The analysis of lipophilic marine toxins : development of an alternative method

Research output: Thesisinternal PhD, WUAcademic

Abstract

Lipophilic marine toxins are produced by certain algae species and can accumulate in filter feeding shellfish such as mussels, scallops and oysters. Consumption of contaminated shellfish can lead to severe intoxications such as diarrhea, abdominal cramps and vomiting. Methods described in European Union (EU) legislation to test for the presence of these toxins are based on a mouse or rat bioassay. These assays are unethical and have a poor sensitivity and selectivity. The aim of this thesis is to develop an alternative method based on liquid chromatography - tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of lipophilic marine toxins.
LC-MS/MS methods described in literature for the determination of lipophilic marine toxins used an acidic chromatographic system. Under acidic conditions peak shape and separation of a number of toxins preferably analyzed in electrospray ionization negative (ESI–) and positive (ESI+) were poor. A LC-MS/MS method with alkaline chromatographic conditions in which we were able to analyze 28 different toxins in a single analysis in separated retention time windows operating in either ESI– or ESI+ was developed. Furthermore, a clean up procedure based on solid phase extraction (SPE) was developed to reduce the amount of matrix effects (ion suppression and enhancement). A combination of SPE clean up and alkaline chromatographic conditions resulted in reduced matrix effects for all matrices tested (mussel, scallop and oyster).
The developed SPE & LC-MS/MS method was in-house validated at regulatory limits based on EU Commission Decision 2002/657/EC. With respect to accuracy, repeatability, reproducibility, decision limit, specificity and ruggedness the method performed well. The method also performed excellently in view of possible new limits that are four- to five-fold lower than current limits for some toxins.
Finally a screening method based on LC orbitrap MS was developed for 85 marine toxins of which most are not stated in EU legislation. The screening used in-house developed software which made it possible to reduce the complex data files and screen for a large number of toxins within seconds.
This thesis will contribute to the replacement of the animal assays that are still prescribed in EU legislation for the determination of lipophilic marine toxins in shellfish.

LanguageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Rietjens, Ivonne, Promotor
  • de Boer, J., Promotor, External person
  • Mulder, Patrick, Co-promotor
Award date15 Oct 2010
Place of Publication[S.l.
Publisher
Print ISBNs9789085857198
Publication statusPublished - 2010

Fingerprint

Marine Toxins
Shellfish
Electrospray ionization
Assays
Screening
Bioassay
Liquid chromatography
Algae
Mass spectrometry
Rats
Animals
Ions
European Union
Chemical analysis

Keywords

  • toxins
  • marine environment
  • algae
  • animal testing alternatives
  • liquid chromatography-mass spectrometry

Cite this

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title = "The analysis of lipophilic marine toxins : development of an alternative method",
abstract = "Lipophilic marine toxins are produced by certain algae species and can accumulate in filter feeding shellfish such as mussels, scallops and oysters. Consumption of contaminated shellfish can lead to severe intoxications such as diarrhea, abdominal cramps and vomiting. Methods described in European Union (EU) legislation to test for the presence of these toxins are based on a mouse or rat bioassay. These assays are unethical and have a poor sensitivity and selectivity. The aim of this thesis is to develop an alternative method based on liquid chromatography - tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of lipophilic marine toxins. LC-MS/MS methods described in literature for the determination of lipophilic marine toxins used an acidic chromatographic system. Under acidic conditions peak shape and separation of a number of toxins preferably analyzed in electrospray ionization negative (ESI–) and positive (ESI+) were poor. A LC-MS/MS method with alkaline chromatographic conditions in which we were able to analyze 28 different toxins in a single analysis in separated retention time windows operating in either ESI– or ESI+ was developed. Furthermore, a clean up procedure based on solid phase extraction (SPE) was developed to reduce the amount of matrix effects (ion suppression and enhancement). A combination of SPE clean up and alkaline chromatographic conditions resulted in reduced matrix effects for all matrices tested (mussel, scallop and oyster). The developed SPE & LC-MS/MS method was in-house validated at regulatory limits based on EU Commission Decision 2002/657/EC. With respect to accuracy, repeatability, reproducibility, decision limit, specificity and ruggedness the method performed well. The method also performed excellently in view of possible new limits that are four- to five-fold lower than current limits for some toxins. Finally a screening method based on LC orbitrap MS was developed for 85 marine toxins of which most are not stated in EU legislation. The screening used in-house developed software which made it possible to reduce the complex data files and screen for a large number of toxins within seconds. This thesis will contribute to the replacement of the animal assays that are still prescribed in EU legislation for the determination of lipophilic marine toxins in shellfish.",
keywords = "toxinen, marien milieu, algen, alternatieven voor dierproeven, lc-ms, toxins, marine environment, algae, animal testing alternatives, liquid chromatography-mass spectrometry",
author = "A. Gerssen",
note = "WU thesis 4904",
year = "2010",
language = "English",
isbn = "9789085857198",
publisher = "S.n.",
school = "Wageningen University",

}

Gerssen, A 2010, 'The analysis of lipophilic marine toxins : development of an alternative method', Doctor of Philosophy, Wageningen University, [S.l..

