Bioanalysis[JOURNAL]
Sun
200 papers
A simple, sensitive microsample LC-MS assay for quercetin and isorhamnetin in mouse and human plasma: application to EMIQ treatment in myotonic dystrophy type 1.
Saravanan Vanaja PS, Mishra S, Cleary JD … +4 more , Nakamori M, Berglund JA, Johnson NE, Gerk PM
Bioanalysis · 2026 Jul · PMID 42394568 · Publisher ↗
BACKGROUND: Quercetin, a dietary flavonoid with emerging therapeutic relevance in myotonic dystrophy type 1 (DM1), has low solubility and poor oral bioavailability. Enzymatically modified isoquercitrin (EMIQ), a water-so...
BACKGROUND: Quercetin, a dietary flavonoid with emerging therapeutic relevance in myotonic dystrophy type 1 (DM1), has low solubility and poor oral bioavailability. Enzymatically modified isoquercitrin (EMIQ), a water-soluble prodrug, raises systemic quercetin exposure. Pharmacokinetic studies require a sensitive assay that uses minimal sample volume. RESEARCH DESIGN AND METHODS: We developed a single-quadrupole liquid chromatography-mass spectrometry (LC-MS) assay for free quercetin, total quercetin (after enzymatic hydrolysis of glucuronide and sulfate conjugates), and the methylated metabolite isorhamnetin in mouse and human plasma. The method used protein precipitation, 10 µL of plasma, reversed-phase C18 separation, and single-ion recording of [M+H]+ adducts. Validation followed a fit-for-purpose approach consistent with M10 guidelines, and the assay was applied to plasma from EMIQ-treated DM1 and wild-type mice (15 g/L for 6 and 12 weeks). RESULTS: Calibration curves showed r > 0.99, with an LLOQ of 0.070 µM for quercetin in both matrices. The assay was successfully validated for quercetin in mouse and human plasma. Total quercetin and isorhamnetin were quantifiable in all treated mice. Exploratory analysis suggested glucuronidation as the major conjugation pathway. CONCLUSIONS: This simple, cost-effective microsampling assay suits preclinical and translational studies of EMIQ in DM1, though the conjugation findings remain exploratory.
ADA assays for high-dose biologics: redefining drug tolerance through clinical insights.
Saxena M, Janik D, Krantz C … +4 more , St-Pierre A, Jadhav M, Michaut L, Bender F
Bioanalysis · 2026 Jul · PMID 42390068 · Publisher ↗
The validation of anti-drug antibody (ADA) assays is vital in biologics development, with regulatory bodies like EMA and FDA emphasizing drug tolerance. In patient care, drug tolerance assessments should reflect actual c...
The validation of anti-drug antibody (ADA) assays is vital in biologics development, with regulatory bodies like EMA and FDA emphasizing drug tolerance. In patient care, drug tolerance assessments should reflect actual clinical use. We developed an ADA assay for a fully human monoclonal antibody used in oncology, addressing the challenges posed by high circulating drug levels and target biology. Various assay formats were tested using multiple positive controls and drug concentrations that mimic real-world exposure and drug-to-ADA ratios. Assessing different positive controls at various concentrations was key to characterizing sensitivity and drug tolerance. An assay initially showing low drug tolerance with one monoclonal control performed adequately with others. Rather than limiting assessments to a single sensitivity level, we evaluated assay performance using clinically relevant drug and ADA concentrations. This approach ensures the assay's sensitivity and drug tolerance are meaningful for patient management and therapeutic decisions. Early and ongoing collaboration with health authorities supported alignment of clinically relevant performance criteria and interpretation strategies. Ultimately, this patient-oriented strategy guarantees ADA results that inform patient safety and treatment effectiveness for high-dose biologic therapies.
Comparison of SERS spectral data sets of blood serum samples of hypopharyngeal cancer using silver and gold nanoparticles as substrates.
Tariq MM, Ali K, Nawaz H … +10 more , Majeed MI, Rashid N, Ditta A, Khan K, Shamim F, Khan R, Zulfiqar R, Nawaz A, Ullah A, Ali M
Bioanalysis · 2026 Jun · PMID 42376845 · Publisher ↗
BACKGROUND: Early detection of hypopharyngeal cancer is crucial for a good prognosis and survival, as it is mostly diagnosed at an advanced stage due to subtle early symptoms. Blood serum contains valuable diagnostic bio...
