Analysis Of Microcystins Research Articles

Analysis of Total Microcystins using LC-MS/MS: Method Improvements and Sample Applications

Microcystin variants (MCs), including microcystin-LR (MC-LR) and related variants, are produced by cyanobacteria and are regulated due to their potential health risks. Current methods for MC analysis predominantly employ LC-MS/MS, limited by the availability of reference materials necessary for quantification. As an alternative method for total MC quantification, the analysis of 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), formed by oxidation of the common ADDA structure in MCs, offers economic and practical advantages by avoiding the need for multiple MC reference materials. This method allows total MC quantification across various matrices, independent of specific MC variants. In this study, we evaluated the utility of MMPB-d3 as a surrogate to assess recovery rates throughout the processes critical for MMPB quantification. Comparative analysis with 4-phenylbutyric acid (4-PB) highlighted the stability of MMPB-d3 under oxidative conditions, which is critical for its application as a surrogate or internal standard. The oxidation process was optimized to achieve consistent recovery rates, averaging 101%, ensuring reliable conversion of MCs to MMPB for subsequent analysis. The limit of quantification (LOQ) has been improved due to the large volume injection of up to 0.1 µg/L. Comparison of MMPB concentrations from field samples with conventional LC-MS/MS results showed a significant correlation (r = 0.80, p < 0.005), but with variations in the absolute concentrations. Calculations using a formulation model with MMPB concentrations allowed us to estimate total MC levels, demonstrating the potential of the proposed method to complement existing target measurement techniques.

Analysis of Total Microcystins using LC-MS/MS: Assessment of Pretreatment Technique for MMPB(2-methyl-3-methoxy-4-phenylbutyric acid)

Algal toxin analysis, particularly for microcystin (MC) variants, is commonly performed using LC-MS/MS as the standard method. In 2016, the environmental protection agency (EPA) published method 546 for the determination of total MCs by ELISA. However, the limited availability of commercial algal toxin standards limits the efficacy of the LC-MS/MS method. This study proposes a novel approach using the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) structure generated by oxidizing MCs with potassium permanganate and sodium periodate. MMPB is formed through the oxidation of the common (all-S,all-E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) structure, eliminating the need for specific standard materials for each MC variant. This provides a method for measuring the total MCs. This study focused on optimizing sample pretreatment before oxidation, salt removal as a post-oxidation step, and the quantitative method to establish a comprehensive total MC detection approach applicable to surface water samples. The whole process included pretreatment using a freeze-drying method, purification using liquid-liquid extraction (LLE) for salt removal, and a comparison of quantitative methods using internal standards. Additionally, an evaluation of internal standards was conducted to assess availability during the oxidation process. In this study, an MMPB recovery rate of up to 50% was achieved, with an analysis precision of 14%. According to the results of QAQC, the satisfactory linearity of the calibration curve was a precision and the performance of the method for spiked samples was verified. The MMPB analysis method developed in this study showed relatively lower quantification limits and recovery rates than individual MC analysis methods. We believe these results stem from the inherent sensitivity differences between MCs and MMPB, as well as the complexity of the pre-treatment process centered around the oxidation process.

Analysis of microcystins in alum water treatment sludges: holding times, temperatures, linearity of response, and sensitivity to pre-coagulation cell titers

ABSTRACT ELISA assays are a potential tool to screen for dissolved or cell bound microcystins in drinking water treatment sludges. In order to evaluate this potential more thoroughly, experiments were performed in alum sludges to: (1) evaluate the impacts of sample storage times, temperatures, and sludge composition on spiked microcystin-LR recovery by ELISA; (2) examine the linearity of ELISA responses to spiked microcystin-LR as a function of sludge composition; and (3) examine the sensitivity ELISA and LC/MS/MS to five different concentrations of microcystin-producing cyanobacteria entrained in sludges of two different compositions. During storage experiments, microcystin recovery efficiencies ranged from 85% to 125% across the range of 12 storage time and temperature combinations with recovery efficiencies in 7 of the 12 combinations falling into the 90% to 110% range. During the linearity experiments, linear models fit ELISA responses in all sludge compositions with R2 values ≥ 0.95. During the sensitivity studies, simple freeze/thaw/centrifugation processing combined with ELISA or LC/MS/MS analyses resulted in detection of microcystins in thickened sludges derived from pre-coagulation cell suspensions of 102–106 cells/mL. In addition, the relationships between toxin concentrations in sludges and the original cell titers were linear regardless of analytical method.

