Search Results (104)

Background/Objectives: Selinexor is a selective nuclear-export inhibitor approved for hematologic malignancies, characterized by extensive plasma protein binding (>95%). However, a validated analytical method to accurately measure the clinically relevant unbound fraction of selinexor in human plasma has not yet been established. This study aimed to develop a fully validated bioanalytical assay for simultaneous quantification of total and unbound selinexor concentrations in human plasma. Methods: We established and fully validated an analytical method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) capable of quantifying total and unbound selinexor concentrations in human plasma. Unbound selinexor was separated using ultrafiltration, and selinexor was efficiently extracted from 50 μL of plasma by liquid–liquid extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase (0.1% formic acid:methanol, 12:88 v/v) with a relatively short runtime of 2.5 min. Results: Calibration curves showed excellent linearity over a range of 5–2000 ng/mL for total selinexor (r² ≥ 0.998) and 0.05–20 ng/mL for unbound selinexor (r² ≥ 0.995). The precision (%CV ≤ 10.35%) and accuracy (92.5–104.3%) for both analytes met the regulatory criteria. This method successfully quantified selinexor in plasma samples from renally impaired patients with multiple myeloma, demonstrating potential inter-individual differences in unbound drug concentrations. Conclusions: This validated bioanalytical assay enables precise clinical pharmacokinetic assessments in a short runtime using a small plasma volume and, thus, assists in individualized dosing of selinexor, particularly for renally impaired patients with altered protein binding. Full article

Background: “CFTR modulators” (also named “caftor”) have been developed and introduced into clinical practice to improve the functionality of defective CFTR protein. Therapeutic drug monitoring (TDM) is not currently used for CFTR modulators in routine clinical practice and there is still much to learn about the pharmacokinetic/pharmacodynamic (PK/PD) and the safety profiles of these drugs in a real-world setting. Moreover, therapeutic ranges are not yet available for both pediatric and adult cystic fibrosis (CF) patients. Methods: A new and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for contemporary quantification of ivacaftor (IVA), tezacaftor (TEZ) and elexacaftor (ELX) in plasma samples has been developed and validated. The clinical performance of our method has been tested on samples collected during the routine clinical practice from n = 25 pediatric patients (aged between 7 and 17 years) affected by cystic fibrosis. This LC-MS/MS method has been validated according to ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines for the validation of bioanalytical methods. Results: Our method fulfilled ICH guidelines in terms of accuracy, precision, selectivity, specificity and carry-over. Intra- and inter-day accuracy and precision were ≤15%. The 9-day autosampler stability was 90–100% for TEZ and ELX; meanwhile, it fell to 76% for IVA. An injection volume of 1 µL and a wider quantification range (0.1–20 µg/mL) represent a novelty of our method in terms of sensitivity and fields of application. Finally, the evaluation of PK exposure parameters for IVA revealed strong agreement with previously published reports and with results from the summary of product characteristics (SmPCs). Conclusions: This method could be adopted to contemporarily measure ELX/TEZ/IVA plasma levels for both PK studies and monitor therapy compliance, especially in case of poor or partial responses to treatment, or to evaluate drug–drug interactions when multiple concomitant medications are required. Considering also the high cost burden of these medications to the health system, a TDM-based approach could facilitate more cost-effective patient management. Full article

Background: Method quantification limits are typically determined by measuring variability at blank level only, without accounting for the uncertainties associated with the parameters of the calibration function applied. Methods: A method for the determination of artificial sweeteners and caffeine in soft drinks was validated. The effect of chosen regression function on quantification limits was assessed, considering both trueness and precision. Results: The validated method exhibited heteroscedasticity for all analytes, which is common in experimental methods. A linear response was observed within the working range for sweeteners, while a quadratic regression was required for caffeine. Due to the heteroscedasticity nature of the responses, weighted regressions were necessary to obtain the lowest method quantification limits, allowing for accurate (i.e., unbiased and precise) estimates at the lower end of the calibration range. Under weighted conditions, the regression equations obtained, with an upper range set at 600 mg·L⁻¹, were as follows: y = 3.9 + 58.9x for acesulfame K; y = 0.8 + 185.1x for saccharin; y = 3.5 + 43.3x for aspartame, and y = −7 + 159x − 0.242x² for caffeine. The method quantification limits determined using weighted regressions were 2 mg·L⁻¹ for each analyte, whereas these limits increased to 20 mg·L⁻¹ when non-weighted regressions were applied. Conclusions: The choice of regression function for transforming instrumental signals into analyte concentrations significantly affects the determination of quantification limits, owing to the inherent heteroscedasticity of analytical and bioanalytical calibrations. Weighted regressions are essential for producing accurate estimates at lower concentration levels. Applying weighted regression in the context of heteroscedastic calibrations can lead to quantification limits that are more than 10 times lower than unweighted approaches. Full article

