Determination Of Microcystins Research Articles

Development and validation of an immunochromatographic test system for the determination of microcystins in water and tissues of freshwater fish

Development and validation of an immunochromatographic test system for the determination of microcystins in water and tissues of freshwater fish

The occurrence and human health risk assessment of microcystins in diverse food matrixes during production

Globally, the presence of cyanotoxins in water supplies and food has been widely investigated for over a decade. Cyanotoxins are harmful metabolites produced by toxic cyanobacterial genera. These metabolites belong to diverse chemical classes, with a variety of physicochemical properties, chemical structures, and toxic activities. The present study seeks to investigate the occurrence of cyanotoxins in water supplies destined for food processing and assess the human health risk from exposure to cyanotoxins. To achieve this, a simple, sensitive, and reliable analytical method was developed for the determination of microcystins (MC-RR, MC-LR, MC-YR) in process water, raw maize meal, and cooked maize (porridge) at ppb (parts per billion) levels. These compounds were extracted using Solid Phase Extraction (SPE) with optimized parameters; thereafter, Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) was used for the rapid determination of the analytes selected for the present study. The method developed was applied to samples collected from the meal grinding station located in Mawoni village in South Africa; and was able to detect and quantify all the target cyanotoxins. MC-LR, MC-YR and MC-RR were detected at concentrations ranging from 10 to 11.2 μg/L, 9.1–9.4 μg/L, and 2.3–3.5 μg/L, in water samples, respectively. However, MC-YR was not detected in ground water sample. Moreover, MC-LR, MC-YR, and MC-RR concentrations in maize and porridge samples ranged between 9.2 and 11.2, 5.5–8.6, and 6.3–9.3 μg/kg dry weight, respectively. The hazard quotient index (HQi) levels found in the present study ranged between 2.2 – 8.4 and 0.11–8.9 for adults and children, respectively, representing potential risks to human health. Findings from LC-MS/MS reveal that cyanotoxins can be transferred from water to food during food processing using cyanotoxins contaminated water. Furthermore, the methods developed can be used by environmental and health agencies to strengthen the monitoring of cyanotoxins in water and food.

Determination of Microcystins in Fish Tissue by ELISA and MALDI-TOF MS Using a Highly Specific Single Domain Antibody.

The development of simple, reliable, and cost-effective methods is critically important to study the spatial and temporal variation of microcystins (MCs) in the food chain. Nanobodies (Nbs), antigen binding fragments from camelid antibodies, present valuable features for analytical applications. Their small antigen binding site offers a focused recognition of small analytes, reducing spurious cross-reactivity and matrix effects. A high affinity and broad cross-reactivity anti-MCs-Nb, from a llama antibody library, was validated in enzyme linked immunosorbent assay (ELISA), and bound to magnetic particles with an internal standard for pre-concentration in quantitative-matrix-assisted laser desorption ionization-time of flight mass spectrometry (Nb-QMALDI MS). Both methods are easy and fast; ELISA provides a global result, while Nb-QMALDI MS allows for the quantification of individual congeners and showed excellent performance in the fish muscle extracts. The ELISA assay range was 1.8-29 ng/g and for Nb-QMALDI, it was 0.29-29 ng/g fish ww. Fifty-five fish from a MC-containing dam were analyzed by both methods. The correlation ELISA/sum of the MC congeners by Nb-QMALDI-MS was very high (r Spearman = 0.9645, p < 0.0001). Using ROC curves, ELISA cut-off limits were defined to accurately predict the sum of MCs by Nb-QMALDI-MS (100% sensitivity; ≥89% specificity). Both methods were shown to be simple and efficient for screening MCs in fish muscle to prioritize samples for confirmatory methods.

Untargeted and targeted LC-MS and data processing workflow for the comprehensive analysis of oligopeptides from cyanobacteria

Cyanobacteria produce a plethora of structurally diverse bioactive secondary metabolites, including cyanotoxins which pose a serious threat to humans and other living organisms worldwide. Currently, a wide variety of mass spectrometry-based methods for determination of microcystins (MCs), the most commonly occurring and studied class of cyanotoxins, have been developed and employed for research and monitoring purposes. The scarcity of commercially available reference materials, together with the ever-growing range of mass spectrometers and analytical approaches, make the accuracy of quantitative analyses a critical point to be carefully investigated in view of a reliable risk evaluation. This study reports, a comparative investigation of the qualitative and quantitative MCs profile obtained using targeted and untargeted liquid chromatography–mass spectrometry approaches for the analyses of cyanobacterial biomass from Lake Kastoria, Greece. Comparison of the total MCs content measured by the two approaches showed good correlation, with variations in the range of 3.8–13.2%. In addition, the implementation of an analytical workflow on a hybrid linear ion trap/orbitrap mass spectrometer is described, based on combining data-dependent acquisition and a powerful database of cyanobacterial metabolites (CyanoMetDB) for the annotation of known and discovery of new cyanopeptides. This untargeted strategy proved highly effective for the identification of MCs, microginins, anabaenopeptins, and micropeptins. The systematic interpretation of the acquired fragmentation patterns allowed the elucidation of two new MC structural variants, MC-PrhcysR and MC-Prhcys(O)R, and proposal of structures for two new microginins, isomeric cyanostatin B and MG 821A, and three isomeric micropeptins at m/z 846.4715, 846.4711 and 846.4723.

