Lipid Extraction, Separation, and GC-MS Analyses of Sterols in Cells

Verification of the hits obtained from the initial screening was carried out with varying concentrations of the compounds of interest. In addition, a more complete sterol profile was determined. The experiment was carried out in 96 well plates as described above for the 384 well plates. After counting cells on ImageXpress Micro XL (10X objective), the medium was removed, the cells were washed with PBS, and the buffer was removed. The plates were stored at −80 °C. To each well was added 10 μL of BHT/TPP solution (2.5 mg of TPP and 1 mg of BHT in 1 mL of EtOH), 10 μL of internal standard solution (0.87 nmol for d₇-Chol, 0.033 nmol for d₇-7-DHC, 0.25 nmol for ¹³C₃-Des, and 0.23 nmol for ¹³C₃-Lan/well), and 200 μL of MeOH. The plate was placed on a rotary shaker for 20 min at room temperature. An aliquot (100 μL) of the supernatant was transferred to a PTAD-predeposited plate, sealed with Easy Pierce Heat Sealing Foil, followed by 20 min of shaking at room temperature, and analyzed by LC-MS as described above. The flow rate was increased to 500 μL/min and run time to 1.5 min to include cholesterol analysis (Chol 369 → 369 and d₇-Chol 376 → 376). The remaining sample in each well was transferred to vials and concentrated on a SpeedVac concentrator for GC-MS analysis. To each vial was added N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA, 50 μL), vortexed well, and allowed to react for 30 min. The sample (5 μL) was injected onto the column (SPB-5, 0.25 μm, 0.32 mm × 30 m) with a temperature program of 220–300° (5 min) at 20°/min and helium flow rate of 2.0 mL/min. The data were collected in full scan mode and the following ions extracted for quantitation relative to d₇-Chol: 458 for cholesterol, zymostenol, lathosterol; 456 for zymosterol, dehydrolathosterol; 349 (M-105) for dehydrodesmosterol; 393 (M-105) for lanosterol; 395 (M-105) for dihydrolanosterol; 486 for dimethylzymostenol; and 465 for d₇-cholesterol. Desmosterol and 7-DHC were not analyzed by GC-MS because they coelute. The remaining sterol intermediates were not detected. All data were normalized to cell count.