Search Results (270)

A comprehensive metabolic profiling of Catharanthus roseus (L.) G. Don was performed using tandem mass spectrometry, along with an evaluation of the biological activities of its various solvent extracts. Among these, the methanolic leaf extract exhibited mild radical scavenging activity, low to moderate antimicrobial activity, and limited cytotoxicity in both the brine shrimp lethality assay and MTT assay against HeLa and A549 cell lines. High-performance liquid chromatography–electrospray ionization–high-resolution tandem mass spectrometry (HPLC-ESI-HRMS/MS) analysis led to the annotation of 34 metabolites, primarily alkaloids. These included 23 indole alkaloids, two fatty acids, two pentacyclic triterpenoids, one amino acid, four porphyrin derivatives, one glyceride, and one chlorin derivative. Notably, two metabolites—2,3-dihydroxypropyl 9,12,15-octadecatrienoate and (10S)-hydroxypheophorbide A—were identified for the first time in C. roseus. Furthermore, Global Natural Products Social Molecular Networking (GNPS) analysis revealed 18 additional metabolites, including epoxypheophorbide A, 11,12-dehydroursolic acid lactone, and 20-isocatharanthine. These findings highlight the diverse secondary metabolite profile of C. roseus and support its potential as a source of bioactive compounds for therapeutic development. Full article

Rouxiella badensis DSM 100043^T had been previously proven to produce a novel glucoselipid biosurfactant which has a very low critical micelle concentration (CMC) as well as very good stability against a wide range of pH, temperature, and salinity. In this study, we performed a function-based library screening from a R. badensis DSM 100043^T genome library to identify responsible genes for biosynthesis of this glucoselipid. The identified open reading frames (ORFs) were cloned into several constructs in Escherichia coli for gene permutation analysis and the individual products were analyzed using high-performance thin-layer chromatography (HPTLC). Products of interest from positive expression strains were purified and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) for further structure elucidation. Function-based screening of 5400 clones led to the identification of an operon containing three ORFs encoding acetyltransferase GlcA (ORF1), acyltransferase GlcB (ORF2), and phosphatase/HAD GlcC (ORF3). E. coli pCAT2, with all three ORFs, resulted in the production of identical R. badensis DSM 100043^T glucosedilipid with Glu-C_10:0-C_12:1 as the main congener. ORF2-deletion strain E. coli pAFP1 primarily produced glucosemonolipids, with Glu-C_10:0,3OH and Glu-C_12:0 as the major congeners, predominantly esterified at the C-2 position of the glucose moiety. Furthermore, fed-batch bioreactor cultivation of E. coli pCAT2 using glucose as the carbon source yielded a maximum glucosedilipid titer of 2.34 g/L after 25 h of fermentation, which is 55-fold higher than that produced by batch cultivation of R. badensis DSM 100043^T in the previous study. Full article

A sensitive method was developed for detecting diazepam residues in aquatic products using magnetic dispersive solid-phase extraction (MDSPE) coupled with ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). Samples extracted with 1% ammonia–acetonitrile were purified using synthesized Fe₃O₄@SiO₂-PSA nanoparticles via MDSPE before UPLC-MS/MS analysis. Separation was performed on a C₁₈ column with gradient elution using 0.1% formic acid–2 mM ammonium acetate/methanol. Detection employed positive electrospray ionization (ESI⁺) in multiple reaction monitoring (MRM) mode. Characterization confirmed Fe₃O₄@SiO₂-PSA’s mesoporous structure with excellent adsorption capacity and magnetic properties. The method showed good linearity (0.1–10 μg/L, r > 0.99) with an LOD and LOQ of 0.20 μg/kg and 0.50 μg/kg, respectively. Recoveries at 0.5–15.0 µg/kg spiking levels were 74.9–109% (RSDs 1.24–11.6%). This approach provides rapid, accurate, and high-precision analysis of diazepam in aquatic products, meeting regulatory requirements. Full article

