Oxysterol Quantification by HPLC-MS

Oxysterols were analyzed using a validated HPLC–MS method (Mutemberezi et al, 2016b (link)). Briefly, 200 μl of cell supernatants was placed in glass vials containing d₇‐4β‐hydroxycholesterol (133.3 pmol) and d₇‐24‐hydroxycholesterol (200 pmol) as internal standards (Avanti Polar Lipids) as well as dichloromethane, methanol (containing 10 μg of butylated hydroxytoluene), and bidistilled water (containing 20 ng ethylenediaminetetraacetic acid; 8:4:2 v/v/v). After mixing and sonication, samples were centrifuged and the organic phase was recovered and dried under a nitrogen stream. The organic residue was resuspended and prepurified by solid phase extraction over silica. The eluate containing oxysterols was analyzed by HPLC‐MS using an LTQ‐Orbitrap XL MS (Thermo Fisher Scientific) coupled to an Accela HPLC system (Thermo Fisher Scientific). Chromatographic separation was performed using an Ascentis Express C‐18 column (2.7 μm, 150 × 4.6 mm, Sigma), kept at 15°C. Mobile phase was a gradient of methanol and water containing acetic acid. MS analyses were performed using an atmospheric pressure chemical ionization source in the positive mode. Data are expressed in nanomolar.