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Anthocyanins are natural pigments responsible for the typical color of red wines and are critically associated with the wine commercial value and longevity. Thus, understanding the molecular bases of the anthocyanin chemical behavior in the complex wine environment is a central issue to the enological sector. By setting up a nuclear-magnetic-resonance-based experimental protocol, new important insights into the chemistry of malvidin-3- O-glucoside, a major wine anthocyanin, in model solution have been provided. Two new equilibrating isomers of the anthocyanin have been identified and quantified at typical wine pH values. More importantly, the key role of pH in determining the solubility and co-pigmentation process of malvidin-3- O-glucoside has been clarified. Consequently, a value of pH of about 3.3 or lower favors co-pigmentation while inhibiting the precipitation of significant quantities of anthocyanins during the maceration-extraction phase of the winemaking.

Background: Numerous oenological practices can cause an excess of dissolved oxygen in wine, thus determining sensory and chromatic defects in the short‐ to long‐term. Hence, it is necessary to manage the excess of oxygen before bottling. Methods: In this study, the management of the dissolved oxygen content by a polypropylene hollow fiber membrane contactor apparatus was performed in two wines from different grape varieties (Aglianico and Falanghina). The wines were analyzed after an 11‐month aging. Anthocyanins and acetaldehyde content were evaluated by HPLC. In addition, other phenolic compounds and chromatic characteristics were analyzed by spectrophotometric methods. NMR and HR ESIMS analyses were conducted to evaluate the amount of pyranoanthocyanins and polymeric pigments. Results: After 11 months of aging, in both wines a decrease of free and total SO2 with respect to initial values was detected. In the wines with the highest dissolved oxygen levels, a more remarkable loss was observed. No significant differences in terms of color parameters were detected. In red wine with the highest oxygen content, a massive formation of polymeric pigments and BSA reactive tannins was observed, as opposed to wines with lower oxygen levels. Conclusion: The study demonstrated that the membrane contactor can prove a successful tool to manage dissolved oxygen in wines as to prevent their oxidative spoilage. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Acetaldehyde is a key compound in determining wine color evolution and sensory properties. Major wine metabolites reactive to acetaldehyde are phenolic compounds, mainly flavan-3-ols and anthocyanins. Many studies have been conducted with the purpose of investigating acetaldehyde reactivity in model solutions, but very poor are the reports of its fate in real wines. By means of LC-HRESIMS and UV/Vis HPLC, red and white wines exposed to an excess of acetaldehyde were analyzed with a specific focus on low molecular weight phenolics. The chemical behavior of acetaldehyde turned out to be different in white and red wines. In white wines, it mainly mediated the formation of vinyl-flavan-3-ol derivatives, while in red wines it led to the formation of ethylidene-bridged red pigments. These latter positively enhanced the color properties of red wines. Conversely, in white wines, the formation of compounds, such as xanthylium ions, causing the undesired browning effects were not detected.

Wines produced from Sangiovese (sg) grapes, the most cultivated red grape variety in Italy and widely grown across the world, is often subjected to loss of clarity due to the formation of a deposit constituted by fine needle-shaped crystals. In this work, a qualitative study by (1)H-NMR and (13)C-NMR analysis of the deposit obtained by filtering cloudy sg wines showed that it was constituted by crystals of quercetin (Q). The analysis of hydro-alcoholic solutions (12% ethanol and pH 3.2.) and red wines added with increasing amounts of Q showed that, above 3 mgL(−1) of Q, a deposit can be detected and, the time necessary for its formation depends on the medium. The comparison among sg and other 11 monovarietal wines showed that sg was the richest in Q and quercetin glycosides (GQ). Both Q and GQ decreased in the analyzed solutions over time and the decrease was faster for Q than for GQ. The controlled exposure to oxygen determined a decrease of Q higher than the 50% of the initial values. Data obtained in this study suggested that practices as micro-oxygenation and wood aging could help to decrease the amount of Q in sg wines. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-020-04296-7) contains supplementary material, which is available to authorized users.

The reactivity of malvidin-3-O-glucoside with acetaldehyde, pyruvic and p-coumaric acids, was separately studied in wine model solution in the presence and absence of catechin, by UV–Visible spectrophotometry, NMR and Mass spectrometry. These naturally occurring metabolites were selected on account of their role in determining the red wine color. The scientific bases underlying the wine color are far from being fully clarified. Nonetheless, understanding the factors that govern the wine color is a crucial prerequisite for winemakers to implement appropriate technological practices to produce naturally stable high-quality wines. Among the investigated wine metabolites, acetaldehyde turned out to be the most reactive towards malvidin-3-O-glucoside by forming, as major products, ethylene-linked polymeric pigments that affected chromatic properties of the wine-like model solutions. Pyruvic acid and p-coumaric acid did not react with malvidin-3-O-glucoside, but in combination with catechin, they both determined a significant hypsochromic effect. Unexpectedly, in our model solutions, the formation of pyranoanthocyanins was not observed.

