Monitoring of regulated and non-regulated biotoxins in marine organisms using liquid-phase microextraction and liquid...

The increase in marine biotoxins produced by microalgae has resulted in a significant rise in their presence in marine organisms. Although regulations have been established to control these compounds, identifying new emerging non-regulated biotoxins is complicated by the lack of available analytical standards. This study aimed to determine different biotoxins in marine organisms by liquid chromatography coupled with high-resolution mass spectrometry using a hybrid quadrupole-time-of-flight analyzer for both targeted and untargeted approaches. Solid-liquid extraction was used to isolate the analytes from the marine organisms, followed by dispersive liquid-liquid microextraction for cleaning and preconcentration purposes. Limits of detection in the 0.002-12.2 ng g^-1 range were obtained for the target analytes, eight regulated biotoxins (azaspiracids 1-5, AZAs 1-5; okadaic acid, OA; dinophysistoxins 1-2, DTXs 1-2) and other six non-regulated biotoxins (gymnodimine, GYM; 13-desmethylspirolide C, 13-SPX C; 13,19-didesmethylspirolide C, 13,19-SPX C; 20-methylspirolide G, 20-SPX G; pinnatoxin G, PnTX G; pectenotoxin 2, PTX-2). Five target analytes, GYM, 13-SPX C, PnTX G, OA and DTX-2, were found in several samples. Additionally, a suspect screening was developed in the untargeted approach considering a database of 456 compounds belonging to same target biotoxins groups. Several biotoxins (16-GYM, GYMs E and a 13-SPX C isomer) were identified in the studied samples. The presence of GYMs E in marine animal tissues has been reported for the first time in this study to the best of our knowledge. Furthermore, in vitro cytotoxicity assays were performed with OA and 13-SPX C on SAF-1 cells as marine organism model.