13 research outputs found
Nouvelles stratĂ©gies pour lâanalyse des cyanotoxines par spectromĂ©trie de masse
Les cyanobactĂ©ries ont une place trĂšs importante dans les Ă©cosystĂšmes aquatiques et un nombre important dâespĂšces considĂ©rĂ© comme nuisible de par leur production de mĂ©tabolites toxiques. Ces cyanotoxines possĂšdent des propriĂ©tĂ©s trĂšs variĂ©es et ont souvent Ă©tĂ© associĂ©es Ă des Ă©pisodes dâempoisonnement. Lâaugmentation des Ă©pisodes dâefflorescence dâorigine cyanobactĂ©riennes et le potentiel quâils augmentent avec les changements climatiques a renchĂ©ri lâintĂ©rĂȘt de lâĂ©tude des cyanobactĂ©ries et de leurs toxines. ConsidĂ©rant la complexitĂ© chimique des cyanotoxines, le dĂ©veloppement de mĂ©thodes de dĂ©tection simples, sensibles et rapides est toujours considĂ©rĂ© comme Ă©tant un dĂ©fi analytique.
ConsidĂ©rant ces dĂ©fis, le dĂ©veloppement de nouvelles approches analytiques pour la dĂ©tection de cyanotoxines dans lâeau et les poissons ayant Ă©tĂ© contaminĂ©s par des efflorescences cyanobactĂ©riennes nuisibles a Ă©tĂ© proposĂ©. Une premiĂšre approche consiste en lâutilisation dâune extraction sur phase solide en ligne couplĂ©e Ă une chromatographie liquide et Ă une dĂ©tection en spectromĂ©trie de masse en tandem (SPE-LC-MS/MS) permettant lâanalyse de six analogues de microcystines (MC), de lâanatoxine (ANA-a) et de la cylindrospermopsine (CYN). La mĂ©thode permet une analyse simple et rapide et ainsi que la sĂ©paration chromatographique dâANA-a et de son interfĂ©rence isobare, la phĂ©nylalanine. Les limites de dĂ©tection obtenues se trouvaient entre 0,01 et 0,02 ÎŒg L-1 et des concentrations retrouvĂ©es dans des eaux de lacs du QuĂ©bec se trouvaient entre 0,024 et 36 ÎŒg L-1. Une deuxiĂšme mĂ©thode a permis lâanalyse du b-N-mĂ©thylamino-L-alanine (BMAA), dâANA-a, de CYN et de la saxitoxine (STX) dans les eaux de lac contaminĂ©s. Lâanalyse de deux isomĂšres de conformation du BMAA a Ă©tĂ© effectuĂ©e afin dâamĂ©liorer la sĂ©lectivitĂ© de la dĂ©tection. Lâutilisation dâune SPE manuelle permet la purification et prĂ©concentration des Ă©chantillons et une dĂ©rivatisation Ă base de chlorure de dansyle permet une chromatographie simplifiĂ©e. Lâanalyse effectuĂ©e par LC couplĂ©e Ă la spectromĂ©trie de masse Ă haute rĂ©solution (HRMS) et des limites de dĂ©tections ont Ă©tĂ© obtenues entre 0,007 et 0,01 ”g L-1. Des Ă©chantillons rĂ©els ont Ă©tĂ© analysĂ©s avec des concentrations entre 0,01 et 0,3 ”g L-1 permettant ainsi la confirmation de la prĂ©sence du BMAA dans les efflorescences de cyanobactĂ©ries au QuĂ©bec.
