LC-QTOF MS Research Articles

Simultaneous determination of multi-class antibiotics and steroid hormones drugs in livestock and poultry faeces using liquid chromatography–quadrupole time-of-flight mass spectrometry

ABSTRACT A method for simultaneous determination of multi-class antibiotics and steroid hormone analysis in faeces of livestock and poultry was developed using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF MS). An in-house database was built for 156 detected drugs using Personal Compound Database Library software (PCDL) including compound name, monoisotopic mass, chemical formula, RT, chemical structure and three CID MS/MS spectra. The linearity result showed that all the drugs exhibited good linearity with determination coefficients (R2) higher than 0.99. The drug recoveries and their RSDs for all three faeces samples (pig, cattle and chicken) were tested and 81, 96 and 92 drugs were chosen for analysis in pig, cattle and chicken faeces, respectively. Further validation showed that 73 veterinary drugs in all three kinds of faeces samples can be quantified in one analytical run. This work shows that qualitative and quantitative analysis using LC-QTOF MS represents a simple, sensitive, low-cost and high-throughput methodology in routine laboratory analyses.

Degradation of a mixture of pharmaceuticals in hospital wastewater by a zero-valent scrap iron (ZVSI) combined reduction-oxidation process

Zero-valent scrap iron (ZVSI) combined with Fenton-based oxidation reaction (reduction-oxidation process) was studied under natural sunlight conditions to degrade a mixture of six pharmaceuticals. ZVSI is a waste product from metalworking industries, which present excellent reactivity. The morphological and structural properties of ZVSI were evaluated using different techniques. The parent compounds and their transformation products (TPs) were monitored using Liquid Chromatographic coupled with Quadrupole-Time of Flight mass spectrometer (LC-QTOF MS). The in silico (quantitative) structure-activity relationship ((Q)SAR) predictions of toxicity and biodegradability were predicted for each TP by using different freely available models. According the characterization ZVSI consisted mainly of carbon (48.5 %) and iron (43.6 %) with 0.6922 m2/g of surface area. Hematite and magnetite were the main crystalline phases ZVSI. Doehlert design and desirability profile were used to select the experimental conditions. Higher degradation rates were achieved at pH 3.0, 32.5 mg L−1 H2O2 and 2 g L−1 ZVSI. Under the optimal conditions, pharmaceuticals removal was as high as 87 % in all matrixes evaluated, following a pseudo-first order kinetic model behavior. Three reuse cycles were performed using real hospital wastewater (RHWW) achieving degradation percentages >70% and no substantial variation in the crystalline phases of the ZVSI was observed. Twenty-three TPs were detected. According to in silico (Q)SAR predictions, the integrated system decreased the toxicity of TPs. Moreover, most of TPs were predicted as persistent compounds. The results indicated that ZVSI combined with solar Fenton-based oxidation is an interesting alternative for the degradation of pharmaceuticals in RHWW allowing the reuse of catalyst.

Optimization of an MMPB Lemieux Oxidation method for the quantitative analysis of microcystins in fish tissue by LC-QTOF MS

Microcystins are toxic heptapeptides produced by cyanobacteria in marine and freshwater environments. In biological samples such as fish, microcystins can be found in the free form or covalently bound to protein phosphatases type I and II. Total microcystins in fish have been quantified in the past using the Lemieux Oxidation approach, where all toxins are oxidated to a common fragment (2-methyl-3-methoxy-4-phenylbutyric acid, MMPB) regardless of their initial amino acid configuration or form (free or protein bound). These studies have been carried out using different experimental conditions and employed different quantification strategies. The present study has further investigated the oxidation step using a systematic approach, to identify the most important factors leading to a higher, more robust MMPB generation yield from fish tissue in order to reduce the method detection limit. Field samples were quantified using an in-situ generated MMPB matrix matched calibration curve by isotope dilution with d3-MMPB via liquid chromatography coupled to time-of-flight mass spectrometry (LC-QTOF MS). This approach improves method's accuracy by taking into account of potential matrix effects that could affect the derivatization, sample prepation and instrumental analysis steps. The validated method showed 16.7% precision (RSD) and +6.7% accuracy (bias), with calculated method detection limits of 7.28 ng g−1 Performance of the method was assessed with the analysis of laboratory exposed Rainbow Trout (Oncorhynchus mykiss) to cyanobacteria as a positive control, where no microcystins were detected in the pre-exposure fish liver and fillet, low levels in the exposed fillet (65.0 ng g−1) and higher levels in the exposed liver (696 ng g−1). Finally, the method was employed for the analysis of 26 fillets (muscle) and livers of Walleye (Sander vitreus) and Yellow Perch (Perca flavescens) from Lake Erie, showing very low concentrations of microcystins in the fillet and higher concentrations in liver, up to 3720 ng g−1.

