7α hydroxycholesterol

The contents of cholesterol and oxysterols were determined according to the slightly improved method of Czauderna et al. [32 (link)]. The improvements consisted of silylation with a BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide, 99%; Sigma Aldrich, St. Louis, MO, USA) procedure. Derivatized analytes were separated on a GC-TOFMS Pegasus^® BT (LECO Corporation, St. Joseph, MI, USA) chromatograph equipped with a capillary column (30 m × 0.25 mm × 0.25 μm film thickness, Rxi^®-17SilMS, Restek, Bellefonte, PA, USA). Identification was made on the basis of mass spectra and by a comparison of retention times of analytes with the following standards: cholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, cholesterol 5α,6α-epoxide, 7-ketocholesterol; Sigma, USA). For recoveries, 5α-cholestane (Sigma, St. Louis, MO, USA) as an IS was used.

Olein, stigmasterol, β-sitosterol, campesterol, brassicasterol, cholesterol, 7α-Hydroxycholesterol, 7β-Hydroxycholesterol, 7-Ketocholesterol, 5α,6α-Epoxy-cholesterol, 5β,6β-Epoxycholesterol, cholestanetriol, 19-Hydroxycholesterol, and 5α-cholestane were purchased from Sigma–Aldrich. The derivatization reagents N-methyl-N-(trimethylsilyl) heptafluorobutyramide and 1-methyl imidazole were ordered from Sigma–Aldrich. Acetone, diethyl ether, dichloromethane, hexane, and methanol were obtained from Merck & Co, Inc. FeSO4·7H₂O, CuSO₄·5H₂O, ZnSO₄, MgSO₄, Na₂SO₄, and MnSO₄ were analytically pure.