In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 µg kg-1 for OA, AZA1 and PTX2 and 1,000 µg kg-1 for YTX. For SPX1, 400 µg kg-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 µg kg-1 OA equivalents and 30 µg kg-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests

Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2.

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Azaspiracid Toxins: Toxicological Profile

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 studie