Isolation of ovatoxin-a from ostreopsis cf. ovata cultures. A key step for hazard characterization and risk management of ovatoxins.

Ostreopsis cf. ovata, a benthic/epiphytic marine dinoflagellate, is currently spreading in tropical, sub-tropical and temperate areas, causing periodic Harmful Algal Blooms (HABs). It produces a wide array of palytoxin-like compounds named ovatoxins (OVTXs), with OVTX-a generally the most abundant congener. Despite numerous cases of human poisonings and environmental damage associated with the presence of OVTXs and O. cf. ovata proliferations, a complete characterization of the toxicity of this class of molecules cannot be performed until Reference Material (RM) for individual congeners is available. This, in turn, requires the availability of sufficient amounts of toxin at a high purity grade. To achieve this goal, herein an analytical re-evaluation of critical-steps of OVTX-a isolation from O. cf. ovata cell pellets is reported. The overall procedure consists of four steps, namely an extraction, a Medium Pressure Liquid Chromatography (MPLC) separation, and two preparative High Performance Liquid Chromatography (HPLC) steps. Particular attention was paid to the extraction step to evaluate the repeatability in OVTX-a yields. For subsequent steps, loading sample preparation and chromatographic conditions were refined. As a result, a significant increase in recovery yields (from 12.5 to 20 ± 3%) and in purity grade (from 51% to 94%) of the isolated OVTX-a was achieved in comparison to previous studies. The improved procedure can easily be applied to isolate sufficient quantities of a good candidate RM for OVTX-a.

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.