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

Abstract

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.

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

“…The LC – HRMS method has a LOD of 0.1 ng/mL in culture extracts and excellent resolution of the ATXs from each other ( Figure S1 ) and potential matrix interferences such as phenylalanine (Phe). 22 In order to evaluate the suitability of the FS DART method for analysis of ATXs in a more complex matrix, a preliminary set of 10 cyanobacterial field samples was analyzed by both DART – HRMS and LC–HRMS. This analysis showed that in several cases, high concentrations of ATXs were determined by DART – HRMS in the absence of detection of ATXs by LC – HRMS ( Figure S2 ).…”

“…17 , 18 High-resolution mass spectrometry (HRMS) is particularly effective at resolving ATX from its common isobaric interference phenylalanine, which can cause interference and ionization suppression in some MS-based methods. 16 , 19 − 21 The improved selectivity of HRMS over low-resolution MS instruments has also enabled the use of direct ionization techniques for ATX analysis including direct analysis in real time (DART), 22 matrix-assisted laser desorption ionization, 20 and laser-induced thermal desorption ionization. 21 , 23 These techniques offer the potential for higher throughput than LC–MS because of their lack of chromatographic separation and reduced requirement for sample preparation but also pose challenges in selectivity and matrix effects.…”

“…Recently, excellent selectivity and sensitivity were demonstrated for analysis of ATX and hATX by full-scan DART–HRMS in cultured cyanobacteria with a limit of detection (LOD) of 1 ng/mL and excellent quantitative agreement with LC–HRMS. 22 However, the significant impact of matrix effects on accuracy (50% suppression) and high sample-to-sample variability (30% RSD) of the method, which used single-point matrix-matched calibration, were highlighted as limitations in need of further study.…”

“…Here, we describe the development and validation of a DART–HRMS/MS method for the analysis of ATXs in complex cyanobacterial mat field samples. This includes refinement of our earlier full-scan method 22 to gain the selectivity needed for analysis in a complex cyanobacterial mat field sample matrix using HRMS/MS, the quantitation of another common ATX analogue, H 2 -ATX, and mitigation of matrix effects using an isotopically labeled ATX standard. We also present the development of a simple sample preparation method for cyanobacteria that is amenable to higher throughput analysis.…”

Applications of ambient ionization mass spectrometry in 2021: An annual review

“…DART-MS has been a popular choice for the analysis of water samples, and was recently applied to the detection of anatoxins, a type of cyanobacterial neurotoxins sometimes found in drinking water reservoirs. 112 and clay, containing 11 different PFAS compounds. 115 The method was first validated using materials spiked with analytical standards before being tested with genuine soil samples collected from an aqueous film forming foams site.…”