Intralaboratory validation of a fast and sensitive UHPLC/MS/MS method with fast polarity switching for the analysis of lipophilic shellfish toxins.

This paper shows the results of an intralaboratory validation of a fast method for the determination of lipophilic shellfish toxins working under acidic conditions using ultra-high performance LC (UHPLC) with MS/MS. Fourteen lipophilic marine toxins and domoic acid were acquired with fast polarity switching. Whereas azaspiracids (AZAs), pecenotoxins, 13-desmethyl spirolide C (SPX1), and gymnodimine were analyzed in the positive mode, yessotoxins (YTXs) were measured in negative mode. The okadaic acid (OA) group compounds were analyzed in both positive and negative ionization modes, and the accuracy of the results for both were compared. When using dynamic multiple reaction monitoring (MRM) in fast polarity switching, LODs were lower and reproducibility and linearity were better compared to static MRM. The UHPLC separation allowed for higher sample throughput in routine use. Compared to the previously used HPLC/MSIMS method, LODs were improved up to a factor of 10 in mussel extract. Matrix effects were evaluated by comparing standards prepared in solvent with matrix-matched calibrations in blank mussel extract. For accurate quantification matrix-matched calibrations were used when analyzing reference mussel materials, providing recoveries for OA, Dynophysis toxins (DTX)1, DTX2, YTX, AZA1, and SPX1 between 80 and 120% with RSDs below 8% over a 3-day validation procedure.

Geographical, temporal, and species variation of the polyether toxins, azaspiracids, in shellfish.

Azaspiracid Poisoning (AZP) is a new toxic syndrome that has caused human intoxications throughout Europe following the consumption of mussels (Mytilus edulis), harvested in Ireland. Shellfish intoxication is a consequence of toxin-bearing microalgae in the shellfish food chain, and these studies demonstrated a wide geographic distribution of toxic mussels along the entire western coastal region of Ireland. The first identification of azaspiracids in other bivalve mollusks including oysters (Crassostrea gigas), scallops (Pecten maximus), clams (Tapes phillipinarium), and cockles (Cardium edule) is reported. Importantly, oysters were the only shellfish that accumulated azaspiracids at levels that were comparable with mussels. The highest levels of total azaspiracids (microg/g) recorded to-date were mussels (4.2), oysters (2.45), scallops (0.40), cockles (0.20), and clams (0.61). An examination of the temporal variation of azaspiracid contamination of mussels in a major shellfish production area revealed that, although maximum toxin levels were recorded during the late summer period, significant intoxications were observed at periods when marine dinoflagellate populations were low. Although human intoxications have so far only been associated with mussel consumption, the discovery of significant azaspiracid accumulation in other bivalve mollusks could pose a threat to human health.

Detection of five new hydroxyl analogues of azaspiracids in shellfish using multiple tandem mass spectrometry.

The polyether dinoflagellate toxins, azaspiracids, are responsible for azaspiracid poisoning (AZP), a new human toxic syndrome arising from the consumption of shellfish. To date, five azaspiracids have been isolated and fully structurally elucidated, including, AZA1, its 8-methyl and 22-demethyl analogues, AZA2 and AZA3, respectively, and two hydroxyl derivatives of AZA3, named AZA4 and AZA5. Using a recently developed method involving liquid chromatography with multiple tandem mass spectrometry (LC-MS(n)), five new azaspiracids, AZA7-AZA11, have been found in mussels (Mytilus edulis). AZA6 is a positional isomer of AZA1 and four of the new compounds are isomers with a mass of 857.5 amu. AZA7 and AZA8 are hydroxyl analogues of AZA1 while AZA9 and AZA10 are hydroxyl analogues of AZA6. AZA11 is a hydroxyl analogue of AZA2. The separation of all 11 azaspiracids was achieved using isocratic reversed phase liquid chromatography using a combination of eluent additives, trifluoroacetic acid and ammonium acetate. The ion-trap MS experiments, with electrospray ionisation, involved the fragmentation of the protonated molecule [M+H](+), trapping and fragmenting the product ions due to the loss of a water molecule [M+H-H(2)O](+), together with mass spectral data analysis that included the characteristic A-ring fragmentation for each compound.

Comparison of solid-phase extraction methods for the determination of azaspiracids in shellfish by liquid chromatography-electrospray mass spectrometry.

Azaspiracids have been identified as the cause of a new toxic syndrome called azaspiracid poisoning (AZP) that has led to incidents of human intoxications throughout Europe following the consumption of mussels. Although five AZP toxins have been structurally elucidated to-date, azaspiracid (AZA1), 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3) are the predominant toxins. Separation of the three main AZP toxins was achieved using reversed-phase liquid chromatography (LC) and coupled to an electrospray ionisation source of an ion-trap mass spectrometer. Five reversed-phase (C18) and three diol solid-phase extraction (SPE) cartridges were compared for their efficacy in the cleanup of shellfish matrix. The comparison was based on the optimum recoveries of AZA1, AZA2 and AZA3 from extracts of mussel tissues. LC-electrospray MS3 analysis was used to quantify the AZP toxins in wash and eluate fractions in the SPE studies. Good recovery and reproducibility data were obtained for one diol SPE cartridge and two C18 SPE cartridge types.

Determination of azaspiracids in shellfish using liquid chromatography/tandem electrospray mass spectrometry.

Azaspiracid (AZA1), a recently discovered marine toxin, is responsible for the new human toxic syndrome, azaspiracid poisoning (AZP), which is caused by the consumption of contaminated shellfish. A new, sensitive liquid chromatography/mass spectrometry (LC/MS) method has been developed for the determination of AZA1 and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3). Separation of these toxins was achieved using reversed-phase LC and coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer. Spectra showed the protonated molecules, [M + H]+, and their major product ions, due to the sequential loss of two water molecules, [M + H - H2O]+, [M + H - 2H2O]+, in addition to fragment ions that are characteristic of these cyclic polyethers. A highly specific and sensitive LC/MS(3) analytical method was developed and, using shellfish extracts containing AZA1, the detection limit (S/N = 3) was 4 pg on-column, corresponding to 0.8 ng/mL. Using the protocol presented here, this is equivalent to 0.37 ng/g shellfish tissue and good linear calibrations were obtained for AZA1 in shellfish extracts (average r2 = 0.9988). Good reproducibility was achieved with % RSD values (N = 5) ranging from 1.5% (0.75 microg/mL) to 4.2% (0.05 microg/mL). An efficient procedure for the extraction of toxins from shellfish aided the development of a rapid protocol for the determination of the three predominant azaspiracids.