Extraction Methods for Cyanotoxins

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  • Topic: High performance liquid chromatography, Chromatography, Borosilicate glass
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  • Published : February 4, 2013
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SOP: Solid phase extraction of cylindrospermopsin in filtered water samples

Document identifier: SOP_TOXIC_UDU_05F

Prepared by: James S. Metcalf and Geoffrey A. Codd, UDU Date: 7 July 2005 1 Introduction

In order to determine the concentration of cylindrospermopsin in the extracellular fraction of filtered environmental waters, solid phase extraction (SPE) is necessary to concentrate the toxin to concentrations capable of being detected by HPLC.

2 Experimental

2.1 Materials

Use analytical reagent grade reagents.

(a) Polygraphite carbon solid phase extraction cartridges (PGC), e.g. Thermo Hypersil keystone Hypersep Hypercarb 200 mg, 3 ml cartridges, from Thermo Hypersil Keystone, UK

(b)C18 solid phase extraction cartridges, e.g. Isolute C18(EC) solid phase extraction columns, size 1 g sorbent in 6 ml reservoir from Argonaut Technologies, Mid Glamorgan, UK

(c) Glass-fibre filters (e.g. Whatman GF/C), diameter 25-70mm

(d) Argon or nitrogen, >99.99%

(e) Borosilicate test tubes or vials, >3 ml capacity

(f) Water purified to 18.2 M cm (e.g. Millipore Milli-Q water)

(g) Methanol HPLC grade, e.g. HPLC grade methanol from Rathburn (Walkerburn, Scotland, UK)

(h) Trifluoroacetic acid (TFA), protein sequence analysis grade. TFA should be stored under argon in a desiccator.

2.2 Special equipment

(a) -20 °C freezer

(b)Vacuum manifold, preferably transparent, equipped with stopcocks, vacuum source and vacuum control

(c)LVE (large volume extraction) kit for unattended loading of large sample volumes, made of PTFE tubing and adapters for column connection.

(d) Filtration unit (minimum 500 ml)

(e) Rotary evaporator or hot block and gas apparatus

(f) Microcentrifuge

2.3 Solutions

(a) TFA, 0.1% (v/v) solution in methanol

2.4 Procedure

(a)Filter a minimum of 1 litre of water through a GF/C filter (if not already performed as in SOP_TOXIC_UDU_02F). Volumes may have to be reduced in the case of dense bloom/scum samples.

(b)Prepare a combined “in series” SPE system with a C18 cartridge connected to a PGC cartridge (C18 before PGC).

(c)Condition the combined SPE system with 10 ml methanol containing 0.1% (v/v) TFA followed by 10 ml of water. Apply the sample to the cartridges. Do not let the cartridges dry during condition and sample application.

(d) Dry the PGC cartridge by passing air through it.

(e)Elute cylindrospermopsin with 3 ml 0.1% (v/v) TFA in methanol from the PGC cartridge and collect in a suitable borosilicate container.

(f)Evaporate the methanolic solution at 50 °C under argon or nitrogen.

(g) Resuspend the residue in 500 µl of Milli-Q water.

(h)Centrifuge at full speed (e.g. 10,000 g, 10 mins). Analyse the supernatant for cylindrospermopsin according to SOP_TOXIC_UDU_09F.

3 References

Metcalf, J. S., Beattie, K. A., Saker, M. L., Codd, G. A.: Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction. FEMS Microbiol. Lett. 216, 159-164 (2002).

Norris, R. L. G., Eaglesham, G. K., Shaw, G. R., Senogles, P., Chiswell, R. K., Smith, M. J., Davis, B. C., Seawright, A. A., Moore, M. R.: Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii. Environ. Toxicol. 16, 391-396 (2001).

SOP: Solid phase extraction of anatoxin-a in filtered water samples

Document identifier: SOP_TOXIC_UDU_04F

Prepared by: James S. Metcalf and Geoffrey A. Codd, UDU Date: 7 July 2005 1 Introduction

In order to determine the concentration of anatoxin-a in the extracellular fraction of filtered environmental waters, solid phase extraction (SPE) is necessary to concentrate...
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