Dereplication of Gambierdiscusbalechii extract by LC-HRMS and in vitro assay: First description of a putative ciguatoxin and confirmation of 44-methylgambierone.

Marine toxins have a significant impact on seafood resources and human health. Up to date, mainly based on bioassays results, two genera of toxic microalgae, Gambierdiscus and Fukuyoa have been hypothesized to produce a suite of biologically active compounds, including maitotoxins (MTXs) and ciguatoxins (CTXs) with the latter causing ciguatera poisoning (CP) in humans. The global ubiquity of these microalgae and their ability to produce (un-)known bioactive compounds, necessitates strategies for screening, identifying, and reducing the number of target algal species and compounds selected for structural elucidation. To accomplish this task, a dereplication process is necessary to screen and profile algal extracts, identify target compounds, and support the discovery of novel bioactive chemotypes. Herein, a dereplication strategy was applied to a crude extract of a G. balechii culture to investigate for bioactive compounds with relevance to CP using liquid chromatography-high resolution mass spectrometry, in vitro cell-based bioassay, and a combination thereof via a bioassay-guided micro-fractionation. Three biologically active fractions exhibiting CTX-like and MTX-like toxicity were identified. A naturally incurred fish extract (Sphyraena barracuda) was used for confirmation where standards were unavailable. Using this approach, a putative I/C-CTX congener in G. balechii was identified for the first time, 44-methylgambierone was confirmed at 8.6 pg cell-1, and MTX-like compounds were purported. This investigative approach can be applied towards other harmful algal species of interest. The identification of a microalgal species herein, G. balechii (VGO920) which was found capable of producing a putative I/C-CTX in culture is an impactful advancement for global CP research. The large-scale culturing of G. balechii could be used as a source of I/C-CTX reference material not yet commercially available, thus, fulfilling an analytical gap that currently hampers the routine determination of CTXs in various environmental and human health-relevant matrices.

Untargeted and targeted LC-MS and data processing workflow for the comprehensive analysis of oligopeptides from cyanobacteria.

Cyanobacteria produce a plethora of structurally diverse bioactive secondary metabolites, including cyanotoxins which pose a serious threat to humans and other living organisms worldwide. Currently, a wide variety of mass spectrometry-based methods for determination of microcystins (MCs), the most commonly occurring and studied class of cyanotoxins, have been developed and employed for research and monitoring purposes. The scarcity of commercially available reference materials, together with the ever-growing range of mass spectrometers and analytical approaches, make the accuracy of quantitative analyses a critical point to be carefully investigated in view of a reliable risk evaluation. This study reports, a comparative investigation of the qualitative and quantitative MCs profile obtained using targeted and untargeted liquid chromatography-mass spectrometry approaches for the analyses of cyanobacterial biomass from Lake Kastoria, Greece. Comparison of the total MCs content measured by the two approaches showed good correlation, with variations in the range of 3.8-13.2%. In addition, the implementation of an analytical workflow on a hybrid linear ion trap/orbitrap mass spectrometer is described, based on combining data-dependent acquisition and a powerful database of cyanobacterial metabolites (CyanoMetDB) for the annotation of known and discovery of new cyanopeptides. This untargeted strategy proved highly effective for the identification of MCs, microginins, anabaenopeptins, and micropeptins. The systematic interpretation of the acquired fragmentation patterns allowed the elucidation of two new MC structural variants, MC-PrhcysR and MC-Prhcys(O)R, and proposal of structures for two new microginins, isomeric cyanostatin B and MG 821A, and three isomeric micropeptins at m/z 846.4715, 846.4711 and 846.4723.

Development of a data dependent acquisition-based approach for the identification of unknown fast-acting toxins and their ester metabolites.

