Search Results (419)

A comprehensive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous quantification of 50 pesticide biomarkers across nine current-use chemical classes in human urine. These classes include organophosphorus insecticides (which encompass dialkyl phosphates and specific metabolites), pyrethroid insecticides, fungicides, neonicotinoid insecticides, herbicides, insect repellents, organochlorine pesticide metabolites, and plant growth regulators. The method employs solid-phase extraction (SPE) for sample preparation, requiring only 0.2 mL of urine. Chromatographic separation was optimized using a Hypersil Gold AQ column, achieving a total run time of 18 min. Mass spectrometric detection utilized polarity switching in electrospray ionization mode with multiple reaction monitoring. Method validation demonstrated satisfactory linearity (R² > 0.99), high sensitivity with limits of detection ranging from 0.01 to 0.88 ng/mL, and extraction efficiencies between 85% and 113%. Precision and accuracy were within acceptable ranges, with relative standard deviations generally below 15%. The method’s robustness was confirmed through participation in external quality assessment schemes. Application to real samples revealed significant inter-individual variability in pesticide biomarker concentrations, with total measured biomarker levels ranging from 89 to 1242 ng/mL across the 10 individuals analyzed. This method offers comprehensive coverage of current-use pesticide chemical classes, including 30 biomarkers from the U.S. National Health and Nutrition Examination Survey (NHANES) biomonitoring program, and demonstrates improved sensitivity and broader analyte coverage compared to existing methods. The developed assay provides a valuable tool for large-scale biomonitoring studies and environmental health research. Full article

Although the genus Rhododendron is globally distributed and rich in bioactive constituents, the metabolomic landscapes of most species remain unexplored, hampering elucidation of their adaptive strategies and pharmaceutical potential. Objectives: This study sought to construct comprehensive metabolic atlases of four representative yet understudied Rhododendron species—R. triflorum, R. faucium, R. nivale, and R. strigillosum—and to quantify inter-specific metabolic divergence by UPLC-MS/MS-based, widely targeted metabolomics. Methods: The petals of four Rhododendron species were freeze-dried, pulverised, and extracted with 70% methanol (containing an internal standard). Metabolites were separated on an SB-C18 column (2.1 × 100 mm, 1.8 µm) using a 0–95% acetonitrile gradient (flow rate 0.35 mL min⁻¹, 40 °C) and analysed by tandem mass spectrometry. Reliable quantification was ensured by molecular weight database matching, ion source standardisation, and quality control (QC), achieving a coefficient of variation (CV) < 15%. Principal component analysis (PCA) and optimised partial least squares discriminant analysis (OPLS-DA) were performed on standardised data with unit variance. Results: A total of 3705 metabolites were confidently identified, dominated by flavonoids (870), terpenoids (572), phenolic acids (394), and amino-acid derivatives (332). PCA and OPLS-DA models revealed clear species-specific clustering (R²Y ≥ 0.98, Q² ≥ 0.95; permutation test p < 0.01). Comparative analysis yielded 1495 significantly differential metabolites; R. triflorum exhibited the highest cumulative abundance, followed by R. faucium, R. nivale, and R. strigillosum. KEGG enrichment highlighted “metabolic pathways” as the most significantly over-represented, together with flavonoid biosynthesis, phenylpropanoid metabolism, and terpenoid backbone biosynthesis. Conclusions: The study delivers the first high-coverage metabolomic reference for four neglected Rhododendron species, evidencing profound inter-specific metabolic differentiation centred on flavonoids, terpenoids, and phenolic acids. The data provide a robust foundation for understanding molecular adaptation to alpine environments and for accelerating targeted drug discovery from Rhododendron resources. Full article

