Exploring cyanotoxin diversity in field samples – an untargeted HRMS/MS based approach

Robust cyanotoxin analysis is challenging because of the existence of multiple cyanotoxin classes with differing chemical properties, a large number of structural analogues, the scarcity of chemical standards and the complex matrices in which cyanotoxins occur. Studies of cyanotoxin occurrence often rely on class-specific and/or targeted analytical methods, unlikely to detect whole toxin classes or unexpected variants. Here, cyanobacterial field samples from different environments were prepared with a simple methanolic extraction and then analyzed using a comprehensive strategy including both LC-MS/MS and LC-HRMS/MS methods. A multi-class LC-MS/MS method for microcystins, anatoxins, saxitoxins and cylindrospermopsins was used for initial screening, and then untargeted LC-HRMS/MS methods designed to detect a broad range of toxin variants and other cyanobacterial metabolites were employed. The downstream untargeted data processing included metabolomics data analysis software, which has been adapted to recognize several classes of cyanobacterial secondary metabolites. With the targeted LC-MS/MS methods we identified various toxins from all major classes, while the untargeted methods provided in-depth information for the specific classes in conjunction with showing the presence of other notable cyanobacterial secondary metabolites. This comprehensive workflow will be used for ongoing analyses of cyanobacterial field samples from temperate to polar environments and assessment of cyanotoxin diversity.