Search Results (115)

Heterozygous mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), are major risk factors for Parkinson’s Disease (PD). Ambroxol, a small chaperone originally used as a mucolytic agent, has been shown to cross the blood–brain barrier, enhance GCase activity, and reduce α-synuclein levels, making it a promising therapeutic candidate for disease-modifying effects in GBA1-associated PD (GBA1-PD). This study aimed to develop a method to quantify ambroxol levels in human plasma and cerebrospinal fluid (CSF) using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Ambroxol was determined by online solid-phase extraction (SPE), coupled with LC-MS/MS, by gradient elution on a monolithic column. Detection employed a 3200 QTRAP tandem mass spectrometer in the positive electrospray ionization mode. Calibration curves exhibited linearity across the analyzed ranges in both plasma and CSF. The recovery rate ranged from 106.7% to 113.5% in plasma and from 99.0% to 103.0% in CSF. No significant matrix effect was observed. Intra-day and inter-day precisions were below 11.8% in both matrices, and accuracy ranged from 89.9% to 103.1% in plasma and from 96.3% to 107.8% in CSF. We evaluated and confirmed the stability of the analyte in plasma and CSF across various storage conditions. The method was successfully validated according to European Medicine Agency (EMA) guidelines and its applicability was confirmed in the context of a multicenter, randomized, double-blind, placebo-controlled, phase II study, designed to monitor the ambroxol levels in the plasma and CSF of GBA1-PD. Full article

Trimethylamine N-oxide (TMAO) is a gut microbial metabolite of dietary precursors, including choline and carnitine. Elevated levels of TMAO in human plasma have been associated with several diseases such as cardiovascular, diabetes mellitus, chronic kidney disease, neurological disorders, and cancer. This has led to an increased interest in the accurate determination of TMAO in human blood, for which a reliable, cost-effective and sensitive analytical method should be established. LC-MS/MS has emerged as a powerful tool for the determination of TMAO due to its high sensitivity, specificity, and ability to handle complex matrices. Herein, we describe the development and validation of an LC-MS/MS method for the determination of TMAO in human blood plasma. Our method involves a simple sample preparation protocol, involving a protein precipitation step along with a non-deuterated IS, followed by a Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis using a triple quadrupole mass spectrometer. Additionally, the method was adapted and implemented on an UPLC-QTOF/MS. The method was validated using the guidelines set by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for assay performance and robustness in human plasma and successfully applied to plasma derived from healthy and hyperlipidemic volunteers. The developed method was found to be specific, sensitive, and accurate for the determination of TMAO in human plasma, with a lower limit of quantification of 0.25 µM. The intra- and inter-assay precision and trueness were within acceptable limits. Full article

Background/Objectives: Brown algae, particularly Eisenia bicyclis, produce various bioactive chemicals with significant application potential in the food, cosmetics, and pharmaceutical industries. This study aimed to evaluate the antibacterial, antibiofilm, and antivirulence properties of the ethyl acetate fraction (EA) of E. bicyclis and its synthesized gold nanoparticles (EA-AuNPs), with a focus on their potential applications against both Gram-positive and Gram-negative bacteria. Methods: The bioactive component in the ethyl acetate fraction was identified using a gas chromatography-mass spectroscopy (GC-MS) device and a liquid chromatography-mass spectrometer/mass spectrometry (LC-MS) system. The crystal violet method was utilized to evaluate the biofilm inhibition experiments. Several instruments, including dynamic light scattering, Fourier transform infrared, X-ray diffraction, field emission transmission electron microscopy, and energy-dispersive spectroscopy, were employed to completely characterize the produced EA-AuNPs. The cytotoxicity of the EA-AuNPs was determined using the MTT assay, and the expression of genes linked with biofilm and virulence in Pseudomonas aeruginosa and Staphylococcus aureus was investigated using real-time polymerase chain reaction (RT-PCR). Results: Various bioactive compounds were identified from the EA using GC-MS and LC-MS, including fatty acids and phlorotannins such as eckol, dieckol, 6,6’-bieckol, and phlorofucofuroeckol in high amounts, highlighting EA as a phlorotannin-rich fraction. The EA also demonstrated significant antibiofilm activity, with 79.86% inhibition at 512 μg/mL against P. aeruginosa and 87.00% at 64 μg/mL against S. aureus. EA was then used in the synthesis of gold nanoparticles (AuNPs) to improve their stability and safety. The synthesized EA-AuNPs were determined to have an average size of 165.04 nm, with a zeta potential of −29.86 mV, indicating good stability. In antibiofilm activity assays, EA-AuNPs demonstrated 45.76% inhibition against P. aeruginosa at 1024 μg/mL and 44.64% inhibition against S. aureus at 128 μg/mL. At sub-MIC levels, EA-AuNPs significantly inhibited biofilm formation and virulence factors, including the motility of P. aeruginosa and staphyloxanthin synthesis in S. aureus. The RT-PCR analysis revealed the downregulation of key genes involved in biofilm formation and virulence in P. aeruginosa and S. aureus. Conclusions: These findings highlight the potential of E. bicyclis solvent-soluble extracts and EA-AuNPs as effective antibacterial, antibiofilm, and antivirulence agents, with significant application potential in the pharmaceutical and food industries. To the best of our knowledge, this is the first report of antibiofilm activity against both Gram-positive and Gram-negative bacteria using EA-AuNPs. Full article