BACKGROUND: Early detection of hypopharyngeal cancer is crucial for a good prognosis and survival, as it is mostly diagnosed at an advanced stage due to subtle early symptoms. Blood serum contains valuable diagnostic biomarkers for disease screening as it contains metabolites from various tissues and organs in the body and represents the biochemical changes occurring due to disease development. RESEARCH DESIGN AND METHODS: Surface-enhanced Raman spectroscopy (SERS) was applied to blood serum from healthy individuals and patients at different stages of hypopharyngeal cancer. Both silver (AgNPs) and gold nanoparticles (AuNPs) were used as SERS substrates to compare their SERS features. Principal component analysis and linear discriminant analysis (PCA-LDA), along with support vector machine (SVM) were performed to analyze and differentiate the features of both substrates. RESULTS: The analysis revealed complementary SERS spectral features associated to specific nanoparticles as a substrate for cancer diagnostics and metabolic profiling. The SVM classifier achieved an overall accuracy of >90%, with a macro-averaged AUC of 0.98. The class-wise performance metrics (precision, recall, and F1-score) approached unity, which confirms the robust discrimination across all diagnostic groups. CONCLUSIONS: The comparison of SERS spectra of both substrates identified that both substrates differentiate the healthy from cancerous ones while providing different and complementary spectral features.
The Gyrolab platform for immunogenicity assessment and biotherapeutic and biomarker analysis: technical advances and bioanalytical applications.
Mano Y, Nabeshima K, Kojima T
Bioanalysis · 2026 Jun · PMID 42350354 · Publisher ↗
Gyrolab is an automated ligand‑binding assay platform designed for the quantification of proteins with high sensitivity and broad dynamic range. Its low minimum required dilution and microfluidic disc format enable effic...
Gyrolab is an automated ligand‑binding assay platform designed for the quantification of proteins with high sensitivity and broad dynamic range. Its low minimum required dilution and microfluidic disc format enable efficient sample processing while maintaining assay robustness, making it a valuable tool across drug‑development stages. This review summarizes published applications of Gyrolab for pharmacokinetic and toxicokinetic analysis and immunogenicity assessment through anti‑drug antibody detection. In addition, the platform's high-volume assay discs have facilitated its use in biomarker studies, allowing quantification of low‑abundance analytes. Representative examples from the literature are presented together with key assay elements, including analytes, matrices, dynamic ranges, and critical reagents. Because assay replicates remain an important consideration for Gyrolab workflows, recent publications addressing singlet versus duplicate strategies and their impact on data interpretation are also discussed. Finally, we provide real analytical data visualized using a three‑dimensional Gyrolab viewer to illustrate variability in duplicate measurements and to highlight future opportunities for improving assay reliability.
Simultaneous quantification of D-penicillamine, D-penicillamine disulfide, and L-cysteine-D-penicillamine disulfide in human plasma: optimization of sample preparation and mass spectrometry procedures to support bioequivalence studies.
Gu J, Li S, Zou G … +8 more , Tampal N, Xia L, Kaur M, Oh DS, Xu X, Faustino PJ, Keire DA, Shakleya D
Bioanalysis · 2026 Jun · PMID 42348023 · Publisher ↗
AIM: D-penicillamine (PSH) is an active pharmaceutical ingredient used for the treatment of various diseases, such as Wilson's disease, rheumatoid arthritis, cystinuria, and heavy metal poisoning. However, evaluating PSH...
AIM: D-penicillamine (PSH) is an active pharmaceutical ingredient used for the treatment of various diseases, such as Wilson's disease, rheumatoid arthritis, cystinuria, and heavy metal poisoning. However, evaluating PSH in plasma poses substantial analytical challenges due to drug instability, thiol-disulfide exchange reactions with endogenous thiols, and the potential formation of multiple chemical forms of PSH. This study describes the development and validation of an analytical method to ensure the stability of PSH during analysis and enable accurate bioavailability measurements and bioequivalence assessments in human plasma. METHODS: An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to simultaneously determine PSH and its two major metabolites, D-penicillamine disulfide and L-cysteine-D-penicillamine disulfide, in human plasma. PSH stability was investigated under various experimental conditions to identify the optimal sample preparation procedure. The method was then applied to conduct a 90-day stability study of PSH in human plasma stored at -80°C. RESULTS AND CONCLUSION: A sensitive and specific analytical method was developed and validated in accordance with the US FDA M10 guidance. PSH remained stable under optimized conditions for at least 90 days. This method provides pharmaceutical researchers with a standardized approach for PSH pharmacokinetic analysis and bioequivalence evaluations.
Development and preliminary clinical application of a time-resolved fluoroimmunoassay for anti-rituximab antibodies in membranous nephropathy.