The aim of this study was to develop and characterize UV-crosslinked hydrogel matrices based on polyethylene glycol diacrylate (PEGDA), gum arabic, betaine, and sodium alginate for potential bioanalytical applications. Various physicochemical analyses were performed, including pre-polymerization emulsion stability (Multiscan), FT-IR spectroscopy, swelling behavior in physiological buffers, pH monitoring, contact angle measurements, and morphological assessment via SEM and optical microscopy. The results demonstrated that both alginate content and UV exposure time significantly influence the structural and functional properties of the hydrogels. The highest swelling ratio (2.32 g/g) was observed for the formulation containing 5% sodium alginate polymerized for 5 min (5SA_5), though this sample showed mechanical fragmentation during incubation. In contrast, the most balanced performance was achieved for the 10SA_15 formulation, which maintained structural integrity and exhibited a swelling ratio of 1.92 g/g after 9 days. The contact angle analysis revealed a surface hydrophilicity range from 50° to 100°, with the lowest angle (50°) recorded for 10SA_5, indicating high surface wettability. These findings confirm the suitability of such hydrogels for biomedical applications, particularly as absorbent, stable platforms for drug delivery or wound healing. Full article

Recombinant monoclonal antibody (mAb) botulinum neurotoxin (BoNT) antitoxins, consisting of three mAbs that bind non-overlapping epitopes, are highly potent. However, the three-mAb mixtures pose unique development and manufacturing challenges. Combining even more mAbs to create multivalent antitoxin drugs multiplies those challenges. We previously reported that a single tri-epitopic IgG1-based mAb (TeAb) containing the variable domains of the three parental BoNT/A mAbs and an Fc was as potent as the combination of three IgGs in the mouse neutralization assay (MNA). Here, we extended the tri-epitopic strategy to three other BoNT serotypes. Each TeAb (TeAb-B for BoNT/B, TeAb-E for BoNT/E, and TeAb-F for BoNT/F) binding was measured using fluorescence-activated cell sorting and flow fluorimetry, and the potency was tested in the MNA. The three TeAbs displayed binding affinities that were the same within error of the parental IgGs for each epitope, and all had higher avidity to each serotype of BoNT than that of the parental mAbs. The potency of the BoNT/B, BoNT/E, and BoNT/F TeAbs was similar to the combinations of the three parental IgGs binding BoNT/B, BoNT/E, and BoNT/F in the MNA. We now have four examples of a single TeAb recapitulating the affinity and in vivo potency of a three-mAb antitoxin. The tri-epitopic strategy could be applied to streamline the production and bioanalytics of antibody drugs where three-mAb binding is required for activity. Full article

Microbial biosensors are bioanalytical devices that can measure the toxicity of pollutants or detect specific substances. This is the greatest advantage of microbial biosensors which use whole cells of microorganisms as powerful tools for measuring integral parameters of environmental pollution. This review explores the core principles of microbial biosensors including biofuel devices, emphasizing their capacity to evaluate biochemical oxygen demand (BOD), toxicity, heavy metals, surfactants, phenols, pesticides, inorganic pollutants, and microbiological contamination. However, practical challenges, such as sensitivity to environmental factors like pH, salinity, and the presence of competing substances, continue to hinder their broader application and long-term stability. The performance of these biosensors is closely tied to both technological advancement and the scientific understanding of biological systems, which influence data interpretation and device optimization. The review further examines cutting-edge developments, including the integration of electroactive biofilms with nanomaterials, molecular biology techniques, and artificial intelligence, all of which significantly enhance biosensor functionality and analytical accuracy. Commercial implementations and improvement strategies are also discussed, providing a comprehensive overview of the state-of-the-art in this field. Overall, this work consolidates recent progress and identifies both the potential and limitations of microbial biosensors, offering valuable insights into their future development for environmental monitoring. Full article

