Evaluation of various pH and temperature conditions on the stability of azaspiracids and their importance in preparative isolation and toxicological studies.

Azaspiracids (AZAs) are a group of shellfish toxins that were discovered in mussels from Irish waters in 1995. Because of the rare occurrence of poisoning incidents, the toxicity of the compounds is a continued matter of debate. Neither their mechanism of action nor their pharmacokinetic behavior has been elucidated, principally because of the lack of standards and reference tissues. Procedures to isolate AZAs from contaminated shellfish or to synthesize them have been developed; in particular, the procedures used for the preparative isolation of these toxins are currently being improved. The present paper describes the stability of AZAs in an array of pH and temperature conditions in methanolic solution, in shellfish tissue, and in aqueous mixtures of acids and shellfish tissues. Strong acids such as hydrochloric and formic acid led to rapid degradation of AZA1 at mM concentration, while the weaker acetic acid required harsher temperature conditions (70 degrees C) and greater concentrations to show similar effects. AZAs showed much greater stability in aqueous acidic mixtures with shellfish tissues, suggesting a significant protective effect of the matrix. A mechanism for the acid-catalyzed degradation is proposed, supported by mass spectral evidence from some of the degradation products. Strong bases (sodium hydroxide) also showed a detrimental effect on AZA1; however, weaker bases (ammonium hydroxide) did not lead to degradation over 24 h at room temperature. Finally, the toxic potential of acid degradation products of AZAs was found to be dramatically reduced compared to the parent compounds, as assessed through cytotoxicity.