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

Abstract

Cyclic imines (CIs) are being considered as emerging toxins in the European Union, and a scientific opinion has been published by the European Food Safety Authority (EFSA) in which an assessment of the risks to human health related to their consumption has been carried out. Recommendations on the EFSA opinion include the search for data occurrence of CIs in shellfish and using confirmatory methods by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which need to be developed and optimized. The aim of this work is the application of LC-MS/MS to the analysis of gymnodimines (GYMs), spirolides (SPXs), pinnatoxins (PnTXs), and pteriatoxins (PtTXs) in mussels from Galician Rias, northwest Spain, the main production area in Europe, and therefore a representative emplacement for their evaluation. Conditions were adjusted using commercially available certified reference standards of GYM-A, SPX-1, and PnTX-G and evaluated through quality control studies. The EU-Harmonised Standard Operating Procedure for determination of lipophilic marine biotoxins in molluscs by LC-MS/MS was followed, and the results obtained from the analysis of eighteen samples from three different locations that showed the presence of PnTXs and SPXs are presented and discussed. Concentrations of PnTX-G and SPX-1 ranged from 1.8 to 3.1 µg/kg and 1.2 to 6.9 µg/kg, respectively, and PnTX-A was detected in the group of samples with higher levels of PnTX-G after a solid phase extraction (SPE) step used for the concentration of extracts.

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

“…Mass spectrometry detection was operated in positive mode and the product ion spectra of [M + H] + ions obtained for m / z 694.4 at three different collision energies (CE) are shown in Figure 2 B (CE 40 eV), Figure 2 C (CE 54 eV), and Figure 2 D (CE 60 eV). Fragmentation pathways are in concordance with those previously reported [ 11 , 13 , 24 ] in which the m / z 164.1 fragment ion (C 11 H 18 N + ) is also common for some SPXs, including SPX-13, 13,19-didesmethyl spirolide C (SPX-13,19), spirolide C (SPX-C), spirolide-D (SPX-D), spirolide H (SPX-H), and spirolide I (SPX-I). Analysis also showed fragmentation clusters at m / z 458.3 and m / z 676.3 ( Figure 2 D).…”

“…Results evidenced the presence of PnTX-G (RT = 3 min) in all samples from brand A ( Figure 3 A) and brand B ( Figure 3 B) of M. chilensis . PnTXs, produced by Vulcanodinium rugosum dinoflagellate, have been detected in seafood from the Mediterranean Sea in 2013 and 2018 [ 10 , 24 ] and in the Atlantic coast of Spain last year [ 11 , 12 , 13 ]. These results confirm also the presence of these toxins in mussels from Chile.…”

“…These toxins were found in Pacific oysters ( Crassostrea gigas ) and razor fish ( Pinna bicolor ) from South Australia and Northland New Zealand [ 31 , 32 ], mussels from Norway [ 7 ], Canada [ 33 ], and clams ( Venerupis decussata ) from France [ 24 ]. Recently, PnTXs were also detected in mussels ( M. galloprovincialis ) from the Atlantic Coast of Spain [ 11 , 12 , 13 ] and in other countries from the Atlantic and Mediterranean coastline including Scotland, Northern Ireland, Ireland, Italy, Norway, and Portugal [ 34 ]. Concentrations of PnTXs in shellfish are generally low, however, PnTX-G was found to reach the concentration of 1200 µg/kg in mussels collected in the coast of France [ 24 , 29 ].…”

“…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 ].…”

Purification, Toxicity and Functional Characterization of a New Proteinaceous Mussel Biotoxin from Bizerte Lagoon

“…Among the vast family of marine biotoxins, some of them have been reported to target voltage-gated Na channels of excitable cells, such as saxitoxin and its isomers, brevetoxins, tetrodotoxins and ciguatoxins [4]. Other marine biotoxins inhibit nicotinic acetylcholine receptors, such as gymnodimines, spirolides and pinnatoxins [5,6].…”