Sample preparation is minimal for all samples. Algal samples are lysed by freezing and thawing and then taken up in water and filtered. Water samples are concentrated by rotary evaporation at 45°C, lysed by freezing and thawing and filtered. These extracts are then ready for analysis.
An Altima C18 column (150 x 2 mm, 5µ) is used with an Omniguard guard column. Both are contained in a column oven maintained at 40°C . For algal samples the isocratic solvent used is methanol / water (7.5 / 92.5) containing ammonium acetate (5mM). As concentrated water samples contain more early eluting interfering compounds a gradient starting at 2.5% methanol in 5mM ammonium acetate is preferred to give better resolution of the cylindrospermopsin.
The Finnigan electrospray interface used can deal with high solvent flow rates and a flow rate of 0.3 ml/min enables rapid analysis with good sensitivity. The mass spectrometer is normally operated in the positive ion scanning mode over a narrow range encompassing the molecular weight of cylindrospermopsin and related compounds present in the algae. At normal voltages molecular ion adducts are the predominant ions in the mass spectra of these compounds. By increasing the extracting voltages collision induced dissociation can be promoted in the source and characteristic fragments of the compounds in the extracts are formed.
The mass spectrometer used in this analysis is a single stage quadrupole instrument. The detection limit for cylindrospermopsin using this instrument is 0.2 mg/Litre injected. Preliminary work using multiple reaction monitoring on a triple stage mass spectrometer has indicated a possible 30 fold increase in sensitivity.
Other compounds are present in the algae which from mass spectral evidence appear to be related to cylindrospermopsin. Further work is being carried out to purify and fully characterise these compounds.