The analysis of lipophilic marine toxins : development of an alternative method. / Gerssen, A.

[S.l. : S.n., 2010. 199 p.

Research output: Thesisinternal PhD, WUAcademic

TY - THES

T1 - The analysis of lipophilic marine toxins : development of an alternative method

AU - Gerssen, A.

N1 - WU thesis 4904

PY - 2010

Y1 - 2010

N2 - Lipophilic marine toxins are produced by certain algae species and can accumulate in filter feeding shellfish such as mussels, scallops and oysters. Consumption of contaminated shellfish can lead to severe intoxications such as diarrhea, abdominal cramps and vomiting. Methods described in European Union (EU) legislation to test for the presence of these toxins are based on a mouse or rat bioassay. These assays are unethical and have a poor sensitivity and selectivity. The aim of this thesis is to develop an alternative method based on liquid chromatography - tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of lipophilic marine toxins. LC-MS/MS methods described in literature for the determination of lipophilic marine toxins used an acidic chromatographic system. Under acidic conditions peak shape and separation of a number of toxins preferably analyzed in electrospray ionization negative (ESI–) and positive (ESI+) were poor. A LC-MS/MS method with alkaline chromatographic conditions in which we were able to analyze 28 different toxins in a single analysis in separated retention time windows operating in either ESI– or ESI+ was developed. Furthermore, a clean up procedure based on solid phase extraction (SPE) was developed to reduce the amount of matrix effects (ion suppression and enhancement). A combination of SPE clean up and alkaline chromatographic conditions resulted in reduced matrix effects for all matrices tested (mussel, scallop and oyster). The developed SPE & LC-MS/MS method was in-house validated at regulatory limits based on EU Commission Decision 2002/657/EC. With respect to accuracy, repeatability, reproducibility, decision limit, specificity and ruggedness the method performed well. The method also performed excellently in view of possible new limits that are four- to five-fold lower than current limits for some toxins. Finally a screening method based on LC orbitrap MS was developed for 85 marine toxins of which most are not stated in EU legislation. The screening used in-house developed software which made it possible to reduce the complex data files and screen for a large number of toxins within seconds. This thesis will contribute to the replacement of the animal assays that are still prescribed in EU legislation for the determination of lipophilic marine toxins in shellfish.

AB - Lipophilic marine toxins are produced by certain algae species and can accumulate in filter feeding shellfish such as mussels, scallops and oysters. Consumption of contaminated shellfish can lead to severe intoxications such as diarrhea, abdominal cramps and vomiting. Methods described in European Union (EU) legislation to test for the presence of these toxins are based on a mouse or rat bioassay. These assays are unethical and have a poor sensitivity and selectivity. The aim of this thesis is to develop an alternative method based on liquid chromatography - tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of lipophilic marine toxins. LC-MS/MS methods described in literature for the determination of lipophilic marine toxins used an acidic chromatographic system. Under acidic conditions peak shape and separation of a number of toxins preferably analyzed in electrospray ionization negative (ESI–) and positive (ESI+) were poor. A LC-MS/MS method with alkaline chromatographic conditions in which we were able to analyze 28 different toxins in a single analysis in separated retention time windows operating in either ESI– or ESI+ was developed. Furthermore, a clean up procedure based on solid phase extraction (SPE) was developed to reduce the amount of matrix effects (ion suppression and enhancement). A combination of SPE clean up and alkaline chromatographic conditions resulted in reduced matrix effects for all matrices tested (mussel, scallop and oyster). The developed SPE & LC-MS/MS method was in-house validated at regulatory limits based on EU Commission Decision 2002/657/EC. With respect to accuracy, repeatability, reproducibility, decision limit, specificity and ruggedness the method performed well. The method also performed excellently in view of possible new limits that are four- to five-fold lower than current limits for some toxins. Finally a screening method based on LC orbitrap MS was developed for 85 marine toxins of which most are not stated in EU legislation. The screening used in-house developed software which made it possible to reduce the complex data files and screen for a large number of toxins within seconds. This thesis will contribute to the replacement of the animal assays that are still prescribed in EU legislation for the determination of lipophilic marine toxins in shellfish.

KW - toxinen

KW - marien milieu

KW - algen

KW - alternatieven voor dierproeven

KW - lc-ms

KW - toxins

KW - marine environment

KW - algae

KW - animal testing alternatives

KW - liquid chromatography-mass spectrometry

M3 - internal PhD, WU

SN - 9789085857198

PB - S.n.

CY - [S.l.

ER -