We continue to work on the CRONIC-DSP project -Evaluation of the effects of diarrheal toxins of marine origin (DSP) after continuous ingestion and their neurological implications-, which aims to study the evaluation of the effects of DSP toxins (Diarrhetic Shellfish Poisoning), administered orally through in vivo experiments in mice. Through the analysis of samples, the variation of toxins over time in the organs, tissues or fluids analyzed can be determined, therefore reflecting their bioavailability and excretion.

The detection of DSP toxins, specifically okadaic acid, dinophysistoxin-2 and dinophysistoxin-1 present in blood, urine, feces, brain, stomach, liver, kidney, small intestine and large intestine samples will be carried out by applying the proper extraction protocol.

  1. First, it is necessary to properly homogenize the sample.
  2. Next, a solid-liquid extraction is carried out by adding a solvent affinity to the analyte under study, in this case, methanol. In this step, we manage to extract the analyte and through agitation and centrifugation, the liquid-phase (upper layer) and the matrix (lower layer) are separated. The supernatant rich in the compound of interest is evaporated to dryness. Subsequently, it is reconstituted in an appropriate solvent to promote protein precipitation.
  3. Finally, the extract is filtered through a 0.22 µm centrifugal filter before being analysed by UPLC-MS/MS.

In the analytical process, sample preparation is a crucial stage, because the analysis depends on correct and efficient extraction. The images show the extraction of okadaic acid for a small intestine sample.

This project has been funded by the Ministry of Science and Innovation (MCIN), the State Research Agency (AEI 10.13039/501100011033) and by the European Union EU Next Generation EU-Recovery, Transformation and Resilience Plan (PRTR) through the call for Public-Private Collaboration Projects (CPP) for the year 2021, of the State Program to Promote Scientific-Technical Research and its Transfer, within the framework of the State Plan for Scientific and Technical Research and Innovation 2021-2023.

For the implementation of the activities necessary to achieve the proposed objectives, CRONIC-DSP has brought together an interdisciplinary consortium made up of the SME Cifga Laboratory and the University of Santiago de Compostela (USC).