Severe browning often occurs in Multivitamin Iron Oral Solution during storage, which directly leads to the decline of product quality. To clarify the main mechanism of browning in this preparation, the contents of 5-hydroxymethylfurfural (5-HMF) and carbohydrates, as well as the relevant characteristic parameters such as color and fluorescence, were determined at different storage times in this study. Subsequently, four reaction models, namely sucrose-lysine, sucrose-citric acid, sucrose-niacin, and sucrose-folic acid, were constructed according to the formulation of the preparation to systematically investigate the effects of each system on browning. The results showed that the sucrose-lysine model was the main color-forming reaction system of the preparation. Citric acid could significantly promote the hydrolysis of sucrose to produce two reducing sugars, glucose and fructose, which not only provided sufficient substrates for the Maillard reaction (MR), but also led to the massive accumulation of 5-HMF. Further analysis revealed that heating temperature and heating time were significantly positively correlated with the contents of 5-HMF, browning index (BI), color density (CD), and reducing sugars in the solution, while significantly negatively correlated with sucrose content (p < 0.05). Two fractions, P1 and P2, were isolated by Sephadex LH-20 column chromatography. Among them, P1 with a molecular weight of 61,660 Da was identified as the key fluorescent color-forming component, whose ultraviolet and fluorescence characteristics were basically consistent with those of Multivitamin Iron Oral Solution. Ultra-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) analysis confirmed that P1 contained characteristic fragments of conjugated unsaturated structure, which was the key chromophore responsible for its fluorescence properties. In summary, this study explored the main browning mechanism of Multivitamin Iron Oral Solution. It was found that after citric acid catalyzed the hydrolysis of sucrose, the generated reducing sugars underwent Maillard reaction with lysine to produce fluorescent color-forming substances, and heat treatment significantly aggravated the browning process. The results of this study not only provide a solid theoretical basis for optimizing the preparation process and improving the storage stability of Multivitamin Iron Oral Solution, but also offer an important reference for the research on the browning mechanism and stability of other sugar-containing liquid preparations. Full article

Background: low-quantity or degraded samples are often studied in forensic genetics. Therefore, it is important to efficiently obtain all the available DNA from the biological sample analyzed to provide the most reliable results. This is particularly challenging in bone marrow processing due to its hydrophobic molecular structure, as for other lipid-rich tissues, especially if rancid. In fact, during adipose tissue decomposition, the putrefaction of fatty acids can in some instances give a compact cerous consistency to the lipidic tissue, hardly susceptible to the nucleic acid extraction mechanisms. According to environmental circumstances, this condition is notably observable in submerged bodies or in putrefied bone marrow. Thus, this study is focused on developing an optimized nucleic acids extraction protocol for putrefied bone marrow. Methods: genetic analyses were performed on putrefied yellow bone marrow collected from 20 human femora recovered from bodies in different decomposition stages. The optimized method was developed by integrating additional steps, reagents and time intervals on a silica-based column commercial kit. This strategy was compared in DNA yield to a standard extraction protocol, represented by the same commercial kit, but following the manufacturer’s directions. Both these strategies were tested in nucleic acid isolation efficiency by performing DNA typing, including real-time PCR quantification, Short Tandem Repeats (STR) amplification and fragments analysis steps. The analytical parameters evaluated were allele count, DNA concentration (ng/µL) and Degradation Index (DI). Results: for allele count and DNA concentration parameters, the optimized protocol showed clear and significant qualitative and quantitative improvements compared with the standard protocol, supporting its potential applicability in forensic casework and laying the foundation for future studies. Conclusions: prior to appropriate laboratory internal validation, the optimized protocol can be used for tough lipid-rich tissues processing without the need to purchase a dedicated system and using a same commercial kit routinely adopted for other forensic genetics matrices. Full article

