Search Results (301)

The ongoing substance use crisis in the United States involves a broad range of illicit and prescription drugs, including opioids, stimulants, sedatives, and various psychoactive and non-psychoactive compounds. Traditional surveillance methods rely on self-reported data, which could lead to bias and recall inconsistencies. Wastewater-based epidemiology has emerged as a powerful, non-invasive tool for monitoring community-level drug use, offering near real-time estimates and the potential to serve as an early warning system. However, challenges such as analyte degradation, wastewater variability, and matrix effects can affect data quality and comparability across regions. This study presents a standardized, practical workflow for multi-drug (n = 52) detection in wastewater, aiming to minimize analyte loss and improve reproducibility. Composite samples were collected from multiple U.S. cities, transported on ice, and extracted using solid-phase extraction. Extraction efficiencies were compared using Oasis Hydrophilic-Lipophilic-Balanced and Mixed-mode Cation-Exchange (MCX) cartridges, with the MCX sorbent providing complementary reversed-phase and cation-exchange interactions that enabled the retention of chemically diverse compounds across multiple drug classes. Analysis was performed with an Ultra-High-Performance Liquid Chromatography system coupled to a triple quadrupole mass spectrometer, in which the instrument parameters and critical methodological considerations, including sample handling, transport, column selection, and method validation, are detailed. This work contributes to the development of a robust, scalable protocol for multi-drug surveillance in wastewater, supporting timely, data-driven public health responses and informing national drug policy efforts. Full article

Background/Objective: Immunoglobulin G (IgG) N-glycosylation is an important regulator of immune function and systemic inflammation and has been associated with cardiometabolic diseases. However, little is known about how IgG glycosylation changes during the course of acute coronary syndrome (ACS) and whether these alterations relate to lipid-lowering response after the initiation of statin therapy. The primary aim of this study was to investigate IgG N-glycosylation following ACS and evaluate its association with response to atorvastatin therapy defined as baseline LDL cholesterol reduction of ≥50%. Methods: In this prospective cohort study, 79 statin-naïve patients hospitalized for the first episode of ACS and treated with atorvastatin 80 mg daily after percutaneous coronary intervention were followed longitudinally. Plasma samples were collected at admission (acute phase), discharge (subacute phase), and follow-up (chronic phase). A control group of 21 individuals received atorvastatin for primary prevention. IgG was isolated from plasma, and N-glycans were released, fluorescently labeled with 2-aminobenzamide, and analyzed using hydrophilic interaction-based ultra-high-performance liquid chromatography with fluorescence detection. Derived glycan traits were calculated, including agalactosylated (G0), monogalactosylated (G1), digalactosylated (G2), core fucosylated (F), bisected (B), and sialylated (S) glycans. Results: No significant differences in derived IgG glycan traits were observed between ACS patients and controls at baseline or follow-up. Within the ACS group, a longitudinal analysis revealed significant increases in G0 and F and a decrease in G2 between the acute and chronic phases. A total of 65% of patients achieved ≥50% reduction in LDL cholesterol (LDL-C), whereas only 22% reached the guideline-recommended LDL-C target of <1.4 mmol/L. Patients achieving ≥50% LDL-C reduction exhibited consistently higher G0 and lower G2 and S across disease phases. In a subgroup of patients with baseline LDL-C >3.9 mmol/L, those who failed to achieve ≥50% LDL-C reduction had significantly lower G0 and higher S across all time points. Conclusions: Specific glycan traits are associated with the degree of LDL-C reduction achieved during statin therapy, particularly in patients with high baseline LDL-C. These findings suggest that IgG glycosylation patterns may reflect biological phenotypes associated with differential lipid-lowering responsiveness after ACS. Full article

