Search Results (304)

A novel, sensitive, and robust LC-MS/MS method was developed and fully validated for the simultaneous determination of fumonisins (FB₁, FB₂, FB₃) and their hydrolyzed metabolites (HFB₁, HFB₂, HFB₃) in donkey plasma, urine, and feces—three critical matrices for toxicokinetic studies. Sample preparation was optimized for each matrix: salting-out assisted liquid–liquid extraction (SALLE) with perchloric acidification for urine and feces, and a dilute–evaporate–shoot (DES) approach for plasma. Chromatographic separation was achieved on a BEH C₁₈ column with water-ACN containing 0.5% formic acid. The method demonstrated excellent linearity (R² ≥ 0.99), acceptable accuracy (mean recoveries: 73.3–111.5%), and good precision (intra- and inter-day RSDs < 20%). The limits of quantification (LOQ) for FBs and HFBs were 0.1–0.15 μg/L in plasma, 1.0 μg/L in urine, and 60 μg/kg in feces. To our knowledge, this is the first reported method capable of quantifying this comprehensive panel of analytes across multiple biological matrices in donkeys, providing an essential tool for future exposure assessments and pharmacokinetic research in this species. Full article

A method for quantifying tebufenpyrad residues in greenhouse sandy loam soils was developed and validated. Given the strong sorption (high K_oc) of tebufenpyrad to mineral–organic domains in soils, desorption-limited and partially bound residues may occur, so sample preparation methods should actively promote desorption to minimize underestimation. The QuEChERS extraction procedure was optimized by adjusting pre-wetting volume and aqueous medium to enhance desorption prior to salt-induced acetonitrile partitioning. Pre-wetting volume markedly affected phase separation and recovery: acceptable ranges were 80.2–82.0% at 5–10 mL, 94.6% at 15 mL, and 99.1% at 20 mL, while a supra-quantitative value of 119.6% was observed at 25 mL, likely due to salt-induced contraction of the acetonitrile layer, which artificially concentrates tebufenpyrad. Among pre-wetting reagents, 15 mL of 0.05% HCl yielded the highest desorption in field soil (0.20 mg/kg), compared with distilled water (0.13 mg/kg), formic acid (0.16 mg/kg), and EDTA (0.14–0.17 mg/kg). The final method employed 15 mL of 0.05% HCl for pre-wetting, followed by acetonitrile extraction and MgSO₄/NaCl partitioning. Linearity (r² = 0.9990) was achieved over 1.25 to 100 ng/mL, with an LOQ of 0.005 mg/kg and average recoveries of 86.7%, 99.8%, and 98.5% at 0.01, 0.1, and 30 mg/kg, respectively (RSD ≤ 6.2%), satisfying SANTE criteria. In greenhouse soil, residues declined from 1.9 to 0.3 mg/kg at the recommended rate (1×) and from 4.8 to 0.7 mg/kg at the doubled rate (2×) within 46 d (DT₅₀ ≈ 20 d). This validated QuEChERS method provides a reliable analytical basis for evaluating tebufenpyrad dissipation in soil. Full article

This study aimed to establish a fast and efficient method for the determination of N-nitrosamines (NAs) in meat products by integrating two sample preparation techniques—QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) and FaPEx (Fast Pesticide Extraction)—with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Chromatographic separation was performed on a Poroshell 120 Phenyl Hexyl column using a gradient elution of acetonitrile and 0.01% formic acid at a flow rate of 0.3 mL/min and a column temperature of 25 °C. Under these conditions, nine NAs and one internal standard were completely separated within 11 min with selective reaction monitoring mode (SRM) for detection. Samples were first extracted with QuEChERS powder using acetonitrile containing 0.1% formic acid, followed by purification with a FaPEx-Chl cartridge. This combined approach demonstrated superior performance compared with traditional solvent extraction or QuEChERS extraction alone. The recoveries of the developed method ranged from 76% to 111% and 52% to 103% at spiking levels of 50 ng/g and 20 ng/g, respectively. The limits of detection (LOD) and quantification (LOQ) were 0.002–0.3 ng/g and 0.006–1.00 ng/g, respectively. The inter-day and intra-day precisions (RSD%) ranged from 2.7% to 17% and 2.9% to 17%, respectively. These results indicate that the proposed method is among the most time-efficient and effective analytical approaches currently available for the determination of NAs in meat products. Full article

