Isolation and preparative purification of microcystin variants

Preparative reversed-phase liquid chromatography was successfully used to purify two microcystins (microcystin LR and microcystin LA) from a cyanobacterial process waste. The separation protocol involved extraction of lyophilized cells by methanol, isolation and concentration by solid-phase extraction, and purification by reversed-phase HPLC. Milligram-level loading of microcystins was obtained on a solid-phase extraction cartridge packed with 0.5 g of C18 stationary phase. The separations were first carried out on an analytical column and then scaled-up to a preparative column. The microcystins were quantified by HPLC and enzyme-linked immunosorbent assay. A method to remove microcystins rapidly and economically from the cyanobacterial process waste is also described.     

High-Throughput Screening of Ten Microcystins and Nodularins, Cyanobacterial Peptide Hepatotoxins, by Reversed-Phase Liquid Chromatography-Electrospray Ionisation Mass Spectrometry

Liquid chromatography-electrospray ionisation mass spectrometry was evaluated in the high-throughput analysis of microcystins and nodularins, cyanobacterial peptide hepatotoxins. Extracts originating from cyanobacterial strains and field material were separated on a 30 mm × 4 mm I.D. Merck Purospher STAR RP-18e column using a rapid gradient of aqueous formic acid and acetonitrile, ionised by electrospray technique and analysed on a Micromass Quattro II triple-quadrupole mass spectrometer operated in the selected ion recording (SIR) mode. The total analysis time per sample was 2.8 min corresponding to 514 samples a day. The system showed good robustness during a series of 320 repetitive injections of a field sample containing three major microcystins.     

Characterization and Diversity of Cyano- bacterial Hepatotoxins (Microcystins) in Blooms from Polish Freshwaters Identified by Liquid Chromatography-Electrospray Ionisation Mass Spectrometry

Microcystins were determined in 36 cyanobacterial (blue-green algal) bloom samples dominated by Microcystis aeruginosa, Aphanizomenon flos-aquae and Nostoc spp. The analysed samples were collected from 8 water bodies in Poland during summers of 1996–2001. Ten microcystins (MC), three major and seven minor variants, were detected in natural cyanobacterial samples by high-performance liquid chromatography (HPLC). The structures of the microcystins were assigned on the basis of liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS). The main microcystins detected in the blooms from Polish freshwaters were MC-LR, MC-RR and MC-YR. Microcystin content of the blooms varied from a few μg g^?1 to 1687 μg g^?1 freeze-dried material. Also some monodesmethyl (dm) and didesmethyl (dmdm) variants of MC-LR, MC-RR, MC-YR were found in a few analysed samples. Furthermore, two compounds were tentatively characterised as MC-(H₄)YR and dm-MC-FR.     

A novel approach for monitoring of cyanobacterial toxins: development and evaluation of the passive sampler for microcystins

We have investigated the ability of an integrative sampler for polar organic chemicals to sequestrate a group of common and highly hazardous cyanobacterial toxins—microcystins. In a pilot experiment, commercially available passive samplers were shown to effectively accumulate microcystins after 7 days’ exposure in the field. To find the most efficient configuration for sequestration of microcystins, four different porous membranes (polycarbonate, polyester, polyethersulfone and nylon) and two sorbents (Oasis HLB and Bondesil-LMS) were evaluated in the laboratory experiments, where samplers of different configuration were exposed to microcystins (microcystin-RR and microcystin-LR) for 14 days under steady conditions. We observed differences in sampling rates and amounts of accumulated microcystins depending on the sampler configurations. The samplers constructed with the polycarbonate membrane and Oasis HLB sorbent (2.75 mg/cm²) provided the highest sampling rates (0.022 L/day for microcystin-RR and 0.017 L/day for microcystin-LR). To the best of our knowledge, the present study is the first reporting application of passive samplers for microcystins, and our results demonstrate the suitability of this tool for monitoring cyanotoxins in water.     

Simultaneous determination of cyanobacterial hepato-and neurotoxins in water samples by ion-pair supported enrichment and HPLC-ESI-MS-MS

Summary Cyanobacterial neurotoxins, such as anatoxin-a and saxitoxin, as well as hepatotoxins including microcystins and nodularin were simultaneously determined in water samples by ion-pair supported, solid phase extraction (SPE) and reversed-phase liquid chromatography coupled to UV and tandem mass spectrometry (RP-LC-UV; MS-MS). With quantification limits in water samples of approximately 50 ng L⁻¹ for the microcystins (MC-LR,-YR,-RR,-LA), nodularin, and anatoxin-a and 630 ng L⁻¹ for saxitoxin the method is well suited for surveillance of the proposed WHO guidelines for cyanobacterial toxins. MS detection permits, unlike the commonly used UV detection, unambiguous identification and accurate quantification of cyanobacterial toxins even in highly matrix-polluted, water samples.     