Mitochondrial dysfunction profoundly alters cellular metabolism, yet its systems-level consequences remain incompletely characterized. Here, we present a comprehensive untargeted metabolomics analysis of respiratory-deficient (ρ⁰) and competent (ρ⁺) Saccharomyces cerevisiae prototrophic cells using ultra-high-performance liquid chromatography coupled to Orbitrap Fusion™ Tribrid™ high-resolution mass spectrometry. By integrating hydrophilic interaction and reversed-phase chromatography in both ionization modes, we detected ~7000 features per chromatographic condition, of which ~12% were structurally annotated through MSⁿ fragmentation and in silico spectral matching. Principal component analysis revealed distinct metabolic signatures between ρ⁰ and ρ⁺ cells, with ~73% of total variance explained by the first two components. Volcano plot and hierarchical clustering analyses identified a marked accumulation of phosphate-containing metabolites, sphingolipids, ceramides, and fatty acid residues in ρ⁰ cells, whereas amino acids, excluding arginine, cysteine, and aromatics, were enriched in ρ⁺ cells. Notably, branched-chain amino acid depletion in ρ⁰ cells correlated with impaired growth and mitochondrial stress. Pathway enrichment analysis, supported by transcriptomic integration, prompted us to further investigate reprogramming of polyamine biosynthesis and aromatic amino acid metabolism. Calibration curves constructed from certified standards validated the accuracy of the LC–MS platform and reinforced annotation confidence. Our findings demonstrate that advanced untargeted metabolomics, coupled with MS³ fragmentation and multi-omics integration, enables high-resolution mapping of metabolic reconfiguration under mitochondrial dysfunction, offering mechanistic insights into mitochondrial retrograde signaling and adaptation. Full article

Background: Pantoprazole is a widely used proton pump inhibitor that is highly unstable under acidic conditions. This limits the performance of conventional formulations and typically requires enteric-coated dosage forms or alternative modified-release approaches. This study reports the development of polymeric matrix mini-tablets designed to protect pantoprazole during gastric exposure and to enable pH-dependent release under intestinal conditions. The formulations combine Eudragit^® S 100, a pH-dependent polymer, with HPMC, a hydrophilic matrix former that modulates drug release through hydration and swelling. Methods: Matrix mini-tablets were prepared by blending pantoprazole with selected excipients at optimised proportions and compressing the blends by direct compression using an eccentric tablet press. Powder blends and mini-tablets were characterised according to pharmacopoeial specifications. Analytical techniques—including High-Performance Liquid Chromatography (HPLC), Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Absorption Spectroscopy (FT-IR), Powder X-Ray Diffraction (PXRD), and Scanning Electron Microscopy (SEM)—were employed to evaluate drug content uniformity, thermal behaviour, and potential drug–excipient interactions. In vitro dissolution studies were performed under sequential pH conditions, and the release kinetics were analysed using mathematical models. Results: Dissolution testing identified formulations F2 and F6 as providing the most suitable gastro-resistant performance in the acidic stage, together with sustained release up to 24 h. Kinetic modelling supported formulation-dependent release mechanisms, and multivariate analysis (PCA) highlighted relationships between physico-mechanical attributes and drug-release behaviour. Conclusions: The proposed matrix system shows potential as a robust, coating-free platform for the modified delivery of acid-labile drugs using direct compression, simplifying manufacturing. These findings support the rational design of oral modified-release formulations based on polymeric matrices. Full article

Untargeted metabolomics faces significant challenges in standardization due to variability introduced by sample preparation and analytical workflows. We systematically evaluated the impact of biological matrices, extraction protocols, and chromatographic configurations to establish a mechanism-informed framework aimed at improving reproducibility in large-scale clinical and epidemiological studies. Three extraction protocols were compared using an in-house pooled heparin plasma: monophasic protein precipitation with isopropanol (IPA), methanol:acetonitrile (MeOH:ACN), and a modified Matyash biphasic method. The most reproducible protocol was then applied to four blood matrices. Samples were analysed using untargeted metabolomics on hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) HPLC columns, with mass spectrometry data processed using Compound Discoverer. Both IPA and MeOH:ACN extractions achieved over 80% of features with coefficient of variation (CV%) ≤ 30% for both RP and HILIC, whereas the Matyash method showed higher variability, with a larger proportion of metabolites exhibiting CV% > 30%. Across matrices, RP chromatography detected over 80% of metabolites with CV% < 30%, while HILIC showed higher variability, with at least 20% of metabolites above this threshold. Among matrices, serum and heparin plasma outperformed EDTA and citrate in reproducibility. We propose a standardized workflow in which monophasic extractions combined with RP chromatography maximize reproducibility and metabolite coverage, minimizing methodological artefacts and providing a reliable framework for robust biological discovery in large-scale untargeted metabolomics studies. Full article

