Application Of Lc-ms Technology For The Detection Of Emerging Marine Toxins From The European Region
Authors:   ,   Cork Institute of Technology, Bane Vaishali 
Published: August 2024

"The seafood industry is economically one of the biggest food industries in the world. Seafood is a rich source of high quality proteins (16% of animal protein consumed by world’s population). Therefore, the contamination of sea food with marine toxins is a major concern for businesses worldwide and for consumer public health. Since 2004, the European Union monitors and legislates for the most common types of biotoxins found in European waters; diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP) and paralytic shellfish poisoning (PSP). However, over the past decade new (emerging) classes of marine toxins has started appearing in European water and shellfish produce. The emerging class of toxins includes tetrodotoxin (TTX), palytoxin (PITX) and cyclic imine (Cl) toxins. The emergence of these toxins in the Europe has been attributed to global warming, Lessepsian migration or from ballast water discharge. These emerging toxins are not monitored in Europe from the prospective of seafood safety. Also, the regulatory limits are not established for these toxins. Mouse bioassay was the traditional reference method for monitoring native toxins in Europe. However, since Jan 2015, the EU Commission Regulations No 15/2011 of 10 Jan 2011, states that Liquid chromatography- mass spectrometry (LC-MS) is the preferred reference method for marine toxin detection. The main reason for avoiding use of the mouse bioassay is ethical concerns regarding the use of animals and its lack of robustness, toxin group selectivity and sensitivity. LC-MS analysis of marine toxins offers selective identification and sensitive, yet robust, quantification abilities. The main objective of this Ph.D. is the development of robust LC-MS methods for detection of tetrodotoxin and cyclic imine toxins. Quantitative LC-MS methods were developed and validated for tetrodotoxin, cyclic imine and other common lipophilic toxin groups on a triple quadrupole (QqQ) and Orbitrap Fourier Transform mass spectrometers (FT-MS). The major challenge in the development of multi-toxin LC-MS methods is the lack of certified reference materials for minor analogues of toxins. This problem was circumvented with the use of Orbitrap FT-MS. The minor analogues of toxins were detected using untargeted full scan and data dependent scanning modes of Orbitrap FT-MS. Thus, the methods presented in this thesis have the capability to screen untargeted toxin compounds such as structural isomers or the metabolites of the toxins. The high resolution accurate mass measurements of the precursor and fragment ions of the toxins and, their natural isotopic compounds give a higher degree of confidence in the detection of toxins (especially isobaric toxins), which often occur at very low levels in seafood samples. These methods were applied to variety of samples; puffer fish and trumpet shell, phytoplankton, sea water and solid phase adsorption toxin tracking (SPATT) samples collected from the European region. The extraction protocols were also validated taking into account matrix ion suppression effects. The methods presented in this thesis have the potential to be use as reference methods for the official control and assessment of new and emerging toxin groups."