Total oxidizable precursor (TOP) assay is an in-lab method which oxidatively converts precursor compounds of poly- and perfluoroalkyl substances (PFASs) into measurable perfluorinated alkyl acids (PFAAs). The method is a very strong tool to semi-quantify PFASs that would otherwise be missed in conventional targeted analysis using liquid chromatography - tandem mass spectrometry (LC-MS/MS). However, there are still challenges for it to be a fully quantitative tool. This report aims to increase the knowledge in using measures to improve the quality assurance of the assay, as well as investigate potential limitations of applying TOP assay outside the scope of what it was originally developed for. Furthermore, the objective was also to apply an evaluated TOP assay method on sample extracts from influent and effluent water from wastewater treatment plants (WWTPs) in China to investigate the occurrence of both legacy and novel PFASs. To achieve improved quality assurance of the method, the use of isotopically labelled standards in the TOP assay were investigated. 13C8-FOSA was used as a model compound to measure the oxidation efficiency in the assay; the results showed that the molar yield of 13C8-FOSA to 13C8-PFOA was 99% ± 2% (n=12) for the WWTP samples. This indicated it to be a good tool to monitor the oxidation, but more research is needed to understand the fate and reaction rate of other precursors. Oxidation of sample extracts were performed without evaporating solvent before oxidation to minimize loss of volatile precursors. This procedure was shown to be possible but require additional oxidation agent and base and may lead to different product pattern after oxidation. Further, performing TOP assay on sample extracts will introduce bias in which types of precursors are extracted and may lead to an underestimation of total PFAAs precursor present, depending on the extraction method or solvent type used. Additionally, the stability of two novel compound groups in the assay were investigated: 1) The Pre 2002 and Post 2002 formulas of Scotchgard™ fluorinated side-chain polymers were both degraded in the assay. For the Pre 2002 formula the primary degradation product was PFOA and for the Post 2002 formula, PFBA was the degradation product. Additionally, both the Pre 2002 and Post 2002 formulas were shown to undergo hydrolysis in high pH conditions, producing perfluoroalkyl sulfonic acids (PFSAs). 2) Per- and polyfluorinated ether acids (PFEAs) were also investigated in the TOP assay, which indicated that perfluorinated ether acids are stable in high pH oxidative conditions but may degrade in low pH oxidative conditions, while polyfluorinated ether acids may be degraded under oxidative conditions in both high and low pH. The analysis of influent and effluent water from Chinese WWTPs was done by screening for 14 legacy PFASs, 16 novel PFASs, and performing TOP assay on sample extracts as well as analysis of extractable organic fluorine for mass balance analysis. Perfluorobutanoic acids (PFBA) had the highest detection rate among the legacy PFASs, and hexafluoropropylene oxide dimer acid (HFPO-DA) was the most prevalent novel PFASs detected. TOP assay on the anionic fraction lead to varying degree increase in PFAS concentration (0-15x) while performing the oxidation on the neutral/cationic fraction did not lead to any observable increase in PFASs. Even with TOP assay, the proportion of extractable organic fluorine still unidentified was 11% to 98%.

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Using ion pair chromatography, separation and analysis of shorter mRNA fragments was possible. The aim of the study was to develop a separation method for quantitation of 5’capped versus uncapped mRNA. Cleavage of mRNA was performed by complementary probes and the enzyme RNase H which created shorter mRNA fragments containing the 5’ end. During this study, several separation methods have been tested where the amount organic solvent proved to be a crucial factor for elution of the fragments. To identify the peaks during the method development, LC-MS had to be used which resulted in an adjustment of the methods parameters. With the mass spectrometric analyses could a capped fragment as well as a capped fragment containing an extra guanine nucleotide be identified, however, there was no presence of the uncapped fragment. Enzymatic protocols have been investigated where the purpose was to create uncapped RNA fragments to be used as a reference. A minor validation of the final separation method has been carried out where the generated data can be used to establish acceptable system suitability test criteria for separation methods when analyzing mRNA drug products. / Genom användandet av jonparskromatografi var separation och analys av kortare mRNA fragment möjlig. Syftet med studien var att sätta upp en kvantifieringsmetod för analys av förhållandet 5’cappat kontra icke-cappat mRNA. Klyvning skedde med hjälp av en komplementär probe och enzymet RNase H som skapade korta mRNA fragment innehållande cap strukturen. Under studiens gång har flertalet separationsmetoder testats, där halt organiskt lösningsmedel var en viktig faktor för eluering. För att identifiera topparna behövdes masspektrometri användas vilket resulterade i en justering av metodens parametrar. Med hjälp av LC-MS kunde det cappade fragmentet detekteras, dock fanns ingen närvaro av ett icke-cappat mRNA. Enzymatiska undersökningar har utförts där målet var att skapa icke-cappat RNA för att använda som referens. En mindre validering av den finala separationsmetoden har genomfört där de genererade datavärdena sedan kan användas för att fastställa accepterbara kriterier för separationsmetoder vid analys av läkemedelsprodukter baserade på mRNA.

