Understanding fungal functional biodiversity during the mitigation of environmentally dispersed pentachlorophenol in cork oak forest soils.

Pentachlorophenol (PCP) is globally dispersed and contamination of soil with this biocide adversely affects its functional biodiversity, particularly of fungi - key colonizers. Their functional role as a community is poorly understood, although a few pathways have been already elucidated in pure cultures. This constitutes here our main challenge - elucidate how fungi influence the pollutant mitigation processes in forest soils. Circumstantial evidence exists that cork oak forests in N. W. Tunisia - economically critical managed forests are likely to be contaminated with PCP, but the scientific evidence has previously been lacking. Our data illustrate significant forest contamination through the detection of undefined active sources of PCP. By solving the taxonomic diversity and the PCP-derived metabolomes of both the cultivable fungi and the fungal community, we demonstrate here that most strains (predominantly penicillia) participate in the pollutant biotic degradation. They form an array of degradation intermediates and by-products, including several hydroquinone, resorcinol and catechol derivatives, either chlorinated or not. The degradation pathway of the fungal community includes uncharacterized derivatives, e.g. tetrachloroguaiacol isomers. Our study highlights fungi key role in the mineralization and short lifetime of PCP in forest soils and provide novel tools to monitor its degradation in other fungi dominated food webs.

Mixed-mode liquid chromatography coupled to tandem mass spectrometry for the analysis of aminoglycosides in meat.

A novel LC-MS/MS method has been developed for the determination of 13 aminoglycoside antibiotics in meat products. Among the chromatographic columns tested, the mixed-mode Obelisc R provided the best performance. Electrospray has been used for the coupling of the LC and the effect of temperature on the ionization has been evaluated. The mass spectra of AGs have been studied in order to select the most adequate precursor and product ions for quantitation and confirmation in SRM mode, showing that the single charged [M+H](+) provided better precisions than the double charged [M+2H](2+). Accurate mass measurements have been performed in order to confirm the molecular composition of the product ions, allowing the establishment of a new mechanism for some product ions of STR and DHSTR. A sample treatment based on an extraction and a SPE clean-up has been applied to a wide variety of meat products such as frankfurters; sausages; and minced meat of pork, veal, and chicken. Method limits of quantitation in the low microgram per kilogram level (1-50 µg kg(-1)), precisions %RSD below 15 % and accuracies expressed as relative errors below 23 % have been obtained, making the proposed method suitable for routine analysis.

Survey of the occurrence of pharmaceuticals in Spanish finished drinking waters.

Fifty samples of finished drinking waters (FDWs) from Spain covering 12 million inhabitants were tested for 53 pharmaceuticals pertaining to 12 different Anatomical Therapeutic Chemical (ATC) classification system codes. The studied compounds are a combination of most commonly consumed pharmaceuticals with other barely reported in the literature. Five compounds, azithromycin, clarithromycin, erythromycin, sulfamethoxazole, and ibuprofen were tentatively identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in some samples (2 to 15 %), but only ibuprofen and azithromycin could be confirmed when analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) with a quadrupole-Orbitrap instrument. Concentration levels of ibuprofen in the positive samples ranged from 12 to 17 ng/L (n = 6) while for azithromycin values from 5 to 9.5 ng/L (n = 3) were found. Ibuprofen fragmentation behaviour in different mass spectrometry instrument configurations (triple quadrupole, quadrupole-ion trap, and quadrupole-Orbitrap) was evaluated.

Atmospheric pressure ionization-tandem mass spectrometry of the phenicol drug family.

In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H](+) dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M-H](-), while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high-resolution mass spectrometry in a quadrupole-Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N-(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS(3) and MS(4) spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high-performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg.

Field amplified sample injection-capillary zone electrophoresis for the analysis of amprolium in eggs.