Background: Human proteins exist in numerous modifications—proteoforms—which are promising targets for biomarker studies. In this study, we aimed to generate comparative proteomics data, including proteoform patterns, from hepatocellular carcinoma (HCC) and nonmalignant liver tissues. Methods: To investigate protein profiles and proteoform patterns, we employed a panoramic, integrative top-down proteomics approach: two-dimensional gel electrophoresis (2DE) coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). Results: We visualized over 2500 proteoform patterns per sample type, enabling the identification of distinct protein signatures and common patterns differentiating nonmalignant and malignant liver cells. Among these, 1270 protein patterns were uniformly observed across all samples. Additionally, 38 proteins—including pyruvate kinase PKM (KPYM), annexin A2 (ANXA2), and others—exhibited pronounced differences in proteoform patterns between nonmalignant and malignant tissues. Conclusions: Most proteoform patterns of the same protein were highly similar, with the dominant peak corresponding to theoretical (unmodified) protein parameters. However, certain proteins displayed altered proteoform patterns and additional proteoforms in cancer compared to controls. These proteins were prioritized for further characterization. Full article

Androgenetic alopecia (AGA) is associated with dihydrotestosterone (DHT)-induced apoptosis in human dermal papilla cells (HDPCs) via androgen receptor (AR) upregulation. This study aimed to evaluate the potential of Cucumis melo var. makuwa leaf extract (CLE) to attenuate these DHT-mediated effects in HDPCs. HDPCs were treated with CLE, and DHT-induced apoptosis and AR expression were assessed. High-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC–ESI–MS) identified Meloside A as the principal bioactive constituent within CLE. CLE significantly attenuated DHT-induced apoptosis in HDPCs, demonstrating a 57.74% reduction at 1000 ppm. Mechanistically, Meloside A inhibited DHT-stimulated AR nuclear translocation and reduced AR protein expression. Furthermore, Meloside A decreased the expression of downstream target genes at 100 ppm, showing a 16.27% reduction in IL-6, a 26.55% reduction in TGF-β1, and a 35.38% reduction in DKK-1. Additionally, Meloside A significantly inhibited ROS generation within DHT-stimulated HDPCs by 45.45% at 100 ppm. These findings suggest that Meloside A, isolated from CLE, exerts anti-AGA effects by modulating AR nuclear translocation and gene expression. This highlights its potential as a therapeutic agent for AGA and provides a basis for developing novel therapeutic strategies for hair loss. Full article

This study established a dual analytical workflow integrating thermal desorption–electrospray ionization–tandem mass spectrometry (TD-ESI-MS/MS) for rapid qualitative screening and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) for confirmatory quantification of 17 psychoactive substances and metabolites across six classes (opioids, amphetamine-type stimulants, cocaine, ketamine-type drugs, cannabinoids, and etomidate analogs) in hair matrices. Validation of the TD-ESI-MS/MS method demonstrated its sensitivity (limits of detection: 0.1–0.2 ng/mg) and precision (<19.3%), with matrix effects controlled to <19.6%. The TD-ESI-MS/MS method achieved an analysis time of 1 min per sample, enabling high-throughput screening with a sensitivity >85.7% and a specificity >89.7% for the 17 analytes. UPLC-MS/MS confirmation validated the screening results with accuracy rates of 89.7–99.8%. An analysis of specimens confirmed positive identified etomidate analogs as the predominant psychoactive substances (73.6%), with a lower prevalence of amphetamine-type stimulants (12.5%), ketamine-type drugs (9.0%), and opioids (2.8%). The polydrug use patterns identified concurrent etomidate–amphetamine consumption (n = 5) and complex analog combinations (etomidate–isopropoxate–metomidate, n = 13), suggesting evolving abuse trends. Despite limitations in the temporal resolution and representativeness of the cohort, this study demonstrated the viability of TD-ESI-MS/MS for bridging forensic and public health priorities. Future work should focus on optimizing the durability of the ion source for TD-ESI and validating this method across diverse populations to enhance its generalizability. Full article

