The separation of sterol intermediates in cholesterol biosynthesis by high pressure liquid chromatography

A three-step procedure has been developed for the separation of complex mixtures of sterol intermediates in cholesterol biosynthesis. The method has been applied to the separation of sterol intermediates formed from [14C]mevalonate by normal rat hepatocyte culture cells. In Step 1, a short gravity-flow silicic acid column (1.2 x 6.5 cm) separates the incubation products into four classes consisting of A) squalene + squalene oxide, B) methyl sterol precursors, C) C27 sterols, and D) polar compounds. In Step 2, the components of classes B) and C) are further resolved by reverse-phase high pressure liquid chromatography (HPLC) on a microBondapak-C18 column. In Step 3, (after acetylation) high pressure liquid chromatography on a microPorasil column of peaks obtained from Step 2 is conducted. This last step resolves mixtures which may be present in peaks resulting from Step 2. The relative retention times of unknown radioactive sterols are compared with authentic co-chromatographed reference sterols in both Steps 2 and 3. Relative retention time factors for several functional groups encountered in sterol intermediates in cholesterol biosynthesis have been determined for both reverse-phase and silicic acid HPLC systems. The use of these functional group factors allows one to calculate a predicted relative retention time for a variety of structural possibilities. The HPLC techniques described utilize single columns, isocratic solvent systems, and comparatively short (< 30 min) elution times, and the three-step procedure is capable of resolving complex mixtures of sterol intermediates.