LC-MS analyses of microcystins in fish tissues overestimate toxin levels—critical comparison with LC-MS/MS

Microcystins are cyclic peptide toxins with hepatotoxic and tumour-promoting properties which are produced in high quantities in freshwater cyanobacterial water blooms, and several studies have reported microcystin accumulation in fish with possible food transfer to humans. In this study, we provide the first comparison of liquid chromatography with single mass-spectrometric and with tandem mass-spectrometric detection for analyses of microcystins in complex fish tissue samples. Use of traditional single mass spectrometry (i.e. monitoring of ions with m/z 519.5 for microcystin-RR and m/z 995.5 for microcystin-LR) was found to provide false-positive responses, thus overestimating the concentrations of microcystins in the tissue samples. More selective tandem mass spectrometry seems to provide more reliable results. The concentrations of microcystins detected by tandem mass spectrometry in fish from controlled-exposure experiments were more than 50% lower in comparison with concentrations obtained by single mass spectrometry. Extensive analyses of edible fish parts—muscles (148 fish specimens from eight different species from five natural reservoirs with dense cyanobacterial water blooms)—showed negligible microcystin concentrations (all analyses below the limit of detection; limit of detection of 1.2–5.4 ng/g fresh weight for microcystin-RR, microcystin-YR and microcystin-LR in multiple reaction monitoring mode). Our findings have practical consequences for critical re-evaluation of the health risks of microcystins accumulated in fish.     

Screening for cyanobacterial hepatotoxins, microcystins and nodularin in environmental water samples by reversed-phase liquid chromatography-electrospray ionisation mass spectrometry

Water samples taken from 93 freshwater and brackish water locations in Aland (SW Finland) in 2001 were analysed for biomass-bound microcystins and nodularin, cyanobacterial peptide hepatotoxins, by liquid chromatography-mass spectrometry (LC-MS) in selected ion recording (SIR) and multiple reaction monitoring modes, HPLC-UV, and enzyme-linked immunosorbent assay (ELISA). The extracted toxins were separated on a short C18 column with a gradient of acetonitrile and 0.5% formic acid, and quantified on a Micromass Quattro Micro triple-quadrupole mass spectrometer with an electrospray ion source operated in the positive SIR or scan mode. An injection of 50 pg of microcystin-LR, m/z 995.5, on column gave a signal-to-noise ratio of 17 (peak-to-peak) at the chosen SIR conditions. In-source or MS-MS fragmentation to m/z 135.1, a fragment common to most microcystins and nodularin, was used for confirmatory purposes. Microcystins with a total toxin concentration equal to or higher than 0.2 microg l(-1) were confirmed by all three methods in water samples from 14 locations. The highest toxin concentration in a water sample was 42 microg l(-1). The most common toxins found were microcystins RR, LR and YR with different degrees of demethylation (non-, mono- or didemethylated). Parallel results achieved with ELISA and HPLC-UV were generally in good agreement with the LC-MS SIR results.     

Dissociation of deprotonated microcystin variants by collision-induced dissociation following electrospray ionization

Microcystins (MC) are a family of hepatotoxic cyclic heptapeptides produced by a number of different cyanobacterial species. Considering the recent advances in the characterization of deprotonated peptides by mass spectrometry, the fragmentation behavior of four structurally related microcystin compounds was investigated using collision-induced dissociation (CID) experiments on an orbitrap mass spectrometer. It is demonstrated in this study that significant structural information can be obtained from the CID spectra of deprotonated microcystins. A predominant ring-opening reaction at the isoMeAsp residue, as well as two major complementary fragmentation pathways, was observed, reducing the complexity of the product ion spectra in comparison with spectra observed from protonated species. This proposed fragmentation behavior was applied to characterize [Leu(1)]MC-LR from a cyanobacterial cell extract. In conclusion, CID spectra of microcystins in the negative ion mode provide rich structurally informative mass spectra which greatly enhance confidence in structural assignments, in particular when combined with complementary positive ion CID spectra.     

液相色谱-离子阱质谱对水华蓝藻提取物中微囊藻毒素的定性研究

本文研究了野外蓝藻水华样品甲醇提取物的液相色谱和电喷雾质谱特征图谱,利用电喷雾离子化-串联质谱(ESI-MSn)离子阱技术对特征图谱中主要微囊藻毒素的分子离子峰进行了二级质谱分析,获得相应的子离子质谱图,并对其进行了结构解析,确定了野外蓝藻水华样品中微囊藻毒素种类,为准确鉴定和分析水华蓝藻中微囊藻毒素提供了基础.     

Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins

A liquid chromatography (LC)-based method with mass spectrometric (MS) detection was developed for simultaneous determination of various algal and cyanobacterial toxins extracted from phytoplankton occurring world-wide in marine waters and lakes. The method enables quantification of saxitoxin, anatoxin-A, domoic acid, nodularin, microcystins, okadaic acid and dinophysistoxin-1 with a single chromatographic run. In addition, the applied chromatographic conditions allow isolation and identification of substances suspected to be "new" microcystins (cyclic peptides) by fraction collection, hydrolysis, derivatisation of resulting free amino acids with the modified chiral Marfey's reagent N-alpha-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) and enantioselective analysis of the amino acid derivatives by LC-ESI-MS.     

Low-energy collisionally activated decomposition and structural characterization of cyclic heptapeptide microcystins by electrospray ionization mass spectrometry

Characteristics of electrospray ionization mass spectrometry/collision-induced dissociation (ESIMS/CID) mass spectra of microcystins, cyanobacterial cyclic heptapeptide hepatoxins, were examined. The collision conditions showed remarkable effects on the quality of the CID mass spectra, which were divided into three patterns according to the number of Arg residues. A characteristic cleavage reaction and neutral losses of MeOH, NH3 and guanidine group(s) from the (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4 E,6E-dienoic acid (Adda) and Arg residues were observed in the ESI and ESIMS/CID mass spectra, suggesting the most probable protonation sites in [M + H]+ and [M + 2H]2+ ions of microcystins. Microcystins with no Arg residue showed only [M + H]+ ions with a proton reacting at the methoxyl group in the Adda residue, and the ESIMS/CID/MS data revealed their structures unambiguously. The protonation site in [M + H]+ ions of microcystins with Arg residue(s) was the guanidine group. The [M + 2H]2+ ions of microcystins possessing one Arg residue had one proton on the Arg residue and probably another proton on the Adda residue, while the [M + 2H]2+ ions of microcystins having two Arg residues showed protonation at both Arg residues and the ESIMS/CID/MS data assigned their sequences. Structures of microcystins possessing one Arg residue can be assigned by ESIMS/CID/MS of [M + H]+ ions combined with those of [M + 2H]2+ ions.     

Electrospray ionization mass spectrometric analysis of microcystins, cyclic heptapeptide hepatotoxins: modulation of charge states and [M+H]+ to [M+Na]+ ratio

Electrospray ionization mass spectrometry was used to develop a rapid, sensitive, and accurate method for determination and identification of hepatotoxic microcystins, cyanobacterial cyclic heptapeptides. To optimize the electrospray ionization conditions, factors affecting charge state distribution, such as amino acid components of sample, proton affinity of the additives, and additive concentration, were investigated in detail and a method for controlling charge states was developed to provide molecular-related ions for assignment of molecular weight and reasonably abundant precursor ions for MS/MS analysis. A procedure for identification of microcystins consisting of known amino acids was proposed: for microcystins giving abundant [M + 2H]2+ ions, the addition of nitrogen-containing bases to the aqueous sample solution is effective to obtain an increased intensity of [M + H]+ ions, whereas the addition of Lewis acids containing nitrogen can produce increased abundances of [M + 2H]2+ ions for microcystins giving weak [M + 2H]2+ ions. Microcystins possessing no arginine residue always give sodium adduct ions [M + Na]+ as the base peak, and these are difficult to fragment via low energy collision-induced dissociation to yield structurally informative products; the addition of oxalic acid increases [M + H]+ ion abundances, and these fragment readily.     

水环境中微囊藻毒素分析技术新进展

微囊藻毒素是常见的蓝藻毒素,具有很强的肝脏神经系统和肾脏毒性. 由于水的富营养化,蓝藻会爆发产生大量的微囊藻毒素,进而对水生生物和食用它们的人类构成巨大威胁. 随着浓缩、富集、分离方法和仪器技术的进步,定量分析微囊藻毒素的方法也在不断进步,且应用越来越广泛. 综述了水、沉积物和生物中微囊藻毒素的富集和检测方法,结果显示:目前常见的采样方法是主动采样法,开发简便可靠和实用的被动采样方法是急需的研究方向之一. 衍生化方法可以降低基质效应,有利于使用不同的检测方法和试验观察,因此开发一种高效、灵敏的衍生化方法检测微囊藻毒素将是重要的研究方向之一.     

Comparison of Fluorescence Immunochromatographic Assay Strip and Gold Colloidal Immunochromatographic Assay Strip for Detection of Microcystin

Abstract

Microcystins are a family of cyclic polypeptides produced by different species of cyanobacteria (blue green algae), which can form blooms in lakes and water reservoirs. However, it is difficult to detect microcystins directly in the water since the concentration of the toxins in water is usually too low. It is necessary to develop a simple and quick method to detect microcystins. In this paper, different detection characteristics of fluorescence immunochromatography and gold colloidal immunochromatography for analysis of cyanobacterial toxins were studied. These two immunochromatography assays are easy to perform, rapid, sensitive, and their quantitative range is within detectable microcystin concentrations in water samples. The fluorescence immunochromatographic system has the unique advantages of low detection limit, and satisfactory accurate results are obtained. The gold colloidal immunochromatographic system has the strong advantage of direct detection of microcystins at the test site without having to bring the samples back to the laboratory. Therefore, these two techniques supplement each other.     

蓝藻水华衍生的微囊藻毒素污染及其对水生生物的生态毒理学研究

富营养化导致的蓝藻水华频发,引发各种衍生物污染,严重时造成重大生态灾害事件,甚至危及人类健康。其中微囊藻毒素以其毒性大、分布广和结构稳定的特点,成为水环境中常见的潜在危害物质,它主要由微囊藻产生,是一类具有多种异构体的环状七肽物质。本文根据微囊藻毒素污染现状及其水生生态毒理学研究的最新研究进展,介绍了微囊藻毒素的理化性质及其产生、迁移和转化,在我国天然水体、水库源水和饮用水中的污染现状以及部分水产品中的微囊藻毒素累积情况,较全面地评述了微囊藻毒素的分子致毒机理以及对水生生态系统的重要组成成分--常见水生植物和鱼类的生态毒理学效应,并提出了该领域未来研究的主要方向。     

Accurate LC-MS analyses for microcystins using per-15N-labeled microcystins

Per-¹⁵N-labeled microcystins were prepared for use as surrogates for accurate liquid chromatography–mass spectrometry analysis. Two strains of Microcystis aeruginosa were cultured in ¹⁵NO₃-containing TS-15 medium. To change from the incorporation of ¹⁴N to ¹⁵N into all cell components, cells of Microcystis aeruginosa were precultured in Na¹⁵NO₃-containing medium for more than 6 months. After mass cultivation of the strains, cells of each strain were harvested and lyophilized. Microcystin variants were extracted from the lyophilized cells and per-¹⁵N-labeled microcystin variants were purified using high-performance liquid chromatography and high-performance thin-layer chromatography. The structures of per-¹⁵N-labeled microcystin variants were confirmed by their mass spectrometry spectra and NMR spectra. When per-¹⁵N-labeled microcystins were used as surrogates for quantitative analysis of these toxins in cyanobacterial cells, excellent accuracy (98–106%) was obtained, with the m/z of M⁺, [M+1]⁺, and [M+2]⁺ of both microcystins and the per-¹⁵N-labeled microcystins as surrogates being completely separated. In conclusion, per-¹⁵N-labeled microcystins are excellent surrogates for microcystin analysis using liquid chromatography–mass spectrometry.     

Detection of microcystins with protein phosphatase inhibition assay, high-performance liquid chromatography-UV detection and enzyme-linked immunosorbent assay: Comparison of methods

A colorimetric protein phosphatase inhibition assay (PPI assay), a commercial enzyme-linked immunosorbent assay (ELISA) test and different HPLC methods using UV detection were compared for the detection of cyanobacterial hepatotoxins, microcystins (MCYST) and nodularin. The suitability of the methods to detect different toxin variants was evaluated by using pure toxins and laboratory cultures as well as water and bloom samples of toxic cyanobacteria. The emphasis of the study was on the analysis of polar demethyl microcystin variants that are common in nature but for which there exist no commercial standards. The IC₅₀ values of MCYST-LR for the PPI assay and the ELISA test were 2.2-2.5 and 0.26-0.38 μg l⁻¹, respectively. The most important factors that decreased toxin recovery in sample treatment were the use of C₁₈ cartridges and polypropylene containers. Good recoveries of toxins were obtained by using hydrophilic-lipophilic balanced (Oasis HLB, Waters) cartridges for concentrating the samples. The results obtained with the PPI assay, the ELISA test and HPLC correlated quantitatively well with the exception of [d-Asp³] microcystins. Concentrations of [d-Asp³]MCYST-RR measured with the PPI assay were only 5% of those obtained by the ELISA test and HPLC. Concentrations of hydrophobic microcystin variants were lower when analysed with ELISA than with the other methods. The World Health Organisation (WHO) has set a guideline value of 1 μg l⁻¹ for the world-wide most common microcystin variant, MCYST-LR in drinking water. Since the quantitative ranges of the PPI assay and the ELISA test are within microcystin concentrations in natural waters, and both tests are easy to perform, they show potential for routine use in the screening and monitoring of microcystins from drinking water supplies and from recreational waters.     