This study compares the chemical composition, antimicrobial effects, and antibiotic-potentiating capacity of three Lauraceae essential oils (EO): Cryptocarya agathophylla (CAEO), Litsea cubeba (LCEO), and Laurus nobilis (LNEO). Gas chromatography–mass spectrometry (GC–MS) analysis revealed distinct chemotypes: CAEO and LCEO were dominated by oxygenated monoterpenes, while LNEO contained the highest levels of monoterpene hydrocarbons. Antibacterial testing against nine bacterial strains showed strain-dependent growth suppression trends, while true minimum inhibitory concentrations (MICs) were reached only in selected cases. EO–ampicillin interactions were evaluated using MIC-based checkerboard criteria, whereas OD-derived inhibition parameters were used exclusively to describe sub-MIC potentiation trends. In combination assays, LNEO exhibited the most pronounced potentiating effects against Streptococcus pyogenes, Shigella flexneri, and Haemophilus influenzae, while CAEO and LCEO showed moderate or strain-dependent enhancement. Hierarchical clustering highlighted distinct oil- and strain-specific interaction profiles. Overall, although CAEO displayed stronger intrinsic antibacterial effects when tested alone, LNEO emerged as the most effective potentiator of ampicillin activity in a strain-dependent manner. The effects of the major compounds identified in the Lauraceae EO were assessed in silico against protein targets of some microorganisms using the AutoDock software version 4.2.6. The docking scores revealed binding affinities of the bioactive compounds towards Dpr protein (4.3–5.8 kcal/mol), DNA gyrase (4.7–7.1 kcal/mol), mono- diacylglycerol lipase (4.4–6.2 kcal/mol), CYP51 (5.8–8.0 kcal/mol), phage-encoded quorum sensing anti-activator (5.8–8.0 kcal/mol) and Chondroitin ABC lyase I (4.8–6.3 kcal/mol). Two (2) hit compounds (α-Citral, β-Citral) were finely defined by strong hydrophobic and hydrophilic interactions with the bacterial and fungal protein targets, respectively. Full article

The gut microbiome evolves in response to host development, health state, lifestyle, nutrition, and microbial interactions. The survival of gut microbiota depends on its ability to utilize its host-indigestible complex oligosaccharides. Certain gut microbes produce glycosidases that cleave N-glycoproteins to release N-glycans that are then used as a carbon source. However, commercial glycosidases are inefficient and, thus, require improved deglycosylation strategies to study their functions and scale up their production. Therefore, the main objective of this study was to recombinantly produce and characterize the novel endo-β-N-acetylglucosaminidase 2 (EndoBI-2) from Bifidobacterium longum subsp. infantis (B. infantis) and to evaluate its enzymatic performance for controlled N-glycan release. Furthermore, the optimum reaction conditions for EndoBI-2 were investigated on model glycoprotein RNAse B using model glycoprotein. The released N-glycans were profiled by hydrophilic interaction liquid chromatography-fluorescence detection-quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). We demonstrated that EndoBI-2 possesses a strong temperature tolerance and efficiently cleaves N-glycans under mild reaction conditions, exhibiting high activity at pH 5. These findings highlight EndoBI-2 as a robust and efficient biocatalyst for the production of bioactive N-glycans from diverse N-glycoproteins, with potential applications in glycobiotechnology. Full article