Kao S, Li D, Xin M … +7 more , Liu D, Zheng T, Qin Y, Zhou X, Zhang N, Wang L, Huang B
Bioanalysis · 2026 Jun · PMID 42345648 · Publisher ↗
OBJECTIVE: In some patients with membranous nephropathy (MN) receiving rituximab therapy, treatment failure may be partly related to the development of anti-rituximab antibodies (ADAs). This study aimed to establish a hi...
OBJECTIVE: In some patients with membranous nephropathy (MN) receiving rituximab therapy, treatment failure may be partly related to the development of anti-rituximab antibodies (ADAs). This study aimed to establish a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for detecting ADAs in these patients. METHOD: A streptavidin-coated microplate was used as the solid phase, with biotinylated rituximab as the capture antibody and Eu -labeled rituximab as the detection antibody. A bridging assay was developed to detect ADAs, and the method was optimized, validated, and preliminarily applied in clinical testing. RESULTS: The developed TRFIA exhibited a linear detection range of 12.5 ng/mL to 800 ng/mL, with a detection limit (LOD) of 3.93 ng/mL. The intra‑assay coefficient of variation (CV) ranged from 3.07% to 7.90%, and the inter‑assay CV ranged from 2.57% to 11.32%. Recovery rates spanned 87.06% to 104.11%, and no cross‑reactivity was detected with anti‑obinutuzumab or anti‑eculizumab. Compared to enzyme‑linked immunosorbent assay (ELISA), the TRFIA showed superior sensitivity (3.9 ng/mL vs. 10 ng/mL) and maintained strong agreement in sample detection ( < 0.0001). CONCLUSION: This assay demonstrates specific potential clinical utility in monitoring resistance to rituximab, providing valuable guidance for optimizing anti-CD20 monoclonal antibody therapy and developing personalized treatment plans.
Advancing beyond stand-alone NAb assays: perspectives and regulatory impact of applying integrated immunogenicity assessment in drug development.
Martin K, Lichtfuss M, Mehta D
Bioanalysis · 2026 Jun · PMID 42345601 · Publisher ↗
Neutralizing antibody (NAb) activity assessment is a regulatory expectation in the clinical development of therapeutic proteins, traditionally addressed through standalone NAb assays. However, evolving regulatory guidanc...
Neutralizing antibody (NAb) activity assessment is a regulatory expectation in the clinical development of therapeutic proteins, traditionally addressed through standalone NAb assays. However, evolving regulatory guidance and industry experience increasingly recognize that integrated analyses of anti-drug antibodies (ADA), pharmacokinetics (PK), pharmacodynamics (PD), efficacy, and safety may provide a more clinically meaningful evaluation of neutralizing activity. This perspective examines the scientific and regulatory rationale for moving beyond routine reliance on standalone NAb assays and describes a risk-based, integrated immunogenicity assessment framework. Two case studies involving low-risk monoclonal antibodies-garadacimab and clazakizumab-are presented, in which validated standalone NAb assays were available but not relied upon for primary clinical interpretation due to limited sensitivity and lack of added clinical value. Instead, longitudinal ADA characterization integrated with PK and PD or efficacy data was used to assess clinically meaningful neutralizing activity. In both cases, this approach enabled clear differentiation between detectable but clinically irrelevant immunogenicity and immunogenicity with clinical consequence and was accepted by regulatory authorities. These examples illustrate how integrated immunogenicity assessments can replace standalone NAb assays while remaining scientifically rigorous, clinically informative, and aligned with regulatory expectations.
Fit-for-purpose application of LC-MS in protein bioanalysis, case studies shared within the European Bioanalysis Forum Hybrid MS team.
Wilson A, Lucey R, Ediage EN … +16 more , Schroeter K, Szarka S, Blattmann P, Boersema P, Bronsema K, Jordan G, Malchow S, Pekar D, Wiese H, Marks B, Schalk F, Reille-Seroussi M, Garea AV, van de Merbel N, Dillen L, Timmerman P
Bioanalysis · 2026 Jun · PMID 42340185 · Publisher ↗
The use of liquid chromatography-mass spectrometry for quantitative large-molecule bioanalysis has increased substantially over the past decade, driven by its ability to address analytical challenges that may be difficul...
