Interlaboratory comparison of two AOAC liquid chromatographic fluorescence detection methods for paralytic shellfish toxin analysis through characterization of an oyster reference material.

An interlaboratory ring trial was designed and conducted by the Centre for Environment, Fisheries, and Aquaculture Science to investigate a range of issues affecting the analysis of a candidate Pacific oyster paralytic shellfish toxin reference material. A total of 21 laboratories participated in the study and supplied results using one or more of three instrumental methods, specifically precolumn oxidation (Pre-COX) LC with fluorescence detection (FLD; AOAC Official Method 2005.06), postcolumn oxidation (PCOX) LC-FLD (AOAC Official Method 2011.02), and hydrophilic interaction LC/MS/MS. Each participant analyzed nine replicate samples of the oyster tissue in three separate batches of three samples over a period of time longer than 1 week. Results were reported in a standardized format, reporting both individual toxin concentrations and total sample toxicity. Data were assessed to determine the equivalency of the two AOAC LC methods and the LC/MS/MS method as well as an assessment of intrabatch and interbatch repeatability and interlaboratory reproducibility of each method. Differences among the results reported using the three methods were shown to be statistically significant, although visual comparisons showed an overlap between results generated by the majority of tests, the exception being the Pre-COX quantitation of N-hydroxylated toxins in post ion-exchange fractions. Intralaboratory repeatability and interlaboratory reproducibility were acceptable for most of the results, with the exception of results generated from fractions. The results provided good evidence for the acceptable performance of the PCOX method for the quantitation of C toxins. Overall the study showed the usefulness of interlaboratory analysis for the characterization of paralytic shellfish poisoning matrix reference materials, highlighting some issues that may need to be addressed with further method assessment at individual participant laboratories.

A feasibility study into the production of a freeze-dried oyster reference material for paralytic shellfish poisoning toxins.

Matrix reference materials are an essential component for the validation and quality control of analytical methodologies for the quantitation of marine biotoxins in shellfish. Given the potential advantages of reference materials in powder form, a study was conducted to assess the feasibility for the production of a freeze-dried oyster tissue reference material containing a range of important paralytic shellfish poisoning toxins. One bulk sample of a wet oyster tissue homogenate was generated following mass culturing of toxic Alexandrium and oyster feeding experiments. The bulk tissue was used to prepare untreated wet frozen aliquots with the remainder being freeze-dried and processed into appropriately-sized powder samples. A pre-column oxidation LC-FLD analysis was used to confirm the absence of any chromatographic artefacts resulting from the processing and to confirm acceptable homogeneity of the tissues. Excellent stability over both the short-term (1 month) and long-term (1 year) of the freeze-dried material was demonstrated as compared with the stability of the untreated wet tissue. A post-column oxidation LC-FLD method was used to confirm the absence of toxin epimerisation in freeze-dried tissues which were observed in the wet tissues. Overall the work showed the feasibility of an approach to produce a homogenous freeze-dried oyster matrix material with enhanced stability in comparison to the untreated wet tissue. The potential for use of the process for preparation of large scale production batches of a freeze-dried CRM for paralytic shellfish poisoning toxins has therefore been demonstrated.

Testing and application of a refined rapid detection method for paralytic shellfish poisoning toxins in UK shellfish.

The Scotia Rapid Test for PSP is designed for qualitative identification of saxitoxins at levels in shellfish equivalent to the limit of detection of the biological reference method. However, issues with the method have been reported, including the low assay cross reactivity for some toxins, high numbers of false positive results and the subjective test interpretation. This study focussed on approaches to improve each of these issues. A refined test was found to improve GTX1&4 test sensitivity in samples containing high proportions of GTX1&4. The subjectivity of the test was successfully eliminated through use of an automated scanner, which enabled both the reliable identification of test results as well as the provision of a numerical result which could be utilised for more refined results interpretation. Finally the high proportion of false positive results in comparison with the LC-FLD was investigated, with a modified approach incorporating an additional extract dilution applied to a range of shellfish samples with different toxicities. Results showed highly variable limits of detection of the method and no significant reduction in false positive results when applying the additional dilution, which may be of concern to laboratories in receipt of high numbers of samples containing low concentrations of toxins.

Feasibility studies into the production of gamma-irradiated oyster tissue reference materials for paralytic shellfish poisoning toxins.

