Determination of microcystins in water using integrated solid-phase microextraction with microbore high-performance liquid chromatography-electrospray quadruple time-of-flight mass spectrometry

The development of a technique combining solid-phase microextraction (SPME) with microbore high-performance liquid chromatography (micro-HPLC)-tandem quadrupole time-of-flight (QTOF) mass spectrometry (MS) for determination of dissolved microcystins in water is reported. Several important parameters affecting the efficiency of SPME extraction of microcystins are investigated. A microbore C18 column HPLC coupled with tandem QTOF-MS with information-dependent acquisition (IDA) is developed to effectively analyze microcystins in microliter volumes of SPME extracts. The micro-HPLC-QTOF-MS with IDA technique provides comprehensive information, including a survey chromatogram (total ion chromatogram), full scan mass spectrum, and product ion scan mass spectra at different collision energies for individual analytes, which allows for both identification and quantitation in the same run. Linear calibration curves of microcystin standard [microcystin (MC)-arginine (R)R] 1-100 microg/L and of microcystin standard [MC-leucine (L)R] 1-250 microg/L are obtained with a correlation coefficient of 0.996. The combination of SPME with HPLC-QTOF-MS and IDA offers limits of detection of 0.6 pg for MC-RR and 1.6 pg for MC-LR. Analysis of spiked lake-water samples shows a recovery of > 86% for MC-RR and > 70% for MC-LR. This technique requires small sample volumes, minimizes the use of organic solvents, and provides sensitive and information-rich analysis of unknown samples.     

ELISA and LC-MS/MS methods for determining cyanobacterial toxins in blue-green algae food supplements

The use of natural products as a diet supplement is increasing worldwide but sometimes is not followed by adequate sanitary controls and analyses. Twenty samples of pills and capsules of lyophilised cyanobacteria (blue-green algae), commercialised in Italy as dietary supplements, were found positive at the Vibrio fischeri bioassay. Further analyses with ELISA and LC-MS/MS methods revealed the presence of four microcystin (MC) analogues, MC-LR, -YR, -LA, -RR and two demethylated forms of MC-RR. The highest total microcystin content was 4.5 and 1.4 microg g-1 in pills and capsules, respectively. The ELISA measurements, compared to the LC-MS/MS analyses, showed significantly lower concentrations of microcystins in pills, this confirming a possible ELISA underestimate of mixed microcystins, due to different sensitivities for some toxic analogues.     

甲基丙烯酸酯毛细管整体柱分离微囊藻毒素的研究

以甲基丙烯酸丁酯为单体,乙二醇二甲基丙烯酸酯为交联剂,在致孔剂存在的条件下原位聚合制备了甲基丙烯酸丁酯毛细管整体柱(150 μm i.d.)。实验中优化了用此整体柱分离3种微囊藻毒素(MC-LR,-YR和-RR)的色谱条件(流动相种类、缓冲溶液浓度、pH、流动相流速),建立了微囊藻毒素的整体柱毛细管液相色谱分离方法,该法可以在9 min之内实现3种微囊藻毒素的基线分离。将该方法应用于实际水样中微囊藻毒素的分析,成功实现了培养水样和巢湖水样中微囊藻毒素的快速分离,两种样品中均检测到MC-LR。结果表明,所制备的甲基丙烯酸酯毛细管整体柱具有良好的重现性、渗透性,在微囊藻毒素的常规检测中具有很好的应用前景。     

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.     

