Search Results (9,878)

Bioanalytical methods to quantitate conjugated payloads are essential for assessing antibody-drug conjugate (ADC) stability and pharmacokinetics (PK). Dual-payload ADCs present analytical challenges; different linker chemistries can require complex digestion conditions to perform the cleavage. Developing separate methods for each linker combination can be time and resource demanding. Rat tritosomes—purified lysosomal fractions from Triton-treated rat liver—provide a comprehensive enzymatic mixture that mimics the lysosomal environment. The presented bioanalytical method combines immunoaffinity purification with tritosome-mediated digestion for simultaneous quantitation of dual-conjugated payloads. The method was applied to a model dual-payload ADC containing two different cytotoxic payloads, conjugated using different enzymatically cleavable linkers, with an unrelated DAR (drug-to-antibody ratio). Method validation in mouse plasma demonstrated excellent accuracy (bias ± 20%, LLOQ and ULOQ ± 25%) and precision (coefficient of variation CV% ≤ 20%, LLOQ and ULOQ ± 25%) across all concentration levels (lower to upper limit of quantitation, LLOQ to ULOQ) for both payloads, with 100% of quality control samples (QCs) meeting acceptance criteria for hybrid LC-MS/MS quantitation methods. This tritosome-based approach provides a unified, efficient platform for multi-payload ADC bioanalysis, eliminates linker-specific method optimization, and enables robust support for preclinical studies. The method has been tested for accuracy and precision on 4 different model ADCs and employed to quantify the conjugated payloads in in vivo samples from a homozygous hFcRn transgenic mouse model (Tg32) PK study, resulting in reliable data in accordance with total antibody measurements. Full article

Background/Objectives: Rumex obtusifolius L. (Polygonaceae) has been traditionally used to treat various disorders, including hepatic and gastrointestinal diseases. However, the phytochemical constituents of its roots and their potential protective effects against skeletal muscle atrophy remain poorly understood. This study aimed to isolate and characterize bioactive constituents from R. obtusifolius roots and evaluate their protective effects against dexamethasone (DEX)-induced muscle atrophy in C2C12 myotubes. Methods: LC–MS-guided phytochemical investigation of the ethanol extract of R. obtusifolius roots, followed by successive column chromatography and HPLC purification, resulted in the isolation of four naphthalene-type glycosides. Their structures were elucidated using 1D and 2D NMR spectroscopy, HR-ESIMS, and chemical transformation. The protective effects of compounds 1 and 4 against dexamethasone (DEX)-induced muscle atrophy were evaluated by assessing myotube morphology, myogenic and atrophy-related protein expression, and PI3K/Akt/mTOR signaling. Results: A new naphthalene malonylglucoside, nepodin-8-O-β-D-(6′-O-malonyl)-glucopyranoside (1), together with three known glycosides (2–4), was identified. Among the isolated compounds, compound 1 significantly attenuated DEX-induced muscle atrophy in a concentration-dependent manner by increasing myotube diameter and improving myotube morphology. It restored the expression of the myogenic markers MyoD and myogenin while suppressing the atrophy-related proteins MuRF1 and MAFBX. Furthermore, compound 1 reversed DEX-induced suppression of the PI3K/Akt/mTOR signaling pathway, indicating recovery of anabolic signaling. Conclusions: This study reports a new naphthalene malonylglucoside (1) from R. obtusifolius roots and demonstrates that compound 1 protects against DEX-induced skeletal muscle atrophy through restoration of myogenic differentiation and activation of the PI3K/Akt/mTOR pathway. These findings suggest that compound 1 is a promising natural lead compound for the development of therapeutics targeting muscle wasting disorders. Full article