Rhamnolipids and siderophores from Pseudomonas protegens strain BNJ-SS-45 isolated from wheat rhizosphere

Pseudomonas protegens strain BNJ-SS-45 possesses potential to secrete multiple antifungal molecules and other metabolites. To expand its metabolic potential, herein, an attempt has been made to characterize rhamnolipid and siderophore molecules. The analysis of purified biosurfactants using liquid chromatography-tandem mass spectrometry (LC–MS/MS) revealed different congeners where mono-rhamnolipids Rha-C10-C14:1/Rha-C12-C12:1, Rha-C10-C12/Rha-C12-C10, Rha-C12-C12/Rha- H2O C10-C14/Rha-C14-C10, Rha-C10-C10:1/Rha-C10:1-C10, Rha-C10-C12:1/Rha-C12:1-C10 are present. Structural investigation of XAD-4 purified siderophores using liquid chromatography quadrupole time-of-flight mass spectrometry (LC–MS-QTOF) analysis revealed the presence of isoquinoline N oxide as a chromophore. Using response surface methodology for optimization a sustainable increase in the level of metabolites is achieved and resulted in 81% and 66% enhancement in rhamnolipids and siderophores level in a 7 L fermentor respectively. Rhamnolipids obtained also exhibited antagonistic activity against both pathogenic fungi and bacteria. These are promising candidates to be used as biocontrol agents especially due to their biodegradable and eco-friendly nature.

LC-QTOF-MS and 1H NMR Metabolomics Verifies Potential Use of Greater Omentum for Klebsiella pneumoniae Biofilm Eradication in Rats.

Bacterial wound infections are a common problem associated with surgical interventions. In particular, biofilm-forming bacteria are hard to eradicate, and alternative methods of treatment based on covering wounds with vascularized flaps of tissue are being developed. The greater omentum is a complex organ covering the intestines in the abdomen, which support wound recovery following surgical procedures and exhibit natural antimicrobial activity that could improve biofilm eradication. We investigated changes in rats’ metabolome following Klebsiella pneumoniae infections, as well as the greater omentum’s ability for Klebsiella pneumoniae biofilm eradication. Rats received either sterile implants or implants covered with Klebsiella pneumoniae biofilm (placed in the peritoneum or greater omentum). Metabolic profiles were monitored at days 0, 2, and 5 after surgery using combined proton nuclear magnetic resonance (1H NMR) and high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC–QTOF-MS) measurements of urine samples followed by chemometric analysis. Obtained results indicated that grafting of the sterile implant to the greater omentum did not cause major disturbances in rats’ metabolism, whereas the sterile implant located in the peritoneum triggered metabolic perturbations related to tricarboxylic acid (TCA) cycle, as well as choline, tryptophan, and hippurate metabolism. Presence of implants colonized with Klebsiella pneumoniae biofilm resulted in similar levels of metabolic perturbations in both locations. Our findings confirmed that surgical procedures utilizing the greater omentum may have a practical use in wound healing and tissue regeneration in the future.

Compound Identification and In Vitro Cytotoxicity of the Supercritical Carbon Dioxide Extract of Papaya Freeze-Dried Leaf Juice

Carica papaya leaves are used as a remedy for the management of cancer. Freeze-dried C. papaya leaf juice was extracted using a supercritical fluid extraction system. Compound identification was carried out using analytical techniques including liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry (LC–QToF-MS) and gas chromatography–mass spectrometry (GC–MS). The cytotoxic activities of the scCO2 extract and its chemical constituents were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on squamous cell carcinoma (SCC25) and human keratinocyte (HaCaT) cell lines. The chemical constituents were quantified by QToF-MS. The supercritical carbon dioxide (scCO2) extract of papaya freeze-dried leaf juice showed cytotoxic activity against SCC25. Three phytosterols, namely, β-sitosterol, campesterol, and stigmasterol, together with α-tocopherol, were confirmed to be present in the scCO2 extract. Quantitative analysis showed that β-sitosterol was the major phytosterol present followed by α-tocopherol, campesterol, and stigmasterol. β-Sitosterol and campesterol were active against SCC25 (half maximal inhibitory concentration (IC50) ≈ 1 µM), while stigmasterol was less active (~33 µM) but was biologically more selective against SCC25. Interestingly, an equimolar mixture of phytosterols was not more effective (no synergistic effect was observed) but was more selective than the individual compounds. The compounds identified are likely accountable for at least part of the cytotoxicity and selectivity effects of C. papaya.