Background/Objectives: According to recent guidelines, including the guideline on bioanalytical method validation issued by the European Medicine Agency, the stability of clinical analytes should be known. We summarize human α₁-proteinase inhibitor (A1PI) and urea stability data in relevant matrices, as these analytes are usually measured in clinical A1PI studies. Methods: Stability samples with appropriate A1PI concentrations were prepared in a citrated human reference plasma pool and a matrix mimicking bronchoalveolar lavage (BAL) solution. These samples were kept at −20 °C and −60 °C for up to 24 months. A1PI protein was measured with a nephelometric method and an enzyme-linked immunosorbent assay using paired commercially available polyclonal antibodies. A1PI elastase inhibitory activity was determined with an elastase complex formation immunosorbent assay that combines A1PI complex formation with a solid phase-immobilized elastase and immunological detection of the formed A1PI-elastase complex and urea in samples kept at −20 °C only with an enzymatic assay. Results: Overall, the stability criterion (100 ± 20%) was met for the analytes A1PI protein and A1PI activity at both temperatures during storage of BAL-mimicking and plasma samples for 15 and 24 months, respectively; urea was stable in both matrices at −20 °C for 18 months. Plasma samples showed smaller drops in concentration over storage time than BAL-mimicking samples. As expected, the reduction of A1PI elastase inhibitory activity was more pronounced than that of A1PI protein. Interestingly, the analyte concentration did not significantly influence the results in either of the sample matrices. Conclusions: The data confirmed the appropriate stability of the three analytes in the matrices of citrated plasma and BAL-mimicking samples for at least up to 15 months. Full article

Vitexin and isovitexin are natural flavone C-glucosides that have numerous benefits for human health. However, their low oral bioavailability and poor gastrointestinal absorption dramatically restrict their potential medicinal uses. To overcome this challenge, chitosan-coated alginate microcapsules were prepared for intragastrical administration to rabbits. An LC-MS/MS method was developed and validated for the simultaneous determination of vitexin and isovitexin in the plasma of treated rabbits, using salicylic acid as the internal standard. Raw rabbit plasma samples were deproteinized using acetonitrile as a precipitation agent. Chromatographic separation was performed on a reversed-phase C18 column (100 mm × 4.6 mm, 3.5 µm), with an isocratic mobile solvent system comprising methanol and 0.1% acetic acid (40:60) as the mobile phase. All the analytes and the internal standard were ionized on a triple quadrupole mass spectrometer and electrospray ionization, operating in negative mode and multiple reaction monitoring. The analytical approach developed underwent validation in terms of system suitability, specificity, selectivity, LLOQ of 2 ng/mL, linearity (2.0–200 ng/mL, R² > 0.99), accuracy (the intra- and inter-day from 94 to 110% with the relative standard deviations no more than 8.7%, precision with the recoveries from 97% to 102%, matrix effect (90–100%), carry-over, dilution integrity (2 times), and stability at room and frozen temperature for up to 1 month, and all the parameters met FDA and EMA requirements for bioanalytical methods. The validated procedure was applied to measure the absorption of vitexin and isovitexin from encapsulated extracts in a pilot pharmacokinetic study on rabbit plasma. Compared to the raw traditional extracts, the microcapsules enhanced the bioavailability of vi-texin and isovitexin regarding C_max and AUC values. Full article

Uric acid (UA), the final metabolic product of purines, plays a crucial role in human health monitoring. The UA concentration in biological fluids serves as a diagnostic marker for various disorders, particularly kidney diseases, and represents a potential therapeutic target. Given the growing emphasis on preventive healthcare, developing methods for real-time UA detection has become increasingly significant. Here, we demonstrate the synthesis of novel tumbleweed-like molybdenum diselenide (MoSe₂) nanostructures through a single-step hydrothermal process. The synthesized MoSe₂ was subsequently hybridized with reduced graphene oxide (rGO) to construct electrodes for UA sensing. Differential pulse voltammetry (DPV) measurements revealed that the MoSe₂/rGO-modified glassy carbon electrode (GCE) exhibited excellent UA detection capabilities under optimized conditions. The sensor demonstrated a remarkably low limit of detection (LOD) of 28.4 nM and maintained linearity across a wide concentration range (40 nM to 200 μM). Notably, the sensor showed high selectivity for UA detection even in the presence of common interfering species, including citric acid (CA), dopamine (DA), ascorbic acid (AA), cysteine (Cys), glucose (Glu), oxalic acid (OA), sodium ions (Na⁺), and potassium ions (K⁺). The developed sensor displayed outstanding selectivity, stability, and reproducibility characteristics. This synthetic approach offers promising opportunities for developing MoSe₂-based electrochemical sensing platforms suitable for diverse bioanalytical applications. Full article