Background: Tear fluid, rich in proteins, is a promising source of novel biomarkers for ocular and systemic health. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the primary method for biomarker discovery. Still, factors such as limited sample volume, extracellular protein contamination, and reflex tearing can significantly impact results. Glass microcapillary tubes minimize these issues. Schirmer strips remain the most common collection method due to existing LC-MS/MS protocol optimization. Methods: In this study, we evaluated multiple digestion protocols for the shotgun quantitative LC-MS/MS analysis of small-volume tear fluid samples collected using glass capillary tubes. Protocol optimization was performed using pooled samples and then compared with the analysis of individual samples. Results: Using the optimized protocol, one μL samples were processed using a timsTOF Pro 2 mass spectrometer (Bruker) coupled online with an Evosep One liquid chromatography system (Evosep), leading to the identification of an average of 361 ± 63 proteins in pooled samples and 525 ± 123 proteins in individual small-volume tear fluid samples. Conclusions: This protocol highlights the practicality of using glass capillary tubes for comprehensive LC-MS/MS-based tear proteomics analysis, paving the way for detailed proteomics characterization of individual tear fluid samples rather than pooled samples. By shifting from pooled to individual samples, this approach greatly accelerates tear biomarker discovery, advancing precision and personalized medicine. Full article

Aryl polyene (APE) are bacterial pigments which show great biotechnological potential because of their biological activities. In this study, the presence of gene clusters associated with APE synthesis was investigated in the genome of Chryseobacterium sp. kr6 and Lysobacter sp. A03. The pigments extracted from strains kr6 and A03 were further characterized by liquid chromatography coupled to a high-resolution mass spectrometer (LC-DAD-MS). These bacteria harbor the relevant genes for APE biosynthesis; while kr6 may produce flexirubin pigments and have a 75% similarity with the flexirubin cluster from Flavobacterium johnsoniae UW101, Lysobacter sp. A03 showed a 50% similarity with the xanthomonadin I gene cluster from Xanthomonas oryzae pv. oryzae. A comparison with the gene clusters of APE-producing bacteria revealed that kr6 and A03 harbor genes for key proteins that participate in APE biosynthesis, such as acyl carrier proteins, acyl dehydratases and acyl reductases. The LC-DAD-MS analysis revealed that kr6 produces a possible mixture of flexirubins, whereas the yellow pigment from A03 is proposed to be a xanthomonadin-like pigment. Although the fine molecular structure of these pigments are not yet fully elucidated, strains kr6 and A03 present great potential for the production of natural bioactive pigments. Full article