Geranium wilfordii Maxim. is a traditional medicinal plant rich in ellagitannins such as corilagin (CG) and geraniin (GR), which possess antioxidant and anti-inflammatory properties. However, accurate quantification of CG and GR in complex herbal matrices is hindered by co-eluting impurities and poor UV resolution. Here, we developed and validated a simple isocratic HPLC–UV method for their simultaneous determination in G. wilfordii extract. Separation was achieved on a Polaris 3 C18-A column (250 mm × 4.6 mm, 3 µm) using acetonitrile/0.2% formic acid in water (11:89, v/v) with UV detection at 270 nm. The method showed excellent linearity (25–300 µg/mL, R² > 0.995), precision (RSD < 2.7%), accuracy (recovery 99.5–101.2%), and low detection limits (<3 µg/mL). Previous approaches have relied on gradient HPLC or MS-based techniques, often requiring long run times, costly instrumentation, or additional purification (e.g., HSCCC). In contrast, this study demonstrates a validated isocratic method that enables baseline separation and simultaneous quantification of CG and GR in a single run. This robust and simplified analytical strategy provides a practical tool for routine quality control and phytochemical standardization, with potential applications across pharmaceutical, food, and cosmetic industries. Full article

Background/Objectives: The inappropriate use of veterinary medicines in feed for food-producing animals can compromise food safety. Intensive animal production is associated with the inappropriate use of antibiotics in feed, at subtherapeutic concentrations, to promote animal growth. It is therefore crucial to develop an effective multi-detection method to ensure that this feed complies with the requirements of European Commission Regulations. This control is essential to ensure consumer protection, as adequate supervision contributes to reducing antimicrobial resistance, a growing concern worldwide. Methods: A literature search was conducted using scientific databases, namely PubMed, ScienceDirect, Scopus and Google Scholar, as well as European Union Regulations. Results: It was observed that the most used standard solution solvents are methanol, acetonitrile, ultrapure water, or mixtures of these solvents. For extraction, the most frequently used solvents include trichloroacetic acid combined with McIlvaine buffer or with acetonitrile, and acetonitrile or methanol combined with formic acid or with ethylenediaminetetraacetic acid disodium (Na₂EDTA). For extraction and purification of the analyte, several steps were verified, such as solid-phase extraction (SPE), dispersive solid-phase extraction (d-SPE), liquid–liquid extraction (LLE), Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS), protein precipitation through freezing and dilution prior to analysis. Liquid chromatography coupled with mass spectrometry is the preferred choice, especially for multiple detection methods. Conclusions: Based on this data, the foundation is established for the development of an appropriate method for the simultaneous extraction of multiple classes of antibiotics, which is applicable to feed different food-production animals. Full article