Comparison of liquid chromatography/mass spectrometry, ELISA, and phosphatase assay for the determination of microcystins in blue-green algae products

More than 100 samples of blue-green algae products (consisting of Aphanizomenon, Spirulina, and unidentified blue-green algae) in the form of pills, capsules, and powders were collected from retail outlets from across Canada. The samples were extracted with 75% methanol in water and centrifuged to remove solids. Aliquots of the extracts along with spiked blank sample extracts were sent to each participating laboratory and independently analyzed for microcystins by enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay, and by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after sample cleanup using C18 solid-phase extraction. The results obtained by ELISA and LC-MS/MS agreed very well over a concentration range of about 0.5-35 microg/g. The colorimetric phosphatase results generally agreed with the other 2 methods. While the 2 biochemical assays measured total microcystin content compared with a standard of microcystin LR, the LC-MS/MS method measured specific microcystins (LA, LR, RR, YR) using external standards of these for identification and quantitation. Microcystin LR was found in all positive samples by LC-MS/MS. Microcystin LA was the only other microcystin found in the samples analyzed. These 2 microcystins represent essentially all the microcystins that were present in the extracts. Otherwise, the LC-MS/MS results would have been significantly lower than the results of the biochemical assays had other unknown microcystins been present.     

An enhanced LC-MS/MS method for microcystin-LR in lake water

A LC-MS/MS method with enhanced sensitivity and specificity was established for monitoring microcystin-LR (MC-LR) in drinking water supplies in southern Taiwan. The enhanced sensitivity was achieved by the selection of a doubly charged MC-LR as the precursor ion to result in an multiple reaction monitoring (MRM) pair ions of m/z 498.6 --> 135.0. Using this ion pair, a record low detection limit of 2 pg was achieved on column, found in the available literature. A sample preparation method involving C8 solid-phase extraction gave satisfactory recoveries of the analyte. Nodularin, with structural similarity to MC-LR, was used as an internal standard to minimize matrix effects of water samples collected from six different water reservoirs in southern Taiwan, where MC-LR was detected at sub-ppb levels in all the reservoirs. The best precision and accuracy of this method were found with samples prepared to contain MC-LR at 0.1 and 1 microg l(-1). This new method requires considerably smaller water sample volumes because of enhanced quantification sensitivity and hence reduces the time needed for analysis. It should serve as a useful example for method development for monitoring other members of the microcystin family in drinking water supplies.     

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.     

微囊藻毒素在单波长紫外光照射下的光降解动态研究

研究了两种微囊藻毒素在两种紫外光照射下的光降解行为,研究结果表明:UV-C是降解微囊藻毒素较好的光源;光照强度是影响毒素降解重要的因素,其次是温度和酸度;在UV-C的照射下,水体腐殖质对光降解具有抑制作用;微囊藻毒素的光解反应符合准一级动力学模型.同时本工作还按实际环境水体中毒素的含量水平进行了模拟研究,发现紫外光UV-C对环境水体中低含量的微囊藻毒素具有很强的去除能力.为今后发展无毒、高效、经济实用的饮用水处理技术做了有益的探索.     

Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.     

Issues arising when interpreting the results of the protein phosphatase 2A inhibition assay for the monitoring of microcystins

Microcystins, cyclic heptapeptidic hepatotoxins produced by a number of bloom forming freshwater cyanobacteria, are considered to represent a serious risk to public health through drinking and recreational water. A highly sensitive bioassay relying on the specific inhibition of the human protein phosphatase 2A was applied to the quantification of microcystins. A systematic approach based on the rational testing of seven purified mcyst variants as well as characterized environmental samples allowed to point out the limits and experimental bias associated with this assay. All the seven microcystin variants known as microcystins RR, YR, LR, LY, LA, LW and LF strongly inhibited the enzyme with IC₅₀ ranging between 0.29±0.02 and 0.84±0.07 nM for microcystins LW and YR, respectively. Using the model system of Microcystis aeruginosa PCC7820 axenic cultures and within the 1-year study of a Planktothrix agardhii bloom, the PP2A assay was shown to be strongly correlated to high-performance liquid chromatography (HPLC) coupled to ultra violet diode array detection. However the slope of the linear regression was significantly influenced by the sample composition, as confirmed by HPLC coupled to electrospray ionization mass spectrometry. A model based on pure additivity of mcyst effects was established to describe PP2A inhibition by standard mcyst mixtures, and fully agreed with experimental observations.     

