Rapid determination of polyether marine toxins using liquid chromatography–multiple tandem mass spectrometry

Abstract

The diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxins (DTX); pectenotoxin-2 (PTX2) and pectenotoxin-2 seco acids, were determined in marine phytoplankton, Dinophysis acuta, and mussels ( Mytilus edulis) collected along the southwest coast of Ireland. Liquid chromatography–multiple tandem mass spectrometry (LC–MS/MS) was employed for the simultaneous determination of a series of marine toxins with large polarity differences. Separation of five DSP toxins was achieved on a C 18 column (Luna-2, 150 mm × 2.1 mm, 5 μm) using an acetonitrile–water gradient with ammonium acetate as an eluent modifier. Electrospray ionisation (ESI) in negative mode, was used to generate the molecule related ion, [M − H] −, for each toxin. To develop a multiple reaction monitoring (MRM) method, fragmentation studies were performed to determine the optimum precursor-product ion combinations: OA (803/255), DTX2 (803/255), DTX1 (817/255), PTX2SAs (875/137) and PTX2 (857/137). This highly sensitive method had detection limits better than 1 pg (on-column). Linear calibrations were obtained for shellfish extracts that were spiked with toxins, OA, 0.007–1.00 μg/ml ( r 2 = 0.9993, N = 3) and DTX2, 0.054–8.5 μg/ml ( r 2 = 0.9992, N = 3). Good reproducibility data were also achieved with %RSD values ( N = 3) ranging from 3.15% (0.56 μg DTX2/ml) to 5.71% (0.14 μg DTX2/ml), for shellfish extracts. The method was sufficiently sensitive to permit the determination of DSP toxins in small numbers of picked phytoplankton cells ( N = 12–40). In one sample of D. acuta the average toxin composition per cell was: OA (7.0 pg), DTX2 (11 pg) and PTX2 (7.2 pg).