LC–MS/MS Analysis of the Emerging Toxin Pinnatoxin-G and High Levels of Esterified OA Group Toxins in Galician Commercial Mussels

Abstract

The occurrence of marine harmful algae is increasing worldwide and, therefore, the accumulation of lipophilic marine toxins from harmful phytoplankton represents a food safety threat in the shellfish industry. Galicia, which is a commercially important EU producer of edible bivalve mollusk have been subjected to recurring cases of mussel farm closures, in the last decades. This work aimed to study the toxic profile of commercial mussels (Mytilus galloprovincialis) in order to establish a potential risk when ingested. For this, a total of 41 samples of mussels farmed in 3 Rías (Ares-Sada, Arousa, and Pontevedra) and purchased in 5 local markets were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS). Chromatograms showed the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2), azaspiracid-2 (AZA-2), and the emerging toxins 13-desmethyl spirolide C (SPX-13), and pinnatoxin-G (PnTX-G). Quantification of each toxin was determined using their own standard calibration in the range 0.1%–50 ng/mL (R2 > 0.99) and by considering the toxin recovery (62–110%) and the matrix correction (33–211%). Data showed that OA and DTX-2 (especially in the form of esters) are the main risk in Galician mollusks, which was detected in 38 samples (93%) and 3 of them exceeded the legal limit (160 µg/kg), followed by SPX-13 that was detected in 19 samples (46%) in quantities of up to 28.9 µg/kg. Analysis from PTX-2, AZA-2, and PnTX-G showed smaller amounts. Fifteen samples (37%) were positive for PTX-2 (0.7–2.9 µg/kg), 12 samples (29%) for AZA-2 (0.1–1.8 µg/kg), and PnTX-G was detected in 5 mussel samples (12%) (0.4 µg/kg–0.9 µg/kg). This is the first time Galician mollusk was contaminated with PnTX-G. Despite results indicating that this toxin was not a potential risk through the mussel ingestion, it should be considered in the shellfish safety monitoring programs through the LC–MS/MS methods.

Analysis of Cyclic Imines in Mussels (Mytilus galloprovincialis) from Galicia (NW Spain) by LC-MS/MS

“…The variation of the concentration levels of PnTXs in relation to spatio-temporal changes in shellfish from the Atlantic and Cantabrian coasts of Spain was recently evaluated, and the maximum concentrations did not exceed 15 µg PnTXs/kg [19]. In addition, another recent work also reported the presence of PnTX-G in mussels from Galicia at levels similar to those found in this study [20]. In relation to the analysis of hydrolyzed extracts, the concentration of PnTX-G increased after alkaline hydrolysis within 3% to 22%, suggesting the presence of esterified forms of the toxin in samples.…”

“…CIs are distributed worldwide in diverse geographical regions, including European waters, where compounds that belong to the GYM, SPX, and PnTX groups have been detected in shellfish from several countries [9][10][11][12][13][14][15]. In Spain, the third largest mussel producer of the world in 2017 [16], PnTX-G and 13-desmethylSPX-C (SPX-1) were found in samples from Catalonia (northeast Spain, Mediterranean Sea) [17] while in Galicia (northwest Spain, Atlantic Ocean), the main production area in the country, SPX-1 was detected [18] and PnTXs have been recently reported [19,20]. The European Food Safety Authority (EFSA) has developed a series of scientific opinions related to marine biotoxins, both for regulated and for emerging groups.…”

“…This work was focused on the application of LC-MS/MS for the analysis of GYMs, SPXs, PnTXs, and PtTXs in mussels (Mytilus galloprovincialis) from Galician Rias in order to allow for the confirmation of PnTX-G and SPX-1 using standard solutions, as well as the detection of PnTX-A based on fragmentation pathways and key m/z ions. Catalonia (northeast Spain, Mediterranean Sea) [17] while in Galicia (northwest Spain, Atlantic Ocean), the main production area in the country, SPX-1 was detected [18] and PnTXs have been recently reported [19,20]. The European Food Safety Authority (EFSA) has developed a series of scientific opinions related to marine biotoxins, both for regulated and for emerging groups.…”

Acute Oral Toxicity of Pinnatoxin G in Mice

“…PnTx-E, -F, and -G have been subsequently isolated from oysters (Crassostrea gigas) collected in South Australia and New Zealand [5], whereas PnTx-H has been purified from cultured Vulcanodinium rugosum collected in South China Sea [6]. During the last years, these toxins, including their fatty acid ester derivatives, have also been detected in microalgae and/or other shellfish species from different geographical areas [7][8][9][10][11][12][13][14][15][16][17][18][19]. Moreover, strains of dinoflagellates producing PnTx-E and -F in New Zealand [7], have been shown to be identical to those producing PnTx-E, -F, and -G in Australia [8] or PnTx-G in Japan [9].…”

Detection of Cyclic Imine Toxins in Dietary Supplements of Green Lipped Mussels (Perna canaliculus) and in Shellfish Mytilus chilensis

“…In the past decade, evidence has grown for the occurrence of non-regulated toxins, spirolides (SPXs) and pinnatoxins (PnTXs), in mollusc from EU waters [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. These compounds belong to the cyclic imine (CIs) group and chemistry, they are macrocyclic molecules with imine and spiro-linked ether moieties [ 15 , 16 ].…”