Elucidation of the mass fragmentation pathways of the polyether marine toxins, dinophysistoxins, and identification of isomer discrimination processes.

RATIONALE: Most of the liquid chromatography/mass spectrometry (LC/MS) methods that have been developed for the analysis of Diarrhetic Shellfish Poisoning (DSP) toxins in shellfish and algae samples have been unable to differentiate the isomers okadaic acid (OA) and dinophysistoxin-2 (DTX2), unless separated by chromatography. Since there are many bioconversion products of these compounds it is imperative to determine characteristic product ions, which can provide unequivocal identification of OA and DTX2 and their analogs. METHODS: Using electrospray ionization, the fragmentation processes for two types of precursor ions, [M+Na](+) and [M-H](-), of the polyether marine toxins, dinophysistoxins (DTXs), were studied using a hybrid linear ion trap Orbitrap mass spectrometer which provided high mass accuracy data in combination with multiple tandem mass (MS(n)) spectra. Three structurally related toxins were compared; okadaic acid (OA), dinophysistoxin-2 (DTX2) and dinophysistoxin-1 (DTX1). A quick multiple reaction monitoring (MRM) LC/MS/MS method was developed utilizing the characteristic precursor/product ion mass transitions. RESULTS: Comparison of the high-resolution product ion, [M-H](-), spectra of these toxins featured dominant signals that resulted from two six-centered rearrangements and previously proposed fragmentation pathways for the ion of m/z 321 and 293 have been corrected and identified. By contrast, the [M+Na](+) product ion spectra only revealed distinctive ions for the isomers, OA (m/z 595, 443 and 151) and DTX2 (m/z 581, 429 and 165). To illustrate the benefits of this study, a mass selective LC/MS/MS method was developed in which the isomers OA and DTX2 co-eluted but were distinguished using the mass transitions, m/z 827/595, 827/443 (OA) and m/z 827/581, 827/429 (DTX2). CONCLUSIONS: Comparison of OA, DTX2 and DTX1 led to the correction of proposed negative ion mode fragmentation pathways. Through extensive study and comparison of the [M+Na](+) product ion spectra, distinctive product ions were identified which allowed for these compounds to be identified and distinguished without separation for the first time.

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

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 C18 column (Luna-2, 150 mm x 2.1 mm, 5 microm) 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 microg/ml (r2 = 0.9993, N = 3) and DTX2, 0.054-8.5 microg/ml (r2 = 0.9992, N = 3). Good reproducibility data were also achieved with %RSD values (N = 3) ranging from 3.15% (0.56 microg DTX2/ml) to 5.71% (0.14 microg 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).

Liquid chromatography--multiple tandem mass spectrometry for the determination of ten azaspiracids, including hydroxyl analogues in shellfish.

Azaspiracids (AZAs) are a group of polyether toxins that cause food poisoning in humans. These toxins, produced by marine dinoflagellates, accumulate in filter-feeding shellfish, especially mussels. Sensitive liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS(n)) methods have been developed for the determination of the major AZAs and their hydroxyl analogues. These methods, utilising both chromatographic and mass resolution, were applied for the determination of 10 AZAs in mussels (Mytilus edulis). An optimised isocratic reversed phase method (3 microm Luna-2 C18 column) separated 10 azaspiracids using acetonitrile/water (46:54, v/v) containing 0.05% trifluoroacetic acid (TFA) and 0.004% ammonium acetate in 55 min. Analyte determination using MS3 involved trapping and fragmentation of the [M + H]+ and [M + H - H2O]+ ions with detection of the [M + H - 2H2O]+ ion for each AZA. Linear calibrations were obtained for AZA1, using spiked shellfish extracts, in the range 0.05-1.00 microg/ml (r2 = 0.997) with a detection limit of 5 pg (signal : noise = 3). The major fragmentation pathways in hydroxylated azaspiracids were elucidated using hydrogen/deuterium (H/D) exchange experiments. An LC-MS3 method was developed using unique parent ions and product ions, [M + H - H2O - CgH10O2R1R3]+, that involved fragmentation of the A-ring. This facilitated the discrimination between 10 azapiracids, AZA1-10. Thus, this rapid LC-MS3 method did not require complete chromatographic resolution and the run-time of 7 min had detection limits better than 20 pg for each toxin.