The use of liquid chromatography-mass spectrometry for quantitative large-molecule bioanalysis has increased substantially over the past decade, driven by its ability to address analytical challenges that may be difficult to resolve using traditional ligand-binding assays. In this European Bioanalysis Forum perspective, seven case studies from pharmaceutical companies and contract research organizations illustrate the fit-for-purpose application of LC-MS across different stages of drug development. These include isoform-specific quantification, subclass-resolved immunoglobulin profiling, characterization of post-translational modifications, assessment of biotherapeutic integrity, and multiplexed analysis in complex biological matrices. Collectively, the examples highlight how LC-MS can support selectivity, structural insight, and analytical flexibility when aligned with the specific context of use. The EBF aims to foster continued scientific exchange by encouraging the sharing of such case studies across its community.
Laser-printed microfluidic paper analytical device for rapid separation of whole blood and hematocrit estimation by smartphone readout.
Ogunmolasuyi AM, Oloyede CD, Archibong EU … +2 more , Irhodia JK, Ige PI
Bioanalysis · 2026 Jun · PMID 42333407 · Publisher ↗
In contrast to the widely used centrifugation and hematocrit method, this report presents a simple-to-fabricate paper microfluidic device (µPAD) for the estimation of RBC and hematocrit using µPAD dimensions, histogram,...
In contrast to the widely used centrifugation and hematocrit method, this report presents a simple-to-fabricate paper microfluidic device (µPAD) for the estimation of RBC and hematocrit using µPAD dimensions, histogram, relative colorimetric intensity, and threshold algorithms. With laser printing (LP) technology, LP-µPAD was fabricated to fractionate freshly collected whole blood from male Wistar rats and human blood samples. Fluid flow on the LP-µPAD was tested to assess the capability of the toner-fortified hydrophobic barrier to confine fluid within designated hydrophilic regions. Taking 30 µl of different percentages of whole blood from rats, the LP-µPAD was evaluated to concentrate RBCs at sample zones to estimate hematocrit levels in rat and human blood samples. The LP-µPAD demonstrated promising capability to confine fluids within the hydrophobic barrier and separate blood samples. Whilst the histogram revealed one peak in whole blood, relative colorimetric intensity methods predicted a critical point (32.00%) useful for determining anemic condition, and the threshold algorithm was employed for the visualization of the captured RBCs at the sample zone. Likewise, with the dimension of the µPAD geometry, hematocrit levels were determined in all the samples on a par with the hematocrit method. Thus, LP-µPAD is a frugal alternative to wax printing and effective for hematocrit estimation at the point of care.
Vaccine Ligand Binding Assay Life Cycle Management, Assay Maintenance, Monitoring and Transfer.
Stoop J, Huleatt J, Gagnon L … +40 more , Buoninfante A, Wadhwa M, Irwin C, Mayer C, Neto JT, Agnes J, Aksyuk A, Baclin A, Bonhomme M, Cloney-Clark S, Corsaro B, Dessy F, Fries L, Garofolo F, Giardina P, Green T, Guimera N, Harris S, Helmy R, Ishii-Watabe A, Jaeger R, Jani D, Janssen S, Joseph J, Kierstead L, Makar K, Marshall JC, Mendes DN, Murphy R, Nadarajah S, Nolan K, Plested J, Scully I, Sonderegger I, Tan C, Verch T, Wilkins D, Xu A, Zheng L, Zhu M
Bioanalysis · 2026 Jun · PMID 42329232 · Publisher ↗
This white paper focuses on maintenance, monitoring and transfer of Ligand Binding Assays (LBAs) supporting vaccine immunogenicity studies and is part 3 of a series to harmonize method validation and bioanalysis. The obj...
This white paper focuses on maintenance, monitoring and transfer of Ligand Binding Assays (LBAs) supporting vaccine immunogenicity studies and is part 3 of a series to harmonize method validation and bioanalysis. The objective of this series was identified during the drafting of the 2020 White Paper in Bioanalysis. The Workshop on Recent Issues in Bioanalysis (WRIB) produces the White Paper in Bioanalysis yearly, which is one of the high-profile articles published in the journal, providing detailed discussions and recommendations on Bioanalytical Method Development and Validation. Since 2017, participation by vaccine assay validation experts and regulators in the WRIB working groups has rapidly increased due to its unique format where industry leaders and regulators can meet and exchange ideas on topics of mutual interest. In early 2021, vaccine manufacturers approached WRIB to sponsor and support the authorship and publication of an overarching vaccine assay validation document based on the 2017-2020 discussions and consensus, starting with LBA, then followed by future papers on Molecular Diagnostics (MDx including both PCR and NGS), Biofunctional Assays (BFA) and Cell-Mediated Immunity (CMI) assays validation. Using the industry and WRIB vaccine network, a vaccine immunogenicity assay validation expert working group was assembled representing 16 global companies. The work on the first 3 white papers officially began in April 2021 focusing on vaccine immunogenicity ligand binding assays (Part 1 LBA Validation, Part 2 Assay Development, Part 3 Assay Lifecycle Management, Assay Maintenance, Monitoring and Transfer). In this publication (Part 3), the activities that are required to ensure consistency of assay performance following validation are discussed. In contrast to bioanalytical methods evaluating chemical and biological drugs (ICH-M10), there are no clear guidelines for methods supporting vaccine immunogenicity studies. Hence, the authors of this White Paper hope that this common effort will help close this regulatory gap. These harmonization white papers will collectively be pivotal for whole vaccine development process.