Un deuxiĂšme volet du projet consiste en lâutilisation dâune technologie dâintroduction dâĂ©chantillon permettant des analyses ultra-rapides (< 15 secondes/Ă©chantillons) sans Ă©tape chromatographique, la dĂ©sorption thermique Ă diode laser (LDTD) couplĂ©e Ă lâionisation chimique Ă pression atmosphĂ©rique (APCI) et Ă la spectromĂ©trie de masse (MS). Un premier projet consiste en lâanalyse des MC totales par lâintermĂ©diaire dâune oxydation de Lemieux permettant un bris de la molĂ©cule et obtenant une fraction commune aux multiples congĂ©nĂšres existants des MC. Cette fraction, le MMPB, est analysĂ©e, aprĂšs une extraction liquide-liquide, par LDTD-APCI-MS/MS. Une limite de dĂ©tection de 0,2 ”g L-1 a Ă©tĂ© obtenue et des concentrations entre 1 et 425 ”g L-1 ont Ă©tĂ© trouvĂ©es dans des Ă©chantillons dâeau de lac contaminĂ©s du QuĂ©bec. De plus, une analyse en parallĂšle avec des Ă©talons pour divers congĂ©nĂšres des MC a permis de suggĂ©rer la possible prĂ©sence de congĂ©nĂšres ou dâisomĂšres non dĂ©tectĂ©s. Un deuxiĂšme projet consiste en lâanalyse directe dâANA-a par LDTD-APCI-HRMS pour rĂ©soudre son interfĂ©rence isobare, la phĂ©nylalanine, grĂące Ă la dĂ©tection Ă haute rĂ©solution. La LDTD nâoffre pas de sĂ©paration chromatographique et lâutilisation de la HRMS permet de distinguer les signaux dâANA-a de ceux de la phĂ©nylalanine. Une limite de dĂ©tection de 0,2 ”g L-1 a Ă©tĂ© obtenue et la mĂ©thode a Ă©tĂ© appliquĂ©e sur des Ă©chantillons rĂ©els dâeau avec un Ă©chantillon positif en ANA-a avec une concentration de 0,21 ”g L-1. Finalement, Ă lâaide de la LDTD-APCI-HRMS, lâanalyse des MC totales a Ă©tĂ© adaptĂ©e pour la chair de poisson afin de dĂ©terminer la fraction libre et liĂ©e des MC et comparer les rĂ©sultats avec des analyses conventionnelles. Lâutilisation dâune digestion par hydroxyde de sodium prĂ©cĂ©dant lâoxydation de Lemieux suivi dâune purification par SPE a permis dâobtenir une limite de dĂ©tection de 2,7 ”g kg-1. Des Ă©chantillons de poissons contaminĂ©s ont Ă©tĂ© analysĂ©s, on a retrouvĂ© des concentrations en MC totales de 2,9 et 13,2 ”g kg-1 comparativement aux analyses usuelles qui avaient dĂ©montrĂ© un seul Ă©chantillon positif Ă 2 ”g kg-1, indiquant la possible prĂ©sence de MC non dĂ©tectĂ©s en utilisant les mĂ©thodes conventionnelles.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 ”g L-1 and concentrations in lake waters were found between 0.024 and 36 ”g 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 ”g L-1. The analysis of bloom water samples detected concentrations of cyanotoxins between 0.01 and 0.3 ”g 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 ”g L-1 was obtained for detection limit and concentrations between 1 and 425 ”g L-1 have been found in contaminated water samples. In addition, a comparison with a parallel analysis using MCs congenersâ 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 ”g L-1 was obtained as detection limit and the method has been applied on water bloom samples with a positive concentration of 0.21 ”g 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 ”g kg-1. Total MCs concentrations were found between 2.9 and 13.2 ”g 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 ”g kg-1. This indicates the possible presence of undetected MCs using conventional analytical methods
Biodegradation of microcystin-LR using acclimatized bacteria isolated from different units of the drinking water treatment plant
