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Although boronates bind reversibly to many vic-diols, and vic-diols are a common structural feature of many algal toxins, this boronate-diol interaction has not been exploited for clean-up and concentration of algal toxins. We have developed methodology for cleaning up and concentrating azaspiracids from mussel extracts using a polymer-bound boronic acid. The method removed the colour from the extract, as well as the majority of interferences and matrix effects normally seen in LC-MS analyses of azaspiracids in mussels. We then developed a modified approach using aryl boronates to selectively capture tetrodotoxin, which also contains a vic-diol, and many of its congeners. The tetrodotoxin analogues were selectively released from the boronate material to yield very clean extracts containing tetrodotoxin analogues and very little else. Potential interferences in LC-MS analyses, such as arginine and other amino acids, were completely eliminated, resulting in a simplified analysis. Boronate technology may also be useful for preparative purification of larger amounts of algal toxins for the preparation of reference materials and toxicological studies. We are now examining the applicability of boronate techniques to the analysis and purification of a range of other algal toxins that contain vic-diols, including ciguatoxins, palytoxins, pectenotoxins and the okadaic acid group., The 18th International Conference on Harmful Algae, Oct. 21-26, 2018, Nantes, France

NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Chromatography A. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Chromatography A, [Volume 1217, Issue 45 (5 November 2010)] doi:10.1016/j.chroma.2010.09.020 http://www.sciencedirect.com/science/article/pii/S0021967310012409, peer-reviewed, Considerable efforts are being made worldwide to replace in vivo assays with instrumental methods of analysis for the monitoring of marine biotoxins in shellfish. Analysis of these compounds by the preferred technique of LC-MS/MS is challenged by matrix effects associated with shellfish tissue components. In methods validation, assessment of matrix interferences is imperative to ensure the accuracy of analytical results. We evaluated matrix interferences in the analysis of okadaic acid (OA) and azaspiracid 1 (AZA1) in mollucscan shellfish by using a conventional acidic method on electrospray triple stage quadrapole (TSQ) and hybrid quadrupole time of flight (QToF) instruments, with matrix matched standards for several species. Using the acidic method, we found no matrix interferences for OA, and matrix suppression for AZA1, with the TSQ instrument; in contrast, we found matrix enhancement for OA, and no matrix interference for AZA1, with QToF. The suppression of AZA1 signal on the TSQ instrument was due to interfering compounds carried over from previous injections. The degree of suppression was dependent on the tissue type, ranging from 20 to 70%. Several strategies were evaluated to eliminate these interferences, including the partitioning of the extract with hexane, optimization of the chromatographic

Since 1995, when several people became ill following consumption of shellfish from Ireland, azaspiracids (AZAs) have been known as shellfish toxins, causing symptoms associated with gastro-intestinal disorders, including diarrhoea, vomiting, headaches and others. The aims of the ASTOX-project were to provide control tools for the analysis of AZAs in shellfish, i.e. calibration standards and tissue reference materials (RMs), and to clarify the toxicity of AZAs in qualitative and quantitative terms, i.e. to understand the mode of action of AZAs and to derive a No Observable Adverse Effect Level (NOAEL) for safe consumption of shellfish., Funder: Marine Institute

To date, the putative shellfish toxin azaspiracid 59 (AZA-59) produced by Azadinium poporum (Dinophyceae) has been the only AZA found in isolates from the Pacific Northwest coast of the USA (Northeast Pacific Ocean). Anecdotal reports of sporadic diarrhetic shellfish poisoning-like illness, with the absence of DSP toxin or Vibrio contamination, led to efforts to look for other potential toxins, such as AZAs, in water and shellfish from the region. A. poporum was found in Puget Sound and the outer coast of Washington State, USA, and a novel AZA (putative AZA-59) was detected in low quantities in SPATT resins and shellfish. Here, an A. poporum strain from Puget Sound was mass-cultured and AZA-59 was subsequently purified and structurally characterized. In vitro cytotoxicity of AZA-59 towards Jurkat T lymphocytes and acute intraperitoneal toxicity in mice in comparison to AZA-1 allowed the derivation of a provisional toxicity equivalency factor of 0.8 for AZA-59. Quantification of AZA-59 using ELISA and LC-MS/MS yielded reasonable quantitative results when AZA-1 was used as an external reference standard. This study assesses the toxic potency of AZA-59 and will inform guidelines for its potential monitoring in case of increasing toxin levels in edible shellfish.