Simplified validation of the ELISA kit determination of Microcystins in surface water.

The enzyme-linked immunosorbent assay (ELISA), as a universal method for the determination of Microcystins, is of great significance for the rapid detection of Microcystins pollution. This study aimed to propose a simplified validation method for Microcystins ELISA kit by summarizing related documents and guidelines. After summarizing and clarifying from 20 validation parameters, 11 parameters were selected to simplify the validation of Microcystins ELISA kit. In addition, the acceptable range and validation details of each parameter were analyzed. The results indicated that the coefficient of determination of the Microcystin-LR standard curve was higher than 0.99. The concentration of quality control samples was within control limits. The accuracy of spiked and proficient samples was within 70%-130%. The variability of intra-assay, inter-assay, and reproducibility was less than 11, 15 and 21%, respectively. The LOD and LLOQ were 0.002 μg/L and 0.05 μg/L, respectively. When the concentration of Microcystins exceeded 5 μg/L, it was recommended to dilute the samples to the working range before detection. The specificity was estimated with seven Microcystin analogues and three amino acids, indicating that the cross-reactivity was less than 30%. These results revealed that the ELISA kit was satisfactory for detecting Microcystins in water.

Rapid detection and quantification of microcystins in surface water by an impedimetric immunosensor

Exposure to cyanotoxins, particularly microcystins, in water reservoirs, is associated with hepatotoxicity and carcinogenesis and leads to acute and chronic damages. Consequently, many countries have imposed stringent regulations, limiting the allowed microcystins concentration in drinking water to < 1 μg/L. Current methods for microcystins detection rely on laboratory-based methods requiring skilled personnel and labor-intensive preparation, which cannot provide a real-time measurement at the required sensitivity. Therefore, there is an imperative need for an on-site diagnostic tool providing a rapid quantitative determination of microcystins in surface water. We have developed an electrochemical biosensor based on the integration of specific antibodies with a biochip and a measurement platform and applied it in the detection of microcystin-LR, among the most toxic microcystins, by electrochemical impedance spectroscopy. Using the miniaturized platform, quantitative detection of microcystin-LR was feasible, exhibiting a broad dynamic range of five logarithmic concentrations and a detection limit of 3 ng/L, which is superior to currently employed surface-based immunoassays. More importantly, specific detection of microcystin-LR from models of cyanobacteria-contaminated water was also demonstrated. Based on our findings, we anticipate that electrochemical biosensors would be an essential tool in water monitoring and environmental diagnostics in the near future.

Rapid determination of microcystins and nodularin in surface water by on-line solid-phase extraction (SPE) with ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS)

A method for determining seven microcystins and nodularin in surface water at nanogram per liter concentrations was developed based on on-line solid-phase extraction ultra-performance liquid chromatography – electrospray ionization tandem mass spectrometry (on-line SPE-UPLC-MS/MS). Aliquots of 5 mL water sample were directly injected into on-line SPE after passage through a membrane filter. The water sample was enriched and purified by a hydrophilic–liphophilic balance (HLB) polymeric column and eluted by mobile phase. The analytes were separated by Acquity BEH130 column, detected using tandem mass spectrometry, and quantified by external standardization. Under the optimized conditions, the method detection limits for all of the targets ranged from 1.4 to 3.3 ng/L with the recoveries from 85.5 to 121%. The precision of the method, indicated by the relative standard deviation, was 3.87 to 13.9%. The results of the analysis of surface water samples demonstrated that the proposed method has the ability to determine microcystins and nodularin without the need for complex manual pretreatment.

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.

