Liquid Chromatography Coupled To Quadrupole Time-of-flight Mass Spectrometry Research Articles

Identification of candidate exposure biomarkers for acetyl tributyl citrate and acetyl triethyl citrate using suspect screening in human liver microsomes

Acetyl tributyl citrate (ATBC) and acetyl triethyl citrate (ATEC) are increasingly used as alternatives to phthalates in various products, including food packaging, medical devices, and personal care items, raising concerns about their potential health impacts. This study aimed to investigate the in vitro human metabolism of ATBC and ATEC and identify potential exposure biomarkers applicable in human biomonitoring. Pooled human liver microsomes were utilized to conduct in vitro metabolism assays of deuterium labeled ATBC (ATBC-d3) and ATEC, and liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (LC-qToF/MS) was employed for analysis. Suspect screening workflow and confidence level assignment were applied for metabolite identification. Time-course analysis revealed rapid metabolism of both compounds, with estimated apparent half-lives of approximately 5 min for ATBC-d3 and less than 15 min for ATEC. Eleven metabolites were identified for ATBC-d3 and six for ATEC. The predominant chemical reactions observed were carboxylic ester hydrolysis, deacetylation, and hydroxylation. Based on their abundance and specificity, MB1 (hydroxylated) and MB11 (hydrolyzed and hydroxylated) were proposed as candidate exposure biomarkers for ATBC, and ME1 (hydrolyzed and deacetylated) for ATEC. The identified metabolites and proposed sequences of kinetic process enhance our understanding of the fate of these compounds in the human body, potentially informing future toxicological assessments and guiding the development of more comprehensive human biomonitoring strategies.

In vivo profiling of phytocannabinoids in Cannabis spp. varieties via SPME-LC-MS analysis

BackgroundIn vivo solid-phase microextraction (SPME) is a minimally invasive, non-exhaustive sample-preparation technique that facilitates the direct isolation of low molecular weight compounds from biological matrices in living systems. This technique is especially useful for the analysis of phytocannabinoids (PCs) in plant material, both for forensic purposes and for monitoring the PC content in growing Cannabis spp. plants. In contrast to traditional extraction techniques, in vivo SPME enables continuous tracking of the changes in the level of PCs during plant growth without the need for plant material collection. In this study, in vivo SPME utilizing biocompatible C18 probes and liquid-chromatography coupled to quadrupole time-of flight mass spectrometry (LC-Q-TOF-MS) is proposed as a novel strategy for the extraction and analysis of the acidic forms of five PCs in growing medicinal cannabis plants. ResultsThe SPME method was optimized by testing various parameters, including the extraction phase (coating), extraction and desorption times, and the extraction temperature. The proposed method was validated with satisfactory analytical performance regarding linearity (10–3000 ng/mL), limits of quantification, and precision (relative standard deviations below 5.5 %). The proposed method was then successfully applied for the isolation of five acidic forms of PCs, which are main components of growing medicinal cannabis plants. As a proof-of-concept, SPME probes were statically inserted into the inflorescences of two varieties of Cannabis spp. plants (i.e., CBD-dominant and Δ9-THC-dominant) cultivated under controlled conditions for 30 min extraction of tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabiviarinic acid (CBVA), and tetrahydrocannabivarinic acid (THCVA). Significance and noveltyThe results confirmed that the developed SPME-LC-Q-TOF-MS method is a precise and efficient tool that enables direct and rapid isolation and analysis of PCs under in vivo conditions. The proposed methodology is highly appealing option for monitoring the metabolic pathways and compositions of multiple PCs in medicinal cannabis at different stages of plant growth.

Untargeted metabolomics-based identification of bioactive compounds from Mangifera indica L. seed extracts in drug discovery through molecular docking and assessment of their anticancer potential.