Total Microcystin Concentration Variability in Water Samples and Recommended Minimum Volume (20 mL) for Freeze Thaw Cycles

AbstractCyanobacterial harmful algal blooms (cyanoHABs) continue to be a monitoring and research focus, particularly on the occurrence of toxins like total microcystins. The objectives of this study were to evaluate sampling and analytical variability in measured total microcystin concentrations and then to evaluate the volume of raw water needed in the freeze thaw cycle to reduce sampling variability. Water samples were collected from a recreational lake with annual cyanoHABs, and then 2 mL was used in freeze thaw cycles before total microcystin analysis. Then, sample volumes used in the freeze thaw cycles varied from 2 to 300 mL for total microcystin analysis. With three separate experiments, we observed a great deal of sampling variability (when using 2 mL in the freeze thaw cycles) while analytical variability was much less. In fact, sampling variability could potentially account for temporal variability observed in the routine monitoring. However, when sample volume used in the freeze thaw cycles increased, total microcystin variability decreased. We recommend at least 20 mL to be used in the freeze thaw cycles when analyzing total microcystins in environmental samples.

Water Quality Characteristics and Microcystin Analysis of Cultured Cyanobacteria

Water Quality Characteristics and Microcystin Analysis of Cultured Cyanobacteria

Isolation and Identification of Tersakan Stream (Amasya, Suluova TÜRKİYE) Cyanobacteria and Investigation of the Presence of the Microcystin-LR

Cyanobacteria are a concern worldwide due to their blooms and the toxins they produce. The aim of this study is to reveal the cyanobacterial diversity of Tersakan Stream and toxin analysis. For these purpose, water samples were taken from Tersakan Stream in 2018 and cyanobacterial diversity was determined by culture-dependent and independent methods. PCR amplification of microcystin genes (McyB and McyE) and Microcystin-LR (L:Leucine, R: Arginine) (MC-LR) analysis in water sample was performed. While 10 different Cyanobacteria were identified by light microscopy examination and 16S rRNA + ITS sequence, less diversity was revealed by culture-independent methods. Sequences of the isolates were associated with Pseudanabaena lonchoides, Leptolyngbya sp, Toxifilum sp, Cylindrospermum moravicum, Nostoc sp, Oscillatoria sp, Synechocystis sp, Synechococcus sp, Cyanobacterium aponinum and Cyanobium sp. Culture-independent sequences were associated with Microcytis sp, Nostoc sp, Cyanobium sp, Dolichospermum sp, Anabaena sp, Leptolyngbya sp and Uncultured cyanobacterium. Microcystin analysis was performed by both molecular and analytical methods. PCR results of McyB and McyE genes supported the HPLC-DAD analysis result. 0.467 μg L-1 microcystin LR was detected in Tersakan Stream by HPLC-DAD analysis. Although the microcystin concentration is low, Tersakan Stream is an important area to be monitored with its eutrophic character and cyanobacterial diversity.

Examination of Microcystin Adsorption by the Type of Plastic Materials Used during the Procedure of Microcystin Analysis.