Determination of microcystins in environmental water samples with ionic liquid magnetic graphene

Microcystins is a class of monocyclic of heptapeptides with many different isomerides. It has become potential hazardous material in water environment for its toxic, distribution and stability. This project worked on a method for determination of trace microcystin (MC-LR and MC-RR) in environmental waters. The ionic liquid magnetic graphene (IL@MG) was prepared and applied to the concentration and determination of microcystins, based on magnetic solid phase extraction (MSPE), and coupled with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The ionic liquid magnetic graphene was prepared by coprecipitatial synthesis and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), specific surface area (BET), pore size distribution (BJH) and magnetic hysteresis loop. The experimental parameters of magnetic solid phase extraction, including amount of IL@MG, pH, extraction time and elution solvent were investigated by a univariate method and orthogonal screening. The method showed good linearity in the range of 0.01–10.0 g/L and 0.005–10.0 μg/L for MC-LR and MC-RR, when the pH of water samples was 4.00 and 10.0 mg adsorbents were used to extract targets for 18 min. The lowest detection limit was 0.414 ng/L and 0.216 ng/L for MC-LR and MC-RR respectively. The recoveries of the microcystins were in the range of 83.6–100.9%, and the relative standard deviation was less than 7.59%. The trace amount of MC-LR (0.020 μg/L) and MC-RR (0.003 μg/L and 0.021 μg/L) was detected in actural water samples. Attributed to its simple operator, low detection limit and high sensitivity, this method could be used for the detection of trace microcystins in water samples.

Determination of microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin in water and fish tissue using isotope dilution liquid chromatography tandem mass spectrometry

Cyanobacteria can form dense blooms under specific environmental conditions, and some species produce secondary metabolites known as cyanotoxins, which present significant risks to public health and the environment. Identifying toxins produced by cyanobacteria present in surface water and fish is critical to ensuring high quality food and water for consumption, and protectionn of recreational uses. Current analytical screening methods typically focus on one class of cyanotoxins in a single matrix and rarely include saxitoxin. Thus, a cross-class screening method for microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin was developed to examine target analytes in environmental water and fish tissue. This was done, due to the broad range of cyanotoxin physicochemical properties, by pairing two extraction and separation techniques to improve isolation and detection. For the first time a zwitterionic hydrophilic interaction liquid chromatography column was evaluated to separate anatoxin-a, cylindrospermopsin, and saxitoxin, demonstrating greater sensitivity for all three compounds over previous techniques. Further, the method for microcystins, nodularin, anatoxin-a, and cylindrospermopsin were validated using isotopically labeled internal standards, again for the first time, resulting in improved compensation for recovery bias and matrix suppression. Optimized extractions for water and fish tissue can be extended to other congeners in the future. These improved separation and isotope dilution techniques are a launching point for more complex, non-targeted analyses, with preliminary targeted screening.

Determination of microcystins in water samples by deep eutectic solvent-based vortex-assisted liquid-liquid microextraction coupled with ultrahigh-performance liquid chromatography-high resolution mass spectrometry.

Rapid screening of two microcystins (i.e., microcystin-YR (MC-YR) and microcystin-LR (MC-LR)) in surface water samples was performed by a simple and eco-friendly procedure using deep eutectic solvent-based vortex-assisted liquid–liquid microextraction (DES-based VALLME) combined with ultrahigh-performance liquid chromatography and electrospray ionization (+)-quadrupole time-of-flight mass spectrometry (UHPLC-ESI(+)-qTOF-MS) detection. To obtain an efficient water-miscible DES, choline chloride and phenol at a molar ratio of 1 : 2 were used as an extractant for VALLME. To optimize factors of DES-based VALLME, response surface design alongside Box–Behnken design was used. The limits of quantitation (LOQs) were 0.5 ng mL−1 and 0.4 ng mL−1 for MC-YR and MC-LR, respectively, which is sensitive enough to meet the World Health Organization (WHO) maximum guideline level for MC-LR in water of 1.0 ng mL−1. Moreover, satisfactory precision with relative standard deviations (RSD) for both intra- and inter-day analysis lower than 11%, and trueness (also known as mean extraction recovery) ranged from 85.5 to 113% based on the ICH method validation guideline.

New Method for Simultaneous Determination of Microcystins and Cylindrospermopsin in Vegetable Matrices by SPE-UPLC-MS/MS.