Mangifera indica L. (mango), a medicinal plant rich in biologically active compounds, has potential to be used in disease-preventing and health-promoting products. The present investigation reveals and uncovers bioactive metabolites with remarkable therapeutic efficiency from mango (family: Anacardiaceae) seeds. Biological activity was determined by antimicrobial, antioxidant and anticancer assays, and metabolite profiling was performed on gas chromatography coupled to quadrupole time-of-flight mass spectrometry (GC-QTOF-MS) and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) platforms. Validation of active metabolites was carried out by in silico molecular docking (Molinspiration Cheminformatics Server and PASS). Extracted and identified metabolites were screened; 54 compounds associated with various groups were selected for the in silico interaction study. Molecular docking revealed lead molecules with a potential binding energy score, efficacy and stable modulation with a selected protein domain. Investigation, directed by in vitro and in silico analysis, confirms mango seeds as an excellent source of potential metabolites as a therapeutic agent. © 2024 Society of Chemical Industry.

A Bacillus-based biofungicide agent prevents ochratoxins occurrence in grapes and impacts the volatile profile throughout the Chardonnay winemaking stages

Bacillus-based biocontrol agents have emerged as a strategy to eliminate or reduce the use of synthetic fungicides that are detrimental to health and the environment. In vineyards, a special concern arises from the control of Aspergillus carbonarius, a fungus known for its potential to produce ochratoxins. Ochratoxin A (OTA) is the most toxic form among ochratoxins and its maximum limit in wine has been established in Europe and Brazil as 2 μg/kg. Wine quality, especially the volatile profile, may be influenced by the antifungal strategies, since fungicide residues are transferred from grapes to must during winemaking. The objective of this study was to evaluate, for the first time, the impact of a biocontrol strategy containing Bacillus velezensis P1 on the volatile profile and occurrence of ochratoxins when grapes infected with A. carbonarius were used in winemaking. The evaluation of ochratoxins was carried out by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS), and volatile compounds were analyzed using comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry (GC × GC/qMS). Six ochratoxins were identified in must prepared with Chardonnay grapes inoculated with A. carbonarius (ochratoxin α, ochratoxin β, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide and OTA). Although winemaking causes a decrease in the levels of all forms of ochratoxins, the co-occurrence of these mycotoxins was verified in wine made with grapes containing A. carbonarius. B. velezensis P1 prevented the occurrence of ochratoxins in must, ensuring the safety of wines. Regarding the volatile profile, a predominant presence of terpenic compounds was verified in samples treated with B. velezensis when compared with those not treated with the biocontrol strategy, whereas the presence of A. carbonarius resulted in a higher concentration of volatile compounds with an odor described as fatty/waxy, possibly compromising wine quality. Therefore, B. velezensis P1 is a new biofungicide possibility to produce ochratoxin-free grapes and high-quality wines.

Identification of the organic compounds in surface water: Suspect screening using liquid chromatography high-resolution mass spectrometry and in silico toxicity evaluation

The suspect screening approach was used to investigate the presence of organic compounds in Lake Guaíba – Brazil based on Liquid Chromatography coupled to Quadrupole-Time of Flight mass spectrometry (LC-QTOF-MS). The library suspect screening consisted of 1450 compounds, which was selected based on the presence of commercially available pesticides (which are not part of the ordinances), pharmaceuticals, drugs of abuse, and their main metabolites. Out of these 1450 compounds, 16 were identified with a suspect screening method in sixty-nine samples carried out for two years in the Lake. The most frequently detected compounds were venlafaxine and its metabolites; desmethylvenlafaxine, benzoylecgonine and its metabolites; cocaine; CGA 321113 (trifloxystrobin metabolite); 4-amino-antipyrine; and quinclorac. Principal Component Analysis (PCA) was used as an exploratory multivariate investigation of data on the detected compounds, and samples were tagged according to site, month, and year of collection to identify temporal and spatial patterns. Results obtained from in silico QSAR models evince the need of performing risk assessment of suspect compounds, for many compounds were found to be mutagenic, toxicant, carcinogenic, and non-readily biodegradable.

Development and Validation of LC-Q-TOF-MS Methodology to Determine Mycotoxin Biomarkers in Human Urine.