Background: In the wine industry, in addition to condensed tannins of grape origin, other commercial tannins are commonly used. However, the influence of oxygen uptake related to different tannin additions during the post fermentative phase in wine has not been completely investigated. In this study, we evaluated the influence of four different commercial tannins (namely, condensed tannins, gallotannins, ellagitannins and tea tannins) during four saturation cycles. Method: Wine samples were added with four different tannin classes (30 g/hL) as to have 5 different experimental samples: control, gallotannins (GT), condensed tannins (CT), ellagitannins (ET), and tea tannins (TT). The chemical composition of the four commercially available tannin mixtures was defined by means of NMR and high-resolution mass spectrometry. After the addition of tannins, each wine sample was oxidized by air over four cycles of saturation. During the experiment oxygen consumption rate (OCR), sulfur dioxide consumption, acetaldehyde production, phenolic compounds, chromatic characteristics, astringency measured by the reactivity towards saliva proteins and astringency subqualities were evaluated. Results: The experiment lasted 52 days. The addition of tannins influenced the oxygen consumption on the 1st day of the saturation cycles and, in the case of TT, a higher total consumption of oxygen was also detected. Acetaldehyde increased during the experiment while the native anthocyanins decreased throughout the oxidation process. Conclusion: Wines added with tannins featured improved color intensities with respect to the control, the addition of TT, GT and ET slightly promoted the formation of short polymeric pigments, the astringency, determined before and at the end of the experiment, decreased in all the samples, including the control wine, and mostly in the ET and GT samples.

Slight variations of pH affect the sensory properties and stability of wines with important implications for the winemaking practices. Preliminary studies in wine-like solutions with pH values ranging from 3 to 4 showed that anthocyanin solubility decreased as the pH increased. A significant drop of solubility was observed at pH 3.3. In order to verify these results in real wines, an experimental protocol was designed with the purpose of investigating the pH influence on the anthocyanin extractability in three musts differing from each other only by the pH levels adjusted to 3.2, 3.5 and 3.9, respectively. The NMR-based analysis of the wines, deriving from the three musts, provided data consistent with those observed in wine-like solutions. The extraction of anthocyanins turned out to be increasingly lower as the pH increased even if the effect of pH was more contained than expected on the basis of the data obtained from model solutions. Other molecules of enological interest, mainly catechins and cinnamates, were proven to be affected by the must pH. Additionally, tannins slightly increased as the pH increased but the reactivity of tannins towards saliva decreased confirming the decisive role of pH also on wine astringency.

The Alpine wormwood Artemisia vallesiaca All. was considered the most valuable ingredient of vermouth, a celebrated aromatized wine. A. vallesiaca has a very limited geographical distribution, and the booming market of vermouth decimated its natural population, resulting in the eventual replacement of this rare species with more common and less expensive wormwoods like A. absinthium L.. Over the past years, attempts to revive the original recipe(s) of vermouth have fostered the establishment of cultivations of A. vallesiaca in pre-montane settings. In order to assist these projects, the phytochemical profile of cultivated plants and of several native populations of A. vallesiaca from the Swiss Valais were comparatively evaluated, focusing on sesquiterpene lactones and on lipophilic flavonoids, the hallmark constituents of Artemisia species. Remarkably, no significant difference was detected between the samples, despite the different origins. The lipophilic flavonoids of A. vallesiaca were similar to those of related species used in the production of vermouth, but the presence of C-9 oxygenated 11β-methyl germacranolides and eudesmanolides (herbolides) made its sesquiterpene lactone profile peculiar. In addition to known compounds, two novel germacranolides were also characterized (herbolides J and K), and the major sesquiterpene lactone from the plant, the bitter germacranolide herbolide D (4), was detected and quantified by 1H NMR in a bitter liqueur aromatized with A. vallesiaca. Taken together, these observations qualify herbolides as marker to identify A. vallesiaca in aromatized alcohol matrixes.

The present work aims to characterize and investigate the anti-inflammatory activity of hop extracts (cv. Cascade) in an in vitro model of gastric inflammation. The biological activities of hydroalcoholic and aqueous extracts from cones were evaluated by comparing IL-8 inhibition induced by TNFα. The hydroalcoholic extract demonstrated a higher inhibitory effect, which was just slightly affected by an in vitro simulated gastric digestion. The identification of active compounds was performed by a bio-guided fractionation which afforded 11 fractions, one of which inhibited IL-8 release in a concentration-dependent fashion in human gastric epithelial AGS cells. Phytochemical analysis revealed xanthohumol A and xanthohumol D as the main active components. The present study provides some experimental evidences that Humulus lupulus L. may exert an anti-inflammatory activity on the gastric district by the inhibition of the IL-8 secretion, partially due to its prenylated chalcones content.

Palytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTX-contaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 × 10(-10)-2.5 × 10(-8) M) in a K(+)-free phosphate buffered saline solution, employing a 5 h incubation at 41 °C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD = 1.4 × 10(-10) M and quantitation, LOQ = 3.4 × 10(-10) M), accuracy (bias = -0.8%), repeatability (RSDr = 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 μg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects.

Biological properties of fruits of Lycium barbarum (goji berries) have been ascribed to their high content of nutrients and phenolics. Comprehensive studies aimed at unambiguously identifying the phenolic components in goji berries are still lacking. In this paper, we report on the isolation and NMR-based identification of the major phenolics in commercially available goji berries. Together with already known phenolics, including caffeic acid, p-coumaric acid, rutin, scopoletin, N-trans-feruloyl tyramine, and N-cis-feruloyl tyramine, an unreported N-feruloyl tyramine dimer was characterized as the most abundant polyphenol isolated from the berries. Usually divalent molecules show enhanced biological activities than their corresponding monomers.

