Search Results (66)

Background/Objectives: Therapeutic drug monitoring (TDM) of β-lactams (BL), BL/β-lactamase inhibitor (BLI) combinations (BL/BLIc), and of fosfomycin may play a key role in optimizing antimicrobial therapy and in preventing resistance development, especially when used by continuous infusion in critically ill or immunocompromised patients. Unfortunately, analytical methods for simultaneously quantifying multiple BL/BLIc in plasma are still lacking. Methods: The aim of this study was to develop and validate two rapid, sensitive, and accurate UPLC–qTOF–MS/MS methods for the simultaneous quantification of five novel β-lactam or β-lactam/β-lactamase inhibitor combinations (ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, cefiderocol, and ceftobiprole) along with fosfomycin. Methods: Human plasma samples were prepared by protein precipitation using methanol containing isotopically labeled internal standards. Chromatographic separation was achieved within 10–12 min using two Agilent Poroshell columns (EC-C18 and PFP) under positive and negative electrospray ionization modes. The method was validated according to the EMA guidelines by assessing selectivity, linearity, precision, accuracy, matrix effects, extraction recovery, and stability. Results: The methods exhibited excellent linearity (R² ≥ 0.998) across the calibration ranges for all of the analytes (1.56–500 µg/mL), with limits of quantification ranging from 1.56 to 15.62 µg/mL. Intra- and inter-day precision and accuracy were always within ±15%. Extraction recovery always exceeded 92%, and the matrix effects were effectively corrected through isotopic internal standards. No carry-over or isobaric interferences were observed. All the analytes were stable for up to five days at 4 °C, but the BL and BL/BLIc stability was affected by multiple freeze–thaw cycles. Conclusions: These UPLC-qTOF-MS/MS multi-analyte methods enabled a simultaneous, reliable quantification in plasma of five novel beta-lactams and of fosfomycin. Robustness, high throughput, and sensitivity make these multi-methods feasible for real-time TDM, supporting personalized antimicrobial dosing and improved therapeutic outcomes in patients with severe or multidrug-resistant infections. Full article

Micro- and nanoplastics (MNPs) are increasingly recognized as pervasive environmental contaminants with profound implications for ecosystems and human health. Their small size, compositional diversity, and occurrence across complex matrices—including water, soil, food, and biological samples—pose substantial analytical challenges. Conventional techniques such as vibrational spectroscopy, chromatographic analysis, and electron microscopy have yielded critical insights into MNP composition, morphology, and distribution; however, these methods often face limitations in sensitivity, throughput, and adaptability to real-world samples. Recent advances in nanotechnology have catalyzed the emergence of nanodevices—encompassing nanosensors, nanopore systems, integrated lab-on-a-chip platforms and nanostructured capture materials—that promise enhanced sensitivity, specificity, and the capacity for real-time, in situ detection. These innovations not only facilitate high-throughput analysis but also provide novel opportunities for integrated characterization of MNPs across diverse matrices. This review synthesizes the current state of nanodevice-based MNP detection, critically examining their principles, performance, and limitations relative to conventional approaches, and outlining the key needs for standardization, matrix-specific adaptation, and regulatory harmonization. Full article

Modern pesticide risk assessment relies on data on bioaccumulation and acute aquatic toxicity, yet generating such data is labour-intensive and animal-demanding. This study evaluated whether phospholipid affinity of pesticides, quantified by the chromatographic hydrophobicity index CHI_IAM obtained from high-throughput gradient biomimetic chromatography, can serve as a surrogate descriptor of these endpoints. Nineteen pesticides representing different chemical and functional classes were analyzed on IAM.PC.DD2 columns, and CHI_IAM values were determined. Bioconcentration factors (BCF) in fish and acute toxicity data (96 h LC₅₀ for fish, 48 h EC₅₀ for Daphnia magna) were retrieved from the Pesticide Properties DataBase. CHI_IAM ranged from −12.1 to 54.8 and correlated strongly with log₁₀BCF (r = 0.84) and log₁₀LC₅₀ in fish (r = −0.84), and moderately with log₁₀EC₅₀ for Daphnia (r = 0.76). Highly lipophilic pesticides with high CHI_IAM showed elevated BCF and low LC₅₀/EC₅₀ values, whereas polar compounds with low CHI_IAM exhibited negligible bioconcentration and low acute toxicity. Deviations from these trends, for compounds with specific modes of action, highlighted the contribution of mechanisms beyond membrane toxicity. Overall, CHI_IAM measured under high-throughput conditions retains prognostic value for ecotoxicological assessment and may serve as a rapid experimental descriptor to support preliminary screening. Full article

