Metabolomic analysis of the effects of cadmium and copper treatment in Oryza sativa L. using untargeted liquid chromatography coupled to high resolution mass spectrometry and all-ion fragmentation.

While the knowledge of plant metabolomes has increased in the last few years, their response to the presence of toxicants is still poorly understood. Here, we analyse the metabolomic changes in Japanese rice (Oryza sativa var. Japonica) upon exposure to heavy metals (Cd(ii) and Cu(ii)) in concentrations from 10 to 1000 µM. After harvesting, rice metabolites were extracted from aerial parts of the plants and analysed by HPLC (HILIC TSK gel amide-80 column) coupled to a mass spectrometer quadrupole-Orbitrap (Q-Exactive). Full scan and all ion fragmentation (AIF) mass spectrometry modes were used during the analysis. The proposed untargeted metabolomics data analysis strategy is based on the application of the multivariate curve resolution alternating least squares (MCR-ALS) method for feature detection, allowing the simultaneous resolution of pure chromatographic profiles and mass spectra of all metabolites present in the analysed rice extracts. All-ion fragmentation data were used to confirm the identification of MCR-ALS resolved metabolites. A total of 112 metabolites were detected, and 97 of them were subsequently identified and confirmed. Pathway analysis of the observed metabolic changes suggested an underlying similarity of the responses of the plant to Cd(ii) and Cu(ii), although the former treatment appeared to be the more severe of the two. In both cases, secondary metabolism and amino acid-, purine-, carbon- and glycerolipid-metabolism pathways were affected, in a pattern consistent with reduction in plant growth and/or photosynthetic capacity and with induction of defence mechanisms to reduce cell damage.

Furan in commercial baby foods from the Spanish market: estimation of daily intake and risk assessment.

The occurrence of furan in commercial baby food samples from the Spanish market was evaluated using an automated headspace solid-phase microextraction method coupled to gas chromatography-mass spectrometry (HS-SPME-GC-MS). A total of 76 baby food samples including infant formula, baby cereals, fruit in cans and/or jars, vegetables, meat, and fish, were surveyed for furan content. The lowest concentration of this compound was found in infant formula (<0.02-0.33 ng ml-1), and cereal-based food (0.15-2.1 ng g-1) while baby food containing fish showed the highest concentrations (19-84 ng g-1). Following recommendation of the European Food Safety Authority (EFSA), the effect on furan content was evaluated of consumer home preparation of foods, heating and handling. Furan concentrations were reduced by up to 35% when samples were heated in a dish using microwave oven and by up to 53% when a hot water bath was used. Finally, we estimated the furan intake from baby food consumption (0.002-1.18 µg kg-1 body weight day-1) and we calculated the margin of exposure (MOE) from samples as purchased and also after home preparation of the food. For infant formula and cereal baby foods, the MOEs (26,278-412,776) indicated no infant health concern or priority, while for meat and fish-based baby foods the values pointed to a potential public health risk, even considering the furan losses during preparation at home.

Transcriptomic and metabolomic profiling of ionic liquid stimuli unveils enhanced secondary metabolism in Aspergillus nidulans.

BACKGROUND: The inherent potential of filamentous fungi, especially of Ascomycota, for producing diverse bioactive metabolites remains largely silent under standard laboratory culture conditions. Innumerable strategies have been described to trigger their production, one of the simplest being manipulation of the growth media composition. Supplementing media with ionic liquids surprisingly enhanced the diversity of extracellular metabolites generated by penicillia. This finding led us to evaluate the impact of ionic liquids' stimuli on the fungal metabolism in Aspergillus nidulans and how it reflects on the biosynthesis of secondary metabolites (SMs). RESULTS: Whole transcriptional profiling showed that exposure to 0.7 M cholinium chloride or 1-ethyl-3-methylimidazolium chloride dramatically affected expression of genes encoding both primary and secondary metabolism. Both ionic liquids apparently induced stress responses and detoxification mechanisms but response profiles to each stimulus were unique. Primary metabolism was up-regulated by choline, but down-regulated by 1-ethyl-3-methylimidazolium chloride; both stimulated production of acetyl-CoA (key precursor to numerous SMs) and non proteinogenic amino acids (building blocks of bioactive classes of SMs). In total, twenty one of the sixty six described backbone genes underwent up-regulation. Accordingly, differential analysis of the fungal metabolome showed that supplementing growth media with ionic liquids resulted in ca. 40 differentially accumulated ion masses compared to control conditions. In particular, it stimulated production of monodictyphenone and orsellinic acid, otherwise cryptic. Expression levels of genes encoding corresponding polyketide biosynthetic enzymes (i.e. backbone genes) increased compared to control conditions. The corresponding metabolite extracts showed increased cell polarity modulation potential in an ex vivo whole tissue assay (The lial Live Targeted Epithelia; theLiTE™). CONCLUSIONS: Ionic liquids, a diverse class of chemicals composed solely of ions, can provide an unexpected means to further resolve the diversity of natural compounds, guiding discovery of fungal metabolites with clinical potential.

