Formation of Azaspiracids-3,-4,-6, and-9 via Decarboxylation of Carboxyazaspiracid Metabolites from Shellfish

The azaspiracid (AZA) class of phycotoxins has been responsible for extended closures of shellfisheries in various locations around Europe, where levels of AZA1-3 are regulated in shellfish. Since their discovery in 1995, AZAs have been the focus of much research, resulting in the discovery of numerous analogues. During studies of procedures for processing of AZA-contaminated mussels (Mytilus edulis), an unusual phenomenon was observed involving AZA3. In uncooked tissues, AZA3 levels would increase significantly when heated for short periods of time in the absence of water loss. A similar increase in AZA3 concentrations occurred during storage of shellfish tissue reference materials for several months at temperatures as low as 4 degrees C. Concentrations of AZA1 and AZA2 did not change during these experiments. Several possible explanations were investigated, including an AZA3-specific matrix effect upon heating of tissues, release of AZA3 from the matrix, and formation of AZA3 from a precursor. Preliminary experiments indicated that toxin conversion was responsible, and more detailed studies focused on this possibility. LC-MS analysis of heating trials, deuterium labeling experiments, and kinetic studies demonstrated that a carboxylated AZA analogue, AZA1 7, undergoes rapid decarboxylation when heated to produce AZA3. Heat-induced decarboxylation of AZA19, AZA21, and AZA23 to form AZA6, AZA4, and AZA9, respectively, was also demonstrated. This finding is of great significance in terms of procedures used in the processing of shellfish for regulatory analysis, and it exemplifies the role that chemical analysis can play in understanding the contribution of metabolic processes to the toxin profiles observed in shellfish samples.