This review highlights the growing relevance of ion-exchange nanofibrous membranes (IEX-NFMs) in membrane chromatography (MC) for protein purification, emphasising their structural advantages such as high porosity, tunable surface functionality, and low-pressure drops. While the adsorption of IEX-NFMs in MC is expanding due to their potential for high throughput and rapid mass transfer, a critical limitation remains: the precise binding capacity of these membranes is not well understood. Traditional experimental methods to evaluate protein–membrane interactions and optimise binding capacities are labour-intensive, time-consuming, and costly. Therefore, this review underscores the importance of computational modelling as a viable predictive approach to guide membrane design and performance prediction. Yet major obstacles persist, including the challenge of accurate representation of the complex and often irregular pore structures, as well as limited and/or oversimplified adsorption models. Along with molecular-scale simulations such as molecular dynamics (MD) simulations and quantum simulations, meso-scale simulations can provide insight into protein–fibre and protein–protein interactions under varying physicochemical conditions for larger time scales and lower computational burden. These tools can help identify key parameters such as binding accessibility, ionic strength effects, and surface charge density, which are essential for the rational design and performance prediction of IEX-NFMs. Moreover, integrating simulations with experimental validation can accelerate optimisation process while reducing cost. This technical review sets the foundation for a computationally driven design framework for multifunctional IEX-NFMs, supporting their use in next-generation chromatographic separations and broadening their applications in bioprocessing and analytical biotechnology. Full article

Plasmid DNA (pDNA) purification plays a key role in the development of vaccines and gene therapies. Affinity chromatography stands out as a promising method for plasmid purification, leveraging a range of biological and synthetic ligands to achieve selectivity. This study investigates the potential of a synthetic ligand library consisting of triazine-based bifunctional compounds designed to mimic the side chains of amino acids that are known to bind nucleic acids. A high-throughput screening method was employed to assess the binding ability of 158 ligands within the library to single-stranded, FITC-labeled homo-oligonucleotides (G and T), each comprising 20 nucleotides, under both hydrophilic and hydrophobic conditions. High-affinity ligands were identified for both T and G oligonucleotides. Follow-up microscale chromatographic screening uncovered some false positives from the initial FITC-based screening, narrowing the selection to 22 ligands for further investigation. In the next phase of the study, the binding affinity of these ligands towards double-stranded oligonucleotides (AT and CG) was assessed. Ligand 1/2, a mimic of Ala-Lys or Gly-Lys, and ligand 2/3, a mimic of Lys-Tyr, were chosen as initial candidates for evaluating plasmid DNA purification from an Escherichia coli crude extract. The results obtained with 0.4 M ammonium sulfate in 20 mM Tris-HCl (pH 8.0) as the binding buffer were similar to those observed when purifying plasmid DNA from E. coli clarified lysates by hydrophobic interaction chromatography. The affinity resins retained RNA, while the less hydrophobic plasmid DNA was excluded in the initial fractions. Future research will be directed towards exploring the potential of the most promising ligands to separate pDNA isoforms. Full article

