Microcystine, determination

Kaya, K. and Sano, T. 1999. Total microcystin determination using e yf/ ro-2-methyl-3- (methoxy- d(3))-4-phenylhutyric acid (MMPB-d(3)) as the internal standard. Anal ChimActa 386 107—112. 

At the molecular level, microcystins are potent inhibitors of protein phosphatases 1 and 2A. The activity of protein phosphatases can be determined by measuring... 

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.

H. S. Eee, C. K. Jeong, H. M. Eee, S. J. Choi, K. S. Do, K. Kim and Y. H. Kim, On-line trace emicliment foi the simultaneous determination of microcystins in aqueous samples using high peifoi mance liquid cliromatography with diode-aixay detection , /. Chromatogr. 848 179-184 (1999). 

BHA, BHT, PG, TBHQ and tocopherols) a variety of stationary phases, mobile phases and detectors can be used [711]. Common antibacterials such as carba-dox, thiamphenicol, furazolidone, oxolinic acid, sul-fadimethoxine, sulfaquinoxaline, nalidixic and piromidic acid can be analysed by GE-RPLC-UV (at 254 nm). Collaborative studies have been reported for the HPLC determination of the antimicrobial sodium benzoate in aqueous solutions [712], Plastics devices used for field collection of water samples may contain polymer additives (such as resorcinol monobenzoate, 2,4-dihydroxybenzophenone or bisphenol A) or cyanobac-terial microcystins [713],... 

Significant concentrations of cyanotoxins have been found to accumulate in the tissues of macroinvertebrates such as mollusks and crustaceans, presenting an indirect route of exposure for invertebrates, fish, and aquatic mammals at higher trophic levels (Negri and Jones 1995). In natural systems, mortality among benthic invertebrate herbivores is probably low because most bloom-forming bacteria are planktonic and only periodically come into contact with the benthos. Nevertheless, Kotak et al. (1996) determined that enhanced mortality of snails at the end of a bloom cycle in Canadian lakes was due to consumption of Microcystis cells that had formed a scum on the surface of macrophytes. Oberemm et al. (1999) found that aqueous microcystins, saxitoxins, and anatoxin-a all resulted in developmental delays in fish and salamander embryos. Interestingly, more severe malformations and enhanced mortality were observed when larvae were exposed to crude cyanobacterial extracts than to pure toxins applied at natural concentrations (Oberemm et al. 1999). 

Dahlmann, J., Budakowski, W.R. and Luckas, B., 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, /. Chromatogr., 994, 1-2, 45, 2003. 

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],... 

L.A. Lawton, C. Edwards and G.A. Codd, Extraction and high-performance liquid chromatography method for the determination of microcystins in raw and treated waters, Analyst, 119 (1994) 1525-1530. 

G. Vasas, D. Szydlowska, A. Gaspar, M. Welker, M. Trojanowicz and G. Borbely, Determination of microcystins in environmental samples using capillary electrophoresis, J. Biochem. Biophys. Methods, 66 (2006) 87-97. 

R. W. Moollan, B. Rae and A. Verbeek, Some comments on the determination of microcystin toxins in water by high performance liquid chromatography. Analyst 121 233 - 238(1996). 

Zhao, Y.-Y., S. Hrudey, and X.-F. Li. 2006. Determination of microcystins in water using integrated sobd-phase microextraction with microbore high-performance liquid chromatography-electrospray quadruple time-of-flight mass spectrometry../. Chromatogr. Sci. 44 359-365. 

Fig. 7.2 Tlie crystal structure of mammalian Ser/Thr protein phosphatase-1, complexed with the toxin mycrocystin was determined at 2.1 A resolution. PPl has a single domain with a fold, distinct from that of the protein tyrosine phosphatases. The Ser/Thr protein phosphatase-1, is a metalloenzyme with two metal ions positioned at the active site with the help of a p-a-p-o-p scaffold. A dinuclear ion centre consisting of Mn2+ And Fe2+ g situated at the catalytic site that binds the phosphate moiety of the substrate. Ser/Thr phosphatases, PPl and PP2A, are inhibited by the membrane-permeable ocadaic acid and by cyclic hexapeptides, known as microcystins. The toxin molecule is depicted as a ball-and-stick structure. On the left and on the ri t, two different views of the same molecule are shown. Microcystin binds to three distinct regions of the phosphatase to the metaLbinding site, to a hydrophobic groove, and to the edge of a C-terminal groove in the vicinity of the active site. At the surface are binding sites for substrates and inhibitors. These ribbon models are reproduced vnth permission of the authors and Nature from ref. 9.