Experimental evidence suggests that moderate consumption of wine has health promoting properties that have been mainly attributed to the wine polyphenol content. However, a systematic analysis of the major healthy molecules contained in wines has not been conducted yet. Our study explored the potential arsenal of beneficial molecules contained in wine from both a qualitative and quantitative perspective. The experimental approach was based on chromatography and untargeted NMR spectroscopy. In addition to already known bioactive molecules, for the first time, xanthurenic acid and oleanic acid were identified in wine in relatively high concentrations. On account of their many biological activities, these two molecules widen the range of potential beneficial effects of wine and pave the way toward the evaluation of their still unexplored sensory properties.

Vitis vinifera cv Falanghina is an ancient grape variety of Southern Italy. A thorough phytochemical analysis of the Falanghina leaves was conducted to investigate its specialised metabolite content. Along with already known molecules, such as caftaric acid, quercetin-3-O-β-D-glucopyranoside, quercetin-3-O-β-D-glucuronide, kaempferol- 3-O-β-D-glucopyranoside and kaempferol-3-O-β-D-glucuronide, a previously undescribed biflavonoid was identified. For this last compound, a moderate bioactivity against metastatic melanoma cells proliferation was discovered. This datum can be of some interest to researchers studying human melanoma. The high content in antioxidant glycosylated flavonoids supports the exploitation of grape vine leaves as an inexpensive source of natural products for the food industry and for both pharmaceutical and nutraceutical companies. Additionally, this study offers important insights into the plant physiology, thus prompting possible technological researches of genetic selection based on the vine adaptation to specific pedo-climatic environments.

Sorbus domestica fruits (sorbs) are commonly harvested and consumed for their nutritious qualities and have long been used as natural remedy against diabetes in the popular medicine. Recently, sorbs have been described as sources of antioxidant compounds including polyphenols. Chemical analyses carried out on sorbs collected in Southern Italy have led to the isolation of a chlorogenic acid isomer as the main antioxidant compound contained in the fruit butanol extract. NMR studies have identified the isolated compound as (1S,3R,4S,5R)5-O-Caffeoylquinic acid. In comparison to chlorogenic acid, the isolated isomer features an inverted configuration at C4. Such configurational inversion causes the chlorogenic acid stereoisomer acid to assume a preferential conformation remarkably different from that of chlorogenic acid. This seems to account for the enhanced potency of (1S,3R,4S,5R)5-O-Caffeoylquinic acid to reduce both the glucose and cholesterol uptakes by the cell line HepG2 when compared to chlorogenic acid.

Lycium barbarum (Solanaceae), long known to the traditional Chinese medicine because of its many health-promoting effects, has of late spread widely across the Western hemisphere, mainly on account of the nutritional richness in vitamins, minerals and antioxidant metabolites of its fruits. Data on bioactive metabolites from fruits and leaves, which are commonly consumed in soups and salads, are scarce and sometimes even contradictory. By means of NMR, the present study identified the specialised products contained in an Italian cultivar of L. barbarum. Kaempeferol, caffeic acid, 3,4,5-trihydroxycinnamic acid and 5-hydroxyferulic acid were found in fresh fruits; rutin and chlorogenic acid were detected in leaves and flowers; also, a previously undescribed N,N-dicaffeoylspermidine derivative was identified in flowers, while N-feruloyltyramine derivatives, for which interesting anti-inflammatory properties have been reported, turned out to be the major bioactive molecules in stems. The plethora of the detected bioactive molecules amplifies the nutraceutical value of berries and leaves and prompts the exploitation of L. barbarum flowers and pruned stems as sources of beneficial compounds.

Ziziphus jujuba Mill., jujube, leaf infusions are popular bedtime beverages as they improve sleep by soothing the nerves. With the aim of providing further insights into the polyphenol content of jujube leaf infusion and their antioxidant activities, a 2,2-diphenyl-1-picrylhydrazyl (DPPH)-guided purification of the aqueous extract of Z. jujuba Mill. leaves was conducted. Three major antioxidant polyphenols (i.e. quercitrin, catechin and gallocatechin) of the leaves were identified. In addition, a significant antibiofilm bioactivity against Streptococcus mutans, a causative agent of human dental caries, was detected for the aqueous leaf extract and its bio-guided separation resulted in the identification of the lupane triterpenoid alphitolic acid as the main antibiofilm metabolite. The results herein presented further promote the consumption of jujube leaf infusion as a healthy antioxidant bedtime beverage, and associate it to an unreported anti-caries activity.