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.     

Generic microcystin immunoassay based on monoclonal antibodies against Adda.

A monoclonal antibody (clone AD4G2) was generated against a common part of microcystins and nodularins, the unusual amino acid Adda [(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid]. A direct competitive ELISA based on this antibody was developed and the cross-reactivity pattern was measured. Different toxins showed a very similar response. The assay provides therefore a sum parameter of microcystins, nodularins and peptide fragments containing Adda. The IC50 for microcystin-LR was 0.33 microg L(-1) which leads to a detection limit of 0.07 microg L(-1). This is well below the concentration of 1 microg L(-1) proposed by the World Health Organisation (WHO) as the limit for drinking water. Microcystin-LR spiked water samples in the concentration range between 0.1 and 1 microg L(-1) were measured and a mean recovery of 113+/-23% was found. The antibody is well suited for the determination of microcystins in drinking as well as surface water.     

Determination of cyanobacterial cyclic peptide hepatotoxins in drinking water using CE

Four cyanobacteria hepatotoxins microcystin LR, microcystin RR, microcystin YR, and nodularin were simultaneously determined in drinking water using CZE and MEKC coupled with UV detection. The toxins were satisfactorily separated in both CZE and MEKC modes. Detection limits were in the range of 0.82–4.81 μg/mL, with R² values of 0.994–0.999. The linearity range tested for the standards was 5–100 μg/mL and RSD percentages were in the range of 1.0–2.5% for retention time and 3.0–10.2% for peak area. When a known amount of standard was spiked into a known volume of water and extracted, recoveries were 90.3% (RR), 101.5% (nodularin), 90.6% (YR), and 88.2% (LR). The use of SPE enabled cleanup and pre‐concentration of a real sample to achieve a 100‐fold concentration factor. Detection limits after SPE of the real sample spiked with microcystins were 0.090 μg/L (RR), 0.076 μg/L (YR), and 0.110 μg/L (LR), with RSD percentage values of 9.9–11.7% for peak area and 2.2–3.3% for retention time. The technique developed provides an alternative method for determining microcystins at levels of concentration that will be able to meet WHO drinking water guidelines for microcystins.     

Development and application of ultra performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry for determination of seven microcystins in water samples

In the present work, an ultra performance liquid chromatography (UPLC)-electrospray ionization tandem triple quadrupole/mass spectrometry (ESI-MS/MS) method was developed in selected reaction monitoring (SRM) mode to determine the amounts of seven/microcystin variants (MCYST-RR, -YR, -LR, -LA, -LY, -LW, -LF) in water samples. Solid phase extraction (SPE) or porous filter was used for sample cleaning-up and analyte enrichment. A C18 column enabled separation of 7 microcystins within 10 min. Translation reaction ions including 995.6 > 134.9 (RR), 1045.8 > 135.1 (YR), 910.9 > 375.7 (LA), 1002.3 > 375.6 (LY), 520.1 > 135.0 (LR), 1025.8 > 891.7 (LW), 986.8 > 852.6 (LF) were set as quantitative ions and the enkephalin was the internal standard. Upon method validation, the limits of detection (LODs) and limits of quantification (LOQs) were found to be 0.06 μg L⁻¹ and 0.2 μg L⁻¹ (injection volume of 10 μL), respectively, for all seven microcystin variants. The correlation coefficients were larger than 0.994 in all linear ranges and recoveries were in the range of 66.24-99.22%. The developed approach was highly sensitive and easy to perform. It generated accurate quantitative results for the analysis of microcystins in environmental water samples with a wide concentration range. The results showed that MC-RR and MC-LR widely existed in polluted water, while MC-LA and MC-LY were detected sporadically.     

