| dc.description.abstract | Obesity and inactivity can lead to a cluster of conditions called metabolic syndrome, which again can lead to non-alcoholic fatty liver disease (NAFLD). NAFLD consists of several stages, where the liver accumulates fats, potentially leading to liver fibrosis and cirrhosis. Cirrhosis is considered a terminal state and requires liver transplantation to save the life of the patient. The only diagnostic tool for NAFLD today is liver biopsy. Hence, less invasive diagnostic tools are currently lacking, like biomarkers, which can be advantageous for early diagnosis. Liver organoids are a promising research model used for disease modeling and recapitulate the human NAFLD in vivo environment. Oxysterols are endogenous cholesterol metabolites seen in some of the NAFLD pathways and are proposed as potential biomarker candidates. However, oxysterols are low abundant in biological samples and need a highly sensitive LC-MS method for quantification. Miniaturizing the inner diameter (ID) of the LC column is one way to enhance the method sensitivity. Additionally, a trap column can further enhance method sensitivity by allowing large volume injections, and an automatic filtration and filter backflushing system (AFFL) enhances the robustness of the platform. Few studies have investigated the use of miniaturized LC columns in oxysterol determination. The aim of this study was to develop a highly sensitive nLC-MS method with core-shell particles, combined with an on-line sample clean-up in the form of a trap column and an AFFL-system, towards quantification of the oxysterols 22R-, 24S-, 25-, and 27-OHC in healthy and NAFLD-induced liver organoids. An automated nLC-MS method combined with an on-line sample clean-up was successfully assembled. 2.5 m core-shell Super Phenyl-Hexyl (SPH) and 5 m phenyl-hexyl (PH) particles were chosen and successfully packed in fused silica capillaries (75 m ID) as the analytical- and trap column, respectively. Significant band broadening was a problem, and a Butterfly Portfolio column heater was included in the platform to enhance column efficiency. It was discovered that the 5 m PH particles in the trap column were partly responsible for the band broadening observed due to incompatibility with the 2.5 m SPH particles. Hence, the 2.5 m SPH particles were found to be suitable in the trap column instead. Relatively efficient nLC columns were packed; however, they proved to be challenging to reproduce and lacked robustness due to short lifetimes, making the oxysterol isomer separation challenging. Core-shell particles continue to prove to be challenging to pack in narrow bore capillaries. Therefore, customizing the packing procedure for these particles should be performed further by, e.g., adding a frit on both ends of the column and optimizing the slurry concentration. | eng |