Accelerating drug discovery with mass spectrometry-driven bioanalytical innovations and beyond.
Mwangi JN, Hill R, Runnebohm A … +2 more , Czerwieniec G, Berna M
Bioanalysis · 2026 Jun · PMID 42324885 · Publisher ↗
The emergence of structurally complex therapeutic modalities, including bispecific antibodies, antibody-drug conjugates, fusion proteins, incretins, radioligand therapeutics, antibody-oligonucleotide conjugates, and smal...
The emergence of structurally complex therapeutic modalities, including bispecific antibodies, antibody-drug conjugates, fusion proteins, incretins, radioligand therapeutics, antibody-oligonucleotide conjugates, and small interfering RNAs, demands advanced mass spectrometry workflows across the ADME pipeline. This forward-looking perspective examines the bioanalytical challenges these next-generation therapeutics present, from absorption and distribution through metabolism and elimination. Current MS platforms, LC-MS/MS, high-resolution MS, quantitative mass spectrometry imaging, and ICP-MS, while highly capable, present opportunities for sensitivity enhancement to fully characterize low-dose therapeutics through the elimination phase. To quantify this need, we developed a pharmacokinetic-driven prediction framework and applied it to FDA-approved complex therapeutics, demonstrating that enhanced sensitivity would enable comprehensive metabolite characterization, particularly during the elimination phase. We identify five convergent innovation priorities: (1) addressing modality complexity through integrated ADME workflows; (2) continued advancement of instrument sensitivity for low-dose therapeutics; (3) resolving biotransformation challenges through enhanced analytical resolution; (4) improving qMSI capabilities for therapeutic-level tissue detection; and (5) implementing AI/ML-driven automation for data complexity management. Rather than advocating for MS-only solutions, we recommend integration of mass spectrometric structural specificity with complementary high-throughput technologies; immunoassays, hybridization ELISA, qPCR, and element-specific detection, to enable comprehensive bioanalysis across all modalities, accelerating the path from discovery to development.
Targeted LC-MS/MS profiling of serum amino acids reveals type 2 diabetes-specific alterations in advanced gastric cancer.
Yaman ME, Kocak OF, Topdagi O … +1 more , Kadioglu Y
Bioanalysis · 2026 Jun · PMID 42307968 · Publisher ↗
BACKGROUND: Altered amino acid metabolism is a feature of gastric cancer. Type 2 diabetes (T2D), a prevalent metabolic comorbidity, may affect circulating amino acid profiles; this study aimed to quantify its impact. RES...
BACKGROUND: Altered amino acid metabolism is a feature of gastric cancer. Type 2 diabetes (T2D), a prevalent metabolic comorbidity, may affect circulating amino acid profiles; this study aimed to quantify its impact. RESEARCH DESIGN AND METHODS: A validated targeted LC-MS/MS assay profiled 22 serum amino acids in 156 participants, including gastric cancer patients with and without type 2 diabetes and controls. RESULTS: Adjusted models showed that many observed differences were attributable to type 2 diabetes and body mass index. Type 2 diabetes was associated with lower ornithine (β = -1.16; q = 0.0019) and interacted with gastric cancer for arginine (β_int = -1.10; q = 0.0044) and phenylalanine (β_int = +1.05; q = 0.0112). After adjustment, only valine, taurine, and total amino acids remained lower in gastric cancer. Body mass index was inversely associated with glutamine and positively associated with glutamate. CONCLUSIONS: Covariate-adjusted analysis distinguishes cancer-related from comorbidity-driven alterations and identifies type 2 diabetes as a key modifier of amino acid metabolism. Metabolic covariates should be considered when interpreting amino acid alterations in advanced gastric cancer biomarker studies. However, the findings should be interpreted in light of the predominantly advanced-stage gastric cancer cohort and require validation in larger prospective studies.
Dilution displacement enabled measurement of total ligand levels in the presence of an anti-ligand antibody using LBA.
