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Summary of investigations conducted at Cefas into the effects of oyster matrix on HPLC and MBA PSP results
Project Code: FS235002S;
1. Turner, A.D., Norton, D.M., Hatfield, R.G., Rapkova-Dhanji, M., Algoet M. and Lees, D.N (2010). Single laboratory validation of a refined AOAC LC method for Oysters, Cockles and Clams in UK shellfish. J. of AOAC Internat. 93(5), 1482-1493.
2. Turner, A.D., Hatfield, R.G., Rapkova, M., Higman, W., Algoet, M., Suarez-Isla, B.A., Cordova, M., Caceres, C., van de Riet, J., Gibbs, R., Thomas, K., Quilliam, M. and Lees, D.N. (2011. Comparison of AOAC 2005.06 LC official method with other methodologies for the quantitation of paralytic shellfish poisoning toxins in UK shellfish species. Anal. Bioanal. Chem.399, 1257-1270.
3. Turner, A.D., Dhanji-Rapkova, M., Algoet, M., Suarez-Isla, B.A., Cordova, M., Caceres, C., Murphy, C.J., Casey, M. & Lees, D.N. (2012) Investigations in matrix components affecting the performance of the official bioassay reference method for quantitation of paralytic shellfish poisoning toxins in oysters. Toxicon, 59(2), 215-230
See also: http://www.foodbase.org.uk/results.php?f_category_id=&f_report_id=612 (previous method validation reports)
http://cot.food.gov.uk/pdfs/cotdraftmins28jun2011.pdf (Minutes of COT review)
Initial results showed no evidence for any artificial signal enhancement in the HPLC analysis of oysters, which may occur through the effects of co-extracted matrix components. Similarly, no effects were attributable to the variable toxin concentrations extracted using either acetic acid or hydrochloric acid extraction solvents. Additional MBA conducted at a separate international laboratory showed there were no issues relating to the reproducibility of the bioassay which may have affected the observed differences.
A period of comparative testing on a range of oyster and non-oyster samples involved the use of four different methodologies in comparison to the MBA. The AOAC 2005.06 pre-column oxidation HPLC method, a post-column oxidation HPLC method (now AOAC 2011.02), an LC-MS/MS method and an electrophysiological assay were all utilised. On average results between the four non-bioassays agreed well and all showed significantly higher total toxicity results than the MBA. As such, there was good evidence for the potential under-estimation of toxicities by MBA as opposed to over-estimation of toxicity by HPLC.
Further work examined the potential presence of matrix components which may affect either of the assays. Whilst the overall salinity and nutritional content of the sample extracts showed no correlation with the observed effects, concentrations of some metals (notably zinc and manganese) were shown to be present at much higher levels in the oyster samples which exhibited the largest differences in toxicity results between the two assays. Further experimental work involving the removal of metals from oyster samples and the addition of zinc to mussel and cockle samples provided strong evidence for the suppressive effect of zinc and potentially other metals on the performance of the bioassay, whilst not affecting the performance of the HPLC.
Overall, the work has demonstrated the accuracy of the official reference MBA method to be compromised in samples containing high concentrations of metals, typically observed naturally in both UK species of oysters. As such the recommendation is to replace the oyster MBA test with the HPLC method in the UK official control monitoring programme.
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