Mycotoxin contamination of foodstuffs is a health concern worldwide and monitoring human exposure to mycotoxins is a key concern. Most mycotoxins and their metabolites are excreted in urine, but a reliable detection method is required, considering the low levels present in this biological sample. The aim of this work is to validate a sensitive methodology capable of simultaneously determining ten targeted mycotoxins as well as detecting untargeted ones by using Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry (LC-Q-TOF-MS). The targeted mycotoxins were: enniatin A, B, A1, and B1, beauvericine, aflatoxin B1, B2, G1 and G2, and ochratoxin A. Several extraction procedures such as liquid-liquid extraction, dilute and shoot, and QuEChERS were assessed. Finally, a modified simple QuEChERS extraction method was selected. Creatinine adjustment and matrix-matched calibration curves are required. The limit of detection and limit of quantification values ranged from 0.1 to 1.5 and from 0.3 to 5 ng/mL, respectively. Recoveries achieved were higher than 65% for all mycotoxins. Later, the method was applied to 100 samples of women’s urine to confirm the applicability and determine their internal exposure. The untargeted mycotoxins most found were trichothecenes, zearalenones, and ochratoxins.

Suspect screening for pesticides in rain and snow using liquid chromatography high-resolution mass spectrometry

Pesticides are one of the only chemical contaminants that are purposefully introduced to the environment in large quantities. After application to crops, pesticides can be volatilized into the atmosphere and undergo medium to long-range transport. Through wet deposition, pesticides can be removed from the atmosphere far from their initial application site, posing a variety of environmental and health hazards, such as toxicity to terrestrial and aquatic species, crop damage, and poor air quality. Identifying pesticides and their degradation products in wet deposition is crucial to understanding how they are transported in the atmosphere. High-resolution mass spectrometry is a useful tool to carry out suspect screening on complex mixtures. However, suspect screening for pesticides in atmospheric waters is lacking. In this work, liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS) is used for the first time to screen for suspect pesticides in wet deposition. The composition of precipitation samples collected in the central United States from 2018 to 2021 was screened against a library of thousands of compounds, and the presence of 23 pesticides was confirmed using reference standards, including common herbicides such as atrazine, metolachlor, and alachlor, as well as three species that had never before been quantified in wet deposition: diphenamid, octhilinone, and desthio-prothioconazole. A significant increase in the deposition flux of many pesticides was observed at the start of planting season, suggesting local agricultural sources to the atmosphere. In addition, pesticides not commonly used locally were also detected, suggesting regional atmospheric transport. Using statistical analyses and air mass back trajectory calculations, the seasonal trends, physicochemical properties, and potential origins of pesticides were examined. Overall, this work highlights the power of liquid chromatography high-resolution mass spectrometry for identifying both known and unexpected pesticides in precipitation, allowing for more accurate modeling of the atmospheric transport and fate of these compounds.

Stereoselective bioaccumulation and dissipation of pyrisoxazole in earthworm-soil microcosm

Pyrisoxazole is a chiral fungicide with high sterilizing activity to the plant pathogenic bacteria. It has two chiral C atoms, which bring four stereoisomers. The present work was the first time to explore the stereoselective bioaccumulation behavior of pyrisoxazole in earthworms by chiral liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). The absolute configurations of pyrisoxazole stereoisomers were confirmed by circular dichroism (CD) coupled with calculated electronic circular dichroism (ECD) method, and the elution order in Lux Cellulose-3 column was as follows: (−)-3S, 5R-pyrisoxazole, (+)-3R, 5S-pyrisoxazole, (+)-3S, 5S-pyrisoxazole and (−)-3R, 5R-pyrisoxazole. The recoveries of pyrisoxazole stereoisomers in earthworm and soil samples ranged from 80.8 % to 101 % with the RSD lower than 6.3 %. In bioaccumulation progress, (+)-3R, 5S-pyrisoxazole was accumulated preferentially in earthworms, and the bioaccumulation concentrations of high-activity (−)-3S, 5R-pyrisoxazole were the lowest. There were no stereoselective bioaccumulation between (+)-3S, 5S-pyrisoxazole and (−)-3R, 5R-pyrisoxazole, while there was diastereoselectivity between Z-pyrisoxazole and E-pyrisoxazole with higher E-pyrisoxazole concentrations. In the whole bioaccumulation process, the BAF values of (+)-3R, 5S-pyrisoxazole were significantly higher than (−)-3S, 5R-pyrisoxazole, and the BAF values of (−)-3S, 5R-pyrisoxazole were the lowest. The dissipation of pyrisoxazole stereoisomers in the artificial soil was very slow and had no stereoselectivity, and the existence of earthworms had little effects on the dissipation of pyrisoxazole stereoisomers, which indicated that the stereoselective behaviors of pyrisoxazole in earthworms were caused by the stereoselective enrichment and dissipation of earthworms themselves. Taken together, (−)-3S, 5R-pyrisoxazole was recommend as a commercial product. This study played a positive role in guiding the development of environmentally friendly pesticides and provided database for the environmental and biological risk assessment of pyrisoxazole.