Background: Nintedanib (NIN), an intracellular inhibitor of tyrosine kinases that inhibits processes fundamental to the progression of pulmonary fibrosis (PPF), is used in the treatment of patients with PPF associated with systemic sclerosis. During NIN therapy, adverse events lead to a permanent dose reduction and treatment discontinuation. Therapeutic drug monitoring (TDM) can be used to manage and optimize drug administration based on the measurement of drug concentrations. Therefore, TDM can be helpful in minimizing the impact of adverse events and help patients remain in therapy. The aim of this study was to develop and validate a new bioanalytical UPLC-MS/MS method enabling the determination of NIN and its active metabolite in the plasma of patients with PPF associated with systemic sclerosis. Methods: Sample preparation was carried out using protein precipitation with an extraction mixture: acetonitrile neutralized with 2 M sodium carbonate. Analytes and the internal standard (intedanib-d3) were monitored using mass spectrometry (MS) and positive-ion-mode electrospray ionization by MRM. Chromatographic analysis was performed on a Zorbax SB-C18 column kept at 40 °C using isocratic elution. The mobile phase contained 0.1% formic acid in water; acetonitrile (35:65 v/v) was pumped at a flow rate of 0.3 mL/min. The analysis time was 5 min. Results: The method was verified according to the EMA guidelines over a concentration range of 2.00–200.00 ng/mL. The correlation coefficients for the calibration curves were found to be 0.9991 and 0.9957 for NIN and its metabolite BIBF 1202, respectively. The within- and between-run precision and accuracy of LLOQ were evaluated for NIN and BIBF 1202 to be within RSD 2.96%, 4.53%, 5.51%, and 6.72% and in the ranges of 102.2–107.3%, 98.0–101.8%, 104.3–114.2%, and 99.1–104.9, respectively. The stability of the analytes in plasma after 4 h at 30 °C was found to be satisfactory, meeting the assumed bias criteria below 15%. Conclusions: The proposed method was successfully applied to analyze two active compounds—NIN and BIBF 1202—in plasma samples at two time points: trough (pre-dose concentration) and 2–3 h (maximum concentration) after the administration of NIN. Full article

A method combining magnetic solid-phase extraction (MSPE) with ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of diazepam residues in aquatic products. A novel magnetic nanoparticle material, Fe₃O₄@SiO₂@DVB-NVP, was synthesized and applied as an adsorbent for sample cleanup. The sample preparation procedure involved extraction with 1% ammonia–acetonitrile, followed by purification using the MSPE technique to efficiently remove matrix interferents. Chromatographic separation was achieved on an ACQUITY UPLC BEH C₁₈ column with a gradient elution program using a mobile phase composed of 0.1% formic acid–2 mM ammonium acetate solution and methanol. Detection was performed under multiple-reaction monitoring (MRM) mode with positive electrospray ionization (ESI⁺). Quantification was carried out using the external standard method. The synthesized magnetic material was characterized using SEM, TEM, FTIR, XRD, BET, and VSM, confirming its mesoporous structure, strong adsorption capacity, and excellent magnetic responsiveness. The method demonstrated good linearity over the concentration range of 0.25–50 μg/L (r² = 0.997). The limits of detection and quantification were 0.20 μg/kg and 0.50 μg/kg, respectively. Average recoveries from spiked blank matrices at three levels (0.5, 2.5, and 5.0 μg/kg) ranged from 89.3% to 119.7%, with relative standard deviations (RSDs) between 0.8% and 10.2%. The proposed method is highly selective, exhibits minimal matrix interference, and provides reliable quantitative performance, making it suitable for the qualitative and quantitative analysis of diazepam residues in aquatic products. Full article

