Characterization of ovatoxin-h, a new ovatoxin analog, and evaluation of chromatographic columns for ovatoxin analysis and purification

Abstract

The presence of Ostreopsis cf. ovata on the Mediterranean coast represents a serious concern to human health due to production of toxins - putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d, -e, -f and -g). However, purified ovatoxins are not widely available and their toxicities are still unknown. In the present study, we report on HR LC-MS/MS analysis of a French O. cf. ovata strain (IFR-OST-0.3V) collected at Villefranche-sur-Mer (France) during a bloom in 2011. Investigation of this strain of O. cf. ovata cultivated in our laboratory by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS) confirmed the production of ovatoxins-a to -e and revealed the presence of a new ovatoxin analog, named ovatoxin-h. O. cf. ovata extracts were pre-purified by Sephadex LH-20 to obtain a concentrated fraction of ovatoxins (OVTXs). This method provided a recovery of about 85% of OVTXs and a cleanup efficiency of 93%. Different stationary phases were tested with this fraction of interest to elucidate the structure of the new OVTX congener and to obtain purified ovatoxins. Eight reversed phase sorbents were evaluated for their capacity to separate and purify ovatoxins. Among them Kinetex C18, Kinetex PFP and Uptisphere C18-TF allowed for best separations almost achieving baseline resolution. Kinetex C18 is able to sufficiently separate these toxins, allowing us to identify the toxins present in the extract purified by Sephadex LH-20, and to partly elucidate the structure of the new ovatoxin congener. This toxin possesses one oxygen atom less and two hydrogens more than ovatoxin-a. Investigations using liquid chromatography coupled to high resolution tandem mass spectrometry suggest that the part of the molecule where ovatoxin-h differs from ovatoxin-a is situated between C42 and C49. Uptisphere C18-TF was proposed as a first step preparative chromatography as it is able to separate a higher number of ovatoxins (especially ovatoxin-d and ovatoxin-e) and because it separates ovatoxins from unknown compounds, identified using full scan single quadrupole mass spectrometry. After pre-purification with Sephadex LH-20, purification and separation of individual ovatoxins was attempted using an Uptisphere C18-TF column. During recovery of purified toxins, problems of stability of OVTXs were observed, leading us to investigate experimental conditions responsible for this degradation.

Toxicity and Growth Assessments of Three Thermophilic Benthic Dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) Developing in the Southern Mediterranean Basin

“…Our OOBZT14 produced only OVTX-a and -b, and the Italian strain CBA29-2012 was found not to produce OVTX-b and -c [82]. Recently, the presence of a new ovatoxin analog, named ovatoxin-h, was reported for the French strain IFR-OST-03V [65]. Concerning the mascarenotoxins (McTX-a and McTX-c), their presence was only observed for the strain D483 originating from the Gulf of Naples [28,83].…”

“…and Acetabularia sp . )Brissard et al (2015) a [65]6 strains including IRTA-SMM-12-62 (Spain, South Catalonia, Ebro River Delta)2436100 (12L:12D)****OVTX-a,-b, -c, -d, -e,-g, IsobPLTX Total toxin content = 50–250 Jania rubens García-Altares et al (2015) a [99]CBA29-2012 (Italy, Quarto dei Mille Genoa)20 ± 0.5**85–135 (16L:8D)F/2**OVTX-a = 33.5, OVTX-d+e = 9, pPLTX = 1.5. Total toxin content in cell pellets = 44Toxic to Artemia salina nauplii ** Giussani et al (2015) a [82]OOBZT14 (Tunisia, Bizerte Bay)253680 (12L:12D)ENSW0.59OVTX-a = 15.56–18.7, OVTX-b = 3.4–4.6 Cymodocea nodosa This study a ATLANTIC WATERS Isolate 538 (Carribean Sea, Leeward Islands, IIe St. Barthelemy, Port de Gustavia)2835300 ft-c (12L:12D)GPM**Mouse bioassay: lack of water or lipid soluble ciguatera toxins in O. ovata extractsTide-poolBesada et al (1982) b [137]IEO-OS06BR, IEO-OS15BR (Brazil, Rio de Janeiro)17 ± 1**100 (14L:10D)K, F/20, F/2**Presence of palytoxin Hemolytic activity on human erythrocytes…”

“…Recent works allowed a better understanding of the taxonomy, phylogeny, toxicity and autoecology of these three potentially toxic benthic dinoflagellates [3,15,23,58,59,60,61,62,63,64,65]. Nevertheless, more research on toxic strains is needed, particularly in the Southern Mediterranean basin.…”

High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

“…Alexandrium ostenfeldii (A. ostenfeldii) extract containing 13,19-didesmethyl spirolide C (13,19-didesme-SPX-C) and Ostreopsis ovata (O. ovata) extract containing ovatoxin-a (OvTX-a) were obtained from Ifremer as previously described [33,37]. Those extracts were mixed with some of the abovementioned certified and in-house reference toxin calibration solutions as well as the mussel extract from Bruckless to obtain a composite multi-toxin sample, used for optimization of chromatographic separation.…”

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods

“…PLTX and 42-hydroxy-palytoxin (42-OH-PLTX) were identified from different species of zoanthids, together with other minor toxins [7][8][9]. PLTX and ovatoxins (from -a to -h, and some isomers) were detected in different strains of Ostreopsis ovata [10][11][12][13][14]. In O. siamensis ostreocin-d was described as the major toxin [15], while mascarenotoxins (-a and -b) were extracted from O. mascarenensis [6].…”