Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii

Abstract

The hepatotoxin cylindrospermopsin (CYN) has been isolated from the cyanobacterium Cylindrospermopsis raciborskii (C. raci.). Efforts to study this toxin have been hampered by the time-consuming requirement to extract it from cultures of the organism. It is usually extracted from lyophilized cells collected from a laboratory culture. Our preliminary work suggested far more of the toxin is available in solution in the culture media than in the cells collected. We have therefore investigated the use of commercially available solid phase extraction sorbents to extract CYN from culture media in which C. raci. has been grown. A range of reverse phase and ion-exchange sorbents were tested across a range of pHs for their ability to retain CYN without success. Subsequently, graphitized carbon cartridges were found to retain CYN strongly. Elution with 5% formic acid in methanol allowed the CYN to be regained for final purification by HPLC. Deoxy-CYN, an analog of CYN can also be extracted using this procedure.

Advances in solid phase extraction of the cyanobacterial toxin cylindrospermopsin

“…At this stage, attention should be paid to the differences in the elution volume for methanol obtained in our work when compared with those from Norris et al (2001) and Metcalf et al (2002), where total recovery was obtained in about 8 mL, half the volume observed in the present study. An explanation to this different behavior may be found in the bed weight of the cartridges.…”

“…While quantification methods are well developed Törökné et al 2004), concentration and extraction of dissolved CYN from natural samples has not been widely developed or published. Norris et al (2001) completed a first approach toward an effective SPE method by selecting graphitized carbon as the most suitable compound when compared with a wide range of the most usual SPE sorbents. Also, in their work, the need for acidification of the solvents was established.…”

“…An explanation to this different behavior may be found in the bed weight of the cartridges. We used cartridges of 500 mg graphitized carbon, while Norris et al (2001) used 300 mg. In Metcalf et al (2002), no indication about the quantity used could be found.…”

“…Development of the SPE method: selection of solvents and sample preparation- Norris et al (2001) suggested methanol as the solvent selected for elution of CYN and emphasized the importance of acidification of the solvent. In the present study, we also tested the suitability of acetone and combinations of dichloromethane:methanol (4:1, 1:1, 1:4) as solvents.…”

“…Norris et al (2001) performed a complete study with a range of SPE cartridges and observed best results when using Advances in solid phase extraction of the cyanobacterial toxin cylindrospermopsin graphitized carbon. Metcalf et al (2002) combined graphitized carbon with C18 and obtained good recoveries when working with high concentrations of CYN.…”