Aflatoxins and fumonisins are two of the most prevalent and toxicologically significant mycotoxins contaminating global food supplies, particularly maize and groundnuts. Although several regulated mycotoxins contribute to food safety concerns, this review focuses on aflatoxins and fumonisins because they frequently co-occur in maize and maize products. Their widespread prevalence, distinct toxicological mechanisms, and combined health effects necessitate an integrated exposure and risk assessment. This review critically evaluates the current state of exposure assessment and its implications for human health. We examine the evolution of sample preparation techniques, highlighting the transition from traditional liquid–liquid extraction to advanced approaches such as QuEChERS and green extraction technologies that can handle the divergent physicochemical properties of lipophilic aflatoxins and hydrophilic fumonisins. Analytical methods are compared, from the robust but limited HPLC-FLD to the multi-analyte capabilities of LC-MS/MS and the emerging potential of aptamer-based biosensors. Furthermore, the review addresses the critical challenge of modified mycotoxins that evade routine detection yet may contribute to total toxicity. By synthesizing data on biomarkers of exposure and the mechanisms of co-exposure, we discuss the complex interplay between these toxins in the etiology of hepatocellular carcinoma and neural tube defects. The review concludes that mitigating the public health burden of mycotoxins requires a holistic strategy that integrates HRMS for non-targeted analysis with human biomonitoring to capture the accurate individual-level exposure. Full article

Influenza virus outbreaks remain a persistent public health concern, yet traditional metabolomics methods are inadequate for addressing key analytical challenges of “dark matter” in influenza research. By integrating quantitative MS¹ data, MS²-derived fragmentation trees and molecular fingerprints, structure-based comparative metabolomics enhances predictive capability for chemical structures, and enables the discovery of candidate metabolic markers without the need for database spectra. In this study, we established a C57BL/6J mouse model of H1N1 infection (with PBS as control) and performed structure-based comparative metabolomics on fecal samples using liquid chromatography–mass spectrometry (LC-MS). Quantitative analysis of MS¹ data identified 40 differential metabolites, while qualitative analysis of MS² data enabled their structural annotation. A candidate metabolite marker, LysoPE 15:0, along with other potential metabolic markers, was annotated and validated using Mirror plot, CFM-ID, and sim-Rank-Network. Our findings demonstrate that structure-based comparative metabolomics enables library spectra-free annotation of metabolomic “dark matter” and provides a methodological workflow for discovering candidate metabolite markers in other diseases. Full article

This study presents an integrated evaluation of the organ-specific phenolic composition and antioxidant activity of Cicer isauricum. Extracts obtained from leaves, stems, and roots were analyzed using liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) and multiple in vitro antioxidant assays. LC–MS/MS analysis revealed a distinct organ-dependent distribution of phenolic compounds. Stem extracts were characterized by high levels of hyperoside (2227.97 µg/g extract), luteolin (298.22 µg/g), and eriodictyol (434.03 µg/g), while leaves were rich in hyperoside (1162.42 µg/g), hesperidin (459.40 µg/g) and kaempferol (182.88 µg/g). Root extracts were dominated by flavan-3-ols, particularly (+)-catechin (355.93 µg/g) and (–)-epicatechin (59.58 µg/g), indicating a differentiated metabolic profile. Antioxidant assays, including cupric reducing antioxidant capacity (CUPRAC), ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, demonstrated that root extracts exhibited the strongest activity, with the lowest IC₅₀ values (DPPH: 4.15 mg/mL; ABTS: 1.41 mg/mL) and highest reducing power (FRAP EC₅₀: 0.41 mg/mL; CUPRAC EC₅₀: 1.78 mg/mL). Correlation analysis confirmed strong associations between total phenolic content and antioxidant capacity, while compound-level evaluation highlighted flavan-3-ols as major contributors. These findings identify roots of C. isauricum as a promising source of natural antioxidants. Full article

