Andrew D. Turner

Development of a sensitive and selective liquid chromatography–mass spectrometry method for high throughput analysis of paralytic shellfish toxins using graphitised carbon solid phase extraction

Routine regulatory monitoring of paralytic shellfish toxins (PST) commonly employs oxidative derivitisation and complex liquid chromatography fluorescence detection methods (LC-FL). The pre-column oxidation LC-FL method is currently implemented in New Zealand and the United Kingdom. When using this method positive samples are fractionated and two different oxidations are required to confirm the identity and quantity of each PST analogue present. There is a need for alternative methods that are simpler, provide faster turnaround times and have improved detection limits. Hydrophilic interaction liquid chromatography (HILIC) HPLC-MS/MS analysis of PST has been used for research purposes, but high detection limits and substantial sample matrix issues have prevented it from becoming a viable alternative for routine monitoring purposes. We have developed a HILIC UPLC-MS/MS method for paralytic shellfish toxins with an optimised desalting clean-up procedure on inexpensive carbon solid phase extraction cartridges for reduction of matrix interferences. This represents a major technical breakthrough and allows sensitive, selective and rapid analysis of paralytic shellfish toxins from a variety of sample types, including many commercially produced bivalve molluscan shellfish species. Additionally, this analytical approach avoids the need for complex calculations to determine sample toxicity, as unlike other methods each PST analogue is able to be quantified as a single resolved peak. This article presents the method development and optimisation information. A thorough single laboratory validation study has subsequently been performed and this data will be presented elsewhere.

Detection of the pufferfish toxin tetrodotoxin in European bivalves, England, 2013 to 2014.

We report the first detection of tetrodotoxins (TTX) in European bivalve shellfish. We demonstrate that TTX is present within the temperate waters of the United Kingdom, along the English Channel, and can accumulate in filter-feeding molluscs. The toxin is heat-stable and thus it cannot be eliminated during cooking. While quantified concentrations were low in comparison to published minimum lethal doses for humans, the results demonstrate that the risk to shellfish consumers should not be discarded.

Comparison of AOAC 2005.06 LC official method with other methodologies for the quantitation of paralytic shellfish poisoning toxins in UK shellfish species

AbstractA refined version of the pre-column oxidation liquid chromatography with fluorescence detection (ox-LC-FLD) official method AOAC 2005.06 was developed in the UK and validated for the determination of paralytic shellfish poisoning toxins in UK shellfish. Analysis was undertaken here for the comparison of PSP toxicities determined using the LC method for a range of UK bivalve shellfish species against the official European reference method, the PSP mouse bioassay (MBA, AOAC 959.08). Comparative results indicated a good correlation in results for some species (mussels, cockles and clams) but a poor correlation for two species of oysters (Pacific oysters and native oysters), where the LC results in terms of total saxitoxin equivalents were found to be on average more than double the values determined by MBA. With the potential for either LC over-estimation or MBA under-estimation, additional oyster and mussel samples were analysed using MBA and ox-LC-FLD together with further analytical and functional methodologies: a post-column oxidation LC method (LC-ox-FLD), an electrophysiological assay and hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. Results highlighted a good correlation among non-bioassay results, indicating a likely cause of difference was the under-estimation in the MBA, rather than an over-estimation in the LC results. FigureTotal saxitoxin equivalents in oysters (Pacific and native) quantified by ox-LC-FLD, LC-ox-FLD, HPLC-MS/MS and electrophysiological assay as compared with the MBA PSP toxicity reference method

Single-Laboratory Validation of a Multitoxin Ultra-Performance LC-Hydrophilic Interaction LC-MS/MS Method for Quantitation of Paralytic Shellfish Toxins in Bivalve Shellfish.