Veterinary medicines are widely administered to farm animals since they keep animals healthy at overcrowded conditions. Nevertheless the continuous administration of medicines to farm animals can frequently lead to the presence of residues of veterinary drugs in consumption products. Amprolium is a quaternary ammonium compound used in the treatment of coccidiosis. In this paper, a method based on CZE to analyze residues of amprolium in eggs was developed and validated for the first time. Parameters such as electrolyte type, concentration, and pH were optimized. In order to improve sensitivity, field-amplified sample injection (FASI) was used for in-line preconcentration after a quick and simple sample treatment based on SPE (Envi-Carb). During method-validation studies using egg samples, a matrix interference was found at the migration time of amprolium. This compound was identified as thiamine and confirmed by MS(n) experiments using CE coupled to MS (CE-MS) with an ion-trap mass analyzer. CZE conditions were reoptimized to separate thiamine from amprolium allowing the quantification of amprolium in eggs at concentrations down to 75 µg/kg, which are far below the MRL-legislated values.

Ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of phenicol drugs and florfenicol-amine in foods.

A method based on ultra-high performance liquid chromatography (UHPLC) is proposed for the determination of chloramphenicol (CAP), its related compounds, thiamphenicol (TAP) and florfenicol (FF), and the polar metabolite florfenicol-amine (FFA), in animal-derived foods (chicken and pork meat, fish, prawns and honey). For the retention of FFA and its simultaneous analysis with the parent compounds a phenyl-hexyl column is proposed. A fast separation is achieved in less than 2 minutes using a methanol : acetic acid-ammonium acetate buffer (5 mM, pH 5) and gradient elution. Under these conditions, the FFA is retained at more than twice the dead volume, as recommended by the legislation. For the coupling with mass spectrometry, heated-electrospray (H-ESI) is used as ionisation source improving vaporization efficiency. To prevent interferences using selected-reaction monitoring (SRM) both quantitation and confirmation transitions were carefully selected. Two different sample treatments based on solid-phase extraction with mixed-mode cartridges for fish and meat products and hydrophilic-lipophilic-balanced cartridges for honey are proposed, providing limits of quantitation (LOQs) below µg kg(-1) level.

Non-aqueous capillary electrophoresis separation of fullerenes and C60 fullerene derivatives.

As the interest in the use of fullerene compounds in biomedical and cosmetic applications increases, so too does the need to develop methods for their determination and quantitation in such complex matrices. In this work, we studied the behavior of C60 and C70 fullerenes in non-aqueous capillary electrophoresis, as well as two C60 fullerene derivatives not previously reported by any electrophoretic method, N-methyl-fulleropyrrolidine and (1,2-methanofullerene C60)-61-carboxylic acid. The separation was performed using fused-silica capillaries with an I.D. of 50 µm and tetraalkylammonium salts, namely tetra-n-decylammonium bromide (200 mM) and tetraethylammonium bromide (40 mM), in a solvent mixture containing 6 % methanol and 10 % acetic acid in acetonitrile/chlorobenzene (1:1 v/v) as the background electrolyte. Detection limits, based on a signal-to-noise ratio of 3:1, were calculated, and values between 1 and 3.7 mg/L were obtained. Good run-to-run and day-to-day precisions on concentration were achieved with relative standard deviation lower than 15 %. For the first time, an electrophoretic technique has been applied for the analysis of C60 fullerene in a commercial cosmetic cream. A standard addition method was used for quantitation, and the result was compared with that obtained by analyzing the same cream by liquid chromatography coupled to mass spectrometry.

Evaluation of the presence of drugs of abuse in tap waters.

A total of seventy samples of drinking water were tested for non-controlled and illicit drugs. Of these, 43 were from Spanish cities, 15 from seven other European countries, three from Japan and nine from seven different Latin American countries. The most frequently detected compounds were caffeine, nicotine, cotinine, cocaine and its metabolite benzoylecgonine, methadone and its metabolite EDDP. The mean concentrations of non-controlled drugs were: for caffeine 50 and 19 ng L(-1), in Spanish and worldwide drinking water respectively and for nicotine 13 and 19 ng L(-1). Illicit drugs were sparsely present and usually at ultratrace level (<1 ng L(-1)). For example, cocaine has mean values of 0.4 (Spain) and 0.3 ng L(-1) (worldwide), whereas for benzoylecgonine, these mean values were 0.4 and 1.8 ng L(-1), respectively. Higher concentrations of benzoylecgonine were found in Latin American samples (up to 15 ng L(-1)). No opiates were identified in any sample but the presence of methadone and EDDP was frequently detected. Total mean values for EDDP were 0.4 ng L(-1) (Spain) and 0.3 ng L(-1) (worldwide). Very few samples tested positive for amphetamines, in line with the reactivity of chlorine with these compounds. No cannabinoids, LSD, ketamine, fentanyl and PCP were detected.

