Bengtström, Linda

Papir og pap anvendes til en række forskellige materialer i kontakt med fødevarer, såsom bagepapir, mikrobølge-popcorns poser og emballager til morgenmadsprodukter og fastfood. Trods den omfattende anvendelse, mangler der viden både om den kemiske sammensætning af forskellige fødevarekontaktmaterialer af pap og papir og de toksikologiske effekter af disse forbindelser.Formålet med dette PhD-studie var at udvikle en rationel interdisciplinær strategi til screening og identifikation af kemiske stoffer i materialer af pap og papir med potentielt sundhedsskadelige effekter. Det første skridt i vores foreslåede strategi er at udvikle en omfattende ekstraktionsteknik, der er kompatibel med både analytisk kemiske og toksikologiske analyser. Til dette formål har vi udviklet en aktiv prøveudtagningsmetode til opsamling af små flygtige organiske forbindelser. Semi- og ikkeflygtige forbindelser blev ekstraheret i et reflux system med kogende ethanol.Efter en indledende in vitro screenings-runde af 20 forskellige pap- og papirprøver blev fem prøver med positive virkninger udvalgt til fraktionering. Sure og basiske HPLC-fraktioner blev derefter testet i cellebaserede assays i en anden screeningsrunde. Fraktioneringen blev anvendt til at reducere antallet af forbindelser, der skal identificeres, samt at reducere eventuel matrix effekt i den massespektrometriske detektion.Derefter blev fraktionerne analyseret ved gaskromatografi og væskekromatografi koblet til højtopløseligt massespektrometri. Disse to teknikker blev designet til at være så komplementære som muligt. Ved også at inkludere væskekromatografi i stedet for udelukkende at anvende gaskromatografi, er muligheden for at identificere forbindelser med potentielt sundhedsskadelige effekter styrket. Flere trin i den tentative gaskromatografiske identifikation kan automatiseres og på grund af standardiseringen muliggøres søgninger i store massespektrometriske biblioteker. Disse biblioteker mangler i væskekromatografi, og en stor del af den tentative identifikation skal derfor udføres manuelt. For at lette den tentative identifikation ved væskekromatografi opbyggede vi en database indeholdende præcise masser for ca. 2.100 kemiske forbindelser rapporteret at være forekommende i pap og papir. Resultater fra undersøgelsen viser, at både isotopforholdet og hits for de nøjagtige masser i databasen i høj grad forbedrer muligheden for en tentativ identifikation.Ud fra en liste af tentativt identificerede forbindelser udarbejdet til de specifikke assays blev udvalgt stoffer til yderligere verifikation af identitet. Denne udvælgelse var baseret på tidligere rapporterede effekter, strukturelle ligheder med kendte ligander, og tilgængeligheden af standarder for identificerede forbindelser, Da enhver positiv identifikation fra databaser bør betragtes som tentativ, blev kromatografisk og massespektrometrisk sammenligning med analytiske standarder anvendt til at verificere identifikationen.Ækvivalensfaktorer for de oprindeligt observerede toksikologiske effekter og fra summen af alle de verificerede forbindelser fra samme assay blev beregnet. Ud fra disse resultater, konkluderede vi at de oprindeligt observerede effekter på metabolismen af miljøfremmede stoffer kun i mindre grad kunne henføres til tre farvestoffer. De hormonforstyrrende effekter kunne i vid udstrækning kunne forklares ved tilstedeværelse af henholdsvis blødgørere i genbrugsfibre og ved lim anvendt i nye fibre.Paper and board are used for a variety of food contact materials, such as baking paper, microwave popcorn bags and packaging for cereals as well as fast foods. Despite this extensive use, there are currently large data gaps about the chemical composition of different paper and board food contact materials and the toxicological effects of these compounds.The aim of this study was to develop a rationalised interdisciplinary strategy for the screening and identification of compounds with potential adverse health effects in paper and board materials. The first step in the proposed strategy was to develop a comprehensive extraction process that is compatible with both chemical and toxicological analyses. For this purpose, a purge-and-trap method was developed for the collection of small volatile organic compounds; in addition semi- and nonvolatile compounds were extracted by a boiling ethanol reflux system.After an initial in vitro screening of 20 different paper and board samples for endocrine disruptive effects, mutagenicity and effects on metabolism of foreign compounds, five samples with adverse effects were selected for fractionation. The fractions were tested in cell assays in a second screening. The fractionation was used to reduce the number of compounds to be identified as well as the matrix effect.Next, the fractions were analysed by gas chromatography and liquid chromatography coupled to high resolution mass spectrometry. These two techniques were designed to be as complimentary as possible and by them in combination increased the possibility to identify compounds with potential adverse health effects. Several steps in the tentative identification by gas chromatography can be automated, due to the standardisation of this technique that enables searches in vast mass spectral libraries. Such libraries are missing for liquid chromatography, and a large part of the tentative identification must be performed manually. To facilitate the tentative identification by liquid chromatography, an accurate mass database containing approximately 2100 entries of compounds with reported use in paper and board was built. The results from this study indicate that both isotope ratio and hits in the accurate mass database greatly increases the possibility of a correct tentative identification.After lists of tentatively identified compound had been produced for a certain toxicological assay, compound were selected for further testing based on previously reported effects, structural similarities to known ligands, and availability of analytical standards for identified compounds. Any positive annotation through databases should be regarded as tentative, and therefore analytical standards were used to confirm the identification.After confirmation, equivalence factors for the initially observed toxicological effect and from all the confirmed compounds tested in the same toxicological assay were calculated. The initially observed effects on the metabolism of xenobiotics could to a minor extent, though not fully, be attributed to dyes used in printing inks. In addition, it was concluded that the endocrine disruptive effects could largely be explained by monomers and plasticisers present in a recycled fibre sample and by sizing agents in virgin fibres.

Online Research Database In Technology

Chemical identification of contaminants in paper and board food contact materials

http://orbit.dtu.dk/ws/files/121618742/Thesis_Linda_Bengtstr_m_1.pdf

Chemical identification of contaminants in paper and board food contact materials

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Online Research Database In Technology