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Agrochemicals frequently undergo various chemical and metabolic transformation reactions in the environment that often result in a wide range of derivates that must be comprehensively characterized to understand their toxicity profiles and their persistence and outcome in the environment. In the development phase, this typically involves a major effort in qualitatively identifying the correct chemical isomer(s) of these derivatives from the many isomers that could potentially be formed. Liquid chromatography-mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy are often used in attempts to characterize such environment transformation products. However, challenges in confidently correlating chemical structures to detected compounds in mass spectrometry data and sensitivity/selectivity limitations of NMR frequently lead to bottlenecks in identification. In this study, we use an alternative approach, infrared ion spectroscopy, to demonstrate the identification of hydroxylated derivatives of two plant protection compounds (azoxystrobin and benzovindiflupyr) contained at low levels in tomato and spinach matrices. Infrared ion spectroscopy is an orthogonal tandem mass spectrometry technique that combines the sensitivity and selectivity of mass spectrometry with structural information obtained by infrared spectroscopy. Furthermore, IR spectra can be computationally predicted for candidate molecular structures, enabling the tentative identification of agrochemical derivatives and other unknowns in the environment without using physical reference standards.

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PURPOSE: HPV-associated head and neck squamous cell carcinoma (HPV+HNSCC) is the most common HPV-associated malignancy in the United States and continues to increase in incidence. Current diagnostic approaches for HPV+HNSCC rely on tissue biopsy followed by histomorphologic assessment and detection of HPV indirectly by p16 IHC. Such approaches are invasive and have variable sensitivity. EXPERIMENTAL DESIGN: We conducted a prospective observational study in 140 subjects (70 cases and 70 controls) to test the hypothesis that a noninvasive diagnostic approach for HPV+HNSCC would have improved diagnostic accuracy, lower cost, and shorter diagnostic interval compared with standard approaches. Blood was collected, processed for circulating tumor HPV DNA (ctHPVDNA), and analyzed with custom ddPCR assays for HPV genotypes 16, 18, 33, 35, and 45. Diagnostic performance, cost, and diagnostic interval were calculated for standard clinical workup and compared with a noninvasive approach using ctHPVDNA combined with cross-sectional imaging and physical examination findings. RESULTS: Sensitivity and specificity of ctHPVDNA for detecting HPV+HNSCC were 98.4% and 98.6%, respectively. Sensitivity and specificity of a composite noninvasive diagnostic using ctHPVDNA and imaging/physical examination were 95.1% and 98.6%, respectively. Diagnostic accuracy of this noninvasive approach was significantly higher than standard of care (Youden index 0.937 vs. 0.707, P = 0.0006). Costs of noninvasive diagnostic were 36% to 38% less than standard clinical workup and the median diagnostic interval was 26 days less. CONCLUSIONS: A noninvasive diagnostic approach for HPV+HNSCC demonstrated improved accuracy, reduced cost, and a shorter time to diagnosis compared with standard clinical workup and could be a viable alternative in the future.

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Negative chemical ionization (NCI) and electron-capture negative ionization (ECNI) are gas chromatography-mass spectrometry (GC-MS) techniques that generate negative ions in the gas phase for compounds containing electronegative atoms or functional groups. In ECNI, gas-phase thermal electrons can be transferred to electrophilic substances to produce M-• ions and scarce fragmentation. As a result of the electrophilicity requirements, ECNI is characterized by high-specificity and low background noise, generally lower than EI, offering lower detection limits. The aim of this work is to explore the possibility of extending typical advantages of ECNI to liquid chromatography-mass spectrometry (LC-MS). The LC is combined with the novel liquid-EI (LEI) LC-EIMS interface, the eluent is vaporized and transferred inside a CI source, where it is mixed with methane as a buffer gas. As proof of concept, dicamba and tefluthrin, agrochemicals with herbicidal and insecticidal activity, respectively, were chosen as model compounds and detected together in a commercial formulation. The pesticides have different chemical properties, but both are suitable analytes for ECNI due to the presence of electronegative atoms in the molecules. The influence of the mobile phase and other LC- and MS-operative parameters were methodically evaluated. Part-per-trillion (ppt) detection limits were obtained. Ion abundances were found to be stable with quantitative linear detection, reliable, and reproducible, with no influence from coeluting interfering compounds from the sample matrix.

