Sterol synthesis.: A timely look at the capabilities of conventional and silver ion high performance liquid chromatography for the separation of C27 sterols related to cholesterol biosynthesis

Sterol intermediates in the biosynthesis of cholesterol have recently assumed a very prominent position in a number of important problems in medicine and biology. In studies of these matters, the separation and identification of the sterol intermediates present formidable challenges, a situation which does not appear to be generally appreciated. High performance liquid chromatography (HPLC) is a simple and rapid approach for the separation of the concerned compounds. Reversed phase HPLC is very commonly used for this purpose. In the present studies, we have evaluated the capabilities of reversed phase, normal phase, and silver ion HPLC for the separation of sterols. Using an extensive collection of authentic sterols, our studies indicate very limited capabilities of reversed phase and normal phase HPLC for the separation of C-27 sterols differing in the number and location of olefinic double bonds. In contrast, silver ion HPLC provided remarkable separations of the same compounds, either as the free sterols or their acetate derivatives. These findings, coupled with the results of recent studies of the properties of the same compounds by gas chromatography and by nuclear magnetic resonance and mass spectroscopy, have important implications regarding current application of methodologies for the separation, identification, and quantitation of sterol intermediates in cholesterol biosynthesis as critical portions of investigations on a number of current and emerging problems in biology and medicine.