Evaluation of Synthetic Cannabinoid Metabolites in Human Blood in the Absence of Parent Compounds: A Stability Assessment.

Synthetic cannabinoids represent a chemically diverse class of novel psychoactive substances (NPS) responsible for large analytical and interpretative challenges for forensic toxicologists. Between 2016 and 2019, the three most prevalent synthetic cannabinoids in the United States were MMB-FUBINACA (FUB-AMB), 5F-MDMB-PINACA (5F-ADB) and 5F-MDMB-PICA, based on results from seized drug and toxicology testing. In 2018, accurate determination of synthetic cannabinoid positivity was brought into question as it was determined that the metabolites of these drug species were present in the absence of parent compounds in forensically relevant blood samples. During this study, the stability of MMB-FUBINACA, 5F-MDMB-PINACA and 5F-MDMB-PICA was evaluated, as well as the characterization of breakdown products. A liquid-liquid extraction method was assessed for recovery of basic parent compounds and acidic metabolites and deemed fit for use in this study. Analysis was performed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) using a SCIEX TripleTOF® 5600+. All three synthetic cannabinoids were found to be unstable when stored in blood at either room temperature or refrigerated; all analytes were considerably more stable when stored in the freezer. All three synthetic cannabinoids degraded to their respective butanoic acid metabolites: MMB-FUBINACA 3-methylbutanoic acid, 5F-MDMB-PINACA 3,3-dimethylbutanoic acid and 5F-MDMB-PICA 3,3-dimethylbutanoic acid. All three of these metabolites were studied and determined to be stable in blood at all storage conditions. Considering these results, our laboratory continued testing for synthetic cannabinoid metabolites in blood samples and found 83 positives (21%) for only a synthetic cannabinoid metabolite. A case report is presented herein where 5F-MDMB-PINACA 3,3-dimethylbutanoic acid was identified in the absence of 5F-MDMB-PINACA. Forensic toxicologists should be aware of the results of this study as they directly impact analytical consideration for test development and implementation, as well as interpretation of findings.

Gut-Derived Serotonin Contributes to the Progression of Non-Alcoholic Steatohepatitis via the Liver HTR2A/PPARγ2 Pathway.

The precipitous increase in occurrence of non-alcoholic steatohepatitis (NASH) is a serious threat to public health worldwide. The pathogenesis of NASH has not yet been thoroughly studied. We aimed to elucidate the interplay between serotonin (5-hydroxytryptamine, 5-HT) and NASH. The serum 5-HT levels in patients with non-alcoholic fatty liver disease (NAFLD) and a rat fed with high fat-sucrose diet (HFSD) were evaluated using liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF MS)/MS. The peripheral Tph1 inhibitor, LP533401, and a tryptophan (TRP)-free diet were administered to rats with NASH, induced by HFSD. BRL-3A cells were treated with 1 mM free fatty acids (FFAs) and/or 50 μM 5-HT, and then small interfering RNA (siRNA) targeting the 5-HT2A receptor (HTR2A) and the PPARγ pharmaceutical agonist, pioglitazone, were applied. We found a marked correlation between 5-HT and NASH. The absence of 5-HT, through the pharmaceutical blockade of Tph1 (LP533401) and dietary control (TRP-free diet), suppressed hepatic lipid load and the expression of inflammatory factors (Tnfα, Il6, and Mcp-1). In BRL-3A cells, 50 μM 5-HT induced lipid accumulation and upregulated the expression of lipogenesis-ralated genes (Fas, Cd36, and Plin2) and the inflammatory response. Specifically, HTR2A knockdown and evaluation of PPARγ agonist activity revealed that HTR2A promoted hepatic steatosis and inflammation by activating PPARγ2. These results suggested that duodenal 5-HT was a risk factor in the pathological progression of NASH. Correspondingly, it may represent an attractive therapeutic target for preventing the development of NASH via the regulation of the HTR2A/PPARγ2 signaling pathway.

