Analytical And Synthetic Methods For Anatoxins.
Authors:   ,   Cork Institute of Technology, Crowley Janet 
Published: June 2024

This thesis is focused on the potent cyanobacterial neurotoxins anatoxin-a (AN), its methyl analogue homoanatoxin-a (HMAN) and their non-toxic dihydro- and epoxydegradation products. In commencing the investigation into this area a literature review was performed in order to identify areas where more research and method development was required (Chapter 1). The review highlighted the fact that although a number of analytical methods had been developed for the analysis of AN, there were few methods for the determination of its potent homologue HMAN and their non-toxic degradation products. As these neurotoxins degrade quickly it is important to monitor their degradation products, particularly if there has been a suspected toxic incident. The first experimental study combined the information from a nano electrospray hybrid quadrupole time-of-flight (QqTOF) mass spectrometer and a quadrupole ion-trap (QIT) mass spectrometer in order to elucidate the fragmentation patterns of the six anatoxins and to confirm formulae assignments for major product ions (Chapter 2). Using these complimentary instruments, comparisons between the spectra of compounds that differ in side-chain length (the AN and HMAN series) were used to identify ions that are characteristic of the homologues. From this information, a new liquid chromatography- multiple tandem mass spectrometry (LC-MS") method was developed for the determination of the six anatoxins and applied to environmental and forensic samples. One of the main problems encountered in the method development of anatoxins has been the lack of availability of standards. AN may be purchased but is expensive and certified reference material is not available, HMAN has to be isolated and the degradation products need to be synthesised from the parent compound. Preliminary studies were undertaken to develop a simple route to synthesise (+/-)-AN, that in turn could be adapted to synthesise HMAN and other analogues as well as an internal standard. It is also envisaged that this synthetic route may also be modified to obtain (-i-)-AN in large enough quantities to allow future research into toxicological activity. While still in the early stages the proposed route offers promise in terms of both cost and yield. During these research studies, the misidentification of AN in a sample from a young man that died mysteriously after being in contact with cyanobacteria highlighted a new problem with the analysis of AN. In addition to difficulties in detecting this toxin due to its rapid decay, detection of AN using single quadrupole mass spectrometry is questionable due to the presence of the amino acid, phenylalanine (Phe). These compounds are isobaric and elute similarly in reversed phase liquid chromatography. Another incident in France was also reported where the presence of Phe prevented the quantitation of AN in a forensic sample taken from a dead dog. Consequently, chapter 3 looked at approaches to prevent the misidentification of AN including, (a) fluorimetric liquid chromatography (LC-FLD) following derivatisation using 4-fluoro-7-nitro-2,l,3- benzoxadiazole (NBD-F); (b) methylation using diazomethane prior to liquid chromatography single stage MS (LC-MS) determination; (c) LC-MS" using a QIT and (d) high resolution MS using a QqTOF. Interference from Phe was not observed in any of procedures, (a) - (c), and the high mass accuracy obtained in method (d), readily distinguished between the two compounds. MS" was also employed to study the fragmentation pathway of Phe provide characteristic fragmentation information that clearly distinguished between AN and Phe. The difficulties associated with the over reliance on low resolution MS data in forensic toxicology were highlighted and this led to the development of a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method on a triple quadrupole (QqQ) mass spectrometer, operating in multiple reaction monitoring (MRM) mode. Using the optimised conditions and the structural information obtained from LC-MS", chromatographic separation was achieved and precursor/product ion pairs were chosen to prevent misidentification of AN and Phe. During this time samples from another canine fatality were obtained and AN was identified upon analysis of the stomach contents. Phe was also present in this sample but by optimising the parameters it did not interfere with quantitation.