Sitagliptin is an orally bioavailable selective DPP4 inhibitor that reduces blood glucose levels without significant increases in hypoglycemia. The aim of this study was to design and validate an innovative, rapid, and highly sensitive LC–MS/MS assay for the precise measurement of sitagliptin concentrations in human plasma. This analytical method, utilizing sitagliptin-d4 as the internal standard, is performed using only 100 μL of plasma and a liquid–liquid extraction procedure based on methyl tert-butyl ether (MTBE). Chromatographic separation is expertly achieved with a Kinetex^® C18 column under isocratic elution, employing a perfect 1:1 blend of 5 mM ammonium acetate (with 0.04% formic acid) and acetonitrile, and maintaining an efficient flow rate of 0.2 mL/min. Detection occurs in positive ionization mode through multiple reaction monitoring, precisely targeting transitions of m/z 408.2 → 193.0 for sitagliptin and 412.2 → 239.1 for the IS. The total runtime of this assay is under 2 min. Comprehensive validation in line with MFDS and FDA criteria demonstrates outstanding linearity (5–1000 ng/mL, r² > 0.998), alongside impressive levels of accuracy, precision, recovery and sample stability. Due to its minimal sample requirement and high-throughput capability, the validated approach is highly appropriate for pharmacokinetic and bioequivalence assessments involving sitagliptin. Full article

A high-throughput method for the determination of a variety of chemical hazards in poultry muscle and egg samples was established via ultra-performance liquid chromatography–tandem triple quadrupole mass spectrometry (UPLC–QqQ-MS). The sample preparation procedure was developed based on this quick, easy, cheap, effective, rugged, and safe (QuEChERS) method and validated for 280 chemical hazards potentially present in poultry products. The target compounds in poultry samples were extracted with a 1% formic acid–acetonitrile solution (15:85, v/v), and the metal ions in the matrix were chelated by adding ethylenediaminetetraacetic acid disodium salt (Na₂EDTA). The supernatant was purified using Enhanced Matrix Removal (EMR) lipid sorbent. Chromatographic gradient separation was performed on an ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) column with multiple reaction monitoring (MRM) under both negative- and positive-ion mode. Internal standard calibration or matrix-matched calibration was used for the quantitation. The results showed that good linearity was achieved for each target compound with correlation coefficients (R²) ≥ 0.99. The limits of detection (LODs) ranged from 0.05 to 10 µg/kg, and the acceptable limits of quantification (LOQs) were determined to be 0.1–20 µg/kg for all 280 compounds. Approximately 90% of the target compounds exhibited mean recoveries ranging from 60% to 120%, with relative standard deviations (RSDs) within 16.2%. This method can be used for the high-throughput rapid detection of prohibited drug residues in poultry eggs due to its easy operation and high accuracy. It was applied in real sample detection, and 43 chemicals including metronidazole were found in 211 poultry samples, with a concentration range of 0.11–638 μg/kg. Full article

Chinese herbal medicines have played a significant role in the development of new and effective drugs, but how to identify the active ingredients from complex extracts of traditional Chinese herbal medicines was a research difficulty. In recent years, few studies have focused on high-efficiency identification of small-molecule inhibitors of Programmed Death Ligand 1 with lower antigenicity and flexible structure tunability. In order to identify small molecule inhibitors of PD-L1 from complex Chinese herbal extracts, this study established a protein–ligand trapping system based on high-performance liquid chromatography coupled with a photo-diode array detector, ion trap/quadrupole time-of-flight tandem mass spectrometry, and a Programmed Death Ligand 1 affinity chromatography unit (ACPD-L1-HPLC-PDA-IT-TOF (Q-TOF)-MS) to rapidly screen and identify small-molecule inhibitors of Programmed Death Ligand 1 from Toddalia asiatica (L.) Lam. Fourteen components were then identified as PD-L1 binders, and surface plasmon resonance (SPR) validation results showed that six of them—magnoflorine (6), nitidine (22), chelerythrine (24), jatrorrhizine (13), toddaculin (68), and toddanol (45)—displayed PD-L1 binding activity. Laser scanning confocal microscopy results demonstrated that these compounds effectively inhibited the binding of PD-1 to PD-L1 in a dose-dependent manner. Additionally, flow cytometry analysis indicated they could promote human lung cancer cell line (A549) apoptosis when co-cultured with Peripheral Blood Mononuclear Cells (PBMCs). The system’s innovation lies in its first integration of dynamic protein–ligand trapping with multi-dimensional validation, coupled with high-throughput screening capacity for structurally diverse natural products. This workflow overcomes traditional phytochemical screening bottlenecks by preserving native protein conformations during affinity capture while maintaining chromatographic resolution, offering a transformative template for accelerating natural product-derived immunotherapeutics through the PD-1/PD-L1 pathway. Full article