In Vitro, In Silico, and In Vivo Assessments of Pharmacokinetic Properties of ZM241385.

Parkinson’s disease is one of the most common neurodegenerative diseases. Adenosine regulates the response to other neurotransmitters in the brain regions related to motor function. In the several subtypes of adenosine receptors, especially, adenosine 2A receptors (A2ARs) are involved in neurodegenerative conditions. ZM241385 is one of the selective non-xanthine A2AR antagonists with high affinity in the nanomolar range. This study describes the in vitro and in vivo pharmacokinetic properties of ZM241385 in rats. A liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qToF MS) method was developed for the determination of ZM241385 in rat plasma. In vivo IV administration studies showed that ZM241385 was rapidly eliminated in rats. However, the result of in vitro metabolic stability studies showed that ZM241385 had moderate clearance, suggesting that there is an extra clearance pathway in addition to hepatic clearance. In addition, in vivo PO administration studies demonstrated that ZM241385 had low exposure in rats. The results of semi-mass balance studies and the in silico PBPK modeling studies suggested that the low bioavailability of ZM241385 after oral administration in rats was due to the metabolism and by liver, kidney, and gut.

Structure Elucidation of Urinary Metabolites of Fentanyl and Five Fentanyl Analogs using LC-QTOF-MS, Hepatocyte Incubations and Synthesized Reference Standards.

Fentanyl analogs constitute a particularly dangerous group of new psychoactive compounds responsible for many deaths around the world. Little is known about their metabolism, and studies utilizing liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) analysis of hepatocyte incubations and/or authentic urine samples do not allow for determination of the exact metabolite structures, especially when it comes to hydroxylated metabolites. In this study, seven motifs (2-, 3-, 4- and β-OH as well as 3,4-diOH, 4-OH-3-OMe and 3-OH-4-OMe) of fentanyl and five fentanyl analogs, acetylfentanyl, acrylfentanyl, cyclopropylfentanyl, isobutyrylfentanyl and 4F-isobutyrylfentanyl were synthesized. The reference standards were analyzed by LC–QTOF-MS, which enabled identification of the major metabolites formed in hepatocyte incubations of the studied fentanyls. By comparison with our previous data sets, major urinary metabolites could tentatively be identified. For all analogs, β-OH, 4-OH and 4-OH-3-OMe were identified after hepatocyte incubation. β-OH was the major hydroxylated metabolite for all studied fentanyls, except for acetylfentanyl where 4-OH was more abundant. However, the ratio 4-OH/β-OH was higher in urine samples than in hepatocyte incubations for all studied fentanyls. Also, 3-OH-4-OMe was not detected in any hepatocyte samples, indicating a clear preference for the 4-OH-3-OMe, which was also found to be more abundant in urine compared to hepatocytes. The patterns appear to be consistent across all studied fentanyls and could serve as a starting point in the development of methods and synthesis of reference standards of novel fentanyl analogs where nothing is known about the metabolism.

Degradation of the anticancer drug flutamide by solar photo-Fenton treatment at near-neutral pH: Identification of transformation products and in silico (Q)SAR risk assessment

Flutamide (FLUT) is a non-steroidal drug mainly used in the treatment of prostate cancer and has been detected in the aquatic environment at ng L−1 levels. The environmental fate and effects of FLUT have not yet been studied. Conventional treatment technologies fail to completely remove pharmaceuticals, so the solar photo-Fenton process (SPF) has been proposed as an alternative. In this study, the degradation of FLUT, at two different initial concentrations in ultra-pure water, was carried out by SPF. The initial SPF conditions were pH0 5, [Fe2+]0 = 5 mg L−1, and [H2O2]0 = 50 mg L−1. Preliminary elimination rates of 53.4% and 73.4%. The kinetics of FLUT degradation could be fitted by a pseudo-first order model and the kobs were 6.57 × 10−3 and 9.13 × 10−3 min−1 t30W and the half-life times were 95.62 and 73.10 min t30W were achieved for [FLUT]0 of 5 mg L−1 and 500 μg L−1, respectively. Analysis using LC-QTOF MS identified thirteen transformation products (TPs) during the FLUT degradation process. The main degradation pathways proposed were hydroxylation, hydrogen abstraction, demethylation, NO2 elimination, cleavage, and aromatic ring opening. Different in silico (quantitative) structure-activity relationship ((Q)SAR) freeware models were used to predict the toxicities and environmental fates of FLUT and the TPs. The in silico predictions indicated that these substances were not biodegradable, while some TPs were classified near the threshold point to be considered as PBT compounds. The in silico (Q)SAR predictions gave positive alerts concerning the mutagenicity and carcinogenicity endpoints. Additionally, the (Q)SAR toolbox software provided structural alerts corresponding to the positive alerts obtained with the different mutagenicity and carcinogenicity models, supporting the positive alerts with more proactive information.

