LC-HRMS Profiling of Paralytic Shellfish Toxins in Mytilus galloprovincialis after a Gymnodinium catenatum Bloom

Abstract

Saxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels' whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs' profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicity.

Keywords: European waters; GC-toxins; Gymnodinium catenatum; LC-HRMS; M-toxins; emergent toxins; marine biotoxins; occurrence data; seafood safety; shellfish poisoning.

Scheme 1

Chemical structures of classical and emerging characterized PST analogues. See Material and Methods Section for toxin abbreviations.

Figure 1

LC-HRMS full-scan positive (ESI⁺) (A) and negative (ESI⁻) (B) mode profiles of mussels (M. galloprovincialis) extract and the correspondent XIC taken at the m/z values 273.13058 at ESI⁺ (C), 394.07810 at (D), 305.12041 at ESI⁺ (E), and 316.13639 at ESI⁺ (F), with a ± 5 ppm window. * GTX6 and M1 signals at m/z 316.13639 ± 5 ppm at ESI⁺ correspond to in-source fragment ions. GTX6 and M1 were quantified under ESI⁻, m/z 394.07810. Red indicates analysis under ESI⁺ and blue under ESI⁻.

Figure 2

Concentrations of classical and emerging PST analogues in naturally contaminated mussels (M. galloprovincialis) extract. Error bars show the standard deviations of three replicate injections. The molar percentage (%) of each toxin is given above the bars. Note: classical GTX1, GTX2, and GTX3 were not detected.