ABSTRACT
This paper shows the detection of Diarrhetic Shellfish Poison (DSP) phycotoxins, using HPLC-FLD with pre-column derivatization procedure and HPLC-MS methods, in the analysis of shellfish extracts tested positive with the official DSP mouse bioassay. The shellfish samples were collected in Chiloe Island, Southern of Chile. The amount of Dinophysistoxin-3 (DTX-3) measured in the shellfish extracts were in average above the international safe limits for DSP content in the shellfish extracts analyzed. As internal control of detection and recovery, DTX-1 analytical standard was spiked into dichloromethane-clean shellfish extracts in order to calculate de extraction recovery of DTX-1. The average recovery was 97%. From all DSP toxins analyzed, the hydrolyzed extract samples appeared mainly DTX-3 in concentrations ranging from 99.40 +/- 1.22 to 257.73 +/- 12.46 ng/g digestive-glands. The acyl-Okadaic Acid (acyl-OA) was also detected in some samples, ranging from 1.02 +/- 1.4 to 3.07. +/- 1.6 ng of DSP toxin/g digestive-glands. This is the first report of acyl-OA ever found in Chilean shellfish samples. This data shows that shellfish samples were contaminated with a complex DSP toxins profile, in which DTX-3 is the major DSP toxin component, followed by DTX-1 and the acyl-OA as the minor one. The important findings showed in this study are the presence of both acyl-derivates (DTX-3 and Acyl-OA) which are the product of a main metabolic biotransformation that occurred inside the shellfish, in order to chelate DTX-1 and OA, transforming them into DTX-3 and the acyl-OA respectively. This metabolic biotransformation must be performed to avoid self-inhibition of their Protein Phosphatase 2A done by DTX-1 and OA, since both acyl-derivates (DTX-3 and acyl-OA) do not inhibit Protein Phosphatase 2A. This complex DSP toxins profile and the permanent presence of both acyl-derivates (DTX-3 and Acyl-OA) could explain the permanent diarrhea symptoms that experience patients who have ingested cooked shellfish in the southern of Chile. This diarrhea is not associated to Vibrio parahaemolyticus or other enteropathogens as had been suggested before. The massive shellfish consumption is an important Chilean cultural habit and now has become a major health issue in the southern of Chile.