Chromatographic Separation of Bioactive Oxycholesterols by GC, HPLC and SFC

In this paper we report the development of chromatographic methods for the separation of 8 biologically active hydroxycholesterols (OHC’s) which include the single-hydroxyl addition species 7α-OHC, 7β-OHC, 25-OHC and 27- OHC, together with the double-hydroxyl addition species 7α, 25-OHC, 7β, 25-OHC, 7α, 27-OHC, and 7β, 27-OHC. Four complementary techniques were employed (gas chromatography, normal phase and reversed phase high performance liquid chromatography, and supercritical fluid chromatography), and for each of the techniques, an optimized method for the separation of all eight compounds in a mixture is presented.