Bioanalysis最新文献

Aims: To develop a UPLC-MS/MS method for quantifying tigecycline (TIG), meropenem (MER), polymyxin B1 (PB1), and PB2 in human plasma, and analyze their unbound fractions and influencing factors in HSCT patients.

Methods: Plasma samples from HSCT patients were analyzed using the developed UPLC-MS/MS technique.

Results: The method showed high accuracy, precision, and stability. Protein binding rates were 82.4% for TIG, 83.7%-79.3% for PB1, and 86.7%-82.6% for PB2, with lower binding at trough vs. peak concentrations.

Conclusions: Preliminary analysis suggests that BMI and serum albumin may influence PB1/PB2 binding in CRE-infected HSCT patients though larger cohorts are needed for confirmation. (clinical trial registration # 2021-0938-01).

Background: Background: An oligonucleotide drug named nusinersen sodium is used to treat Spinal Muscular Atrophy (SMA), requires accurate detection for therapeutic research. There are no published reports on liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for detecting nusinersen in rat cerebrospinal fluid (CSF).

Methods: An LC-MS/MS method has been created and verified to detect nusinersen in Sprague-Dawley (SD) rat CSF. The method employed solid-phase extraction for post-extraction analysis and used dT20 as an internal standard. Negative ion multiple reaction monitoring (MRM) mode scanning and the electrospray ionization (ESI) source were used. The method was validated over a concentration range of 5-2000 ng/mL with a Lower Limit of Quantification (LLOQ) of for nusinersen at 5 ng/mL.

Results and conclusions: The method achieves extremely high accuracy and precision, good linearity, high extraction recovery, and provides a useful approach for evaluating the pharmacokinetics of nusinersen in rats.

Background: Cytokines are critical biomarkers, but their accurate measurement is susceptible to pre-analytical variables. This study investigated the effect of delayed blood processing on the quantitation of eight cytokines (CCL2, CXCL10, IFN-γ, IL-2Ra, IL-6, IL-7, IL-18, TNFα) in matched serum and plasma from healthy volunteers. Methods: Blood was processed immediately or after 3, 6, 24, and 48 hours, stored at room temperature or 4°C. Cytokines were quantified using a multiplexed Ella assay.

Results: Our data showed only IL-2Ra remained stable across all conditions. The remaining cytokine concentrations were significantly impacted by processing delay and storage temperature, with changes evident as early as 3 hours and more pronounced at 24 and 48 hours (p < 0.05). Storing blood at 4°C generally mitigated changes compared to room temperature, but analyte-specific and matrix-dependent variations persisted.

Conclusion: These findings emphasize delayed processing significantly alters cytokine levels, highlighting the importance of prompt, standardized processing and careful consideration of pre-analytical factors for reliable cytokine quantitation in clinical studies.

Aim: Oral peptide therapeutics typically have short half-lives due to rapid degradation by digestive enzymes. Systematic peptide engineering and formulation optimization led to the development of a clinical candidate MEDI7219, an orally bioavailable glucagon-like peptide 1 (GLP-1) peptide, with greater stability than wild-type GLP-1 or semaglutide:~60% of MEDI7219 remained intact after 2 h in vitro incubation with simulated intestinal fluid. This study further investigates proteolytic stability by elucidating biotransformation products of MEDI7219 using liquid chromatography-mass spectrometry (LC-MS) methods.

Method: Peptide metabolism was assessed using in vitro pancreatin assay followed by analysis utilizing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID) and electron-activated dissociation (EAD) approaches.

Results: We have confidently identified 13 metabolites. Time course profiles of parent and metabolite peaks are consistent with sequential enzymatic cleavage pattern. The 13 metabolites mapped to 8 cleavage sites. Most of these cleavage sites can be explained by the specificity of digestive enzymes, e.g. trypsin, pepsin, and elastase. However, α-methyl-L-phenylalanine appeared to be well protected from chymotrypsin and pepsin digestion since no cleavage peptides ending with α-methyl-L-phenylalanine were observed.

Conclusion: These study results provide further structural details explaining previously published stability data and provide new insights into potential GLP1 proteolytic liabilities for future engineering.

Background: Oligonucleotide therapeutics (ONTs) are a rapidly growing class of drug, with 20+ approved drugs on the market and more undergoing preclinical and clinical investigation for various indications. Many groups in the field are appending chemical modifications to modulate tissue specificity. Conjugation of long-chain fatty acids to siRNA molecules increases the hydrophobicity of the analyte and poses analytical challenges for extraction and LC-MS.

Results: We report the development and optimization of an SPE extraction method for a lipid-conjugated siRNA. To improve assay quantitation by LC-MS, a stable isotope label internal standard (SILIS) was evaluated that enabled robust quantitation with high accuracy and precision (±5% in most cases).

Conclusion: We demonstrate the performance of the assay in mouse plasma and tissue homogenates and apply the assay to the determination of tissue exposure and plasma PK profile for a novel lipid-conjugated siRNA molecule and suggest that a SILIS quantitation approach should be standard practice in siRNA bioanalysis.

Aims: This study aims to address the clinical need for managing secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD) patients on dialysis and hypercalcemia in individuals with parathyroid carcinoma or primary hyperparathyroidism (PHPT) ineligible for parathyroidectomy. Cinacalcet, a key calcimimetic agent, targets the calcium-sensing receptor (CaSR) in the parathyroid gland to lower elevated parathyroid hormone (PTH) levels. The study focuses on developing a robust high-performance liquid chromatography tandem mass spectrometry method for cinacalcet quantification to support therapeutic monitoring and pharmacokinetics.

Methods: A bioanalytical method using cinacalcet-D3 (CCT-D3) as an internal standard and tandem mass spectrometry in positive ion mode for accurate quantification employing Triple Quad Mass spectrometer in normal mode and a Zorbax Eclipse XDB-C18 column.

Results: The method was comprehensively validated, demonstrating reliability through high precision, accuracy, linearity within the range of 0.300-150.00 ng/mL, and stability, all in compliance with established bioanalytical guidelines. Furthermore, its environmental sustainability was assessed using modern green chemistry evaluation metrics.

Conclusion: This study presents a robust, selective LC-MS/MS assay for quantifying cinacalcet in human plasma. The assay demonstrates excellent linearity, precision, and recovery, making it suitable for pharmacokinetic studies, therapeutic drug monitoring, and bioequivalence or bioavailability assessments.

The European Bioanalysis Forum has observed increasing misclassification of pharmacokinetic, anti-drug antibody, and biomarker research assays not used for patient management under the European Union's In Vitro Diagnostic Regulation, despite their non-diagnostic intent in early clinical development. This misinterpretation, fueled by ambiguous protocol language, limited cross-functional awareness, and inconsistent national implementation, is leading to unnecessary delays in clinical trials and increased and non-added value regulatory burden. Through a structured evaluation involving a focus workshop and regional roadshows, the European Bioanalysis Forum identified some manageable origins of the issue and its operational consequences. This recommendation paper outlines these observations and wants to propose a pragmatic path forward. This includes clearer regulatory guidance to exempt noncommercial, non-diagnostic assays from In Vitro Diagnostic Regulation when not developed or intended as registered diagnostics. We also highlight the importance of stakeholder education and coordinated regulatory dialogue. These steps aim to preserve the regulator's intent of patient protection while enabling timely and efficient clinical research.

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.