RESUMO
Severe food poisoning events after the consumption of sharks have been reported since the 1940s; however, there has been no clear understanding of their cause. Herein, we report for the first time the presence of ciguatoxins (CTXs) in sharks. The identification by mass spectrometry of CTXs, including two new analogues, in a bull shark (Carcharhinus leucas) that was consumed by humans, causing the poisoning and death of 11 people in Madagascar in 2013 is described. Typical neurotoxic ciguatera symptoms were recorded in patients, and toxicological assays on extracts of the shark demonstrated CTX-like activity. These results confirm this episode as a ciguatera poisoning event and expand the range of pelagic fish species that are involved in ciguatera in the Indian Ocean. Additionally, gambieric acid D, a molecule originally described in CTX-producing microalgae, was identified for the first time in fish. This finding can contribute to a better understanding of trophic relations within food webs. The present work confirms that consumption of sharks from the Indian Ocean should be considered a ciguatera risk, and actions should be taken to evaluate its magnitude and risk in order to manage shark fisheries.
Assuntos
Ciguatera/epidemiologia , Ciguatera/etiologia , Ciguatoxinas/análise , Ciguatoxinas/toxicidade , Tubarões , Animais , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Ciguatera/mortalidade , Surtos de Doenças , Humanos , Oceano Índico , Madagáscar/epidemiologia , Espectrometria de Massas , Camundongos , Vigilância em Saúde Pública , Alimentos Marinhos/toxicidade , Tubarões/metabolismoRESUMO
Although consumption of Tetraodontidae species is prohibited in the EU, intoxications are still reported. The evaluation of tetrodotoxins (TTXs) by mass spectrometry (LC-MS/MS and LC-HRMS) and a screening immunoassay (mELISA) in tetraodontid fishes caught along the Western Mediterranean Sea revealed high concentrations of TTXs in Lagocephalus sceleratus while no TTXs were identified in L. lagocephalus and Sphoeroides pachygaster individuals. The high TTXs content found in the L. sceleratus analysed herein demonstrate the occurrence of highly toxic puffer fish in the Western Mediterranean Sea. Being L. sceleratus a recent invasive species in the Mediterranean, surveillance, risk assessment and risk management measures are necessary. The strategy used within this research work could be a valuable tool for future food safety monitoring.
Assuntos
Tetraodontiformes/metabolismo , Tetrodotoxina/análise , Animais , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Feminino , Espécies Introduzidas , Masculino , Espectrometria de Massas , Mar Mediterrâneo , Espanha , Especificidade da Espécie , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.
Assuntos
Bioensaio/métodos , Cromatografia Líquida/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Ressonância de Plasmônio de Superfície/métodos , Espectrometria de Massas em Tandem/métodos , Tetrodotoxina/análise , Animais , Calibragem , Reações Cruzadas , Limite de Detecção , Camundongos , TetraodontiformesRESUMO
The evaluation of the temperature effect on the competitiveness between the amine addition and the amidation reaction in a model cure acid-catalysed reaction between the epoxidized methyl oleate (EMO), obtained from high oleic sunflower oil, and aniline is reported. The study was carried out analysing the kinetic profiles of the chemical species involved in the system, which were obtained applying multivariate curve resolution-alternating least squares (MCR-ALS) to the Fourier transform infrared spectra data obtained from the reaction monitoring at two different temperatures (60°C and 30°C). At both experimental temperatures, two mechanisms were postulated: non-autocatalytic and autocatalytic. The different behaviour was discussed considering not only the influence of the temperature on the amidation reaction kinetic, but also the presence of the homopolymerization of the EMO reagent.
