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

Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been adopted as the EU reference method (No.15/2011) for detection and quantitation of lipophilic toxins. Traditional LC-MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers. Matrix effects are an important issue to address in LC-MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce matrix effects for all compounds, and was found to be particularly useful for the non-targeted approach. Limits of detection and method accuracy were comparable between the systems tested, demonstrating the applicability of HRMS as an effective tool for screening and quantitative analysis. HRMS offers the advantage of untargeted analysis, meaning that datasets can be retrospectively analysed. HRMS (full scan) chromatograms of passive samplers yielded significantly less complex data sets than mussels, and were thus more easily screened for unknowns. Consequently, we recommend the use of HRMS in combination with passive sampling for studies investigating emerging or hitherto uncharacterised toxins.

Bacterial community structure of the marine diatom Haslea ostrearia

The marine diatom Haslea ostrearia produces a water-soluble blue-pigment named marennine of economic interest (e.g. in aquaculture for the greening of oysters). Up to date the studies devoted to ecological conditions under which this microalga develops never took into account the bacterial-H. ostrearia relationships. In this study the bacterial community was analysed by PCR-TTGE before and after H. ostrearia isolation cells recovered from 4 localities, to distinguish the relative part of the biotope and the biocenose and eventually to describe the temporal dynamic of the structure of the bacterial community. The bacterial structure of the phycosphere differed strongly from that of the bulk sediment. The similarity between bacteria recovered from the biofilm and the suspended bacteria did not exceed 10% (vs. > 90% amongst biofilms). The differences in genetic fingerprints, more especially high between two H. ostrearia isolates showed also the highest differences in the bacterial structure as the result of specific metabolomics profiles. The non-targeted metabolomic investigation showed that these profiles were more distinct in case of bacteria-alga associations than for the H. ostrearia monoculture. At the scale of a culture cycle in laboratory conditions, the bacterial community was specific to the growth stage. When H. ostrearia was subcultured for 9 months, a shift in the bacterial structure was shown from 3-months subculturing and the bacterial structure stabilized afterwards (70-86% similarities). A first insight of the relationships between H. ostrearia and its surrounding bacteria was shown for a better understanding of the ecological feature of this diatom.

First metabolomic approach of the epiphytic bacteria-marine diatom Haslea ostrearia relationships

The marine diatom Haslea ostrearia [1] produces a water-soluble blue-pigment named marennine [2] of economic interest. But the lack of knowledge of the ecological conditions, under which this microalga develops in its natural ecosystem, more especially bacteria H. ostrearia interactions, prevents any optimization of its culture in well-controlled conditions. The structure of the bacterial community was analyzed by PCR-TTGE before and after the isolation of H. ostrearia cells recovered from 4 localities, to distinguish the relative part of the biotope and the biocenose and eventually to describe the temporal dynamic of the structure of the bacterial community at two time-scales. The differences in genetic fingerprints, more especially high between two H. ostrearia isolates (HO-R and HO-BM) showed also the highest differences in the bacterial structure [3] as the result of specific metabolomics profiles. The non-targeted metabolomic investigation showed that these profiles were more distinct in case of bacteria-alga associations than for the H. ostrearia monoculture Here we present a Q-TOF LC/MS metabolomic fingerprinting approach [3]: - to investigate differential metabolites of axenic versus non axenic H. ostrearia cultures. - to focus on the specific metabolites of a bacterial surrounding associated with the activation or inhibition of the microalga growing. The Agilent suite of data processing software makes feature finding, statistical analysis, and identification easier. This enables rapid transformation of complex raw data into biologically relevant metabolite information.

Preliminary metabolomic approach on the bacterial structure community of Haslea ostrearia

The marine diatom Haslea ostrearia [1] produces a water-soluble blue-pigment named marennine [2] of economic interest. But the lack of knowledge of the ecological conditions, under which this microalga develops in its natural ecosystem, more especially bacteria H. ostrearia interactions, prevents any optimization of its culture in well-controlled conditions. The structure of the bacterial community was analyzed by PCR-TTGE before and after the isolation of H. ostrearia cells recovered from 4 localities, to distinguish the relative part of the biotope and the biocenose and eventually to describe the temporal dynamic of the structure of the bacterial community at two time-scales. The differences in genetic fingerprints, more especially high between two H. ostrearia isolates (HO-R and HO-BM) showed also the highest differences in the bacterial structure [3] as the result of specific metabolomics profiles. The non-targeted metabolomic investigation showed that these profiles were more distinct in case of bacteria-alga associations than for the H. ostrearia monoculture Here we present a Q-TOF LC/MS metabolomic fingerprinting approach [3]: - to investigate differential metabolites of axenic versus non axenic H. ostrearia cultures. - to focus on the specific metabolites of a bacterial surrounding associated with the activation or inhibition of the microalga growing. The Agilent suite of data processing software makes feature finding, statistical analysis, and identification easier. This enables rapid transformation of complex raw data into biologically relevant metabolite information.