Bioanalysis最新文献

Introduction: Selecting the optimal platforms to quantitate cytokines is challenging due to varying performance and the plethora of options available.

Aims: To compare performance of three highly sensitive, multiplex assays on three different platforms - MSD S-plex, Olink Target 48, and Quanterix SP-X - to MSD V-plex which is widely used for quantitative cytokine assay.

Methods: Serum and stimulated plasma samples were analyzed across each platform. The proportion of quantifiable samples was compared for each analyte and correlation analyses were performed to relate the data. For MSD S-plex, parallelism and antibody pair knockdown experiments gauged specificity of the kit.

Results: MSD S-plex was the most sensitive multiplex platform followed by Olink Target 48, Quanterix SP-X, and MSD V-plex. Concentrations across platforms differed greatly for some cytokines, but all platforms showed strong correlation. Results for MSD S-plex were confirmed by parallelism and knockdown.

Conclusion: MSD S-plex should be a priority platform for ultra-sensitive assay. Olink Target 48 offers an enticing combination of sensitivity and multiplex capability that warrants consideration when many cytokines require quantitation. MSD V-plex, MSD S-plex and Olink quantitative assays offer high utility across drug development programs, but fit-for-purpose performance should be assessed on a per-analyte basis.

Aims: Circulating total desmosine, representing endogenous systemic elastin degradation activity, is an emerging biomarker for mortality risk in several diseases and aging. However, the existing analytical method takes more than 23 hours to complete, limiting its potential applications. The objective of this study was to shorten the turnover time of a stable isotope dilution liquid chromatogram mass spectrometry-based desmosine assay.

Materials & methods: Plasma samples were analyzed using acid hydrolysis followed by solid-phase extraction and LC-MS. Two approaches to reduce assay time were tested: microwave-assisted acid hydrolysis and direct injection following solid-phase extraction.

Results: The combination of acid hydrolysis at 180°C for 8 minutes and a low-volume elution design for solid-phase extraction reduced the overall assay time to ~ 30 minutes. The assay was validated with intra-day precision and accuracy ranging from 4% to 14%, and -7% to 9%, respectively, while inter-day precision and accuracy were 0% to 9% and 1% to 3%, respectively. The assay was tested in a cohort of patients with acute aortic dissection and control subjects, where desmosine concentrations were approximately three-fold higher in patients.

Conclusions: These results demonstrated that rapid desmosine analysis can be achieved with the use of both microwave-assisted hydrolysis and streamlined solid-phase extraction.

Following up on our most recent discussion paper focussing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated Bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e., the what) and another on the practical considerations (i.e., the how). In this paper, we discuss 'the how'. Both papers should be helpful for the bioanalytical community to better understand the challenges and provide an insight on why bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS should not be compared with the more traditional LC-MS/MS assay for small molecules or ligand binding assays for biotherapeutics.

Within the bioanalytical community, the use of blank matrix from preclinical animals for bioanalytical method validation and sample analysis is common practice and required in the context of guidelines for bioanalytical method validation. At the same time, its use has been challenged by the scientific community for decades, since there is ample scientific evidence to allow the use surrogate matrices for this purpose. Nevertheless, legacy and current regulatory thinking continues to be reluctant to allow the use of surrogate matrices in bioanalytical testing except for so-called rare matrices. As part of ongoing discussions in relation to the ICH M10 Guideline, the European Bioanalysis Forum re-challenges the unnecessary use of blank matrices from preclinical animals and believes that, as part of community responsibility and ethical standards and when supported by data, the use of surrogate matrices should become widely accepted. It is in this context that targeted experiments were conducted within the European Bioanalysis Forum to gather additional data and re-open the discussions with all involved and that it should become acceptable to use surrogate matrices wherever possible.

Iron oxide nanozymes (IONzymes) have become fundamental components in various analyte detection methodologies such as colorimetric, electrochemistry, fluorescence and luminescence. Their tunability, stability and the possibility of modification, alongside their ability to mimic the catalytic properties of natural enzymes like peroxidase, render them invaluable in analytical chemistry. This review explores the diverse applications of IONzymes across analytical chemistry, with a particular highlighting on their roles in different detection techniques and their potential in biomedical and diagnostic applications. This information would be valuable for researchers and practitioners in the fields of analytical chemistry, biochemistry, biotechnology and materials science who are interested in applying IONzymes in their work. In essence, this review article on iron oxide nanozymes in analytical chemistry would serve as a valuable resource for researchers, educators and industry professionals, offering insights, guidance and inspiration for further study and application of this promising class of nanomaterials.

