| Background: Oxysterols are an important class of cholesterol metabolites that have roles in regulation of cholesterol homeostasis, immune signaling and apoptosis. Liquid chromatography-mass spectroscopy (LC-MS) has become the method of choice for oxysterol measurement in biological fluids and tissues. This thesis focuses on the development and evaluation of chemical derivatization techniques for enhanced mass spectrometric detection of oxysterols.;Methods and Materials: Three derivatization schemes (picolinyl esters, dansyl chloride and dimethylglycine) were performed on authentic oxysterols to create electrospray ionization (ESI) active compounds. The mass spectrum of derivatization products were characterized and preliminary chromatographic separation was developed for seven oxysterols by selected ion monitoring LC-MS. The optimum derivatization scheme was selected and the chromatographic method was evaluated for calibration linearity, accuracy, limits of detection and quantitative recovery.;Results: Dansylated oxysterols did not generate predictable ESI fragmentation patterns. Oxysterol picolinyl esters were predictably ionized however isomeric compounds including 7alpha and 7beta-hydroxycholesterol could not be chromatographically resolved. Dimethylglycine derivatization provided predictable ESI fragments, which were completely resolved HPLC. The preliminary method was linear and accurate between 6 ng/mL to 500 ng/mL with a LOD 1-8 ng/mL. Recovery estimates were between 80 and 120% for 7alpha-hydroxycholesterol (HC). Poor recovery was observed for 25-hydroxycholesterol (HC) and 27-hydroxycholesterol. This poor recovery for 25-hydroxycholesterol and 24-hydroxycholesterol was encountered due to incomplete resolution of 25-HC secondary peak from 24-HC and for 27-HC due to the concentrations being present beyond the range of the calibration.;Conclusion: Oxysterol dimethylglycine derivatives were easily formed and efficiently ionized by ESI interface with either single quadrupole or tandem mass spectrometry. High Pressure (rather than ultra-pressure) LC separation was sufficient to achieve complete separation of major plasma oxysterols. The preliminary method described merits for full-method development and validation. If successful, this would represent the first multi-oxysterol methodology employing DMG derivatization. |
