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- Nilsson Broberg, Malin
(författare)
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Metabolite Profiling of Drugs using Mass Spectrometry : Identification of analytical targets for doping control and improvements of the metabolite search process
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Doping is defined as the use of prohibited substances or methods by the World Anti-Doping Agency and the aim with doping control analysis is to detect the use of these illicit substances or methods. Substances that are prohibited in human or equine sports have either a positive or negative impact on the performance. Since administered drugs generally are metabolized to a varying degree and thereby not only excreted in their original form, their metabolite profiles are of high interest because drug metabolites may be present in the body for a longer time than the administered drug itself. Thereby detection of metabolites can improve the window of detection. Unfortunately, the metabolite profiles of non-approved drugs that are mainly available on the Internet, such as Selective Androgen Receptor Modulators (SARMs) are often unknown. This thesis consists of four papers that all encompass drug metabolite profiling either in vivo, in vitro or in a combination, utilizing separation with liquid chromatography and detection with high resolution mass spectrometry. In paper I and II, the equine in vivo metabolite profiles of the two SARMs ACP-105 and LGD-3303 were investigated and the results showed that using drug metabolites as analytical targets can prolong the detection time. For ACP-105, the in vivo metabolite profile was compared with different incubation models such as liver microsomes, S9 fractions and the fungus Cunninghamella elegans. The in vivo and in vitro metabolite profiles showed an interesting overlap for several metabolites, demonstrating the importance and usefulness for in vitro methods in doping control, especially since microsome incubates are allowed as reference material. An optimization of microsome incubation conditions utilizing experimental design was presented in paper III and IV, showing that the optimized conditions greatly impacted the yield of drug metabolites, but also that the optimal conditions are substance dependent. In paper III, a multivariate data analysis search tool utilizing OPLS-DA was presented, which greatly simplified the in vitro drug metabolite identification process of ACP-105 and the results showed relevance in comparison with human in vivo metabolites.In conclusion, several new analytical targets with improved detectability for equine and human doping control have been presented, where the drug metabolite profile showed to be of great importance. All together, these new analytical targets, the optimized microsome incubation conditions for improved metabolite yield and the search tool that aids the metabolite investigation through multivariate data analysis, have made a positive contribution to the doping control area.
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| 2. |
- Pirttilä, Kristian, 1986-
(författare)
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Development of analytical methods for the determination of the small molecule component of complex biological systems
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The research field of untargeted metabolomics aims to determine the relative abundance of all small metabolites in a biological system in order to find biomarkers or make biological inference with regards to the internal or external stimuli. This is no trivial aim, as the small metabolites are both vast in numbers and extremely diverse in their chemical properties. As such, no single analytical method exist that is able to capture the entire metabolome on its own. In addition, the data generated from such experiments is both immense in volume and very complex. This forces researchers to use algorithmic data processing methods to extract the informative part of this data. Such algorithms are, however, both difficult to parametrize and designed to be highly inclusive, the combination of which often leads to errors. One such algorithm is the peak picking procedures used to find chromatographic peaks in liquid chromatography-mass spectrometry (LC-MS) data.In this thesis, four papers are included that focus both on the development of new methods for sample analysis and data processing as well as the application of such, and other, methods in two interdisciplinary research projects. The first paper describes the development and application of a protocol for LC-MS based untargeted analysis of guinea pig perilymph. The focus of the study was to investigate the biochemical processes underlying the protective effect of hydrogen gas on noise-induced hearing loss (NIHL) in guinea pigs exposed to impulse noise. This study sparked two research projects based on limitations observed during the analytical work. The first limitation was that of limited chemical coverage in the analysis when sample volumes are highly limited. The second paper describes the design and validation of a novel separation method for the sequential analysis of both hydrophilic and lipophilic compounds in biological samples. The second limitation observed was the abundance of false peaks reported by peak picking software. These have a negative effect on both downstream data processing as well as data analysis and metabolite identification. The third paper describes the development of a new algorithm for comprehensive peak characterization in untargeted analytical data with the purpose of filtering such false peaks. Both methods presented in the second and third paper were applied to the analysis of guinea pigs perilymph samples in a follow-up study on the attenuating effect of hydrogen gas on NIHL in guinea pigs exposed to broad band continuous noise.
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| 3. |
- Erngren, Ida, 1989-
(författare)
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Analytical method development in liquid chromatography- mass spectrometry based metabolomics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Metabolomics is the analytical field which aims at analyzing all small molecules, metabolites, in a biological system simultaneously. Currently no analytical methods are able to capture the entire metabolome, therefore, the analytical methods are often developed to be as general as possible. However, as research within the metabolomics field is generally driven by biological questions method development is often overlooked. Moreover, method development in metabolomics is very challenging, as evaluation of the methods are difficult since they are not developed for any particular metabolites. Method development is very important though, data quality and accuracy of relative quantitations is paramount if metabolomics is to be used to answer the biological questions at hand.The articles included in the thesis focus around both analytical method development and applications of metabolomics. In the first paper, head and neck cancer cell lines with different sensitivity to ionizing radiation was investigated using LC-MS based metabolomics. A theory on how the radiation resistant (UM-SCC-74B) cell line could alter its metabolism to handle redox status, DNA repair and DNA methylation was formulated. In the second article the sampling of sponge samples (Geodia barretti) was investigated with regard to its effects on detected metabolite profiles and data quality. It was found that freezing the samples directly was the best alternative which allowed for analysis of most metabolite classes. Storing the samples in solvent lead to a substantial extraction of metabolites to the solvent. For metabolomics, the solvents were more useful than the actual sponge samples that had been stored in solvent. In article three the problems caused by high concentrations of inorganic ions in biological samples in HILIC-ESI-MS analyses was described. The inorganic ions can affect relative quantitation and lead to erroneous results and overly complicated datasets inflated by the extra signals caused by cluster formation. To mitigate the problems caused by the inorganic ions a sample preparation method was developed in article four. The method used cation exchange SPE to trap alkali metal ions which, resulted in less ion-suppression, higher signal intensities of relevant metabolites as well as reduced adduct and cluster formation.In conclusion, this thesis have described projects where metabolomics have been applied to answer biological questions as well as analytical method development in LC-MS based metabolomics. Limitations with current methods was described and possible solutions to improve the methods has been presented.
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