Methenamine, a urinary antiseptic with antimicrobial properties, decomposes into toxic formaldehyde under acidic conditions. Its use is prohibited in dairy cattle in Korea to prevent harmful residues in milk. This study was designed to develop and validate a sensitive and reliable LC–MS/MS method for determining methenamine in raw milk and bovine muscle in compliance with the Positive List System (PLS) regulations. Samples were extracted with acetonitrile (ACN)–methanol (MeOH) (7:3, v/v) containing ammonia water, followed by defatting with n-hexane and purification with primary secondary amine (PSA). Chromatographic separation was performed on a hydrophilic interaction liquid chromatography (HILIC) column, and quantification was conducted using matrix-matched calibration to minimize matrix effects. The method showed excellent linearity (R² > 0.999), low limits of quantification (LOQ) (0.49 μg/kg for raw milk; 0.64 μg/kg for bovine muscle), and acceptable recoveries (78.1–102.8%) with precision (CV ≤ 8.75%), meeting Codex CAC/GL 71-2009 criteria. Stability studies demonstrated that methenamine remained stable in stock solutions, working standards and processed extracts under the storage and handling conditions used. Application to incurred samples resulted in the detection of methenamine in 2 of 32 raw milk samples (0.65 and 1.14 μg/kg) but in none of the 25 bovine muscle samples, with all detected levels below the Korean PLS limit. These findings confirm that the developed method is accurate, sensitive, and applicable for routine surveillance of methenamine residues to ensure consumer safety. Full article

Many polar compounds of biochemical and pharmaceutical relevance exhibit low retention in reversed-phase liquid chromatography (RPLC), making their separation challenging. While hydrophilic interaction liquid chromatography (HILIC) columns are commonly used for such analyses, they require mobile phases with high organic solvent content. This work explores an alternative approach using RPLC with conventional C18 columns and mobile phases containing low percentages of acetonitrile, along with small amounts of the surfactant sodium dodecyl sulfate (SDS). This combination significantly enhances the retention of highly polar compounds. When the SDS concentration is sufficiently low, below the critical micellar concentration in water (8 mM), the retention increase follows a linear pattern. The retention behavior of polar compounds with different properties (nucleosides, methylxanthines, sulfonamides, and the diuretic hydrochlorothiazide) is examined using mobile phases in the submicellar region, with SDS concentrations ranging from 0 to 0.3 mM, acetonitrile contents between 10 and 20% (v/v), and temperatures varying from 25 to 55 °C. Changes in peak half-widths are also analyzed. Since SDS adsorbs onto the stationary phase, modifying its surface, the equilibration time has been investigated as a critical factor affecting retention reproducibility, influenced by the SDS concentration, acetonitrile content, and temperature. The results emphasize the need for complete equilibration to ensure reliable and consistent results. Full article

Hydrophilic interaction liquid chromatography (HILIC) is widely used for the analysis of glycans and oligosaccharides, yet the molecular basis of retention remains incompletely understood. In this study, we investigated dextran ladders labelled with 2-aminobenzamide (2-AB) and Rapifluor-MS™ (Waters, Milford, MA, USA) across a wide range of degrees of polymerization (DP 2–15), temperature conditions (10 °C to 70 °C), and gradient programs using a Acquity™ Premier Glycan BEH Amide column (Bridged Ethylene Hybrid, Waters, Milford, MA, USA). Van’t Hoff analysis revealed distinct enthalpic and entropic contributions to retention, allowing identification of a mechanistic transition from enthalpy-dominated docking interactions at low DP to entropy-driven dynamic adsorption at higher DP. This transition occurred reproducibly between DP 4–6, depending on the fluorescent label, while gradient steepness primarily influenced the location of the minimum enthalpy. Molecular dynamics simulations provided additional evidence, showing increased conformational flexibility and end-to-end distance variability for longer oligomers. This finding is consistent with entropy-dominated adsorption accompanied by displacement of structured interfacial water. Together, these results establish a molecular-level framework linking retention thermodynamics, conformational behavior, and solvation effects, thereby advancing our mechanistic understanding of glycan separation in HILIC. Full article

