Abstract
The diarrhoeic shellfish poisoning (DSP) toxins, okadaic acid (OA) and its isomer, dinophysistoxin-2 (DTX-2), were determined in the marine phytoplankton, Dinophysis acuta, harvested in Ireland. Unialgal samples (22–100 cells) were extracted and derivatised using 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP) and analysed by liquid chromatography (LC). Isocratic elution on a C18 reversed-phase column, with fluorimetric detection, was used to determine OA (58±7 pg/cell) and DTX-2 (78±14 pg/cell). The detection limit was 0.1 ng OA/20 μl injection using ADAM. Gradient LC, using a polymeric bonded phase, successfully separated mixtures containing both the ADAM and BAP derivatised toxins. Identification of DSP toxins was confirmed using isocratic micro LC with tandem mass spectrometric (μLC–MS–MS) analysis of the free toxins and μLC–MS of the BAP-derivatised toxins with an ionspray (IS) interface, coupled to an atmospheric pressure ionisation (API) source. Collision induced dissociation (CID) ion mass spectra of the protonated molecule, [M+H]+, at m/z 805 for OA and DTX-2, identified three diagnostic fragment ions for each analyte which were used for selected reaction monitoring (SRM) LC–MS–MS analysis. The detection limit for OA and DTX-2 was 0.025 ng/0.2 μl injected. These studies showed that D. acuta was the progenitor of DTX-2 in shellfish.
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