| dc.description.abstract | Non-alcoholic fatty liver disease (NAFLD) is a disease caused by the accumulation of fat in the liver, mainly due to obesity and lifestyle. The disease is a worldwide health issue, with high prevalence and a lack of non-invasive biomarkers. To understand the disease, a model system is needed. The use of organoids, stem cell-derived three-dimensional cultures, is suggested as a model reflecting human physiology better than animal models. NAFLD can be induced in liver organoids. This enables the use of “Organ-in-a-Column” technology for studying the disease. Previous research has suggested that oxysterols are potential biomarkers of NAFLD. To make detection easier, oxysterols are usually derivatized in advance of analysis. “Organ-in-a-column” is an on-line system, in which organoids are coupled with Liquid Chromatography – Mass Spectrometry. The on-line approach makes sample preparation in the form of derivatization difficult. Therefore, this study has focused on method development for detecting underivatized oxysterols, with the aim of using the method in an “organ-in-a-column”-setup. Without a derivatization step in the sample preparation, the detection of oxysterols was challenging due to low sensitivity. Attempts of coeluting the groups of hydroxycholesterols and dihydroxycholesterols to establish a steatotic and control fingerprint from organoids with enhanced detection limit were partly successful for the analyte group of dihydroxycholesterols, but not the group of hydroxycholesterols. Sample clean-up was performed on-line using an automated filtration and filter flush solid phase extraction. This ensured robust analysis by the removal of particles before analysis. The optimized experimental parameters included a 5 𝜇L injection volume, a gradient elution utilizing isopropanol (IPA) as the organic modifier (20-65 %), 0.1 % formic acid for pH control in the mobile phase, and the use of a SuperPhenyl hexyl (2.1 mm x 5 cm) column at a temperature of 40 °C. The method allowed for detection of underivatized oxysterols in concentrations down to 0.050 𝜇g/mL in 10:90 IPA:Cell medium. The detection limit was too high to detect oxysterols secreted from liver organoids and the method needs further development before the organ-in-a-column approach can be used for disease monitoring. Also, adjustments to reduce the carry-over would be important to achieve a method providing reliable results. | eng |