Zhang C, Sheridan JP, Boehm N … +2 more , Keller S, Fang Y
Bioanalysis · 2026 Jun · PMID 42290408 · Publisher ↗
BACKGROUND: Measurement of soluble ligand targets of biotherapeutics can be challenging, especially when non-competing reagents are unavailable. This study aimed to develop a simple assay to quantify total soluble B-cell...
BACKGROUND: Measurement of soluble ligand targets of biotherapeutics can be challenging, especially when non-competing reagents are unavailable. This study aimed to develop a simple assay to quantify total soluble B-cell Maturation Antigen (sBCMA), including antibody-bound sBCMA, in serum when anti-BCMA antibody is present. RESEARCH DESIGN AND METHODS: A sample predilution strategy was coupled with a ligand-binding assay (LBA) to dissociate sBCMA from anti-BCMA antibody complexes and enable measurement of total sBCMA. RESULTS: A lower limit of quantitation (LLOQ) of 3 ng/mL in neat serum was achieved in the presence of clinically relevant concentrations of anti-BCMA antibody less than 30 µg/mL. sBCMA serum levels were elevated in patients with multiple myeloma (MM) (mean 198.3 ng/mL; range 3.3-2035.7 ng/mL) compared with healthy subjects (mean 12.4 ng/mL; range 2.9-18.4 ng/mL). CONCLUSIONS: This dilution-based approach enabled accurate measurement of total sBCMA despite circulating anti-BCMA antibody. However, this sample predilution approach may or may not have general applicability for similar bioanalytical purposes, when candidate biotherapeutic levels are moderate, assay is highly sensitive, and the target protein levels are substantially elevated, as described in this case.
Pharmaceutical drug monitoring: the role of oral fluid sampling.
Soares S, Rosado T, Barroso M … +1 more , Gallardo E
Bioanalysis · 2026 Jun · PMID 42283120 · Publisher ↗
Oral fluid has attracted increasing interest as an alternative biological matrix for monitoring pharmaceutical compounds in clinical and forensic toxicology. Its noninvasive nature, simple collection procedures, and suit...
Oral fluid has attracted increasing interest as an alternative biological matrix for monitoring pharmaceutical compounds in clinical and forensic toxicology. Its noninvasive nature, simple collection procedures, and suitability for repeated sampling make it particularly attractive for large-scale drug screening, medical diagnosis, long-term treatment monitoring, and point-of-care applications. Compared with blood sampling, oral fluid collection is better accepted by patients and does not require trained personnel, facilitating its use in decentralized or real-time testing scenarios. In addition, drug concentrations in oral fluid frequently reflect the pharmacologically active unbound fraction of compounds, which may enhance its clinical relevance for individualized dosing and evaluation of therapeutic response. Several studies have reported meaningful correlations between drug concentrations measured in oral fluid and those determined in blood (plasma or serum) for different therapeutic classes, including antiepileptics, antidepressants, analgesics, antibiotics, and immunosuppressants. These characteristics support the growing interest in oral fluid as a useful matrix for therapeutic drug monitoring strategies aimed at optimizing treatment while reducing the risk of adverse effects. This review summarizes and critically discusses recent developments in analytical approaches for the detection of pharmaceutical compounds acting on the central nervous system in oral fluid and their potential application in drug monitoring.
Advances in the use of DNA aptamers for bioanalysis of antibody-based therapeutics.
Todoroki K
Bioanalysis · 2026 Jun · PMID 42272446 · Publisher ↗
Nucleic acid aptamers are single-stranded oligonucleotides that fold into defined three-dimensional structures and bind molecular targets with high affinity and specificity. Because aptamers are sequence-defined, chemica...
Nucleic acid aptamers are single-stranded oligonucleotides that fold into defined three-dimensional structures and bind molecular targets with high affinity and specificity. Because aptamers are sequence-defined, chemically synthesized reagents, they offer high purity, minimal lot-to-lot variability, and flexible functionalization, positioning them as practical alternatives or complements to antibody-based critical reagents in regulated bioanalysis. This review summarizes the current landscape of DNA aptamer-enabled bioanalysis for therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), with emphasis on three application areas: (i) ELISA-compatible ligand-binding assays, (ii) immunocapture workflows coupled to liquid chromatography-tandem mass spectrometry or LC-based measurements, and (iii) aptamer-based biosensors for bioanalysis and process analytics. This review highlights key considerations in aptamer acquisition (SELEX design, partitioning, and counter-selection to improve anti-idiotype selectivity) and illustrates representative assay configurations, including drug-to-antibody ratio-responsive formats for ADCs and ratiometric/electrochemical and fluorescence resonance energy transfer-based aptasensors for on-site monitoring. Finally, this review discusses future directions, notably coupling aptamer binding to nucleic acid amplification for ultra-sensitive quantification, and practical challenges, such as nuclease susceptibility, nonspecific interactions with serum proteins, and structure-dependent robustness that must be addressed under fit-for-purpose validation frameworks.