The antiproliferative and apoptotic potential of Clinacanthus nutans against human breast cancer cells through targeted apoptosis pathway.

Clinacanthus nutans dichloromethane fraction (CN-Dcm) extract has previously been proven to suppress breast cancer (MCF7) cell proliferation. Despite this, the extrinsic and intrinsic apoptosis mechanisms involved in C. nutans extract-treated MCF7 cells are still unknown. This study was intended to subfractionate CN-Dcm extract using column chromatography and analyse the treated MCF7 cells using the CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay, Annexin V/propidium iodide (PI) assay, western blot, and reverse transcription-qualitative polymerase chain reaction (RT-qPCR). Out of nine subfraction extracts (SF1 to SF9), SF2 extract strongly inhibited MCF7 cells with the lowest IC50 value (23.51 ± 1.00µg/mL) and substantially induced apoptosis in the MCF7 cells. In treated MCF7 cells, SF2 extract significantly upregulated the expression of P53, BAX, BID, caspase-8, caspase-9, and caspase-3, while downregulating the expression of BCL2. The presence of potential bioactive chemical compounds in the SF2 extract was identified using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Thus, the SF2 extract has the potential to induce apoptosis in MCF7 cells through intrinsic and extrinsic pathways.

Blood-Brain Barrier Permeability Study of Potential Neuroprotective Compounds Recovered From Plants and Agri-Food by-Products.

Plants and agri-food by-products represent a wide and renewable source of bioactive compounds with neuroprotective properties. In this research, various green extraction techniques were employed to recover bioactive molecules from Kalanchoe daigremontiana (kalanchoe), epicarp of Cyphomandra betacea (tamarillo), and cooperage woods from Robinia pseudoacacia (acacia) and Nothofagus pumilio (lenga), as well as a reference extract (positive control) from Rosmarinus officinalis L. (rosemary). The neuroprotective capacity of these plant extracts was evaluated in a set of in vitro assays, including enzymatic [acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and lipoxygenase (LOX)] and antioxidant [ABTS, and reactive oxygen and nitrogen species (ROS and RNS)] bioactivity tests. Extracts were also submitted to a parallel artificial membrane permeability assay mimicking the blood–brain barrier (PAMPA-BBB) and to two cell viability assays in HK-2 and SH-SY5Y cell lines. Comprehensive phytochemical profiling based on liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) analysis showed enriched content of phenolic and terpenoid compounds in the target extracts. Moreover, in vitro bioactivity tests showed promising neuroprotective capacity, particularly for supercritical-fluid extraction (SFE) extract from acacia (ABTS IC50 = 0.11 μg ml−1; ROS IC50 = 1.56 μg ml−1; AChE IC50 = 4.23 μg ml−1; BChE IC50 = 1.20 μg ml−1; and LOX IC50 = 4.37 μg ml−1), whereas PAMPA-BBB assays revealed high perfusion capacity of some representative compounds, such as phenolic acids or flavonoids. Regarding cytotoxic assays, tamarillo and rosemary SFE extracts can be considered as non-toxic, acacia SFE extract and lenga pressurized liquid extraction (PLE) extract as mild-cytotoxic, and kalanchoe as highly toxic extracts. The obtained results demonstrate the great potential of the studied biomass extracts to be transformed into valuable food additives, food supplements, or nutraceuticals with promising neuroprotective properties.

