Workflow of the pre-chromatographic 'Lawrence' method for bivalves contaminated with Gymnodinium catenatum's paralytic shellfish poisoning toxins

Saxitoxin and its analogues are potent neurotoxins that originate the acute human neurological syndrome of paralytic shellfish poisoning (PSP) via bivalve vectors. The prechromatographic oxidation method, known commonly as the ?Lawrence method?, was approved as the official testing method to replace mouse testing in the European Union. For solving the complex overlap of toxin oxidation products, it resorts to two solid-phase extraction steps (SPE) and two oxidation steps, making it a very labour-intensive method. For bivalves contaminated with Gymnodinium catenatum, hydrogen peroxide oxidation can quantify at once several N1?H analogues: dcGTX2+3, dcSTX, C1+2 and B1 toxins. For the remaining N1?OH analogues: dcNEO, C3+4 and B2, separate SPE and oxidation steps are required, leading to sample dilution and often adding a null result to the total quantifiable toxicity. In order to refine the routine application of the Lawrence method, a PSP episode that took place in the Autumn of 2018 in Portugal due to a G. catenatum bloom was studied in detail. A workflow was prepared based on total detectable toxin concentration obtained by the peroxide oxidation to investigate whether the steps leading to the quantification of dcNEO, C3+4 and B2 will be further required or not. For samples containing a concentration of N1?H toxins below 1.6 ?mol/kg, no further processing was required. For all samples above 3 ?mol/kg, B2 and dcNEO should be quantified. For all samples above 10 ?mol/kg, C3+4 should be quantified. Between 1.6 and 3.2 ?mol/kg, although B2 could be quantified in more than 65% of the samples, its low toxicity would add to total toxicity an average of only 42 ?g STX.2HCl equiv./kg. This workflow is not valid for species with extensive carbamoylase activity or in the case of Alexandrium profiles.

Automated summary

Saxitoxin and its analogues are neurotoxins causing paralytic shellfish poisoning, and the Lawrence method is a labor-intensive prechromatographic oxidation technique approved in the EU to replace mouse testing. The method uses two solid-phase extraction steps and two oxidation steps to quantify various analogues in contaminated bivalves. A study on a PSP episode in Portugal in 2018 led to a workflow based on total detectable toxin concentration, determining the need for further quantifying certain analogues. Samples below 1.6 μmol/kg N1?H toxins required no further processing, while samples above 3 μmol/kg needed quantification of B2 and dcNEO, and above 10 μmol/kg required quantification of C3+4. Samples between 1.6 and 3.2 μmol/kg showed that although B2 could be quantified in most samples, its low toxicity only added a small amount to the total toxicity.