Palytoxin ranks among the most potent marine biotoxins. Its lethality was well known to native Hawaiians that used to smear a "moss" containing the toxin on their spears to cause instant death to their victims. Human intoxications due to exposure to palytoxin and to its many congeners have been reported worldwide. Currently, palytoxins constitute the main threat to public health across the Mediterranean Sea. In the present work we report on the isolation and stereostructural determination of a new palytoxin analogue from a Hawaiian Palythoa tuberculosa sample. This new toxin is a stereoisomer of 42-hydroxypalytoxin isolated from Palythoa toxica. The whole absolute configuration of this latter toxin is also reported in the paper. Interestingly, the two 42-hydroxypalytoxins do not share the same biological activity. The stereoisomer from P. tuberculosa showed cytotoxicity toward skin HaCaT keratinocytes approximately 1 order of magnitude lower than that of 42-hydroxypalytoxin from P. toxica and about 2 orders of magnitude lower than that of palytoxin itself. This finding holds the prospect of interesting structure-activity relationship evaluations in the future.

Palytoxin is one of the most potent non protein marine toxins known and it is suspected to cause severe, and sometimes even lethal intoxications in humans. Its origin has long been controversial and different potential production sources have been proposed. Nowadays, dinoflagellates belonging to the genus Ostreopsis are the most accredited producers of palytoxins. Since the isolation of palytoxin in 1971, a number of palytoxin congeners have been isolated so far from different biological sources. In this chapter the main aspects of chemistry of palytoxins are reported.

Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant extracts as well as to the anti-inflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. "Cascade" has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low μM concentrations.

Anecdotal reports exist of aquarium hobbyists that experienced severe respiratory distress and/or skin injury following cleaning operation of home aquaria containing Palythoa sp. soft corals. Hundreds of cases of respiratory illness and/or dermatitis have been recorded in proximity to the sea concomitantly with algal blooms of Ostreopsis spp. in the Mediterranean area. Both Palythoa spp. and Ostreopsis spp. contain congeners of palytoxin, a highly potent toxin whose inhalation hazard is however unknown. In this study, we demonstrate the presence of high levels of palytoxins (palytoxin and hydroxypalytoxin) in both soft coral and seawater from a home marine aquarium involved in the poisoning of a whole family. Due to the high toxin levels found in seawater, a procedure for a rapid and efficient determination of palytoxin in seawater was set-up. A comparison of symptoms of Palythoa- and Ostreopsis-related inhalatory poisonings showed many similarities including fever, respiratory distress, nausea, and flu-like symptoms. From the chemical and symptomatological data reported herein it is reasonable to hold palytoxins responsible for respiratory disorders following inhalation. Although the exact mechanism through which palytoxin congeners exert their inhalatory toxicity is still unknown, this represents a step toward demonstrating that palytoxin congeners exert toxic effects through inhalation both in natural environments and in the surroundings of private and public aquaria.

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.

Palytoxins are a class of extremely potent non-proteic marine biotoxins, whose main biological target is the Na+/K+-ATPase. Since its isolation in 1971 from samples of Hawaiian Palythoa spp., palytoxin has drawn scientists’ attention from across the world because of its high toxicity, intriguing chemical structural architecture, and involvement in fascinating ancient Hawaiian folklore. Palytoxins have recently spread also to more temperate areas, such as the Mediterranean Sea causing severe human intoxications. Over the past years our scientific work has brought to light the occurrence of new palytoxin analogs by extensive NMR investigation and a new liquid chromatography tandem mass spectrometry method set up following the Mediterranean toxic outbreaks.

Blooms of benthic dinoflagellates of the genus Ostreopsis (mainly O. cf. ovata and occasionally O. cf. siamensis) represent a serious concern for humans in the Mediterranean area, due to production of palytoxin-like compounds listed among the most potent marine toxins known. In this work, six strains of Ostreopsis sp. from Cyprus Island were analyzed through an integrated approach based on molecular, chemical, and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis, belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced ovatoxin-a (OVTX-a), OVTX-d, OVTX-e, and isobaric palytoxin, so far found only in O. cf. ovata; the other three strains produced only new palytoxin-like compounds, which we named ovatoxin-i, ovatoxin-j(1), ovatoxin-j(2), and ovatoxin-k. The new ovatoxins present the same carbon skeleton as ovatoxin-a, differing primarily in an additional C2H2O2 moiety and an unsaturation in the region C49-C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j(1) and OVTX-k) and the lack of a hydroxyl group in the region C53-C78 (in OVTX-i and OVTX-j(1)). The toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06-2.8 pg cell(-1), definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, an eco-toxicological test on Artemia salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata. The whole of these data suggest that Ostreopsis sp. from Cyprus Island poses a relatively low risk to humans.

Palytoxins from Ostreopsis cf. ovata (a putative palytoxin and ovatoxins) are emerging toxins in the Mediterranean basin and are not yet regulated, although there is evidence that they can accumulate in seafood and thus enter the human food chain. This poses serious concerns for human health, because palytoxin itself is among the most potent marine toxins known. In 2009, the European Food Safety Authority (EFSA) announced the need for optimization of efficient analytical methods for detecting palytoxins and for preparing standards. Herein, we propose a procedure including a one-step extraction, solid-phase-extraction (SPE) clean-up, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) detection of individual palytoxins in mussels. The method enabled efficient chromatographic separation of individual compounds, including structural isomers, with good sensitivity, reproducibility, and linearity in a large dynamic range (14-1000 ng mL(-1) in matrix). As a result, the putative palytoxin from Ostreopsis cf. ovata was identified as an isomer of palytoxin itself and re-named isobaric palytoxin. The whole procedure (sample preparation and LC-HRMS analysis) proved able to detect palytoxins in both spiked and natural mussel samples at levels as low as 70 μg kg(-1) in crude mussel extracts and 15 μg kg(-1) after SPE clean-up. Although full validation of the method is currently prevented by the unavailability of palytoxin(s) certified standards and reference material, this study constitutes a first step towards achieving this.