Evaluation of the novel passive sampler for cyanobacterial toxins microcystins under various conditions including field sampling

In the present study, we have evaluated the effectiveness of a passive sampler for polar organic chemicals to accumulate a group of widespread and hazardous tumor-promoting toxins produced in cyanobacterial water blooms—microcystins (MC). The previously optimized configuration of the sampler based on polycarbonate membrane and Oasis HLB sorbent (2.75 mg/cm²) was validated under various exposure scenarios in laboratory and field. Calibration of the passive sampler conducted under variable conditions and concentrations of MC revealed linearity of the sampling up to 4 weeks. The sampling rates of microcystins for two different exposure scenarios were derived (e.g., MC-LR: R _s = 0.017 L/day under static and 0.087 L/d under turbulent conditions). R _s values were further used for calculations of time-weighted average concentrations in natural water. Improved sensitivity and selectivity of the in-house-made sampler was observed in comparison with the commercially available Polar Organic Compound Integrative Sampler (POCIS). Comparisons of grab and passive sampling methods were performed during cyanobacterial water bloom season in the Brno reservoir, Czech Republic in 2008. Data obtained by passive sampling provided a more relevant picture of the situation and enabled better assessment of potential risks. The present study demonstrated that the modification of POCIS is suitable for monitoring of occurrence and retrospective estimations of microcystin water concentrations, especially with respect to the control of drinking water quality.     

Regioselective Cleavage of Thioether Linkages in Microcystin Conjugates

Microcystins are cyanobacterial toxins that can be found in fresh and coastal waters during algal blooms. Microcystin contamination of water can cause severe poisoning of animals and humans. Quantification of these toxins in biological samples is complicated because a major proportion of microcystins is covalently linked to proteins through thioether bonds formed through a Michael‐type addition of cysteine residues of proteins to an N‐methyldehydroalanine residue in the microcystins. We investigated chemical methods that can be used to cleave such thioether bonds by means of an elimination reaction that leaves the microcystin backbone intact for subsequent analysis. The known reagent O‐mesitylenesulfonylhydroxylamine (MSH) led to regioselective thioether cleavage, but a large excess of reagent was needed, thus making purification challenging. An unexpected side reaction observed during the investigation of the base‐induced elimination inspired us to develop a new thioether‐cleavage methodology based on the addition of propargylamine as a nucleophile that can trap the elimination product. This methodology could be successfully applied to the quantitative cleavage of a microcystin‐LF–glutathione conjugate. The alkyne moiety introduced by this procedure offers the possibility for further reactions with azides by using click chemistry, which might be useful for the derivatization or isolation of microcystins.     

Selective determination of total normal microcystin by colorimetry, LC/UV detection and/or LC/MS

Microcystins, hepatotoxic cyclic heptapeptides, are produced by freshwater cyanobacteria, and are classified four groups according to the amino acid structure at unit 7. Normal microcystins contain N-methyldehydroalanine (Mdha) or dehydroalanine (Dha) at unit 7, and command the great part of all microcystins. As unusual microcystin classes, [Dhb⁷]microcystins, [ - and -Ala⁷, or N-MeAla⁷]microcystins and [ -Ser⁷]microcystins have been found.

On tumor initiation and/or promotion activities of microcystins, the tumor promotion activity of normal microcystins has been found, but cancer-related activities of microcystins belonging in the other classes have not been clear.

To determine normal microcystins as hepatotoxic tumor promoters, a selective determination method was developed. Only Mdha or Dha in normal microcystins was reacted with glutathione (GSH). The GSH-normal microcystins conjugates were reacted with trinitrobenzene sulfonate (TNBS). The TNB–GSH-normal microcystin conjugate can be determined as the total normal microcystin by colorimetry. After methanolysis of the conjugate, dimethyl TNB–glutamate from the conjugate was determined by liquid chromatography/ultraviolet detection (LC/UV) and/or liquid chromatography/mass spectrometry (LC/MS). The detection limits of the total normal microcystin by colorimetry, LC/UV and/or LC/MS were 1 μg, 10 and 0.1 ng, respectively.