Production of a saxitoxin standard from cyanobacteria.

Abstract

A methodology for the production of a saxitoxin (STX) standard from cyanobacteria (Raphydiopsis brookii strain SPC 338) was developed. The cyanobacterium was cultured in 5-litre culture bottles in ASM-1 medium, pH 7.4 at 22±1°C, under continuous light at an irradiance of 45-50 mmol m-2 s-1 and moderate aeration rate. The cells were grown until the late-exponential growth phase (approximately 3 weeks), at which point the culture was harvested. The cultured material was lyophilized, extracted with 0.1 M acetic acid and ultrasonication, and centrifuged. The supernatant was lyophilized and subjected to purification by chromatographic methods. STX purity was determined by HPLC-FLD analysis. Male Swiss-Webster mice were injected intraperitoneally with crude extract and with fractions obtained from all purification steps, all diluted in sterile 0.9% NaCl solution (the bio-guided assay). Time to death, signs of poisoning, and other symptoms were observed up to 72 h after injection. STX extracted from R. brookii showed 95% purity, which is similar to STX standards obtained from mussels. The cyanobacterium standard contains only STX, whereas the standard obtained from mollusks contains both STX and an analog. By selecting another cyanobacteria species, it may be possible to obtain different analogues at a lower price. The biomass is easily obtained in culture, its production is not seasonal, and no special storage equipment is required. Thus, this method represents an excellent option for obtaining STX standards.