A new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish is proposed herein. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-MS). The high percentage of organic modifier in the mobile phase and the omission of ion pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on a ionspray (ISI)-ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray (TSI)-triple quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. Multiple reaction monitoring experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit of DA in tissue (20 ug/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004, demonstrated for the first time in Italy the presence of domoic acid as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.

A detailed analysis of toxic shellfish collected in the Adriatic sea in October 2000 allowed us to isolate a new cytotoxic chlorosulfolipid (3). Its gross structure has been elucidated through an extensive NMR analysis including various 2D techniques; the relative stereochemistry has been solved by applying the Murata's method. Compound 3 showed to posses cytotoxic activity against WEHI 164 and J774 cells. The presence of chlorosulfolipids in toxic mussels from the northern Adriatic sea has not to be considered incidental as we have been detecting these cytotoxic compounds since 1998. Their simultaneous and constant presence together with typical marine biotoxins represents a further risk both to consumers' health and aquacultures economic proceeds. © 2004 Elsevier Ltd. All rights reserved.

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata are a concern in the Mediterranean Sea, since this species produces a wide range of palytoxin-like compounds listed among the most potent marine toxins. This study focused on two analogs of palytoxin found in cultures of six strains of Ostreopsis cf. ovata isolated from the south of Catalonia (NW Mediterranean Sea). In addition to some already known ovatoxins, our strains produced two minor compounds, ovatoxin-g and the so far called putative palytoxin, whose structures had not been elucidated before. Insufficient quantity of these compounds impeded a full Nuclear Magnetic Resonance (NMR)-based structural elucidation, thus we studied their structure in crude algal extracts through Liquid Chromatography Electro Spray Ionization High Resolution Mass Spectrometryn (LC-ESI-HRMSn) in positive ion mode. Under the used MS conditions, the molecules underwent fragmentation at many sites of their backbone and a large number of diagnostic fragment ions were identified. As a result, tentative structures were assigned to both ovatoxin-g and the putative palytoxin, the latter being identified as a palytoxin isomer and re-named isobaric palytoxin.

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

A clonal culture of Alexandrium andersoni, obtained from germination of a resting cyst, collected from the Gulf of Naples, was found positive for PSP toxicity by mouse bioassay. The toxicity profile of this dinoflagellate consists mainly of toxins belonging to the saxitoxin class, in particular of Saxitoxin (STX) and Neosaxitoxin (NEO), as determined by a wide MS and (1)H NMR analysis. This represents the first report of the presence of A. andersoni in the Mediterranean Sea, as well as of its toxicity.

A new analogue of yessotoxin (YTX), carboxyYTX, 3, was isolated from the digestive glands of mussels cultured on the Italian Adriatic coast. Its structure was determined by MS and NMR spectroscopy.

This study investigated the composition of diarrhoetic shellfish toxins in the hepatopancreas of mussels from the northern Adriatic Sea. The major toxins were shown to be yessotoxin, identified by its chromatographic properties and spectral data, and okadaic acid, detected both by fluorometric high-performance liquid chromatography and by comparison of its spectral properties with those of an authentic sample.

More than 40 years after its isolation, the understanding of how palytoxin interacts with biological systems has yet to be fully determined. The Na(+),K(+)-ATPase pump constitutes a molecular receptor for palytoxin that is able to convert the pump into an open channel, with consequent loss of cellular K(+) and remarkable rise of cytosolic Na(+) levels. In addition, a slight permeability to Ca(2+) is detected when palytoxin binds to the pump. It has been demonstrated that the increase of cytosolic free Ca(2+) concentration gives rise to downstream events ultimately leading to cell death. The widely accepted recognition of the dependence of important cellular events on calcium ion concentration propelled us to investigate the occurrence of palytoxin-Ca(2+) complex in aqueous solution by NMR- and molecular modeling-based approach. We identified two specific regions of palytoxin where Ca(2+) is preferentially coordinated. This study constitutes the first characterization of a calcium complex with palytoxin and, as such, is expected to support the investigation of the toxin molecular bioactivity.

Blooms of Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to more temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe. However, while O. confronta (cf.) ovata has caused several toxic outbreaks, the presence of O . cf. siamensis has been reported rather occasionally and in very few regions; as a consequence, O . cf. ovata toxin profile has been in-depth studied while poor information exists on toxicity of the Mediterranean and Atlantic O . cf. siamensis . In the present study toxin profile of Mediterranean and Atlantic O . cf. siamensis isolates also phylogenetically related has been studied through liquid chromatography–high resolution mass spectrometry (LC–HRMS) versus a palytoxin standard, a crude extract of O . cf. ovata containing all the ovatoxins so far known (ovatoxin-a to -f), and a Japanese O. siamensis extract which contained ostreocin-d and ostreocin-b. The Mediterranean and Atlantic O . cf. siamensis strains were shown not to produce either ostreocins, which are produced by the Japanese O. siamensis strain, or ovatoxins, which are produced by the Mediterranean O . cf. ovata. Only sub-fg levels of palytoxin on a per cell basis were detected in the Mediterranean strain. This study demonstrates that the Mediterranean and the Atlantic O . cf. siamensis strains are devoid of any appreciable toxicity. Thus, at least in the European area, O . cf. siamensis seems to present a much lower risk to human health than O . cf. ovata .