Biotreatment efficiency, degradation mechanism and bacterial community structure in an immobilized cell bioreactor treating triclosan-rich wastewater

ABSTRACT Biotreatment of triclosan is mainly performed in conventional activated sludge systems, which, however, are not capable of completely removing this antibacterial agent. As a consequence, triclosan ends up in surface and groundwater, constituting an environmental threat, due to its toxicity to aquatic life. However, little is known regarding the diversity and mechanism of action of microbiota capable of degrading triclosan. In this work, an immobilized cell bioreactor was setup to treat triclosan-rich wastewater. Bioreactor operation resulted in high triclosan removal efficiency, even greater than 99.5%. Nitrogen assimilation was mainly occurred in immobilized biomass, although nitrification was inhibited. Based on Illumina sequencing, Bradyrhizobiaceae, followed by Ferruginibacter, Thermomonas, Lysobacter and Gordonia, were the dominant genera in the bioreactor, representing 38.40 ± 0.62% of the total reads. However, a broad number of taxa (15 genera), mainly members of Xanthomonadaceae, Bradyrhizobiaceae and Chitinophagaceae, showed relative abundances between 1% and 3%. Liquid Chromatography coupled to Quadrupole Time-Of-Flight Mass Spectrometry (LC-QTOF-MS) resulted in the identification of catabolic routes of triclosan in the immobilized cell bioreactor. Seven intermediates of triclosan were detected, with 2,4-dichlorophenol, 4-chlorocatechol and 2-chlorohydroquinone being the key breakdown products of triclosan. Thus, the immobilized cell bioreactor accommodated a diverse bacterial community capable of degrading triclosan.

Effect of cultivation media and temperature on metabolite profiles of three nematicidal Bacillus species

Summary Globally, root-knot nematode (RKN) infestations cause great financial losses. Although agrochemicals are used to manage these pests, there is increased interest in using biocontrol agents based on natural antagonistic microorganisms, such as Bacillus. These nematicidal bacteria demonstrate antagonism towards RKN through different modes of action, including specialised metabolite production. The aim of this study was to compare metabolite profiles of nematicidal Bacillus species and assess the influence of cultivation conditions on these profiles. Two hyphenated metabolomics platforms, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), were employed to profile and compare metabolite features produced during the cultivation of three nematicidal Bacillus species (Bacillus firmus, B. cereus and B. soli) in complex Luria-Bertani broth (LB) and a simpler minimal broth (MB), at three different temperatures (25, 30 and 37°C). Cultivation in complex LB as opposed to simpler MB resulted in the production of more statistically significant metabolite features. Selected temperatures in this study did not have a significant influence on metabolite profiles. Moreover, media-specific influences outweighed temperature-specific influences on metabolite profiles. Results from this study are a valuable first step in establishing suitable cultivation conditions for the production of Bacillus metabolites of interest.

Aspergillus carbonarius-derived ochratoxins are inhibited by Amazonian Bacillus spp. used as a biocontrol agent in grapes