Ion-molecule adduct formation in tandem mass spectrometry.

Nowadays most LC-MS methods rely on tandem mass spectrometry not only for quantitation and confirmation of compounds by multiple reaction monitoring (MRM), but also for the identification of unknowns from their product ion spectra. However, gas-phase reactions between charged and neutral species inside the mass analyzer can occur, yielding product ions at m/z values higher than that of the precursor ion, or at m/z values difficult to explain by logical losses, which complicate mass spectral interpretation. In this work, the formation of adduct ions in the mass analyzer was studied using several mass spectrometers with different mass analyzers (ion trap, triple quadrupole, and quadrupole-Orbitrap). Heterocyclic amines (AαC, MeAαC, Trp-P-1, and Trp-P-2), photo-initiators (BP and THBP), and pharmaceuticals (phenacetin and levamisole) were selected as model compounds and infused in LCQ Classic, TSQ Quantum Ultra AM, and Q-Exactive Orbitrap (ThermoFisher Scientific) mass spectrometers using electrospray as ionization method. The generation of ion-molecule adducts depended on the compound and also on the instrument employed. Adducts with neutral organic solvents (methanol and acetonitrile) were only observed in the ion trap instrument (LCQ Classic), because of the ionization source on-axis configuration and the lack of gas-phase barriers, which allowed inertial entrance of the neutrals into the analyzer. Adduct formation (only with water) in the triple quadrupole instruments was less abundant than in the ion trap and quadrupole-Orbitrap mass spectrometers, because of the lower residence time of the reactive product ions in the mass analyzer. The moisture level of the CID and/or damper gas had a great effect in beam-like mass analyzers such as triple quadrupole, but not in trap-like mass analyzers, probably because of the long residence time that allowed adduct formation even with very low concentrations of water inside the mass spectrometer.

Aggregation behavior of fullerenes in aqueous solutions: a capillary electrophoresis and asymmetric flow field-flow fractionation study.

In this work, the electrophoretic behavior of hydrophobic fullerenes [buckminsterfullerene (C60), C70, and N-methyl-fulleropyrrolidine (C60-pyrr)] and water-soluble fullerenes [fullerol (C60(OH)24); polyhydroxy small gap fullerene, hydrated (C120(OH)30); C60 pyrrolidine tris acid (C60-pyrr tris acid); and (1,2-methanofullerene C60)-61-carboxylic acid (C60CHCOOH)] in micellar electrokinetic capillary chromatography (MECC) was evaluated. The aggregation behavior of the water-soluble compounds in MECC at different buffer and sodium dodecyl sulfate (SDS) concentrations and pH values of the background electrolyte (BGE) was studied by monitoring the changes observed in the electrophoretic pattern of the peaks. Broad and distorted peaks that can be attributed to fullerene aggregation were obtained in MECC which became narrower and more symmetric by working at low buffer and SDS concentrations (below the critical micelle concentration, capillary zone electrophoresis (CZE) conditions). For the characterization of the suspected aggregates formed (size and shape), asymmetrical flow field-flow fractionation (AF4) and transmission electron microscopy (TEM) were used. The results showed that the increase in the buffer concentration promoted the aggregation of the particles, while the presence of SDS micelles revealed multiple peaks corresponding to particles of different aggregation degrees. Furthermore, MECC has been applied for the first time for the analysis of C60 in two different cosmetic products (i.e., anti-aging serum and facial mask).

Characterization of aggregates of surface modified fullerenes by asymmetrical flow field-flow fractionation with multi-angle light scattering detection.