Fungicide contamination is an increasing global environmental concern, due to the harm they may pose to non-target organisms, their contribution to antimicrobial resistance, and the potential risks to human health when drinking water (DW) sources are impacted. Many fungicides used in agriculture are chiral and may exist as racemates, or a combination of diastereoisomers and/or enantiomers. Since enantiomers can differ in environmental fate, distribution, and toxicity, enantioselective analysis of chiral fungicides is crucial. The aim of this study was to develop and validate an analytical method for the determination of azole chiral and achiral fungicides in DW using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS). Chromatographic separation of one achiral fungicide and five chiral fungicides was achieved using a polysaccharide chromatographic column under reverse elution mode. The validated method demonstrated high sensitivity with method detection limits (MDL) below 0.86 ng L⁻¹ and was successfully applied to 13 DW samples collected from various supply networks across Portugal. Seven out of the 15 targeted analytes were found at trace concentrations (>MDL). Fluconazole was the most frequently detected (~87% of the samples). The hazard quotients (HQs) for individual compounds for each individual fungicide (sum of the enantiomers for those chiral) and the hazard index (HI, sum of the individual HQ values) were calculated in each DW sample, indicating no significant health risks to consumers, since it is well below 0.1 for all compounds. Full article

This study investigates the axial–flexural behavior of steel fiber–reinforced concrete (SFRC) columns under combined constant axial load and monotonic lateral loading. Nine column specimens with different axial load ratios (0.0, 0.10, and 0.20) and steel fiber contents (0.0%, 0.5%, and 1.0%) were tested under monotonic loading to evaluate their failure modes, load–deflection behavior, ductility, and energy absorption capacity. In addition, a sectional P–M interaction analysis was performed to examine the influence of steel fiber inclusion on flexural strength under different axial compression levels. The interaction diagrams indicated that steel fibers expanded the flexural strength envelope, with a more pronounced enhancement in the low-axial-load region. The test results revealed that increasing the axial load ratio enhanced the specimens’ peak load capacity but reduced their ductility, leading to a brittle failure mode. Conversely, the incorporation of steel fiber improved the crack distribution, delayed crack propagation, and enhanced both ductility and energy absorption, particularly under moderate axial load conditions. The failure modes were characterized generally by flexural cracking and localized crushing in the compression zone, with the specimens that contained steel fiber exhibiting a more gradual post-peak load response than the specimens without steel fiber. The energy absorption capacity, quantified as the area under the load–deflection curve, was maximized when the axial load ratio of 0.10 was used in tandem with steel fiber reinforcement, indicating an optimal balance between strength and ductility. Overall, steel fiber inclusion improved deformation capacity and energy absorption under monotonic loading, particularly at low-to-moderate axial load ratios. Full article

Tobacco smoke exposure not only increases the risks of lung cancer and cardiovascular disease, but can be a stressor contributing to mental illness. It is important to clarify the relationship between chronic tobacco smoke exposure and mental stress from the perspective of disease prevention. We developed a simple and highly sensitive method for simultaneously analyzing nine biomarkers: nicotine and cotinine (tobacco smoke exposure markers); cortisol, testosterone, and dehydroepiandrosterone (stress-related markers); and serotonin, melatonin, dopamine, and oxytocin (relaxation-related markers). Biomarkers were extracted and concentrated by in-tube solid-phase microextraction with a Supel-Q PLOT capillary, followed by separation and detection within 7 min using liquid chromatography–tandem mass spectrometry on a Discovery HS F5 column. Calibration curves using stable isotope-labeled internal standards showed good linearity (0.005–100 ng mL⁻¹) with detection limits of 0.09–13.5 pg mL⁻¹. Intra-day and inter-day precision had relative standard deviations below 7.2% and 15.5% (n = 6), respectively, with recovery rates of 84.0–108.8%. The automated method requires only ultrafiltration of hair methanol extract, enabling non-invasive pg-level analysis using just a few milligrams of hair. Hair analysis reflects an association between chronic tobacco smoke exposure and stress. This method is effective for analyzing the relationship between long-term tobacco smoke exposure and chronic stress. Full article