The incidence of eutrophication is increasing due to fertilizer abuse and global warming. Eutrophication can induce the proliferation of cyanobacteria such as Microcystis, which produces microcystins. Microcystins are toxic to specific organs such as the liver and the heart. Thus, monitoring of microcystins is strongly required to control drinking water and agricultural product qualities. However, microcystins could be adsorbed by plastic materials during sample storage and preparation, hindering accurate analysis. Therefore, the current study examined the recovery rate of microcystins from six plastics used for containers and eight plastics used for membrane filters. Among the six plastics used for containers, polyethylene terephthalate showed the best recovery rate (≥81.3%) for 48 h. However, polypropylene, polystyrene, and high- and low-density polyethylenes showed significant adsorption after exposure for 1 hr. For membrane materials, regenerated cellulose (≥99.3%) showed the highest recovery rate of microcystins, followed by polyvinylidene fluoride (≥94.1%) and polytetrafluoroethylene (≥95.7%). The adsorption of microcystins appeared to be strongly influenced by various molecular interactions, including hydrophobic interaction, hydrogen bonding, and electrostatic interaction. In addition, microcystins’ functional residues seemed to be critical factors affecting their adsorption by plastic materials. The present study demonstrates that polyethylene terephthalate and regenerated cellulose membrane are suitable plastic materials for the analysis of microcystins.

Microwave cell lysis for ADDA‐ELISA analysis of total microcystins and nodularins

Microwave cell lysis for <scp>ADDA‐ELISA</scp> analysis of total microcystins and nodularins

Seasonal Occurrence of Cyanobacteria and First Detection of Microcystin-LR in Water Column of Foum-Gleita Reservoir, Mauritania

This work was carried out to study the seasonal occurrence of cyanobacteria and their microcystin-LR in the water column of Foum-Gleita reservoir (Mauritania). Limnological and biological factors were investigated at three depths (surface, − 3, and − 6 m) in this reservoir during a full year. Nutrients were analyzed by Spectrophotometry, phytoplankton was analyzed by Inverted Microscopy, microsystins were analyzed by High Performance Liquid Chromatography-tandem Mass Spectrometry, and environmental factor relationships were analyzed by Pearson’s correlation and Multiple Linear Regression. Physicochemical analyses have shown that this reservoir is hypertrophic with dissolved inorganic nitrogen and total phosphorus concentrations relatively high, varying from 1.39 to 6.53 and 0.21 to 0.57 mg/L, respectively. The annual surface water temperature was exceptionally high (27.8 ± 3.6 °C), characterizing Sahelian climatic conditions. Phytoplankton analyses have shown dominance of two potentially toxic cyanobacteria species, Microcystis aeruginosa and Dolichospermum flos-aquae, during the warm season (May-September). Microcystin analysis revealed only the presence of the most toxic variant, microcystin-LR. Microcystin-LR concentration in the surface water samples, during cyanobacterial blooms, was consistently high (5.638 µg/L), exceeding 5-times the World Health Organization drinking water limit (1 µg/L); however, it was much lower (0.83 µg/L) at depth (− 6 m). Analysis of environmental factor relationships showed that the most influential factors on abundance of Microcystis aeruginosa and Dolichospermum flos-aquae and variability of microcystin-LR concentrations were total phosphorus, dissolved inorganic nitrogen, iron, temperature and pH. Finally, the study clearly demonstrated the need for regular monitoring of cyanobacteria and cyanotoxins in the reservoir.

First occurrence of Cylindrospermopsin in the Azores (Lake São Brás, S. Miguel Island)

Anthropogenic impacts have been reported as one of the main causes of eutrophication in freshwater systems and nutrient availability, mainly from discharges, are key to cyanobacteria development and harmful cyanobacteria blooms (cyanoHABs) upsurge. As these scenarios are common in the Azores, a cyanobacteria monitoring program was implemented in the lakes of the archipelago, which comprises cyanobacteria identification and cyanotoxin (only microcystin) analysis in over 15 lakes. However other cyanotoxins should be searched as is the case of cylindrospermopsin (CYN). This cyanotoxin has been reported more recently in a wider range of countries, because of global warming. Using ESI–LC–MS/MS (electrospray ionization–liquid chromatography–mass spectrometry), the presence of CYN was investigated in samples from four eutrophic lakes. Cylindrospermopsin was detected in one sample from Lake Sao Bras, which represents a threat to environmental and public health. This is the first CYN identification in the Azores reinforcing the necessity for lake monitoring. We propose the use of a polyphasic approach to fully understand CYN presence in the Azorean lakes, investigating the producing taxa, the environmental factors that promote cyanobacteria growth, and CYN toxicity effects.