ABSTRACT Bacillus spp. have been used as a biocontrol strategy to eliminate/reduce toxic fungicides in viticulture. Furthermore, the presence of fungi that are resistant to commonly used products is frequent, highlighting the need for new biocontrol strains. Aspergillus carbonarius can produce ochratoxins, including ochratoxin A (OTA), which has a regulatory maximum allowable limit for grape products. The purpose of this study was to assess the ability of four Amazonian strains of Bacillus (P1, P7, P11, and P45) to biocontrol A. carbonarius and various forms of ochratoxins in grapes. Berries treated with strain P1 presented no fungal colonies (100% reduction), while P7, P11 and P45 strains caused a reduction of 95, 95 and 61% on fungal counts, respectively. Six forms of ochratoxin were found in the grapes inoculated with A. carbonarius, including ochratoxin α, ochratoxin β, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide, and OTA. Four of these ochratoxin forms (ochratoxin β, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide) are reported for the first time in grapes. These ochratoxins were identified using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS). All Bacillus strains inhibited the synthesis of OTA, which is the most toxic form of ochratoxin. No ochratoxin form was found when P1 and P7 were used. Although some forms of ochratoxin were detected in grapes treated with Bacillus spp. P11 and P45, the levels decreased by 97%. To our knowledge, this is the first report on the inhibition of Aspergillus carbonarius-derived ochratoxin by Bacillus species. P1 strain, identified as Bacillus velezensis, was found to be the most promising for completely inhibiting fungal growth and production of all ochratoxins.

Bioprocess performance, transformation pathway, and bacterial community dynamics in an immobilized cell bioreactor treating fludioxonil-contaminated wastewater under microaerophilic conditions

Fludioxonil is a post-harvest fungicide contained in effluents produced by fruit packaging plants, which should be treated prior to environmental dispersal. We developed and evaluated an immobilized cell bioreactor, operating under microaerophilic conditions and gradually reduced hydraulic retention times (HRTs) from 10 to 3.9 days, for the biotreatment of fludioxonil-rich wastewater. Fludioxonil removal efficiency was consistently above 96%, even at the shortest HRT applied. A total of 12 transformation products were tentatively identified during fludioxonil degradation by using liquid chromatography coupled to quadrupole time-of-flight Mass spectrometry (LC-QTOF-MS). Fludioxonil degradation pathway was initiated by successive hydroxylation and carbonylation of the pyrrole moiety and disruption of the oxidized cyanopyrrole ring at the NH-C bond. The detection of 2,2-difluoro-2H-1,3-benzodioxole-4-carboxylic acid verified the decyanation and deamination of the molecule, whereas its conversion to the tentatively identified compound 2,3-dihydroxybenzoic acid indicated its defluorination. High-throughput amplicon sequencing revealed that HRT shortening led to reduced α-diversity, significant changes in the β-diversity, and a shift in the bacterial community composition from an initial activated sludge system typical community to a community composed of bacterial taxa like Clostridium, Oligotropha, Pseudomonas, and Terrimonas capable of performing advanced degradation and/or aerobic denitrification. Overall, the immobilized cell bioreactor operation under microaerophilic conditions, which minimizes the cost for aeration, can provide a sustainable solution for the depuration of fludioxonil-contaminated agro-industrial effluents.

Neuroprotective Assessment of Moringa oleifera Leaves Extract against Oxidative-Stress-Induced Cytotoxicity in SHSY5Y Neuroblastoma Cells.

The current trend worldwide is searching plant extracts towards prevention of neurodegenerative disorders. This study aimed to investigate the neuroprotective effect of Alpinia galanga leaves (ALE), Alpinia galanga rhizomes (ARE), Vitis vinifera seeds (VSE), Moringa oleifera leaves (MLE), Panax ginseng leaves (PLE) and Panax ginseng rhizomes (PRE) ethanolic extracts on human neuroblastoma (SHSY5Y) cells. The 1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging of VSE and MLE were 81% and 58%, respectively. Ferric-reducing antioxidant power (FRAP) of ALE and MLE (33.57 ± 0.20 and 26.76 ± 0.30 μmol Fe(ΙΙ)/g dry wt., respectively) were higher than for the other extracts. Liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) revealed MLE active compounds. Intracellular study by nitro-blue tetrazolium (NBT) test showed that MLE and VSE had high O2− scavenging (0.83 ± 0.09 vs. 0.98 ± 0.08 mg/mL, respectively). MLE had the highest ROS scavenging followed by PRE (0.71 ± 0.08 vs. 0.83 ± 0.08 mg/mL, respectively), by 2,7-dichlorodihydrofluorescein diacetate (DCFHDA) assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and neuroprotection tests on SHSY5Y showed that PRE had a better neuroprotective effect but higher cytotoxicity compared to MLE (viable cells 51% vs. 44%, IC50 1.92 ± 0.04 vs. 2.7 ± 0.2 mg/mL, respectively). In conclusion, among the studied plants, MLE has potential for developing as a neuroprotective agent.

