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.

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.

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).

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.

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.

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.

Field-amplified sample injection-micellar electrokinetic capillary chromatography for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers and derivatives in canned soft drinks.

Conditions were established for the separation and analysis of bisphenol A, bisphenol F, and their diglycidyl ethers by micellar electrokinetic capillary chromatography (MECC). Good resolution was obtained for all compounds, although in order to achieve the separation of ortho-ortho, ortho-para, and para-para isomers of bisphenol F diglycidyl ether (BFDGE), BFDGE x 2H(2)O and BFDGE x 2HCl, it was necessary to use a 25 microm id fused silica capillary. To increase sensitivity, a field-amplified sample injection (FASI)-MECC method was developed using 10 mM SDS solution as injection matrix and a 75 microm id fused silica capillary. Instrumental quality parameters such as LODs (<55 microg/L with standards), linearity (r(2)>0.999), and run-to-run and day-to-day precisions (RSD values lower than 12.5%) were determined. Finally, the suitability of the FASI-MECC method for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers in canned soft drinks was evaluated. Quantitation was performed by matrix-matched calibration using a plastic-bottled isotonic drink as matrix. The results showed that FASI-MECC is an economic method for the screening and quantitation of these kinds of compounds in soft drink beverages, with no loss of reproducibility, and effective at concentrations lower than the specific migration level values established by the European Union.

Fast liquid chromatography/tandem mass spectrometry (highly selective selected reaction monitoring) for the determination of toltrazuril and its metabolites in food.

In this work a fast liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the analysis of toltrazuril, a coccidiostatic drug, and its metabolites in meat food products. The applicability of atmospheric pressure chemical ionization (APCI) and heated electrospray ionization in both positive and negative modes was studied. APCI in negative mode provided the best results and the base peak originated from the loss of CF(3) (toltrazuril and toltrazuril sulfone) and CHF(3)* (toltrazuril sulfoxide) was used as the precursor ion in MS/MS. A fast LC separation on a C(18) Fused-Core column was used together with the APCI-MS/MS method developed using enhanced mass resolution mode (highly selective selected reaction monitoring, H-SRM) to improve the sensitivity and selectivity for the analysis of these compounds in food samples. A simple sample treatment based on an extraction with acetonitrile and a cleanup with a C(18) cartridge was used. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation lower than 10%), accuracy, and linearity and allowed the determination of these compounds in food samples down to the parts per billion level (limits of detection between 0.5 and 5 microg kg(-1)).

Occurrence of psychoactive stimulatory drugs in wastewaters in north-eastern Spain.

The occurrence of several psychoactive drugs in water resources from north-eastern Spain (NE-Spain) has been evaluated. The drugs were analyzed using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-ESI-MS/MS) after enrichment by solid-phase extraction (SPE). Most of the studied controlled drugs (8 out of 11) were found in both influent and effluent samples from several wastewater treatment plants. Cocaine and its metabolite were detected in wastewaters at concentrations ranging from 4 ng/L to 4.7 mug/L and from 9 ng/L to 7.5 mug/L respectively while concentrations of amphetamine type stimulatory drugs ranged from 2 to 688 ng/L. Removal percentages were estimated by sampling eight WWTPs (n=4). Cocaine and benzoylecgonine removal percentages were higher than 88% while those of amphetamine type stimulants varied ranging from 40% to more than 99%. Daily variability was also evaluated by performing a sequential survey, which revealed important fluctuations in the concentrations of nicotine, paraxanthine, amphetamine and ecstasy during the week. From the total concentrations found in wastewater influents estimations of the cocaine and ecstasy consumption were performed. For cocaine the results were approximately 14 doses per 1000 inhabitants (15-64 years old) per day and for ecstasy, approximately 4 doses per 1000 young adults (15-34 years old) per day for ecstasy.

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.

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.

Analysis of acrylamide in food products by in-line preconcentration capillary zone electrophoresis.

Two in-line preconcentration capillary zone electrophoresis (CZE) methods (field amplified sample injection (FASI) and stacking with sample matrix removal (LVSS)) have been evaluated for the analysis of acrylamide (AA) in foodstuffs. To allow the determination of AA by CZE, it was derivatized using 2-mercaptobenzoic acid. For FASI, the optimum conditions were water at pH > or = 10 adjusted with NH3 as sample solvent, 35 s hydrodynamic injection (0.5 psi) of a water plug, 35 s of electrokinetic injection (-10 kV) of the sample, and 6s hydrodynamic injection (0.5 psi) of another water plug to prevent AA removal by EOF. In stacking with sample matrix removal, the reversal time was found to be around 3.3 min. A 40 mM phosphate buffer (pH 8.5) was used as carrier electrolyte for CZE separation in both cases. For both FASI and LVSS methods, linear calibration curves over the range studied (10-1000 microg L(-1) and 25-1000 microg L(-1), respectively), limit of detection (LOD) on standards (1 microg L(-1) for FASI and 7 microg L(-1) for LVSS), limit of detection on samples (3 ng g(-1) for FASI and 20 ng g(-1) for LVSS) and both run-to-run (up to 14% for concentration and 0.8% for time values) and day-to-day precisions (up to 16% and 5% for concentration and time values, respectively) were established. Due to the lower detection limits obtained with the FASI-CZE this method was applied to the analysis of AA in different foodstuffs such as biscuits, cereals, crisp bread, snacks and coffee, and the results were compared with those obtained by LC-MS/MS.

Effect of red wine marinades on the formation of heterocyclic amines in fried chicken breast.