Training induces sweat morphology changes in horses from muddy sweat (MS) to foamy sweat (FS) and clear sweat (CS), reflecting physiological adaptation. However, the metabolic mechanisms linking sweat phenotypes to systemic amino acid dynamics remain unclear. This study integrated sweat untargeted metabolomics and plasma amino acid targeted metabolomics to reveal coordinated metabolic remodeling. Six 2-year-old Yili horse stallions underwent 10-week training. Plasma and sweat were sampled pre- and post-race at each stage. LC-MS/MS and UHPLC-MS/MS were used for sweat metabolome and plasma amino acid analysis, followed by multivariate statistics, KEGG enrichment, and correlation network (CNet) analysis. Differential sweat metabolites decreased across stages (45, 127, and 38 for MS vs. FS, MS vs. CS, and FS vs. CS). Pre-race BCAA concentrations were higher in MS than in FS and CS, while glycine was lower. Post-race valine, histidine, and aspartate were elevated only in MS. Pre-race plasma amino acids positively correlated with sweat lipids and organic acids, shifting to negative post-race. ABC transporters, mTOR signaling, and BCAA metabolic pathways were key co-regulators. The MS-to-CS transition reflects metabolic remodeling from acute stress to homeostatic adaptation. Plasma BCAAs and sweat cortisol are potential biomarkers, with ABC transporters and mTOR pathways mediating sweat-plasma metabolic coordination. Full article

The increasing demand for sustainable crop protection strategies has intensified interest in plant-beneficial bacteria as alternatives to synthetic agrochemicals. In this study, the soil-derived bacterium Pseudomonas putida V01 was isolated and characterized for its antifungal and plant growth-promoting potential through an integrated approach combining biological assays, untargeted metabolomics, and in vivo plant experiments. Cell-free culture filtrates exhibited strong antifungal activity against major phytopathogenic fungi, completely inhibiting the growth of Sclerotium rolfsii and significantly reducing mycelial development of Alternaria alternata and Fusarium proliferatum by 40% and 20%, respectively. Volatile organic compounds (VOCs) selectively inhibited Botrytis cinerea and A. alternata by 28% and 10%, respectively, and affected sporulation of F. proliferatum. Metabolomic profiling through LC-qTOF-MS and GC-MS analyses revealed a chemically diverse metabolome, including putatively annotated diketopiperazines, cyclic peptides, phenolic compounds, and fatty acids. VOC profiling indicated ketones and alcohols as the predominant volatile classes, with 2-undecanone and 2-undecanol among the most abundant compounds detected. In vivo assays on wheat seedlings showed significant increases in shoot growth, biomass accumulation, and chlorophyll content compared with untreated controls. These findings indicate that P. putida V01 combines complementary antifungal and plant growth-promoting activities associated with a diverse repertoire of diffusible and volatile metabolites. The integrated biological and metabolomic characterization highlights its potential as a multifunctional microbial inoculant for sustainable crop production and disease management. Full article

Background: We recently synthesized a novel series of triazolyl-oxazolidinones (PH066, PH139, PH162, and PH166) that exhibited significant in vivo anticonvulsant activity in rat models of electrically- and chemically-induced seizures. Objectives: The objectives of the current study were to develop and validate analytical UPLC-MS/MS assays for quantitative determination of these novel compounds in plasma and tissue samples obtained from pharmacokinetic studies to support and/or explain the reported in vivo effects in rats, as well as to study the stability of these compounds in the solid state. Methods: Sensitive, selective, precise, and accurate UPLC-MS/MS methods were developed using a UPLC BEH C₁₈ column for analyte separation. The mobile phase consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid in different ratios depending on the analyte of interest. Quantitative determination was performed using the multiple-reaction monitoring scanning mode. The methods were utilized to determine the concentration of the four compounds in plasma and tissue samples obtained at different time points after intraperitoneal (IP) injection of 100 mg/kg of each compound to rats. The stability of the compounds was investigated under forced degradation conditions. Results: The developed methods were specific for each of the analytes and were found to be linear in the concentration range of 5–30 μg/mL in plasma and 0.5–25 μg/g in tissue samples. The intra-day and inter-day precision and accuracies were within the acceptable range for the four compounds. The methods were applied to quantify these compounds in the plasma and various tissue samples obtained from rats after IP administration of 100 mg/kg of each compound. The maximum plasma concentrations were 14.6, 9.20, 14.5, and 18.1 μg/mL, and those of brain were 2.6, 0.22, 1.9, and 2.8 μg/g for PH066, PH139, PH162, and PH166, respectively. The compounds degraded under forced degradation conditions to various degradation products. Conclusions: The developed methods were selective, linear, accurate, and precise and are suitable for quantitation of the four compounds in plasma and tissue samples obtained during pharmacokinetic investigations, as well as for detecting and quantifying their degradation products. Full article

