Bioanalysis最新文献

Adrenaline, a critical biomarker of stress and autonomic nervous system function, plays a central role in diagnosing and monitoring several medical conditions such as cardiovascular ailments, endocrine illnesses, and neurodegenerative syndromes. Traditional detection methods, including gas chromatography, high-performance liquid chromatography, mass spectrometry, and spectrophotometry, offer high sensitivity and specificity but are limited by complex instrumentation, high cost, and the need for specialized personnel. These constraints hinder their use in rapid, point-of-care, or decentralized settings. This review provides a thorough summary of conventional and emerging adrenaline detection technologies, focusing on recent biosensing platform advancements. Electrochemical and optical sensors enhanced by nanomaterial-based electrode modifications and advanced molecular recognition strategies have improved sensitivity, selectivity, and portability.

Background: The use of duplicate analysis in biomarker assays is a standard practice. While it does not inherently increase variability or contribute to assay failure, it can reveal the high variability associated with manual pipetting, thereby highlighting potential issues within the assy.

Research design and methods: This study evaluated the reliability of singlicate analysis compared to duplicate analysis for interferon-gamma (IFN-γ) assays using ELISA in mouse and non-human primate (NHP) serum samples. Assay performance was assessed across 50 plates, with results analyzed for minimum required dilution (MRD), standard curve linearity, surrogate sample accuracy, recovery, precision, and variability between analysts, including both an experienced and a novice analyst.

Results: There were minimal differences in relative accuracy, and precision between singlicate and duplicate analysis, with CVs less than 5% for both methods. Singlicate and duplicate-generated standard curves were strongly correlated (NHP R² = 0.9995, mouse R² = 1.0000). Analyst variability had less impact on singlicate analysis, with lower inter-analyst CVs (1.0-6.5%) compared to duplicate analysis (5.0-7.5%).

Conclusions: These findings underscore the robustness of singlicate analysis. A workflow for singlicate assay validation is proposed, demonstrating its potential to streamline biomarker assay development while ensuring regulatory compliance. This study supports the adoption of singlicate analysis as a viable alternative to conventional duplicate methods for biomarker assays.

As artificial intelligence enters bioanalysis, traditional validation frameworks, designed for static, deterministic systems are proving unfit for purpose. This paper, developed from the 2025 European Bioanalysis Forum Spring Focus Workshop, challenges the assumption that applications using artificial intelligence should be validated like conventional tools. We propose a shift toward adaptive qualification: an approach rooted in scientific oversight, contextual relevance and earned trust. Reframing artificial intelligence as a learning system, more trainee than tool, we explore how oversight must evolve beyond compliance to ensure transparency, robustness and fitness for purpose. Above all, we argue that scientists must remain at the helm. Not to preserve legacy processes, but to guide this evolving landscape with clarity, collaboration and responsibility, keeping innovation sharp and the patient in focus.

Background: Current HPLC-based methods for doxofylline analysis lack speed and precision. A rapid, specific, and sensitive UPLC-MS/MS method was developed for the determination of doxofylline in this study.

Research design and methods: This method was fully validated and doxophylline-d4 was used as an internal standard. A Kinetex-C18 column (EVO 100Å, 50 × 2.1 mm, 5 μm) was used for the separation procedure, with mobile phases consisting of 0.3% formic acid (A) and 90% acetonitrile solution with 0.3% formic acid (B). The total runtime of the gradient elution procedure was 2.6 minutes. The mass spectrometry analysis was carried out employing a multiple reaction monitoring model and using the transitions of m/z 267.000→181.000 for doxofylline and m/z 271.200→181.100 for the internal standard.

Results: The linear range of detection for doxophylline was between 20.0 to 16,000 ng/mL. The intra-batch accuracy deviations of each concentration level ranged from -8.0% to 2.5%, while the intra-batch precisions ranged from 1.3% to 9.0%. And the inter-batch accuracy deviations were -5.8% ~0.8%, while the inter-batch precisions were 2.2% ~7.0%.

Conclusions: This method was applied to pharmacokinetic clinical trials of single oral administration of doxophylline tablets successfully.

Clinical trial registration: www.clinicaltrials.gov identifier is CTR20240006 and CTR20233665.

The use of blank authentic matrix for bioanalytical method validation and sample analysis has long been standard practice within the bioanalytical community and is considered a requirement under current guidelines for bioanalytical method validation. However, this practice has been increasingly challenged as there are scientifically valid alternatives to replace blank authentic matrices with surrogate matrices. The use of authentic matrices from preclinical animal species conflicts with the ethical 3Rs principles (Replacement, Reduction, and Refinement), a concern that the European Bioanalysis Forum has been addressing for years through data-driven advocacy. In this manuscript, the EBF presents experimental data supporting the use of surrogate matrices in preclinical assays for immunoassay formats. This approach significantly reduces the reliance on authentic matrices, while preserving the quality and integrity of bioanalytical data. The findings advocate for revisiting or clarifying regulatory guidelines, such as ICH M10, to more widely accept the use of surrogate matrices, ensuring alignment with ethical standards without compromising scientific rigor.

