Arsenosugar extracted from algae: Assessment of countercurrent chromatography for isolation.

Arsenosugars are the predominant species of arsenic in most seaweed. The analysis of these compounds is hampered by the lack of calibration standards needed in their unambiguous identification and quantification. This affects the availability of reliable information on their potential toxicity, which is scarce and controversial. Knowing the potential of centrifugal partition chromatography (CPC) as a preparative separation technique applied to a number of natural compounds, the aim of this work is to investigate the feasibility of CPC in the case of isolation and purification of arsenosugars from algae extracts. Several biphasic solvents systems have been studied to evaluate the distribution of the As species. Given the physical characteristics of these compounds, the presence of strong acids, the formation of ionic pairs or the presence of salts at high ionic strength have been considered. System 1-BuOH/EtOH/sat.(NH4)2SO4/water at a volume ratio 0.2:1:1:1 originates adequate distribution constants of analytes that allows the required separation. The total arsenic content and the arsenic speciation of the eluted solutions from CPC were analyzed by ICP-MS and IC-ICP-MS, respectively. The developed CPC procedure allows us to obtain of the three arsenosugars with a purity of 98.7 % in PO4-Sug, 90.4 % in SO3-Sug and 96.1 % in SO4-Sug.

Search for Biomarkers for the LC-ESI-QqQ Determination of Phenoxymethylpenicillin Treatment in Raw or Cooked Chicken Meat Samples.

The high standards required for food safety make it necessary to trace unambiguously raw or cooked food products coming from medicated animals. Nevertheless, considering the lability of ß-lactams and their degradation, the detection of the presence of antibiotics in meat either raw or submitted to a cooking process is not easily affordable. To achieve this goal, an evaluation of the effect of common domestic cooking procedures, such as boiling and grilling, on the fate of phenoxymethylpenicillin (PENV) residues was performed. Finally, in this work, the penilloic acid from PENV (MET02) and the corresponding penicilloic acid (PENV-HYDRO) are suggested as biomarkers. These compounds present the highest relative abundances 5 days after the treatment was stopped (5PT) and show enough thermal stability to be considered suitable biomarker candidates for the pharmacological treatment instead of the parent compound. Nevertheless, the peaks corresponding to MET02 are significantly more intense than those for PENV-HYDRO, which makes preferential the use of MET02 to perform the control of samples.

Survey of arsenic content in edible seaweeds and their health risk assessment.

Since humans are especially sensitive to arsenic exposure, predominantly through diet, a strict control of the most widely consumed seaweeds is mandatory. Total arsenic contents and arsenic species in twenty-five different seaweeds from five different origins were studied. Seaweeds selected, included Phaeophyta (brown seaweed), Chlorophyta (green seaweed) and Rhodophyta (red seaweed) genera. The highest arsenic content appears in the Phaeophyta seaweed in the range from 11 to 162 mg kg-1 dried weight. Arsenosugars were found to be the predominant species of arsenic in most seaweeds, being up to 99.7% of total arsenic in some samples. The arsenic dietary intakes for seaweeds studied were assessed and the Target Hazard Quotients (THQ) and the Target Cancer Risk (TCR) were calculated, taking into account inorganic arsenic contents (iAs). iAs species in seaweeds showed low risk of arsenic intake except for Hizikia fusiforme samples.

Arsenosugar extracted from algae: Isolation by anionic exchange solid-phase extraction.

Obtaining reliable speciation data for evaluating dietary exposure, and increasing understanding of arsenic biochemistry in algae, are hindered by the availability of suitable standards of arsenosugars, the major species in these types of samples. Moreover, chemical syntheses of such compounds have been reported to be complex and tedious. The aim of this work was to investigate the feasibility of the anionic exchange SPE cartridges (SAX and WAX) as an easy and quick alternative for the isolation and preconcentration of arsenosugars. Two commercial silica-based SPE cartridges strong anion exchange sorbent (DSC-SAX) and weak anion exchange sorbent (DSC-NH2) were compared for the SPE of three arsenosugars (PO4-Sug, SO3-Sug and SO4-Sug). The effect of pH, ionic strength, type of salt and elution solvent on the elution protocols of these arsenosugars are studied. Eluted solutions from SPE were analyzed by ICP-MS for total arsenic content and IC-ICP-MS for the study of arsenic speciation. The developed SPE procedure allows to obtain a solution containing the three arsenosugars isolated from other arsenic species with recoveries over 75% for SO3-Sug and SO4-Sug, whereas for PO4-Sug were around 45%.