Preclinical pharmacokinetics of novel MYDGF for human PK and dose prediction.
Myzithras M, Wei W, Bigwarfe T … +4 more , Chen LZ, Gupta P, Li H, Pekcec A
Bioanalysis · 2026 · PMID 42253111 · Full text
AIM: This study aimed to characterize the pharmacokinetics (PK) of recombinant human myeloid-derived growth factor (hMYDGF), a 15.8 kDa novel therapeutic protein for cardiac repair after myocardial infarction, in various...
AIM: This study aimed to characterize the pharmacokinetics (PK) of recombinant human myeloid-derived growth factor (hMYDGF), a 15.8 kDa novel therapeutic protein for cardiac repair after myocardial infarction, in various preclinical animal species (mouse, Yorkshire pig and cynomolgus monkey) to guide human PK and dose prediction. METHODOLOGY: A custom, specific, ultra-sensitive MSD assay was developed to accurately quantitate hMYDGF in plasma. Single-dose pharmacokinetic studies were conducted in mice, Yorkshire pigs, and cynomolgus monkeys, followed by bioanalytical assessment and comprehensive pharmacokinetic analysis. human serum stability was also assessed using a custom LC-MS/MS-based assay. Plasma time-concentration profiles were fitted to a two-compartment PK model, with allometric scaling used to predict human PK parameters. RESULTS: Rapid plasma clearance of hMYDGF was observed across all species, consistent with renal elimination driven by its low molecular weight. Serum stability tests indicate that clearance was not caused by nonspecific proteolytic degradation. CONCLUSION: Pharmacokinetics were accurately characterized in all species, enabling allometric scaling and predicted human PK and efficacious dose. Simulations indicated that a single intravenous bolus of 0.42 mg/kg in humans would achieve plasma levels linked to preclinical efficacy, supporting MYDGF's potential as a protein replacement therapy for acute myocardial infarction.
Quantification of oxaliplatin and lansoprazole in tumor tissue homogenates by LC-MS/MS: a fit-for-purpose bioanalytical approach.
Alves E, Bannimath G, Prabhakaran P
Bioanalysis · 2026 · PMID 42212864 · Full text
BACKGROUND: Quantification of drug concentrations within tumor tissue is essential for evaluating true intratumoral exposure, yet remains analytically challenging due to matrix complexity, protein binding, and drug insta...
BACKGROUND: Quantification of drug concentrations within tumor tissue is essential for evaluating true intratumoral exposure, yet remains analytically challenging due to matrix complexity, protein binding, and drug instability. Existing LC-MS/MS methods for oxaliplatin and lansoprazole are primarily limited to plasma or pharmaceutical matrices. METHODS: This study describes the development and fit-for-purpose evaluation, guided by ICH M10 principles, of LC-MS/MS methods for quantifying oxaliplatin and lansoprazole in murine tumor tissue homogenates. Isocratic separation on a C18 column with an ammonium acetate-acetonitrile mobile phase and compound-specific MRM detection enabled selective analysis. RESULTS: The methods demonstrated acceptable linearity, agreement between nominal and back-calculated concentrations, controlled matrix effects, consistent extraction recovery, and satisfactory autosampler and freeze-thaw stability. The assays were successfully applied to determine intratumoral drug concentrations in control, monotherapy, and combination treatment groups. CONCLUSION: These matrix-matched LC-MS/MS methods provide a fit-for-purpose approach for exploratory intratumoral drug quantification and support more reliable assessment of pharmacologically relevant tumor exposure in preclinical studies.
Validation approach of an LC-MS/MS assay for ADA detection, applied to a Von Willebrand factor-targeted biotherapeutic.
Schalk FB, Shumate K, Nagilla P … +3 more , Lageveen-Kammeijer GSM, Horvatovich P, van de Merbel NC
Bioanalysis · 2026 · PMID 42206838 · Full text
BACKGROUND: The efficiency of ligand binding assay (LBA)-based immunogenicity assessment can be compromised by the presence of high concentrations of soluble pharmacological target. This is particularly challenging for t...
