High Resolution Hybrid Mass Spectrometry For The Determination Of Marine Biotoxins, Azaspiracids, And Their Biotransformation Products.
Authors:   ,   Cork Institute of Technology, Skrabakova Zuzana 
Published: September 2024

Azaspiracids (AZAs), polyether marine toxins, are produced by marine microalgae and accumulate in filter-feeding bivalve molluscs, such as mussels, clams, oysters and scallops, while grazing on phytoplankton, from which they derive nutrition. Although being discovered quite recently, AZAs have caused several human intoxication events and thus became a problem for health authorities and shellfish industries. The rapid development of highly sensitive liquid chromatography multiple tandem mass spectrometry (LC-MS/MS, LC-MS") methods enabled the identification of numerous AZA analogues and added to the control of shellfish contamination. Tandem mass spectrometry techniques, while being highly selective and sensitive for determination of AZAs in complex shellfish matrices, are target methods and therefore not suitable for detection of novel analogues, metabolites and degradation products. The aim of this research project was to develop a non-target mass spectrometric approach for analysis of AZAs in shellfish and apply this to determination of AZAs in shellfish, to elucidate their fragmentation pathways and to study their bioconversion processes in shellfish. The overview of the marine toxins, the latest mass spectrometric, Orbitrap, technology and the results of the exploration of the performance of the Orbitrap mass spectrometer for analysis of the polyether biotoxins, azaspiracids, are described in seven chapters of this Thesis: 1. Firstly, five major human toxic syndromes caused by the consumption of shellfish contaminated by algal toxins have been reviewed. The prevalence of harmful algal blooms (HABs) is possibly a consequence of anthropogenic activities and global climate change. Since the marine toxins have been proved to have negative implications for human health increased toxin surveillance programmes and the development of reliable detection methods are mandatory for limiting human exposure to shellfish toxins. 2. The Orbitrap mass analyser is the most recent development in mass spectrometry technology. Despite of the rather high price of the Orbitrap instruments, those became very popular and have been employed successfully in numerous applications for analysis of both, large and small molecules. The ability of Orbitrap technology to perform equally for proteomics and for micropollutants is a result of multiple ion sources, mass analysers, detectors and scan modes that can be implemented in the instrument. 3. The most recently discovered, and possibly very potent, is the azaspiracic group of polyether marine toxins. Although liquid chromatography coupled with mass spectrometry (LC-MS) has become finally the official method for monitoring of azaspircids, the suitability of the particular MS method should be evaluated as well. Previously employed mass analysers allowed for sensitive and selective determination of azaspiracids i shellfish tissue and phytoplankton. However, for the identification of unexpected azaspiracid analogues are non-target approaches better suited. 4. The LTQ Orbitrap was exploited to develop and validate a full-scan Fourier transform (FT) MS method for analysis of azaspiracids in shellfish. A comparison of this on-target approach with a target method, performed on linear ion trap mass analyser (,IT MS), demonstrated suitability of the full-scan FTMS for analysis of azaspiracids in shellfish tissue. A dependent scan, using higher collisioanally induced dissociation (HCD) U generate product ion spectra from the ions observed in the full-scan, enhanced the identification of the main metabolites of azaspiracid-1 in shellfish. Subsequently, this method enabled for discovery of two major AZA analogues in scallops {Pecten maximus). 5. Finally, the possibility of direct analysis of azaspiracids in shellfish extracts using nanoelectrospray providing automatic system, NanoMate, coupled to the Orbfap mass analyser was explored. This LC free method allowed for development of a rapid;creening method for the determination of AZAs in crude shellfish extracts, which have not been subjected to extensive clean-up procedures, thus preventing unpredictable losses c’ analytes differing in physicochemical properties as well as those at trace concentration level.