Fullerenes are carbon nanoparticles with widespread biomedical, commercial and industrial applications. Attributes such as their tendency to aggregate and aggregate size and shape impact their ability to be transported into and through the environment and living tissues. Knowledge of these properties is therefore valuable for their human and environmental risk assessment as well as to control their synthesis and manufacture. In this work, asymmetrical flow-field flow fractionation (AF4) coupled to multi-angle light scattering (MALS) was used for the first time to study the size distribution of surface modified fullerenes with both polyhydroxyl and carboxyl functional groups in aqueous solutions having different pH (6.5-11) and ionic strength values (0-200mM) of environmental relevance. Fractionation key parameters such as flow rates, flow programming, and membrane material were optimized for the selected fullerenes. The aggregation of the compounds studied appeared to be indifferent to changes in solution pH, but was affected by changes in the ionic strength. Polyhydroxy-fullerenes were found to be present mostly as 4nm aggregates in water without added salt, but showed more aggregation at high ionic strength, with an up to 10-fold increase in their mean hydrodynamic radii (200mM), due to a decrease in the electrostatic repulsion between the nanoparticles. Carboxy-fullerenes showed a much stronger aggregation degree in water (50-100nm). Their average size and recoveries decreased with the increase in the salt concentration. This behavior can be due to enhanced adsorption of the large particles to the membrane at high ionic strength, because of their higher hydrophobicity and much larger particle sizes compared to polyhydroxy-fullerenes. The method performance was evaluated by calculating the run-to-run precision of the retention time (hydrodynamic radii), and the obtained RSD values were lower than 1%. MALS measurements showed aggregate sizes that were in good agreement with the AF4 data. A comparison of the scattering radii from the MALS with the hydrodynamic radii obtained from the retention times in AF4 indicated that the aggregate shapes are far from spherical. TEM images of the fullerenes in the dry state also showed branched and irregular clusters.

Characterisation and determination of fullerenes: A critical review.

A prominent sector of nanotechnology is occupied by a class of carbon-based nanoparticles known as fullerenes. Fullerene particle size and shape impact in how easily these particles are transported into and throughout the environment and living tissues. Currently, there is a lack of adequate methodology for their size and shape characterisation, identification and quantitative detection in environmental and biological samples. The most commonly used methods for their size measurements (aggregation, size distribution, shape, etc.), the effect of sampling and sample treatment on these characteristics and the analytical methods proposed for their determination in complex matrices are discussed in this review. For the characterisation and analysis of fullerenes in real samples, different analytical techniques including microscopy, spectroscopy, flow field-flow fractionation, electrophoresis, light scattering, liquid chromatography and mass spectrometry have been reported. The existing limitations and knowledge gaps in the use of these techniques are discussed and the necessity to hyphenate complementary ones for the accurate characterisation, identification and quantitation of these nanoparticles is highlighted.

Wide-range screening of psychoactive substances by FIA-HRMS: identification strategies.

Recreational drugs (illicit drugs, human and veterinary medicines, legal highs, etc.) often contain lacing agents and adulterants which are not related to the main active ingredient. Serious side effects and even the death of the consumer have been related to the consumption of mixtures of psychoactive substances and/or adulterants, so it is important to know the actual composition of recreational drugs. In this work, a method based on flow injection analysis (FIA) coupled with high-resolution mass spectrometry (HRMS) is proposed for the fast identification of psychoactive substances in recreational drugs and legal highs. The FIA and HRMS working conditions were optimized in order to detect a wide range of psychoactive compounds. As most of the psychoactive substances are acid-base compounds, methanol-0.1 % aqueous formic acid (1:1 v/v) as a carrier solvent and electrospray in both positive ion mode and negative ion mode were used. Two data acquisition modes, full scan at high mass resolution (HRMS) and data-dependent tandem mass spectrometry (ddMS/HRMS) with a quadrupole-Orbitrap mass analyzer were used, resulting in sufficient selectivity for identification of the components of the samples. A custom-made database containing over 450 substances, including psychoactive compounds and common adulterants, was built to perform a high-throughput target and suspect screening. Moreover, online accurate mass databases and mass fragmenter software were used to identify unknowns. Some examples, selected among the analyzed samples of recreational drugs and legal highs using the FIA-HRMS(ddMS/HRMS) method developed, are discussed to illustrate the screening strategy used in this study. The results showed that many of the analyzed samples were adulterated, and in some cases the sample composition did not match that of the supposed marketed substance.