Using a straightforward, sensitive and precise liquid chromatographic approach, it is now possible to concurrently measure the amounts of ibuprofen (IBU) and paracetamol (PAR) in human plasma. A µ BondapakTM C18 column (300 mm × 3.9 mm, 15-20 μm) demonstrated acceptable separation when utilizing a mobile phase of 10 mM disodium hydrogen orthophosphate solution and acetonitrile at an 80:20, v/v ratio. The elution was isocratic at room temperature and a flow rate of 1.0 milliliters per minute. The UV detector was set to monitor PAR and IS (tinidazole) for 6.5 min at 254 nm, then IBU for the next 3 min at 220 nm. PAR and IBU showed linearity across the 0.05 to 100 µg/ml concentration range. The precision of the measurements ranged from 98.5% to 105% for PAR and from 95.1% to 102.8% for IBU. The average drug recovery rate was 100% for PAR and 98.9% for IBU. This method was effectively utilized to assess samples from an actual population administered PAR and IBU (325/200 mg) for pharmacokinetic research. The technique employs green and white tools to evaluate their environmental sustainability and efficacy. The suggested strategy was implemented utilizing the Six Sigma method.

Aim: JBD0131, a novel anti-multidrug-resistant tuberculosis (MDR-TB) drug, can target and inhibit the synthesis of mycolic acids, which are crucial components of the cell wall of the Mycobacterium tuberculosis complex. To support the results of this clinical trial in healthy subjects, development of a specific and accurate quantification method for detecting JBD0131 and its metabolite DM131 in human plasma is needed.Materials & methods: Samples with prior added stabilizer were pretreated by protein precipitation method and the extracts were subjected to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The m/z transitions for the precursor/product ion pairs were 402.1/273 for JBD0131, 333.1/273 for DM131 and 386.1/257 for the internal standard (IS).Results: This method showed good linearity from 1 to 2000 ng/ml for JBD0131 and 0.25 to 500 ng/ml for DM131 and was validated in terms of selectivity, linearity, accuracy, precision, matrix effect, recovery of pretreament and stability.Conclusion: This method was sensitive and specific for measuring the plasma concentrations of JBD0131 and its metabolites. And it was applied for the investigation of the pharmacokinetics of JBD0131 and DM131 in a clinical trial.

The process of developing new drugs in the pharmaceutical industry is both time-consuming and costly, making efficiency crucial. Recent advances in hardware and computational methods have led to the widespread application of computational science approaches in drug discovery. These approaches, including artificial intelligence and molecular simulations, span from target identification to pharmacokinetics research, aiming to reduce the likelihood of failure and present lower costs. Machine learning-based methods predict new applications for developing new drugs based on accumulated knowledge, while molecular simulations estimate interactions between drugs and target proteins at the atomic level based on physical laws. Each approach has its advantages and disadvantages, and they complement each other. As a result, the future of computational science approaches in drug discovery is expected to focus on developing new methodologies that integrate these two techniques to enhance the efficiency of drug discovery.

Assessing the pharmacokinetics of biotherapeutics is essential across all phases of drug development to understand drug exposure. At early stages, platform drug quantification immunoassays offer a versatile method for evaluating exposure for diverse biotherapeutics when specific reagents are not yet available, providing fast data for molecules with common structural features. To ensure clearly defined bioanalytical data, it is essential to conduct interference testing for anti-drug antibodies (ADA) or soluble target (starget), although these assays measure total exposure. In this study, a novel platform immunoassay has been developed that detects a variety of multi-domain drugs with the common structural feature of glycine-serine (G/S) linkers. The assay was successfully qualified and is suitable for early total drug exposure analysis of compounds with G/S linkers. Qualification parameters, including accuracy and precision, were successfully determined, and interference from ADAs and starget was assessed. ADA and starget interference tests showed no impact for one compound, but may affect total drug detection for another. Therefore, it remains recommended to assess the impact of ADAs and starget on assay performance. Overall, the platform G/S linker assay provides a rapid alternative for total exposure analysis in early development when specific assays are unavailable.