Arsenic is a highly toxic element that occurs naturally and widely in the environment. Its toxicity depends on the forms in which it occurs, with inorganic arsenic species considered more toxic than organic ones. However, besides the commonly analyzed arsenic species [As(III), As(V), AsB, DMA, and MMA], there are also fat-soluble organic species, arsenolipids, which can be as toxic as or even more toxic than inorganic species. Therefore, to accurately assess the health risks resulting from the consumption of foods containing arsenic, a speciation analysis is needed. Taking this into account, the study aimed to develop a method for the simultaneous determination of both water-soluble [As(III), As(V), DMA, and AsB] and fat-soluble (AsHC 360 and AsFA 362) arsenic species using HPLC-ICP-MS. The present study reports the results of a preliminary optimization investigation. The method development and analyses were conducted qualitatively using standards and food samples—algae, salmon and tuna. The developed method allowed for the full separation of arsenolipids and the partial separation of water-soluble arsenic species in a single run. As there are no commercially available arsenolipid standards, the syntheses of AsHC 360 and AsFA 362 were also a part of the study. Additionally, the synthesized arsenolipids were analyzed by LC-MS. Full article

Osteosarcoma remains the most common primary malignant bone tumor in adolescents and is characterized by aggressive metastasis, resistance to therapy, and extensive bone microenvironment remodeling. Therefore, the identification of novel multi-target therapeutic agents capable of simultaneously inducing ferroptosis and disrupting tumor-supportive signaling is urgently needed. This study employed a multi-omics-guided approach to investigate the anti-osteosarcoma potential of metabolites derived from the sea cucumber Holothuria scabra. LC–MS/MS profiling identified major bioactive constituents, including holothurins, scabrasides, fucosterol, desmosterol, and 24-methylenecholesterol. Integrated transcriptomic analysis of the GSE42352 dataset revealed key ferroptosis- and bone microenvironment-associated targets, including CXCR4, CTSK, RUNX2, VEGFA, and TFRC. In silico pharmacological prediction and molecular docking demonstrated favorable anticancer properties and strong binding affinities of several metabolites toward these targets, with fucosterol and holothurin A exhibiting the most promising interactions. Functional validation in MG-63 osteosarcoma cells showed concentration-dependent reductions in cell viability and migration following H. scabra treatment. Furthermore, treatment decreased GPX4, NRF2, and GSH levels while increasing TFRC and MDA, indicating activation of ferroptotic cell death. In a MG-63/RAW264.7 co-culture model, H. scabra suppressed RANKL, VEGFA, MMP9, and TRAP-positive osteoclast formation, suggesting inhibition of osteoclastogenesis, angiogenesis, and metastatic potential. Collectively, these findings identify H. scabra as a promising marine source of multi-target compounds for osteosarcoma management through coordinated induction of ferroptosis and remodeling of the bone tumor microenvironment. Full article