Ovatoxin-a is the main toxin produced by Ostreopsis ovata, a benth- ic dinoflagellate that has bloomed mas- sively across the Mediterranean basin over the past years, inflicting both human and environmental suffering. Ovatoxin-a has recently been isolated from cultures of O. ovata and structur- ally identified as an analogue of paly- toxin: in comparison with palytoxin, ovatoxin-a lacks three hydroxy groups at the 17-, 44- and 64-positions, but fea- tures an extra hydroxy functionality at the 42-position. Herein we report on the NMR-based elucidation of the stereochemistry of ovatoxin-a, which includes 7 stereogenic double bonds and 62 asymmetric carbon atoms. Un- derstanding the full stereochemistry of ovatoxin-a is a step towards the eluci- dation of its mechanism of action on a molecular level.

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.

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported.

In the Mediterranean Sea, blooms of Ostreopsis cf. ovata and Ostreopsis siamensis have become increasingly frequent in the last decade and O. cf. ovata was found to produce palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, -d and -e), a class of highly potent toxins. The environmental conditions seem to play a key role in influencing the abundance of Ostreopsis spp. High cell densities are generally recorded in concomitance with relatively high temperature and salinity and low hydrodynamics conditions. In this study the effects of temperature and salinity on the growth and toxicity of an Adriatic O. cf. ovata isolate were investigated. The highest growth rates of the Adriatic strain were recorded for cultures grown at 20 °C and at salinity values of 36 and 40, in accordance with natural bloom surveys. Toxicity was affected by growth conditions, with the highest toxin content on a per cell basis being measured at 25 °C and salinity 32. However, the highest total toxin content on a per litre basis was recorded at 20 °C and salinity 36, since under such conditions the growth yield was the highest. O. cf. ovata had lethal effects on Artemia nauplii and juvenile sea basses, and produced haemolysis of sheep erythrocytes. A comparison between haemolysis neutralization assay and HR LC-MS results showed a good correlation between haemolytic effect and total toxin content measured through HR LC-MS. Considering the increasing need for rapid and sensitive methods to detect palytoxin in natural samples, the haemolytic assay appears a useful method for preliminary quantification of the whole of palytoxin-like compounds in algal extracts.

Mass appearances of the toxic dinoflagellate genus Ostreopsis are known to cause dangerous respiratory symptoms in humans exposed to aerosols. The outbreaks can appear in shallow marine waters of temperate regions around the globe. We followed a massive bloom event on a public beach on the northern Adriatic coast near Rovinj, Croatia. We identified the responsible species and the produced toxins as well as the dynamics of the event with respect to environmental conditions. Ostreopsis cf. ovata appeared in masses from September through October 2010 on a public beach near Rovinj, Croatia but stayed undetected by public health organizations. Respiratory symptoms were observed whenever humans were exposed to substrate samples containing large numbers of Ostreopsis cells. During the mass abundance of O. cf. ovata also exposure to the aerosols on the beach evoked respiratory symptoms in humans. Our measurements showed high cell abundances and high toxin contents with a stable relative contribution of putative Palytoxin and Ovatoxins a-e. Artificial beach structures proved to dramatically reduce settling of the observed Ostreopsis biofilm. Blooms like those reported herein have a high potential to happen undetected with a high potential of affecting the health of coastal human populations. Increased monitoring efforts are therefore required to understand the ecology and toxicology of those bloom events and reduce their negative impact on coastal populations.

Palytoxin is a potent marine toxin and one of the most complex natural compounds ever described. A number of compounds identified as palytoxin congeners (e.g., ovatoxins, mascarenotoxins, ostreocins, etc.) have not been yet structurally elucidated due to lack of pure material in quantities sufficient to an NMR-based structural investigation. In this study, the complex fragmentation pattern of palytoxin in its positive high resolution liquid chromatography tandem mass spectra (HR LC-MS(n)) was interpreted. Under the used conditions, the molecule underwent fragmentation at many sites of its backbone, and a large number of diagnostic fragment ions were identified. The natural product itself was used with no need for derivatization. Interestingly, most of the fragments contained calcium in their elemental formula. Evidence for palytoxin tendency to form adduct ions with calcium and other divalent cations in its mass spectra was obtained. Fragmentation pattern of palytoxin was used as template to gain detailed structural information on ovatoxin-a, the main toxin produced by Ostreopsis ovata, (observe correct font) a benthic dinoflagellate that currently represents the major harmful algal bloom threat in the Mediterranean area. Either the regions or the specific sites where ovatoxin-a and palytoxin structurally differ have been identified.