A gas chromatographic (GC) method has been described in the literature. GC is based on the oxidation of microcystins which splits the Adda side chain to produce 3-methoxy-2-metlyl-4-pheitylbu-tyric acid (MMPB), which is then determined, either by GC or GC/MS (as its methyl ester) (Sano 1992 Kaya and Sano 1999) or by HPLC/fluoiescence detection (after conversion to a fluorescent derivative) (Sano 1992). GC/MS has been used to monitor microcystins in Japanese lakes (Tanaka 1993) and in sediments (Tsuji 2001). A similar method was developed by Harada (1996), but in this case the MMPB was determined directly without derivatization using GC/MS or LC/MS. The results of this approach ate given in terms of total toxin concentration, which then can be expressed in terms of microcystin-LR. However, individual toxins ate not determined and consequently it is not possible to produce a result in terms of microcystin-LR toxicity equivalents. This procedure cannot therefore be used to monitor water samples in relation to the proposed guideline. 

Fluorescence has been also selected as a detection mode for an improved sensitivity and selectivity in the HPLC determination of microcystins and nodnlarin (James and James 1991). This method uses a post-colunm system, whereby the arginine residne of microcystin-LR was derivatized with a fluorescent reagent. This approach obviously hmits the application to toxins containing atgitune, e g., nticrocystin-LR, and toxins snch as microcystin-LA. 

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). 

A microcystin can be identified according to its mass spectram, as long as a standard is available. In MS/MS, the fragmentation pattern can be used to greatly assist in determining the identities of unknown microcystins (Bateman 1995 Lawton 1995 Yuan 1998, 1999). However, the problem of the lack of standards is still present as with any other analytical procedure since standards are necessary for accurate quantification. In addition toxicity data are required in order that toxicity equivalents can be calculated. 

Kondo, E, Ikai, Y, Oka, H., Matsumoto, H., Yamada, S., Ishikawa, N., Tsuji, K., Harada, K.-l.,Shimada, T, Oshikata, M., and Suzuki, M. 1995. Reliable and sensitive method for determination of microcystins in complicated matrices by frit-fast atom bombardment liquid chromatography/mass spectrometry. Natural Toxins 3 41 9. 

Lawrence, IF, and Menard. C. 2001. Determination of microcystins in blue-green algae, fish and water using liquid chromatography with ultraviolet detection after sample clean-up employing immunoafiBnity chromatography. Journal of... 

Sano, T, Nohara, K., Shiraishi, E, and Kaya, K. 1992. A method for micro-determination of total microcystin content in waterblooms of cyanobacteria (blue-green algae). International Journal of Environmental Analytical Chemistry 49 163-170. 

Dongjin Pyo also reported some other procedures The extraction by SFE was performed with a Beckman 116 pump (System Gold programmable solvent module 126), a 15 cm X 10 mm I.D. ODS column and Agilent HPLC 1100 series diode-array detector. Methanol/0.05 M phosphate buffer (52 48), pH 3, was used as a mobile phase at a flow rate of 2 mL/min. The analytical results obtained from real lake samples indicate the viability of the method for the determination of microcystins in real samples. 

In order to achieve the rapid and precise determination of microcystins in complicated matrices, a systematic procedure is seriously required. This may include screening, sample purification, identification, and quantification... 

Tsutsumi, T. Nagata, S. Hasegawa, A. Ueno, Y. Im-munoaffinity column as clean-up tool for determination of trace amounts of microcystins in tap water. Food Chem. Toxicol. 2000, 38, 593-597. 

The first example in which MS was utilized to determine the specificity of an A domain from an NRPS gene cluster was published in a joint effort by the Moore and Kelleher laboratories. Hicks et al. 97 investigated the substrate specificity of the loading module McyG of microcystin synthetase. Microcystin is a cyclic NRPS-PKS hybrid toxin derived from various cyanobacteria genera. The initiation module McyG comprises an A-T didomain (Figure 14(b)), which was predicted based on the 10 letter code to activate and load... 

As well portrayed in Fig. 9, in our distance geometry-derived stmctures of microcystine-LR the Adda side-chain retains sufficient conformational space for relatively large movements despite the restricted rotational freedom imparted by the double bonds. In family A the Adda side-chain is bent over the ring structure and in family B it is directed away from the ring, an effect even more pronounced in family C. The latter position as well as the relative flexibility of the terminal portion of the side-chain compares well with the microcystin-LR structure determined by Trogen et al (110) in DMSO and in DMSO/water. Moreover, this hydrophobic side-chain is at some distance from the sequentially adjacent Arg side-chain. 

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