Analysis microcystin

In the Slimmer of 1989, Rutland Water, the largest man-made lake in Western Europe and which supplies potable water to approximately 500 000 people in the East of England, contained a heavy bloom of Microcystis aeruginosa. By the end of the summer, a number of sheep and dogs had died after drinking from the bloom and concentrated scum. Analysis revealed that the cyanobacterial bloom material was toxic to laboratory mice, and that rumen contents from a poisoned sheep contained fivemicrocystin variants.Microcystins were detected in waters used for recreation in Australia at concentrations greater than 1 mg per... 

A powerful tool now employed is that of diode array detection (DAD). This function allows peaks detected by UV to be scanned, and provides a spectral profile for each suspected microcystin. Microcystins have characteristic absorption profiles in the wavelength range 200-300 nm, and these can be used as an indication of identity without the concomitant use of purified microcystin standards for all variants. A HPLC-DAD analytical method has also been devised for measurement of intracellular and extracellular microcystins in water samples containing cyanobacteria. This method involves filtration of the cyanobacteria from the water sample. The cyanobacterial cells present on the filter are extracted with methanol and analysed by HPLC. The filtered water is subjected to solid-phase clean-up using C g cartridges, before elution with methanol and then HPLC analysis. 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

Figure 5.3 Analysis of 100 ml of (a) surface water and (b) drinking water sample spiked with 0.1 pig/ml of microcystins, using column-switching HPLC 1, microcystin-RR 2, microcystin-YR 3, microcystin-LR. Reprinted from Journal of Chromatography A, 848, H. S. Lee et al, On-line trace enrichment for the simultaneous determination of microcystins in aqueous samples using high performance liquid chromatography with diode-array detection , pp 179-184, copyright 1999, with permission from Elsevier Science.

An international intercomparison exercise in the determination of microcystin, carried out by using the most common methods (LC/DAD, ELISA and LC/MS) indicated that LC/DAD is affected by lower precision [234], while the coupling of the LC technique with ELISA permit the achievement of high sensitivity and specificity in the determination of microcystins and nodularin [235] without the need of pre-concentration the method meets the World Health Organization guidelines (1 pg L ). The combination of ELISA characterization and LC analysis with fluorescence, UV, and tandem MS detections, allowed the first identification of cylindrospermopsin, an algal toxin that caused the poisoning of up to 148 persons in Australia [236],... 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

However, problems associated with the reproducibility between electrodes derived from the screen-printing process and the partial electrode fouling have compromised the sensitivity of the biosensors. Work is in progress to improve both the reproducibility and the limits of detection by the use of new types of electrodes. The toxin overestimation observed with the amperometric biosensor, in the case of the microcystin analysis, suggests the use in parallel to other analytical techniques in order to minimise the risk of false-positive results. Nevertheless, the electrochemical strategy is appropriate to discriminate between toxic/non-toxic samples. 

C.J. Ward, K.A. Beattie, E.Y.C. Lee and G.A. Codd, Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria comparisons with high-performance liquid chromatography analysis for microcystins, FEMS Microbiol. Lett., 153 (1997) 465-473. 

K. Tsuji, S. Naito, F. Kondo, M.F. Watanabe, S. Suzuki, H. Nakazawa, M. Suzuki, T. Shimada and K.-I. Harada, A clean-up method for analysis of trace amounts of microcystins in lake waters, Toxicon, 32 (1994) 1251-1259. 

C. Edwards, L.A. Lawton, K.A. Beattie, G.A. Codd, S. Pleasance and G.J. Dear, Analysis of microcystins from cyanobacteria by liquid... 

K. Tsuji, H. Masui, H. Uemura, Y. Mori and K.-I. Harada, Analysis of microcystins in sediments using MMPB method, Toxicon, 39 (2001) 687-692. 

T Nishizawa, M Asayama, K Fujii, K Herada, M Shirai. Genetic analysis of the peptide synthetase genes for the cyclic heptapeptide microcystin in Microcystis spp. J Biochem (Tokyo) 126 520-529, 1999. 

Saxitoxin has been labeled with fluorescamine, o-phthaldialdehyde (OPA) and dansyl chloride and detection limits as low as 0.1 attomole were reported for the OPA derivative of saxitoxin (26). Labeling, separation, and analysis of saxitoxin was best accomplished using fluorescamine, which produces ionic derivatives that can be separated from other fluorescently labeled marine toxins, such as tetrodotoxin and microcystin. However, the precolumn labeling methods required xM concentrations of analyte, limiting the utility of the technique for trace analysis. 

Analytical standards are frequently not available for algal toxins, and analysis of the toxins is slow and difficult (although rapid methods are becoming available for a few of these toxins, such as the microcystins). Therefore, the preferred approach is to monitor source water for evidence of blooms, or bloom-forming potential, and to increase vigilance where such events occur, Chemical analysis of cyanotoxins is not the preferred focus of routine monitoring, and it is used primarily in response to bloom events. 

