Structural investigation of not-derivatized palytoxins in crude extracts by a new HR LC-MS/MS-based approach

Palytoxin (C129H223N3O54) and its analogues (ovatoxins and ostreocins) fall into the same group of marine biotoxins considered highly dangerous for human health through ingestion, skin contact and/or inhalation [1]. They were found in several marine organisms: zoanthids belonging to the genus Palythoa [2] as well as in the sub-tropical microalga Ostreopsis spp, mainly O. cf. ovata [3] which has recently spread across the Mediterranean and Southern-Atlantic coasts of Europe, and in marine cyanobacterium Trichodesmium spp. [4]. Several cases of inhalatory poisonings and/or skin injuries have been reported in beachgoers concomitantly with massive blooms of O. cf. ovata [5] as well as in aquarium hobbyists from incidental contact with palytoxin-producing Palythoa spp. [1]. Symptom similarities between Ostreopsis- and Palythoa-related poisonings suggest that the etiological agent is the same. From a chemical standpoint, all palytoxin congeners share the same long polyhydroxylated aliphatic chain with the parent compound and differ little in structural details – generally number, position and/or stereochemistry of a few methyl, methylene and/or hydroxyl groups. Interestingly, for this group of toxins a characteristic LC-HRMS and MS/MS behavior has been identified: all of them form a wide variety of multiply charged ions, including protonated ions, adducts with cations, and ions due to multiple subsequent water losses. Furthermore, these molecules share also the same fragmentation behavior: they undergo fragmentation at several sites of their backbone, generating diagnostic multiply charged ions each one accompanied by several water losses. HR LC-MS and MS/MS spectra of palytoxins can be used as fingerprints for their confident identification directly in the crude extracts even at ng/mL levels. Since natural extracts are usually contaminated by a complex mixture of congeners, this approach revealed effective for preliminary structural characterization of unknown minor congeners present at levels too low for NMR studies.