The mouse bioassay and high performance liquid chromatography (HPLC) post-column oxidation method are different methods of quantifying paralytic shellfish poisoning toxins. In this study, we compared their ability to accurately quantify the toxicity levels in two types of field sample (oysters and m...
The mouse bioassay and high performance liquid chromatography (HPLC) post-column oxidation method are different methods of quantifying paralytic shellfish poisoning toxins. In this study, we compared their ability to accurately quantify the toxicity levels in two types of field sample (oysters and mussels) with different toxin profiles for routine regulatory monitoring. A total of 72 samples were analyzed by both methods, 44 of which gave negative results, with readings under the limit of detection of the mouse bioassay ($40{\mu}g/100g$ saxitoxin [STX] eq). In 14 oysters, the major toxin components were gonyautoxin (GTX) 1, -2, -3, -4, -5, decarbamoylgonyautoxin-2 (dcGTX2), and decarbamoylsaxitoxin (dcSTX), while 14 mussels tested positive for dcSTX, GTX2, -3, -4, -5, dcGTX2, neosaxitoxin (NEO), STX, and dcSTX. When the results obtained by both methods were compared in two matrices, a better correlation ($r^2=0.9478$) was obtained for mussels than for oysters ($r^2=0.8244$). Additional studies are therefore needed in oysters to investigate the differences in the results obtained by both methods. Importantly, some samples with toxin levels around the legal limit gave inconsistent results using HPLC-based techniques, which could have a strong economic impact due to enforced harvest area closure. It should therefore be determined if all paralytic shellfish poisoning toxins can be quantified accurately by HPLC, and if the uncertainties of the method lead to doubts regarding regulatory limits.
The mouse bioassay and high performance liquid chromatography (HPLC) post-column oxidation method are different methods of quantifying paralytic shellfish poisoning toxins. In this study, we compared their ability to accurately quantify the toxicity levels in two types of field sample (oysters and mussels) with different toxin profiles for routine regulatory monitoring. A total of 72 samples were analyzed by both methods, 44 of which gave negative results, with readings under the limit of detection of the mouse bioassay ($40{\mu}g/100g$ saxitoxin [STX] eq). In 14 oysters, the major toxin components were gonyautoxin (GTX) 1, -2, -3, -4, -5, decarbamoylgonyautoxin-2 (dcGTX2), and decarbamoylsaxitoxin (dcSTX), while 14 mussels tested positive for dcSTX, GTX2, -3, -4, -5, dcGTX2, neosaxitoxin (NEO), STX, and dcSTX. When the results obtained by both methods were compared in two matrices, a better correlation ($r^2=0.9478$) was obtained for mussels than for oysters ($r^2=0.8244$). Additional studies are therefore needed in oysters to investigate the differences in the results obtained by both methods. Importantly, some samples with toxin levels around the legal limit gave inconsistent results using HPLC-based techniques, which could have a strong economic impact due to enforced harvest area closure. It should therefore be determined if all paralytic shellfish poisoning toxins can be quantified accurately by HPLC, and if the uncertainties of the method lead to doubts regarding regulatory limits.
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제안 방법
, 2013). The aim of this study was to compare the MBA and HPLC PCOX methods, and evaluate their ability to accurately assess the toxicity levels of two types of field sample with different toxin profiles for routine regulatory monitoring.
All individual stock solutions were prepared following the NRC instructions. Two working solutions were then prepared, the first containing dcGTX2, dcGTX3, dcSTX, GTX1, GTX2, GTX3, GTX4, GTX5, NEO, and STX, and the second containing C1 and C2.
The amount (μmoles of STXeq) of each toxin in the sample extracts was calculated using the linear regression of the calibration graph, and the specific relative toxicity of each individual PSP toxin was determined (Table 2).
대상 데이터
A Finnigan Surveyor Plus HPLC system, equipped with a Finnigan Surveyor FL Plus Detector (Thermo Electron, San Jose, CA, USA), was operated at an excitation wavelength of 330 nm and an emission wavelength of 390 nm. The Post-column Derivatization (Pickering Laboratories, Mountain View, USA) was capable of maintaining temperature at 85°C.
Table 3 lists the 28 samples that gave results above the MBA LOD, including the description of the shellfish species, the scientific name, results obtained by both methods, and the toxin composition. The major toxins in the oysters were GTX1, -2, -3, -4, -5, dcGTX2, and dcSTX, whereas GTX2, -3, -4, -5, dcGTX2, NEO, STX, and dcSTX were detected in mussels. C toxins were not detected in any of the samples.
이론/모형
10 sieve, and drained for 5 min. PSP toxins were extracted from 100-g samples of homogenized shellfish tissue following the AOAC MBA method 959.08 (Association of Official Analytical Chemists, 2005) using 0.1 M HCl. To deproteinate the samples for high performance liquid chromatography (HPLC) analysis, 25 μL 30% (w/v) trichloroacetic acid (TCA) were added to 500 μL shellfish extract in a microcentrifuge tube, which was then mixed in a vortex mixer and centrifuged at 16,000 g for 5 min.
Paralytic shellfish toxins toxin concentrations in mussel obtained by mouse bioassay and high performance liquid chromatography postcolumn oxidation (HPLC PCOX) methods (samples with results <200 μg/100 g saxitoxin [STX] eq by both method).
1. Paralytic shellfish poisoning toxin concentrations in oyster obtained by mouse bioassay and high performance liquid chromatography post-column oxidation (HPLC PCOX) methods. STX, saxitoxin; MBA, mouse bioassay.
3. Paralytic shellfish toxins concentrations in mussel obtained by mouse bioassay and high performance liquid chromatography postcolumn oxidation (HPLC PCOX) methods. STX, saxitoxin; MBA, mouse bioassay.
성능/효과
In 8 of 14 oysters (sample codes 2, 3, 4, 5, 6, 10, 11, and 13), a large positive or negative bias (relative standard deviations greater than 15%) was obtained using the MBA or HPLC PCOX (Fig. 1). A comparison of the results of both methods for oysters indicated a linear correlation of r2 = 0.
4). When the results obtained by both methods were compared in two matrices, a good correlation for mussels was obtained, but a relatively poor correlation for oysters. Turner et al.
(2010) previously analyzed some oyster samples using HPLC PCOX, an electrophysiological assay, and a hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. The results obtained using the other methods showed a good correlation, suggesting that the difference may be due to either an under- or overestimation by MBA and/or the HPLC method. Turner et al.
2) HPLC mobile phases for C toxins—solvent C, 2 mM tetrabutyl ammonium phosphate solution adjusted to pH 5.8 with 1% ammonium hydroxide; solvent D, 2 mM tetrabutyl ammonium phosphate solution adjusted to pH 5.8 with 4% acetonitrile.
후속연구
In cases, such as these, that are close to the regulatory limit, the uncertainty of both methods should therefore be considered before decisions allowing or banning the harvesting of shellfish are made. In addition, whether all relevant PSP toxins can be detected by the HPLC PCOX method, and whether the uncertainty of both methods would lead to doubts over the regulatory limits, should be assessed. Investigations of the occurrence of phytoplankton and long-term and short-term monitoring of each specific area would also be required, and precautionary steps should be taken if necessary.
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