Depression and anxiety are two common mental health issues that require serious attention, as they have significant impacts on human well-being, with both being emotionally and physically reflected in the increasing number of suicide cases globally. The World Health Organization (WHO) estimated that about 322 million people around the world experienced mental illnesses in 2017, and this number continues to increase. Cortisol is a major stress-controlled hormone that is regulated by the hypothalamic–pituitary–adrenal (HPA) axis. The HPA axis has three main components, including the hypothalamus, pituitary gland, and adrenal gland, where cortisol, the primary stress hormone, is released. It plays crucial roles in responding to stress, energy balance, and the immune system. The cortisol level in the bloodstream usually increases when stress develops. Molecularly imprinted polymers (MIPs) have been highlighted in terms of creating artificial bioreceptors by mimicking the shape of detected biomolecules, making natural bioreceptor molecules no longer required. MIPs can overcome the limitations of chemicals and physical properties reducing over time and the short-time shelf life of natural bioreceptors. MIPs’ benefits are reflected in their ease of use, high sensitivity, high specificity, reusability, durability, and the lack of requirement for complicated sample preparation before use. Moreover, MIPs incur low costs in manufacturing, giving them a favorable budget for the market with simple utilization. MIPs can be formulated by only three key steps, including formation, the polymerization of functional monomers, and the creation of three-dimensional cavities mimicking the shape and size of targeting molecules. MIPs have a high potential as biosensors, especially working as bioanalytics for protein, anti-body, antigen, or bacteria detection. Herein, this research proposes an MIP-based cortisol biosensor in which cortisol is imprinted on methyl methacrylate (MMA) and methacrylic acid (MAA) produced by UV polymerization. This MIP-based biosensor may be an alternative method with which to detect and monitor the levels of hormones in biological samples such as serum, saliva, or urine due to its rapid detection ability, which would be of benefit for diagnosing depression and anxiety and prescribing treatment. In this study, quantitative detection was performed using an electrochemical technique to measure the changes in electrical signals in different concentrations of a cortisol solution ranging from 0.1 to 1000 pg/mL. The MIP-based biosensor, as derived by calculation, achieved its best detection limit of 1.035 pg/mL with a gold electrode. Tests were also performed on molecules with a similar molecular structure, including Medroxyprogesterone acetate and drospirenone, to ensure the sensitivity and accuracy of the sensors, demonstrating a low sensitivity and low linear response. Full article

Background: The combination of ivacaftor, tezacaftor and elexacaftor (ETI) is approved for patients with cystic fibrosis (CF) aged two years and older and at least one F508del mutation in the CFTR gene. Variability in ETI treatment response has been repeatedly reported, and its reasons are unclear and understudied. Objectives: We present a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid and simultaneous quantification of ETI in plasma, dried plasma spots (DPS), and whole blood volumetric absorptive microsampling (VAMS). Methods: The method utilizes a rapid extraction protocol with 200 μL methanol after the addition of deuterated internal standards. Chromatographic separation was achieved using a reversed-phase Hypersil Gold aQ column (Thermo Fisher Scientific). The method was validated according to ICH (International Council on Harmonisation) guidelines M10 for bioanalytical method validation, demonstrating linearity in the concentration range 0.020–12.000 µg/mL. It was also proved accurate and reproducible with no matrix effect. This method was applied to anonymized samples from patients undergoing ETI treatment: eight plasma and DPS and five VAMS samples were analyzed. Results: ETI concentrations measured in plasma and DPS were interchangeable, whereas ETI concentrations in VAMS were lower than in plasma, as expected for molecules with high plasma protein binding (99%). A correction factor based on the hematocrit value was used to calculate the equivalent plasma concentration from VAMS concentrations. Conclusions: This method is suitable for pharmacokinetic (PK) studies and could facilitate the centralization of samples to specialized laboratories, supporting multicenter studies. Full article