Cyanotoxins are a large group of noxious metabolites with different chemical structure and mechanisms of action, with a worldwide distribution, producing effects in animals, humans, and crop plants. When cyanotoxin-contaminated waters are used for the irrigation of edible vegetables, humans can be in contact with these toxins through the food chain. In this work, a method for the simultaneous detection of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR), and Cylindrospermopsin (CYN) in lettuce has been optimized and validated, using a dual solid phase extraction (SPE) system for toxin extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Results showed linear ranges (5–50 ng g−1 f.w.), low values for limit of detection (LOD) (0.06–0.42 ng g−1 f.w.), and limit of quantification (LOQ) (0.16–0.91 ng g−1 f.w.), acceptable recoveries (41–93%), and %RSDIP values for the four toxins. The method proved to be robust for the three variables tested. Finally, it was successfully applied to detect these cyanotoxins in edible vegetables exposed to cyanobacterial extracts under laboratory conditions, and it could be useful for monitoring these toxins in edible vegetables for better exposure estimation in terms of risk assessment.

Solid-phase extraction, quantification, and selective determination of microcystins in water with a gold-polypyrrole nanocomposite sorbent material

A novel sorbent material, gold-polypyrrole (Au-PPy) nanocomposite-coated silica, is described for the efficient solid-phase extraction (SPE) of six common microcystins (MCs) well below the recommended United States EPA and World Health Organization (WHO) guidelines. With the optimized SPE protocol, samples spiked with MCs were determined at ng/L concentrations by liquid chromatography-mass spectrometry (LC–MS) in different aqueous sample matrices, including HPLC-grade, tap, and lake water. The average recoveries for all MCs tested in the three water matrices ranged from 94.1–103.2% with relative standard deviations (RSDs) of 1.6–5.4%, which indicated excellent extraction efficiency and reproducibility. Limits of detection (LODs) and limits of quantification (LOQs) for all MCs in both tap and lake water samples were determined to be ≤1.5 ng/L and 5.0 ng/L, respectively. The Au-PPy nanocomposite-coated sorbent material was reusable for at least three independent MC extractions with a single SPE cartridge in the concentration range of 10–500 ng/L. Importantly, off-column selective separation at the sample preparation and preconcentration stage between more hydrophilic and more hydrophobic MCs was achieved by sequential elution through changes in the solvent composition and SPE bed size. Therefore, the Au-PPy nanocomposite-coated silica sorbent is a promising new material for the quantification of MC variants in water samples.

Mass Spectrometric Analysis of Microcystins from Cyanobacterial Biomass: Optimization of the Sample Preparation Procedure

The sample preparation procedure for determination of microcystins from biomass by the LC–MS method was optimized. Treatment of freeze-dried biomass samples with acetonitrile prior to the main extraction was effectively used for decreasing the amount of protein-like matrix compounds in extracts. Use of acetonitrile–water mixtures during the main ultrasound-assisted extraction allowed increasing the total microcystins amount in the resultant extracts compared to the traditional extraction using 75% methanol.

Determination of microcystins in algal bloom samples by ultra performance liquid chromatography-tandem mass spectrometry and elimination of their matrix effects

A rapid and simple method was commonly used for the determination of microcystins by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The reliability, accuracy and sensitivity of LC-MS/MS determination were affected by the matrix effect because of the complex matrices in algal bloom samples. In this study, the matrix interference and matrix effect problems were effectively reduced or eliminated by diluting samples, optimizing gradient elution programs, and reducing injection volumes. The correlation coefficients (r2) of the seven microcystins were ≥ 0.99. The limits of detection and limits of quantification ranged from 0.064 μ g/L to 0.103 μ g/L and 0.213 μ g/L to 0.343 μ g/L, respectively. Algal bloom samples were determined by diluting 10 times and injecting 5 μ L of sample volume. The method provides some references for the determination of microcystins. It is of great significance for ensuring the safety of drinking water source.

Rapid and simultaneous determination of seven microcystins in fish meat by liquid chromatography-tandem mass spectrometry coupled with pass-through solid phase extraction

An analytical method was developed for the simultaneous and rapid determination of seven microcystins in fish meat by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with pass-through solid phase extraction (SPE). The samples were extracted with methanol-water (90:10, v/v) after heat treatment by water bath at 80℃, and then cleaned up with an Oasis PRiME HLB pass-through SPE column. The samples were analyzed directly on a Waters XSelect HSS T3 column using 0.1% (v/v) aqueous formic acid and acidified acetonitrile (0.1% formic acid, v/v) as mobile phases. Qualitative and quantitative analysis of the analytes was carried out under the multiple reaction monitoring mode with positive electrospray ionization. The matrix matching external standard method was used for quantitation analysis. To solve the problem of parent ion selection of the microcystins, the ionization characteristics of microcystins were evaluated under different mobile phase conditions. Finally, the results showed that the acid could promote the intensity of the doubly charged ions significantly. The calibration curves were linear well in the corresponding concentration ranges, with correlation coefficient ≥ 0.99. The limits of quantification ranged from 0.30 to 2.0 μ g/kg. The average spiked recoveries for the seven microcystins were between 70.6% and 96.1% with the relative standard deviations of 3.4%-9.6%. The proposed method is sensitive, accurate, and efficient. It is applicable for the determination of microcystins in fish meat.

