Rapid quantitative screening of cyanobacteria for production of anatoxins using direct analysis in real time high-resolution mass spectrometry

Rationale Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins that are increasingly problematic in drinking water reservoirs and recreational water bodies worldwide. Because of their high polarity and low molecular weight, analysis of ATXs is challenging and they can be considered underreported compared with other classes of cyanobacterial toxins. Improved screening methods are therefore needed to effectively assess their occurrence and concentrations in the environment. Methods A rapid screening method was developed for ATXs in cyanobacteria using direct analysis in real time combined with high-resolution mass spectrometry (DART-HRMS), requiring less than 2 min per sample for triplicate analysis. The developed method was evaluated for its quantitative capabilities, applied to the screening of 30 cyanobacterial culture samples for the presence of anatoxin-a, homoanatoxin-a and dihydroanatoxin-a, and compared with a more typical liquid chromatography (LC)/HRMS method. Results Excellent linearity was observed in the analysis of a matrix-matched calibration curve using DART-HRMS, with ionization suppression of about 50% and relative standard deviations between replicate analyses of approximately 30%. Limits of detection for both anatoxin-a and homoanatoxin-a were estimated as 1 ng/mL. Excellent agreement was observed between DART-HRMS and LC/HRMS with all ATX-producing cultures correctly identified and only one false positive culture by DART-HRMS. Conclusions DART-HRMS shows excellent promise for the rapid, quantitative screening of ATXs in cyanobacteria and could be expanded in the future to include the analysis of field samples and drinking water, as well as additional ATX analogues.

Automated summary

A rapid screening method using DART-HRMS was developed for the detection of ATXs in cyanobacteria, showing excellent promise for quantitative analysis. This method could be expanded in the future for analyzing field samples, drinking water, and other ATX analogues.