Molecular Level Analysis Reveals Changes in Chemical Composition of Dissolved Organic Matter From South Texas Rivers After High Flow Events

Riverine dissolved organic matter (DOM) is a major source of reduced carbon exported from land to marine environments, and the inflow of riverine organic matter greatly affects biogeochemical cycling in estuaries and bays. Thus, any change in DOM composition, such as changes caused by flood waters as a result of storms and hurricanes, can subsequently affect estuarine environments. To investigate the impact of high flow events on riverine DOM, multidimensional molecular level information of DOM from four south Texas Rivers (Aransas, Lavaca, Mission, and Nueces Rivers) was acquired using high-resolution Ion Mobility Quadrupole Time of Flight Liquid Chromatography Mass Spectrometry (IM Q-TOF LCMS). Base-flow samples were collected in May, July and October of 2016, June of 2017, and March of 2018, while high-flow samples were collected in September of 2017, and June and September of 2018. Based on the molecular formula assigned from IM Q-TOF LCMS, H/C ratio decreased during high-flow event (1.52 to 1.51 in ESI+; 1.19 to 1.07 in ESI-), while O/C ratio increased (0.31 to 0.33 in ESI-). Furthermore, DOM shifted from a protein-like and lipid-like dominated community at base flow condition, to a lignin, tannin and condensed aromatic structure dominated one during high flow event based on MS and tandem MS data. These changes in high-flow riverine DOM indicate an increase of terrestrial signal, which likely is a result of mobilization of terrestrial organic matter from the watersheds by flooding. These mobilized DOM, though refractory at high-flow condition in rivers, could be reactive in coastal regions when condition changes, and thus potentially fuel microbial activities downstream. In addition, about 3.76 – 21.8% of DOM molecules contain structural isomers among different flow conditions. This low number of isomer percentages suggests that as the products of various enzymatic biochemical reactions, the number of isomers in DOM is constrained. Taken together, our study provides insights into structural changes of riverine DOM in response to extreme climate events in subtropical regions and have implications in understanding biogeochemical changes in estuaries under a changing climate.

Column bioreactor of immobilized Stropharia sp. ITCC 8422 on natural biomass support of L. cylindrica for biodegradation of anthraquinone violet R

The column bioreactor of fungal biomass Stropharia sp. ITCC - 8422 was immobilized on natural biomass support of L. cylindrica and effectively decolorized anthraquinone violet R (AQVR). The designed column bioreactor successfully decolorized >80% AQVR up to third cycle whereas >55% AQVR decolorization was observed up to 7 cycles. Regenerated column reactor had improved efficiency and was used for multiple cycles. No significant adsorption on immobilized fungal and natural biomass was observed. The crude yellow laccase (YL) decolorized >80% AQVR (100 ppm) at pH 9 and 40 °C after 10 min. Untreated and treated AQVR were examined using spectral studies and the degraded products were confirmed using Q-TOF LCMS and pathway was proposed. AQVR was broken down to low molecular weight products i.e., m/z = 146.0607 and 130.064. The degraded products were less toxic and confirmed using phyto-toxicity test.

Ginger extract activates caspase independent paraptosis in cancer cells via ER stress, mitochondrial dysfunction, AIF translocation and DNA damage

The rhizome of ginger (Zingiber officinale) a common culinary agent is also known for its medicinal activity. We have earlier reported that pure 6-shogaol, an important component of ginger induces paraptosis in triple negative breast cancer (MDA-MB-231) and non small cell lung (A549) cancer cells. However, the chemopreventive potential of the whole ginger extract in food remains to be elucidated. Here, we demonstrate for the first time that ginger extract (GE) triggers similar anticancer activity/paraptosis against the same cell lines but through different molecular mechanisms. Q-TOF LC-MS analysis of the extract showed the presence of several other metabolites along with 6-shogaol and 6-gingerol. GE induces cytoplasmic vacuolation through ER stress and dilation of the ER. Drastic decrease in the mitochondrial membrane potential and ATP production along with the excess generation of ROS contributed to mitochondrial dysfunction. Consequently, GE caused the translocation of apoptosis inducing factor to the nucleus leading to the fragmentation of DNA. Taken together, these show a novel mechanism for ginger extract induced cancer cell death that can be of potential interest for cancer preventive strategies.

