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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Since 2005, the benthic dinoflagellate Ostreopsis cf. ovata has bloomed across the Mediterranean basin, provoking serious toxic outbreaks. LC/MS studies have identified a number of palytoxin-like compounds, termed ovatoxins, along with trace amounts of putative palytoxin as the causative agents of the O. cf. ovata -related human sufferings. So far, any risk assessment for ovatoxins as well as establishment of their allowance levels in seafood has been prevented by the lack of pure toxins. The present paper reports on the isolation, NMR-based structural determination, and preliminary mouse lethality evaluation of ovatoxin-a, the major toxic compound contained in O. cf. ovata extracts. Availability of pure ovatoxin-a will open the double prospect of fully evaluating its toxicity and preparing reference standards to be employed in LC/MS quantitative analyses. Elucidation of ovatoxin-a's complex structure will ultimately herald the understanding of the molecular bases of ovatoxins bioactivity.

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Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.

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The marine diatom Pseudo-nitzschia galaxiae Lundholm et Moestrup has been recently described from Mexican and Australian plankton. In this paper, we illustrate the considerable morphological variability of the species in the Mediterranean Sea and present first evidence of its toxicity. In addition to lanceolate cells 25-41 m long, which fit the original description of the species, markedly larger (<82 m) and smaller (>10 m) specimens are commonly recorded. Cells of the largest size have almost parallel valve margins, while smaller specimens have extremely short rostrate ends and do not form colonies. Despite remarkable differences in shape and size, the typical ultrastructure of the species was observed for the different size classes in culture and in natural samples. In culture, cell length decreased at a rate of 1.1-2.1 m per month. Liquid chromatography-mass spectrometry (LC-MS) analyses revealed the presence of domoic acid (DA) at very low levels in two of seven strains analyzed. LSU rDNA analysis confirmed the identity of the species and showed a very low genetic variability for the strains from the Gulf of Naples, with no relationships with size and overall shape of the cells. A relatively high number (53) of Pseudo-nitzschia sequences were considered in the phylogenetic analysis, yet the relationships among species remain unclear, probably in relation with a recent speciation process in the genus. In natural samples, P. galaxiae populations of different cell sizes occurred at different times over the year, with smaller cells found in winter and early spring, and medium and larger cells peaking in late spring-summer. The maximum concentration value in the Gulf of Naples was recorded in May 1985 (9.4 × 10 6 cells l −1 ). From the analysis of a high number of both natural and culture samples, it is concluded that size and shape variations are indicative of different stages of the life cycle of P. galaxiae, which exhibit a synchronized and seasonal occurrence at the interannual scale.