Nitrofurans are banned veterinary medicinal products due to their carcinogenic and mutagenic properties; however, their protein-bound metabolites (AOZ, AMOZ, AHD, SEM, and DNSAH) may persist in food-producing animals, particularly in eggs. Reliable confirmatory methods are therefore essential for residue monitoring under the stringent requirements of Commission Implementing Regulation (EU) 2021/808. This study reports the development and validation of a sensitive and selective LC–MS/MS method combining acid hydrolysis, 2-nitrobenzaldehyde derivatization, and QuEChERS extraction for the determination of nitrofuran metabolites in eggs. Chromatographic separation was carried out using a phenyl-hexyl column, and detection using a tandem mass spectrometer, supported by isotope-labeled internal standards, ensured robust identification and quantification. Linearity was satisfactory over the investigated concentration range (R² > 0.99), with recoveries between 82 and 109%. The method’s precision was acceptable, with repeatability RSD values below 10% and within-laboratory reproducibility RSD values below 22%. Matrix effects were effectively controlled, remaining within ±20% following internal standard normalization. Decision limits (CCα) ranged from 0.29 to 0.37 µg/kg, well below the EU reference point for action of 0.5 µg/kg. The method’s performance was further confirmed through participation in an accredited proficiency test scheme. Overall, the validated method provides a reliable analytical tool for routine official control laboratories, enabling the sensitive confirmatory detection of banned nitrofuran residues in eggs and supporting food safety and regulatory compliance. Full article

Glucocorticoid (GC) residues present in aquatic products raise food safety concerns, as their chronic dietary intake may pose potential risks of endocrine and metabolic disruption. For the first time, a sensitive and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed and validated herein for the simultaneous determination of 12 GCs residues, including critical isomeric pairs and acetate ester derivatives, in a variety of aquatic foods, employing deuterated isotopic internal standards. Key optimizations included using a pentafluorophenyl column for effective isomer separation, a synergistic extraction system for high recovery, and QuEChERS purification to mitigate matrix effects. The method exhibited excellent linearity (r² > 0.996) and high accuracy (recoveries 97.3–99.3%), and the intra- and inter-day precision values were below 3% in five representative aquatic matrices, with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.5 μg/kg and 0.75 μg/kg, respectively. Animal experiments confirmed the in vivo retention of acetate derivatives, justifying their inclusion in monitoring. Real sample analysis of 18 market samples revealed the presence of cortisone and hydrocortisone in 17 samples. This represents the first reported LC-MS/MS method that provides a sensitive, reliable tool for regulatory monitoring of GC residues in diverse aquatic products, thereby supporting food safety assurance. Full article

The use of lecithin as an emulsifier in food supplements has increased in recent years. However, successful formation of liposomes or micelles requires an appropriate mixture of phospholipids in lecithin. To evaluate the emulsification properties of lecithin for food supplements, a reliable analytical procedure for characterizing phospholipids is necessary. A liquid chromatography–mass spectrometry method was developed to identify phospholipids in lecithin without standard reference materials. For efficient separation of phospholipids before mass spectrometric analysis, a reverse-phase high-performance liquid chromatography method was optimized using a Waters XBridge Protein BEH C4 column. The optimized chromatographic method demonstrated good linearity and precision. Molecular ions were detected in full scan mode to determine accurate mass-to-charge ratios for individual peaks in the chromatogram. A custom Python program was then used to generate a list of possible phospholipid species for each peak based on the measured mass-to-charge ratios. Tandem mass spectrometry was performed to confirm the identity of specific phospholipids by comparing experimental fragmentation patterns with theoretical predictions. Identification of the phospholipids was also confirmed with four commercially available standard reference compounds, demonstrating the reliability of the proposed approach. The developed method offers a practical and cost-effective strategy for identifying phospholipids in complex matrices, especially when standard reference compounds are unavailable. Additionally, it enables targeted selection of standard compounds for future quantitative analyses, making it a valuable tool for comprehensive lipid profiling. Full article

