Detection of okadaic acid esters in the hexane extracts of Spanish mussels

Two types of low polar derivatives of OA and dinophysitoxins have been reported in shellfish or in phytoplankton: 7-O-acyl esters containing a fatty acyl group attached through the 7-OH group and diol esters in which the carboxylic group of the toxins has been esterified. These compounds cannot be directly detected by liquid chromatography and fluorimetric detection as 9-anthryldiazomethane derivatives, owing in the first case to their low polarity and high molecular weight, and in the second case because they have the carboxylic group esterified. All of them must be hydrolysed before derivatization to be detected as Adam derivatives of the corresponding non-acylated toxins. In the Lee procedure, after extraction of the shellfish digestive glands with 80% methanol, a liquid-liquid partition with a non-polar solvent such as hexane is carried out in order to remove non-polar lipids. The presence of non-polar toxins was investigated in Spanish mussels and confirmed in the hexane layer, usually discarded in conventional extraction procedures, by analysis of the alkaline hydrolysis products. A preferred solubilization of these toxins in a non-polar solvent like hexane is reported. The inclusion of a hydrolytic step of the hexane extract in the general procedure is suggested in order to monitor the contribution of non-polar diarrhoetic shellfish poisons (DSPs) to the total DSP shellfish toxicity. This is the first report of DSPs other than OA and DTX2 in Spanish mussels.