A new method for sensitive, specific, and direct determination of palytoxin is proposed herein. It is based on combination of reversed-phase liquid chromatography with mass spectrometry (LC-MS). The new method was set up on a turbo ion spray-triple quadrupole MS instrument operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes (positive ions). The minimum detection levels for matrix-free toxin on column were thus estimated from the data to be 200 and 125 pg in SIM and MRM modes, respectively. Spiking experiments before and after extraction allowed us to assess limits of detection and quantitation for palytoxin in matrix, accuracy, and intraday and interday reproducibility of the method. The developed method was decisive for the analysis of a plankton sample collected along Genoa coasts in July 2005 when respiratory illness in people exposed to marine aerosols occurred. It is suggested that putative palytoxin was the causative agent responsible for patients' symptoms and demonstrated for the first time the presence of such a toxin in Italian waters.

In this article we report on the liquid chromatography tandem mass spectrometry (LC-MS) investigation of plankton samples collected in the summer of 2006 along the Ligurian coasts, coinciding with a massive bloom of the tropical microalga Ostreopsis ovata. LC-MS analyses indicated the occurrence of putative palytoxin along with a much more abundant palytoxinlike compound never reported so far, which we named ovatoxin-a. On the basis of molecular formula, fragmentation pattern, and chromatographic behavior, the structure of ovatoxin-a appeared to be strictly related to that of palytoxin. We report also on the analysis of cultured O. ovata, which was necessary to unequivocally demonstrate that putative palytoxin and ovatoxin-a contained in field samples were actually produced by O. ovata itself. They have only occasionally caused cases of human sufferings, until, in summer 2005, a massive proliferation of O. ovata broke out with alarming proportion in Liguria, nearby Genoa, Italy. This phenomenon caught the attention of both national and international media, which delivered breaking news on the event, as hundreds of people required medical attention after exposure to marine aerosol. The symptoms shown by all the patients included fever associated with serious respiratory distress and, in some cases, conjunctivitis. The toxic outbreak reached its climax in coincidence with the highest concentration of O. ovata in seawater and disappeared as the population of this microalga started fading away.We collected a concentrated plankton sample off Genoa coasts during the toxic outbreak and analyzed it for the most common phycotoxins found in the Mediterranean sea, such as okadaic acid, spirolides, azaspiracids, yessotoxins, paralytic shellfish poisoning toxins, and domoic acid [3,4]. We also investigated the occurrence of brevetoxins which, although never reported in the Mediterranean sea, are usually associated with poisonings due to marine aerosols [5]. None of the above toxins was detected in the plankton sample [4].Some Ostreopsis species produce palytoxin (Figure 1) [6 -8], a complex polyhydroxylated compound and one of the most potent marine biotoxins, or palytoxin analogues such as ostreocin-D [9 -11] and mascarenotoxins [12]. Structure of ostreocin-D was assigned to based on NMR data [9,10], and verified by negative-ion fast-atom bombardment collision-induced dissociation tandem mass spectrometry [11]. The charge-remote fragmentations were facilitated by a negative charge introduced to the terminal amino group or to a hydroxyl group at the other terminus of the molecule by reaction of pure ostreocin-D with 2-sulfobenzoic acid cyclic anhydride. Mascarenotoxins [12] were purified and identified as palytoxin-like compounds based on comparison of their mass spectrum profiles and fragmentation patterns with those of a reference standard of palytoxin. Other analogues of palytoxin, namely homo-, bishomo-, neo-, and deoxy-palytoxin were isolated from the soft coral Palythoa tuberculosa [13] but theywere not yet proven to...

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