An LC-MS method for simultaneous determination of cholesterol, oxysterol and vitamin D levels in human plasma and cerebrospinal fluid.

Background: Cholesterol is synthesized de novo in the brain and influences the etiology of neurodegenerative disorders. Oxysterols are side chain oxidized forms of cholesterol and partially regulate cholesterol homeostasis. Oxysterols produced by neurons are potential biomarkers for brain disorders. To assess oxysterols in neurological disease we have developed and validated a Liquid Chromatography-Mass Spectrometry (LC-MS) method for measuring oxysterol levels and related analytes in human plasma. Method: Serum and plasma samples were prepared by saponification and reverse phase (C18) solid phase extraction. Reconstituted samples were analyzed by LC-MS using a reverse phase column and a binary gradient of methanol and water. Internal standard calibration curves were prepared using a blank matrix stripped of cholesterol. Cholesterol, Oxysterols and 25-hydroxy Vitamin D3 (Cholecalciferol) levels were simultaneously determined in multiple sclerosis patients. Total cholesterol was simultaneously detected using diode array detection. Results: The LC-MS method achieved resolution for 20 oxysterols and vitamin D3 and D2, an total cholesterol in human plasma. The limit of detection (LOD) was 5-12 ng/ml for oxysterols and vitamin D analytes while the LC coupled with photo diode array (PDA) detected cholesterol with an LOD of 5 μg/mL. Analysis of plasma from 152 multiple sclerosis patients indicates detectable levels of 5 oxysterols, vitamin D3 and cholesterol in 100% of samples. Descriptive statistics of analytes levels in the patient sample are presented. Conclusion: The LC-MS method described here is suitable for the analysis of selected oxysterols, vitamin D3 and total cholesterol levels in human health and disease