Bacterial community isolated from different units of a Drinking Water Treatment Plant (DWTP) including pre-ozonation unit (POU), the effluent-sludge mixture of the sedimentation unit (ESSU) and top-sand layer water sample from the filtration unit (TSFU) were acclimatized separately in the microcystin-leucine arginine (MC-LR)-rich environment to evaluate MC-LR biodegradation. Maximum biodegradation efficiency of 97.2 ± 8.7% was achieved by the acclimatized-TSFU bacterial community followed by 72.1 ± 6.4% and 86.2 ± 7.3% by acclimatized-POU and acclimatized-ESSU bacterial community, respectively. Likewise, the non-acclimatized bacterial community showed similar biodegradation efficiency of 71.1 ± 7.37%, 86.7 ± 3.19% and 94.35 ± 10.63% for TSFU, ESSU and POU, respectively, when compared to the acclimatized ones. However, the biodegradation rate increased 1.5-folds for acclimatized versus non-acclimatized conditions. The mass spectrometry studies on MC-LR degradation depicted hydrolytic linearization of cyclic MC-LR along with the formation of small peptide fragments including Adda molecule that is linked to the reduced toxicity (qualitative toxicity analysis). This was further confirmed quantitatively by using Rhizobium meliloti as a bioindicator. The acclimatized-TSFU bacterial community comprised of novel MC-LR degrading strains, Chryseobacterium sp. and Pseudomonas fragi as confirmed by 16S rRNA sequencing. Biodegradation of microcystin-LR by in-situ bacterial community present in the drinking water treatment plant without formation of toxic by-product.Fil: Kumar, Pratik. UniversitĂ© du QuĂ©bec a Montreal; CanadĂĄFil: Hegde, Krishnamoorthy. UniversitĂ© du QuĂ©bec a Montreal; CanadĂĄFil: Brar, Satinder Kaur. UniversitĂ© du QuĂ©bec a Montreal; CanadĂĄFil: CledĂłn, Maximiliano. Universidad Nacional del Comahue; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Kermanshahi-pour, Azadeh. Dalhousie University Halifax; CanadĂĄFil: Roy-Lachapelle, Audrey. University of Montreal; CanadĂĄFil: Galvez-Cloutier, Rosa. Laval University; Canad
A data-independent methodology for the structural characterization of microcystins and anabaenopeptins leading to the identification of four new congeners
Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu(1), Ser(7)] MC-HtyR, [Asp(3)]MC-RHar, AP731, and AP803). The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L(-1). To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems
Evaluation of ELISA-based method for total anabaenopeptins determination and comparative analysis with on-line SPE-UHPLC-HRMS in freshwater cyanobacterial blooms
Detection of Cyanotoxins in Algae Dietary Supplements
Algae dietary supplements are marketed worldwide as natural health products. Although their proprieties have been claimed as beneficial to improve overall health, there have been several previous reports of contamination by cyanotoxins. These products generally contain non-toxic cyanobacteria, but the methods of cultivation in natural waters without appropriate quality controls allow contamination by toxin producer species present in the natural environment. In this study, we investigated the presence of total microcystins, seven individual microcystins (RR, YR, LR, LA, LY, LW, LF), anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, cylindrospermopsin, saxitoxin, and ÎČ-methylamino-l-alanine in 18 different commercially available products containing Spirulina or Aphanizomenon flos-aquae. Total microcystins analysis was accomplished using a Lemieux oxidation and a chemical derivatization using dansyl chloride was needed for the simultaneous analysis of cylindrospermopsin, saxitoxin, and ÎČ-methylamino-l-alanine. Moreover, the use of laser diode thermal desorption (LDTD) and ultra-high performance liquid chromatography (UHPLC) both coupled to high resolution mass spectrometry (HRMS) enabled high performance detection and quantitation. Out of the 18 products analyzed, 8 contained some cyanotoxins at levels exceeding the tolerable daily intake values. The presence of cyanotoxins in these algal dietary supplements reinforces the need for a better quality control as well as consumerâs awareness on the potential risks associated with the consumption of these supplements
A data-independent acquisition approach based on HRMS to explore the biodegradation process of organic micropollutants involved in a biological ion-exchange drinking water filter
A Data-Independent Methodology for the Structural Characterization of Microcystins and Anabaenopeptins Leading to the Identification of Four New Congeners
