Detection of Hypoglycin A and MCPrG Metabolites in the Milk and Urine of Pasture Dairy Cows after Intake of Sycamore Seedlings.

Hypoglycin A (HGA), methylenecyclopropylglycine (MCPrG), hypoglycin B (HGB), and γ-glutamyl-α-(methylenecyclopropyl) glycine (γ-glutamyl-MCPrG) are secondary plant metabolites occurring in sycamore maple (Acer pseudoplatanus) as well as several other Sapindaceae (e.g., Blighia sapida). By interfering with energy metabolism, they may cause severe intoxication in humans and other species. However, to date, there is not enough data available concerning the intake, metabolism, or excretion of sycamore maple toxins in dairy cows. In May 2022, five cows were observed over four days, when they had first access to a pasture with two sycamore maples. Grazing of their seedlings that grew numerously in between the pasture plants was monitored by direct observation. Milk samples were drawn both from individual cows and from the bulk tank. Spontaneous urine samples were collected from all cows on day 3 after access to the pasture. Seedlings (100 g) were sampled on the pasture and analyzed, together with milk and urine samples, for sycamore toxins and their metabolites using liquid chromatography-tandem mass spectrometry and liquid chromatography-high-resolution mass spectrometry. Cows ingested sycamore seedlings while grazing. Values of HGA in milk were below the limit of quantification. However, metabolites of HGA and MCPrG were detected in individual milk samples already at the end of the first day of grazing. Urine samples of all five cows showed higher concentrations of conjugated HGA and MCPrG metabolites than in milk. Observations suggest that dairy cows may have a low susceptibility toward sycamore maple toxins. However, whether this could be attributed to foregut fermenting species in general requires further elucidation.

Origanum majorana L. protects against neuroinflammation-mediated cognitive impairment: a phyto-pharmacological study.

BACKGROUND: Neuroinflammation and oxidative stress are critical players in the pathogenesis of numerous neurodegenerative diseases, such as Alzheimer's disease (AD) which is responsible for most cases of dementia in the elderly. With the lack of curative treatments, natural phenolics are potential candidates to delay the onset and progression of such age-related disorders due to their potent antioxidant and anti-inflammatory effects. This study aims at assessing the phytochemical characteristics of Origanum majorana L. (OM) hydroalcohol extract and its neuroprotective activities in a murine neuroinflammatory model. METHODS: OM phytochemical analysis was done by HPLC/PDA/ESI-MSn. Oxidative stress was induced in vitro by hydrogen peroxide and cell viability was measured using WST-1 assay. Swiss albino mice were injected intraperitoneally with OM extract at a dose of 100 mg/kg for 12 days and with 250 µg/kg LPS daily starting from day 6 to induce neuroinflammation. Cognitive functions were assessed by novel object recognition and Y-maze behavioral tests. Hematoxylin and eosin staining was used to assess the degree of neurodegeneration in the brain. Reactive astrogliosis and inflammation were assessed by immunohistochemistry using GFAP and COX-2 antibodies, respectively. RESULTS: OM is rich in phenolics, with rosmarinic acid and its derivatives being major constituents. OM extract and rosmarinic acid significantly protected microglial cells against oxidative stress-induced cell death (p < 0.001). OM protected against the LPS-induced alteration of recognition and spatial memory in mice (p < 0.001) and (p < 0.05), respectively. Mice that received OM extract prior to the induction of neuroinflammation showed comparable histology to control brains, with no overt neurodegeneration. Furthermore, OM pre-treatment decreased the immunohistochemistry profiler score of GFAP from positive to low positive and COX-2 from low positive to negative in the brain tissue, compared to the LPS group. CONCLUSION: These findings highlight the potential preventive effects of OM phenolics against neuroinflammation and pave the way toward drug discovery and development for neurodegenerative disorders.

A sensitive LC-MS/MS method for the quantification of the plant toxins hypoglycin A and methylenecyclopropylglycine and their metabolites in cow's milk and urine and application to farm milk samples from Germany.

