scholar13 Barnaby OS, Benitex Y, Cantone JL et al. Beyond classical derivatization: analyte 'derivatives' in the bioanalysis of endogenous and exogenous compounds. Bioanalysis 7(19), 2501–2513 (2015).Link, CAS, Google Scholar14 Marcos J, Pozo OJ. Derivatization of steroids in biological samples for GC–MS and LC–MS analyses. Bioanalysis 7(19), 2515–2536 (2015).Link, CAS, Google Scholar15 Athanasiadou I, Kiousi P, Kioukia-Fougia N, Lyris E, Angelis YS. Current status and recent advantages in derivatization procedures in human doping control. Bioanalysis 7(19), 2537–2556 (2015).Link, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByA review of bioanalytical applications of microextraction techniques combined with derivatizationNatalia Manousi, Marianna Ntorkou, Paraskevas D Tzanavaras & Constantinos K Zacharis28 August 2023 | Bioanalysis, Vol. 15, No. 15Chemical tagging mass spectrometry: an approach for single-cell omics19 July 2023 | Analytical and Bioanalytical Chemistry, Vol. 75NMR-based isotope editing, chemoselection and isotopomer distribution analysis in stable isotope resolved metabolomicsMethods, Vol. 206Extraction of High-Value Chemicals from Plants for Technical and Medical Applications7 September 2022 | International Journal of Molecular Sciences, Vol. 23, No. 18Derivatization, an Applicable Asset for Conventional HPLC Systems without MS Detection in Food and Miscellaneous Analysis24 February 2022 | Critical Reviews in Analytical Chemistry, Vol. 34Recent Applications of Derivatization Techniques for Pharmaceutical and Bioanalytical Analysis through High-performance Liquid ChromatographyCurrent Analytical Chemistry, Vol. 18, No. 2Derivatization procedures and their analytical performances for HPLC determination in bioanalysis24 November 2020 | Biomedical Chromatography, Vol. 35, No. 1The role of gut microbiota (GM) and GM-related metabolites in diabetes and obesity. A review of analytical methods used to measure GM-related metabolites in fecal samples with a focus on metabolites' derivatization stepJournal of Pharmaceutical and Biomedical Analysis, Vol. 191Strategies for effective development of ultra-sensitive LC–MS/MS assays: application to a novel STING agonistKasie Fang & Hermes Licea-Perez28 April 2020 | Bioanalysis, Vol. 12, No. 7The importance of evaluating the chemical structures and strategies to avoid pitfalls in quantitative bioanalysisHermes Licea-Perez, Christopher A Evans & Scott G Summerfield26 November 2018 | Bioanalysis, Vol. 11, No. 2Sample preparation for large‐scale bioanalytical studies based on liquid chromatographic techniques30 November 2017 | Biomedical Chromatography, Vol. 32, No. 1New insights into supercritical fluid chromatography for chiral separations1 January 2017 | Analytical Methods, Vol. 9, No. 17Application of Cookson-type reagents for biomedical HPLC and LC/MS analyses: a brief overview12 September 2016 | Biomedical Chromatography, Vol. 31, No. 1From patient to tube: the importance of physiologically relevant quantitative bioanalytical assaysScott Summerfield, Matthew Barfield, Neil Spooner & Steve White25 November 2016 | Bioanalysis, Vol. 8, No. 24 Vol. 7, No. 19 STAY CONNECTED Metrics History Published online 3 November 2015 Published in print October 2015 Information© Future Science LtdKeywordschemical derivatizationchiral analysischromatographic separationionization efficiencypolar compoundsselectivityFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event - a full immersion bioanalytical week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged 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 advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 1 covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (hybrid LBA/LCMS and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will also be published in volume 7 of Bioanalysis, issues 23 and 24, respectively.

BACKGROUND:Camphanic acid chloride has proven to be an efficient chiral derivatization reagent for determination of stereoisomers.RESULTS:The utility of chemical derivatization of various stereoisomers containing hydroxy functional groups with camphanic acid chloride in the presence or absence of a base is highlighted. This procedure is shown to be relatively simple, fast and a cost-effective method of separating racemic drugs and stereoisomeric metabolites in biological matrices. Camphanic derivatives contain two additional chirogenic centers, which are found to enhance stereoisomeric separation on both traditional and chiral stationary phases.CONCLUSION:Four methodologies described herein for separation of multiple stereoisomers in biological samples confirm camphanic acid chloride to be a powerful chiral reagent for stereoisomeric resolution for drug metabolism and PK applications.

An integrated capillary scale (300 μm id) ceramic microfluidic LC system combined with MS/MS has been successfully employed for the quantitative analysis of pharmaceutical compounds in human plasma. The capillary ceramic microfluidic LC/MS/MS system showed an approximate 20-fold (range 11-38-fold) increase in sensitivity compared with a standard 2.1 mm scale UPLC/MS/MS system for a broad range of analytes. The loading capacity of the devices capillary separations channel allowed injection of 2 μL of an aqueous solution, and up to 1.2 μL of a typical protein-precipitated plasma sample, onto the reversed-phase chromatography system. The system also showed excellent chromatographic performance and robustness, with no deleterious effects on the chromatography observed over the course of 1000 injections of protein-precipitated plasma. The ability of the ceramic microfluidic LC/MS/MS system to deliver this level of sensitivity and performance enables the routine quantification of pharmaceutical compounds from small format samples, such as those obtained by dried blood spot or other blood microsampling approaches, to be performed.