L-Carnitine (L-CAR) and acetyl-L-carnitine (Acetyl L-CAR) are the essential cofactor compounds in lipid metabolism and are used in the treatment of various diseases. The European Food Safety Authority (EFSA) has reported that Acetyl-L-CAR contributes to normal cognitive function and has a beneficial physiological effect. Therefore, the sensitive separation and determination of L-CAR and Acetyl-L-CAR in foodstuffs can provide critical information. A notable trend in modern food analysis is the increasing use of miniaturized analytical columns with a narrow inner diameter (ID). In this study, a new, green analytical method for food analysis was developed to analyze L-CAR and Acetyl-L-CAR in food samples by nano-LC/UV with a hydrophilic monolithic 100 µm ID capillary. This is the first time that the preparation and application of a hydrophilic monolithic nano-column for the analysis of L-CAR and Acetyl-L-CAR in food samples by nano LC/UV has been reported. The hydrophilic monolith was prepared using in situ co-polymerization of glyceryl methacrylate (GMM) and ethylene dimethacrylate (EDMA). Following preparation and characterization, the hydrophilic monolith was used to analyze L-CAR and Acetyl-L-CAR in food samples, including three infant powdered milk samples and five supplements using nano LC/UV. The developed method was validated in terms of precision, sensitivity, linearity, recovery, and repeatability. The LOD and LOQ values were found to be in the range of 0.04–0.09 µg/kg, respectively. In short, the proposed method proved to be suitable for the routine analysis of L-CAR and Acetyl-L-CAR in food samples. Full article

The detection of tetrodotoxins (TTXs) in European shellfish led the European Union to request a risk assessment from the European Food Safety Authority (EFSA). EFSA set a reference limit of 44 µg TTX equivalents kg⁻¹ and called for more data on TTX occurrence, especially in gastropods, which can accumulate in TTXs but remain poorly studied. Recently, preliminary monitoring has revealed the recurrent presence of TTXs in mussels in three areas along the North–Central Adriatic coast of Italy, while research on non-bivalve organisms has not yet been carried out. This study presents a preliminary survey, conducted from January 2023 to March 2025, on the presence of TTXs in gastropods, echinoderms, and arthropods collected from this area. A method in Hydrophilic Interaction Liquid Chromatography coupled with tandem Mass Spectrometry (HILIC-MS/MS) for detecting TTXs in bivalve mollusks was first tested through an international proficiency test, then optimized for the other invertebrates, the object of this study. TTX levels in all gastropods and arthropod samples were undetectable, while traces (~5 µg kg⁻¹) were found in one echinoderm sample (Paracentrotus lividus), marking the first reported occurrence of TTX in this species. Sea urchins are widely consumed in Italy; therefore, this finding is of particular importance from a public health perspective and deserves further investigation. Some gastropod genera or species sampled (e.g., Nassarius, Rapana venosa) have been identified as TTX carriers in other regions; therefore, the negative results obtained in this study may be related to seasonal or geographic variability. These results provide valuable data to EFSA’s call for monitoring emerging risks, particularly as climate change may increase TTX prevalence in European waters as well as worldwide. Full article