Total Analysis of Microcystins in Fish Tissue Using Laser Thermal DesorptionâAtmospheric Pressure Chemical IonizationâHigh-Resolution Mass Spectrometry (LDTD-APCI-HRMS)
Microcystins (MCs) are cyanobacterial
toxins encountered in aquatic
environments worldwide. Over 100 MC variants have been identified
and have the capacity to covalently bind to animal tissue. This study
presents a new approach for cell-bound and free microcystin analysis
in fish tissue using sodium hydroxide as a digestion agent and Lemieux
oxidation to obtain the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB)
moiety, common to all microcystin congeners. The use of laser diode
thermal desorptionâatmospheric pressure chemical ionization
coupled with Q-Exactive mass spectrometry (LDTD-APCI-HRMS) led to
an analysis time of approximately 10 s per sample and high-resolution
detection. Digestion/oxidation and solid phase extraction recoveries
ranged from 70 to 75% and from 86 to 103%, respectively. Method detection
and quantification limits values were 2.7 and 8.2 ÎŒg kg<sup>â1</sup>, respectively. Fish samples from cyanobacteria-contaminated
lakes were analyzed, and concentrations ranging from 2.9 to 13.2 ÎŒg
kg<sup>â1</sup> were reported
Novel fluidized-bed biofilm reactor for concomitant removal of microcystin-LR and organics
Fluidized bed biofilm reactor (FBBR) was evaluated for the removal of microcystin-LR (MC-LR) from drinking water-sludge (0.3% w/v). Biofilm formed inside the solid media carriers (biocarriers) were studied for the MC-LR degradation in FBBRs via known MC-LR degraders: Arthrobacter ramosus (reactor A: RA) and Bacillus sp. (reactor B: RB), along with the heterogeneous bacterial community (HBC) present in the sedimentation-unit sludge as a background matrix. Their ability to form biofilm inside the immobilized biocarriers was periodically quantified for over 300 days to determine the duration of mature biofilm growth, sloughing event and then re-maturation. The bioreactor performance was mainly evaluated in terms of MC-LR, nitrate, nitrite, ammonia removal, and soluble-chemical oxygen demand (s-COD) removal. Biological degradation of MC-LR showed significant role over the physical adsorption, as the removal efficiency increased by around 30% and 26% for RA and RB respectively, as compared to the control bioreactor RD (without any bacterial cells) and an increase by over 15% and 11% when compared to reactor RC (contained only HBC). Mass spectra analysis for RA, RB, and RC strengthen the possibility of a toxic-free degradation mechanism. Overall, RA showed the best MC-LR removal efficiency of around 93.7%, which comprised no MC-LR in the supernatant phase and around 3 ”g/L in the sludge-mixture phase. Toxicity assessment of biodegraded sample (using bioindicator) further revealed the toxic-free nature by RA with >80% removal for ammonia, nitrate, and nitrite. Scale-up of laboratory scale FBBR (2 L) is also proposed to handle 200 m3 of feed water per day based on a similar volumetric mass transfer coefficient (kLa) to study the feasible process economics.Fil: Kumar, Pratik. Centre Eau Terre Environnement; CanadĂĄFil: Hegde, Krishnamoorthy. Centre Eau Terre Environnement; CanadĂĄFil: Brar, Satinder Kaur. Centre Eau Terre Environnement; CanadĂĄFil: CledĂłn, Maximiliano. Universidad Nacional del Comahue. Centro de InvestigaciĂłn Aplicada y Transferencia TecnolĂłgica en Recursos Marinos "Almirante Storni". - Provincia de RĂo Negro. Ministerio de Agricultura, GanaderĂa y Pesca. Centro de InvestigaciĂłn Aplicada y Transferencia TecnolĂłgica en Recursos Marinos "Almirante Storni". Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet Centro Nacional PatagĂłnico. Centro de InvestigaciĂłn Aplicada y Transferencia TecnolĂłgica en Recursos Marinos "Almirante Storni"; ArgentinaFil: Kermanshahi pour, Azadeh. Dalhousie University Halifax; CanadĂĄFil: Roy Lachapelle, Audrey. University of Montreal; CanadĂĄ. Environment Canada; CanadĂĄFil: Galvez Cloutier, Rosa. Laval University; Canad