The state of the art of LC-MS of palytoxin and its analogues is reported in the present review. MS data for palytoxin, 42-hydroxy-palytoxin, ostreocin-D, mascarenotoxins, and ovatoxins, obtained using different ionization techniques, namely fast-atom bombardment (FAB), matrix assisted laser desorption ionization (MALDI), and electrospray ionization (ESI), are summarized together with the LC-MS methods used for their detection. Application of the developed LC-MS methods to both plankton and seafood analysis is also reported, paying attention to the extraction procedures used and to limits of detection (LOD) and quantitation (LOQ) achieved. In a research setting, LC-MS has shown a good potential in determination of palytoxin and its analogues from various sources, but, in a regulatory setting, routine LC-MS analysis of palytoxins is still at a preliminary stage. The LOQ currently achieved in seafood analysis appears insufficient to detect palytoxins in shellfish extract at levels close to the tolerance limit for palytoxins (30 μg/kg) proposed by the European Food Safety Authority (EFSA, 2009). In addition, lacking of certified reference standard of palytoxins as well as of validation studies for the proposed LC-MS methods represent important issues that should be faced for future perspectives of LC-MS technique.

Over the past decades, Italian coastlines have been plagued by recurring presence of the benthic dinoflagellate Ostreopsis ovata. Such an alga has caused severe sanitary emergencies and economic losses due to its production of palytoxin-like compounds. Previous studies have confirmed the presence of ovatoxin-a (OVTX-a) as the major toxin of the algal toxin profile together with small amounts of putative palytoxin (PLTX). In our ongoing research on O. ovata toxins we report herein on in-depth investigation of an O. ovata culture carried out by high-resolution (HR) liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS2). Particularly, the presence of putative PLTX and OVTX-a was confirmed and the occurrence in the extract of four new palytoxin-like compounds, OVTX-b, -c, -d, and -e, was highlighted. Elemental formulae have been assigned to the new ovatoxins and information has been gained about their structural features. A quantitative study of the O. ovata culture extract indicated that the whole of the new ovatoxins represents about 46% of the total toxin content and, thus, their presence has to be taken into account when LC/MS-based monitoring programs of either plankton or contaminated seafood are carried out. Copyright © 2010 John Wiley & Sons, Ltd.

Since its isolation dated back to as far as 1971, palytoxin has all along drawn scientists' attention from across the world because of its high toxicity and fascinating chemical architecture. Commitment of the international scientific community to the study of this extremely potent non-proteic toxin has led to discover quite a number of palytoxin analogues. Once confined only to tropical and subtropical areas, palytoxins have recently spread also to more temperate regions, such as the Mediterranean Sea where they have caused severe human intoxications. Studies on the Mediterranean toxic outbreaks brought to light the existence of further palytoxin-like compounds, ovatoxins, never reported elsewhere in the world.

This paper reports on the determination of toxin profile of mussels (Mytilus galloprovincialis) collected in November-December 2003 along the Emilia Romagna coasts (Italy) when a high concentration of Alexandrium ostenfeldii cells was detected in sea water. Detailed liquid chromatography-mass spectrometry (LC-MS) analyses were performed on the crude extracts in both selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. They revealed that M. galloprovincialis had accumulated the three major spirolides produced by the alga, namely 13-desMethyl spirolide C, 13,19-didesMethyl spirolide C and 27-hydroxy-13,19-didesMethyl spirolide C, which fully accounted for toxicity of lipophilic extracts shown in mouse bioassay. Interestingly, yessotoxin (YTX) and its analogues were still present in mussel polar extracts but YTX itself was not the major toxin contained in mussels. The presence of pectenotoxin-2 seco acid (PTX-2sa) and its putative epimer was also assessed. The presence of azaspiracids, never reported from the Adriatic sea, as well as of diarrhetic shellfish poisoning toxins (okadaic acid, dinophysistoxins and OA esters) and domoic acid, long known as contaminants of Adriatic mussels, was also investigated.

This paper reports on the analysis of the toxin content from Palythoa tuberculosa and Palythoa toxica samples collected off of the Hawaiian coast. Our work, based on in-depth high-resolution liquid chromatography-mass spectrometry analysis along with extensive NMR study, led us to structurally characterize 42-hydroxy-palytoxin, a new palytoxin congener. This toxin and palytoxin itself appeared to be the major components of toxic extract from a P. tuberculosa sample, while 42-hydroxy-palytoxin was proven by far to be the main palytoxin derivative in P. toxica. Functional studies on this new palytoxin-like compound suggest that the new palytoxin analogue and palytoxin itself present similar biological activities.