Analysis of C60-fullerene derivatives and pristine fullerenes in environmental samples by ultrahigh performance liquid chromatography-atmospheric pressure photoionization-mass spectrometry.

In this work, a method is proposed for the simultaneous analysis of several pristine fullerenes (C60, C70, C76, C78, and C84) and three C60-fullerene derivatives (N-methyl fulleropyrrolidine, [6,6]-phenyl C61 butyric acid methyl ester and [6,6]-phenyl C61 butyric acid butyl ester) in environmental samples. The method involves the use of ultrahigh performance liquid chromatography coupled to atmospheric pressure photoionization mass spectrometry (UHPLC-APPI-MS) and allowed the chromatographic separation in less than 4.5min. The product ions from tandem mass spectrometry studies of fullerene derivatives were characterized and the most abundant one (m/z 720), corresponding to [C60](-), was selected for quantitation. Selected reaction monitoring (SRM at 0.7m/z FWHM) by acquiring two transitions using both isotopic cluster ions [M](-) and [M+1](-) as precursor ions was proposed for quantitation and confirmation purposes. For pristine fullerenes, highly selective selected ion monitoring (H-SIM) acquisition mode by monitoring the isotopic cluster ions [M](-) and [M+1](-) was used. Pressurized solvent extraction conditions were optimized in order to improve recoveries of the studied fullerene compounds from sediment samples. Values up to 87-92% for C60-fullerene derivatives and lower but still acceptable, 70-80%, for pristine fullerenes were obtained. Method limits of quantitation (MLOQs) ranging from 1.5pgL(-1) to 5.5ngL(-1) in water samples and from 0.1ngkg(-1) to 523ngkg(-1) in sediments were obtained with good precision (relative standard deviations always lower than 13%). The applicability of the developed method was evaluated by analyzing several environmental samples such as sediments and pond water and the detected levels for C60-fullerene derivatives were of 0.1-2.7ngkg(-1) and 1.5-8.5pgL(-1), respectively. C60 and C70 were the only pristine fullerenes detected in the analyzed samples (0.1-7.2ngkg(-1) in sediments and 9-330pgL(-1) in water pond samples).

New branches in the degradation pathway of monochlorocatechols by Aspergillus nidulans: a metabolomics analysis.

A collective view of the degradation of monochlorocatechols in fungi is yet to be attained, though these compounds are recognised as key degradation intermediates of numerous chlorinated aromatic hydrocarbons, including monochlorophenols. In the present contribution we have analysed the degradation pathways of monochlorophenols in Aspergillus nidulans using essentially metabolomics. Degradation intermediates herein identified included those commonly reported (e.g. 3-chloro-cis,cis-muconate) but also compounds never reported before in fungi revealing for 4-chlorocatechol and for 3-chlorocatechol unknown degradation paths yielding 3-chlorodienelactone and catechol, respectively. A different 3-chlorocatechol degradation path led to accumulation of 2-chloromuconates (a potential dead-end), notwithstanding preliminary evidence of chloromuconolactones and protoanemonin simultaneous formation. In addition, some transformation intermediates, of which sulfate conjugates of mono-chlorophenols/chlorocatechols were the most common, were also identified. This study provides critical information for understanding the role of fungi in the degradation of chlorinated aromatic hydrocarbons; furthering their utility in the development of innovative bioremediation strategies.

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls.

