2.8. Oxysterol extraction

The dried DCM lower phase was reconstituted in 500μl of MEOH and 1.5ml of LC-MS grade water containing 0.1% formic acid and mixed well. A vacuum manifold connected to a multichannel filtration apparatus was used for the extraction of oxysterols. Oasis HLB solid phase extraction (SPE) cartridges (Waters) were used for selective separation of oxysterols from the lipid extracts. Cartridges were fitted into the vacuum manifold and primed with 800μl MEOH and 600μl H₂O + 0.1% formic acid. Samples were added slowly to the cartridges and the flow through was discarded. Samples were washed with 600μl H₂O + 0.1% formic acid and 600μl hexane to remove polar non-binding analytes and excess cholesterol. Butyl acetate (1ml) was added to the cartridges and eluates were collected in a fresh tube with a controlled flow of 1 drop per second. Butyl acetate eluates were dried under N₂ gas and reconstituted in 40μl of 40% MEOH containing 0.1% formic acid.