TOWARD QUANTITATIVE ISOLATION OF PALYTOXINS. PRELIMINARY STABILITY AND PURIFICATION STUDIES

Palytoxin is one of the most potent marine toxins and may be produced by several different species of the zoanthid Palythoa. Ovatoxins are palytoxin analogues produced by the sub-tropical microalga Ostreopsis cf. ovata, which has recently spread across the Mediterranean and Southern-Atlantic coasts of Europe. Several cases of inhalatory poisonings and/or skin injuries have been reported in beachgoers concomitantly with massive blooms of O. cf. ovata as well as in aquarium hobbyists from incidental contact with palytoxin-producing Palythoa spp. Symptom similarities between Ostreopsis- and Palythoa-related poisonings suggest that the etiological agent is the same. As a matter of fact, palytoxins and ovatoxins differ little in structural details- a few methyl, methylene and/or hydroxyl groups over a long polyhydroxylated aliphatic chain. So, they are likely to cause the same overall symptomatology although their relative potencies might be different. The increasing spread of the Ostreopsis phenomenon and the ever-growing number of palytoxin congeners being discovered makes the need of evaluating their toxicity urgent. The availability of sufficient amounts of well characterized reference material is the cornerstone for the achievement of toxicity data. To achieve this goal, we are developing an isolation procedure for quantitative recovery of individual palytoxin congeners from Palythoa spp. grown in marine aquarium and we are evaluating the stability of palytoxins under different conditions. In this work, different types of stationary phases (HLB, STRATA-X, SP-850, SP-207, HP-20, RP-18, Carbograph) and elution conditions were evaluated in terms of recovery yield and reproducibility for isolation of palytoxins from both soft-coral and aquarium water. Best recoveries were obtained by using HP-20 and RP-18. Furthermore, it emerged that Carbograph is a stationary phase able to completely retain palytoxin. For this reason it could be used in the detoxification procedures of the home aquaria. Several HPLC analytical columns (Poroshell 120 EC C18 and C8, TSK gel ODS 120A, Jupiter C18, ODS-3 prodigy, among others) are being tested for base-line chromatographic separation of individual congeners. The stability and recovery of palytoxin during different evaporation procedures (nitrogen stream, freeze drying, vacuum concentration, and speed-vac) has been also investigated. Strong acid negatively affected recovery of palytoxin during evaporation procedures, while acetic acid seemed to slightly facilitate recovery. A strong influence was also observed for the materials of the containers used in evaporation. Silanised glass and Teflon surfaces yielded significantly higher recoveries than unsilanised glass or polypropylene tubes. Optimised evaporation appears to be a critical step to minimise losses of palytoxin and analogues in preparative isolation procedures.