Background: Currently there are no clinically validated biomarkers recommended for prostate cancer (PCa) risk stratification other than prostate-specific antigen (PSA). Objective: This study aimed to identify urine metabolites that are associated with the presence of high-grade PCa at the time of radical prostatectomy. Methods: Urine samples were collected from patients who underwent radical prostatectomy. High-resolution metabolomics were implemented using liquid chromatography mass spectrometry (LC-MS). To enhance metabolic feature identification, sample extracts were analyzed in two modes, C18 chromatography [reverse-phase (RP)] and hydrophilic interaction chromatography (HILIC). Results: This analysis included a total of 22 patients with PCa (10 high-grade and 12 low-grade) and identified 52 differential metabolites, 40 in RP and 12 in HILIC, at the p-value 0.05 level. Among these, methyl alpha-aspartyl phenylalaninate was most significantly differentiated, while 3-methylbutanoicacid had the largest difference (slope −3.488). In the pathway analysis, the histidine metabolism pathway was significantly enriched (p < 0.05) with an enrichment factor of 3.5. Although not statistically significant, alterations were also observed in the vitamin B12, B7 (biotin), B6, and B3 (niacin) pathways. Conclusions: These findings suggest that urinary metabolites may have the potential to differentiate high-grade from low-grade PCa. Our study also highlights the metabolic reprogramming that occurs as PCa becomes more aggressive and potential differences in dietary patterns. Full article

Industrial hemp (Cannabis sativa L.; Cannabaceae), traditionally cultivated for fiber, also represents a valuable source of nutrient-rich seed oil. In this study, cold-pressed hemp seed oils from three Romanian varieties (Teodora, Silvana, and Armanca) were evaluated for their fatty acid composition, minor bioactive constituents, antioxidant activity, growth-promoting property toward probiotic strains in vitro, and molecular docking interactions with probiotic targets. Gas chromatography revealed a fatty acid profile dominated by linoleic (49.4–51.9%), oleic (16.3–22.8%), and α-linolenic acids (9.8–14.4%), resulting in favorable PUFA/SFA ratios (5.17–6.39) and ω-6/ω-3 ratios (3.93–5.53). The oils also contained phenolics (118–160 mg GAE/kg), chlorophylls (6.18–8.31 mg/kg), and carotenoids (2.58–3.37 mg/kg), which contributed to their antioxidant activity (DPPH inhibition 35.92 µM TE/100 g–43.37 µM TE/100 g). Broth microdilution assays against Lacticaseibacillus rhamnosus GG, L. paracasei ATCC BAA-52, and L. acidophilus ATCC 4356 demonstrated strain- and dose-dependent potential to promote probiotic growth under in vitro conditions. While L. rhamnosus and L. paracasei were inhibited at low concentrations and only mildly stimulated at higher levels, L. acidophilus showed robust growth promotion, reaching +54.7% effect and CP = 1.55 with Teodora oil at 16 mg/mL. Molecular docking highlighted strong binding affinities of γ-linolenic and linoleic acids with key metabolic enzymes involved in probiotic metabolism (hydratase, enolase, glyceraldehyde-3-phosphate dehydrogenase, ribonucleoside hydrolase), forming stable hydrophilic and hydrophobic interactions which are explored in defining the stability of the ligand-protein complexes. These results indicate that both major fatty acids and minor bioactive constituents contribute to the nutritional and antioxidant value of Romanian hemp seed oils and reveal a potential to promote probiotic growth under in vitro conditions, as supported by complementary in silico evidence. Full article

Background/Objectives: The multi-attribute method (MAM) has found diverse use in the analytical characterization of therapeutic protein products during their development and production. As the MAM matures it has the potential to enter quality control (QC) laboratories, consolidating and replacing many less informative chromatographic techniques; however, this requires an appropriate risk assessment and understanding of method capability. Methods: A validated MAM approach was used to quantify product quality attributes (PQAs) using three different mass spectrometers across two laboratories; the results were compared to conventional hydrophilic interaction chromatography–fluorescence detection (HILIC-FLD) and cation exchange chromatography–ultraviolet (CEX-UV) techniques. Results: Stressed, long-term, and accelerated stability studies were performed, and their effects on glycosylation, deamidation, oxidation and N- and C-termini were quantified. Conclusions: Overall, the inter-instrument inter-laboratory data provided here showed important considerations for transferring methods between laboratories and establishing the correlation between the MAM and conventional data, elements which are necessary to transition the MAM to the QC environment and ultimately achieving the goal of replacing orthogonal QC methods. Full article