Background: Multifocal osteonecrosis involving three or more anatomical sites is an uncommon but severe manifestation of glucocorticoid-associated osteonecrosis and may be associated with systemic clinical backgrounds. This study investigated the clinical characteristics and exploratory serum proteomic profiles of multifocal osteonecrosis using clinical and proteomic analyses. Methods: We analyzed 107 patients who underwent surgery for osteonecrosis of the femoral head between 2019 and 2024. Whole-body MRI was used to detect multifocal lesions. Patients were classified into glucocorticoid-related osteonecrosis of the femoral head (GO) and multifocal glucocorticoid-related osteonecrosis (MGO). Clinical variables were compared, and multivariate logistic regression identified clinical factors associated with multifocal osteonecrosis. Serum proteomic profiling using nanoLC–MS/MS was performed as an exploratory analysis to compare protein expression among GO, MGO, and osteoarthritis controls. Results: Multifocal osteonecrosis was identified in 31 patients (29.0%). Patients with MGO were younger (42.6 vs. 50.9 years, p = 0.021) and had higher glucocorticoid doses (59.5 vs. 48.5 mg, p = 0.005). Hematologic diseases (OR 14.51, 95% CI 3.42–86.69, p < 0.001) and skin manifestations (OR 3.17, 95% CI 1.05–10.44, p = 0.046) were independently associated with multifocal osteonecrosis. Exploratory proteomic analysis showed protein expression patterns related to fibrinolysis, coagulation, inflammation, and vascular homeostasis in MGO. Conclusions: Multifocal osteonecrosis was associated with systemic clinical backgrounds and showed exploratory vascular- or coagulation-related proteomic patterns within glucocorticoid-associated osteonecrosis. Full article

The COVID-19 pandemic stimulated the widespread use of traditional medicinal plants in Brazil, particularly in regions with limited access to healthcare and approved therapies. In this context, medicinal plants became accessible alternatives for symptom management and disease prevention, highlighting the value of traditional health systems as sources of biologically relevant species for further investigation. This study documented medicinal plants used to prevent and treat COVID-19 in São Luís, Maranhão, Brazil, and evaluated the inhibitory activity of Uncaria tomentosa (Willd. Ex Schult)extracts, the most frequently cited species, against the SARS-CoV-2 RBD:ACE2 interaction. An ethnopharmacological survey was initially conducted with 400 participants between November 2022 and March 2023 where a total of 38 medicinal ethnospecies were reported, with an overall prevalence of medicinal plant use of 22.75%. Considering that aqueous preparations were the predominant form of use reported by participants, both aqueous and ethanolic extracts were prepared from the stem bark and leaves of U. tomentosa and evaluated in an in vitro Spike (RBD) inhibition assay. The highest inhibitory activity was observed for stem bark extracts of U. tomentosa, which achieved 98.09% inhibition for the ethanolic extract and 73.40% for the aqueous extract. Preparations obtained from the leaves showed lower activity, with inhibition values of 41.12% and 19.74%, respectively. Chemical profiling was performed by LC-MS/MS combined with molecular networking. Chemical analysis enabled the annotation of oxindole alkaloids and flavonoids previously reported in the literature as exhibiting relevant biological activities in models of viral infections. These findings highlight the ethnopharmacological relevance of U. tomentosa, support its potential as a source of bioactive metabolites, and reinforce the value of ethnopharmacological approaches in identifying promising species for further biological investigation. Full article

The red imported fire ant (Solenopsis invicta Buren) is a destructive invasive pest, and conventional chemical control faces challenges related to environmental contamination and resistance development, highlighting the need for novel control agents and greener management strategies. In this study, pyrethrins-mediated silver nanoparticles (Pyr-AgNPs) were synthesized via a green route, characterized, and evaluated for their insecticidal activity, environmental stability, and metabolic effects on S. invicta workers. Bait bioassays showed that Pyr-AgNPs exhibited high toxicity to S. invicta, causing 100% cumulative corrected mortality at 500 mg·kg⁻¹ after 9 days of feeding, with a 5-d LC₅₀ of 116.83 mg·kg⁻¹. Exposure assays further demonstrated that Pyr-AgNPs had good environmental stability and residual efficacy, as bait containing 1000 mg·kg⁻¹ Pyr-AgNPs still caused 100% cumulative corrected mortality after 9 days following 96 h of outdoor exposure, significantly outperforming the pyrethrins treatment. LC-MS-based untargeted metabolomic analysis revealed that treatment with Pyr-AgNPs markedly altered the metabolic profile of S. invicta workers, with 607 differential metabolites identified, mainly belonging to organic acids and derivatives, lipid and lipid-like molecules, amino acids and peptides, cofactors, and redox-related metabolites. Pathway enrichment analysis indicated that these metabolic disturbances were primarily associated with energy metabolism, redox homeostasis, and membrane lipid metabolism. Overall, these findings provide preliminary mechanistic clues into the toxicity of Pyr-AgNPs and support their potential application in the sustainable management of S. invicta. Full article