Genotoxic heterocyclic amines (HAs) are formed via the Maillard reaction and free radical reaction mechanisms when meat or fish is cooked at usual cooking conditions. In this paper, the effect of the addition of red wine was tested to study if it interferes in HA formation. Fried chicken breast was the food item chosen, and three different red wines, characterized in terms of grape varieties, free amino acids, antioxidant properties, and metallic composition, were used to marinate meat prior to the heating process. Unmarinated samples and samples marinated with an ethanol-water mixture provided reference HA levels. The frying experiments were performed under well-controlled temperature and time conditions. The samples were analyzed for HA content using solid-phase extraction and LC-MS/MS. DMIP, PhIP, MeIQx, 4,8-DiMeIQx, IFP, TMIP, harman, and norharman were identified in fried chicken breast. Red wine marinades were found to reduce the formation of some of the HAs formed. PhIP, with a reduction of up to 88%, was the most minimized amine, although the formation of harman was enhanced.

Determination of odorous mixed chloro-bromoanisoles in water by solid-phase micro-extraction and gas chromatography-mass detection.

A headspace-solid-phase micro-extraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) method has been proposed for the simultaneous determination of odorous trihalogenated anisoles in water. Parameters affecting efficiency of HS-SPME procedure, such as the selection of the SPME coating, extraction time, temperature and ionic strength were optimized. The commercially available polydimethylsiloxane (PDMS 100 microm) fiber appears to be the most suitable for the simultaneous determination of these compounds. Run-to-run precision with relative standard deviations (R.S.D.s) between 5 and 15% were obtained for most of the compounds except for 2,5-dicloro-6-bromo-anisole, 2,3-dibromo-6-chloroanisole, pentachloro- and pentabromoanisole (>20%). The method was linear over two orders of magnitude, and detection limits were compound dependent and ranged from 0.03 ng/L for 2,4,6-trichloroanisole to 0.25 ng/L for 2,3-dibromo-6-chloroanisole. The HS-SPME-GC-MS procedure was tested using real samples and relatively good standard deviations were obtained when using p-iodoanisole as internal standard for quantification. This is the first time that the individual identification of odorous trihalogenated chloro-bromoanisoles has been reported, being HS-SPME-GC-MS a suitable method for simultaneous determination of these compounds in water at concentration levels below their odor limit of detection.

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.

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.

High mass accuracy in-source collision-induced dissociation tandem mass spectrometry and multi-step mass spectrometry as complementary tools for fragmentation studies of quaternary ammonium herbicides.

Fragmentation studies using both an ion-trap mass analyzer and a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer were performed in order to establish the fragmentation pathways of organic molecules. A general strategy combining MSn data (n = 1-4) in an ion-trap analyzer with tandem mass spectrometry and in-source collision-induced dissociation tandem mass spectrometry (CID MS/MS) in a Q-TOF instrument was applied. The MSn data were used to propose a tentative fragmentation pathway following genealogical relationships. When several assignments were possible, MS/MS and in-source CID MS/MS (Q-TOF) allowed the elemental compositions of the fragments to be confirmed. Quaternary ammonium herbicides (quats) were used as test compounds and their fragmentation pathways were established. The elemental composition of the fragments was confirmed using the TOF analyzer with relative errors <0.0023 Da. Some fragments previously reported in the literature were reassigned taking advantage of the high mass resolution and accuracy of the Q-TOF instrument, which made it possible to solve losses where nitrogen was involved.

Multistep mass spectrometry of heterocyclic amines in a quadrupole ion trap mass analyser.

The fragmentation of heterocyclic amines (HAs) in an ion trap was studied by means of the infusion of methanolic solutions containing the compounds under assay, and using an atmospheric pressure chemical ionization (APCI) as ion source. The MS(n) spectra obtained for compounds included in the same family, either aminoimidazoazaarenes (AIAs) or carbolines, were compared in order to propose fragmentation pathways for each HA. Moreover, labelled AIAs were used to establish the mechanisms. The protonated molecule was always obtained, but subsequent fragmentation was different for both families. In the case of AIAs, major product ions came from the fragmentation of the aminoimidazole moiety, thus the base peak in MS(2) corresponded to the loss of the methyl group, and losses of C(2)NH(3) and CN(2)H(2) were also observed. Further fragmentation occurred in the heterocyclic rings, mainly with losses of HCN and CH(3)CN. For carbolines, the most important product ions came from the loss of ammonia, except for harman and norharman, the loss of a methyl group for methylated carbolines or the loss of diverse fragments from the heterocyclic rings. In some cases, ion-molecule reactions into the ion trap were observed. For instance, for AalphaC or MeAalphaC one ion originating from these reactions corresponded to the base peak.

Identification of 2,3-butanedione (diacetyl) as the compound causing odor events at trace levels in the Llobregat River and Barcelona's treated water (Spain).

A study of organic compounds imparting sweet and buttery odor problems in the Llobregat River (northeast Spain) and in treated water was conducted. Solid-phase microextraction (SPME), gas chromatography-olfactometry, and flavor profile analysis (FPA) were used as analytical methodologies to identify the compound responsible for odor incidents. 2,3-Butanedione (diacetyl) with a concentration range of 0.90-26 microg/l in river water samples entering the water treatment plant was identified as the compound causing the odor events. Flavor profile analysis establishes 0.05 microg/l as its odor threshold concentration (OTC) in water, with an odor recognition concentration of 0.20 microg/l. The analyses were carried out with SPME-GC-MS and parameters affecting SPME extraction such as selection of the fiber (carboxen-polydimethylsiloxane), extraction time (30 min), temperature (60 degrees C), and ionic strength were evaluated. Quality parameters of the optimized method gives good linearity (r2 > 0.999), a limit of detection (0.08 microg/l) similar to the OTC of the compound, and good reproducibility (R.S.D. < 20%). The SPME method was applied to identify the compound causing the odor.