Investigation of pharmaceuticals and their metabolites in Brazilian hospital wastewater by LC-QTOF MS screening combined with a preliminary exposure and in silico risk assessment

This work evaluates the occurrence of pharmaceuticals, with special emphasis on their metabolites, in raw hospital wastewater (HWW) using wide-scope screening based on liquid chromatography coupled to high resolution mass spectrometry. The applied strategy uses an extended purpose-built database, containing >1000 pharmaceuticals and 250 metabolites. Raw HWW samples from a hospital located in south Brazil were collected over six months, with a monthly sampling frequency. Accurate-mass full-spectrum data provided by quadrupole-time of flight MS allowed the identification of 43 pharmaceuticals and up to 31 metabolites in the samples under study. Additionally, other four metabolites not included in the initial database could be identified using a complementary strategy based on the common fragmentation pathway between the parent compound and its metabolites. Nine metabolites derived from four pharmaceuticals were identified in the raw HWW samples, whereas their parent compounds were not found in these samples. The results of this work illustrate the importance of including not only parent pharmaceuticals but also their main metabolites in screening analysis. Besides, the inclusion of in silico QSAR predictions allowed assessing the environmental fate and effect of pharmaceuticals and metabolites in terms of biodegradability, as possible Persistent, Bioaccumulative and Toxic (PBT) compounds, and their potential hazard to the aquatic environment.

Investigation of pesticides and their transformation products in the Júcar River Hydrographical Basin (Spain) by wide-scope high-resolution mass spectrometry screening

The Water Framework Directive 2000/60/EC implemented by the European Union established as the main objectives to achieve a “good ecological and chemical status” of the surface water and a “good quantitative and chemical status” of groundwater bodies. One of the major pressures affecting water bodies comes from the use of pesticides and their potential presence in the water ecosystems. For this purpose, the reliable determination of pesticides and their transformation products (TPs) in natural waters (both surface and groundwater) is required. The high number of compounds potentially reaching the aquatic environment makes extraordinary difficult, if not impossible, to investigate all these compounds even using the most powerful analytical techniques. Among these, liquid chromatography coupled to high-resolution mass spectrometry is emphasized due to its strong potential for detection and identification of many organic contaminants thanks to the accurate-mass full spectrum acquisition data. This work focuses on wide-scope screening of many pesticides and their TPs in surface water and groundwater samples, collected between March and May 2017, in the Júcar River Hydrographical Basin, Spain. For this purpose, a home-made database containing more than 500 pesticides and TPs was employed. Analyses performed by liquid chromatography coupled to quadrupole-time of flight mass spectrometry (LC-QTOF MS) allowed the identification of up to 27 pesticides and 6 TPs. The most detected compounds in groundwater were the herbicides atrazine, simazine, terbuthylazine, and their TPs (atrazine-desethyl, terbumeton-desethyl and terbuthylazine-desethyl). Regarding surface water, the fungicides carbendazim, thiabendazole and imazalil, the herbicide terbutryn and the TP terbumeton-desethyl were also detected. These results illustrate the wide use of these compounds (in the present or in the recent past) in the area under study and the vulnerability of the water bodies, and are in accordance with previous findings in other water bodies of the different Spanish Hydrographic systems.

LC–QQQ and LC–QTOF MS methods for comprehensive detection of potential allergens in various propolis extracts

Propolis is a highly complex matrix, in which phenolics are the mostly analyzed constituents. However, detailed propolis analysis is often lacking due to the high price of the commercially available phenolic standards and the lack of standards of phenols that are causing allergic reaction to propolis. Therefore, it is crucial to develop methods for detection and quantification of these constituents so that, afterwards, propolis extractions can be adjusted to the desired final application. Developed mass spectrometry-based methods were used for the quantification of 25 known phenols as well as for the identification and quantification of 27 additional phenolic derivatives, for which the standards are not readily available. We demonstrated that these newly discovered phenolic derivatives may have a high share in propolis, among which hydroxycinnamic are the most important ones as they are responsible for allergenic properties of propolis-based products. To track the changes in hydroxycinnamic acid derivatives, different propolis extract were prepared and analyzed with developed protocol. All obtained data were processed using chemometric tools to gain insight into allergens content in different propolis extracts. The presented protocol is in line with the food and pharma industry’s growing needs for a comprehensive knowledge on the propolis composition, as it is used in a variety of functional and medicinal products. Knowing that some phenolic components may exert unwanted side effects, a precise selection of a fraction of compounds from propolis is essential for the development of safe-end products.

