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.