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

Characterization of F-actin depolymerization as a major toxic event induced by pectenotoxin-6 in neuroblastoma cells

Pectenotoxins are a group of marine toxins produced by dinoflagellates and formerly included within the group of diarrhetic shellfish poison or toxins (DSP or DST) because of their physico-chemical properties. However, toxicological data on pectenotoxins are still very scarce and its mechanism of action is largely unknown, but toxicity in laboratory animals has been demonstrated by intraperitoneal injection. In this report, we present results of in vitro toxicological assessment of pectenotoxin-6, a derivative of the parental toxin pectenotoxin-2 first isolated from toxic scallops. Results obtained demonstrate an specific time- and dose-dependent depolymerization of F-actin in neuroblastoma cells exposed to pectenotoxin-6 (half-maximal effect about 700 nM at 24 hr). The change in the state of polymerization of actin was not accompanied by other major effects on specific signal transduction pathways or cell survival rate. Pectenotoxin-6 does not modify cytosolic calcium levels either in a calcium containing or calcium-free medium in human lymphocytes. Only when capacitative calcium influx was first activated, the toxin addition significantly decreased the following calcium influx. In these cells, pectenotoxin-6 only modifies cAMP (adenosine 3',5'-cyclic monophosphate) levels in calcium-free conditions. In addition, no effect on cell attachment or apoptosis induction was observed at micromolar concentrations of pectenotoxin-6. Therefore, we conclude that cytoskeletal disruption is a key mechanism of PTX6-induced toxicity in eukaryotic cells. (C) 2002 Elsevier Science Inc. All rights reserved