Quantitative determination by screening ELISA and HPLC-MS/MS of microcystins LR, LY, LA, YR, RR, LF, LW, and nodularin in the water of Occhito lake and crops.

The occurrence of harmful cyanobacterial blooms in surface waters is often accompanied by the production of a variety of cyanotoxins, and these toxins are designed to target in humans specific organs on which they act. When introduced into the soil ecosystem by spray irrigation of crops, they may affect the same molecular pathways in plants having identical or similar target organs, tissues, cells, or biomolecules. There are also several indications that terrestrial plants, including crops, can bioaccumulate cyanotoxins and present, therefore, potential health hazards for humans. During this project, for monitoring purposes, water samples were collected from lake Occhito, in which there was an algal bloom (Planktothrix rubescens) in 2009, and from three tanks which acted as hydraulic junctions. In addition, crop samples irrigated with water from the three tanks mentioned above were also picked. Finally, the characterization of principal cyanobacteria was performed, to determine the presence of cyanotoxins such as microcystins and validate a method of screening ELISA for the determination of microcystins in vegetable samples and a confirmatory method by HPLC-ESI-MS/MS. Graphical abstract Occhito lake (left), microcystin LR (center), Tomato field in Foggia (right); figures below: ELISA (left), HPLC-MS/MS (right).

Opredelenie mikrozistinov i anatoksina-a metodom zhidkostnoy chromato-mass- spektrometrii nizkogo razresheniya

Opredelenie mikrozistinov i anatoksina-a metodom zhidkostnoy chromato-mass- spektrometrii nizkogo razresheniya

Solid-phase extraction based on mesoporous Fe 3 O 4 @mSiO 2 @Cu 2+ magnetic nanoparticles coupled with high performance liquid chromatography for the determination of microcystins in water samples

Mesoporous magnetic nanoparticles of Fe3O4 @ mSiO2 @ Cu2+ were synthesized by two methods of hydrothermal synthesis and room temperature synthesis. The synthesized mesoporous Fe3O4 @ mSiO2 @ Cu2+ nanoparticles presented uniform sizes, good magnetic properties and specific selectivity. In this work, a novel magnetic solid-phase extraction (MSPE) method based on mesoporous Fe3O4 @ mSiO2 @ Cu2+ nanoparticles (NPs) coupled with high performance liquid chromatography (HPLC) was developed for the simultaneous extraction of trace amount of microcystin-LR (MC-LR) in water samples. The MSPE extraction conditions and HPLC conditions were optimized. The results showed that good linear relationship (r = 0.999 4) was obtained between peak area and mass concentration of MC-LR in the range of 0.1-15 µg/L. The limit of detection was 0.025 µg/L, and the limit of quantification was 0.082 µg/L. Furthermore, this method was used for the analysis of algal toxins in water samples with the recovery of 78%. The results indicated that Fe3O4 @ mSiO2 @ Cu2+ magnetic nanoparticles were good pretreatment alternatives for the concentration of microcystin-LR in water samples.

Microcystin-(Leucine-Arginine) Immunosensor Based on Iron(II, III) Magnetic Nanoparticles

An electrochemical immunosensor for microcystin-(leucine-arginine) based on magnetic bionanoparticles and iron(II, III) oxide was developed. The bionanoparticles were prepared by cross-linking antibodies of microcystin-(leucine-arginine) and amino-functionalized magnetic iron(II, III) oxide nanoparticles with glutaraldehyde, followed by immobilization on the surface of a magnetic electrode. The immunosensor was based on the model of direct competition, as microcystin-(leucine-arginine) and horseradish peroxidase-conjugated microcystin-(leucine-arginine) competitively combined with immobilizing antibodies. The peak current of differential pulse voltammetry (DPV) decreased with an increase of microcystin-(leucine-arginine) concentration after antigen-antibody reaction. When the background current was stabilized, the anodic peak current response and change in peak response were recorded. Under the optimized conditions, the change in response was proportional to the microcystin-(leucine-arginine) concentration between 0.010 and 100 µg/L with a limit of detection equal to 0.009 µg/L. Amperometry was adopted to determine microcystin-(leucine-arginine); the linear dynamic range was 0.10 to 100 µg/L with a detection limit of 0.08 µg/L. The method was successfully applied in the determination of microcystin-(leucine-arginine) in river water and the recoveries were between 90.2% and 110.5%.