Comparison of cholesterol oxidation product preparation methods for subsequent gas chromatographic analysis

The stability of cholesterol hydroperoxides (CHPs) under the analytical conditions and preparation methods commonly used for the determination and quantification of cholesterol oxidation products (COP), was evaluated. CHPs were prepared by photoxidation and separated by silica thin-layer chromatography (TLC). CHPs were individually collected by normal-phase high-performance liquid chromatography (HPLC) and then subjected either to reduction or to cold saponification. The corresponding hydroxyl derivatives were generated by reduction, whereas cold saponification gave rise predominantly to 7-ketocholesterol. In another test, silylated and non-silylated CHPs were separately injected into a gas chromatograph at 310C, collected and reinjected into a gas chromatography-mass spectrometry (GC-MS). The silylated CHPs were more stable than the non-silylated ones, giving 7-ketocholesterol, 7- and 7-hydroxycholesterol as main degradation products. Two unknown degradation peaks were detected in both silylated and non-silylated CHPs, having 384 as main m/z fragment. The study of their mass spectra lead to the conclusion that peaks A and B correspond to 6- and 6-hydroxycholesterol, respectively.