Myriam Algoet

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

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.

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.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis) common mussels (Mytilus edulis) and hard clams (Mercenaria mercenaria), but not found in cockles (Cerastoderma edule), razors (Ensis species) or scallops (Pecten maximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrum cordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

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.

A Rapid Method for the Analysis of Paralytic Shellfish Toxins Utilizing Standard Pressure HPLC: Refinement of AOAC 2005.06.

Superficially porous column technologies have previously been shown to provide faster chromatographic analysis of toxin oxidation products when analyzing shellfish for paralytic shellfish toxins. While sub 3 μm fused core columns have facilitated enhanced method performance, including significantly lower analysis times and lower LOD, they were also found to last for only a few hundred injections before pressure increases rendered them unusable with standard HPLC. Recently 5 μm superficially porous columns have become commercially available. In this study, a 5 μm fused core column was used to develop a fast chromatographic method for the analysis of paralytic shellfish toxins, with performance characteristics and column lifetime being assessed. The 5 μm column was found to be able to perform approximately 3000 injections without significant increases in back pressure or reduction in performance. Data generated using the column were found to be equivalent to that determined using current HPLC column technologies for both screening and quantitation methods. Furthermore, an increase in sensitivity for all toxins tested under the routine monitoring program for British waters was observed and the overall run time of the analysis halved. Overall, the 5 μm fused core column provided a significant increase in sample throughput, a reduction in mobile phase consumption, and an increase in method sensitivity.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: Okadaic acid, dinophysis toxins and pectenotoxins

Official control biotoxin testing of bivalve molluscs from Great Britain has been conducted by Cefas for over a decade. Reflecting the changes in legislation, bioassays were gradually replaced by analytical methods, firstly for analysis of Paralytic shellfish toxins, followed by introduction of liquid chromatography tandem mass spectrometric (LCMS/MS) method for lipophilic toxins (LTs) in 2011. Twelve compounds, representing three main groups of regulated lipophilic toxins, as well as two non-regulated cyclic imines were examined in over 20,500 samples collected between July 2011 and December 2016. The toxins belonging to Okadaic acid (OA) group toxins were the most prevalent and were quantified in 23% of samples, predominantly from Scotland. The temporal pattern of OA group occurrences remained similar each year, peaking in summer months and tailing off during autumn and winter, however their abundance and magnitude varied between years significantly, with concentrations reaching up to 4993 μg OA eq./kg. Three toxin profiles were identified, reflecting the relative contribution of the two main toxins, OA and dinophysis toxin-2 (DTX2). Dinophysis toxin-1 (DTX1) was less common and was never detected in samples with high proportions of DTX2. Inter-annual changes in profiles were observed within certain regions, with the most notable being an increase of DTX2 occurrences in north-west Scotland and England in the last three years of monitoring. In addition, seasonal changes of profiles were identified when OA, the dominant toxin in early summer, was replaced by higher proportions of DTX2 in late summer and autumn. The profile distribution possibly reflected the availability of individual Dinophysis species as a food source for shellfish, however persistence of DTX2 during autumn and winter in mussels might have also been attributed to their physiology. Mussels were the only species with higher average proportions of non-esterified toxins, while Pacific oysters, cockles, surf clams, razors and queen scallops contained almost exclusively ester forms. In addition, a temporal change in proportion of OA and DTX2 free form was observed in mussels. Pectenotoxin-2 (PTX2) was quantified only on rare occasions.