Nouvelles stratégies pour l’analyse des cyanotoxines par spectrométrie de masse

Cyanobacteria have a very important place in aquatic ecosystems and a significant number of species are considered harmful given their production of toxic metabolites. These cyanotoxins have various chemical proprieties and have often been associated with poisoning episodes. The frequency of cyanobacterial blooms is increasing and the study of cyanobacteria and their toxins is of increasing interest, especially considering the potential increase associated with climate changes. Given the chemical complexity of the cyanotoxins, the development of simple, sensitive and fast detection methods is an analytical challenge. Considering these issues, the development of new analytical approaches for the detection of cyanotoxins in water and fish samples contaminated with harmful cyanobacterial blooms have been proposed. A first approach consists of the use of an on-line solid phase extraction coupled to liquid chromatography and tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of six microcystins (MCs), anatoxin-a (ANA-a) and cylindrospermopsin (CYN). This method allows a simple and rapid analysis and enables the chromatographic separation of ANA-a and its isobaric interference, phenylalanine. The detection limits ranged from 0.01 to 0.02 µg L-1 and concentrations in lake waters were found between 0.024 and 36 µg L-1. A second method consists of using manual solid phase extraction (SPE) coupled to high resolution mass spectrometry (HRMS) for the determination of b-N-methylamino-L-alanine (BMAA), ANA-a, CYN and saxitoxin (STX) in contaminated lake water. The analysis of two conformational isomers of BMAA was done to improve the selectivity. Dansyl chloride-based derivatization allows simplified chromatography. The detection limits were obtained between 0.007 and 0.01 µg L-1. The analysis of bloom water samples detected concentrations of cyanotoxins between 0.01 and 0.3 µg L-1 allowing the confirmation of the presence of BMAA in algal blooms in Québec. A second part of the project consists in the use of an alternative sample introduction technology for MS analysis. It enables ultra-fast analysis (< 15 seconds/sample) without the use of a chromatographic step, and is called laser diode thermal desorption (LDTD) coupled with atmospheric pressure chemical ionization (APCI). The first LDTD project consists of the analysis of total MCs via Lemieux oxidation in order to obtain a common moiety of all MCs existing congeners. This fraction, the MMPB, is analyzed after a liquid-liquid extraction step, with the LDTD-APCI-MS/MS. A value of 0.2 µg L-1 was obtained for detection limit and concentrations between 1 and 425 µg L-1 have been found in contaminated water samples. In addition, a comparison with a parallel analysis using MCs congeners’ standards suggested the possible presence of undetected MCs or isomers. A second project involves the direct analysis of ANA-a using LDTD-APCI-HRMS in order to solve the isobaric interference, phenylalanine, which is possible due to the high resolution detection. The LDTD offers no chromatographic separation and by using HRMS, we can distinguish ANA-a signals from those of phenylalanine. A value of 0.2 µg L-1 was obtained as detection limit and the method has been applied on water bloom samples with a positive concentration of 0.21 µg L-1. Finally, using the LDTD-APCI-HRMS combination, analysis of total MCs has been adapted to fish tissues to determine the unbound and bound MCs and compare the results with standard analysis. The use of digestion with sodium hydroxide prior to Lemieux oxidation followed by SPE purification yielded a detection limit of 2.7 µg kg-1. Total MCs concentrations were found between 2.9 and 13.2 µg kg-1 in real field-collected contaminated fish samples and comparison was made with standard analysis which yield a single positive sample with a concentration of 2 µg kg-1. This indicates the possible presence of undetected MCs using conventional analytical methods.

Keyphrases: Anatoxine-a, Cyanobacteria, Cyanotoxins, Microcystins, Anatoxin-a, Cylindrospermopsin, Saxitoxin, b-N-methylamino-L-alanine, Laser diode thermal desorption, Liquid chromatography, Mass spectrometry