Current Issue : July-September Volume : 2025 Issue Number : 3 Articles : 5 Articles
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 Cmax and AUC values....
To develop a cell-based in vitro thyroid-stimulating hormone (TSH) biological activity assay that can simulate in vivo pharmacodynamic mechanisms, we constructed two HEK293-TSHR cell lines based on two main cell signaling pathways (Gαs-cAMP-PKA and Gαq/11-PLC-Ca2+) that TSH depends on for its in vivo physiological function. These cell lines stably expressed the luciferase reporter driven by the cAMP response element (CRE) and nuclear factor of activated T cells (NFAT) response element, and two reporter-gene assays (RGAs) were correspondingly established and validated. The two transgenic genes could measure signals produced from the simulation of the in vivo effects of TSH from the Gαs-cAMP and Gαq/11-PLC pathways after TSH activation. TSH showed a good dose–response relationship in these two cell lines and conformed to the four-parameter model. We optimized the critical experimental parameters of these two methods and performed comprehensive methodological validation according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, the Chinese Pharmacopoeia, and the United States Pharmacopoeia. The two methods showed good specificity, accuracy, precision, and linearity and can be used to aid in assessments of the biological activity of TSH drugs, product characterization, final product release, stability studies, and comparability studies for biosimilar applications....
Replicating in vivo conditions is essential for understanding immune responses and measuring immune biomarkers in blood. Sampling immune biomarkers in plasma or serum often fails to detect disease-relevant signals, possibly because these markers are sequestered in immune cells or extracellular vesicles. Furthermore, traditional whole blood cultures using external media may not accurately mimic the physiological environment of blood cells. To address these limitations, we developed a strategy using whole blood cell lysates and supernatants to optimize biomarker detection. Additionally, we employed neat whole blood culture methods, preserving the natural cellular and biochemical environment to assess sensitivity to immune modulators, such as lipopolysaccharide (LPS). This cost-effective approach minimizes variability and contamination risks. By utilizing Luminex multiplex immunoassays, we profiled immune biomarkers with higher sensitivity and efficiency than traditional ELISAs. Blood samples from individuals with high alcohol consumption validated our method by assessing biomarker levels before and after LPS stimulation, providing insights into intracellular responses and inflammatory pathways. This method enhances our understanding of inflammatory processes in blood cells, demonstrating the advantages of cell lysates, supernatants, and advanced multiplex assays in immunological research....
Extracellular vesicles (EVs) have a key role in intercellular communication. We hypothesized that EVs are biomarkers of nephropathy or kidney allograft rejection. We screened patients with chronic kidney disease (CKD) and kidney transplant (KT) recipients. We measured the urine and plasma levels of total EVs overall and EV subpopulations (positive for podocalyxin, aquaporin-1, CD133, CD144, CD19, CD3, CD16, CD56, or CD41). We included 92 patients with CKD, 70 KT recipients, and 33 healthy volunteers. In CKD, the total urine EV concentration was correlated positively with the estimated glomerular filtration rate (eGFR), but none of the subpopulations was identified as a potential biomarker of nephropathy. Among the KT recipients, 30 had good allograft function and 40 had allograft disease (13 with antibody-mediated rejections (ABMR), 12 with T-cell-mediated rejection (TCMR), and 15 with allograft dysfunction). Patients with ABMR had low plasma levels of EVs derived from B-cells, T-cells, and endothelium (p = 0.003, 0.009, and 0.005, respectively). Patients with TCMR had a low urine level of EVs derived from endothelium (p = 0.05). EVs derived from B-cells, T-cells, and endothelium might be biomarkers of kidney allograft rejection. However, we did not identify biomarkers of nephropathy in CKD....
Objectives: This study aimed to develop and validate a sensitive and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of flualprazolam and isotonitazene in serum samples to address critical gaps in forensic and clinical toxicology. Materials and Methods: A single-center validation study was conducted using serum samples spiked with analyte standards. Analytical parameters, including linearity, precision, recovery, the limit of detection (LOD), and the limit of quantification (LOQ), were evaluated in accordance with international guidelines. The LC-MS/MS method employed a Shimadzu Triple Quadrupole™ MS 8045 system with solid-phase extraction (SPE) for sample preparation. Results: The method exhibited high linearity for flualprazolam (r2 = 0.997) and isotonitazene (r2 = 0.999) over a concentration range of 1–100 ng/mL. The LODs were determined as 0.608 ng/mL and 0.192 ng/mL, and the LOQs were 1.842 ng/mL and 0.584 ng/mL for flualprazolam and isotonitazene, respectively. Recovery tests yielded results within the acceptable range of 70–120%. Flualprazolam demonstrated recovery rates of 98.0% and 97.0% at theoretical concentrations of 10 ng/mL and 50 ng/mL, respectively. In contrast, the isotonitazene recovery rates were slightly lower, measuring 75.5% at 10 ng/mL and 71.9% at 50 ng/mL, suggesting minor matrix effects that could influence its quantification. Precision analysis, including both repeatability and reproducibility, highlighted the reliability of the method. The %RSD values for flualprazolam were consistently below 7.07%, with mean concentrations closely aligning with theoretical values across fortification levels. For isotonitazene, the %RSD values remained below 6.24%, although recoveries at higher concentrations indicated potential challenges in matrix interaction. Conclusions: This validated LC-MS/MS method offers high sensitivity, precision, and recovery for detecting flualprazolam and isotonitazene in serum, filling a critical need in toxicological investigations. Further validation in other biological matrices is recommended to broaden its applicability....
Loading....