5-Hydroxymethylfurfural content in foodstuffs determined by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) has been applied for the determination of 5-hydroxymethylfurfural in several foodstuffs. A 75mM phosphate buffer solution at pH 8.0 containing 100mM sodium dodecylsulphate was used as background electrolyte (BGE), and the separation was performed by applying +25kV in a 50µm I.D. uncoated fused-silica capillary. Good linearity over the range 2.5-250mgkg(-1) (r(2)⩾0.999) and run-to-run and day-to-day precisions at low and medium concentration levels were obtained. Sample limit of detection (0.7mgkg(-1)) and limit of quantification (2.5mgkg(-1)) were established by preparing the standards in blank matrix. The procedure was validated by comparing the results with those obtained with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Levels of HMF in 45 different foodstuffs such as breakfast cereals, toasts, honey, orange juice, apple juice, jam, coffee, chocolate and biscuits were determined.

Analysis of amprolium by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

We present a fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of the coccidiostat amprolium in food samples. Tandem mass spectrometry in a triple quadrupole was used for quantitative purposes, and the information from multiple-stage mass spectrometry in an ion-trap mass analyzer contributed to fragmentation studies. Hydrophilic interaction liquid chromatography (HILIC) in a Fused-Core column using isocratic elution (acetonitrile:formic acid/ammonium formate buffer pH 4, 50 mM (60:40)) successfully analyzed this compound in less than 3 min. The HILIC system was coupled to heated electrospray-MS/MS using highly selective-selected reaction monitoring (H-SRM) to improve sensitivity and selectivity for the analysis of amprolium, after a simple sample treatment based on an "extract and shoot" strategy. Accurate mass measurements were performed to identify the interfering compound responsible for causing matrix ion enhancement in the signal of amprolium. The addition of l-carnitine (the interfering compound) (1 microg L(-1)) to standards and sample extracts allowed the use of the external calibration method for quantitative purposes. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation, RSD, lower than 13%), accuracy and linearity and allowed the determination of amprolium down to the ppb level (LODs between 0.1 and 0.6 microg kg(-1)).

Liquid chromatography multi-stage mass spectrometry for the analysis of 5-hydroxymethylfurfural in foods.

A new, simple and selective method for the analysis of 5-hydroxymethylfurfural (HMF) in foods by liquid chromatography coupled to ion trap multi-stage mass spectrometry (LC-MS(n)) is proposed in the present study. Several chromatographic columns were tested and the best results were obtained using a phenyl fluorinated column. MS conditions were established using an atmospheric pressure chemical ionisation (APCI) source in the positive ionisation mode. MS/MS was used for quantitative analysis while MS(3) was required for confirmation purposes. Quality parameters such as day-to-day and run-to-run precision (RSD<10%) and detection limit (133 ng g(-1), 333 pg injected) were established. This method was successfully applied to the analysis of HMF content in several Spanish food samples from a local market.

Ion-trap tandem mass spectrometry for the analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in food.

This paper reports on the applicability of gas chromatography coupled to ion-trap tandem mass spectrometry (GC/ITMS/MS) for the analysis of polychlorinated dibenzo- p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in food. MS/MS parameters were selected to achieve the high sensitivity and selectivity required for food analysis. Good precision (RSD=5-18% for PCDD/Fs and 6-14% for dl-PCBs) and low limits of detection for PCDD/Fs (0.1-0.93 pg/g of fat) and dl-PCBs (0.1-0.89 pg/g of fat) were obtained. A comparative study of the congener-specific determination using both GC/ITMS/MS and GC-high resolution mass spectrometry (GC/HRMS) was performed by analyzing several matrices such as milk, fish oil, chicken, pork, fish, eggs, and a chicken compound feed, at low pg/g levels. The results using GC/ITMS/MS were in good agreement with those obtained by GC/HRMS. Consequently, GC/ITMS/MS is proposed for the analysis of PCDD/Fs and dl-PCBs in food and feed samples.

