Isolation of ovatoxin-a from ostreopsis cf. ovata cultures. A key step for hazard characterization and risk management of ovatoxins

Ostreopsis cf. ovata, a benthic/epiphytic marine dinoflagellate, is currently spreading in tropical, sub-tropical and temperate areas, causing periodic Harmful Algal Blooms (HABs). It produces a wide array of palytoxin-like compounds named ovatoxins (OVTXs), with OVTX-a generally the most abundant congener. Despite numerous cases of human poisonings and environmental damage associated with the presence of OVTXs and O. cf. ovata proliferations, a complete characterization of the toxicity of this class of molecules cannot be performed until Reference Material (RM) for individual congeners is available. This, in turn, requires the availability of sufficient amounts of toxin at a high purity grade. To achieve this goal, herein an analytical re-evaluation of critical-steps of OVTX-a isolation from O. cf. ovata cell pellets is reported. The overall procedure consists of four steps, namely an extraction, a Medium Pressure Liquid Chromatography (MPLC) separation, and two preparative High Performance Liquid Chromatography (HPLC) steps. Particular attention was paid to the extraction step to evaluate the repeatability in OVTX-a yields. For subsequent steps, loading sample preparation and chromatographic conditions were refined. As a result, a significant increase in recovery yields (from 12.5 to 20 +/- 3%) and in purity grade (from 51% to 94%) of the isolated OVTX-a was achieved in comparison to previous studies. The improved procedure can easily be applied to isolate sufficient quantities of a good candidate RM for OVTX-a.