The cuprizone (CPZ) model of multiple sclerosis (MS) is excellent for studying the molecular differences behind the damage caused by poisoning. Metabolic differences in the kynurenine pathway (KP) of tryptophan (TRP) degradation are observed in both MS and a CPZ mouse model. Our goal was to analyze the kynurenine, serotonin, and indole pathways of TRP degradation on the periphery, in the neurodegenerative processes of inflammation. In our study, mice were fed with 0.2% CPZ toxin for 5 weeks. We examined the metabolites in the three pathways of TRP breakdown in urine, plasma, and relevant visceral organs with bioanalytical measurements. In our analyses, we found a significant increase in plasma TRP, 5-hydroxytryptophan (5-HTP), and indole-3-acetic acid (IAA) levels, while a decrease in the concentrations of 3-hydroxy-L-kynurenine (3-HK), xanthurenic acid (XA), kynurenic acid (KYNA), and quinaldic acid in the plasma of toxin-treated group was found. A marked decrease in the levels of 3-HK, XA, KYNA, quinaldic acid, and indole-3-lactic acid was also observed in the visceral organs by the end of the poisoning. Furthermore, we noticed a decrease in the urinary levels of the TRP, KYNA, and XA metabolites, while an increase in serotonin and 5-hydroxyindoleacetic acid in the CPZ group was noticed. The toxin treatment resulted in elevated tryptamine and indoxyl sulfate levels and reduced IAA concentration. Moreover, the urinary para-cresyl sulfate concentration also increased in the treated group. In the present study, we showed the differences in the three main metabolic pathways of TRP degradation in the CPZ model. We confirmed the relationship and correlation between the content of the kynurenine metabolites in the plasma and the tissues of the visceral organs. We emphasized the suppression of the KP and the activity of the serotonin and indole pathways with a particular regard to the involvement of the microbiome by the indole pathway. Consequently, this is the first study to analyze in detail the distribution of the kynurenine, serotonin, and indole pathways of TRP degradation in the periphery. Full article

Ropivacaine, a widely used regional anesthetic also used for pain management, has been increasingly used in recent years due to its increased efficacy and improved safety compared to similar anesthetics. Biomonitoring of ropivacaine and its metabolites during and after anesthesia is an essential process for ensuring therapeutic efficacy and safe usage for patients. The most useful biomonitoring tool in recent years has been liquid chromatography coupled with mass spectrometry (LC-MS/MS), which offers selectivity, sensitivity, as well as accuracy of measurements. The current manuscript summarizes and discusses the existing liquid chromatographic methods described in the literature, as well as the personal experience with developing bioanalytical and analytical methods for the quantification of ropivacaine in biological samples for clinical applications. It is focused on methodological aspects, recent advancements, challenges, and future perspectives, highlighting the importance of LC-MS/MS techniques in ropivacaine analysis. Full article

Abstract: The introduction of biological therapies has revolutionized inflammatory bowel disease (IBD) management. A critical consideration in developing these therapies is ensuring adequate drug concentrations at the site of action. While blood-based biomarkers have shown limited utility in optimizing treatment (except for TNF-alpha inhibitors and thiopurines), tissue drug concentrations may offer valuable insights. In antimicrobial therapies, tissue concentration monitoring is standard practice and could provide a new avenue for understanding the pharmacokinetics of biological and small-molecule therapies in IBD. Various methods exist for measuring tissue concentrations, including whole tissue sampling, MALDI-MSI, microdialysis, and fluorescent labeling. These techniques offer unique advantages, such as spatial drug-distribution mapping, continuous sampling, or cellular-level analysis. However, challenges remain, including sampling invasiveness, heterogeneity in tissue compartments, and a lack of standardized bioanalytical guidelines. Drug pharmacokinetics are influenced by multiple factors, including molecular properties, disease-induced changes in the gastrointestinal tract, and the timing of sample collection. For example, drug permeability, solubility, and interaction with transporters may vary between Crohn’s disease and ulcerative colitis. Research into the tissue concentrations of drugs like anti-TNF agents, ustekinumab, vedolizumab, and tofacitinib has shown variable correlations with clinical outcomes, suggesting potential roles for tissue concentration monitoring in therapeutic drug management. Although routine clinical application is not yet established, exploring tissue drug concentrations may enhance understanding of IBD pharmacotherapy. Full article

Metformin is the gold standard substrate for evaluating potential inhibitors of the organic cation transporters (OCTs). Here, we established a UPLC-MS/MS assay to quantify metformin in cell pellets with a range of 0.05–50 ng/mL using 6-deuterated metformin as an internal standard. We used an ion-pairing chromatographic approach with heptafluorobutyric acid, making use of a reverse-phase column, and overcame the associated ion-suppression via previously established post-column injection of aqueous ammonia. The assay was validated according to the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommendations for bioanalytical methods. The established extraction procedure was simple, very fast and ensured almost 100% recovery of the analyte. The exceptionally sharp peak form and retention of the ion-pairing chromatography are superior to other methods and allow us to measure as sensitively as 0.05 ng/mL. We used the herein established and validated method to develop a cellular OCT inhibition assay by using metformin as a substrate and human embryonic kidney cells (HEK) overexpressing the OCTs 1-3. The method presented may be useful for identifying new OCT inhibitors, but also for drug–drug interactions and other pharmacokinetic studies, where accurate quantification of low metformin amounts in relevant tissues is mandatory. Full article