A single-laboratory validation study was conducted for the hydrophilic interaction-LC-MS/MS analysis of paralytic shellfish toxins (PSTs) in bivalve shellfish. The method was developed as an alternative to the precolumn oxidation AOAC 2005.06 and postcolumn oxidation AOAC 2011.02 LC with fluorescence detection methods, receptor binding assay AOAC 2011.27, as well as the mouse bioassay AOAC 959.08. PSTs assessed were saxitoxin, neosaxitoxin, deoxydecarbamoylsaxitoxin, decarbamoylsaxitoxin, decarbamoylneosaxitoxin, gonyautoxins 1-6, decarbamoylgonyautoxins 2-3, and N-sulfocarbamoyl gonyautoxins 2&3. The method also included the determination of decarbamoylgonyautoxins 1&4, N-sulfocarbamoyl gonyautoxins 1&4, and M toxins. Twelve commercially produced bivalve species from both New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops, and clams. Validation studies demonstrated acceptable method performance characteristics for specificity, linearity, recovery, repeatability, and within-laboratory reproducibility. LOD and LOQ were significantly improved in comparison to current fluorescence-based detection methods, and the method was shown to be rugged. The method performed well in comparison to AOAC 2005.06, with evidence obtained from both comparative analysis of 1141 PST-contaminated samples and successful participation in proficiency testing schemes. The method is suitable for use in regulatory testing and will be submitted for an AOAC collaborative study.

Occurrence and profiles of lipophilic toxins in shellfish harvested from Argentina.

The presence of phytoplankton responsible for the production of lipophilic marine biotoxins is well recognised throughout parts of South America. To date, the quantitation of lipophilic toxins in Argentinean shellfish has been limited to select and highly focussed geographical studies. This work reports the analysis for lipophilic marine biotoxins in shellfish harvested across five regions of Argentina between 1992 and 2012. LC-MS/MS analysis was used for the quantitation of all regulated lipophilic toxins. High concentrations of okadaic acid group toxins were quantified, with a clear dominance of the parent okadaic acid and more than 90% of the toxin present as esters. Results showed DSP toxins in shellfish from the Buenos Aires Province during 2006 and 2007, earlier than previously described. There was also strong evidence linking the presence of okadaic acid to human intoxications. Other lipophilic toxins detected were yessotoxin, pectenotoxin-2 and 13-desMeC spirolide. With evidence published recently for the presence of azaspiracid producers, this work reports the detection of low concentrations of azaspiracid-2 in shellfish. As such the data provides the first published evidence for yessotoxins and azaspiracids in Argentinean shellfish and further evidence for the continuing presence of lipophilic marine toxins in Argentinean waters.

Investigations into matrix components affecting the performance of the official bioassay reference method for quantitation of paralytic shellfish poisoning toxins in oysters

Significant differences previously observed in the determination of paralytic shellfish poisoning toxins (PSTs) in oysters using official method AOAC 2005.06 and 959.08 were investigated in detail with regard to possible matrix effects. Method AOAC 2005.06 gave results 2-3 times higher than the mouse bioassay method, 959.08, differences thought to be due to underestimation of PSTs by the mouse bioassay. In order to prove the cause of these large differences, work was conducted here to examine the presence and effects of matrix components on the performance of each of the two assays. A range of oyster, cockle and mussel samples were extracted using the AOAC 959.08 hydrochloric acid (HCl) extraction method and analysed for PSP by both MBA and LC-FLD. In addition, extracts were analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for metals as well as being subjected to a range of nutritional testing methods. Whilst there was no evidence for effect of nutritional components on either assay, ICP-MS analysis revealed a relationship between samples exhibiting the largest differences in relative method performance, specifically those with the largest LC-FLD/MBA toxicity ratio, and samples containing the highest concentrations of zinc and manganese. In order to prove the potential effect of the metals on either the LC-FLD and/or MBA assays, HCl extracts of a range of shellfish were subjected to a number of matrix modifications. Firstly, a number of PSP-positive oyster samples were processed to reduce the concentrations of metals within the extracts, without significantly reducing the concentrations of PSTs. Secondly, a range of mussel and cockle extracts, plus a standard solution of saxitoxin di-hydrochloride were spiked at variable concentrations of zinc. All treated and non-treated extracts, plus a number of controls were subjected to ICP-MS, LC-FLD and MBA testing. Results proved the absence of any effect of metals on the performance of the LC-FLD, whilst showing a large suppressive effect of the metals on the MBA. As such, the results show the performance of the official MBA is potentially unsafe for application to the routine monitoring of PSP toxicity in oysters or in any other shellfish found to contain high concentrations of metal ions.