A study was conducted to assess the feasibility for the production of sterile, stable and homogenous shellfish reference materials containing known concentrations of paralytic shellfish poisoning (PSP) toxins. Pacific oysters were contaminated with toxins following mass culturing of toxic algae and shellfish feeding experiments. Live oysters were shucked and tissues homogenised, before measuring into multiple aliquots, with one batch subjected to gamma irradiation treatment and the other remaining untreated. The homogeneity of both batches of samples was assessed using a pre-column oxidation liquid chromatography with fluorescence detection (Pre-COX LC-FLD) method and shown to be within the limits of normal within-batch repeatability. A twelve-month stability experiment was conducted for both untreated and gamma irradiated batches, specifically examining the effects of long term storage at -20 °C, +4 °C and +40 °C. Results indicated mostly good stability of PSP toxins in both materials when stored frozen at -20 °C, but with the instability of GTX2&3 concentrations in the untreated tissues eliminated in the irradiated tissues. Analysis using a post-column oxidation (PCOX) LC-FLD method also showed epimerisation in both GTX1&4 and GTX2&3 epimeric pairs in untreated samples after only 6 months frozen storage. This issue was not present in the tissues irradiated before long term storage. Biological activity testing confirmed the absence of bacteria in the irradiated samples throughout the 12 month study period. With such results there was clear evidence for the potential of increasing the scale of the mass culturing and shellfish feeding for the production of large batches of tissue suitable for the preparation of a certified matrix reference material. Overall results demonstrated the feasibility for production of oyster reference materials for PSTs, with evidence for prolonged stability following gamma irradiation treatment and storage at -20 °C.

Transformation of paralytic shellfish poisoning toxins in Crassostrea gigas and Pecten maximus reference materials.

Matrix reference materials are an important requirement for the assessment of method performance characteristics and for routine quality control. In the field of marine toxin testing where biological assays have been used and where modern analytical testing methods are now becoming available, this requirement has become an urgent one. Various approaches are utilised for preparation of such materials in the absence of available naturally occurring toxic shellfish samples. Toxin-free shellfish may be artificially fortified through the addition of cultured toxic phytoplankton or shellfish may be incurred through natural feeding on toxic algae in a laboratory environment. Both of these approaches may be potentially affected by issues relating to the degradation or transformation of toxin analytes, so studies were conducted to assess these effects within our laboratory. A range of PSP-toxic shellfish tissues were prepared using the two approaches, in both Pacific oyster (Crassostrea gigas) and king scallops (Pecten maximus). Additionally, sub-samples of incurred Pacific oyster tissue were further treated, through addition of artificial chemical stabilisers and gamma irradiation. Two separate month-long stability trials were conducted at +4 °C on each material. Results highlighted clear evidence for improved stability of materials following shellfish feeding experiments in comparison with the tissues which had been spiked with plankton. In addition, there were clear differences in stability of toxins between the two shellfish species studied. There was evidence for good stability of C1&2 toxins in both the incurred tissues and improved stability of some toxins in tissues which had been subjected to either gamma irradiation or treatment with chemical additives. The results therefore highlighted the benefits of conducting shellfish feeding if suitable stable reference materials are to be prepared containing a full range of PSP toxin analytes. The study also highlighted the benefits of post-production treatment to prolong the stability of the materials. Work is ongoing to assess the full characteristics of candidate reference materials prepared with these approaches with the aim of producing a homogenous and stable PSP reference material in Pacific oysters.

A feasibility study into the provision of Paralytic Shellfish Toxins laboratory reference materials by mass culture of Alexandrium and shellfish feeding experiments.

As the official control laboratory for biotoxin testing in England, Wales and Scotland, Cefas employs two approaches for the detection of Paralytic Shellfish Poisons (PSP) in bivalve shellfish: a qualitative HPLC method for oysters, whole king scallops and cockles (with PSP bioassay confirmation of positive HPLC samples) with subsequent quantitation of positive samples by mouse bioassay and a quantitative HPLC method for mussels (no PSP bioassay confirmation required). To aid the validation of the quantitative HPLC method for native oysters, Pacific oysters, cockles and king scallops and ultimately remove the need for the PSP bioassay for these species, appropriate contaminated shellfish matrices were required. As it was not possible to obtain naturally contaminated material for these species, shellfish were contaminated in-house through feeding experiments with high concentrations of Alexandrium species. A number of feeding experiments with two Alexandrium strains were performed successfully. The contaminated shellfish materials generated contained a number of different profiles of PSP toxins. This work has demonstrated the feasibility of these methods for the production of laboratory reference materials in a variety of bivalve shellfish species. Based on this study laboratory reference material production via these methods is now undertaken routinely within Cefas. By running two concurrent feeding trials per year for each species, enough laboratory reference material is produced for approximately 1 year of the programme. This removes the necessity for natural contaminated material which is not always available for reference material production. Additionally, such materials enable both the comparative testing of different PSP methodologies and the ongoing generation of long-term precision data for the HPLC method.