Development and validation of a multiclass LC–MS/MS method for the analysis of cyanotoxins

Cyanobacteria are prokaryotic organisms that can form large monospecific blooms, which pose a risk to human and animal health as some species produce toxic secondary metabolites called cyanotoxins. Multiclass cyanotoxin analysis is challenging due to varying chemical and physical properties between classes, as well as potentially large numbers of analogues within each class. Incorporating anatoxins (ATXs) into multiclass methods can be particularly challenging due to their small molecular size, potential interferences, polarity, and a lack of chemical standards for most analogues. Here, we present the development of a multiclass LC-MS/MS method and a quantitative calibration solution for aetokthonotoxin (AETX), an emerging cyanotoxin linked to mass mortalities of bald eagles in the Eastern United States. The developed method is capable of detecting 17 microcystins (MCs), nodularin-R, three cylindrospermopsins (CYNs), AETX, and 17 ATXs, including recently tentatively identified 10-hydroxy analogues. Analytes were identified by retention time and product ion ratio matching with available standards. The method was evaluated with respect to limits of detection (LODs), linear range, accuracy, and precision using neat and matrix matched standards. LODs in wet cyanobacterial biofilms ranged from 0.14 ng/g for CYN to 2.8 ng/g for [Dha⁷]MC-LR with accuracies ranging from 65% for [Leu¹]MC-LY to 116% for CYN. Finally, the method's application was demonstrated through analysis of cyanobacterial field samples, a dietary supplement matrix reference material, and passive sampler extracts to assess versatility within different matrices.