Background/Objectives: This study provides the first comprehensive phytochemical composition and biological evaluation of Dianthus sylvestris subsp. aristidis (Batt.) Greuter & Burdet, a plant endemic to Algeria with unexplored pharmacological potential. The objective is to identify novel bioactive metabolites in the plant’s extracts and assess their potential applications for skincare and metabolic disorder management, addressing gaps in the current understanding of its medicinal value. Methods: Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) profiling was used to analyze the hydromethanolic (HMeOH) leaf extract and identify bioactive compounds. The biological activities of HMeOH, ethyl acetate (EtOAc), and butanolic (n-BuOH) extracts were tested for cytotoxicity using the brine shrimp lethality test, photoprotective potential by calculating the sun protection factor (SPF), and enzymatic inhibitory activities against alpha-amylase, urease, and tyrosinase. Results: The LC-ESI-MS/MS profiling of the MeOH extract identified 22 bioactive compounds, including phenolic acids and flavonoids, some of which have not been previously reported in this species. Cytotoxicity tests showed that all extracts were non-toxic (half-lethal concentration (LC₅₀) > 100 micrograms per milliliter). The SPF values indicated significant photoprotective potential, with EtOAc (SPF = 45.19 ± 0.73) and n-BuOH (SPF = 43.81 ± 0.59) extracts showing high sun protection activity. The n-BuOH extract exhibited strong alpha-amylase inhibitory activity (half-maximal inhibitory concentration (IC₅₀) = 307.08 micrograms per milliliter), surpassing the standard acarbose (IC₅₀ = 3650.93 micrograms per milliliter), suggesting potential applications in diabetes management. Conclusions: Dianthus sylvestris subsp. aristidis demonstrates significant pharmacological potential as a source of bioactive secondary metabolites for skincare and metabolic disorder management. These findings provide new insights into the plant’s therapeutic potential and set a foundation for future pharmacological and clinical investigations. Full article

Ensuring the authenticity of Mozzarella di Bufala Campana (MdBC), a Protected Designation of Origin (PDO) cheese, is essential for regulatory enforcement and consumer protection. This study evaluates a multi-technology analytical platform developed to detect adulteration due to the addition of non-buffalo milk or non-PDO buffalo milk in PDO dairy buffalo products. Peripheral laboratories use gel electrophoresis combined with polyclonal antipeptide antibodies for initial screening, enabling the detection of foreign caseins, including those originating outside the PDO-designated regions. For more precise identification, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) differentiates species by detecting proteotypic peptides. In cases requiring confirmation, nano-liquid chromatography coupled to electrospray tandem mass spectrometry (nano-LC-ESI-MS/MS) is used in central state laboratories for the highly sensitive detection of extraneous milk proteins in PDO buffalo MdBC cheese. On the other hand, analysis of the pH 4.6 soluble fraction from buffalo blue cheese identified 2828 buffalo-derived peptides and several bovine specific peptides, confirming milk adulteration. Despite a lower detection extent in the pH 4.6 insoluble fraction following tryptic hydrolysis, the presence of bovine peptides was still sufficient to verify fraud. This integrated proteomic approach, which combines electrophoresis and mass spectrometry technologies, significantly improves milk adulteration detection, providing a robust tool to face increasingly sophisticated fraudulent practices. Full article

The detection and quantification of mycotoxins in beer are critical for ensuring consumer safety and regulatory compliance. These contaminants, originating from barley and other grains, persist and potentially transform during the brewing process. This study presents an innovative analytical protocol using liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) for the simultaneous qualitative and quantitative analysis of nine mycotoxins, including aflatoxins (AFB1, AFB2, AFG1, AFG2), Ochratoxin A (OTA), Fumonisins (FB1, FB2), Deoxynivalenol (DON), and HT-2. The method leverages the efficiency of multi-mycotoxin immunoaffinity columns, providing streamlined sample preparation with high specificity and sensitivity. Validation was conducted using craft beers from Calabria, including freeze-dried samples to enhance analytical consistency and stability. The method’s accuracy was confirmed by using spiking samples with mycotoxins at concentrations compliant with the European Commission’s regulations (Recommendation 2024/1038/EU). The developed protocol delivers reliable results with minimized resource consumption, offering a robust tool for quality control and safety assessments in brewing. By addressing knowledge gaps in freeze-dried craft beer, this study contributes to advancing food safety standards in the brewing industry. Full article