The extracellular domain of the neurotrophin receptor p75 has been shown to be a prominent biomarker for both disease diagnosis and progression for amyotrophic lateral sclerosis. This urinary analyte may serve as a valuable fluid biomarker which greatly increases the ease of sample collection in both healthy volunteers and patients. In this paper, the method development and validation for an electrochemiluminescence assay is described. This assay completely uses commercially available reagents and can be performed using common lab equipment found in most bioanalytical labs. This method shows good accuracy and precision, high sensitivity as well as good parallelism illustrating the ability of the method to detect and report on urinary concentrations of neurotrophin receptor p75. The assay can quantitate as low as 78 pg/mL of neurotrophin receptor p75 and > 98% of healthy urine samples tested fell within the dynamic range of the assay.

Accurate measurement of protein concentration is crucial in biological research, where protein-based reagents play a key role in assay performance and reliability. Traditional methods of protein quantification often measure total protein concentration, failing to account for the active portion capable of binding to its intended target. Calibration-free concentration analysis (CFCA), which uses surface plasmon resonance (SPR) technology, offers a solution by specifically measuring the active protein concentration of the sample. CFCA leverages binding under partially mass-transport limited conditions to directly quantify the functional protein in a sample, overcoming variability associated with recombinant protein production. This article provides a background on CFCA and the case for its more widespread use in protein reagent characterization, as it offers a way to reduce lot-to-lot and vendor-to-vendor variability while improving reproducibility and standardization of assays. This perspective was informed by searching and selecting pertinent articles from PubMed (Nov 2024-March 2025) and by examining the reference lists of key papers.

Background: Delta-like protein 3 (DLL3) is considered to inhibit the Notch pathway in the tumorigenesis of small cell lung cancer (SCLC) and other neuroendocrine carcinomas, making it a potential therapeutic target in the treatment of cancer. Since the soluble form (sDLL3) is expected to be useful for predicting the status of DLL3 expression on tumors, analytical methods to measure sDLL3 are required.

Research design and methods: Assay methods using ELISA and the SMCxPRO platform were developed to analyze sDLL3 concentration in human serum. The performance of the ELISA was evaluated and the SMCxPRO assay was fully validated, and the comparability of the 2 assays was assessed.

Results: The performance of the ELISA was acceptable, and in the SMCxPRO assay validation, all pre-defined validation acceptance criteria were met. The 2 assays were comparable within the range of quantification. Concentrations ranged from below the limit of quantification (<1.00 pg/mL) to 18.0 pg/mL for healthy volunteers and from 1.27 pg/mL to 519 pg/mL for SCLC patients by SMCxPRO assay.

Conclusions: Two sensitive assay methods to measure sDLL3 in human serum were successfully established. These assays have potential as novel blood-based assays to assess the status of DLL3 expression on tumors in humans.

Background/aim: The three-tiered testing strategy for an anti-drug antibody (ADA) assay is a common practice for assessing the immunogenicity to therapeutic products. Efforts to streamline the ADA testing process led to proposals of using signal to noise (S/N) as a substitute for titer when determining ADA magnitude. This study aims to identify the critical factors that may influence the correlation of S/N and titer.

Method/result: Experimental and clinical ADA datasets were examined to assess how drug concentration and the assay drug tolerance affect the measurement and correlation of S/N and titer. Under various experimental conditions the influence of drug on titer was minimal across a range of drug concentrations. However, drug presence affected the S/N, particularly when drug concentrations approached the assay drug tolerance. Clinical ADA datasets showed a moderate to strong correlation between S/N and titer, demonstrating similar patterns of drug impact on both measurements, as observed in the experimental data.

Conclusion: The presence of drug in clinical samples and the drug tolerance of the ADA assay simultaneously influence the measurement and correlation of S/N and titer. The fold difference between drug tolerance and the maximum drug concentration in samples is a key factor in determining this correlation.

The 17th GCC Closed Forum was held in San Antonio, TX, USA, on 10^th May 2024. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The topics discussed at the meeting included integrative bioanalysis, patient-centric sampling, emerging technologies, data integrity, sample reconciliation efforts, discrepancies of ELISpot data, cross-validation harmonization, ultrasensitive platforms for immunogenicity assays, remote regulatory assessments, shedding assays by qPCR/dPCR, and biomarker assays. Conclusions and consensus from discussions of these topics are included in this article.