Microwave cell lysis for ADDA‐ELISA analysis of total microcystins and nodularins

AbstractA fast and effective microwave treatment procedure was developed for the ADDA–enzyme‐linked immunosorbent assay analysis of total extracellular and intracellular microcystins and nodularins in drinking water and ambient water. This microwave treatment procedure for a full batch of samples required 10–20 min versus the 3–4 hr typically needed for three freeze/thaw cycles. The obtained lowest‐concentration minimum reporting level and method detection limit were 0.21 and 0.09 μg/L microcystin‐LR (MC‐LR) equivalents, respectively. Mean recoveries of 80%–92% with relative standard deviations of 4.2%–9.0% were obtained for all studied water matrices fortified at concentrations of 0.3–2.0 μg/L MC‐LR. Compared with US Environmental Protection Agency Method 546, this alternate procedure provided more effective cell lysis and subsequently detected significantly higher concentrations of total microcystins and nodularins in a few water samples collected during harmful algal blooms, such as 61.0 versus 34.4 μg/L MC‐LR equivalents in Lake Erie and 11.5 versus 9.1 μg/L MC‐LR equivalents in an Indiana reservoir.

Analysis of total microcystins and nodularins by oxidative cleavage of their ADMAdda, DMAdda, and Adda moieties

Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda5]MCs and [DMAdda5]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda5]- and [ADMAdda5]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were <0.25% when analyzed with an Adda enzyme-linked immunosorbent assay (ELISA). Oxidative cleavage experiments identified compounds that could be used in the analysis of total MCs/NODs in a similar fashion to the 2R-methyl-3S-methoxy-4-phenylbutanoic acid (MMPB) technique. Products from oxidative cleavage of both the 4,5- and 6,7-ene of Adda, DMAdda and ADMAdda were observed, and three oxidation products, one from each Adda variant, were chosen for analysis and applied to three field samples and a Nostoc culture. Results from the oxidative cleavage method for total Adda, DMAdda, and ADMAdda were similar to those from the Adda-ELISA, PP2A inhibition, and LC-MS/MS analyses, except for the Nostoc culture where the Adda-ELISA greatly underestimated microcystin levels. This oxidative cleavage method can be used for routine analysis of field samples and to assess the presence of the rarely reported, but toxic, DMAdda/ADMAdda-containing MCs and NODs.

High Levels of Anabaenopeptins Detected in a Cyanobacteria Bloom from N.E. Spanish Sau-Susqueda-El Pasteral Reservoirs System by LC-HRMS.

The appearance of a bloom of cyanobacteria in the Sau-Susqueda-El Pasteral system (River Ter, NE Spain) in the autumn of 2015 has been the most recent episode of extensive bloom detected in Catalonia. This system is devoted mainly to urban supply, regulation of the river, irrigation and production of hydroelectric energy. In fact, it is one of the main supply systems for the metropolitan area of cities such as Barcelona and Girona. An assessment and management plan was implemented in order to minimize the risk associated to cyanobacteria. The reservoir was confined and periodic sampling was carried out. Low and high toxicity was detected by cell bioassays with human cell lines. Additionally, analysis studies were performed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–high resolution mass spectrometry (LC–HRMS). A microcystin target analysis and suspect screening of microcystins, nodularins, cylindrosperpmopsin and related cyanobacterial peptides by LC–HRMS were applied. The results for the analysis of microcystins were negative (<0.3 μg/L) in all the surface samples. Only traces of microcystin-LR, -RR and -dmRR were detected by LC–HRMS in a few ng/L from both fractions, aqueous and sestonic. In contrast, different anabaenopeptins and oscillamide Y at unusually high concentrations (µg-mg/L) were observed. To our knowledge, no previous studies have detected these bioactive peptides at such high levels. The reliable identification of these cyanobacterial peptides was achieved by HRMS. Although recently these peptides are detected frequently worldwide, these bioactive compounds have received little attention. Therefore, more studies on these substances are recommended, especially on their toxicity, health risk and presence in water resources.