Fumonisins are among the most prevalent mycotoxins in maize and maize-based products, posing significant food safety and public health risks due to their hepatotoxic, nephrotoxic, and potential carcinogenic effects. Given the strict regulatory limits set by the European Commission and Codex Alimentarius, the development of reliable, sensitive, and matrix–robust analytical methods remain a priority for routine monitoring in both food and feed systems. In this study, a reusable immuno-affinity purification methodology for the quantitative determination of fumonisin mycotoxins (FB1, FB2 and FB3) in foods and feeds (maize matrix) was developed. A single extraction protocol using 2% formic acid in water was employed, followed by cleanup with an immuno-affinity purification column and toxin elution by methanol/PBS (1:1, v/v). Detection and quantification of the mycotoxins was achieved by a normal phase ultra-high performance liquid chromatography coupled with electrospray ionisation triple quadrupole mass spectrometry (UHPLC/ESI-MS/MS). The chromatographic mobile phase utilised was a linear gradient of methanol/water containing 0.1% formic acid. The developed method has a limit of detection of 2.5 ng/g and a limit of quantification of 5 ng/g, all well below the European commission’s guidance values of 1000 ng/g for corn destined for human consumption and 800 ng/g for maize-based breakfast cereals and snacks. While the recovery rates of the method in this study ranged from 65–70% for the three fumonisin analogues in solutions, when tested in maize matrix, recoveries were markedly lower (~30%) due to pronounced matrix suppression. Good repeatability (standard deviation <10%) was achieved for all the fumonisin analogues. The developed method, although quick and effective in solvent systems, suffered limitations to its practical usage due to matrix suppression of the extracts derived from the immuno-affinity purification column, thus significantly reducing the application of the method in measuring fumonisin mycotoxins in food and feed samples. Overall, the method was effective in quantification of fumonisin mycotoxins in solvent solutions but not in food and feed matrices, thus necessitating further optimisation for practical usage. The performance of the developed method was compared to a commercial lateral flow immunochromatographic assay which proved to be better than the developed method in the quantification of toxins in food matrices, as the commercial lateral flow immunochromatographic assay outperformed the developed method in maize matrices. These findings highlight the need for matrix-based validation and further refinement of antibody stability to ensure robust application in regulatory monitoring of fumonisins using immunoaffinity purification methods. Full article

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) is a primary electron donor for both antioxidant enzymes, such as glutathione reductase, and pro-oxidant enzymes, such as NADPH oxidases that produce reactive oxygen species (ROS) and nitric oxide synthases that generate nitric oxide which act as signaling molecules. Monitoring NADPH levels, NADPH/NADP⁺ ratio, and especially distinguishing from NADH, provides vital information about cellular redox status, energy generation, survival, lineage specification, and death pathway selection. NADPH detection is key to understanding metabolic reprogramming in cancer, aging, and cardiovascular, hormonal, neurodegenerative, and autoimmune diseases. Liquid chromatography combined with mass spectrometry (LC-MS) is crucial for NADPH detection in redox signaling because it offers the high sensitivity, specificity, and comprehensive profiling needed to quantify this vital but labile redox cofactor in complex biological samples. Using hepatoma cell lines, liver tissues, and primary hepatocytes from mice lacking transaldolase or nicotinamide nucleotide transhydrogenase, or having lupus, this study demonstrates that accurate measurement of NADPH depends on its preservation in reduced form which can be optimally achieved by extraction of metabolites in alkaline solution, such as 0.1 M potassium hydroxide (KOH) in comparison to 80% methanol (MeOH) alone or 40:40:20 methanol/acetonitrile/formic acid solution. While KOH extraction coupled with hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry most reliably detects NADPH, NADP, NADH, NAD, polyamines, and polyols, MeOH extraction is best suited for detection of glutathione and overall discrimination between complex metabolite extracts. This study therefore supports performing parallel KOH and MeOH extractions to enable comprehensive metabolomic analysis of redox signaling. Full article

The demand for sustainable methods to extract bioactive compounds from native fruits is increasing. We evaluated the potential of natural deep eutectic solvents (NaDES) combined with ultrasound-assisted extraction (UAE) to recover phenolic compounds from Ugni molinae Turcz. (murta), a Chilean fruit with recognized ethnopharmacological and antioxidant value. Seven choline chloride-based NaDESs (M1–M7) were assessed and compared with conventional methanol: formic acid extraction (M8). The choline chloride: 1,2-propanediol system (1:2, M2) achieved the highest recovery of total phenolics (64.87 mg GAE/g) and flavonoids (35.38 mg QE/g), together with strong antioxidant activity (DPPH IC₅₀: 1.05 µg/mL; ORAC: 40,291 µmol TE/g). When comparing the different NaDES formulations (M1–M8), M8 displayed superior FRAP and ORAC values, although its phenolic and flavonoid yields were lower, reflecting differences in solvent selectivity. HPLC-DAD analysis further revealed that NaDES, particularly M5 (choline chloride: oxalic acid, 1:1), favored the extraction of flavonoid and anthocyanin-type compounds. Multivariate and PCA analyses showed distinct chemical profiles in NaDES extracts, forming two clusters apart from M8. Pearson correlation analysis linked antioxidant capacity with major flavonoids. Overall, NaDES combined with UAE represents an efficient, green strategy for selectively recovering bioactives, supporting applications in foods, nutraceuticals, and health products from Chilean native fruits. Full article

