Rapid Quantitation of Anatoxins in Benthic Cyanobacterial Mats Using Direct Analysis in Real-Time–High-Resolution Tandem Mass Spectrometry

Abstract

Toxic benthic cyanobacterial mats are increasingly reported worldwide as being responsible for animal mortalities due to their production of the potent neurotoxin anatoxin-a (ATX) and its analogues. Improved analytical methods for anatoxins are needed to address public health and watershed management challenges arising from extremely high spatial and temporal variability within impacted systems. We present the development, validation, and application of a direct analysis in real-time–high-resolution tandem mass spectrometry (DART–HRMS/MS) method for analysis of anatoxins in cyanobacterial field samples, including a simplified sample preparation approach. The method showed excellent sensitivity and selectivity for ATX, homoanatoxin-a, and dihydroanatoxin-a. Isotopically labeled ATX was used as an internal standard for all three analogues and successfully corrected for the matrix effects observed (86 ± 16% suppression). The limit of detection and recovery for ATX was estimated as 5 ng/g and 88%, respectively, using spiked samples. The total analysis time was ∼2 min, and excellent agreement was observed with results from a liquid chromatography–HRMS reference method. Finally, the DART–HRMS/MS method was applied to a set of 45 Microcoleus -dominated benthic cyanobacterial mat samples from the Wolastoq near Fredericton, Canada, demonstrating its power and applicability in enabling broad-scale field studies of ATX distribution.

Analytical Methods for Anatoxin-a Determination: A Review

“…More recently, rapid methods using DART-HRMS have been developed for cyanobloom samples; however, they have not shown good accuracy parameters (RSD% up to 34%) [ 50 , 53 ]. Recently, Cevallos-Cedeño et al [ 54 ] developed an ELISA method for ATX-a.…”

“…Methods that consider the determination of its degradation products allow for more realistic data on the status of this neurotoxin to be obtained. In this regard, there are different methods focused on the determination of ATX-a that can be applied [ 44 , 50 , 53 , 94 ]. However, there are few multitoxin methods that consider the detection of the products derived from ATX-a [ 60 ], meaning that, in cases in which this toxin is thought to be the main cause of poisoning, it may be advisable to use methods that only determine ATX-a and ATX-a-derived compounds.…”

“…This technique can be useful for the analysis of a large number of samples in a short time at the expense of the reproducibility of the method (around 30% RSD), with an LOD = 1 ng/mL [ 50 ]. Shortly afterwards, an improvement to this method was made using DART-HRMS/MS, which also allowed for the analysis of HATX-a and dihydroanatoxin-a, as well as the reduction of the matrix effect due to the use of an isotopically labeled ATX-a standard [ 53 ].…”

Applications of ambient ionization mass spectrometry in 2022: An annual review

“…In a recent study utilizing reactive PS ionization, the authors were able to quickly explore different experimental parameters and reaction products by performing in situ reactions on the PS substrate. 162 The use of AIMS in environmental sciences has also exhibited some produced by cyanobacteria, in field samples, 164 the use of iEESI to detect perfluorooctanoic acid contamination in fish, 165 and the use of a dual plasma ion source (incorporating both corona discharge and a microwave plasma torch) to detect trace sterols in urban water samples. 166 Plasma-based AIMS techniques have also been utilized for the analysis of complex oil products.…”

“…Although a somewhat niche area of study, the research demonstrates the versatility of AIMS for environmental applications and the power of combining techniques to target a broader range of analytes. Other studies have further evaluated AIMS for different applications to environmental samples, including the use of DART to detect anatoxin‐a, a neurotoxin produced by cyanobacteria, in field samples, 164 the use of iEESI to detect perfluorooctanoic acid contamination in fish, 165 and the use of a dual plasma ion source (incorporating both corona discharge and a microwave plasma torch) to detect trace sterols in urban water samples. 166 …”