The essential amino acid tryptophan yields a plethora of secondary metabolites with key roles in plants and animals. Its fate in different living organisms is crucial for their own health, and metabolic profiling is a valuable tool for investigating it. Among the various metabolites, those retaining the indole structure were examined for qualitative and quantitative profiling. Liquid chromatography coupled with a tandem mass spectrometry detector with an electrospray ionization source (HPLC-ESI-MS/MS), acquiring in multiple reaction monitoring (MRM) mode, was used to develop a selective and sensitive method for the simultaneous analysis of tryptophan and 10 indole structure-retaining metabolites of it. Satisfactory values were obtained for linearity (R² ≥ 0.99 for all compounds except two), sensitivity (LOD, within 6–31 ng/mL, and LOQ, within 17–94 ng/mL, where minimum and maximum values were relative to serotonin and 5-methoxytryptamin, respectively), reproducibility (interday and intraday precision and accuracy), and effect of the matrix (recovery and matrix effect). The method was then successfully applied to the analysis of different types of beverage, such as herbal products, like Eschscholzia californica and a sleep herbal tea marketed with added melatonin (consumed to reduce anxiety and improve sleep quality), and fermented beverages, like beer and kefir. High amounts of tryptophan (from 77 ng/mL in kefir to 26,974 ng/g in the sleep herbal tea) followed by lower contents of serotonin (from 29 ng/mL in kefir to 2207 ng/g in the sleep herbal tea), were found in all samples along with the serotonin pathway-related compounds 5-hydroxytryptophan and tryptamine. Melatonin was detected in the plant matrix Eschscholzia c. for the first time to our knowledge (446 ng/g) and in the fermented beverages (96 ng/mL in beer and 39 ng/mL in kefir), regardless of their vegetable or animal origin, along with the melatonin route metabolites 5-methoxytryptamine and tryptophan ethyl ester. The amount of melatonin in the sleep herbal tea (556,464 ng/g) was in strong agreement with the declared content. Suggested applications include the search for biomarkers in phytochemical characterization, mechanistic studies of tryptophan’s chemistry, valorization of foods, beverages, and tryptophan-rich agro-food by-products and waste for nutraceutical and pharmacological purposes. Full article

Anthocyanins constitute the primary pigment components in hawthorn (Crataegus pinnatifida) peel, yet their specific composition and concentration profiles remain poorly characterized. This study employed ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS)-based metabolomics to systematically compare anthocyanin profiles between red-peel (CPR) and yellow-peel (CPY) hawthorn cultivars. Our analysis identified 26 anthocyanin metabolites in CPR and 24 in CPY, with cyanidin-3-O-galactoside and cyanidin-3-O-arabinoside being the predominant compounds in both. Multivariate analysis revealed seven significantly differential metabolites, including cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, pelargonidin-3-O-galactoside, pelargonidin-3-O-glucoside, pelargonidin-3-O-arabinoside, and peonidin-3-O-galactoside. Notably, all the differential metabolites exhibited reductions in CPY compared to CPR. Chromatic analysis demonstrated that CPR possessed highly significantly lower hue angle values (h_ab) than CPY (47.7093 ± 4.1706, 83.6427 ± 1.4604, p < 0.01), showing strong negative correlations with key anthocyanins. These findings enhance the scientific understanding of anthocyanin biosynthesis in hawthorn peel and provide a certain reference for the development and utilization of anthocyanins in hawthorn peel. Full article

This study critically examines the limitations of the official Italian methodology used for detecting bovine adulteration milk in Protected Designation of Origin (PDO) Mozzarella di Bufala Campana (MdBC). This method focuses on the whey fraction of cheese samples, which comprises about 1% of total MdBC proteins, and is based on a high-performance liquid chromatography (HPLC) quantification of the bovine β-lactoglobulin A (β-Lg A) as a marker. Here, we have demonstrated that this official methodology suffers from measurement inconsistencies due to its reliance on raw bovine whey standards, which fail to account for β-Lg genetic polymorphisms in real MdBC samples and protein thermal modifications during cheesemaking. To overcome these limitations, we propose a dual proteomics-based approach using matrix-assisted laser desorption ionization (MALDI-TOF) mass spectrometry (MS) and nano-HPLC-electrospray (ESI)−tandem mass spectrometry (MS/MS) analysis of MdBC extracted whey. MALDI-TOF-MS focused on identifying proteotypic peptides specific to bovine and buffalo β-Lg and α-lactalbumin (α-La), enabling high specificity for distinguishing the two animal species at adulteration levels as low as 1%. Complementing this, nano-HPLC-ESI-MS/MS provided a comprehensive profile by identifying over 100 bovine-specific peptide markers from β-Lg, α-La, albumin, lactoferrin, and osteopontin. Both methods ensured precise detection and quantification of bovine milk adulteration in complex matrices like pasta filata cheeses, achieving high sensitivity even at minimal adulteration levels. Accordingly, the proposed dual proteomics-based approach overcomes challenges associated with whey protein polymorphism, heat treatment, and processing variability, and complements casein-based methodologies already validated under European standards. This integrated framework of analyses focused on whey and casein fraction enhances the reliability of adulteration detection and safeguards the authenticity of PDO buffalo mozzarella, upholding its unique quality and integrity. Full article