Sustainable management of agri-food product safety presents a major challenge requiring extensive action to ensure food safety and consumer health. The pursuit of environmentally friendly solutions that will constitute an alternative to the chemical compounds commonly used in agriculture and the food industries is one of the most important problems. One solution is plant extracts containing various biologically active compounds and exhibiting antimicrobial activity. This study aims to determine the biological activity of extracts obtained from Origanum vulgare L. (leaves) by supercritical CO₂ (SC-CO₂) extraction using different reaction conditions and compositions. In vitro studies revealed antimicrobial activity against selected bacteria (including Salmonella Enteritidis, Listeria monocytogenes, and Staphylococcus aureus) and fungi (Fusarium spp.), depending mainly on the microorganism species; however, extraction conditions also influenced these properties. The microscopic observations established by optical and fluorescence microscopy showed the changes in the fungal cell’s viability and morphology. There was no observed significant release of intracellular material as stated based on ICP-MS analysis of sodium and potassium concentration. Antibiofilm properties of extract obtained by extraction at 40 °C were also demonstrated against S. aureus, P. aeruginosa, and L. monocytogenes, with stronger properties observed against Gram-positive bacteria. Phytochemical characterization of the extracts was determined using a liquid chromatography system with an orbitrap mass spectrometer (LC/MS), identifying, i.e., phenolic acids: protocatechuic, hydroxybenzoic, caffeic, and rosmarinic; flavonoids: luteolin, naringenin, and kaempferol; and terpenoids: oleanolic and ursolic acids. Full article

Background/Objectives: Sorghum bicolor is a source of many bioactive components, such as polyphenols. Those components are present mainly in its bran, often removed in industrial processes through decortication. In that sense, this work aimed to analyze the polyphenol content, especially free flavonoids, from the bran of a newly developed variety compared to other commercially available varieties. Methods: The samples were white sorghum TDN^® Sorgho, red sorghum Mini Sorgho, and the newly developed red sorghum RILN-156. First, decortication was conducted to obtain the bran samples, which were triturated and then sieved. The use of colorimetric methods allowed the general quantification of the polyphenolic components. First, the polyphenol content was extracted using 70% methanol. Then, the samples’ total phenolic content, total flavonoid content, total tannin content, total anthocyanin content, and antioxidant potential were determined. To analyze the different components and identify the free flavonoids, an untargeted metabolomics analysis (with liquid chromatography coupled with mass spectrometer (LC/MS) and capillary electrophoresis coupled with a mass spectrometer (CE/MS)) was performed. Results: The results have shown that apart from anthocyanin and tannin, the newly developed variety, RILN-156, presented the highest concentration of polyphenolic content, including a higher antioxidant capacity. The exploratory analysis identified 19 flavonoids within the samples, with galangin and daidzein being the most abundant ones. Conclusions: These results show a promising finding for using this newly developed sorghum variety (RILN-156) industrially and further investigating its health benefits. They also elucidate the differences between colored sorghum within themselves and with white sorghum varieties. Full article

A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer in multiple reaction monitoring (MRM) mode in electrospray ionization (ESI) positive ionization mode. The transitions were monitored at m/z 310.2 → 96.0 for KTF and m/z 313.2 → 99.1 for IS. KTF and IS were extracted from plasma using liquid-liquid extraction with methyl tertiary-butyl ether and then analyzed for 3 min with extracted samples (7 µL) into the LC–MS/MS system. Analytes were separated on a Luna^® Hilic column (50 × 2.0 mm i.d., 3 μm) using the Nexera X2 HPLC. The mobile phase A consisted of 10 mmol/L ammonium formate (pH 3.0), while mobile phase B consisted of 0.05% formic acid in acetonitrile. The ratio of mobile phase was 5:95 (v/v) at a flow rate of 0.2 mL/min. The method has been thoroughly validated in accordance with the bioanalytical method validation guidelines established by the Ministry of Food and Drug Safety in Korea and the U.S. Food and Drug Administration, addressing selectivity, lower limit of quantification, linearity, carryover, precision, accuracy, recovery, matrix effect, and stability. The developed LC–MS/MS method was effectively utilized for the bioequivalence assessment of ketotifen in Beagle dog plasma following the oral administration of ketotifen syrup. Full article

Ciguatera poisoning (CP) is the most common type of marine biotoxin food poisoning worldwide, and it is caused by ciguatoxins (CTXs), thermostable polyether toxins produced by dinoflagellate Gambierdiscus and Fukuyoa spp. It is typically caused by the consumption of large fish high on the food chain that have accumulated CTXs in their flesh. CTXs in trace amounts are found in natural samples, and they mainly induce neurotoxic effects in consumers at concentrations as low as 0.2 µg/kg. The U.S. Food and Drug Administration has established CTX maximum permitted levels of 0.01 µg/kg for CTX1B and 0.1 µg/kg for C-CTX1 based on toxicological data. More than 20 variants of the CTX1B and CTX3C series have been identified, and the simultaneous detection of trace amounts of CTX analogs has recently been required. Previously published works using LC-MS/MS achieved the safety levels by monitoring the sodium adduct ions of CTXs ([M+Na]⁺ > [M+Na]⁺). In this study, we optimized a highly sensitive method for the detection of CTXs using the sodium or lithium adducts, [M+Na]⁺ or [M+Li]⁺, by adding alkali metals such as Na⁺ or Li⁺ to the mobile phase. This work demonstrates that CTXs can be successfully detected at the low concentrations recommended by the FDA with good chromatographic separation using LC-MS/MS. It also reports on the method’s new analytical conditions and accuracy using [M+Li]⁺. Full article

