Utveckling av metoder för att mäta isotopkvoter i kolesterol med FT-ICR-masspektrometri

Abstract

Cancer remains one of the leading causes of mortality worldwide, highlighting the need for reliable methods for early detection. Metabolic reprogramming in cancer cells, particularly within lipid metabolism, can alter the isotopic composition of cholesterol, potentially giving rise to measurable differences between healthy and malignant tissue. Cholesterol isotope ratios therefore represent a promising class of biomarkers for tumor progression, but their accurate determination requires analytical techniques with exceptionally high mass resolution and precision. The aim of this study was to develop and evaluate a high-resolution method for isotope ratio analysis of cholesterol using Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICR-MS). Particular emphasis was placed on achieving efficient and reproducible ionization of this inherently challenging analyte. Method development was carried out in two stages. In the first phase, analytical parameters were optimized using a structured experimental design, where MALDI with an HCCA matrix was evaluated both with and without chemical derivatization. In the second phase, the optimized conditions were applied to isotopically labeled samples to enable quantitative analysis of isotope patterns. Method performance was assessed in terms of signal intensity, fragmentation behavior, reproducibility, and preservation of isotopic information, allowing for a comprehensive evaluation of analytical reliability and sensitivity to experimental variation. The results demonstrate that both ionization strategy and experimental parame ters critically influence method performance. Derivatization improved ionization efficiency and increased signal intensity, while optimization through experimental design enhanced reproducibility. However, significant limitations remain, particularly related to variability inherent to the MALDI process and uncertainties in model predictability, which affect both precision and generalizability. Overall, the study shows that FT-ICR-MS holds strong potential for accurate isotope ratio analysis of cholesterol, but that its practical reliability is highly dependent on sample preparation, calibration strategies, and control of experimental variability. Further method refinement and validation are therefore required before application to biological samples and diagnostic contexts can be considered, underscoring both the promise and current limitations of the approach.