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Ultraviolet photodissociation (UVPD) has been shown to produce extensive structurally informative data for a variety of chemically diverse compounds. Herein, we demonstrate the performance of the 193 nm UVPD fragmentation technique on structural/moiety characterization of 14 singly charged agrochemicals. Two-dimensional mass spectrometry (2DMS) using infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID) have previously been applied to a select range of singly charged pesticides. The ≥80% moiety coverage achieved for the majority of the species by the UVPD and 2D-UVPD methods was on par with and, in some cases, superior to the data obtained by other fragmentation techniques in previous studies, demonstrating that UVPD is viable for these types of species. A three-dimensional (3D) peak picking method was implemented to extract the data from the 2DMS spectrum, overcoming the limitations of the line extraction method used in previous studies, successfully separating precursor specific fragments with milli-Dalton accuracy. Whole spectrum internal calibration combined with 3D peak picking obtained sub-part-per-million (ppm) to part-per-billion (ppb) mass accuracies across the entire 2DMS spectrum.

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Herein we present an efficient, column-switching method that relies on a custom-made T-union passive diffusion micromixer to assist water dilution and promote trap solute focusing of a high sample volume dissolved in pure organic solvent using a 0.075 mm i.d. nano-LC column. This method allows injecting 20 µL (or higher) of sample volume, speeding up the analysis time, with a 400-fold increase of the limits of quantitation for selected compounds. Five pesticides in different media were used as model compounds, and the analyses were carried out with a triple quadrupole mass spectrometer equipped with a Liquid Electron Ionization (LEI) LC-MS interface working in multiple reaction monitoring (MRM) mode. The system microfluidics were investigated using COMSOL modeling software. Robustness of the entire system was evaluated using a post-extraction addition soil extracts with limits of detection values spanning from 0.10 to 0.45 µg/L. Reproducible results in terms of peak area, peak shape, and retention times were achieved in soil matrix. Repeatability test on peak area variations were lower than 10%.

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Analysis of agrochemicals in an environmental matrix is challenging because these samples contain multiple agrochemicals, their metabolites, degradation products, and endogenous compounds. The analysis of such complex samples is achieved using chromatographic separation techniques coupled to mass spectrometry. Herein, we demonstrate a two-dimensional mass spectrometry (2DMS) technique on a 12 T Fourier transform ion cyclotron resonance mass spectrometer that can analyze a mixture of agrochemicals without using chromatography or quadrupole isolation in a single experiment. The resulting 2DMS contour plot contains abundant tandem MS information for each component in the sample and correlates product ions to their corresponding precursor ions. Two different fragmentation methods are employed, infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID), with 2DMS to analyze the mixture of singly charged agrochemicals. The product ions of one of the agrochemicals, pirimiphos-methyl, present in the sample was used to internally calibrate the entire 2DMS spectrum, achieving sub part per million (ppm) to part per billion (ppb) mass accuracies for all species analyzed. The work described in this study will show the advantages of the 2DMS approach, by grouping species with common fragments/core structure and mutual functional groups, using precursor lines and neutral loss lines. In addition, the rich spectral information obtained from IRMPD and EID 2DMS contour plots can accurately identify and characterize agrochemicals.

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Investigating the structure of active ingredients, such as agrochemicals and their associated metabolites, is a crucial requisite in the discovery and development of these molecules. In this study, structural characterization by electron-induced dissociation (EID) was compared to collisionally activated dissociation (CAD) on a series of agrochemicals. EID fragmentation produced a greater variety of fragment ions and complementary ion pairs leading to more complete functional group characterization compared to CAD. The results obtained displayed many more cross-ring fragmentation of the pyrimidine ring compared to the pyridine ring. Compounds that consisted of one aromatic heterocyclic moiety (azoxystrobin, fluazifop acid, fluazifop-p-butyl, and pirimiphos-methyl) displayed cross-ring fragmentation while compounds with only aromatic hydrocarbon rings (fenpropidin and S-metolachlor) displayed no cross-ring fragmentation. The advantages of high-resolution accurate mass spectrometry (HRAM MS) are shown with the majority of assignments at ppb range error values and the ability to differentiate ions with the same nominal mass but different elemental composition. This highlights the potential for HRAM MS and EID to be used as a tool for structural characterization of small molecules with a wide variety of functional groups and structural motifs.