Hypoglycin A (HGA) and its homologue methylenecyclopropylglycine (MCPrG) are present in ackee and lychee as well as seeds, leaves, and seedlings of some maple (Acer) species. They are toxic to some animal species and humans. The determination of HGA, MCPrG, and their glycine and carnitine metabolites in blood and urine is a useful tool for screening for potential exposure to these toxins. In addition, HGA, MCPrG, and/or their metabolites have been detected in milk. In this work, simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods without derivatization were developed and validated for the quantification of HGA, MCPrG, and their metabolites in cow's milk and urine. An extraction procedure from milk samples has been developed, whereas a dilute-and-shoot approach was implemented for urine samples. For quantification, the MS/MS analysis was performed in multiple reaction monitoring mode. The methods were validated according to the European Union guidelines using blank raw milk and urine as matrices. The limit of quantification presented here for HGA in milk (1.12 µg/L) is noticeably lower than the lowest published limit of detection (9 µg/L). Acceptable values for recovery (89-106% and 85-104% in milk and urine, respectively) and precision (≤ 20%) were obtained for all the quality control levels. The stability of HGA and MCPrG in frozen milk over a period of 40 weeks has been demonstrated. The method was applied to 68 milk samples from 35 commercial dairy farms and showed the absence of any quantifiable amounts of HGA, MCPrG, and their metabolites.

Root uptake and metabolization of Alternaria toxins by winter wheat plants using a hydroponic system.

Fungi of the genus Alternaria are ubiquitous in the environment. Their mycotoxins can leach out of contaminated plants or crop debris into the soil entering the plant via the roots. We aim to evaluate the importance of this entry pathway and its contribution to the overall content of Alternaria toxins (ATs) in wheat plants to better understand the soil-plant-phytopathogen system. A hydroponic cultivation system was established and wheat plants were cultivated for up to two weeks under optimal climate conditions. One half of the plants was treated with a nutrient solution spiked with alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA), whereas the other half of the plants was cultivated without mycotoxins. Plants were harvested after 1 and 2 weeks and analyzed using a QuEChERS-based extraction and an in-house validated LC-MS/MS method for quantification of the ATs in roots, crowns, and leaves separately. ATs were taken up by the roots and transported throughout the plant up to the leaves after 1 as well as 2 weeks of cultivation with the roots showing the highest ATs levels followed by the crowns and the leaves. In addition, numerous AOH and AME conjugates like glucosides, malonyl glucosides, sulfates, and di/trihexosides were detected in different plant compartments and identified by high-resolution mass spectrometry. This is the first study demonstrating the uptake of ATs in vivo using a hydroponic system and whole wheat plants examining both the distribution of ATs within the plant compartments and the modification of ATs by the wheat plants.

Co-Occurrence of Hypoglycin A and Hypoglycin B in Sycamore and Box Elder Maple Proved by LC-MS/MS and LC-HR-MS.

Hypoglycin A (HGA) and methylenecyclpropylglycine (MCPrG) are formed by some maple trees (Acer species) and have been associated with incidences of atypical myopathy among horses in pastures. In this work, a simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method without derivatization was developed for the quantification of HGA and MCPrG in maple samples and validated according to EU guidelines. The LOQ presented here for HGA (16.4 µg/kg) is considerably lower than the lowest published LOQ (500 µg/kg). This method confirms that sycamore and box elder maple contain considerable amounts of HGA and MCPrG. In addition, the presence of the dipeptides hypoglycin B and γ-glutamyl-MCPrG in these two maple species is shown using high-resolution MS. This is the first report on the presence of these dipeptides in maple since 1973. The presence of HGB and γ-glutamyl-MCPrG could change the way we understand animal intoxication following the ingestion of maple.

Procedure providing SI-traceable results for the calibration of protein standards by sulfur determination and its application on tau.

Quantitative proteomics is a growing research area and one of the most important tools in the life sciences. Well-characterized and quantified protein standards are needed to achieve accurate and reliable results. However, only a limited number of sufficiently characterized protein standards are currently available. To fill this gap, a method for traceable protein quantification using sulfur isotope dilution inductively coupled plasma mass spectrometry (ICP-MS) was developed in this study. Gel filtration and membrane filtration were tested for the separation of non-protein-bound sulfur in the protein solution. Membrane filtration demonstrated a better performance due to the lower workload and the very low sulfur blanks of 11 ng, making it well suited for high-purity proteins such as NIST SRM 927, a bovine serum albumin (BSA). The method development was accomplished with NIST SRM 927e and a commercial avidin. The quantified mass fraction of NIST SRM 927e agreed very well with the certified value and showed similar uncertainties (3.6%) as established methods while requiring less sample preparation and no species-specific standards. Finally, the developed procedure was applied to the tau protein, which is a biomarker for a group of neurodegenerative diseases denoted "tauopathies" including, e.g., Alzheimer's disease and frontotemporal dementia. For the absolute quantification of tau in the brain of transgenic mice overexpressing human tau, a well-defined calibration standard was needed. Therefore, a pure tau solution was quantified, yielding a protein mass fraction of (0.328 ± 0.036) g/kg, which was confirmed by amino acid analysis.