Danshen, a well-known traditional Chinese medicine (TCM), has gained increasing attention for its protective effects on nonalcoholic fatty liver disease (NAFLD). However, the molecular mechanisms underlying these effects remain to be elucidated. Niemann-Pick C1-like 1 (NPC1L1), a key transporter mediating intestinal cholesterol absorption, has emerged as a critical target for NAFLD treatment. This study aimed to screen for NPC1L1 inhibitors from Danshen and investigate their therapeutic effects on NAFLD. We established a high-throughput screening platform using stable Caco2 cell lines expressing human NPC1L1 (hL1-Caco2) and discovered that tanshinones (Tans), the liposoluble components of Danshen, inhibited NPC1L1-mediated cholesterol absorption in hL1-Caco2 cells. Additionally, Tans treatment reduced hepatic steatosis in high-fat diet (HFD)-fed mice. To identify the active compounds in Tans, activity-oriented separation was performed by integrating the high-throughput screening platform and two-dimensional chromatographic techniques. Ultimately, cryptotanshinone (CTS) was identified as a novel NPC1L1 inhibitor and significantly decreased hepatic steatosis in HFD-fed mice. Molecular docking and dynamics simulation showed that CTS stably bound with NPC1L1, where TRP383 acted as the key amino acid. Taken together, this study demonstrates, for the first time, that CTS, a liposoluble compound from Danshen, is a novel NPC1L1 inhibitor. Our findings suggest that the inhibitory effect of CTS against NPC1L1-mediated intestinal cholesterol absorption may be a potential mechanism, contributing to its alleviation of NAFLD in mice. Full article

A fast and green approach for the non-chromatographic assaying of volatile metalorganic compounds (VMOCs) is presented, involving the use of thermal desorption microwave-induced plasma optical emission spectrometry for the multi-species simultaneous determination of VMOCs in wastewater plant samples after headspace solid-phase microextraction (HSSPME-TD-MIP-OES), and optimized as a tool for the assessment of ambient exposure to hazardous VMOC pollutants. With the aim of VMOC monitoring, all species are separated and quantified within 10 s in comparison with about 10–20 min required by conventional GC-based procedures. Calibration against aqueous standards was carried out for several metalorganic species. The method was successfully applied for the quantitative extraction of As, Bi, Hg, Sb, Si and Sn compounds. Limits of detection ranging from 5 to 30 ng L⁻¹ and relative standard deviations lower than 4% were obtained. The method is appropriate for high-sample-throughput measurements, and it proved to be suitable for the analysis of wastewater and sewage sludge samples. Full article

Phosphorus is essential to environmental systems because it affects both agricultural productivity and ecological balance. Since it contributes to eutrophication and pollution problems, its existence in a variety of environmental matrices, including soil, water, and air, necessitates precise and effective determination methods for monitoring and managing its levels. This review paper provides an extensive overview of the latest advancements in analytical techniques for measuring phosphorus in environmental samples. We investigate sophisticated spectroscopic, chromatographic, and electrochemical techniques in addition to conventional approaches like colorimetric analysis. Innovative techniques such as mass spectrometry (MS), X-ray fluorescence (XRF) spectrometry, and nuclear magnetic resonance (NMR) spectroscopy are also highlighted in this study, along with newly developed technologies such as biosensors, lab-on-a-chip devices, and nanotechnology-based techniques. Real-time and field-deployable monitoring technologies are also covered, with a focus on their advantages and usefulness. Among the techniques reviewed, XRF and colorimetry methods have proven to be the most reliable due to their precision, cost-effectiveness, and adaptability for different sample matrices. While emerging spectroscopic and electrochemical techniques offer promising alternatives, further validation and standardization are needed for routine environmental monitoring. Future research should focus on integrating automated and high-throughput techniques to enhance monitoring capabilities further. Full article