RESUMO
A spectrophotometric sequential injection analysis (SIA-DAD) method linked to multivariate curve resolution-alternating least squares (MCR-ALS) has been developed for sulphate determination. This method involves the reaction, inside the tubes of the SIA system, of sulphate with barium-dimethylsulphonazo (III) complex, Ba-DMSA (III), displacing Ba(2+) from the complex and forming DMSA (III). When the reaction products reach the detector a data matrix is obtained, which allows a second-order calibration to be developed. The experimental conditions (concentration and sample and reagent volumes) to obtain the highest sensitivity have been chosen applying a 2(4-1) fractional factorial design. The proposed sequential flow procedure permits up to 15 mg SO(4)(2-) L(-1) to be determined with a limit of detection of 1.42 mg L(-1) and it is able to monitor sulphate in samples at a frequency of 15 samples per hour. The method was applied to determine sulphate in natural and residual waters and in biodiesel. The reliability of the method was established for water samples by parallel determination using a standard turbidimetric method for sulphate in natural and residual water samples with results within statistical variation. For biodiesel samples, the method was validated comparing the concentration of some spiked samples with the expected concentration using a test-t.
Assuntos
Biocombustíveis/análise , Nefelometria e Turbidimetria/métodos , Sulfatos/análise , Água/química , Bário/química , Complexos de Coordenação/química , Análise MultivariadaRESUMO
A new concept of flow titration is proposed and demonstrated for the determination of total acidity in plant oils and biodiesel. We use sequential injection analysis (SIA) with a diode array spectrophotometric detector linked to chemometric tools such as multivariate curve resolution-alternating least squares (MCR-ALS). This system is based on the evolution of the basic specie of an acid-base indicator, alizarine, when it comes into contact with a sample that contains free fatty acids. The gradual pH change in the reactor coil due to diffusion and reaction phenomenona allows the sequential appearance of both species of the indicator in the detector coil, recording a data matrix for each sample. The SIA-MCR-ALS method helps to reduce the amounts of sample, the reagents and the time consumed. Each determination consumes 0.413ml of sample, 0.250ml of indicator and 3ml of carrier (ethanol) and generates 3.333ml of waste. The frequency of the analysis is high (12 samples h(-1) including all steps, i.e., cleaning, preparing and analysing). The utilized reagents are of common use in the laboratory and it is not necessary to use the reagents of perfect known concentration. The method was applied to determine acidity in plant oil and biodiesel samples. Results obtained by the proposed method compare well with those obtained by the official European Community method that is time consuming and uses large amounts of organic solvents.
Assuntos
Técnicas de Química Analítica , Óleos de Plantas/análise , Antraquinonas/química , Biocombustíveis , Calibragem , Interpretação Estatística de Dados , Difusão , Desenho de Equipamento , Etanol/química , Ácidos Graxos não Esterificados/química , Análise de Injeção de Fluxo/métodos , Concentração de Íons de Hidrogênio , Óleos de Plantas/química , SoftwareRESUMO
The aza-Michael reaction, a variation of the Michael reaction in which an amine acts as the nucleophile, permits the synthesis of sophisticated macromolecular structures with potential use in many applications such as drug delivery systems, high performance composites and coatings. The aza-Michael product can be affected by a retro-Mannich-type fragmentation. A way of determining the reactions that are taking place and evaluate the quantitative evolution of the chemical species involved in the reactions is presented. The aza-Michael reaction between a modified fatty acid ester with alpha,beta-unsaturated ketone groups (enone containing methyl oleate (eno-MO)) and aniline (1:1) was studied isothermally at 95 degrees C and monitored in situ by near-infrared spectroscopy (NIR). The number of reactions involved in the system was determined analyzing the rank matrix of NIR spectra data recorded during the reaction. Singular value decomposition (SVD) and evolving factor analysis (EFA) adapted to analyze full rank augmented data matrices have been used. In the experimental conditions, we found that the resulting aza-Michael adduct undergoes a retro-Mannich-type fragmentation, but the final products of this reaction were present in negligible amounts. This was confirmed by recording the (1)H NMR spectra of the final product. Applying multivariate curve resolution-alternating least squares (MCR-ALS) to the NIR spectra data obtained during the reaction, it has been possible to obtain the concentration values of the species involved in the aza-Michael reaction. The performance of the model was evaluated by two parameters: ALS lack of fit (lof=1.31%) and explained variance (R(2)=99.92%). Also, the recovered spectra were compared with the experimentally recorded spectra for the reagents (aniline and eno-MO) and the correlation coefficients (r) were 0.9997 for the aniline and 0.9578 for the eno-MO.