Strategies to avoid the mis-identification of anatoxin-a using mass spectrometry in the forensic investigation of acute neurotoxic poisoning

Anatoxin-a (AN) is a potent neurotoxin, produced by a number of cyanobacterial species, and consumption of freshwater contaminated with this toxin has led to animal deaths. Forensic investigations of suspected AN poisonings are frequently hampered by difficulties in detecting this toxin in biological matrices due to its rapid decay. In addition, detection of AN using single quadrupole mass spectrometry (MS) is suspect due to the presence of the amino acid, phenylalanine (Phe), since these compounds are isobaric and elute similarly in reversed phase liquid chromatography (LC). Approaches to prevent the misidentification of AN that have been explored in these studies included: (a) fluorimetric LC following derivatisation using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F); (b) methylation using diazomethane prior to LC-MS determination; (c) multiple tandem MS using a quadrupole ion-trap (LC-MS3); and (d) hybrid quadrupole time-of-flight (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 AN (165.11536) and Phe (165.07898). LC-MSn was also employed to study the fragmentation pathway of Phe and multi-stage MS spectra provided characteristic fragmentation information that clearly distinguished between AN and Phe. The difficulties associated with the over reliance on low resolution MS without MS/MS data in forensic toxicology are discussed.