Perfluorobutanoic acid (PFBA): No high-level accumulation in human lung and kidney tissue.

Perfluorobutanoic acid (PFBA) belongs to the complex group of synthetic perfluoroalkyl substances (PFAS) which have led to ubiquitous environmental contamination. While some of the long-chain compounds accumulate in the human body, the short-chain compound PFBA was found to have a relatively short half-life in blood of a few days, in agreement with relatively low PFBA serum/plasma levels of roughly 0.01 ng/ml in European studies. Surprisingly, very high median levels of PFBA of 807 and 263 ng/g tissue for human lung and kidney autopsy samples, respectively, were reported in a paper of Pérez et al. (2013). This would question the concept of PFAS blood analysis reflecting the body burden of these compounds. To verify the results of high PFBA tissue accumulation in humans, we have analyzed PFBA in a set of 7 lung and 9 kidney samples from tumor patients with a different method of quantification, using high-resolution mass spectrometry with the accurate mass as analytical parameter. The only human sample with a quantifiable amount of PFBA (peak area more than twice above the analytical background signals) contained approximately 0.17 ng/g lung tissue. In the light of our results and considering the analytical problems with the short-chain compound PFBA exhibiting only one mass fragmentation, it appears to be likely that PFBA is not accumulating on a high level in human lung and kidney tissue. In general, the analysis of short-chain PFAS in complex matrices like food or tissue is very challenging with respect to instrumental quantification and possible sample contamination.

Cu, Fe, and Zn isotope ratios in murine Alzheimer's disease models suggest specific signatures of amyloidogenesis and tauopathy.

Alzheimer's disease (AD) is characterized by accumulation of tau and amyloid-beta in the brain, and recent evidence suggests a correlation between associated protein aggregates and trace elements, such as copper, iron, and zinc. In AD, a distorted brain redox homeostasis and complexation by amyloid-beta and hyperphosphorylated tau may alter the isotopic composition of essential mineral elements. Therefore, high-precision isotopic analysis may reveal changes in the homeostasis of these elements. We used inductively coupled plasma-mass spectrometry (ICP-MS)-based techniques to determine the total Cu, Fe, and Zn contents in the brain, as well as their isotopic compositions in both mouse brain and serum. Results for male transgenic tau (Line 66, L66) and amyloid/presenilin (5xFAD) mice were compared with those for the corresponding age- and sex-matched wild-type control mice (WT). Our data show that L66 brains showed significantly higher Fe levels than did those from the corresponding WT. Significantly less Cu, but more Zn was found in 5xFAD brains. We observed significantly lighter isotopic compositions of Fe (enrichment in the lighter isotopes) in the brain and serum of L66 mice compared with WT. For 5xFAD mice, Zn exhibited a trend toward a lighter isotopic composition in the brain and a heavier isotopic composition in serum compared with WT. Neither mouse model yielded differences in the isotopic composition of Cu. Our findings indicate significant pathology-specific alterations of Fe and Zn brain homeostasis in mouse models of AD. The associated changes in isotopic composition may serve as a marker for proteinopathies underlying AD and other types of dementia.

Neuromodulatory Activity of Dietary Phenolics Derived from Corchorus olitorius L.

Dietary phenolics are known for their potent antioxidant and anti-inflammatory activities, making them promising candidates for protection against neuroinflammation and neurodegeneration. Hydroalcohol extract of Egyptian species of Corchorus olitorius L. (Co) leaves was investigated for its neuroprotective effects in a lipopolysaccharide-induced neuroinflammatory mouse model. Twenty five metabolites were characterized from the bioactive extract using high-performance liquid chromatography HPLC/PDA/HRESI/MSn , revealing 1,5-dicaffeoylquinic acid (Co11) as one of the major constituents (5.7%), which was isolated and its identity was confirmed by spectral data as first report. Co significantly protected microglia against H2 O2 -induced cytotoxicity and immunohistochemistry showed reduced expression of the astrocytic marker, glial fibrillary acidic protein, and the inflammatory marker, cyclooxygenase-2. These findings correlated with significant improvement of cognitive functions and reduction of LPS-induced neurodegeneration in Co-treated mice as revealed by histopathology. The current study shows promising effects of Co in limiting neurodegeneration and cognitive impairment caused by neuroinflammation and glial cell activation. PRACTICAL APPLICATION: Information presented here shed light on the promising effects of Corchorus olitorius (Co) for the modulation of neuroinflammatory pathways improving the neuroinflammation-related neurodegeneration and cognitive decline. This makes Co a promising candidate as a nutraceutical supplement to be used against neuroinflammation-related disorders.

Characterization of hepatoprotective metabolites from Artemisia annua and Cleome droserifolia using HPLC/PDA/ESI/MS-MS

ABSTRACT The hepatoprotective activities of two traditionally used plants, Cleome droserifolia (Forssk.) Delile, Cleomaceae, and Artemisia annua L., Asteraceae, were recently reported. However, the biologically active metabolites responsible for this activity were not identified. The aqueous extract of C. droserifolia aerial parts, and the polar fraction of A. annua leaves were screened for their antioxidant activities using the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assay. The in vitro viability of HepG-2 cells treated with CCl4 and the extracts were assessed by MTT assay. The effects of the extracts on the liver enzymes and the total soluble protein in CCl4-intoxicated HepG-2 cells were investigated. An HPLC/PDA/ESI/MS-MS based analysis was carried out for extract of C. droserifolia and polar fraction of A. annua. Both exhibited pronounced free radical scavenging activities (86 and 83%, respectively). Both showed a significant increase in cell viability: 86.43% for the extract of C. droserifolia and 79.32% for polar fraction of A. annua. Only the extract of C. droserifolia (39.6 ± 5.41 and 20.4 ± 6.91 IU/dl, respectively) and polar fraction of A. annua (40.8 ± 2.14 and 24.5 ± 3.11 IU/dl, respectively) restored the levels of liver enzymes (aspartate transaminase and alanine transaminase, respectively) compared to the CCl4 intoxicated group (87.5 ± 4.34 and 34.1 ± 8.12 IU/dl, respectively) and other herbal extracts. More than fifty phenolic secondary metabolites were identified in the extracts under investigation. The significant hepatoprotective activities of both extracts seemed to be strongly connected to their content of hydroxycinnamoyl quinic acids and flavonoids.

Complementarity of molecular and elemental mass spectrometric imaging of Gadovist™ in mouse tissues.

Drug biodistribution analyses can be considered a key issue in pharmaceutical discovery and development. Here, mass spectrometric imaging can be employed as a powerful tool to investigate distributions of drug compounds in biologically and medically relevant tissue sections. Both matrix-assisted laser desorption ionization-mass spectrometric imaging as molecular method and laser ablation inductively coupled plasma-mass spectrometric imaging as elemental detection method were applied to determine drug distributions in tissue thin sections. Several mouse organs including the heart, kidney, liver, and brain were analyzed with regard to distribution of Gadovist™, a gadolinium-based contrast agent already approved for clinical investigation. This work demonstrated the successful detection and localization of Gadovist™ in several organs. Furthermore, the results gave evidence that gadolinium-based contrast agents in general can be well analyzed by mass spectrometric imaging methods. In conclusion, the combined application of molecular and elemental mass spectrometry could complement each other and thus confirm analytical results or provide additional information.

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR.

Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

Polyphenols from Tamarix nilotica: LC⁻ESI-MSn Profiling and In Vivo Antifibrotic Activity.

Tamarix nilotica (Ehrenb.) Bunge (Tamaricaceae), an indigenous plant to the Middle East region, is well-known as a medicinal plant for treating many human ailments. The current study aimed at exploring the polyphenol profile of the alcohol soluble fraction of aqueous T. nilotica extract, assessing its in vivo antifibrotic activity and the possible underlying mechanism, to unravel the impact of quantitative difference of sulphated polyphenols content on the antifibrotic activity of T. nilotca grown in two different habitats. Polyphenol profiling of T. nilotica extracts was performed using HPLC-HRESI-QTOF-MS-MS. The major polyphenol components included sulphated flavonoids, phenolic acids and free aglycones. The antifibrotic activity was evaluated through carbon tetrachloride-induced liver fibrosis in rats. Biochemical evaluations revealed that both fractions ameliorated the increased levels of hepatic aminotransferases, lipid peroxidation, hydroxyproline, α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB). Moreover, both fractions reduced catalase activity (CAT) and enhanced hepatic glutathione (GSH) content. Histopathological imaging undoubtedly confirmed such results. In conclusion, the T. nilotica polyphenol-rich fraction exhibited potential antifibrotic activity in rats. Significant alterations in GSH levels were recorded based on the sulphated polyphenol metabolite content.