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Liquid Electron Ionization (LEI), is an innovative liquid chromatography-mass spectrometry (LC-MS) interface that converts liquid HPLC eluent to the gas-phase in a mass spectrometer equipped with an electron ionization (EI) source. LEI extends the electronic spectra libraries access to liquid chromatography, providing a powerful tool in the untargeted approacssh. Negligible matrix effects allow accurate quantitative information. The purpose of this research was to evaluate the main aspects concerning the interfacing process. These fundamental studies were necessary to understand the mechanism of LEI in details, and improve the interfacing process, especially regarding robustness and sensitivity. Hardware components were installed to prevent analytes precipitation, reduce thermal decomposition of sensitive compounds, and to stabilize the nano-flow delivery with different mobile-phase compositions. Particular attention was devoted to insulating the heated vaporization area from the LC part of the system. Experiments were performed to optimize the interface inner capillary dimensions, and other operative parameters, including temperature, gas and liquid flow rates. Test compounds of environmental interest were selected based on molecular weight, thermal stability, volatility, and polarity. Robustness was evaluated with a set of replicated injections and calibration experiments using a soil matrix as a test sample. MRM detection limits in the low-picogram range were obtained for five pesticides belonging to different classes in a soil sample. High-quality electron ionization mass spectra of a mixture of pesticides were also obtained.

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Lignocellulosic waste (LW) is abundant in availability and is one of the suitable substrates for anaerobic digestion (AD). However, it is a complex solid substrate matrix that hinders the hydrolysis stage of anaerobic digestion. This study assessed various pre-treatment and post-treatments of lignocellulosic waste for anaerobic digestion benefiting from advanced P-graph and GaBi software (Thinkstep, Germany) from the perspective of cost and environmental performances (global warming potential, human toxicity, ozone depletion potential, particulate matter, photochemical oxidant creation, acidification and eutrophication potential). CaO pre-treatment (P4), H2S removal with membrane separation post-treatment (HSR MS) and without the composting of digestate is identified as the cost-optimal pathway. The biological (P7- Enzyme, P8- Microbial Consortium) and physical (P1- Grinding, P2- Steam Explosion, P3- Water Vapour) pre-treatments alternatives have lower environmental impacts than chemical pre-treatments (P4- CaO, P5- NaOH, P6- H2SO4) however they are not part of the near cost optimal solutions. For post-treatment, the near cost optimal alternatives are H2S removal with organic physical scrubbing (HSR OPS) and H2S removal with amine scrubbing (HSR AS). HSR AS has a better performance in the overall environmental impacts followed by HSR MS and HSR OPS. In general, the suggested cost-optimal solution is still having relatively lower environmental impacts and feasible for implementation (cost effective). There is very complicated to find a universal AD solution. Different scenarios (the type of substrate, the scale, product demand, policies) have different constraints and consequently solutions. The trade-offs between cost and environment performances should be a future extension of this work.

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We investigated the metabolic capabilities of C. elegans using compounds whose metabolism has been well characterised in mammalian systems. We find that similar metabolites are produced in C. elegans as in mammals but that C. elegans is deficient in CYP1-like metabolism, as has been seen in other studies. We show that CYP-34A9, CYP-34A10 and CYP-36A1 are the principal enzymes responsible for the metabolism of tolbutamide in C. elegans. These are related to the mammalian enzymes that metabolise this compound but are not the closest homologs suggesting that sequence comparison alone will not predict functional conservation among cytochrome P450s. In mammals, metabolite production from amytryptiline and dextromethorphan is dependent on specific cytochrome P450s. However, in C. elegans we did not find evidence of similar specificity: the same metabolites were produced but in small amounts by numerous cytochrome P450s. We conclude that, while some aspects of cytochrome P450 mediated metabolism in C. elegans are similar to mammals, there are differences in the production of some metabolites and in the underlying genetics of metabolism.