Background: A novel triple therapy regimen for Helicobacter pylori eradication, recently approved by the U.S. FDA, comprises vonoprazan (VPN), a potassium-competitive acid blocker, in combination with amoxicillin (AMX) and clarithromycin (CMN). This study presents the development and full validation of a rapid, selective, and sensitive LC-MS/MS method for the simultaneous quantification of these three drugs in spiked human plasma. Methods: Sample preparation was performed using a simple and efficient liquid–liquid extraction (LLE) technique. Chromatographic separation was achieved within 5 min using a Phenomenex Kinetex C18 column (100 × 4.6 mm, 2.6 µm) and a gradient elution system consisting of 0.1% formic acid in water and acetonitrile. Moreover, diazepam was used as an internal standard. The mass spectrometric detection was conducted in multiple reaction monitoring (MRM) mode using positive electrospray ionization. Results: The method exhibited excellent linearity over the investigated concentration ranges (2, 5, 10, 20, 50, and 100 ng/mL for amoxicillin and clarithromycin and 5, 10, 20, 30, 50, and 100 ng/mL for vonoprazan). Intra- and inter-day precision and accuracy values met FDA bioanalytical method validation guidelines, with relative standard deviations and relative errors below 15%. Mean absolute recoveries were above 93% for all analytes. Conclusions: The developed method was fully validated, rapid, selective, and sensitive LC-MS/MS and was assessed using the AGREE tool as a greenness assessment approach, confirming its environmental friendliness and alignment with green analytical chemistry principles. Full article

(1) Background: Caffeine is one of the most widely consumed psychoactive substances. Its safety profile and short half-life make it an ideal drug model for studying the pharmacokinetics of caffeine. This study aimed to develop a method for determination of caffeine in a small volume of saliva (200 µL). (2) Methods: Solid-phase extraction was employed to isolate caffeine from saliva, followed by quantitative analysis using liquid chromatography coupled with diode-array detection. Chromatographic separation was achieved on a C18 column, using a gradient mobile phase of acetonitrile and 0.1% formic acid. (3) Results: The method was validated for selectivity, linearity, precision, and accuracy. Linearity was established over the range of 10–10,000 ng/mL (R² = 0.995). The coefficients of variation for intra- and inter-day precision for the three tested caffeine concentrations did not exceed 12.11%. Recovery from spiked saliva samples exceeded 90.53%. The developed method was applied to preliminary studies to follow the pharmacokinetics of caffeine in saliva. The concentration of the substance was studied in the saliva obtained from a volunteer after espresso consumption. (4) Conclusions: The developed method will offer a reliable approach for non-invasive caffeine monitoring in clinical and research applications. Full article

