Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America

Abstract

With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have been developed which have been subjected to single-laboratory and multi-laboratory validation studies, gaining acceptance as official methods of analysis and approval for use in some countries as official control testing methods. The majority of validation studies conducted to date do not, however, incorporate shellfish species sourced from Latin America. Consequently, this study sought to investigate the performance of five alternative PSP testing methods together with the MBA, comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD) methods including both pre-column and post-column oxidation, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and a commercial lateral flow assay (LFA) from Scotia. A total of three hundred and forty-nine shellfish samples from Argentina, Mexico, Chile and Uruguay were assessed. For the majority of samples, qualitative results compared well between methods. Good statistical correlations were demonstrated between the majority of quantitative results, with a notably excellent correlation between the current EU reference method using pre-column oxidation LC-FLD and LC-MS/MS. The LFA showed great potential for qualitative determination of PSP toxins, although the findings of high numbers of false-positive results and two false negatives highlighted that some caution is still needed when interpreting results. This study demonstrated that effective replacement methods are available for countries that no longer wish to use the MBA, but highlighted the importance of comparing toxin data from the replacement method using local shellfish species of concern before implementing new methods in official control testing programs.

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis

“…When methods were compared two by two, all three assays were significantly correlated with each other, with the highest correlation coefficient (r 2 = 0.841) found between the fRBA and LC-MS/MS (Figure 8). Similar findings were observed in the detection of paralytic shellfish toxins such as saxitoxins in dinoflagellates and shellfish extracts using these three methodological approaches, with a significant correlation found between rRBA and HPLC, and between rRBA and LC-MS [132,133].…”

“…In this study, despite the use of the same batch of CTX3C standard and the same chemical extract of each sample tested by the three methods, differences were still observed. These findings are of importance, as a lot of studies report differences in discrete numerical values of toxicity estimates [22,133,144]. The most likely explanation for these observations is that comparisons must be relative: the different assays measure different parameters, making cross-comparisons of precise numerical concentrations difficult.…”

“…The effective concentration at 50% (EC 50 , defined as the concentration of competitor at which the binding of the fluorescently labelled PbTx-2 is reduced by half) obtained under the two incubation conditions did not show any significant difference between the two tested temperatures and incubation durations. In most RBA-based studies, the binding reaction usually occurs at a temperature of 4 • C from 60 to 120 min for the detection of both VGSC activators, i.e., CTXs and PbTxs [17,81,[83][84][85]87,89,[117][118][119][120][121][122][123][124][125][126][127], and VGSC inhibitors, i.e., saxitoxins [128][129][130][131][132][133], but also gambierol and gambieric acid-A [54], and brevenal [134,135]. Our findings indicated that an incubation at 37 • C for 30 min showed the same efficacy in the detection and quantification of CTXs in G. polynesiensis, so this protocol was further applied in the following experiments.…”

A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis

“…Certain kits nonetheless performed better on naturally contaminated samples (Pipis only) compared to spiked samples (Neogen and Abraxis PP2A). The reasons for this remain unclear, but support the assertion by Turner et al [ 32 ] that validation studies need to include both relevant shellfish species and naturally contaminated shellfish samples, so that any rapid test kit performance is measured using local toxin profiles.…”

A High Throughput Screening HPLC-FLD Method for Paralytic Shellfish Toxins (PSTs) Enabling Effective Official Control

“…PSTs have been detected in bivalves from all over the world [ 5 , 6 , 7 , 8 ]. In Italy, toxic events have been described since 2000s in mussel farms from Sicily and Sardinia, mainly caused by Alexandrium species (Dinophyceae) [ 9 ].…”