2.11. Sterols and oxysterols quantification by isotope dilution GC-MS

To a screw-capped vial sealed with a Teflon septum, 0.25 mL of plasma were added together with 500 ng of D7-lathosterol, 50 ng of D6-lanosterol, 50 ng of D7-24S-hydroxycholesterol, 50 ng of D6-25-hydroxycholesterol, 50 ng of D6-27-hydroxycholesterol, 50 ng of D7-7-ketocholesterol, 50 ng of D7-7β-hydroxycholesterol (Avanti Polar Lipids), as internal standards, 50 μl of butylated hydroxytoluene (5 g/L) and 50 μl of K3-EDTA (10 g/L) to prevent auto-oxidation. Each vial was flushed with argon for 10 min to remove air. Alkaline hydrolysis was allowed to proceed at room temperature with magnetic stirring for 60 min in the presence of ethanolic 2 M potassium hydroxide solution. After hydrolysis, the sterols were extracted twice with 5 ml hexane. The organic solvents were evaporated under a gentle stream of argon and converted into trimethylsilyl ethers with BSTFA.

Gas chromatography mass spectrometry (GC-MS) analysis was performed on a GC equipped with an Elite column (30 m × 0.32 mmid × 0.25 mm film; PerkinElmer, USA) and injection was performed in splitless mode and using helium (1 ml/min) as a carrier gas. The temperature program was as follows: initial temperature of 180 °C was held for 1 min, followed by a linear ramp of 20 °C/min to 270 °C, and then a linear ramp of 5 °C/min to 290 °C, which was held for 10 min. The mass spectrometer operated in the selected ion-monitoring mode. Peak integration was performed manually, and sterols were quantified from selected-ion monitoring analysis against internal standards using standard curves for the listed sterols. Additional qualifier (characteristic fragment ions) ions were used for structural identification.

Inter-assay CV ranged between 2.3% for lathosterol and 5.3% for 25-hydroxycholesterol. Recovery ranged from 97 up to 104% (5–7).