Background/Objectives: The application of green analytical chemistry (GAC) principles is increasingly important in developing sustainable analytical practices for food safety monitoring. Natural deep eutectic solvents (NADESs) have emerged as green alternatives to conventional organic solvents. This study aimed to develop a sustainable analytical method for determining antibiotic residues in processed meat and frozen poultry products. Methods: A dispersive liquid–liquid microextraction (DLLME) procedure based on a NADES composed of anisaldehyde and decanoic acid (3:1, molar ratio) was coupled with UHPLC–MS/MS for the simultaneous determination of macrolides (clarithromycin, erythromycin), sulfonamides (sulfamethoxazole, sulfadimethoxine), and a fluoroquinolone (enrofloxacin) in food samples. Key extraction parameters, including NADES volume, vortex time, centrifugation time, sample amount, and pH, were optimized. The method was validated for linearity, accuracy, precision, and recovery and applied to real samples from the Saudi market. Results: The method showed excellent analytical performance, with good linearity (R² ≥ 0.9982), recoveries of 84.1–99.4%, and RSDs ≤ 5.75%. The target antibiotics were successfully quantified in processed meat and frozen poultry samples, confirming applicability. In addition, a comprehensive evaluation using eight assessment tools confirmed the method’s environmental sustainability, practicality and innovation. Conclusions: The proposed NADES-based DLLME–UHPLC–MS/MS method is a rapid, sensitive, and eco-friendly alternative to conventional techniques for monitoring antibiotic residues in processed meat and poultry, supporting both food safety and GAC principles. Full article

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by glycolipid dysregulation and hepatic steatosis. Centella asiatica (CA) and its triterpenoid constituents exert metabolic benefits. In addition, previous metabolomics study found that asiatic acid regulated pyrimidine metabolism in obese mice, while the key target and pathway were undefined. This study investigated the regulatory effects of CA and its active constituents on T2DM-related glycolipid disorders, focusing on the pyrimidine metabolism pathway. T2DM mice were established using a high-fat diet combined with streptozotocin (STZ) and treated with Centella asiatica ethanolic extract or asiatic acid (AA), with glibenclamide as a positive control. Then, glycolipid metabolism, hepatic function, pyrimidine metabolites, and related mechanisms were assessed using biochemical assays, LC–MS/MS, cellular experiments, molecular analyses, and molecular docking. CAE and AA significantly reduced FBG (decreased by 51.01% and 53.01%), improved glucose intolerance, corrected dyslipidemia, alleviated hepatic steatosis, and attenuated insulin resistance in T2DM mice. They elevated hepatic uridine, cytidine, and UDP-glucose (UDPG) levels, promoted glycogen synthesis, inhibited uridine phosphorylase 1 (UPP1) activity, upregulated UDPG synthesis genes (PGM1, UGP2), and downregulated lipogenic genes (ACACA, Fasn, SREBP1/2). Molecular docking indicated specific binding of AA and asiaticoside to UPP1. This work distinguishes from our prior research by identifying UPP1 as a functional target and elucidating the detailed molecular mechanism. CA improves T2DM-associated glycolipid disorders and hepatic injury by modulating the pyrimidine metabolism-UDPG-glycogen synthesis pathway and targeting UPP1, highlighting its therapeutic potential for metabolic diseases. Full article