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.

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

A 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.