Extraction recoveries and stability of diarrhetic shellfish poisoning (DSP) toxins in naturally contaminated samples.

Instituto Nacional dos Recursos Biológicos-IPIMAR, Lisbon, Portugal.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment (Impact Factor: 1.8). 03/2009; 26(2):229-35. DOI: 10.1080/02652030802290530
Source: PubMed


During the last few years the occurrence of a high percentage of esters of diarrhetic shellfish poisoning (DSP) toxins has been observed in shellfish from the Portuguese coast. Most of the commercial bivalves contain DSP toxins in ester forms, either acyl derivatives of okadaic acid (OA) or of dinophysistoxin-2 (DTX-2). The stability of these toxins in shellfish tissues and in raw methanol extracts was investigated in two different naturally contaminated species, mussel and carpet shell, over a 4-week period. The results for both species revealed that DSP toxins were more stable in tissue than in raw methanol extracts. Losses of DSP toxins were seen in the first 2 weeks and were more than 30%, but after that a period of stabilization was observed. The decrease was due probably from losses of esters of OA and DTX-2, the free toxins were stable over the period studied. The extraction most commonly used for chemical and biochemical assays relied on methanolic extraction with aqueous 80% methanol. In this work we have tested the extraction solvent on the extractability of DSP toxins from several naturally contaminated species. A single dispersive extraction with methanol, with solvent ratios of 70%, 80%, 90% and 100%, were tested. After alkaline hydrolysis of esterified toxins and clean-up with hexane and dichloromethane, the samples were analysed by liquid chromatography-mass spectrometry (LC-MS). The recovery of DSP toxins increased with increasing percentages of methanol up to 90%. A decrease in recovery with 100% methanol was observed probably due to problems during the liquid-liquid partitioning.

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Available from: Susana Sousa Gomes, Oct 01, 2015
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    ABSTRACT: Portuguese bivalves are recurrently contaminated with okadaic acid (OA) and dinophysistoxin-2 (DTX2), found mainly in esterified forms. Throughout the years different conditions have been reported in the literature for releasing the parent toxins through an alkaline hydrolysis step, in order to simplify their detection by HPLC-FLD or LC-MS. In order to clearly understand toxin stability and reaction end-point the binominous temperature/time course and base concentration were studied using naturally contaminated bivalve samples. The results showed a strong temperature dependence of the reaction. At 60 degrees C and 70 degrees C the hydrolysis was fast, and 40min were sufficient for maximal recovery of OA and DTX2, while at 40 degrees C and 50 degrees C it was only complete after 100min and 60min, respectively. At room temperature the reaction was slow and incomplete even after 2h. Stability of OA and DTX2 in semi-purified bivalve matrix at 70 degrees C for 2h was demonstrated. Concentrations of sodium hydroxide lower than 2.5M, corresponding to a final incubation concentration of 0.23M, resulted in incomplete release of parent toxins, demonstrating that high concentrations are needed when taking into account the dilution in the supernatant extract.
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    ABSTRACT: Okadaic acid (OA), a key diarrheic shellfish poisoning (DSP) toxin will possibly arouses DSP symptoms by consuming the contaminated shellfish. The DSP toxins are stable at high temperatures, and long-term DSP toxicity is carcinogenic. Therefore a fast and reliable analytical method for the detection of OA and analogues in shellfish is worth developing. In this paper, a direct competitive ELISA (dcELISA) for detecting OA in seafood was developed based on monoclonal antibody (McAb). The regression equation of direct competitive ELISA was y=-38.831x+130.25 with a coefficient correlation of R 2 =0.989 8. The linear range and the limit of detection (LOD) were 1.56-75 ng/mL and 1.09 ng/mL, respectively. The average recovery of OA-spiked sample was 86.05% with the coefficient of variation (CV) of 7.83%. The results indicated that the developed dcELISA is a fast, sensitive and convenient assay and could be used for detecting of OA in seafood.
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