Liquid chromatography with fluorimetric, mass spectrometric and tandem mass spectrometric detection for the investigation of the seafood-toxin-producing phytoplankton, Dinophysis acuta

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 C-18 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 mu l 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 (mu LC-MS-MS) analysis of the free toxins and mu LC-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 mit 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 mu l injected. These studies showed that D. acuta was the progenitor of DTX-2 in shellfish. (C) 1997 Elsevier Science B.V.