210
R. Raine et al.
results. However, the non-hydrolysed samples appeared to underestimate levels. All
hydrolysed samples analysed by ELISA during the closure period produced positive
results; no ‘false positives’ were found in hydrolysed samples determined by the
ELISA. However, most non-hydrolysed samples gave results below the MPL during
the closure period. Nevertheless, all positive results (i.e. >EU MPL of 0.16 g OA
eq g
1 ) determined by LC-MS (and the MBA) were also positive using the ELISA
method when the hydrolysis step was employed.
Figure 18.3d–f shows a comparison of DSP toxin levels in mussels flesh collected
during summer 2010 when analysed by DSP ELISA (ABRAXIS), OKATEST and
LC-MS methods. All the data sets show a similar pattern, with the notable exception
of samples analysed by the DSP ELISA kit after the hydrolysis step. During the
sampling period, only one sample from the middle sample site (28 June) gave a
positive result by LC-MS (i.e. >MPL) whereas six positive results by MBA were
recorded. During the 2010 sampling period, 35 samples analysed by LC-MS were
below the limit of detection and/or quantification. However, both the immunoassay
and enzymatic assay were able to detect DSP toxins at levels below the LC-MS
LOD. Non-hydrolysed samples analysed by the ELISA method did not detect any
positive DSP (>MPL) samples. DSP levels recorded by the enzymatic assay were
more similar to the LC-MS data. Although no positive samples were detected
during the closure period, high levels of DSP were detected by the OKATEST kit
which were higher than those detected by the ELISA non-hydrolysed, and on two
occasions slightly higher than the samples analysed by LC-MS. Hydrolysed samples
analysed by ELISA gave significant overestimations of DSP levels in all samples.
This was most likely caused by matrix effects resulting from the hydrolysis. These
matrix effects were evident in samples with high and low concentrations of toxins,
with 23 false positives found.
Table 18.1 shows a comparison of DSP toxin data from samples taken in 2009,
stored and re-analysed using both the Protein Phosphatase (PP2A, Okatest) enzyme
assay and LC-MS methods on non-hydrolysed and hydrolysed extracts from mussel
flesh. Data from 2010 is also included. Good agreement is seen between the two
methods in the 19 samples that were re-analysed. All but two extracts were in
agreement and on both occasions the two errant results were borderline. On a sample
originally taken on 20 July at the middle site, non-hydrolysed extract analysed
by LC-MS gave a negative toxicity result, but when the hydrolysed sample was
analysed a positive result was obtained, agreeing with the original MBA analysis,
and also with the PP2A re-analysis on both hydrolysed and non-hydrolysed extracts.
Discussion
The DSP toxin group consists of the lipophilic toxin okadaic acid and its analogues
dinophysistoxin-1 and -2 (DTX-1, DTX-2) and dinophysistoxin-3, a complex
mixture of 7-O-acyl ester derivatives of OA, DTX-1,-2 (Suzuki and Quilliam 2011).
Until 2011, detection of DSP toxins in shellfish was carried out by the MBA, as the
R. Raine et al.
results. However, the non-hydrolysed samples appeared to underestimate levels. All
hydrolysed samples analysed by ELISA during the closure period produced positive
results; no ‘false positives’ were found in hydrolysed samples determined by the
ELISA. However, most non-hydrolysed samples gave results below the MPL during
the closure period. Nevertheless, all positive results (i.e. >EU MPL of 0.16 g OA
eq g
1 ) determined by LC-MS (and the MBA) were also positive using the ELISA
method when the hydrolysis step was employed.
Figure 18.3d–f shows a comparison of DSP toxin levels in mussels flesh collected
during summer 2010 when analysed by DSP ELISA (ABRAXIS), OKATEST and
LC-MS methods. All the data sets show a similar pattern, with the notable exception
of samples analysed by the DSP ELISA kit after the hydrolysis step. During the
sampling period, only one sample from the middle sample site (28 June) gave a
positive result by LC-MS (i.e. >MPL) whereas six positive results by MBA were
recorded. During the 2010 sampling period, 35 samples analysed by LC-MS were
below the limit of detection and/or quantification. However, both the immunoassay
and enzymatic assay were able to detect DSP toxins at levels below the LC-MS
LOD. Non-hydrolysed samples analysed by the ELISA method did not detect any
positive DSP (>MPL) samples. DSP levels recorded by the enzymatic assay were
more similar to the LC-MS data. Although no positive samples were detected
during the closure period, high levels of DSP were detected by the OKATEST kit
which were higher than those detected by the ELISA non-hydrolysed, and on two
occasions slightly higher than the samples analysed by LC-MS. Hydrolysed samples
analysed by ELISA gave significant overestimations of DSP levels in all samples.
This was most likely caused by matrix effects resulting from the hydrolysis. These
matrix effects were evident in samples with high and low concentrations of toxins,
with 23 false positives found.
Table 18.1 shows a comparison of DSP toxin data from samples taken in 2009,
stored and re-analysed using both the Protein Phosphatase (PP2A, Okatest) enzyme
assay and LC-MS methods on non-hydrolysed and hydrolysed extracts from mussel
flesh. Data from 2010 is also included. Good agreement is seen between the two
methods in the 19 samples that were re-analysed. All but two extracts were in
agreement and on both occasions the two errant results were borderline. On a sample
originally taken on 20 July at the middle site, non-hydrolysed extract analysed
by LC-MS gave a negative toxicity result, but when the hydrolysed sample was
analysed a positive result was obtained, agreeing with the original MBA analysis,
and also with the PP2A re-analysis on both hydrolysed and non-hydrolysed extracts.
Discussion
The DSP toxin group consists of the lipophilic toxin okadaic acid and its analogues
dinophysistoxin-1 and -2 (DTX-1, DTX-2) and dinophysistoxin-3, a complex
mixture of 7-O-acyl ester derivatives of OA, DTX-1,-2 (Suzuki and Quilliam 2011).
Until 2011, detection of DSP toxins in shellfish was carried out by the MBA, as the