Evaluation of Various pH and Temperature Conditions on the Stability of Azaspiracids and Their Importance in Preparative Isolation and Toxicological Studies

Abstract

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.

Effect of the industrial steaming on the toxicity, estimated by LC–MS/MS, of mussels exposed for a long time to diarrhetic shellfish poisoning (DSP) toxins

“…McCarron et al (2009) demonstrated that one involved mechanism was the decarboxylation of several AZA analogues that contain a carboxylic function. Notwithstanding, Alfonso et al (2008) found that protein digestion of the tissues of AZA-containing mussels increases the amount of these toxins that could be extracted with MeOH, suggesting that protein denaturation during cooking could also contribute to the increase of AZAs concentration with this process.…”

“…The second one is the presence of nondetected toxin derivatives that could be converted to the main toxins by the thermal treatment, as was described with azaspiracids (Hess et al, 2005;McCarron et al, 2009). The third one is an incomplete detection of the toxin content in raw or steamed mussel (or in both) by the method used, as previously suggested, to explain the increase of concentration of some azaspiracids with cooking (Alfonso et al, 2008).…”

“…It is known that cooking or, in general, thermal processing of molluscs produces dehydration of the meat and consequently a weight decrease, while the degradation of lipophilic toxins is zero or very limited (Alfonso et al, 2008;Hess et al, 2005;McCarron, Kilcoyne, & Hess, 2008;McCarron, Kilcoyne, Miles, & Hess, 2009;Picot et al, 2012, andadditional comments in EFSA Panel on Contaminants in the Food Chain, 2009b). In this sense, an increase in the concentration of the okadaic acid group of toxins, and other lipophilic toxins such as azaspiracids (AZAs), due to industrial steaming should be expected.…”

High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

“…Due to potential stability problems of AZAs, PnTX-E and PTX2 in acidic conditions [39][40][41] (and the acid present in the certified calibrant to enhance storage capacity of 13-desmeSPX-C), three initial toxin mixtures were prepared in methanol: (i) Mix-1 containing PTX2, AZA1 to 3, OA, DTX1 and 2, PnTX-E, YTX, homo-YTX and DA; (ii) Mix-2 containing 13-desmeSPX-C, GYM-A, PnTX-F, PnTX-G and DA and (iii) BTX1,2-mix with BTX1 and BTX2. These stock solutions were then serially diluted in MeOH using a Hamilton Microlab diluter-dispenser (Hamilton Company, Reno, NV).…”

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids

“…The presence of protein-bound AZAs was further suggested in a later work examining the stability of AZAs [43]. However, in studies conducted as part of this work, there was no evidence of significant amounts of protein-bound toxins in CRM-AZAMus; therefore, there is no perceived risk of changes to the certified concentrations (see Electronic Supplementary Material (ESM)).…”