Abstract
Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.
Highlights
Fractions separated by thin-layer chromatography (TLC) or high performance liquid chromatography (HPLC) were quantitated by gas-liquid chromatography (GLC) using a 1.5 m x 4 mm i.d. column packed with 3% OV-17 at 24OoC, with 5a-cholestane added as an internal standard [6]
We found only 85% of the tritium in the cholesterol fraction from the batch we fractionated on C18 HPLC
For many purposes purification of radiolabeled sterols by C18 HPLC alone would be sufficient; we found that further purification by silica HPLC produced a reliable product for use in enzyme assays and in vivo turnover studies, and as the recovery standard for fractionation of fecal neutral steroids
Summary
HPLC grade solvents were purchased from Fisher Scientific Co. (Pittsburgh, PA). Cholesterol, campesterol, and 0-sitosterol standards were obtained from Applied Science (Deerfield, IL), and all other steroid standards were from Steraloids, Inc. (Wilton, NH). [22,23JH(n)]pSitosterol and [4-14C]cholesterol were purchased from Amersham Corp. (Arlington Heights, IL). HPLC grade solvents were purchased from Fisher Scientific Co. 'To whom correspondence and reprint requests should be addressed. Amounts of [14C]cholesterol greater than 250 pCi were purified on a Magnum 9, Partisil-10 column, 9.4 mm x 50 cm (Whatman Co., Clifton, NJ) by elution with isooctane-isopropanol 97:3 (dv) at 4 ml/min. Relative retention characteristics of steroid standards on radially compressed C18 and silica cartridges were determined with minor modifications of the above procedures. The C18 elution solvent was acetonitrile-isopropanol-methanol 20:3:7 (v/v/v), with a flow rate of 1.4 ml/min. The solvent for silica HPLC was isooctane-isopropanol 99:l (v/v) [4], at a flow rate of 1.5 ml/min.
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