Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria. We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables subcellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.K Borah and HR Griffiths acknowledge INClusilver funded by the European Union, grant number H2020–INNOSUP‐2017‐2017 731349; NeutroCure funded by the European Union, grant number H2020-FETOPEN-01-2018-2019-2020 861878 and Faculty Research Support Fund (FRSF) fund from the University of Surrey 2019–2020. K Borah also acknowledges support of training grant 2019 Ref T022 from VALIDATE network. I Ampong, D Gao and HR Griffiths acknowledge funding from BBSRC (China Partnering Award BB/M028100/2. D Gao acknowledges funding from the National Natural Science Foundation of China(NFSC)(GrantNo.81873665).IHKD acknowledges funding from Alzheimer's
research UK midlands network grant 2019. AH Crosby and EL Baple acknowledge support from the Hereditary Spastic Paraplegia Support Group and The Diamond Jubilee Doctoral Scholarship Fund.published version, accepted version, submitted versio