Cork, the outer bark of Quercus suber, shows a unique compositional structure, a set of remarkable properties, including high recalcitrance. Cork colonisation by Ascomycota remains largely overlooked. Herein, Aspergillus nidulans secretome on cork was analysed (2DE). Proteomic data were further complemented by microscopic (SEM) and spectroscopic (ATR-FTIR) evaluation of the colonised substrate and by targeted analysis of lignin degradation compounds (UPLC-HRMS). Data showed that the fungus formed an intricate network of hyphae around the cork cell walls, which enabled polysaccharides and lignin superficial degradation, but probably not of suberin. The degradation of polysaccharides was suggested by the identification of few polysaccharide degrading enzymes (ß-glucosidases and endo-1,5-α-l-arabinosidase). Lignin degradation, which likely evolved throughout a Fenton-like mechanism relying on the activity of alcohol oxidases, was supported by the identification of small aromatic compounds (e.g. cinnamic acid and veratrylaldehyde) and of several putative high molecular weight lignin degradation products. In addition, cork recalcitrance was corroborated by the identification of several protein species which are associated with autolysis. Finally, stringent comparative proteomics revealed that A. nidulans colonisation of cork and wood share a common set of enzymatic mechanisms. However the higher polysaccharide accessibility in cork might explain the increase of ß-glucosidase in cork secretome. BIOLOGICAL SIGNIFICANCE: Cork degradation by fungi remains largely overlook. Herein we aimed at understanding how A. nidulans colonise cork cell walls and how this relates to wood colonisation. To address this, the protein species consistently present in the secretome were analysed, as well as major alterations occurring in the substrate, including lignin degradation compounds being released. The obtained data demonstrate that this fungus has superficially attacked the cork cell walls apparently by using both enzymatic and Fenton-like reactions. Only a few polysaccharide degrading enzymes could be detected in the secretome which was dominated by protein species associated with autolysis. Lignin degradation was corroborated by the identification of some degradation products, but the suberin barrier in the cell wall remained virtually intact. Comparative proteomics revealed that cork and wood colonisation share a common set of enzymatic mechanisms.

Ultra-high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry for the analysis of benzimidazole compounds in milk samples.

In this work the atmospheric pressure ionization behavior and the tandem mass spectrometry fragmentation of 19 benzimidazoles was studied. Several atmospheric pressure ionization (API) sources (ESI, APCI and APPI) were evaluated showing that APCI was the best option, mainly for KETO-TCB that provided a 20 times improvement versus ESI. From the studies of tandem mass spectrometry, fragmentation pathways were proposed thus characterizing the most abundant and interesting product ions and selecting the most abundant and selective ones for the confirmatory quantitative method performed in SRM mode in a triple quadrupole mass analyzer. For quantitative analysis, the 19 benzimidazoles were separated in less than 7 min using a C18 column packed with superficially porous particles providing high efficiency within the range of UHPLC. A QuEChERS-like sample treatment was applied to milk samples before the UHPLC-APCI-MS/MS determination providing method limits of quantitation that ranged from 0.6 to 3 µg kg(-1) and showing good repeatability (RSD: 2-18%) and accuracy (relative errors: 1-23%) suitable for using this method for routine analysis.

Direct analysis in real time high-resolution mass spectrometry for high-throughput analysis of antiparasitic veterinary drugs in feed and food.

RATIONALE: Direct analysis in real time (DART) is a novel ionization technique that has been demonstrated in numerous applications as a useful tool for fast and convenient mass spectrometry (MS)-based analysis of complex samples. In this study, the feasibility of DART ionization coupled to a high-resolution mass spectrometer utilizing an orbitrap mass analyzer (orbitrap MS) for high-throughput analysis of antiparasitic veterinary drugs was explored. METHODS: To obtain the best DART-orbitrap MS performance, stepwise optimization of instrumental parameter settings, such as ionization gas temperature and mass resolving power, was performed. The optimized method was applied to feed and bovine milk samples previously extracted following a QuEChERS-like strategy. RESULTS: Most antiparasitic drugs could be analyzed following the described method. Positive DART ionization provided the protonated molecules [M+H](+); in negative DART ion mode, deprotonated molecules [M-H](-) were observed. As an exception, polyether ionophores could be observed as the sodiated adducts [M+Na](+). Samples of milk and feed were extracted using a modified QuEChERS method for the determination of benzimidazoles and coccidiostats respectively and quantification was carried out by matrix-matched calibration curves. CONCLUSIONS: The combination of an analysis time of less than 1 min per sample and the possibility to acquire accurate masses under high mass resolving power (HR) makes the DART-HRMS technique an effective tool for rapid qualitative screening of antiparasitic veterinary drugs. Additionally, the results obtained in this study demonstrated the feasibility of this approach to quantify target analytes at levels down to 1 µg kg(-1) for benzimidazolic compounds in milk and 0.25 mg kg(-1) for coccidiostats in chicken feed.

Biomonitoring of dietary heterocyclic amines and metabolites in urine by liquid phase microextraction: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a possible biomarker of exposure to dietary PhIP.