Online immunocapture ICP-MS for the determination of the metalloprotein ceruloplasmin in human serum.

OBJECTIVE: The human copper-protein ceruloplasmin (Cp) is the major copper-containing protein in the human body. The accurate determination of Cp is mandatory for the reliable diagnosis of several diseases. However, the analysis of Cp has proven to be difficult. The aim of our work was a proof of concept for the determination of a metalloprotein-based on online immunocapture ICP-MS. The immuno-affinity step is responsible for the enrichment and isolation of the analyte from serum, whereas the compound-independent quantitation with ICP-MS delivers the sensitivity, precision, and large dynamic range. Off-line ELISA (enzyme-linked immunosorbent assay) was used in parallel to confirm the elution profile of the analyte with a structure-selective method. The total protein elution was observed with the 32S mass trace. The ICP-MS signals were normalized on a 59Co signal. RESULTS: The human copper-protein Cp could be selectively determined. This was shown with pure Cp and with a sample of human serum. The good correlation with off-line ELISA shows that Cp could be captured and eluted selectively from the anti-Cp affinity column and subsequently determined by the copper signal of ICP-MS.

Comparative pharmacokinetics of trandolapril, its active metabolite, and verapamil in human plasma of Egyptian population using HPLC-MS/MS.

Trandolapril and verapamil are commonly used antihypertensive drugs. However, there is a lack of available data on the change of their pharmacokinetics in patients with liver or kidney impairment and hence the need for dose adjustment. In this article, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the monitoring of trandolapril, its active metabolite trandolaprilat, and verapamil in human plasma of patients with renal impairment and/or liver insufficiency. The chromatographic separation was achieved on a Gemini C18 reversed phase column using a gradient elution mode with a run time of 10 minutes. The mobile phase consisted of a mixture of methanol and 2% acetic acid. The electrospray ionization MS/MS analysis was performed in multiple reaction monitoring (MRM) mode. The assay was validated as per Food and Drug Administration (FDA) guidelines for bioanalytical method validation and proved to be suitable for the determination of therapeutic drug levels in plasma. The inter-group changes in pharmacokinetic data were compared to that of healthy volunteers. The comparison showed a significant difference in the pharmacokinetic parameters between the studied groups. The presented results exhibit the benefits of the proposed assay as a validated analytical tool for the continuous drug monitoring.

High resolution UPLC-MS/MS profiling of polyphenolics in the methanol extract of Syzygium samarangense leaves and its hepatoprotective activity in rats with CCl4-induced hepatic damage.

Oxidative stress plays a crucial role in the development of several liver diseases. Many natural polyphenols can attenuate oxidative stress and liver injury. In this study, a phytochemical profiling of a methanol extract from leaves of Syzygium samarangense revealed 92 compounds belonging to flavonoids, phenolic acids, condensed tannins, and ellagitannins. The S. samarangense extract exhibited a noticeable antioxidant activity with an EC50 of 5.80 µg/mL measured by DPPH scavenging capacity assay, 2632 Trolox equivalents, 10 mM Fe2+ equivalents/mg of samples by TEAC and FRAP assays, respectively. The total phenolic content was 419 mg gallic acid equivalent GAE/g extract. In a cell-based model (HaCaT cells), the extract completely inhibited ROS production induced by UVA, and prevented GSH-depletion and p38 phosphorylation. In addition, the extract exhibited a substantial antioxidant and hepatoprotective activities in CCl4-treated rats, with an increase in GSH (reduced glutathione) and SOD (superoxide dismutase) activities by 84.75 and 26.27%, respectively, and a decrease of 19.08, 63.05, 52.21, 37.00, 13.26, and 15.15% in MDA, ALT, AST, TB (total bilirubin), TC (total cholesterol), and TG (total glycerides), respectively. These results were confirmed by histopathological analyses. We believe that Syzygium samarangense is a good candidate for further evaluation as an antioxidant and liver protecting drug.