An investigation of the effect of intense Hot Water Extraction (HWE) on the chemical properties and processability of shredded hemp stalks (Cannabis sativa L.) is presented in this study. The chemical composition of untreated hemp was compared to that of hemp subjected to V and XV successive HWE cycles. This study investigated changes in selected chemical compounds, such as extractives, lignin, cellulose, ash, and monosaccharides such as glucose and xylose. Additionally, post-HWE liquids were analyzed. Lignin content was determined by the UV–VIS spectrophotometry method, whereas monosaccharides (glucose, xylose) and inhibitors (formic acid, acetic acid, levulinic acid, ethanol, 5-(hydroxymethyl)furfural, and furfural) were identified by HPLC. Extractives and ash were effectively removed by the HWE process, decreasing from 3.2 to 2.0% and from 3.9% to 2.7%, respectively. The reduction in acid-soluble lignin was an important finding, indicating a selective modification of the lignin matrix. By the end of V cycles, xylose content in the liquid phase significantly increased from 117.9% to 19.4%, indicating a reduction in hemicelluloses. The cellulose content of the solid material rose from 42.9% to 46.2% at the end of XV cycles. Full article

Background/Objectives: Antibiotic detection in honey is challenging due to the complexity of this product, the typically low levels of residues, and the absence of Maximum Residue Levels (MRLs) for beehive products. The use of antibiotics in apiculture poses potential risks to human health, including antimicrobial resistance and toxic effects. Reliable, sensitive, and selective analytical methods are essential to ensure food safety and enable accurate monitoring of antibiotic contamination in honey. This study aimed to validate a multi-analyte procedure in accordance with the parameters established in Commission Implementing Regulation (EU) 2021/808 for the identification and quantification of antibiotics, including tetracyclines, lincosamides, quinolones, macrolides, β-lactams, sulfonamides, and diaminopyrimidines. Methods: An extraction protocol was developed using 0.1% formic acid in ACN:H₂O (80:20, v/v), followed by a modified QuEChERS with the addition of 1 g NaCl and 2 g MgSO₄. The extracts were analyzed by UHPLC-TOF-MS. Results: The method, validated under CIR (EU) 2021/808, demonstrated robust performance, with recoveries ranging from 80.1% to 117.6%, repeatability between 0.5% and 32.2%, reproducibility between 2.3% and 31.6%, and determination coefficients (R²) ranging from 0.9429 to 0.9982. Validation was achieved for 15 antibiotic residues, with CCβ from 3 to 15 μg·kg⁻¹, LODs between 0.09 and 6.19 μg·kg⁻¹, and LOQs between 0.29 and 18.77 μg·kg⁻¹. Application to 10 commercial Portuguese honey revealed no detectable levels of the target antibiotics. Conclusions: The combination of a simplified extraction with UHPLC-TOF-MS provides a reliable approach for the determination of antibiotics in honey. This validated method represents a valuable tool for food safety monitoring and risk assessment of apiculture practices. Full article

A standardized pretreatment protocol was established for simultaneous determination of diazepam and its metabolites—nordazepam, oxazepam, and temazepam—in aquatic products using liquid chromatography–mass spectrometry. Samples were extracted with 0.2% formic acid in acetonitrile (solid–liquid ratio 1:5, m/v), purified via MCX solid-phase extraction, eluted with 5% ammoniated methanol, and concentrated under reduced pressure. The residue was reconstituted in 0.2% formic acid–50% acetonitrile aqueous solution. Chromatographic and mass spectrometric conditions were optimized on two platforms: UPLC-QE-Orbitrap-MS and UPLC-MS/MS, with quantification based on internal standards. Both platforms showed excellent linearity across 0.2–200 ng/mL (R² > 0.997), with detection and quantification limits as low as 0.1 μg/kg and 0.2 μg/kg, respectively. Following Codex Alimentarius guidelines (CAC/GL-71), 330 matrix samples (intra-batch n = 6, inter-batch n = 5) were validated, showing strong inter-platform agreement (Pearson r > 0.990, p < 0.001). Intra-batch RSDs ranged from 1.86% to 14.64%, and inter-batch RSDs from 1.10% to 11.41%. Recoveries ranged from 73.8% to 117.9% (p > 0.05). The dual-platform detection system developed herein demonstrates high sensitivity, strong matrix interference resistance, and excellent reproducibility, enabling accurate trace quantification of diazepam and its metabolites in heterogeneous aquatic samples. Full article