Detecting the Cylindrospermopsin using HPLC-PDA and NMR - Assignment Example

?Introduction Cylindrospermopsin is a liver toxic alkaloid produced mainly by the cyanobacteria Cylindrospermopsis raciborskii albeit the toxin is also produced by Umezakia natans and other bacterial species (Harada et al., 1994). This toxic alkaloid is of great concern to the public health sector since it is highly soluble in water because it has both positive and negative charges. The toxin has a variant, a deoxy-cylindrospermopsin which has been verified in some strains (Li et al., 2001). This toxin is mainly toxic to the liver at low concentrations and is present on the surface waters used by human and animals for drinking. Due to its presence in surface waters chronic exposure of the water with very low concentrations of cylindrospermopsin will ultimately lead to toxicity. Standard methods used in the detection and quantification of cylindrospermopsin are based on LC-MS (Eaglesham et al., 1999). However as noted by Welker et al. (2002), LC-MS may be expensive and out of reach for countries encountering toxic blooms and High Performance Liquid Chromatography (HPLC) and characterization with nuclear magnetic resonance (NMR) may offer a reliable alternative. High-performance liquid chromatography separates compounds from a mixture and also helps in identifying, quantifying and purifying an individual compound from a mixture of compounds. The basis of separation of compounds in HPLC is pressure which is used to load the compounds into the separation chamber where individual compounds are separated under the influence of the liquid solvent conditions such as temperature and pressure and the chemical attraction of the sample mixture and stationary phase in the separation column. Nuclear magnetic resonance (NMR) on the other hand is an analytical technique that uses magnetic nuclei which absorb and re-emit electromagnetic radiations at a specific resonance frequency. This frequency is however dependent on the strength of the magnetic field. The resonance obtained in a magnetic field for any particular compound analysed is always directly proportional to the strength of the magnetic field. Detection and analysis of cylindrospermopsin using HPLC Cylindrospermopsins have few methods of detection compared to the well known microcystins and saxitoxins. High-performance liquid chromatography - photodiode array (HPLC-PDA) has been shown to be a good method for the detection of cylindrospermopsins and its analogues because of their characteristic UV spectra (?max at 262 nm). The only limitation to this method is that sample purification is necessary because it is normally co-eluted with other contaminants (Welker et al. 2002). Purification of cylindrospermopsin is normally performed using HP-20 resin, which removes most of the ionic components from the fraction. Before the detection of cylindrospermopsins by HPLC they have to be extracted. Water samples containing the cyanobacterial cells are filtered on glass fibre filters and extracted by ultrasonication in 75% methanol. Extraction procedure The air dried frozen filter samples should be placed on the borosilicate glass tubes and freeze-thawed twice to obtain maximum recover after which 1.2ml of methanol is added and mixed in the bath ultrasonicator for 15 minutes. The samples should further be ultrasonicated individually for 1 minute and the aliquots of the extracts centrifuged at 10,000 ? g for 10 min after which 500 µl of the supernatants are transferred to borosilicate vials and evaporated to dryness at 40°C under argon. The dried extracts can then be reconstituted in 100 µl of 75% methanol and centrifuged in vials at 10,000 ? g for 10 min or filtered through the HPLC grade filter. Before running the HPLC, the HPLC system should be set up as described in the manufacturer’s instructions including degassing, priming and changing columns. The column oven should be set at 40?C and the HPLC changed gradually to starting conditions. The chromatogram samples and standards should be set as per the recommended HPLC gradients using 10 µl injections. The eluents to be used include water, methanol, acetonitrile and their mixtures. The chromatogram should then be analysed and the retention times and spectra compared to the standards. Collection of large quantities of cylindrospermopsins can also be obtained through preparative chromatography so as to increase the quantity of the toxin for analysis. Therefore chromatography can be used both for preparation and also purification of the extract. Reversed phase HPLC is normally used. In this method the silica is modified to make it non polar by joining long hydrocarbon chains to its surface to make a strong attraction between the polar solvent and the polar molecules passing through the column. This leads to increased amount of time for the elution of the polar molecules while the less polar compounds will be eluted fast. In conclusion, HPLC-PDA analysis of cylindrospermopsins is a reliable technique for the detection of these toxins due to the fact that the retention time from the HPLC spectrum is reproducible with reasonable range of linearity. However, the detection limit is low especially for environmental samples which have very low cylindrospermopsins. In addition this toxin has to be separated from its analogues which, if not cleaned up, can affect the chromatograms. Nuclear magnetic resonance (NMR) The Nuclear magnetic resonance is a method mainly used for purification of compounds. This method mainly deals with the magnetic properties of certain nuclei such as hydrogen hence the term proton NMR. This method has been used to successfully check the purity of cylindrospermopsins. The principle behind the working of NMR is that there is polarization of the magnetic nuclear spins in an applied constant magnetic field. This is followed by the agitation of this alignment of the nuclear spins by an electromagnetic pulse usually radio frequency. The perturbation frequency required is dependent on the static magnetic field and nuclei of observation. During NMR analysis two fields which are perpendicular to each other are normally chosen so as to maximize the NMR signal strength. It has been noted that the external magnetic field required in bringing hydrogen into resonance decreases if it is attached to a more electronegative element since the hydrogen nucleus feels more of the field. This means that even small changes in the electronegativity of the atoms attached to it will make a difference in the field in order to achieve resonance. Therefore a hydrogen atom will require application of a slightly different magnetic field to achieve resonance depending on the type of atom or atoms which are attached to it. Since the resonance obtained in a magnetic field for any particular compound analysed is always directly proportional to the strength of the magnetic field, then it is easier to determine the purity of a particular compound such as using 500 MHz proton NMR spectroscopy. In a NMR spectrum there will be a number of peaks depending on the environments of hydrogens attached to different compounds. The sizes of the peaks in the spectrum are important because it gives the number of hydrogen atoms in each environment. The height of the peak does not matter much but the important thing is the ratio of the areas under the peaks. In conclusion, it is possible to detect and analyse cylindrospermopsins using HPLC-PDA and NMR techniques. HPLC is mainly used for the separation of the compounds and also for the clean up from its analogues after which its purity can be tested based on the spectrum obtained which is normally reproducible. Furthermore the purity of the extract can be determined using NMR spectrum and is obtained by calculating the ratios of the areas under the spectrum. References Eaglesham, G.K., Norris, R.L., Shaw, G.R., Smith, M.J., Chiswell, R.K., Davis, B.C., Neville, G.R., Seawright, A.A. and Moore M.R. Use of HPLC-MS/MS to monitor cylindrospermopsin, a blue-green algal toxin, for public health purposes. Environ Toxicol 1999; 14:151-154 Harada, K.I., Ohtani, I., Iwamoto, K., Suzuki, M., Watanabe, M.F., Watanabe, M. and Terao, K.(2002). Isolation of cylindrosper-mopsin from a cyanobacteriumUmezakia natans and its screening method. Toxicon, 32:73-84 Li, R., Carmichael, W.W., Brittain, S., Eaglesham, G.K., Shaw, G.R., Li, Y. and Watanabe, M.M. (2001). First report of the cyanotoxins cylindrospermopsin and desoxycylindrospermopsin from Raphidiopsis curvata (cyanobacteria). J Phycol 37:1121-1126 N.A. (2000). Cylindrospermopsin; Review of Toxicological Literature. Toxicological summary for Cylindrospermopsin. Integrated Laboratory systems (ILS) Welker, M., Bickel, H. and Fastner, J. (2002) HPLC-PDA detection of cylindrospermopsin - opportunities and limits. Water Research, 36: 4659-4663. Read More