Azaspiracids (AZAs) are marine algal toxins that can be accumulated by edible shellfish to cause a foodborne gastrointestinal poisoning in humans. In the European Union, only AZA1, -2 and -3 are currently regulated and their concentration in shellfish is determined through their toxic equivalency factors (TEFs) derived from the intraperitoneal lethal potency in mice. Nevertheless, considering the potential human exposure by oral route, AZAs TEFs should be calculated by comparative oral toxicity data. Thus, the acute oral toxicity of AZA1, -2 and -3 was investigated in female CD-1 mice treated with different doses (AZA1: 135-1100 mu g/kg; AZA2 and AZA3: 300-1100 mu g/kg) and sacrificed after 24 h or 14 days. TEFs derived from the median lethal doses (LD50) were 1.0, 0.7 and 0.5, respectively for AZA1, -2 and -3. In fact, after 24 h from gavage administration, LD(50)s were 443 mu g/kg (AZA1; 95% CL: 350-561 mu g/kg), 626 mu g/kg (AZA2; 95% CL: 430-911 mu g/kg) and 875 mu g/kg (AZA3; 95% CL: 757-1010 mu g/kg). Mice dead more than 5 h after the treatment or those sacrificed after 24 h (doses: = 175 mu g AZA1/kg, >= 500 mu g AZA2/kg and >= 600 mu g AZA3/kg) showed enlarged pale liver, while increased serum markers of liver alteration were recorded even at the lowest doses. Blood chemistry revealed significantly increased serum levels of K+ ions (>= 500 mg/kg), whereas light microscopy showed tissue changes in the gastrointestinal tract, liver and spleen. No lethality, macroscopic, tissue or haematological changes were recorded two weeks post exposure, indicating reversible toxic effects. LC-MS/MS analysis of the main organs showed a dose-dependency in gastrointestinal absorption of these toxins: at 24 h, the highest levels were detected in the stomach and, in descending order, in the intestinal content, liver, small intestine, kidneys, lungs, large intestine, heart as well as detectable traces in the brain. After 14 days, AZA1 and AZA2 were still detectable in almost all the organs and intestinal content.

Azaspiracids (AZAs) are a toxin group that originate from marine dinoflagellates of the genera Azadinium and Amphidoma. After accumulation of these toxins in edible marine organisms and their subsequent consumption, humans develop a gastrointestinal syndrome referred to as azaspiracid shellfish poisoning (AZP). This syndrome is very similar to diarrheic shellfish poisoning (DSP), with main symptoms appearing after a few hours from consumption and including diarrhea, vomiting, and stomach cramps. Due to extensive metabolism in shellfish, more than 30 analogues have been reported to date, and purified compounds for selected analogues have recently been made available for toxicological studies. Currently, only AZA1, AZA2, and AZA3 are regulated in Europe and internationally; however, more recent evidence suggests that AZA6, AZA17, and AZA19 may also be analogues of importance for estimating the full risk of seafood. Even though animal studies have pointed out target organs (digestive tract, liver, heart, and lung), mechanism of action studies at cellular level are not yet conclusive. While a number of common targets have been excluded (protein phosphatases, kinases, actin depolymerization, G protein-coupled receptors), some evidence points toward ion channel activity of AZAs. Still, in vitro studies do not correlate well with symptoms observed in humans. Also, while some animal studies point toward longer-term effects, no such evidence has been reported from human poisoning events. However, it should be noted that in-depth epidemiological studies are still lacking. Even though all risk assessments have based their evaluation on a single, relatively early poisoning event in 1997, in Arranmore Island, Ireland, producing organisms and toxin occurrences have been reported worldwide, and further occurrence studies should provide a better base for such epidemiological studies