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Anaerobic digestion (AD) serves as a promising alternative for waste treatment and a potential solution to improve the energy supply security. The feasibility of AD has been proven in some of the technologically and agriculturally advanced countries. However, development is still needed for worldwide implementation, especially for AD process dealing with municipal solid waste (MSW). This paper reviews various approaches and stages in the AD of MSW, which used to optimise the biogas production and quality. The assessed stages include pre-treatment, digestion process, post-treatment as well as the waste collection and transportation. The latest approaches and integrated system to improve the AD process are also presented. The stages were assessed in a relatively quantitative manner. The range of energy requirement, carbon emission footprint and the percentage of enhancement are summarised. Thermal hydrolysis pre-treatment is identified to be less suitable for MSW (-5% to +15.4% enhancement), unless conducted in the two-phase AD system. Microwave pre-treatment shows consistent performance in elevating the biogas production of MSW, but the energy consumption (114.24-8,040 kWeh t-1) and carbon emission footprint (59.93-4,217.78 kg CO2 t-1 waste) are relatively high. Chemical (∼0.43 kWeh m-3) and membrane-based (∼0.45 kWeh m-3) post-treatments are suggested to be a lower energy consumption approach for upgrading the biogas. The feasibility in terms of cost (scale up) and other environmental impacts (non-CO2 footprint) needs to be further assessed. This study provides an overview to facilitate further development and extended implementation of AD.

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To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.

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RATIONALE: This paper describes a strategy for the profiling and identification of metabolites based on chemical group classification using high-resolution accurate mass (HR/AM) full scan mass spectrometry (MS) and All-Ion fragmentation (AIF) MS2 data. METHODS: The proposed strategy uses a hybrid quadrupole Orbitrap (Q-Exactive) employing stepped normalised collision energy (NCE) at 35% and 80% to produce key chemically diagnostic product ions from full coverage of the product ion spectrum. This approach allows filtering of high-resolution AIF MS2 data in order to identify parent-related compounds produced following incubation in rat liver microsomes (RLMs). RESULTS: An antidepressant drug, nefazodone (NEF), was selected as the model test compound to demonstrate the proposed workflow for metabolite profiling. This resulted in the identification of three indicative chemical groups within NEF: triazolone, phenoxy and chlorophenylpiperazine. High-resolution mass spectrometry provides increased specificity to distinguish between two characteristic product ion masses m/z 154.0975 (C7 H12 N3 O) and 154.0419 (C8 H9 NCl), which are not fully resolved by spectrometers operating at nominal mass resolution, indicative of compounds containing the triazolone and chlorophenylpiperazine moieties, respectively. CONCLUSIONS: This post-acquisition processing strategy provides comprehensive detection and identification of high- and low-level metabolites from an 'all-in-one' analysis. This enables functional groups to be systematically traced across a wide range of metabolites, leading to the successful identification of 28 in vitro NEF-related metabolites. In our hands this approach has been applied to agrochemical environmental fate and dietary metabolism studies, as well as metabolomics and biomarker analysis. Copyright © 2015 John Wiley & Sons, Ltd.

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The tripeptide H-Val-Ala-Leu-OH and the N-Ac-tetrapeptide amide Ac-Thr-Lys-Trp-Phe-NH2, and their beta-peptidic counterparts H-beta(3)hVal-beta(3)hAla-beta(3)hLeu-OH and Ac-beta(3)hThr-(S)beta(2)hLys-beta(3)hTrp-beta(3)hPhe-NH2, respectively, have been injected into Heliothis virescens larvae and added to cell cultures of black mexican sweet maize. The body liquids of the larvae and the supernatant of the plant cell cultures were sampled 0, 2, 3, 6, 17, and/or 24 h after application and analyzed by LC/MS. While the two alpha-peptides were degraded rapidly in these environments, the concentration of the beta-peptides was found to decrease very slowly. Thus, ca. 60% of the original amount of the beta-tetrapeptide was detected in the liquids of the insect after 24 h. The plant cells did not seem to make use of the beta-peptides at all, whereas, the alpha-tripeptide completely disappeared from the supernatant after 3 h. Thus, we have demonstrated, for the first time, the high stability of beta-peptides against degradation and metabolism in an insect and a plant. Especially remarkable is the persistence of the beta-tetrapeptide with its functionalised and, thus, 'metabolisable' side chains of Thr, Lys, Trp, and Phe in the insect larvae, which are known to have a high level of activity of oxidizing enzymes. The results described here match those of ADME investigations with radioactively labeled beta-peptides in rats, where essentially complete stability has been observed, while environmental microorganisms have been found to biodegrade beta-peptides, albeit slowly. Possible implications of these findings for biomedical and pest-control applications are proposed.

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