Rhizoma Panacis Japonici (RPJ) is an ancient herbal medicine from China that has long been employed for its medicinal benefits in relieving arthritis physical debility and diverse afflictions. The primary bioactive constituents found in RPJ are triterpene saponins, which exhibit numerous pharmacological actions, including anti-inflammatory, antioxidant, and immunomodulating effects. The present study established a straightforward and effective approach for characterizing triterpene saponins in RPJ. An offline HILIC × RP LC/QTOF-MS method was developed, along with a self-constructed in-house database containing 612 saponins reported in the Panax genus and 228 predicted metabolites. The approach achieved good chromatographic performance in isolating triterpene saponins of RPJ, with the HILIC column as the first dimension (¹D) and the BEH C18 column as the second dimension (²D). The developed two-dimensional liquid chromatography system exhibited an orthogonality of 0.61 and a peak capacity of 1249. Detection was performed using a QTOF mass spectrometer in a data-independent manner (MS^E) in a negative ion mode. Using the in-house database, the collected MS data were processed by an automatic workflow on UNIFI 1.8.2 software, which included data correction, matching of precursor and product ions, and peak annotation. In this study, 307 saponins were characterized from RPJ and 76 saponins were identified for the first time in Panax japonicus. This research not only enhances our understanding of the chemical characteristics of RPJ but also offers a simple and efficient method for analyzing the complex composition of herbal medicine. Full article

The leaching of herbicides into the soil is essential to control germinating seeds and parts of vegetative weeds. However, herbicide transportation to deeper soil layers can result in groundwater contamination and, consequently, environmental issues. In this research, our objective was to investigate differences in herbicide leaching between commercial formulations and analytical standards using three different soils. Leaching experiments were carried out for diuron, hexazinone, and sulfometuron-methyl herbicides isolated and in binary and ternary mixtures. The herbicide residue quantification was performed by ultra-high-performance liquid chromatography coupled to a mass spectrometer (LC-MS/MS). Diuron had less mobility in soils and was retained in the most superficial layers. Hexazinone and sulfometuron-methyl were more mobile and leached into deeper layers. The leaching process was more intense for hexazinone and sulfometuron-methyl. The additives present in the commercial formulation favored the leaching in soils of diuron, hexazinone, and sulfometuron-methyl herbicides isolated and mixture compared to the analytical standard. This fact highlights the importance of considering these effects for the positioning of herbicides in the field to increase the efficiency of weed control and minimize the potential for environmental contamination. Full article

To establish the fingerprint of Cibotii rhizoma using high-performance liquid chromatography (HPLC) and evaluate the quality of Cibotii rhizoma from different regions using chemometrics to identify the potential quality markers, thirteen batches of Cibotii rhizoma samples were analyzed. the similarity evaluation system of TCM chromatographic fingerprint similarity evaluation was used to confirm common peaks. The SPSS 27 software was used for hierarchical cluster analysis (HCA), and SIMCA 14.1 software was used for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Moreover, a batch of Cibotii rhizoma was selected for LC-MS analysis and speculated on 15 common components. HPLC fingerprint were established, 15 common peaks were matched, two chromatographic peaks were identified using standard substances (protocatechuic acid and protocatechuic aldehyde), and 13 common components were inferred through liquid chromatograph-mass spectrometer (LC-MS). The 13 batches of the samples showed good similarities (>0.910). The results of HCA, PCA and OPLS-DA showed that 13 batches of samples were divided into three groups, and different markers were selected. The method is simple, rapid and reproducible, and can provide a reference for the overall quality evaluation of Cibotii rhizoma. Full article