Determination of the odor threshold concentrations of chlorobrominated anisoles in water.

Trihalophenols, which are drinking water disinfection byproducts (DBPs) formed by chlorination or chloramination practices, can be biomethylated into trihalogenated anisoles. These latter compounds have traditionally been suspected of causing odor episodes in drinking water around the world. The odor threshold concentration (OTC) of mixed chlorobrominated anisoles, which were previously synthesized, was determined by flavor profile analysis (FPA) performed by an experienced panel trained to identify odors and tastes in water. The odor threshold amount (OTA) was evaluated by using a gas chromatograph equipped with olfactometry (GC-O) and electron capture detectors (ECD). FPA results for mixed chlorobromoanisoles gave a theoretical OTCs range from 2 to 30 ng/L, the 2,6-diBr-3Cl-anisole being the most odorous compound. Rubber is the general descriptor described by panelists for these compounds, although earthy and musty are the following most cited descriptors.

State-of-the-art of the hyphenation of capillary electrochromatography with mass spectrometry.

The high separation efficiency and loading capacity of capillary electrochromatography (CEC) make it an attractive separation mode for coupling with mass spectrometry (MS), which has the ability to unambiguously identify analytes with high selectivity and sensitivity. We present an overview of recent advances on both instrumentation and separation columns employed in CEC-MS systems. In particular, the main characteristics of the stationary phases, as well as the configurations of the column outlet that are related with the coupling arrangements of the MS ionization sources, are reported. At present, packed columns and conventional electrospray ionization (ESI) sources are mainly employed in CEC-MS. Nevertheless, the use of monolithic capillary columns and nanoelectrospray sources has the potential for wide acceptance in the next future. Moreover, the main features of several mass analyzers including ion trap, quadrupole, time-of-flight, magnetic sector, and Fourier transform-ion cyclotron resonance are examined. Finally, current applications of this technology, mainly in the pharmaceutical field and proteomics, are reviewed.

Optimization of a clean-up procedure for the determination of heterocyclic aromatic amines in urine by field-amplified sample injection-capillary electrophoresis-mass spectrometry.

Heterocyclic amines (HAs), generated when proteinaceous food is cooked, are of special interest since they can be carcinogenic for humans. In this paper, the optimization of a clean-up procedure for the isolation and preconcentration of 15 heterocyclic amines in urine samples is described. The method proposed combines liquid extraction on a solid support of diatomaceous earth with solid-phase extraction in cartridges. Tests were performed on several cartridges containing graphitic carbon or mixed phases, i.e., combining reversed-phase and cation-exchange mechanism, and the best results were obtained with Oasis MCX. The optimized purification method was applied to the quantification of heterocyclic amines in hydrolyzed spiked human urine. The method was carried out by capillary electrophoresis (CE) coupled to mass spectrometry (MS) and applying field-amplified sample injection (FASI) as in-line preconcentration procedure. We obtained detection limits down to 0.3 ng/ml of urine and errors lower than 17%.

Determination of heterocyclic aromatic amines by capillary electrophoresis coupled to mass spectrometry using in-line preconcentration.

This paper shows the potentiality of capillary electrophoresis (CE) coupled to mass spectrometry (MS) for the analysis of heterocyclic aromatic amines obtaining good results in terms of sensitivity and precision. These compounds have a special interest since they can be carcinogenic for humans. The optimization of a CE-MS method was performed and the best conditions were obtained using a 16 mM formic acid/ammonium formate solution at pH 4.5 with 60% methanol as running electrolyte. For CE-MS coupling, a sheath liquid methanol/20 mM formic acid (75/25) solution at a flow rate of 3 microL/min and hydrodynamic injection of methanol mixtures for 10 s were used. Detection limits ranging from 18 ng/g to 360 ng/g and precisions up to 1.4% and 12% for migration time and concentration, respectively, were obtained. In order to improve sensitivity, field-amplified sample injection was applied as an in-line preconcentration method. Methanol/5 mM formic acid (50/50) as a sample solvent, 3 s hydrodynamic injection (0.5 psi) of a methanol plug, and 25 s of electrokinetic injection (10 kV) of the sample were found to be the optimum conditions. Detection limits up to 25 times lower and similar precisions than those reported for hydrodynamic injection were obtained.

