Determination Of Okadaic Acid Research Articles

Characterization of Diarrhetic Shellfish Toxins in the Mizuhopecten yessoensis (Scallop) Midgut Gland by High-Performance Liquid Chromatography – Tandem Mass Spectrometry (HPLC-MS/MS)

Liquid chromatography – tandem mass spectrometry (LC-MS/MS) is routinely applied for the determination of toxins associated with diarrhetic shellfish poisoning (DSP) as a part of the Japanese official method. For scallops, not only the edible part but also the midgut gland may be used for analysis. In this study, the matrix effects in the LC-MS/MS-based determination of okadaic acid (OA) and dinophysistoxin-1 (DTX1) in the scallop midgut gland were evaluated to assess the impact on the accuracy of the results. The analyzed sample in an interlaboratory comparison study was subjected to the Japanese official method. The solutions obtained with and without a cleanup process (combination of hexane washing and solid-phase extraction using a C18 cartridge) were diluted to varying ratios and used for evaluation. For all prepared solutions, the ionization of OA and DTX1 was enhanced compared to the samples in methanol; the cleanup process did not sufficiently reduce the matrix effects. Although the dilution of the sample solution reduced the matrix effect, the improvement was limited. Another pretreatment technique is necessary for the accurate determination of DSP-associated toxins in this matrix.

Determination of Okadaic Acid and Dinophysistoxins-1 in Mussel by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography-tandem mass Spectrometric method for the determination of diarrhetic shellfish poisoning including okadaic acid(OA) and dinophysistoxins-1(DTX-1) in mussel. After being extracted using methanol and water(80:20,V/V),The solution was defatted with n-hexane and extracted in chloroform. After being concentrated ,the residue was dissolved with n-hexane and acetone(20:80,V/V).Then the solution was cleaned-up by solid phase extraction(SPE) on an Si cartridge.The analytes were eluted with methanol - acetone(40:60,V/V). The quantitative and confirmatory determination of OA and DTX-1 were performed by MRM mode. OA and DTX-1 were determined in the negative ion mode. The calibration curves were linear. The method was accurate and reproducible, being successfully applied to the monitoring of OA and DTX-1.The recoveries of standard addition levels were 92.5%-96.7%. The relative standard deviations were all below 6.67%. The limit of quantitation (LOQ) for this method as defined by ten times signal to noise was 20μg/kg.

Determination of Okadaic Acid and Dinophysistoxin-1 in Mussels by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Food poisoning may occur due to ingestion of shellfish contaminated by okadaic acid or dinophysistoxin-1 from toxic dinoflagellates. A rapid and simple method for the determination of okadaic acid and dinophysistoxin-1 is needed to identify food poisoning sources. A high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) method is reported for this analysis in mussels. The separation was completed in six minutes using the selective ion monitoring mode. The limits of detection were 50 ng/mL for okadaic acid and dinophysistoxin-1, and the HPLC-MS results were in agreement with a standard bioassay. Because of its speed and sensitivity, the reported method provides a useful approach to identify sources of food poisoning.

Determination of okadaic acid in shellfish by using a novel chemiluminescent enzyme-linked immunosorbent assay method

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) was developed to determine okadaic acid (OA). Concentrations of the capture monoclonal anti-OA antibodies, conjugate of OA-HRP and a composition of blocking buffers were varied to optimize the assay condition. The values of IC10, IC50 and working range (IC20–IC80) for CL-ELISA were 0.01, 0.07, and 0.03–0.2ng/mL, respectively. Additionally, the analytical recovery values of CL-ELISA from 3 shellfish spiked samples with OA concentrations of 0.03, 0.1 and 0.2ng/mL ranged from 86.7% to 111.2%. Closely examining the OA concentrations in 19 various shellfish products performed by CL-ELISA revealed that OA concentrations in 6 of the 19 examined samples was undetected, whereas the 13 samples were contaminated with low levels of OA ranging from 1.2 to 8.0ng/g.

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H2O2/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.

Determination of okadaic acid, dinophysistoxin-1 and related esters in Greek mussels using HPLC with fluorometric detection, LC-MS/MS and mouse bioassay

An approach involving both chemical and biological methods was undertaken for the detection and quantification of the marine toxins okadaic acid (OA), dinophysistoxin-1 (DTX-1) and their respective esters in mussels from different sampling sites in Greece during the period 2006–2007. Samples were analyzed by means of a) high performance liquid chromatography with fluorometric detection (HPLC-FLD), using 9-athryldiazomethane (ADAM), as a pre-column derivatization reagent, b) liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and c) the mouse bioassay. Free OA and DTX-1 were determined by both HPLC-FLD and LC-MS/MS, while their respective esters were determined only by LC-MS/MS after alkaline hydrolysis of the samples. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the HPLC-FLD method were 0.015 μg/g HP and 0.050 μg/g HP, respectively, for OA. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the LC-MS/MS method were 0.045 μg/g HP and 0.135 μg/g HP, respectively, for OA. Comparison of results between the two analytical methods showed excellent agreement (100%), while both HPLC-FLD and LC-MS/MS methods showed an agreement of 97.1% compared to the mouse bioassay.

Production of monoclonal antibody and development of an enzyme-linked immunosorbent assay for the determination of okadaic acid in shellfish from China

cell-free system mimicking the phagolysosomes of ceUs as well as in human lung epithelial type II A549 cells. Levels of BAI in the US Geological Survey (USGS) database of coal quality were calculated using a model taking into account chemical interactions of pyrite, sulfuric acid, calcite, and total iron. Results: The CWP prevalence in the three c0almine regions ofPA, WV, and UT correlated positively with the content of BAI released in the cell-free system (correlation coefficient r=0.94). The UT coals had very little BAI due to the presence of calcite (CaCO3). Two coal samples with different BAI contents were then selected for cell treatment. The coal from the PA coalmine with a high BAI induced activator protein-I, a transcription factor responsive to oxidative stress. In contrast, the coai from the UT coalmine without BAI had no effect. Intedeukin-6 (IL-6), a pro-inflammatory cytokine, was significantly increased and persisted in cells treated with the PA coal. The IL-6 level in cells treated with UT coal was increased but reverted to the control level shortly. Using the linear fit of CWP prevalence and the calculated BAI in the USGS coal database, we have derived and mapped seven thousands coal samples' pneumoconiotic potencies. Conclusions: Our results suggest that a sustainable level of oxidative stress induced by BAI in the PA and WV coals may be responsible for cellular damage, thus leading to the observed high prevalence of lung disease in the PA and WV coal miners. Levels of BAI in the coals may be used for the prediction of coal's toxicity, even before large-scale mining.

Determination of Okadaic Acid and Related Toxins in Greek Mussels by HPLC with Fluorimetric Detection

The investigation for the presence of diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), and dinophysistoxin‐1 (DTX‐1), in mussels (Mytilus galloprovincialis) harvested in Thermaikos Gulf (northern Aegean Sea) is reported. The digestive glands of mussels were homogenized with methanol–water (80:20 v/v), and OA was extracted from the homogenate with hexane–chloroform (50:50 v/v). OA was reacted with 9‐anthryldiazomethane (ADAM) in methanol for 1 hr at 35°C. The product was purified by passing through a silica solid phase extraction cartridge before being determined by reversed‐phase liquid chromatography with fluorimetric detection (HPLC/FLD). Linear calibration curves (r = 0.9993), in the typical analytical range of 1–10 ng OA‐injected and detection limit of 0.14 ng OA‐injected (S/N 3:1) were obtained. Recovery of the toxin from mussels ranged from 100.4% to 110.4%. Analysis of samples during a DSP episode in 2002 indicated the presence of OA at levels above the regulatory limit, while DTX‐1 was detected in only one sample.

Immunosensor for the Determination of Okadaic Acid Based on Screen-Printed Electrode

A disposable immunosensor for okadaic acid (OA), using a screen-printed electrode (SPE), was developed and characterised. Detection of the product, p-aminophenol, resulting from the reaction catalysed by alkaline phosphatase (AP), was carried out using an amperometric three-electrode system poised at a voltage of + 300 mV versus Ag/AgCl. Alkaline phosphatase was used as a label for the antigen, OA, and two kinds of alkaline phosphatase preparation were studied for the conjugation of okadaic acid. The calibration curve for okadaic acid obtained from the conjugate created from low-activity AP, 969 units/mg, was unsatisfactory in terms of sensitivity, but a high-activity conjugate delivered the required sensitivity and limit of detection. Studies on the stability of the sensor with α-OA antibody and OA-AP conjugate showed that the current response decreased drastically after one day. Stabilisation strategies have been formulated to overcome this problem. The calibration curve obtained with the high activity conjugate was linear up to 40 ng/ml of okadaic acid with a minimum concentration of analyte detected of 5 ng/ml and a detection limit of 2 ng/ml.

Evaluation of the fluorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels

The protein phosphatase inhibition assay for okadaic acid, the major DSP toxin, modified to use the fluorescence substrates methylumbelliferyl phosphate (MUP) and fluorescein diphosphate (FDP), was compared to the assay using p-nitrophenylphosphate ( p-NPP) and the bioluminescence assay using luciferin phosphate (L–P). Under the standard assay conditions used okadaic acid inhibited the enzyme activity dose-dependently with IC 50 values of 1.5 nM (MUP) and 1.2 nM (FDP). This compares to IC 50 values of 0.9 and 6 nM using L–P and p-NPP respectively. CDP- star, a chemiluminescence substrate, was not hydrolysed by the enzyme. Decreasing the enzyme concentration lowered the IC 50 for the colorimetric method (IC 50=2 nM [ p-NPP], 0.75 nM enzyme) but no shift was observed with fluorimetry. However at enzyme concentrations <1.5 nM (standard assay) the error margin was too great for routine analysis. The method using fluorimetry allowed detection of okadaic acid concentrations to levels ≤1 μg/100 g of mussel tissue which is well below the limit of 20 μg/100 g (mouse bioassay) set by some regulatory agencies. Determination of the toxin content in naturally contaminated mussels in three separate experiments gave coefficients of variance ranging from 16 to 29% (MUP) and from 8 to 78% ( p-NPP). Multicomparison studies showed that concentrations of okadaic acid in naturally contaminated mussel samples determined by fluorescence generally agreed with those obtained using ELISA and LC–MS procedures, and with the mouse bioassay. However using the mouse bioassay as the standard, values determined by the ELISA, PP-2A and LC–MS all scored false negative results compared to those for the mouse bioassay in the range 20–40 μg/100 g mussel, and at the limit of the mouse bioassay the values by the other three methods were substantially less. With few exceptions the methods scored okadaic acid with highest to lowest values in the following order: mouse bioassay>ELISA>PP-2A>LC–MS. The fluorimetric assay was both more sensitive and accurate than the colorimetric assay (the latter showed a propensity towards false positives in the region 20 μg/100 g), and the moderate increase in equipment cost appears to be outweighed by the performance of the method.

Determination of okadaic acid by micellar electrokinetic chromatography with ultraviolet detection

Micellar electrokinetic chromatography (MEKC) with ultraviolet (UV) detection was applied for the determination of non-derivatized phycotoxins associated with diarrhoetic shellfish poisoning. A detection limit for 40 pg of okadaic acid (OA) was achieved. The UV intensities of this toxin measured at 200 nm showed good linearity in the range 40–640 pg. OA was detected in mussels spiked with 10 ng/g whole tissue. The presence of OA and dinophysistoxin-2 was observed in the crude extract of the dinoflagellate Prorocentrum lima.