Identification of several Tonalide® transformation products in the environment

This work reports on the survey carried out to determine the presence of microcystins (MCs) by using ELISA tests and HPLC-UV for such determination in different sample sites along the Spanish course of the Guadiana River. The most important cyanobacteria species identified were, Microcystis aeruginosa and Oscillatoria spp. The highest total microcystin content recorded was 6.40?µg?L^?1 in 2002. The main toxins found were microcystins RR and LR, with microcystin YR present at trace levels. Improvements in sample clean up were carried out by using Immunoaffinity solid phase extraction (SPE) and its advantages regarding to conventional SPE were clearly demonstrated. The confirmation of MCs presence in the evaluated water reservoirs, underlines the necessity of monitoring programs as well as the improvement of analytical methodologies to efficiently prevent the human health risks as a consequence of MCs contamination.     

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.     

Determination of microcystins in biological samples by matrix solid-phase dispersion and liquid chromatography-mass spectrometry

A method for the detection and quantification of the microcystins (MCs)-MC-LR, MC-RR and MC-YR-in biological samples by matrix solid-phase dispersion (MSPD) has been developed. The optimum extraction conditions were 500 mg of liver or kidney, C18 bonded silica as dispersant, and a mixture methanol-water (70:30) as eluent. The MCs were determined by liquid chromatography electrospray mass spectrometry (LC/ES/MS). Recoveries of biological extracts at three different spiked levels (1-10 mg kg(-1)) ranged from 40.5 to 87.0% in liver, and from 52.5 to 74.5 in kidney. R.S.D.s were < 15.6% and < 10.6%, respectively. The detection and quantification limits were 0.05 and 0.5 mg kg(-1), for all MCs. The method was applied to MCs detection in liver and kidney of rat previously injected i.p. with MC-LR. Results showed the presence of MC-LR in the liver of the animals injected with the highest dose.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.     

Determination of microcystins in biological samples from freshwater fish

A new HPLC–DAD method has been developed to identify and quantify free microcystins (MC) in biological samples from fish (intestine and liver). The toxins were extracted from 500 mg sample with a mixture of methanol–water (85 : 15, v/v) and the extracts obtained were purified employing immunoaffinity columns (IAC). The purification step was optimised by a full factorial 3² design. MC were separated using conventional C18 column and an acetonitrile-acidified water (pH 3) gradient. Detection and quantification limits resulted equal for the two toxins assayed (MC-RR and MC-LR) and were 0.15 and 0.5 µg g^?1, respectively. The accuracy for each MC in liver samples were 96% (range 80–113%) for MC-RR and 101% (range 93–118%) for MC-LR. The results were slightly lower for intestine samples, with recoveries ranging between 85% (75–93%) for MC-RR and 88% (80–97%) for MC-LR. The proposed method was applied for the determination of free MC in fish intoxicated with these toxins, in order to determine its utility to evaluate the potential risks for human health if MC-contaminated fish are consumed. The results showed the transference of MC-LR from cyanobacterial cells to fish tissues.     

Capacitive sensing of microcystin variants of Microcystis aeruginosa using a gold immunoelectrode modified with antibodies,gold nanoparticles and polytyramine

We report on the application of an automated and easy-to-use device to directly measure the immunoreactions between adda-specific monoclonal antibodies and microcystins. The antibodies were immobilized on a gold electrode whose surface was modified first with polytyramine and then with gold nanoparticles. The immunoreaction leads to a change in the capacitance of the system. Under optimum conditions, the sensor is capable of performing stable regeneration-assay cycles and has a low detection limit at a concentration of 0.01 pM level of microcystin-leucine-arginine (MC-LR). The surface of the biosensor can be regenerated with pH 2.5 glycine buffer which dissociates the antibody-antigen complex. The biosensor was used to monitor the production of microcystins during batch cultivation of Microcystis aeruginosa (isolated from ponds in Botswana). Liquid chromatography coupled to MS/MS detection was used to identify three variants, viz. MC-LR (995.6 Da), DmMC-LR (981.2 Da) and MC-LA (910.5 Da).