RESUMEN
Microcystins (MCs) can be detected in various matrices in two forms: a freely extractable fraction and a total (free and covalently protein-bound) fraction. Although the majority of MCs analyses are limited to the free fraction, they do not allow the analysis of all MCs variants or protein-bound forms. Other methods, known as total MCs analysis methods, enable simultaneous analysis of all MCs variants, as well as bound forms, which may be a major form of toxin accumulation in organisms. Among these techniques, the chemical oxidation method (e.g. Lemieux) allows the detection of total forms of MC (and nodularins) by oxidizing the common part to all MC and nodularins, and analyzing the resultant MMPB product (2-methyl-3-methoxy-4-phenylbutyric acid). However, the execution of this method in the context of health monitoring is challenging due to the variability of the protocols, the recoveries obtained with these protocols, and the important matrix effects associated with the method. The objectives of this study were i) to optimize an existing protocol of chemical oxidation "Lemieux1" on fresh fish fillet matrices, ii) to compare two existing protocols ("Lemieux1" and "Lemieux2"), and iii) apply Lemieux oxidation to fish fillets and livers naturally contaminated with MCs-producing cyanobacteria and to freshwater mussels contaminated with MCs in laboratories. Optimization of the "Lemieux1" protocol, in particular in the oxidation and SPE (solid phase extraction) steps improved the method's yields on the fresh fish fillet matrix (from <5 % to around 40 %). Moreover, several quantification methods have been compared through various calibration techniques (solvent calibration curve, matrix-matched calibration curve, oxidized MC-LR calibration curve and also by testing the addition of d3-MMPB as an internal standard). Comparison with the "Lemieux2" protocol showed the best results on the same matrix, with yields of around 65 %. MMPB was analyzed using this "Lemieux 2" protocol, in livers of carps sampled during an episode of cyanobacteria proliferation, at concentrations ranging from 17.9 to 27.5 µg/kg MMPB and at concentrations ranging from 50 to 2890 µg/kg MMPB in freshwater mussels laboratory contaminated to MCs.
Asunto(s)
Microcistinas , Oxidación-Reducción , Microcistinas/análisis , Animales , Cromatografía Liquida , Peces , Bivalvos , Espectrometría de Masas/métodos , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Microcystins (MCs) are cyclic heptapeptidic toxins produced by many cyanobacteria. Microcystins can be accumulated in various matrices in two forms: a free cellular fraction and a covalently protein-bound form. To detect and quantify the concentration of microcystins, a panel of techniques on various matrices (water, sediments, and animal tissues) is available. The analysis of MCs can concern the free or the total (free plus covalently bound) fractions. Free-form analyses of MCs are the most common and easiest to detect, whereas total-form analyses are much less frequent and more complex to achieve. The objective of this review is to summarize the different methods of extraction and analysis that have been developed for total forms. Four extraction methods were identified: MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) method, deconjugation at basic pH, ozonolysis, and laser irradiation desorption. The study of the bibliography on the methods of extraction and analysis of the total forms of MCs showed that the reference method for the subject remains the MMPB method even if alternative methods and, in particular, deconjugation at basic pH, showed results encouraging the continuation of the methodological development on different matrices and on naturally-contaminated samples.