Glyphosate-based herbicides are the most widely used pesticides in the world; however, the toxicity of glyphosate (GlyP) toward humans, especially its carcinogenicity, is controversial. The aim of this work was to validate a rapid assay for measuring GlyP and its metabolite aminomethylphosphonic acid (AMPA) in urine for human biomonitoring. The analytes were purified via solid-phase extraction in the presence of isotopically labeled internal standards. An LC-MS/MS assay was developed using a column with a novel hybrid stationary phase combined with anion exchange and hydrophilic interaction liquid chromatography. Detection and quantification were performed using negative electrospray ionization in a hybrid triple quadrupole/linear ion trap mass spectrometer. The retention times for AMPA and GlyP were 1.44 and 7.24 min, respectively. Calibration curves showed a linear dynamic range of up to 40 µg/L, inter- and intra-run precisions <7.5%, and accuracies within 10% of the theoretical concentrations. The limits of quantification were 0.1 µg/L and 0.5 µg/L for GlyP and AMPA, respectively. The matrix effect bias was controlled using internal standards. Successful participation in external quality assurance exercises strengthens the validity of the method. The assay was applied to the measurement of GlyP and AMPA in the urine of 9 urban residents, 26 rural residents, and 12 agricultural workers; while AMPA was mostly not quantifiable, the median GlyP values were 0.1 and 0.34 µg/L in rural residents and workers, respectively. The assay is useful to assess GlyP and AMPA in human urine following different exposure scenarios. Full article

This study investigated the antioxidant, antiaging, and antibacterial properties of Gracilaria verrucosa (GV) based on 95% methanol (GVM), ethanol (GVE), and hot water (GVW) extractions. Antioxidant activity assays revealed the total polyphenol and flavonoid contents were highest in GVM and GVE. The 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) activities were highest in GVE and GVM. Furthermore, GVE exhibited the highest ferric-reducing antioxidant power (FRAP) value. In comparison, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were highest in GVM. Collectively, GVE and GVM had stronger antioxidant activities than GVW. Additionally, collagenase, elastase, and tyrosinase inhibition assays showed that GVM exhibited the strongest anti-wrinkle and skin-whitening activities. Liquid chromatography–tandem mass spectrometer (LC–MS/MS) revealed that GVW had the highest 4-hydroxy benzoic acid content, whereas GVE had the highest naringenin and naringin contents. Additionally, GVE exhibited the strongest antimicrobial activity against six foodborne bacteria, with minimum inhibitory and bactericidal concentrations of 0.06–0.3 and 0.1–0.5 μg/μL. Correlation analysis of the GV extracts indicated a strong positive relationship between TPC and ABTS, SOD, and CAT activities (r = 0.760–0.982, p = 0–0.018). Overall, GVE and GVM can be applied to the development of functional agents across diverse industries. Full article

Pyrrolizidine alkaloids are secondary metabolites produced by plants as a defense against insects. These can cause acute or chronic toxicity in humans. Therefore, avoiding potential poisoning from the consumption of tea and culinary plants contaminated with pyrrolizidine alkaloids (PAs), pyrrolizidine alkaloids N-oxides (PANOs), and tropane alkaloids (TAs) is important for human health and food safety. Therefore, it is important to determine the levels of these substances with reliable and highly accurate methods. In this study, the PAs, PANOs, and TAs in herbal teas and culinary herbs sold in Turkish markets were identified and their levels were determined. Thus, the general profiles of herbal teas and culinary herbs in Turkey were revealed, and the compliance of the total amounts of PA and TA with the regulations was examined. The identification and quantification of 25 PAs and N-oxides and 2 TAs (atropine and scopolamine) in the samples was performed with a liquid chromatography-quadrupole time-of-flight tandem mass spectrometer (LC-Q-ToF/MS). At least a few of these substances were detected in all of the tested herbal teas and culinary herbs. The total contents of the black tea, green tea, mixed tea, flavored tea, chamomile tea, sage tea, linden tea, fennel tea, rosehip tea, peppermint, and thyme samples ranged from 4.6 ng g⁻¹ to 1054.5 ng g⁻¹. The results obtained shed light on the importance of analyzing the total dehydro PA, PANO, and TA amounts in plant-based products consumed in diets with sensitive and accurate methods, and they highlight the necessity of performing these analyses routinely in terms of food safety. Full article