Dipeptidyl peptidase-4 (DPP-4) is a protease that degrades incretin and inhibits the secretion of insulin. Consequently, DPP-4 inhibition promotes insulin secretion and prevents the onset of type 2 diabetes. Given the growing interest in food-derived DPP-4 inhibitory peptides as potential functional ingredients, buckwheat (Fagopyrum esculentum) represents a promising source; however, few studies have investigated the bioactivity of peptides derived from buckwheat flour hydrolysates. In this study, two DPP-4 inhibitory peptides, Ile-Pro-Trp and Ile-Pro-Leu, were identified through purification of buckwheat flour hydrolysate and liquid chromatography–tandem mass spectrometry analysis. In a rat oral glucose tolerance test (OGTT), a fraction of buckwheat flour hydrolysate, crudely purified by reverse-phase column chromatography, showed a non-significant trend toward reducing increases in blood glucose. To our knowledge, this study is the first to show that Ile-Pro-Trp isolated from food protein hydrolysates exhibits considerable DPP-4 inhibitory activity. Moreover, this is the first study identifying Ile-Pro-Ile as a DPP-4 inhibitor from a plant source. 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

Aflatoxin M1 (AFM1), a hydroxylated metabolite of aflatoxin B1, is a persistent food safety hazard in the dairy production chain. This study investigated AFM1 occurrence in fermented dairy products collected from the Croatian market in spring 2025 and assessed associated dietary exposure risks. A total of 81 samples were analyzed using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) following immunoaffinity column clean-up. AFM1 was detected in 48.1% of samples, with a mean concentration of 0.015 µg/kg. Products with thermophilic and probiotic bacterial cultures showed the lowest incidence rates, at 33.3% and 40.0%, respectively. Significantly higher AFM1 occurrence was found in Croatian samples than in imported ones (p < 0.05). Exposure assessment, based on estimated daily intake (EDI), hazard index (HI), and margin of exposure (MOE), identified toddlers and children as the most at-risk groups, with EDI ranging from 0.21 to 0.93 ng/kg bw/day, depending on AFM1 concentration. HI exceeded 1 even at mean AFM1 levels, while MOE fell below the safety threshold of 10,000 in worst-case scenarios, indicating potential health concerns. These findings underscore the need for continuous monitoring and targeted risk mitigation strategies for vulnerable populations, and support expanding regulatory limits to include processed dairy products. Full article

A rapid ultra-high-performance liquid chromatography method with diode-array (DAD) and tandem mass spectrometric (MS/MS) detection was developed and, for the first time, applied for the determination of potassium iodide in ophthalmic formulations. The approach is based on an indirect derivatization reaction in which iodide is oxidized by hydrogen peroxide to electrophilic iodine species that react with 4-hydroxybenzoic acid, forming 3-iodo-4-hydroxybenzoic acid as a stable and quantifiable product. Key reaction parameters, including oxidant concentration, temperature, and incubation time, were optimized to ensure selective mono-iodination and consistent analytical response. Chromatographic separation was performed on a C18-DE RP column using gradient elution with aqueous ammonium acetate and acetonitrile, while MS/MS detection was carried out in MRM mode under ESI(−) conditions. Both UHPLC–DAD and UHPLC–MS/MS methods were validated according to ICH Q2(R2), demonstrating linearity with r² ≥ 0.998, recovery values of 97.5–107.1%, and intraday/interday RSD values up to 3.7%. UHPLC–MS/MS provided higher sensitivity (LOD 37.7 ng/mL; LOQ 114 ng/mL), whereas UHPLC–DAD reached LOD and LOQ values of 24.9 and 75.4 µg/mL. Comparative analysis showed that DAD is suitable for routine quantification, while MS/MS allows lower detection limits and improved selectivity. The developed method offers a practical and reliable tool for the quality control of potassium iodide in ophthalmic formulations. 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