Biodegradation and detoxification of malachite green by a newly isolated bioluminescent bacterium Photobacterium leiognathi strain MS under RSM optimized culture conditions

Abstract The isolated bioluminescent bacterium was identified as Photobacterium leiognathi strain MS using 16 S rRNA sequence analysis and subjected to malachite green (MG) dye degradation. The Box-Behnken based response surface methodology (RSM) was employed in optimizing medium conditions exhibiting maximum growth at pH 8.0, temperature 30 °C, NaCl 3.5%, and peptone 6.5%. Photobacterium leiognathi strain MS was capable of tolerating high concentration of MG (1.0 gL-1) with 92.50% decolorization potency within 24 h. UV-Vis, FTIR, and LC-MS QTOF analyses confirmed biodegradation of MG into several metabolites as well as its catabolism pathway. A significant increase in the activity of laccase was obtained which revealed its major involvement in the degradation of MG dye. Moreover, phytotoxicity and cytotoxicity analyses illustrated that the metabolites generated were less toxic than the parental compound.

Study of cyanotoxin degradation and evaluation of their transformation products in surface waters by LC-QTOF MS

In the present work, the degradation of three cyanotoxins from the hepatotoxins group was investigated under laboratory-controlled experiments in water samples. Surface waters spiked with microcystin-LR (MC-LR), nodularin (NOD) and cylindrospermopsin (CYN) were subjected to hydrolysis, chlorination and photo-degradation, under both sunlight (SL) and ultraviolet (UV) radiation. A total of 12 transformation products (TPs) were detected and tentatively identified by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF MS). These comprised: 6 chlorination TPs (3 from CYN and 3 from MC-LR, 2 isomers); 4 UV TPs (all from CYN); and 2 sunlight TPs (one isomer from MC-LR and another from NOD). No TPs were observed under hydrolysis conditions. The chemical structures for all TPs were tentatively proposed based on the accurate-mass QTOF MS full-spectra. Analysis of real-world samples collected from the Peñol reservoir (Antioquia, Colombia) revealed the presence of MC-LR and CYN as well as a sunlight TP identified in the laboratory experiments. Data presented in this article will assist further research on TPs potentially formed in future tertiary degradation processes applied for the removal of organic micro-pollutants in water; as well as improving available knowledge on the toxic implications of cyanobacterial toxins TPs in surface waters.

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F-PY-PICA, 5F-PY-PINACA, and their analogs.

5F-PY-PICA and 5F-PY-PINACA are pyrrolidinyl 1-(5-fluoropentyl)ind (az)ole-3-carboxamides identified in 2015 as putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS). 5F-PY-PICA, 5F-PY-PINACA, and analogs featuring variation of the 1-alkyl substituent or contraction, expansion, or scission of the pyrrolidine ring were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS). In competitive binding experiments against HEK293 cells expressing human cannabinoid receptor type 1 (hCB1 ) or type 2 (hCB2 ), all analogs showed minimal affinity for CB1 (pKi <5), although several demonstrated moderate CB2 binding (pKi 5.45-6.99). In fluorescence-based membrane potential assays using AtT20-hCB1 or -hCB2 cells, none of the compounds (at 10μM) produced an effect >50% of the classical cannabinoid agonist CP55,940 (at 1μM) at hCB1 , although several showed slightly higher relative efficacy at hCB2 . Expansion of the pyrrolidine ring of 5F-PY-PICA to an azepane (8) conferred the greatest hCB2 affinity (pKi 6.99) and activity (pEC50 7.54, Emax 72%) within the series. Unlike other SCRA NPS evaluated in vivo using radio biotelemetry, 5F-PY-PICA and 5F-PY-PINACA did not produce cannabimimetic effects (hypothermia, bradycardia) in mice at doses up to 10mg/kg.