azaspiracid and pectenotoxin-1

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

A liquid chromatography quadrupole linear ion trap mass spectrometry method with fast polarity switching and a scheduled multiple reaction monitoring algorithm mode was developed for multiclass screening and identification of lipophilic marine biotoxins in bivalve molluscs. A major advantage of the method is that it can detect members of all six groups of lipophilic marine biotoxins [okadaic acid (OA), yessotoxins (YTX), azaspiracids (AZA), pectenotoxins (PTX), cyclic imines (CI), and brevetoxins (PbTx)], thereby allowing quantification and high confidence identification from a single liquid chromatography tandem mass spectrometry (LC-MS/MS) injection. An enhanced product ion (EPI) library was constructed after triggered collection of data via information-dependent acquisition (IDA) of EPI spectra from standard samples. A separation method for identifying 17 target toxins in a single analysis within 12min was developed and tested. Different solid phase extraction sorbents, the matrix effect (for oyster, scallop, and mussel samples), and stability of the standards also were evaluated. Matrix-matched calibration was used for quantification of the toxins. The limits of detection were 0.12-13.6μg/kg, and the limits of quantification were 0.39-45.4μg/kg. The method was used to analyze 120 shellfish samples collected from farming areas along the coast of China, and 7% of the samples were found to be contaminated with toxins. The library search identified PbTx-3, YTX, OA, PTX2, AZA1, AZA2, and desmethylspirolide C (SPX1). Overall, the method exhibited excellent sensitivity and reproducibility, and it will have broad applications in the monitoring of lipophilic marine biotoxins.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Food Analysis; Gas Chromatography-Mass Spectrometry; Humans; Hydrophobic and Hydrophilic Interactions; Imines; Limit of Detection; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Ostreidae; Oxocins; Pectinidae; Pyrans; Reference Standards; Reproducibility of Results; Shellfish; Solid Phase Extraction; Spiro Compounds; Tandem Mass Spectrometry

Chinese shellfish samples were harvested from different locations along the Chinese coast. These shellfish were analyzed by liquid chromatography in combination with mass spectrometry to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), petenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirlides (SPXs) and gymnodimines (GYM). The results revealed the lipophilic toxin profiles varied with shellfish sampling locations. In addition to OA, GYM and YTX derivatives, PTX-2 and its derivatives were found for the first time in the following Chinese shellfish: Crassostrea gigas, Mactra chinensis and Mytilus galloprovincialis. The presence of GYM, YTXs, OA and PTXs in Chinese shellfish collected from regions where no previous record of DSP-neutral toxic compounds was reported. Serious efforts should therefore be made to conduct a phycotoxin monitoring program to detect the presence of lipophilic toxins in biological materials of marine origin, which may ensure that Chinese seafood products do not present a health risk. With respect to suspected carcinogenicity, further research on the distribution and concentrations of toxic compounds are needed, in order to carry out long-term risk assessments, particularly sub-acute and chronic toxicity tests associated with of lower doses.

Topics: Chromatography, High Pressure Liquid; Furans; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Macrolides; Marine Toxins; Mass Spectrometry; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Shellfish; Spiro Compounds

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