Steric-hyperlayer sedimentation field flow fractionation and flow cytometry analysis applied to the study of Saccharomyces cerevisiae.

Sedimentation field flow fractionation separation associated with flow cytometry has been used for the characterization of several commercial Saccharomyces cerevisiae yeasts used for wine production. A new type of channel 80 microm thick and new operating conditions, such as sample introduction when field and flow are established and a channel inlet connected to the accumulation wall, were used. Good repeatability (5% RSD) and reduced analysis time (2-10 min) were obtained. The avoidance of the stop-flow relaxation process in conjunction with the use of a channel of reduced thickness has demonstrated that an effective "steric-hyperlayer" mode driving to a major focusing effect of the species in the channel thickness is involved in the elution of the yeast cells. Flow cytometry analyses were performed, and the forward scattering and side scattering yeast characteristics correlation maps were obtained. Field flow fractionation and flow cytometry information obtained indicated that the fractogram profiles of the yeast cell depended not only on the size, but also on the shape and density.

Estimation of the composition of heparin mixtures from various origins using proton nuclear magnetic resonance and multivariate calibration methods.

A multivariate calibration method for the characterization of heparin samples based on the analysis of (1)H nuclear magnetic resonance (NMR) spectral data is proposed. Heparin samples under study consisted of two-component or four-component mixtures of heparins from porcine, ovine and bovine mucosae and bovine lung. Although the (1)H NMR spectra of all heparin types were highly overlapping, each origin showed some particular features that could be advantageously used for the quantification of the components. These features mainly concerned the anomeric H, which appeared in the range 5.0-5.7 ppm and the peaks of acetamidomethyl protons at 2.0-2.1 ppm. The determination of the percentage of each heparin class depended on these differences and was carried out using partial least squares regression (PLS) as a calibration method. Prior to the PLS analysis, the spectral data were standardized using the internal standard peak (sodium 4,4-dimethyl-4-silapentanoate- 2,2,3,3- d (4), TSP) as the reference. The quantification of each heparin type in the samples using PLS models built with 4 or 5 components was satisfactory, with an overall prediction error ranging from 3% to 10%.

Proton nuclear magnetic resonance characterisation of glycosaminoglycans using chemometric techniques.

Various types of glycosaminoglycans (GAGs) including heparins, chondroitin sulfates, dermatan sulfate and hyaluronic acid were studied from their proton nuclear magnetic resonance (1H NMR) spectra using chemometric techniques. Despite the complexity of the 1H NMR signals, data analysis using principal component analysis enabled the different GAG classes to be distinguished and permitted their classification according to their chemical structure. The analysis of the composition of the major disaccharide unit and other relevant chemical structures in the heparin samples was performed using partial least squares regression.

Determination of aldehydes in drinking water using pentafluorobenzylhydroxylamine derivatization and solid-phase microextraction.

A headspace solid-phase microextraction (HS-SPME) procedure followed by gas chromatography and electron capture detection (GC-ECD) has been developed for the determination of aldehydes in drinking water samples at microg/l concentrations. A previous derivatization with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was performed due to the high polarity and instability of these ozonation by-products. Several SPME coatings were tested and the divinylbenzene-polydimethylsiloxane (DVB-PDMS) coating in being the most suitable for the determination of these analytes. Experimental SPME parameters such as selection of coating, sample volume, addition of salt, extraction time and temperature of desorption were studied. Analytical parameters such as precision, linearity and detection limits were also determined. HS-SPME was compared to liquid-liquid microextraction (proposed in US Environmental Protection Agency Method 556) by analyzing spiked water samples; a good agreement between results obtained with both techniques was observed. Finally, aldehydes formed at the Barcelona water treatment plant (N.E. Spain) were determined at levels of 0.1-0.5 microg/l. As a conclusion, HS-SPME is a powerful tool for determining ozonation by-products in treated water.