Flavokavain B (FKB), a hepatotoxic chalcone from Piper methysticum (kava), has raised safety concerns due to its role in disrupting redox homeostasis and inducing apoptosis in hepatocytes. Conventional chromatographic methods for FKB detection, while sensitive, are costly and impractical for field applications. In this work, DNA aptamers were selected using the library-immobilized method and high-throughput sequencing. Three families of aptamers were obtained, and the best one named FKB-S showed a dissociation constant (K_D) of 280 nM using microscale thermophoresis. To demonstrate its practical utility, a rapid and label-free colorimetric aptasensor was developed based on aptamer-induced gold nanoparticle aggregation. This assay achieved a detection limit of 150 nM (43.46 ng/mL) and provided results within 10 min. Compared to traditional chromatographic methods, the aptasensor offers a simple, cost-effective, and equipment-free approach for on-site FKB detection, making it a promising tool for the quality control and safety monitoring of kava-based products in diverse environments. Full article

Background: Testosterone is an androgenic hormone that plays important roles in both males and females. The circulating levels of total testosterone vary from 1 to 1480 ng/dL. High-throughput immunoassays often lack accuracy in lower concentration ranges (below 100 ng/dL), particularly when used for females or children. To address this limitation, we developed a total testosterone LC-MS/MS assay on three instruments. Methods: Sample preparation began with the dilution and conditioning of 200 µL of serum. A supported liquid extraction cartridge was used to extract the analyte from biological matrices. Chromatographic separation was achieved using a C18 column with a runtime of 5 min per sample. This assay was validated on a Triple Quad 6500 and an API 4500 instrument. Results: Method validation was carried out according to the CLSI C62-ED2 guideline and our hospital protocol. The within-day coefficient of variation (CV) was less than 10% and the between-day CV was less than 15%. The assay had a limit of quantitation of 0.5 ng/dL with an analyte measure range of 2–1200 ng/dL. A comparison using Deming regression and Bland–Altman plots showed that this assay correlated well with a reference method. The results from the API 4500 and an Orbitrap were consistent with those from the TQ 6500. Both serum-separator tubes (BD) and serum-activator tubes were found to be suitable. Conclusions: We successfully developed and validated a robust total testosterone LC-MS/MS assay for routine clinical testing. This assay was harmonized across two triple quadrupole instruments and one high-resolution mass spectrometer. Full article

Background: Nicotinamide adenine dinucleotide (NAD⁺), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD⁺ derivatives and it’s reduced forms, with ¹³C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD⁺ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD⁺ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD⁺ metabolism and the potential significance of these metabolites in various biological contexts. Full article

Oxalate, the simplest dicarboxylic acid, is a prevalent antinutrient that chelates with various metals and can lead to the formation of kidney stones in humans. The accurate detection of the oxalate concentration in food and the cultivation of low-oxalate crops are important for enhancing public health. In this study, we established a high-throughput and highly sensitive technique for oxalate detection using ultra-high-performance liquid chromatographic–triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS). Additionally, we overexpressed the gene O7, which encodes oxalyl-CoA synthetase in the maize oxalate degradation pathway, resulting in O7-OE lines. By employing the UPLC-QqQ-MS/MS method to measure oxalate levels in these transgenic lines, we observed that the oxalate content in the kernels of O7-OE lines was reduced by approximately 43%, with a concurrent increase in some micronutrients such as zinc. Importantly, the transgenic maize showed normal seed storage compound accumulation or other agronomic characteristics. In summary, we developed a high-throughput detection method that advances oxalate measurement. Furthermore, by generating new maize germplasm with diminished oxalate, our work offers potential health advantages to consumers. Full article