Optimization of a Methodology for Cell Based Untargeted Metabolomics with UHPLC-Q-ToF-MS

Sammanfattning: Metabolomics is the study of small molecules (molecular weight < 1500 Da) in a biological system and can provide relevant information about endpoints of biochemical pathways by establishing metabolic profiles for different sample cohorts and comparing these through multivariate data analysis. The aim of this thesis was to establish and test a suitable methodology for cell-based untargeted metabolomics, utilizing UHPLC-MS. Two different system setups were established, using one C18 column and one HILIC column. Parameter optimization was carried out with a reference solution, containing a diverse set, with regards to their physical and chemical properties, of seven substances. Cultured HCT 116 cells were chosen as model system. Three different sample preparation procedures were evaluated, based on number of detected unique markers and relative degree of reproducibility. Two of the procedures were based on liquid extraction, Dual Layer Fractionation (DLF) and Consecutive Extraction (CE), and the third was a dilution (DI) of the cell samples. The HILIC system did not achieve an adequate number of detected unique markers for any of the sample preparation procedures and further optimization is required. For the C18 system, CE proved to have the highest degree of reproducibility, while retrieving the next greatest set of detected unique markers.