Phytonutrients and Antimicrobial Activities of Methanolic Extract from Hafr Al-Batin Truffles

The desert truffle is a wild mushroom, also referred to as Kamah or Fagaa. Kamah is a rich source of polysaccharides that have medicinal, antitumoral, antibacterial, and immune-stimulant effects. Studies of hypogeous fungi, especially desert truffles, have recently entered traditional studies of epigeous higher Basidiomycetes. Based on the tasty desert truffle Kamah obtained from Hafr Al-Batin Governorate, Saudi Arabia, as a source of potential antimicrobial agents with both the aim of obtaining novel agents toward bacteria and Fungi of clinical significance. We specifically tested the antibacterial and antifungal efficacy of methanol extracts of Kamah against the Gram-negative bacterial pathogens reference strains E. coli ATCC® 8739, P. Aeruginosa ATCC®9027, S. aureus ATCC®6538, Enterococci NCTC®775 and opportunistic fungus C. albicans ATCC®1231.The extract had MIC (minimum inhibitory concentrations) varying from 100 g/ml to 500 g/ml against the pathogens examined. The LC-QTOF-MS (liquid chromatography coupled to quadrupole time of flight mass spectrometry) phytoconstituents assay chromatogram indicated that the methanol extracts of Kamah comprises 264 with retention periods varying from 1.04 to 18.86, which were categorized as unsaturated and saturated natural ingredients sch as aromatic compounds, carboxylic acids, oxygenated hydrocarbons, fatty acids, amino acids, and vitamins).The main compounds were discovered to be 21 with peak areas larger than 2X10-5 and retention periods varying from 2.3 to 9.13.The main known substances with the maximum peaks were adenosine (11.724), phenylalanine (7.711), phenprobamate (7.711), and 5-hydroxytryptophan (5.711). Such preliminary findings, we assume, are encouraging in terms of obtaining a beneficial antibiotic substitute to battle antibiotic-resistant pathogens especially eye infections.

Assessment of the presence of transformation products of pharmaceuticals in agricultural environments irrigated with reclaimed water by wide-scope LC-QTOF-MS suspect screening

The transformation that pharmaceuticals can undergo during the water reclamation cycle, or by biotic/abiotic reactions when reclaimed water (RW) is used for irrigation, can lead to the presence of transformation products (TPs) in agricultural environments. However, data on TPs in real crops are scarce. Herein, a suspect screening approach was applied for the comprehensive investigation of 262 potential TPs, associated with 20 prioritised pharmaceuticals found in real tomato crops exposed to long-term RW irrigation. The occurrence and fate of the TPs was evaluated by the retrospective analysis of RW, soil, leave and tomato samples from 4 intensive production greenhouses. Sample analysis was accomplished by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Up to 18 TPs were tentatively identified, of which 2 were not previously reported. 7 TPs were finally confirmed with analytical standards. 5 TPs were determined in RW, 15 TPs in soil and 2 TPs in leaves. Remarkably, the investigated TPs were not found in tomato fruits. These results shed light on the variety of TPs that can be found in the water reuse cycle and contribute to the assessment of the global risks of wastewater reuse and the safety of the vegetable and fruit production system.

