| Abstract | Toxins responsible for incidents of shellfish poisoning, including okadaic acid (OA), dinophysistoxins-1 and -2 (DTX1, DTX2), pectenotoxins, brevetoxins and spirolides can occur as acyl esters in plankton and shellfish creating complications during routine analysis. Alkaline hydrolysis is usually used to release free OA/DTX and two analyses are conducted, before and after hydrolysis, to determine the amounts of unesterified and esterified toxin. However, alkaline hydrolysis is not suitable for all acylated compounds such as pectenotoxins or spirolides. Harsh alkaline conditions destroy the parent toxins. An alternative approach to alkaline hydrolysis is the use of enzymes. Two enzymatic methods were developed for the hydrolysis of 7-O-acyl fatty acid esters (“DTX3”) and the carboxylate esters of OA (“diol-esters”). Lipase (EC 3.1.1.3) was found to be specific for complete conversion of DTX3 compounds to OA, DTX1 and DTX2. OAdiol was hydrolyzed completely to OA by an esterase (EC 3.1.1.1). It has also been determined that the lipase is able to hydrolyze acyl esters of spirolides and the esterase is able to convert the acyl esters of pectenotoxins, without any degradation of parent compounds. Applications to contaminated Norwegian shellfish samples containing acyl esters of OA and DTX1 have shown complete hydrolysis within 4 hours. Analytes, including the acyl esters, the released toxins and even the released fatty acids, were monitored using LC-MS. Enzymatic hydrolysis has been shown to have potential as an alternative to the conventional alkaline hydrolysis procedure in preparation of shellfish samples for analysis of toxins. |
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