domoic-acid and yessotoxin

A freeze-dried mussel tissue-certified reference material (CRM-FDMT1) was prepared containing the marine algal toxin classes azaspiracids, okadaic acid and dinophysistoxins, yessotoxins, pectenotoxins, cyclic imines, and domoic acid. Thus far, only a limited number of analogues in CRM-FDMT1 have been assigned certified values; however, the complete toxin profile is significantly more complex. Liquid chromatography-high-resolution mass spectrometry was used to profile CRM-FDMT1. Full-scan data was searched against a list of previously reported toxin analogues, and characteristic product ions extracted from all-ion-fragmentation data were used to guide the extent of toxin profiling. A series of targeted and untargeted acquisition MS/MS experiments were then used to collect spectra for analogues. A number of toxins previously reported in the literature but not readily available as standards were tentatively identified including dihydroxy and carboxyhydroxyyessotoxin, azaspiracids-33 and -39, sulfonated pectenotoxin analogues, spirolide variants, and fatty acid acyl esters of okadaic acid and pectenotoxins. Previously unreported toxins were also observed including compounds from the pectenotoxin, azaspiracid, yessotoxin, and spirolide classes. More than one hundred toxin analogues present in CRM-FDMT1 are summarized along with a demonstration of the major acyl ester conjugates of several toxins. Retention index values were assigned for all confirmed or tentatively identified analogues to help with qualitative identification of the broad range of lipophilic toxins present in the material.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Freeze Drying; Kainic Acid; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Reference Standards; Spiro Compounds; Tandem Mass Spectrometry

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

Topics: Animals; Chromatography, Liquid; Freeze Drying; Furans; Kainic Acid; Macrolides; Marine Toxins; Mass Spectrometry; Mollusk Venoms; Mytilus edulis; Okadaic Acid; Oxocins; Pyrans; Reference Standards; Seafood; Spiro Compounds

Different clinical types of algae-related poisoning have attracted scientific and commercial attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, mainly due to the low sensitivity and the absence of specialized variations. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins. This chapter describes state-of-the-art LC-MS/MS methods for the detection and quantitation of different classes of phycotoxins in shellfish matrices. These classes include the highly hydrophilic paralytic shellfish poisoning (PSP) toxins. Hydrophilic interaction liquid chromatography (HILIC) has been shown to be useful in the separation of PSP toxins and is described in detail within this chapter. Another important class of phycotoxins is diarrhetic shellfish poisoning (DSP) toxins. This group traditionally comprises okadaic acid and dinophysistoxins (DTXs), pectenotoxins (PTXs), and yessotoxins (YTXs). The most recently described shellfish poisoning syndrome, azaspiracid shellfish poisoning (AZP) is caused by azaspiracids, which in turn are diarrhetic, but usually are treated separately as AZP. The last group of regulated shellfish toxins is the amnesic shellfish poisoning (ASP) toxin domoic acid, produced by species of the genus Pseudo-nitzschia.

Topics: Chromatography, Liquid; Kainic Acid; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Shellfish; Spiro Compounds; Tandem Mass Spectrometry

The effect of gamma-irradiation on concentrations of hydrophilic and lipophilic phycotoxins has been investigated by use of HPLC-UV and LC-MS. Pure toxins in organic solvents and toxins in mussel (Mytilus edulis) tissues were irradiated at three different doses. In solution all toxin concentrations were reduced to some extent. Most severe decreases were observed for domoic acid and yessotoxin, for which the smallest dose of irradiation led to almost complete destruction. For pectenotoxin-2 the decrease in concentration was less severe but still continuous with increasing dose. Azaspiracid-1 and okadaic acid were the least affected in solution. In shellfish tissue the decrease in toxin concentrations was much reduced compared with the effect in solution. After irradiation at the highest dose reductions in concentrations were between ca. 5 and 20% for the lipophilic toxins and there was no statistical difference between control and irradiated samples for azaspiracids in tissue. Irradiation of shellfish tissues contaminated with domoic acid led to a more continuous decrease in the amount of the toxin with increasing dose. The effect of irradiation on the viability of microbial activity in shellfish tissues was assessed by using total viable counting techniques. Microbial activity depended on the type of shellfish and on the pretreatment of the shellfish tissues (with or without heat treatment). As far as we are aware this is the first investigation of the effectiveness of irradiation as a technique for stabilising tissue reference materials for determination of phycotoxins. Our results suggest that this technique is not effective for materials containing domoic acid. It does, however, merit further investigation as a stabilisation procedure for preparation of shellfish tissue materials for some lipophilic toxins, in particular azaspiracids. Chemical structures of the toxins investigated in the study.

Topics: Animals; Calibration; Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Chromatography, Liquid; Ethers, Cyclic; Gamma Rays; Kainic Acid; Macrolides; Marine Toxins; Mass Spectrometry; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Reference Values; Shellfish; Spectrophotometry, Ultraviolet; Spiro Compounds

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Topics: Actins; Animals; Biological Assay; Bivalvia; Cell Line, Tumor; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers, Cyclic; Fluorometry; Humans; Kainic Acid; Marine Toxins; Methanol; Mice; Mollusk Venoms; Okadaic Acid; Oxocins; Phosphoprotein Phosphatases; Reproducibility of Results; Saxitoxin; Sensitivity and Specificity; Shellfish