Total oxidizable precursor assay (TOPA) has been used to understand the contribution of unknown precursors to the extractable organofluorine (EOF) compounds. The investigation of compounds containing a CF3 moiety especially connected to an aromatic ring is important because any compounds with such a moiety (pharmaceuticals) may oxidize to form trifluoroacetic acid (TFA). This information may help to increase the understanding of the sources of TFA in the environment. TOPA has been used for this purpose, however, this method has a weakness as the conditions during oxidation results in samples containing high concentrations of sulfates. The sulfates might compete with C2-C3 perfluoroalkyl acids (PFAAs) for binding sites in the solid-phase extraction (SPE), which in turn decreases the recovery of these compounds substantially. This study aimed to optimize the extraction following the TOP assay to increase the recovery of C2-C3 PFAAs, and to apply the improved method to determine whether selected pharmaceuticals (fluoxetine, seproxetine and celecoxib) may oxidize to form TFA. The main parameters affecting the recoveries of C2-C3 PFAAs were found to be pH as well as sorbent volume of the SPE cartridge. The optimized method included adjusting samples to pH 2.0 using formic acid prior to solid phase extraction (SPE), as well as using 500 mg sorbent cartridges for SPE. By optimizing the extraction, it was possible to increase the recovery of TFA from 2% to 54%. All three pharmaceuticals were oxidized into TFA to various extent (molar ratio of TFA from fluoxetine: 35%, seproxetine: 63% and celecoxib: 35%). This knowledge could be used as an indication of potential precursors in a sample and can help understand the contribution of CF3-containing pharmaceuticals to the EOF.

This work was made to discover if greenhouse crops, particularly broccoli and Chinese cabbage, can change their metabolism and therefore their nutritional profile depending on what ultraviolet radiation (UV) they were exposed during growth. This is important since the world’s population increases and therefore, we need to be able to produce foods with increased nutritional quality. To explore whether we can use UV for that, we first analyzed the plant extracts searching for changes in plant metabolism after exposure to UV light and then subjected the plants exposed or not to UV to an in vitro fermentation system that mimics the digestion by the human gut microbiota. The latter was done to understand how our gut microbiota can metabolize plants with distinct metabolite profile and whether this will impact in the levels of metabolites with beneficial health effects. We started by applying different UV treatments during growth of broccoli and Chinese cabbage (i.e., plants were grown using standard growth conditions or they were exposed to supplementary UVA or to supplementary UVB) and for the analysis of plants, we applied a non-targeted approach to explore overall changes in metabolism induced by UV light. The plants were further subjected to an in vitro fermentation approach to simulate human digestion. The obtained fermentation extracts were further analyzed for levels of short-chain fatty acids (SCFA) at different time points during the in vitro fermentation. Major insights from this experiment were that the different light contributes to significant changes in the plant’s metabolic profile depending on what light they were cultured in. However, such changes in plant metabolism does not change how our gut microbiota produce SCFA during fermentation. Another conclusion when exploring broccoli and Chinese cabbage is that UV effects cannot be generalized to different plants. This depends on their different genotypes, even in the same species, which gives different response of light on the plant’s receptors. No differences in the SCFA profile were found after fermentation of control and UV-exposed plants; however, results from plant extracts indicate that a more comprehensive analysis of fermentation extracts might reveal changes in how our gut microbiota metabolizes plant components. Although further analysis is needed to confirm such hypothesis, the pipeline showed that UV light influences metabolism of broccoli and cabbage and therefore modulate plant metabolism and their potential impact on human health by increasing the levels of compounds with antioxidant properties.

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The in-biology novel high-valent manganese cofactor {MnIII MnIV} has been elucidated and characterized in a ribonucleotide reductase from Facklamia ignava, establishing the new radical-free ribonucleotide reductase class; class Id. The cofactor was found to be directly connected to catalytic activity and a mixed-metal cofactor was disproven using a newly formulated in vitro assembly protocol and activity assays. The cofactor was assembled in vitro using hydrogen peroxide, and it was shown that the F. ignava ribonucleotide reductase can perform catalase activity, with the cofactor formation as a side-reaction. Using electron paramagnetic resonance spectroscopy to study the in vitro assembled cofactor, a hydrogen peroxide-driven assembly mechanism was proposed, where the mechanism is like that of manganese catalases. An over-oxidized {MnIV MnIV} intermediate is suggested to be the direct precursor to the {MnIII MnIV} cofactor. The cofactor was also shown to be able to be assembled with superoxide, with in situ generation of superoxide using hydroquinone. An in vivo assembly mechanism was proposed to involve superoxide generated by some unknown accessory enzyme, similar to the NrdI assembly system of class Ib ribonucleotide reductases.