Extraction Methods for Cyanotoxins
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
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
References: Lawton, L.A., Edwards, C., Codd, G.A.: Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analyst (London) 119, 1525-1530 (1994). Fastner, J., Flieger, I., Neumann, U.: Optimized extraction of microcystins from field samples - a comparison of different solvents and procedures. Water Res. 32, 3177-3181 (1998). Spoof, L., Vesterkvist, P., Lindholm, T., Meriluoto, J. Screening for cyanobacterial hepatotoxins, microcystins and nodularin, in environmental water samples by reversed-phase liquid chromatography-electrospray ionisation mass spectrometry. J. Chromatogr. A 1020, 105-119 (2003). N. B. The reader is also advised to follow up the development of the ISO standard for microcystin analysis. ISO/FDIS 20179: Water quality - Determination of microcystins - Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultr