Protein phosphatase inhibition assay adapted for determination of total DSP in contaminated mussels

Mountfort, Douglas O.; Suzuki, Toshiyuki; Truman, Penelope

doi:10.1016/s0041-0101(00)00144-6

Cited by 101 publications

(47 citation statements)

References 14 publications

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“…The alkaline hydrolysis of the samples was performed according to the EURLMB SOP [11] based on the protocol initially developed by Mountfort et al [26]. 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 Toxins were separated on a Waters X-Bridge TM C8 (guard column 2.1 x 10 mm, 3.5 μm particle size, column 2.1 x 50 mm, 3.5 μm particle size; Waters, Milford, MA, USA) in an Agilent 1200 LC system (Agilent Technologies, Santa Clara, CA) consisting of a binary pump (G1312B), four channel degasser (G1379B), thermostated low carry-over autosampler (G1367C + G1330B), and column oven (G1316B • Mobile phases in alkaline conditions (pH 11) according to Gerssen et al [12,13]: Mobile phase A consisted of 6.7 mM of ammonia in ultrapure Milli-Q water.…”

Section: Alkaline Hydrolysismentioning

confidence: 99%

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions

García‐Altares

Diogène

Iglesia

2013

Journal of Chromatography A

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We performed a comprehensive study to assess the fit for purpose of four chromatographic conditions for the determination of six groups of marine lipophilic toxins (okadaic acid and dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins, gymnodimine and spirolides) by LC-MS/MS to select the most suitable conditions as stated by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). For every case, the elution gradient has been optimized to achieve a total run-time cycle of 12 min. We performed a single-laboratory validation for the analysis of three relevant matrices for the seafood aquaculture industry (mussels, pacific oysters and clams), and for sea urchins for which no data about lipophilic toxins have been reported before. Moreover, we have compared the method performance under alkaline conditions using two quantification strategies: the external standard calibration (EXS) and the matrixmatched standard calibration (MMS). Alkaline conditions were the only scenario that allowed detection windows with polarity switching in a 3200 QTrap mass spectrometer, thus the analysis of all toxins can be accomplished in a single run, increasing sample throughput. The limits of quantification under alkaline conditions met the validation requirements established by the EURLMB for all toxins and matrices, while the remaining conditions failed in some cases. The accuracy of the method and the matrix effects where generally dependent on the mobile phases and the seafood species. The MMS had a moderate positive impact on method accuracy for crude extracts, but it showed poor trueness for seafood species other than mussels when analyzing hydrolyzed extracts. Alkaline conditions with EXS and recovery correction for OA were selected as the most proper conditions in the context of our laboratory. This comparative study can help other laboratories to choose the best conditions for the implementation of LC-MS/MS according to their own necessities.

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Section: Alkaline Hydrolysismentioning

confidence: 99%

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions

García‐Altares

Diogène

Iglesia

2013

Journal of Chromatography A

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“…1b) with a peak observed during the same period in January 2006. Generally a good correlation was obtained between OA determined by the PP2A method and the HPLC-ADAM method (Mountfort et al 2001). However, in this study the concentrations of OA for some mussel samples were underestimated by the HPLC-ADAM method compared to the PP2A assay as indicated in Fig.…”

Section: Resultsmentioning

confidence: 45%

First Evidence of Okadaic Acid in Mytilus galloprovincialis Mussels, Collected in a Mediterranean Lagoon, Tunisia

Kacem

Hajjem

Bouaı̈cha³

2009

Bull Environ Contam Toxicol

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Evidence of the presence of okadaic acid (OA) and its monthly fluctuations are reported for the first time in Mytilus galloprovincialis samples collected from June 2005 to May 2006 in the Bizerte Lagoon. All of the samples with the exception of those taken in August 2005 were found to be contaminated. The level of OA in mussels exceeded the regulatory limit of DSP toxins (16 microg OA/100 g mussel meat) within the European Union (2002) in only two cases: in January and February, 2006 with 31.85 +/- 3.06, and 18.86 +/- 1.88 microg OA/100 g mussel meat respectively. During the investigative period, potentially toxic dinoflagellates were observed. The results reported here, although based on limited sampling, demonstrate for the first time the existence of a risk to public health from diarrheic shellfish poisoning in cultivated bivalve mollusc in Tunisia.

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“…To hydrolyze esters of the OA group of toxins, 1 mL aliquots of CRM-AZA-Mus extract (prepared with method D) were placed in an HPLC vial, 125 μL of 2.5 N NaOH was added, and the mixture was placed in a water bath at 76°C for 40 min [34]. The samples were cooled and neutralized with 125 μL of 2.5 N HCl before filtration prior to LC-MS/MS analysis for OA, DTX1, and DTX2.…”

Section: Ester Hydrolysismentioning

confidence: 99%

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids

et al. 2014

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Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.

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Protein phosphatase inhibition assay adapted for determination of total DSP in contaminated mussels

Cited by 101 publications

References 14 publications

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions

First Evidence of Okadaic Acid in Mytilus galloprovincialis Mussels, Collected in a Mediterranean Lagoon, Tunisia

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids

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