Phycotoxins in the marine food-web represent a serious threat to human health. Consumption of contaminated shellfish and/or finfish poses risk to consumer safety: several cases of toxins-related seafood poisoning have been recorded so far worldwide. Cyclic imines are emerging lipophilic toxins, which have been detected in shellfish from different European countries. Currently, they are not regulated due to the lack of toxicological comprehensive data and hence the European Food Safety Authority has required more scientific efforts before establishing a maximum permitted level in seafood. In this work, a novel data dependent liquid chromatography - high resolution mass spectrometry (LC-HRMS) approach has been successfully applied and combined with targeted studies for an in-depth investigation of the metabolic profile of shellfish samples. The proposed analytical methodology has allowed: i) to discover a plethora of unknown fatty acid esters of gymnodimines and ii) to conceive a brand new MS-based strategy, termed as backward analysis, for discovery and identification of new analogues. In particular, the implemented analytical workflow has broadened the structural diversity of cyclic imine family through the inclusion of five new congeners, namely gymnodimine -F, -G, -H, -I and -J. In addition, gymnodimine A (376.5 µg/kg), 13-desmethyl spirolide C (11.0-29.0 µg/kg) and pinnatoxin G (3.1-7.7 µg/kg) have been detected in shellfish from different sites of the Mediterranean basin (Tunisia and Italy) and the Atlantic coast of Spain, with the confirmation of the first finding of pinnatoxin G in mussels harvested in Sardinia (Tyrrhenian Sea, Italy).

HPLC-Based Analysis of Impurities in Sapropterin Branded and Generic Tablets.

This work was aimed at the definition of a chromatographic method able to separate and quantify impurities present in sapropterin-containing drugs during an accelerated stability study. The chromatographic method was applied to the orphan drug Kuvan® and to its corresponding generic sapropterin Dipharma (Diterin®), both of which are approved for the treatment of hyperphenylalaninemia-induced symptoms. The two products tested had a similar manufacture date and both had an approved stability shelf-life of three years. Samples were analyzed by HPLC at T = 0 and after six months of storage at 40 °C and 75% relative humidity. Identification of the impurities was supported by a detailed mass spectrometry and MS/MS profile. The analysis demonstrated an overall higher stability for the Diterin® formulation, which was related to a lower increase of some impurities compared to Kuvan®.

Are Canned Beverages Industries Progressively Switching to Bisphenol AF?

Seven bisphenols, endocrine-disruptor chemicals, were analytically determined for risk assessment in 52 large-consumption beverages collected from the Italian market. The analytes under examination were bisphenol A, bisphenol F, bisphenol E, bisphenol B, bisphenol AF, bisphenol A diglycidyl ether, and bisphenol M. The concentration levels of all bisphenols detected ranged from

Plastic-associated harmful microalgal assemblages in marine environment.

Plastic debris carry fouling a variety of class-size organisms, among them harmful microorganisms that potentially play a role in the dispersal of allochthonous species and toxic compounds with ecological impacts on the marine environment and human health. We analyzed samples of marine plastics floating at the sea surface using a molecular qPCR assay to quantify the attached microalgal taxa, in particular, harmful species. Diatoms were the most abundant group of plastic colonizers with maximum abundance of 8.2 × 104 cells cm-2 of plastics, the maximum abundance of dinoflagellates amounted to 1.1 × 103 cells cm-2 of plastics. The most abundant harmful microalgal taxon was the diatom Pseudo-nitzschia spp., including at least 12 toxic species, and the dinoflagellate Ostreopsis cf. ovata with 6606 and 259 cells cm-2, respectively. The abundance of other harmful microalgal species including the toxic allochthonous dinoflagellate Alexandrium pacificum ranged from 1 to 73 cells cm-2. In the present study, a direct relationship between the abundance of harmful algal species colonizing the plastic substrates and their toxin production was found. The levels of potential toxins on plastic samples ranged from 101 to 102 ng cm-2, considering the various toxin families produced by the colonized harmful microalgal species. We also measured the rate of adhesion by several target microalgal species. It ranged from 1.8 to 0.3 day-1 demonstrating the capacity of plastic substrate colonizing rapidly by microalgae. The present study reports the first estimates of molecular quantification of microorganisms including toxin producing species that can colonize plastics. Such findings provide important insights for improving the monitoring practice of plastics and illustrate how the epi-plastic community can exacerbate the harmful effects of plastics by dispersal, acting as an alien and toxic species carrier and potentially being ingested through the marine trophic web.