Pre- versus post-column oxidation liquid chromatography fluorescence detection of paralytic shellfish toxins

Both pre- and post-column oxidation liquid chromatography methods with fluorescence detection are available for detecting paralytic shellfish toxins. Each method has been evaluated in multiple laboratories and validated as a potential alternative to the mouse bioassay. This communication compares the advantages and limitations of both methods. For a given laboratory, the selection of either method may be based primarily on practicality and less on any deficiencies in scientific merit.

Variability of paralytic shellfish toxin occurrence and profiles in bivalve molluscs from Great Britain from official control monitoring as determined by pre-column oxidation liquid chromatography and implications for applying immunochemical tests.

As the official control monitoring laboratory in Great Britain for the analysis of marine biotoxins in shellfish, Cefas have for the past five years conducted routine monitoring for paralytic shellfish poisoning toxins (PST) using a non-animal alternative method to the mouse bioassay reference method; a refined version of the AOAC 2005.06 pre-column oxidation liquid chromatography method. Application of this instrumental methodology has enabled the generation of data not only on the occurrence and magnitude of PST events, but also the quantitation and assessment of different PST profiles. Since implementation of the method in 2008, results have shown huge variabilities in the occurrence of PSTs, with large spatial and temporal variabilities around the coastline. Mean PST profiles were not found to correlate either with total PST content of the shellfish, the year of sampling or with a few notable exceptions, the shellfish species. Toxin profiles were found to fall into one of four distinct profile types, with one relating solely to the exclusive presence of decarbamoyl toxins in surf clams. The other profile types contained variable proportions of gonyautoxins, N-sulfocarbamoyl toxins, neosaxitoxin and saxitoxin. While some indications of geographical repeatability were noted, this was not observed for all profile types. Consequently, the application of rapid immunochemical testing methods to end product testing would need to be considered carefully given the large differences in PST congener cross-reactivities.

Lipophilic toxins in cultivated mussels (Mytilus galloprovincialis) from Baja California, Mexico.

Here, we report different lipophilic toxins (LTs) detected by LC-MS/MS in Mediterranean mussels (Mytilus galloprovincialis) collected through 2012 in Todos Santos Bay, northwest Baja California, Mexico. The concentration of okadaic acid (OA), dinophysistoxin 2 (DTX2), and pectenotoxin 2 (PTX2) reached 500 μg kg(-1) during July and increased to 1647 μg kg(-1) in October. These toxins were associated with the presence of Dinophysis fortii and Dinophysis acuminata and a strong stratification of the water column. Other LTs present were yessotoxins, with a maximum concentration of 1080 μg kg(-1) in June. Cyclic imines (13-desmethyl spirolide and gymnodimine) and azaspiracid 1 were also detected in the mussels but at low concentrations. Diarrhetic toxins concentrations evaluated by LC-MS/MS were compared with the results of two mouse bioassay protocols. Positive results were obtained with both MBA protocols in several samples that presented toxicities below 160 μg OA-eq kg(-1), as estimated by LC-MS/MS results whereas other samples returned negative MBA results in samples with concentrations above this level. Therefore, analytical methods need to be applied to confirm the presence of regulated LTs. This is the first report of LTs in mussels cultivated in Mexico. The occurrence of these toxins represents an emerging problem in the region.

Potential Threats Posed by Tetrodotoxins in UK Waters: Examination of Detection Methodology Used in Their Control

Tetrodotoxin is a neurotoxin responsible for many human fatalities, most commonly following the consumption of pufferfish. Whilst the source of the toxin has not been conclusively proven, it is thought to be associated with various species of marine bacteria. Whilst the toxins are well studied in fish and gastropods, in recent years, there have been a number of reports of tetrodotoxin occurring in bivalve shellfish, including those harvested from the UK and other parts of Europe. This paper reviews evidence concerning the prevalence of tetrodotoxins in the UK together with methodologies currently available for testing. Biological, biomolecular and chemical methods are reviewed, including recommendations for further work. With the recent development of quantitative chromatographic methods for these and other hydrophilic toxins, as well as the commercial availability of rapid testing kits, there are a number of options available to ensure consumers are protected against this threat.