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.

Genetic diversity analysis of isolates belonging to the Photobacterium phosphoreum species group collected from salmon products using AFLP fingerprinting

An accurate amplified fragment length polymorphism (AFLP) method, including three primer sets for the selective amplification step, was developed to display the phylogenetic position of Photobacterium isolates collected from salmon products. This method was efficient for discriminating the three species Photobacterium phosphoreum, Photobacterium iliopiscarium and Photobacterium kishitanii, until now indistinctly gathered in the Photobacterium phosphoreum species group known to be strongly responsible for seafood spoilage. The AFLP fingerprints enabled the isolates to be separated into two main clusters that, according to the type strains, were assigned to the two species P. phosphoreum and P. iliopiscarium. P. kishitanii was not found in the collection. The accuracy of the method was validated by using gyrB-gene sequencing and luxA-gene PCR amplification, which confirmed the species delineation. Most of the isolates of each species were clonally distinct and even those that were isolated from the same source showed some diversity. Moreover, this AFLP method may be an excellent tool for genotyping isolates in bacterial communities and for clarifying our knowledge of the role of the different members of the Photobacterium species group in seafood spoilage.