Identification of phenolic secondary metabolites from Schotia brachypetala Sond. (Fabaceae) and demonstration of their antioxidant activities in Caenorhabditis elegans.

BACKGROUND: Schotia brachypetala Sond. (Fabaceae) is an endemic tree of Southern Africa whose phytochemistry and pharmacology were slightly studied. The present work aimed at profiling the major phenolics compounds present in the hydro-alcohol extract from S. brachypetala leaves (SBE) using LC/HRESI/MS/MS and NMR and prove their antioxidant capabilities using novel methods. METHODS: In vitro assays; DPPH, TEAC persulfate decolorizing kinetic and FRAP assays, and in vivo assays: Caenorhabditis elegans strains maintenance, Intracellular ROS in C. elegans, Survival assay, GFP expression and Subcellular DAF-16 localization were employed to evaluate the antioxidant activity. RESULTS: More than forty polyphenols, including flavonoid glycosides, galloylated flavonoid glycosides, isoflavones, dihydrochalcones, procyanidins, anthocyanins, hydroxy benzoic acid derivatives, hydrolysable tannins, and traces of methylated and acetylated flavonoid derivatives were identified. Three compounds were isolated and identified from the genus Schotia for the first time, namely gallic acid, myricetin-3-O-α-L-1C4-rhamnoside and quercetin-3-O-L-1C4-rhamnoside. The total phenolics content of SBE was (376 mg CAE/g), followed by flavonoids (67.87 QE/g). In vitro antioxidant activity of SBE was evidenced by DPPH radical scavenging activity (IC50 of 9 µg/mL), FRAP ferric reducing activity (5,000 mol Fe2+ E/mg) and ABTS peroxide inhibiting activity (1,054 mM Trolox E/mg). The tested extract was able to protect the worms against juglone induced oxidative stress, an increased survival rate (up to 41%) was recorded, when compared with the control group (11%) and attenuate the reactive oxygen species (ROS) accumulation in dose-dependent and reached up to 72% for the highest tested concentration. SBE was also able to attenuate the levels of heat shock protein (HSP) expression in dose-dependent up to 60% in the 150 µg SBE/mL group. In DAF-16 Subcellular localization SBE treated worms showed nuclear localization pattern up to 78%, while it was only 5% in the untreated control group. DISCUSSION: A pronounced antioxidant activity in vivo, which can be attributed to its ability to promote the nuclear translocation of DAF-16/FOXO, the main transcription factor regulating the expression of stress response genes. The remarkable antioxidant activity in vitro and in vivo correlates to SBE rich phenolic profile.

Simultaneous determination of eight neonicotinoid insecticide residues and two primary metabolites in cucumbers and soil by liquid chromatography-tandem mass spectrometry coupled with QuEChERS.

A new, rapid, sensitive, precise and validated high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of eight neonicotinoid insecticides with their two primary metabolites in cucumbers and soil based on QuEChERS as a pretreatment procedure. In QuEChERS procedure, cucumber samples were extracted with acetonitrile and cleaned using (C18 sorbent material), while soil samples were extracted with a mixture of acetonitrile:dichloromethane (8.3:16.7v:v). The LC-MS/MS conditions were optimized to provide good selectivity and specificity of the developed method where neonicotinoids were separated using gradient elution of water and acetonitrile both containing 0.1% formic acid with Gemini C18 column where the last compound was eluted at 9.5min. Average recoveries of the eight neonicotinoids and their metabolites ranged between 81.6% and 95.7% in fortified cucumber samples with relative standard deviations (RSDs) lower than 13.18% and between 80.3% and 104% in fortified soil samples with relative standard deviations (RSDs) lower than 8.44%. The limits of detection (LODs) and quantification (LOQs) for the ten compounds were in the ranges of (0.08-6.06ng/g) and (0.26-20ng/g), respectively. The method was applied successfully to determine residues and rate of disappearance of the eight neonicotinoids from cucumber and soil and their half-lives where a safety pre-harvest interval of 5days for acetampirid, 12days for imidacloprid, 15days for nitenpyram, 12days for thiamethoxam, 5days for flonicamid, 8days for clothianidin, 2days for Dinotefuran, and 1day for thiacloprid were suggested.

Bridging the gap between molecular and elemental mass spectrometry: higher energy collisional dissociation (HCD) revealing elemental information.

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS.