High Resolution Mass Spectrometry Methods for High Throughput and Untargeted Analysis of Anatoxins in Cyanobacteria

Anatoxins (ATXs) are increasingly linked to animal fatalities worldwide, but their analysis is challenging due to their polarity, instability, sample complexity and the scarcity of standards for most analogues. Conventional methods often don’t detect or differentiate analogues or are prone to significant matrix interferences in quantitation or detection. Here, we describe two recently developed methods for ATX analysis and demonstrate their application to cyanobacterial field samples from Atlantic Canada. A direct analysis in real time-high resolution tandem mass spectrometry (DART-HRMS/MS) method with limits of detection (LOD) of approximately 5 μg/kg and a total run time of under 2 min per triplicate analysis was suitable for rapid quantitation of anatoxin-a, homoanatoxin-a and dihydroanatoxin-a. Sample preparation was simplified to only require cell lysis, homogenization and centrifugation. An untargeted LC-HRMS/MS is also presented that offered lower LODs (0.1 μg/kg) and the opportunity to differentiate isomeric species and detect unknown ATX conjugates. Both methods used isotope dilution calibration with ¹³C₄-anatoxin-a to correct for the significant and variable matrix effects and showed excellent quantitative performance. They are broadly applicable to other quantitative or screening applications and will allow for more comprehensive study of ATX occurrence including the typically undetected fraction of conjugated ATXs.