Abstract
Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3–8 µg kg−1) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3–320 µg kg−1 in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.
Highlights
Secondary metabolites from marine organisms have raised an uninterrupted interest in numerous fields, including the development of new drugs or drug leads, ecology, marine biology, toxicology and food safety.In food system and nutritional toxicology, the production of stable poisoning metabolites, marine biotoxins (MBTXs, known as phycotoxins), by several harmful microalgae species, and their bioaccumulation in the food chain represent a health and economic concern [1–4]
This study presents a rapid, high throughput and sensitive ultra-high performance liquid chromatography (UHPLC) coupled with electrospray ionization (ESI)—triple quadrupole (QqQ) tandem mass spectrometry (MS/MS) method
In order to enhance the adsorption of Lipophilic marine toxins (LMTs) on the OASIS HLB columns, it was necessary to dilute the methanolic extract with an amount of water higher than 35% [42]
Summary
In food system and nutritional toxicology, the production of stable poisoning metabolites, marine biotoxins (MBTXs, known as phycotoxins), by several harmful microalgae species, and their bioaccumulation in the food chain represent a health and economic concern [1–4]. More than 1000 metabolites correlated with LMTs have been identified; in fulfilment of the European Food Safety Authority (EFSA) reports and studies, the European Commission (EC) set the maximum permitted levels (MPLs) for four subgroups in shellfish. The EC and Member States established the relevant compounds and assigned a toxic equivalency factor (TEF) to them [1,12–15]. They appointed sampling plans, classified the location and boundaries of the production and relaying areas of bivalve molluscs, and planned regulatory controls and monitoring programs, according to Regulations (EC) No 853/2004 and 627/2019 [16–18]
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