Development and validation of a liquid chromatography- mass spectrometry method for analysis of oxysterols in human plasma
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Background: Oxysterols are hydroxy, keto or epoxy oxidation products of cholesterol formed by enzymatic action or auto-oxidation. Oxysterols have been shown to have several biological activities including regulation of cholesterol homeostasis. Oxysterols have also been suggested to be informative biomarkers of oxidative stress. The goal of this project is to develop and validate a liquid chromatography-mass spectrometry (LC-MS) method for determination of multiple oxysterols in human plasma. Methods: A reverse phase LC using a methanol/water gradient was systematically developed and the interface for MS detection was selected by flow injection analysis. Total oxysterols were prepared from plasma by cold saponification with ethanolic sodium hydroxide, followed by reverse phase (C18), solid phase extraction. The effect of sample preparation steps on the stability of oxysterols was determined. A delipidated, blank plasma matrix was prepared by solvent stripping. Method validation was performed to establish linearity, sensitivity, recovery, matrix effects, accuracy and imprecision. Results: Reverse phase LC using a silica based C18 column and a binary gradient of methanol/water achieved resolution of 13 oxysterol compounds and free cholesterol in 35 minutes. MS detection using APCI+ interface and selected ion monitoring yielded limits of detection for individual oxysterols ranging from 5-20 ng/mL. Stability studies indicated no breakdown or formation of oxysterols during sample preparation steps for 6 of 13 oxysterol compounds analyzed. Extraction recoveries were reproducible across a wide range of concentration and were consistently > 77% for all analytes and deuterated internal standards. A blank matrix material consisting of lipid stripped human plasma contained persistently trace levels of free cholesterol and 7-keto-cholesterol. Estimates of imprecision in normal human plasma are <10 % for 24(s)-, 25-, 27 and 7α hydroxycholesterol and ≤15 % for 7-keto-cholesterols. Conclusion: The described method is fully validated for four hydroxycholesterol compounds and partially validated for 7-keto-cholesterol measurement in human plasma and is suitable for studies investigating the oxysterols levels in human health and disease.