A combination of PPIA and microcystin immunoassay was proposed by Carmichael et al. (1999) to indicate the potential toxicity of a bloom sample and the concentration of the microcystins. A combined assay, consistent with this principle, was developed by Metcalf et al. (2001) this includes preexposure of the sample to microcystin antibodies, to make microcystins/nodularins that are present biounavailable to the subsequent addition of protein phosphatase enzyme, before assaying for protein phosphatase inhibitoiy activity. The resulting assay, termed the colorimetric immunoprotein phosphatase inhibition ass (CIPPIA), was found to be specific for microcystins and nodularins since the microcystin antibodies protect the protein phosphatase from inhibition by the toxins. Complete protection from inhibition of protein phospliatase by the antibodies indicates that the inhibition of the protein phosphatase in the sample was due to the cyanobacterial toxins. These colorimetric assays showed a good correlation with the HPLC analysis of extracts cyanobacteria. Immunoassays can also be combined with physicochemical methods such as HPLC (Zeck 2001b). In this case, the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. 

Immnnoassays can also be combined with ply sicochemical methods such as HPLC, whereby the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. Immunoaffinity columns containing microcystin antibodies (Rivasseau 1999a) can also be used to confer specificity to the PPIA for the analysis of the cyanobac-terial toxins. Lin and Chu (1994) reported that polyclonal antibodies raised against microcy stin-LR could successfully be used to protect PP2 A from the action of microcystin in vitro. 

Biochemicals, Nottingham, U.K.). All are advocated by the manufacturers for the analysis of micro-cystins in water, and several microcystin variants have been tested using these systems. 

An ELISA test using monoclonal antibodies against microcystin-LR has been used by Ueno (1996b) to analyze the microcy stin concentration in environmental samples from ponds, lakes, reservoirs, and rivers in Japan, Thailand, Germany, and Portugal. Although microcystins are mainly associated with freshwater cyanobacteria, these results are relevant to the analysis of microcystins and particularly nodularins in brackish water environments. 

Improvements in the selectivity of the separation of microcystins and nodnlarin have been achieved by selecting the most efficient stationary phase, with this aim (Spoof 2002) compared a monolithic C-bonded silica rod colnmn (Merck Chromolith) to particle-based C and antide C 18 18 16 sorbents in the HPLC separation of eight microcystins and nodularin-R. Two gradient mobile phases of aqneons trillnoroacetic acid modified with acetonitrile or methanol, different flow-rates, and different gradient lengths were tested. The performance of the Chromolith colunrn measured the resolution of some microcystin pairs. The selectivity, efficiency (peak width), and peak asymmetry equalled, or exceeded, the performance of traditional particle-based columns. The Chromolith 21 colnmn allowed a shorteiting of the total analysis time to 4.3 minutes with a flow rate of 4 ml/minute. 

LC/MS with various interfaces and different ioitization modes has been reported for the determination of microcystins (Poon 1993 Lawton 1995 Kondo 1992, 1995 Bateman 1995). Derivatiza-tion of microcystins prior to LC/MS analysis has also been reported as a technique to assist in identifying microcystins (Sherlock 1997). 

Pyo, D., Lee, I, and Choi, E. 2005. Trace analysis of microcystins in water using enzyme-linked immunosorbent assay. Microchemical Journal 80 165-169. 

Sherlock, I.R., James, K.J., Caudwell, EB., and MacKintosh, C. 1997. The first identification of microcystins in Irish lakes aided by a new derivatisation procedure for electrospray mass spectrometric analysis. Natural Toxins 5 247-254. 

Yuan, M., Namikoshi, M., Otsuki, A., Watanabe, M.E, and Rinehart, K.L. 1999. Electrospray ionization mass spectrometric analysis of microcystins, cyclic peptapeptide hepatotoxins Modulation of charge states and [M+H] to [M+Na] ratio. Journal of American Society of Mass Spectrometry 10 1138-1151. 

Microcystins were first purified by Botes et al in 1982, and, since then, many different approaches " have been adopted for the isolation of microcystins from cyanobacterial cells. The most widely used proce-dures are as follows The lyophilized cyanobacterial cells which contain microcystins are extracted with organic solvents several times, and then the extracts are applied to multistep column chromatography and thin-layer chromatography.For example, Harada et al. established an effective analysis method for microcystins RR and LR. They used 5% aqueous acetic acid solution as an extracting solvent and isolated microcystins by using preparative or semipreparative liquid chromatography with ODS, silica gel, or gel permeation columns. 

Recently, an immunoaffinity purification method using antimicrocystin-LR monoclonal antibodies (named M8H5) has been developed.This purification method was found to be remarkably effective in the removal of coexisting substances and in the enrichment of microcystins in samples.This work will focus on the immunoaffinity purification methods for microcystins in lake ° and tap water samples, and the analysis methods for microcystins and their metabolites in mouse and rat livers.It will also cover the reuse of the immunoaffinity column. 

Fig. 2 HPLC analysis of lake water extracts. Microcystin-/ree lake water spiked with microcystins-LR, -YR, and -RR (100 ng each) was analyzed (a) before and (b) after purification with immunoaffinity column, (c) A water sample taken from Lake Suwa, Japan, in 1998 was analyzed after purification with the immunoaffinity column.

Fig. 4 HPLC analysis of a c3ftosolic extract from mouse liver spiked with 5 pg each of microcystins-RR and -LR. (a) Before and (h) after purification with immunoaffinity column.

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