The north-western coasts of the Adriatic sea have long been subjected to recurring cases of harmful algae blooms and concomitant mussel contamination. In 1995, yessotoxins (YTXs) were detected for the first time in Adriatic mussels and their presence was subsequently linked to the presence of Protoceratium reticulatum and occasionally Lingulodinium polyedrum in seawater. Since then, farmed molluscs are yearly found to contain YTXs for long periods with a severe impact on aquaculture activities. In 2004, unusually high amounts of homoYTXs were detected in mussels. In this period, a careful look at the phytoplankton composition highlighted that P. reticulatum and L. polyedrum were nearly absent in seawater whereas the dinoflagellate Gonyaulax spinifera was present at high densities. G. spinifera has been recently associated with YTX production in New Zealand. The present work was performed in order to ascertain whether Adriatic G. spinifera was a toxic species. Determination of the toxin content of natural samples (mussels and net haul plankton samples) as well as monitoring of the dinoflagellate species present in seawaters allowed us to correlate the presence of G. spinifera to that of homoYTX in mussels. Cultures of G. spinifera were set up which gave us the opportunity to investigate toxin content and profile of the dinoflagellate, for the first time, by liquid chromatography–mass spectrometry (LC–MS) and to perform a molecular characterization of the species through rDNA sequencing. Two G. spinifera isolates, sampled in different years, were analyzed and resulted to be different in both toxin content and gene sequence: the isolate sampled in 2005 produced only low levels of YTX (5.4 pg cell−1) while that sampled in 2006 contained higher toxin amounts, namely 33.4 pg cell−1 of homoYTX and 3.6 pg cell−1 of YTX; the two isolates also differed in 111 and 176 nucleotide positions, respectively, in the SSU and partial LSU rDNA genes. Comparing similar sequences of G. spinifera strains contained in GenBank, divergences among the Adriatic strains and strains of different geographical origin emerged; on the contrary, a good similarity was evidenced (1.3%) between the Adriatic isolate sampled in 2006 and two New Zealand strains; the similarity was confirmed also by the presence in all strains of YTXs, although the New Zealand strains were analyzed by ELISA which could not distinguish among the different YTX analogues. Due to an originally confusing description of the species and to the availability of very low number of deposited sequences the doubt arises whether the studied G. spinifera strains belong to the same species. However, although the taxonomy of the species complex G. spinifera remains largely unresolved, this study allowed to unambiguously link the presence of G. spinifera to recurring mussel toxicity episodes.

Two new desulfated yessotoxin (YTX) analogues were isolated from a toxic batch of Adriatic mussels collected in October 2004. Their stereostructures were elucidated through extensive NMR and MS-based analysis. The finding of these desulfocarboxyhomoYTXs in shellfish poses additional problems to those institutions entitled to control seafood safety, since desulfated YTXs fail the European Union method currently in force for checking toxicity in mollusks.

Samples of Mytilus galloprovincialis were harvested from five different locations in Thermaikos gulf, Greece after harmful algae bloom. All of the mussel samples were found positive by mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins. Liquid chromatography (LC) coupled with mass spectrometry (MS) was used to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and yessotoxins (YTXs). In order to investigate the presence of okadaic acid esters, alkaline hydrolysis was performed for all the samples, and LC–MS analyses were carried out on the samples before and after hydrolysis. Hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) analyses were also carried out to investigate the presence of domoic acid and paralytic shellfish poisoning (PSP) toxins at trace levels. All of the samples were found to be contaminated only with okadaic acid at levels 0.10–0.20 μg/g.

The structure-activity relationship of yessotoxins (YTX) has been probed by measuring the potency of several YTX analogues to cause the accumulation of a 100 kDa MW fragment of E-cadherin in MCF-7 breast cancer cells. Under our experimental conditions, the EC 5 0 of YTX, the reference compound, was 0.55 nM. The introduction of a methylene unit adjacent to one of the sulfate groups, as is the case with the homoyessotoxin molecule, did not appear to greatly affect the potency of the analogue, as the measured EC 5 0 for this compound was 0.62 nM. The EC 5 0 values we measured for 45-hydroxyhomoyessotoxin and carboxyyessotoxin were about 9.4 and 26 nM, respectively, whereas the EC 5 0 of noroxoyessotoxin, lacking most of the C 9 chain, was about 50 nM. Thus, significant differences in the potencies of YTX analogues were found when structural changes involved the C 9 terminal chain of these compounds, leading to the conclusion that this portion of the molecule is essential for the activity of YTX in MCF-7 cells. A comparison of our findings with available information regarding the potency of YTX and its analogues in other experimental systems shows that the EC 5 0 's we measured for the different compounds are up to 200-fold lower and vary in a wider concentration range. We speculate that YTX effects could involve two separate receptorial systems.

While the occurrence of yessotoxin (YTX) has been reported worldwide from Protoceratium reticulatum, the biogenetic origin of some YTX analogues is still unknown, thus raising an issue whether they are metabolites of YTX formed in mussels or true products of different dinoflagellate species. Findings reported herein suggest that P. reticulatum from the north–western Adriatic sea is responsible for production, together with YTX (1), of homoYTX (2), 45-OHYTX (3), carboxyYTX (5), and noroxoYTX (7). YTX and its analogues have been determined by high performance liquid chromatography coupled with electrospray ion trap mass spectrometry (HPLC–MS and HPLC–MSn experiments). The result is the first to confirm production of these YTX analogues from this species and indicate it as a producing organism of homoYTX, 45-OHYTX, carboxyYTX, and noroxoYTX found in shellfish.

A liquid chromatography mass spectrometry (LC–MS) method is proposed for the sensitive, specific and direct detection of yessotoxin and its analogues, marine biotoxins which are associated with diarrhetic shellfish poisoning (DSP) and which have been found in the North Adriatic sea since 1995. The LC–MS method provided a detection limit of 70 pg for yessotoxin in full scan mode and was applied to determine the toxic profiles of a number of extracts or partially purified fractions of toxic mussels collected along the Emilia Romagna coasts (Italy) in the period 1995–1999. Detection of a desulfo-yessotoxin derivative from Mytilus galloprovincialis collected in 1998 is also reported.