Heterocyclic amines (HCAs) are mutagenic/carcinogenic compounds formed at ng/g levels during frying meat or fish. The effect of the normal intake of dietary HCAs in humans and their involvement in the etiology of cancer are currently unknown. In this work, a new extraction method, liquid phase microextraction (LPME) with hollow fibers, and LC-MS/MS have been used for the first time to determine HCAs and metabolites in nonspiked human urine following a single meal of chicken cooked at 180 °C for 6 min. The total intake of HCAs was estimated to be 6 µg, of which 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) accounted for about 1 µg. The concentrations of PhIP in nonhydrolyzed urine samples ranged from 11.7 to 59.4 pg/g. The total amount of PhIP in urine ranged between 9.3 and 21.1 ng, which corresponds to 0.91-2.1% of the ingested PhIP. In addition, the urine levels of 4'-OH-PhIP (2-amino-1-methyl-6-(4'-hydroxy)phenylimidazo[4,5-b]pyridine) and 5-OH-PhIP (2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine) also showed a narrow variation between the samples. The analysis of urine samples after acid hydrolysis did not give additional information but showed a notable increase in norharman in some cases. The obtained results suggest PhIP in urine as a possible biomarker of exposure to HCAs and the LPME and LC-MS/MS method as an appropriate strategy to biomonitor HCAs in urine.

Strategies for the multi-residue analysis of 100 pesticides by liquid chromatography-triple quadrupole mass spectrometry.

Analytical strategies for screening, quantitation and confirmation of a group of 100 pesticides in fruit and vegetable samples by LC-MS and LC-MS/MS were developed. The pesticides studied belong to different chemical families of herbicides, insecticides and fungicides. A selection of some degradation products was also included. Chromatographic separation was performed using a Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm and 5 µm particle size), and gradient elution with acetonitrile-water (both with 0.1% formic acid) as mobile phase. LC-MS/MS using highly-selective selected reaction monitoring (H-SRM) acquisition mode monitoring two transitions for each compound showed to be the most sensitive methodology. Quantitation was carried out using matrix-matched standard calibration and good linearity of response was demonstrated (r>0.998). Limits of detection (by acquiring two transitions and with the ion-ratio requirements) ranged between 0.01 and 20 µg/kg were obtained. So, in general, the sensitivity achieved meets the maximum residue levels (MRLs) established by the European Union regulation for food monitoring programs. Pesticide confirmation was carried out following European Union guidelines. In order to prevent false-positives, further confirmatory strategies were proposed. LC-MS in highly-selective selected ion monitoring (H-SIM) mode with accurate mass measurement was used to obtain an orthogonal criterion (exact mass) for confirmation. Accurate mass measurements were always bellow 0.9 mDa for almost all pesticides studied (similar to those described with TOF instruments). A user reversed energy ramp (RER) product ion scan spectra library was generated by means of a data dependent analysis for routine library searching of pesticides. The combination of LC-MS/MS in H-SRM mode and the generation of the RER product ion scan spectra and library search were then used to achieve further confirmation on pesticide analysis. The LC-MS and LC-MS/MS strategies developed were successfully applied for the analysis and confirmation of pesticides in different types of fruit and vegetables samples, and examples of the screening, quantitation and confirmation of pesticides in these samples are shown in this work.

Atmospheric pressure photoionization mass spectrometry of fullerenes.

Atmospheric pressure photoionization (APPI) was evaluated for the analysis of fullerenes. An important response improvement was found when using toluene mediated APPI in negative mode if compared with other atmospheric pressure ionization (API) sources (electrospray and atmospheric pressure chemical ionization). Fullerene APPI negative mass spectra were dominated by the isotopic cluster of the molecular ion, although isotopic patterns for M+1, M+2, and M+3 ions showed higher than expected relative abundances. These discrepancies are explained by the presence of two isobaric ions, one due to (13)C and the other due to the addition of hydrogen to a double bond of the fullerene structure. Triple quadrupole tandem mass spectrometry, ultrahigh resolution mass spectrometry, and accurate mass measurements were used to confirm these assignments. Additionally, cluster ions M+16 and M+32 were characterized following the same strategy. Ions due to the addition of oxygen and alkyl additions were attributed to the presence of methanol in the mobile phase. For the fast chromatographic separation of fullerenes (less than 3.5 min), a sub-2 µm C18 column and isocratic elution (toluene/methanol, 45:55 v/v) was used. Highly selective-selected ion monitoring (H-SIM) mode (mass resolving power, >12,500 fwhm) was proposed monitoring the two most intense isotope ions in the [M](-•) cluster. Method limits of quantitation down to 10 pg L(-1) for C(60) and C(70) fullerenes and between 0.75 and 5.0 ng L(-1) for larger fullerenes were obtained. Finally, the ultrahigh performance liquid chromatography (UHPLC)-APPI-MS method was used to analyze fullerenes in river and pond water samples.