Metabolites in Milk after Enrofloxacin Treatment and Their Persistence to Temperature.

In this work, metabolomic profile changes in milk from cows affected by mastitis and treated with enrofloxacin (ENR) have been studied using LC-HRMS techniques. Principal component analysis was applied to the obtained results, and the interest was focused on changes affecting compounds without a structural relationship to ENR. Most of the compounds, whose concentrations were modified as a result of the pharmacological treatment and/or the pathological status, were related to amino acids and peptides. Compounds that may become possible biomarkers for either disease or treatment have been detected. Additionally, the alterations caused by thermal processes, such as those applied to milk before consumption, on the identified metabolites have also been considered.

Determination of quinolone residues in raw cow milk. Application of polar stir-bars and ultra-high performance liquid chromatography-tandem mass spectrometry.

Seventeen quinolone antibiotics were determined in cows' milk. A method of high sensitivity, selectivity and accuracy was developed. Accuracy (trueness and precision), linearity, sensitivity, selectivity, decision limit and detection capability were established following the recommendations of the Commission Decision 2002/657/EC and the Food and Drug Administration (FDA) guideline. The use of polar stir-bar sorptive extraction (SBSE) prior to UHPLC-MS/MS analysis is proposed. The variables that affect SBSE were optimised using multivariate optimisation strategies. The ionic strength, the extraction time and the sample volume were studied. pH and stir-bar coating (polydimethylsiloxane, PDMS, and polyethyleneglycol modified silicone, PEG) were studied. PEG showed the best extraction yield at pH 6. For validation, a matrix-matched calibration and a recovery assay were carried out. Limits of quantification from 0.5 µg kg-1 for nalidixic acid, flumequine and piromidic acid, to 4.0 µg kg-1 for sarafloxacin were calculated. The precision (%, RSD) was lower than 15% for all antibiotics. Recoveries in fortified samples were between 88 and 114%.

Occurrence of veterinary pharmaceuticals in golden eagle nestlings: Unnoticed scavenging on livestock carcasses and other potential exposure routes.

Wildlife exposure to pharmaceuticals can occur through contaminated water, and through the excreta and carcasses of medicated livestock, with potential for bioaccumulation and transfer through food webs. We evaluated whether nestling exposure to pharmaceuticals can occur from food delivered to nests in the golden eagle (Aquila chrysaetos), a top predator and facultative scavenger. Despite the fact that diet analysis suggests an apparently low dependence on livestock carcasses reduced to two piglets remains (1.5% of food remains, n=134), a high proportion of nestlings (71%, n=7) showed fluoroquinolone residues in plasma, mostly enrofloxacin, which is exclusively used in veterinary treatments. The occurrence and concentration (54.5±6.6µg·L-1) of fluoroquinolones in plasma was similar to those found in the nestlings of three vulture species largely dependent on livestock carcasses obtained at supplementary feeding stations, which are managed for the conservation of their populations. Although the number of analysed eaglets is comparatively small, the fact that enrofloxacin was found in all nests sampled in three breeding seasons suggest an exposure to the drugs similar to that of vultures. An underestimation of the role of carrion, especially from small piglets whose consumption may have gone unnoticed, and the predation of semi-domestic prey and generalist prey exploiting carcasses of medicated livestock, can contribute to explaining the unexpectedly high occurrence of these drugs in eaglets.

Food safety in scavenger conservation: Diet-associated exposure to livestock pharmaceuticals and opportunist mycoses in threatened Cinereous and Egyptian vultures.

Pharmaceuticals from veterinary treatments may enter terrestrial food webs when medicated livestock are available to wildlife in supplementary feeding stations aimed at the conservation of endangered scavengers. Here, we hypothesized that the exposure risk to livestock fluoroquinolones, as indicators of pharmaceutical burden in food, is related to the variable reliance of scavengers on domestic versus wild animal carcasses. Since the misuse of broad-spectrum antibiotics is a major predisposing factor for opportunistic mycoses, we evaluated disease signs potentially associated with diet-dependent drug exposure in nestlings of two threatened vultures. A greater occurrence (100%, n=14) and concentration of fluoroquinolones (mean±SD=73.0±27.5µgL-1, range=33.2-132.7), mostly enrofloxacin, were found in Cinereous vultures, Aegypius monachus, due to their greater dependence on livestock carcasses than Egyptian vultures, Neophron percnopterus (fluoroquinolones occurrence: 44%, n=16, concentration: 37.9±16.6µgL-1, range=11.5-55.9), which rely much more on carcasses of wild animals (42% of remains vs. 23% in the cinereous vulture). The chaotic, chronic and pulsed ingestion of these drugs throughout nestling development is proposed as one of the most plausible explanations for the high occurrence and intensity of oral Candida-like lesions in nestling vultures. The high occurrence of fluoroquinolone residues and disease hindered the probing of a cause-effect relationship between both factors in individual vultures. This relationship could be evaluated through a population-based approach by sampling vultures not exposed to these drugs. The high dependence of vultures on domestic animals today compared to past decades and the growing intensification of livestock farming, imply an expected increase in the impact of pharmaceuticals on scavenger populations. This requires further evaluation due to potential consequences in biodiversity conservation and environmental health. We encourage the prioritization of efforts to promote the use of less medicated free-ranging livestock carcasses left in the countryside, rather than stabled stocks made available in vulture restaurants. Additionally, attention should be paid to the population recovery of wild species that dominated scavenger diets in the past.

