Identification of anatoxins in blue-green algae food supplements using liquid chromatography-tandem mass spectrometry

Blue-green algae (cyanobacteria) in tablets and capsules, which are marketed as health food supplements, were investigated for the presence of neurotoxins related to anatoxin-a. These neurotoxins, which are nicotinic agonists, were investigated using isocratic micro-liquid chromatography tandem mass spectrometry (micro-LC-MS-MS). The investigated compounds were anatoxin-a and homoanatoxin-a, together with their degradation products, dihydroantoxin-a, epoxyanatoxin-a, dihydrohomoanatoxin-a and epoxyhomoanatoxin-a which were synthesized from the parent toxins. The analytes were extracted with methanol followed by isocratic chromatography on a micro C-18 reversed-phase column using acetonitrile-water, 50:50 (v/v), containing 20 mM acetic acid at 30 mu-l min(-1). The toxins were ionized in a ionspray (IS) interface operating in the positive ion mode, where the intact protonated molecules, [M+H](+) were generated at m/z 166, m/z 168, m/z 182, m/z 180, m/z 182 and m/z 196, for anatoxin-a, dihydroanatoxin-a, epoxyanatoaxin-a, homoanatoxin-a, diydrohomoanatoxin-a and epoxyhomoanatoxin-a, respectively. These served as precursor ions for collision-induced-dissociation (CID) and diagnostic product ions for these anatoxins were identified to carry out toxin confirmation by selected reaction monitoring (SRM) LC-MS-MS analysis. Dihydrohomoanatoxin-a and a novel isomer of epoxyanatoxin-a were identified in blue-green algae tablets. This finding suggests that a potential human health hazard could be associated with the consumption of these food supplements.