Identification of Anatoxin-a and Related Metabolites in Exposed Mice Samples with a High-Resolution Mass Spectrometry Discovery Workflow

Harmful cyanobacterial blooms (HCBs) are a public health concern and require ongoing surveillance to monitor the negative water quality effects and cyanotoxins associated with these blooms. (+)-Anatoxin-a (ATX) is a potent neurotoxin produced by select cyanobacteria during HCB formation. Many HCB toxins are commonly associated with discolored water; however, ATX can be present in clear water, which results in a high risk of exposure by accidental ingestion for humans and animals. In this work, we used a qualitative, semitargeted liquid chromatography high resolution mass spectrometry (LC-HRMS) method and a discovery data analysis workflow to detect and identify ATX and its predicted mammalian metabolites in urine samples from ATX-dosed mice. Potential compounds were evaluated for identification with product-ion spectral matching to a local library, accurate mass list matching, further data processing and interpretation, and comparison to undosed mice urine samples. As a result, ATX and dihydroanatoxin-a (dhATX) were successfully identified in the dosed mice samples through retention time (RT) and product-ion spectral matching to their respective commercial standards. The positive identification of dhATX suggests its formation as an abundant metabolic product of ATX within mammalian systems. Additionally, multiple chromatographic peaks were observed that matched the exact mass of 3-OH ATX and were further identified by the presence of diagnostic product ions and comparison to a standard synthesized in-house. In total, seven potential ATX metabolites, including dhATX and 3-OH ATX, were detected and characterized in the dosed mice samples. All identified metabolites were either oxidized or reduced forms of ATX, which suggests that oxidation and reduction are the main pathways for endogenous ATX metabolism in mice. These results are among the first reports of metabolic products of ATX in biological samples and provide a metabolic profile of ATX for higher confidence screening for ATX after a suspected exposure event.