Recent developments in the methods of quantitative analysis of microcystins.

Cyanotoxins are produced by the toxic cyanobacterial species present in algal blooms formed in water bodies due to nutrient over-enrichment by human influences and natural environmental conditions. Extensive studies are available on the most widely encountered cyanotoxins, microcystins (MCs) in fresh and brackish water bodies. MC contaminated water poses severe risks to human health, environmental sustainability, and aquatic life. Therefore, commonly occurring MCs should be monitored. Occasionally, detection and quantification of these toxins are difficult due to the unavailability of pure standards. Enzymatic, immunological assays, and analytical techniques like protein phosphatase inhibition assay, enzyme-linked immunosorbent assay, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and biosensors are used for their detection and quantification. There is no single method for the detection of all the different types of MCs; therefore, various techniques are often combined to yield reliable results. Biosensor development offered a problem-solving approach in the detection of MCs due to their high accuracy, sensitivity, rapid response, and portability. In this review, an endeavor has been made to uncover emerging techniques used for the detection and quantification of the MCs.

Optimization of an MMPB Lemieux Oxidation method for the quantitative analysis of microcystins in fish tissue by LC-QTOF MS

Microcystins are toxic heptapeptides produced by cyanobacteria in marine and freshwater environments. In biological samples such as fish, microcystins can be found in the free form or covalently bound to protein phosphatases type I and II. Total microcystins in fish have been quantified in the past using the Lemieux Oxidation approach, where all toxins are oxidated to a common fragment (2-methyl-3-methoxy-4-phenylbutyric acid, MMPB) regardless of their initial amino acid configuration or form (free or protein bound). These studies have been carried out using different experimental conditions and employed different quantification strategies. The present study has further investigated the oxidation step using a systematic approach, to identify the most important factors leading to a higher, more robust MMPB generation yield from fish tissue in order to reduce the method detection limit. Field samples were quantified using an in-situ generated MMPB matrix matched calibration curve by isotope dilution with d3-MMPB via liquid chromatography coupled to time-of-flight mass spectrometry (LC-QTOF MS). This approach improves method's accuracy by taking into account of potential matrix effects that could affect the derivatization, sample prepation and instrumental analysis steps. The validated method showed 16.7% precision (RSD) and +6.7% accuracy (bias), with calculated method detection limits of 7.28 ng g−1 Performance of the method was assessed with the analysis of laboratory exposed Rainbow Trout (Oncorhynchus mykiss) to cyanobacteria as a positive control, where no microcystins were detected in the pre-exposure fish liver and fillet, low levels in the exposed fillet (65.0 ng g−1) and higher levels in the exposed liver (696 ng g−1). Finally, the method was employed for the analysis of 26 fillets (muscle) and livers of Walleye (Sander vitreus) and Yellow Perch (Perca flavescens) from Lake Erie, showing very low concentrations of microcystins in the fillet and higher concentrations in liver, up to 3720 ng g−1.

Synthesis of metal-organic framework-5@chitosan material for the analysis of microcystins and nodularin based on ultra-performance liquid chromatography-tandem mass spectrometry

Microcystins (MCs) and nodularin (NOD) are tumor promoters produced by cyanobacteria and present in surface water. In this work, a novel mesoporous metal-organic framework-5@chitosan (MOF-5@CS) material was synthesized and applied for the enrichment of MCs and NOD in water and fish samples. The mesoporous MOF-5@CS material was firstly synthesized via a one-step hydrothermal method, and the chitosan was combined with MOF-5 via chemical bonding assembly. As a new adsorbent, the as-synthesized material was found having a large specific surface area and good thermal stability. Under the optimized conditions, MCs and NOD were enriched by the MOF-5@CS material and detected by ultra-performance liquid chromatography-tandem mass spectrometry. The limit of detection of the new method for MCs and NOD were in the range of 0.0018-0.077 ng/mL. The value of relative standard deviation for repeatability were 2.69-6.30%, and the recovery of the analytes ranged from 84.36% to 118.51%. Compared with other reported method for MCs and NOD detection in complex matrices, better adsorption performance for MCs and NOD were obtained by our new method, and the sensitivity of MCs-RR and NOD were improved nearly 20 times and 30 times, respectively.