BACKGROUND: The efficiency of ligand binding assay (LBA)-based immunogenicity assessment can be compromised by the presence of high concentrations of soluble pharmacological target. This is particularly challenging for therapies targeting highly abundant multimeric plasma proteins, such as Von Willebrand Factor (VWF). METHOD: We developed and validated a hybrid LBA-LC-MS/MS assay in which a VWF-directed monoclonal antibody is immobilized to selectively extract anti-drug antibodies (ADAs) from plasma. Nonspecific binding was minimized by immunopurification and the ADAs were digested using trypsin. Semi-quantitative LC-MS/MS analysis of signature peptides unique to seven ADA isotypes (IgG1-4, IgM, IgA, and IgE) was performed against isotype-specific reference standards. RESULTS: The method validation was conducted in alignment with current regulatory expectations for immunogenicity assessment, addressing all critical performance characteristics, including cut-point determination, sensitivity, specificity, and selectivity as well as precision, reproducibility, and robustness. The validated assay was successfully implemented as the primary method for assessing immunogenicity in clinical trial samples. CONCLUSION: This study demonstrates that hybrid LBA-LC-MS/MS enables single tier isotype-specific immunogenicity assessment in the presence of extreme target concentrations. The approach provides a viable alternative to traditional LBA-based strategies and can serve as the primary technique for immunogenicity assessment for regulatory-compliant evaluation of ADA responses.
Recent developments in analytical techniques for monitoring anabolic-androgenic steroids in biological and environmental matrices: challenges and future opportunities.
Alourfi NM
Bioanalysis · 2026 · PMID 42200603 · Full text
Anabolic-androgenic steroids (AAS) are widely used in clinical practice but are also frequently misused, necessitating reliable analytical methods for their detection in biological and environmental matrices. This review...
Anabolic-androgenic steroids (AAS) are widely used in clinical practice but are also frequently misused, necessitating reliable analytical methods for their detection in biological and environmental matrices. This review summarizes recent developments in analytical techniques for AAS determination between 2011 and 2024, with emphasis on chromatographic and mass spectrometric approaches. Relevant literature was identified through searches of major scientific databases, including Scopus, Web of Science, and PubMed, using keywords related to anabolic-androgenic steroids and analytical detection methods; studies were selected based on relevance to methodological advances. Gas chromatography-mass spectrometry (GC-MS) remains a robust platform, with high-resolution GC-HRMS enhancing selectivity and retrospective analysis capabilities. Liquid chromatography-mass spectrometry (LC-MS/MS) provides high sensitivity and throughput for targeted quantification, while LC-HRMS enables broader screening through suspect and non-targeted approaches. Advances in sample preparation, including microextraction and automated workflows, have improved analytical efficiency and reduced matrix effects. Emerging platforms such as ion mobility spectrometry and ambient ionization methods offer rapid screening but remain complementary to established techniques. Overall, modern AAS analysis reflects a shift toward integrated analytical strategies combining targeted and high-resolution approaches to address increasing analytical complexity and evolving regulatory demands.
Immunogenicity assays are biomarker assays: is the 3-tiered paradigm fit-for-purpose? An illustrative case study.
Stevenson LF
Bioanalysis · 2026 · PMID 42199061 · Full text
This perspective highlights challenges associated with implementation of the 3-tiered paradigm over the past two-plus decades. A case study is used to illustrate re-analysis of anti-drug antibody (ADA) data from a Phase...
This perspective highlights challenges associated with implementation of the 3-tiered paradigm over the past two-plus decades. A case study is used to illustrate re-analysis of anti-drug antibody (ADA) data from a Phase 1 clinical study of a monoclonal antibody, applying a biomarker-oriented approach. Unlike traditional analyses that focus on "positive" responses defined by statistical cut points, this approach incorporates all data, including "negative" responses, to characterize complete response profiles.By leveraging signal-to-noise (S/N) and raw signal data from the screening tier, the analysis provides a more granular, contextualized view of immunogenicity. Placebo data further inform longitudinal variability within the study population. This approach demonstrably clarifies apparent baseline positivity, distinguishes true treatment-emergent responses, and reveals that some subjects classified as ADA-positive under the traditional paradigm reflect biological variability or low-level, clinically irrelevant responses.Continuous readouts such as S/N enhance interpretation relative to titers and enable earlier insight into response dynamics without requiring additional assay tiers. Collectively, these findings underscore limitations of the current paradigm, including potential inflation of incidence, loss of clinical context, and mischaracterization of program risk. A biomarker-based framework offers an opportunity to generate more informative immunogenicity data more efficiently, supporting improved decision-making and facilitating timely regulatory evaluation.
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