Wildlife contamination with fluoroquinolones from livestock: Widespread occurrence of enrofloxacin and marbofloxacin in vultures.

There is much recent interest in the presence and impact of veterinary pharmaceuticals in wildlife. Livestock carcasses are often disposed of in supplementary feeding stations for avian scavengers, as a management and conservation tool for these species worldwide. In feeding stations, vultures and other scavengers can consume carcasses almost immediately after disposal, which implies the potential ingestion of veterinary pharmaceuticals as a non-target consequence of supplementary feeding. Using UPLC-MS/MS and HPLC-TOF, we evaluated the presence and concentration of fluoroquinolone residues in plasma of nestling vultures feeding on domestic livestock carrion. Three different fluoroquinolones (marbofloxacin, enrofloxacin and its metabolite ciprofloxacin) and a non-targeted ß-lactam (nafcillin) were detected in vulture plasma. The high proportion of individuals (92%) with fluoroquinolone residues at variable concentrations (up to ∼20 µg L(-1) of enrofloxacin and ∼150 µg L(-1) of marbofloxacin) sampled in several geographically distant colonies and on different dates suggests that these and other drugs were potentially ingested throughout nestling development. Contamination with veterinary fluoroquinolones and other pharmaceuticals should be considered as an unintended but alarming consequence of food management in threatened wildlife.

Multiclass method for the determination of quinolones and ß-lactams, in raw cow milk using dispersive liquid-liquid microextraction and ultra high performance liquid chromatography-tandem mass spectrometry.

An analytical method based on a sample treatment by dispersive liquid-liquid microextraction (DLLME) followed by ultra high performance liquid chromatography-tandem mass spectrometry analysis (UHPLC-MS/MS) for the determination of 17 quinolones and 14 ß-lactams (penicillins and cephalosporins) in raw cow milk, was validated according to the European Commission guidelines as cited in the Decision 2002/657/EC. The extraction efficiency of the DLLME depends on several parameters such as the nature and volumes of extractant and dispersive solvents, pH, concentration of salt, shaking time and time of centrifugation. These variables were accurately optimized using multivariate optimization strategies. A Plackett-Burman design to select the most influential parameters and a Doehlert design to obtain the optimum conditions have been applied. Two different pH values were used for the extraction of compounds (pH 3 for acidic quinolones and ß-lactams and pH 8 for amphoteric quinolones). The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. The limits of quantification found ranged from 0.3 ng g(-1) for amoxicillin to 6.6 ng g(-1) for ciprofloxacin, and the precision was lower than 15% in all cases as is required by the European Regulation. The decision limits (CCα) ranged between 4.1 and 104.8 ng g(-1), while detection capabilities (CCß) from 4.2 to 109.7 ng g(-1). These values were very close to the corresponding maximum residue limits (MLRs) for the studied antibiotics. Recoveries between 72 and 110% were also obtained. Finally, in order to evaluate the applicability of the method, 28 raw cow milk samples were analysed and it was observed that 28% of the samples were positive. However, only 11% were considered non-compliant with the current EU legislation (Commission Regulation 37/2010), due to some milk samples corresponded to treated cows with these antibiotics.

Identification of metabolites and thermal transformation products of quinolones in raw cow's milk by liquid chromatography coupled to high-resolution mass spectrometry.