Laser irradiation desorption of microcystins from protein complex in fish tissue and liquid chromatography-tandem mass spectrometry analysis.

Microcystins are a group of cyanotoxins which interact with the C-terminal region of PP1 and PP2A proteins, so denaturation and inactivation are necessary for breaking covalent binding to release microcystins. In this study, a novel extraction method was developed by laser irradiation desorption of microcystins from fish protein. The sample was mixed with aqueous methanol and irradiated by a 450nm laser, with an optimized value of laser power density at 8 W and exposure time at 5min. ThenLC-MS/MS was applied for the determination of microcystins in fish extracts. The ionization behaviors of microcystins were investigated firstly, and doubly charged microcystins were selected as precursor ions in multiple reaction monitoring scan for quantification. This proposed quantitative method was well validated in terms of selectivity, linearity, sensitivity, accuracy, recovery, and stability. The successful application of this LC-MS/MS method showed its ability for the analysis of microcystins in low concentration, and it would be of significant interest for environmental and food safety applications to ensure the safety of fish and related products.

Quantitative analysis of microcystin variants by capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing.

Dynamic pH junction is an online focusing method in CE based on the electrophoretic mobility difference of analytes in the sample matrix and the background electrolyte. An advantage of this method over the conventional CE is that the sensitivity can be significantly improved. By injecting a long sample plug in the capillary and focusing the analytes at the pH boundary between the background electrolyte and sample matrix, the LOD can be improved by 10-100 folds. The dynamic pH junction method can be easily coupled with ESI-MS. In this work, we used this method for the analysis of microcystins (MCs). The detection limits and dynamic ranges were studied. The separation was optimized by adjusting the injection time, and concentrations and pH values of the background electrolyte. The optimization of analyte focusing leads to enhanced detection response compared to conventional injections, achieving 200-400 fold higher averaged peak heights for four microcystin (MC) variants. More importantly, this method was successfully used for the quantitative analysis of microcystins (MCs) in crude algae samples from natural water bodies, making it promising for practical applications.

A Novel Proof-of-Concept Sandwich Immunoassay for Screening Microcystin in Cyanobacteria Based on Michael Addition Reaction.

We present an innovative concept of a screening tool for detecting free microcystin in cyanobacteria using a sandwich immunodetection format, based on Michael addition reaction between α,β-unsaturated carbonyl moiety of microcystin and thiol of coating substance. This proof-of-concept immunoassay was developed using bovine serum albumin as a microcystin-binding model, and was tested with toxic Microcystis samples. The preliminary results indicate that the proposed Michael addition-based immunodetection is promising and can be used as a platform for further development to become a useful tool for free microcystin analysis in various samples in the future.

Occurrence of the First Toxic Microcystis Bloom in a Recent Moroccan Reservoir

In order to understand initial conditions that promote the first occurrence of a Microcystis aeruginosa bloom and to evaluate its toxic potential, we studied spatio-temporal dynamics of phytoplankton, including Microcystis, microcystins concentrations, the relative proportion of potentially microcystin producing cells and physicochemical parameters in the recently setup Moroccan reservoir “Yaacoub Al Mansour.” High summer temperatures, thermal stability, hydrological stability of the water column and the decrease of nutrient contributions seem to be the major environmental factors responsible of this first bloom occurrence. Despite the low cellular abundance, high microcystins concentrations, associated with low percentage of toxic strains, were measured. Thus, microcystins analysis and associated management of the reservoir are necessary from the beginning of the setup and can help managers to find the better environmental strategy against early cyanobacteria proliferations.