Validation of a new tetrodotoxin extraction protocol in samples of mussel, oyster and razor clam for its application in mELISA assay

Identifier:  TFG:2137

Handle: https://hdl.handle.net/20.500.11797/TFG2137

Authors:  Reverté Navarro, Jaume

Abstract:
During the last 10 years, the presence of tetrodotoxin (TTX) in European shellfish has been reported in numerous cases. This situation highlighted an important problem of food safety in all Europe. To date, none of the screening methods for the detection of TTX had been validated for the analysis of shellfish samples. For this reason, the analysis had to be performed with LC-MS/MS technology. The elevated costs and complexities derived from this technique boosted the innovation and the creation of new technologies for detection of TTX in shellfish. From this innovation, the mELISA assay was born. This immunoassay proved to be valid and specific to TTX, however, it also showed a lot of problems with the matrix effects produced by the shellfish extracts. In this study, a new extraction protocol has been evaluated for its application on mELISA assay with the aim of reducing the matrix effects. By the analysis of the extract obtained with the new protocol, we were able to set new working concentrations where the matrix effect disappears. These working concentrations are: 150 mg/mL (razor clam), 25 mg/mL (oyster) and 5 mg/mL (mussel). Working with these concentrations, TTX detection with mELISA was only possible with razor clam and oysters (detection concentrations of 40 μg/kg and 640 μg/kg, respectively). We also appreciated an inefficiency of our extraction method. When working with razor clams, only the 70 % of the total TTX present in the samples was recovered after the extraction process, and with oyster’s, the recovery was only 15 %. Although recoveries were not high in all cases, we proved that the new extraction protocol is still valid for the detection of TTX in the case of razor clam and oyster.