Separation and isolation of β-sitosterol oxides and their non-mutagenic potential in the Salmonella microsome assay

The recent increase in phytosterol-enriched functional food provokes questions concerning the safety of their oxidation products. However, most of the existing toxicity studies have been performed with mixtures instead of single compounds, a consequence of the lack of pure phytosterol oxidation product (POP)-standards. The objectives of this study were to take in use a method for the isolation of β-sitosterol oxidation products and to assess their mutagenic and pro-oxidative potential. Oxides were prepared by thermo-oxidation, purified by column chromatography and separated by a NP-HPLC-UV system. 7-ketositosterol, 7β-OH-sitosterol, 7α-OH-sitosterol, a mixture of 6β-OH-3-ketositosterol/6α-OH-3-ketositosterol (ratio 4:3) and a mixture of polar oxides were fractionated. Yields in the range of several milligrams per fraction were achieved within one HPLC-run. Identification and quantification was done by GC-MS and GC-FID, respectively. In the Ames test the collected fractions failed to show a mutagenic activity towards Salmonella typhimurium strains TA98, TA100 and TA102.