Development of a method for the identification of azaspiracid in shellfish by liquid chromatography–tandem mass spectrometry

Azaspiracid is the main toxin responsible for a number of recent human intoxications in Europe resulting from shellfish consumption. The first micro liquid chromatography–tandem mass spectrometry (micro-LC–MS–MS) method was developed for the determination of this novel shellfish poisoning toxin in mussels. The analyte was extracted from whole mussel meat with acetone and chromatographed on a C18 reversed-phase column (1.0 mm I.D.) by isocratic elution at 30 μl/min with acetonitrile–water (85:15, v/v), containing 0.03% trifluoroacetic acid. The toxin was ionised in an ionspray interface operating in the positive ion mode, where only the intact protonated molecule, [M+H]+, was generated at m/z 842. This served as precursor ion for collision-induced dissociation and three product ions, [M+H−nH2O]+ with n=1–3, were identified for the unambiguous toxin confirmation by selected reaction monitoring LC–MS–MS analysis. A detection limit of 20 pg, based on a 3:1 signal-to-noise ratio, was achieved for the analyte. This LC–MS–MS method was successfully applied to determine azaspiracid in toxic cultivated shellfish from two regions of Ireland.