The presence of residues of antibiotics, metabolites, and thermal transformation products (TPs), produced during thermal treatment to eliminate pathogenic microorganisms in milk, could represent a risk for people. Cow's milk samples spiked with enrofloxacin (ENR), ciprofloxacin (CIP), difloxacin (DIF), and sarafloxacin (SAR) and milk samples from cows medicated with ENR were submitted to several thermal treatments. The milk samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to find and identify TPs and metabolites. In this work, 27 TPs of 4 quinolones and 24 metabolites of ENR were found. Some of these compounds had been reported previously, but others were characterized for the first time, including lactose-conjugated CIP, the formamidation reaction for CIP and SAR, and hydroxylation or ketone formation to produce three different isomers for all quinolones studied.

Modeling the electrophoretic behavior of quinolones in aqueous and hydroorganic media.

The applicability of the classical semiempirical relationships between the electrophoretic mobility and charge-to-mass ratio (m(e) versus q/M(alpha)) has been investigated to describe migration behavior of a series of quinolones in aqueous and hydroorganic media. The Stoke's law (alpha=1/3), the classical polymer model (alpha=1/2) and the Offord's surface law (alpha=2/3) were evaluated in the pH range comprised between 2 and 11 in ACN-water mixtures of 0, 10 and 30% m/m ACN. As accurate acidity constant values were available for charge calculations, correlations were excellent. The results obtained using the Offord's surface law were slightly better and the plots of q/M(2/3) against the separation electrolyte pH were used to select the optimum pH values for separation of a quinolone mixture. The electropherograms simulated considering the accurate charge-to-mass ratio of each quinolone at the selected pH values were in good agreement with the experimental results. The ability of these relationships to predict the separation of the studied quinolones at a certain pH value in methanol-water and THF-water mixtures was also demonstrated.

Determination of penicillins in milk using LC-UV, LC-MS and LC-MS/MS.

The aim of this work is to establish a method for the simultaneous determination of eight penicillins in milk samples by LC-UV, LC-MS and LC-MS/MS. The procedure involves a step for clean-up and to preconcentrate the analytes by SPE and a subsequent chromatographic analysis. LC-UV, LC-MS and LC-MS/MS have been used for the simultaneous quantification of penicillins in milk. The proposed methods have been validated according to the EU guideline and present LOQ below the maximum limits of residues (MRLs) established by the European Union for penicillins in milk. The developed methods were applied to different milk samples obtained from cows medicated with penicillins.

Determination of fluoroquinolones in chicken tissues by LC-coupled electrospray ionisation and atmospheric pressure chemical ionisation.

The aim of this work was to develop a method for determining seven quinolones (ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine) in chicken muscle by LC coupled to MS. Two ionisation techniques, ESI and atmospheric pressure chemical ionisation (APCI) were compared using standard solutions. LOD and LOQwere determined under the optimised conditions for the two sources. The ESI was found the best for the purpose. The optimised method (LC-ESI-MS) was validated for the simultaneous analysis of the quinolones regulated by European Community in spiked chicken tissues, using norfloxacin as internal standard. Recoveries obtained varied in the range 60-109%. This method was compared with LC-UV method established previously.

Effective sorbents for solid-phase extraction in the analysis of quinolones in animal tissues by capillary electrophoresis.

In this work, several commercial sorbents (Zorbax C18, Bond Elut C18, Isolute ENV+, Oasis HLB, Oasis MAX, SDB-RPS, and MPC-SD) were compared for the solid-phase extraction of the series of quinolones regulated by the European Community in chicken tissues in order to establish a method for the determination of this series of quinolones by capillary electrophoresis and diode array detection. Sorbents were chosen in order to achieve maximum recoveries and optimal clean-up efficiency. Better results were obtained using SDB-RPS and Oasis MAX which would provide suitable limits of detection, below of the maximum residue limits (MRLs) regulated.

Determination of residues of enrofloxacin and its metabolite ciprofloxacin in chicken muscle by capillary electrophoresis using laser-induced fluorescence detection.

A method for the residue analysis of the veterinary antimicrobial agent enrofloxacin and its active desethyl metabolite ciprofloxacin in chicken muscle tissue has been developed and validated. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd laser (lambda(ex) = 325 nm) providing an enhancement in sensitivity and selectivity compared to conventional UV detection. The assay has been validated with satisfying results. The limits of quantification for enrofloxacin and ciprofloxacin were 5 microg/kg and 20 microg/kg, respectively, with a fivefold preconcentration yielded by a sample clean-up with a simple liquid-liquid extraction procedure. Calibration graphs were linear from 5 to 1000 microg/kg for enrofloxacin and from 20 to 1000 microg/kg for ciprofloxacin. The assay allows the detection of contaminated muscle samples at the required maximum residue limit of the European Union, which is 100 microg/kg for the sum of enrofloxacin and ciprofloxacin.