New chlorinated amphetamine-type-stimulants disinfection-by-products formed during drinking water treatment.

Previous studies have demonstrated high removal rates of amphetamine-type-stimulants (ATSs) through conventional drinking water treatments; however the behaviour of these compounds through disinfection steps and their transformation into disinfection-by-products (DBPs) is still unknown. In this work, for the first time, the reactivity of some ATSs such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) with chlorine has been investigated under simulated and real drinking water treatment conditions in order to evaluate their ability to give rise to transformation products. Two new DBPs from these illicit drugs have been found. A common chlorinated-by-product (3-chlorobenzo)-1,3-dioxole, was identified for both MDA and MDEA while for MDMA, 3-chlorocatechol was found. The presence of these DBPs in water samples collected through drinking water treatment was studied in order to evaluate their formation under real conditions. Both compounds were generated through treatment from raw river water samples containing ATSs at concentration levels ranging from 1 to 15 ng/L for MDA and from 2.3 to 78 ng/L for MDMA. One of them, (3-chlorobenzo)-1,3-dioxole, found after the first chlorination step, was eliminated after ozone and GAC treatment while the MDMA DBP mainly generated after the postchlorination step, showed to be recalcitrant and it was found in final treated waters at concentrations ranging from 0.5 to 5.8 ng/L.

Degradation pathway of pentachlorophenol by Mucor plumbeus involves phase II conjugation and oxidation-reduction reactions.

Environmental pollution by pentachlorophenol (PCP) is a critical concern worldwide and fungal bioremediation constitutes an elegant and environment-friendly solution. Mucorales from the Zygomycota phylum are often observed to be competitive in field conditions and Mucor plumbeus, in particular, can efficiently deplete PCP from media. The pathway for PCP degradation used by this fungus has not been investigated. In this study, PCP-derived metabolites were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, including tetra- and tri-chlorohydroquinones and phase II-conjugated metabolites. Amongst the latter are the previously reported glucose, sulfate and ribose conjugates, and identified for the first time in fungi sulfate-glucose conjugates. A PCP transformation pathway for M. plumbeus is proposed, which excludes the involvement of cytochrome P-450 and extracellular ligninolytic enzymes.

Analysis of UV ink photoinitiators in packaged food by fast liquid chromatography at sub-ambient temperature coupled to tandem mass spectrometry.

A fast method of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed for the analysis of eleven UV ink photoinitiators in packaged food. Chromatographic separation was achieved in a pentafluorophenylpropyl (PFPP) column at 5°C and acetonitrile:25 mM formic acid-ammonium formate (pH 2.7) in gradient elution. To reduce sample treatment, a QuEChERS (quick, easy, cheap, effective, rugged and safe) method for the extraction and clean-up of UV photoinitiators in packaged foods was evaluated. Triple quadrupole working in H-SRM on Q1 mode was used for both quantitation and confirmation purposes and the most intense and selective transitions were chosen. Quality parameters of the developed QuEChERS LC-MS/MS method were established and applied for the analysis of photoinitiators in food packaged at ng kg(-1) levels.

Occurrence and removal of pharmaceuticals and hormones through drinking water treatment.

The occurrence of fifty-five pharmaceuticals, hormones and metabolites in raw waters used for drinking water production and their removal through a drinking water treatment were studied. Thirty-five out of fifty-five drugs were detected in the raw water at the facility intake with concentrations up to 1200 ng/L. The behavior of the compounds was studied at each step: prechlorination, coagulation, sand filtration, ozonation, granular activated carbon filtration and post-chlorination; showing that the complete treatment accounted for the complete removal of all the compounds detected in raw waters except for five of them. Phenytoin, atenolol and hydrochlorothiazide were the three pharmaceuticals most frequently found in finished waters at concentrations about 10 ng/L. Sotalol and carbamazepine epoxide were found in less than a half of the samples at lower concentrations, above 2 ng/L. However despite their persistence, the removals of these five pharmaceuticals were higher than 95%.