Analysis of seized stanozolol formulations in South Brazil by liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry

Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone which are used medically for several diseases. However, misuse is commonly observed by athletes to promote enhancement of strength and performance. AAS are frequently obtained through online black markets from clandestine drug manufacturing laboratories, without any quality standards, being potentially dangerous for users. The purpose of this work was the development and application of a fast and simple procedure for the quantitation of stanozolol by liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) in tablets packs seized in Rio Grande do Sul state, Brazil. The samples of stanozolol were separated considering its dosage form. The internal standard (methyltestosterone) was added to the aliquots of the samples, dissolved in methanol and 5μL were injected into the analytical system. The newly developed method has been validated for lower limit of quantitation (LLOQ), linearity, accuracy, precision and selectivity. The LLOQ was 0.1 µg/mL. The developed method was successfully applied to 31 samples seized by the Secretaria da Receita Federal do Brasil (a Brazilian federal revenue service agency). According to the results, 90.3% of the suspected medicines (n=31) were adulterated, and 65% exhibited higher concentrations of stanozolol than those indicated in the label. This work successfully established a new method for quantification of stanozolol using LC-QTOF-MS. This method aims at contributing to the identification and quantification of this anabolic androgenic steroid frequently seized by federal inspection agencies.

Hyphenated chromatography detection and compound-target-disease investigation on herb-pair Chuanxiong Rhizoma - Xiangfu Rhizoma

Ethnopharmacology relevanceThe combination of Chuanxiong Rhizoma (Ligusticum chuanxiong Hort., umbelliferae) with Xiangfu Rhizoma (the rhizoma of Cyperus rotundus L., Cyperaceae), is deemed as CR-XR herb-pair (Yaodui) in China. Their compatible mechanism needs a further research using modern analytical techniques and bioinformatic tool. MethodsHead Space- Solid Phase Micro Extraction coupled with Gas Chromatography/Mass Spectrometer detection (HS-SPME-GC/MS) and Liquid Chromatography coupled to quadrupole Time of Flight - Mass Spectrometry (LC-qTOF-MS) were applied in an accurate identification of the absorbed phytochemicals in mice serum; Their potential targets were available after compound-protein interaction (CPI) prediction and molecular docking verification; Then the corresponding disease types, as well as the relevant Traditional Chinese Medicine (Zhongyi) syndromes (Zheng), were matched from databases and references. ResultsResolution from hyphenated chromatographic datasets, thirty-eight phytochemicals were detected in serum samples from mice. Seventy potential target proteins were thereby found through a bioinformatic calculation, which mainly focused on circulatory, endocrine and nervous diseases in Western medicine, also related with Qizhi and Xueyu Zheng from the perspective of Zhongyi. Part of the relationships among compound-Target-Disease have been confirmed by literatures. These virtual data were sketched out as ‘The active Compound - potential Target’ network, ‘Target – Disease’ network and ‘Target - Zhongyi Disease’ network, in which the network topology was used to analyze them. ConclusionsOur work successfully explained the compatible mechanism of CR-XR Yaodui, which exert ‘multi-components, multi targets’ in treating Qizhi and Xueyu Zheng.

Suspect and Nontargeted Strategies to Investigate in Vitro Human Biotransformation Products of Emerging Environmental Contaminants: The Benzotriazoles.

Benzotriazole derivatives (BTRs) are high production volume chemicals involved in a wide range of applications and consumer products resulting in their ubiquitous presence in environmental matrices. Yet, the human exposure assessment to these chemicals is limited since it is based only on the analysis of parent compounds in biological matrices. The objective of this study was to investigate the in vitro human biotransformation for three widely used BTRs and to stepwise examine the role of Phase I and II enzymes (cytochrome P450 (CYP), uridine glucuronic acid transferase (UGT), and sulfotransferase (SULT)) in their biotransformation. Extracts with generated biotransformation products (bioTPs) were analyzed using liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS), followed by their identification based on a workflow combining suspect and nontargeted strategies. Ten bioTPs were identified for 1H-benzotriazole, 14 for tolyltriazole, and 14 for 5-chloro-1H-benzotriazole. Most of the proposed bioTPs were identified and structurally elucidated for the first time. Based on these findings, possible bioTPs and metabolic transformation pathways were subsequently predicted for other structurally close BTR derivatives. Our findings provide new identified in vitro biotransformation products for future biomonitoring studies and emphasize that it is important to investigate the biotransformation pathway to assess overall exposure to xenobiotics.