This study presents a method for the simultaneous determination of six acidic herbicides and their metabolites in various matrices, including fruits, vegetables, grains, and edible oils. The method employs acidified acetonitrile extraction combined with dispersive solid-phase extraction cleanup (dSPE) using MgSO₄, Florisil, and Graphitized carbon black (GCB). The analysis was performed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) with electrospray ionization (ESI) in both positive and negative modes using multiple reaction monitoring (MRM). The mass concentrations of six herbicide pesticides and their metabolites were predominantly within the range of 0.0005~0.050 mg/L and exhibited strong linear relationships with the corresponding peak area, with the coefficient of determination (R²) exceeding 0.993. The limits of detection (LOD) for the method ranged from 0.0001 to 0.008 mg/kg. The recovery rates of adding recovery experiments to cabbage, chives, pear, wheat flour, and soybean oil were 69.8~120%, and the relative standard deviation (RSD) was 0.6~19.5%. The results indicate that this method is efficient and fast, and can be used for the detection of compounds in various actual matrices. Full article

A study was conducted to search for the best separation of eighteen cannabinoids, the maximum number of cannabinoids that have been quantified so far, for potency testing of hemp-based products using liquid chromatography diode array detector (LC-DAD). The investigation utilized four column types, all sharing the same dimension (150 mm × 2.1 mm) and core–shell particle size (2.7 µm), but different stationary phases: dimethyl-octadecyl (Poroshell 120 EC-C18), diisobutyl-octadecyl (Raptor ARC-18), reverse phase (RP)-carbamate (Cortecs Shield RP-18), and RP-amide (Ascentis Express RP-Amide). The resolution of adjacent cannabinoids was kept close to 1.5 or higher, while the separation time was kept as short as possible. The fastest separation was achieved within 15.0 min using two sequential Raptor ARC-18 columns, with a mobile phase consisting of 75.0% acetonitrile and 25.0% aqueous solution of 0.03% formic acid and 0.5 mM ammonium formate at pH 2.97, at a flow rate of 0.5 mL/min. A slightly improved resolution of the eighteen cannabinoids was obtained within 18.5 min using two sequential Poroshell 120 EC-C18 columns under similar conditions, except for a mobile phase containing 77.5% acetonitrile and a reduced flow rate of 0.45 mL/min due to backpressure higher than 600 bars. Furthermore, a rapid 7.0 min separation was achieved for potency testing of hemp-based products by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) using a Cortecs Shield RP-18 column, with a mobile phase consisting of 70.0% acetonitrile and 30.0% aqueous solution of 0.01% formic acid and 1 mM ammonium formate at pH 3.38 at a flow rate of 0.5 mL/min. Full article

Cyanobacteria are an ancient group of prokaryotes capable of oxygenic photosynthesis. Recently, thermal crises symptoms in hot springs have been associated with acute cyanopeptides poisoning. The aim of this work is to develop a fast, easy and reliable method to monitor the presence of toxic cyanopeptides in geothermal waters. The analytical method based on capillary zone electrophoresis coupled with tandem mass spectrometry (CZE-MS/MS) was developed for the simultaneous determination of 14 cyanopeptides in less than 7.5 min. A basic 50 mM ammonium acetate buffer at pH 10.2 was selected as the background electrolyte, positive electrospray ionization (ESI+) was employed for all compounds, and a salting-out assisted liquid–liquid extraction (SALLE) protocol with acetonitrile as an extraction solvent and MgSO₄ as an auxiliary salting-out agent was optimized as sample treatment. Six natural hot springs in the province of Granada (Andalucía, Spain) were sampled at the beginning of the summer season (June) and at the end (September). Biomass collected at two sample points (Santa Fe and Zújar) contained cyanobacteria cells from the genera Phormidium, Leptolyngbya, and Spirulina. Nevertheless, cyanotoxins covered by this work were not found in any of the water samples analyzed. The greenness and transferability of the method was evaluated highlighting its sustainability and applicability. Full article