High-speed counter-current chromatographic separation of phytosterols

Phytosterols are bioactive compounds which occur in low concentrations in plant oils. Due to their beneficial effects on human health, phytosterols have already been supplemented to food. Commercial phytosterol standards show insufficient purity and/or are very expensive. In this study, we developed a high-speed counter-current chromatography (HSCCC) method for the fractionation and analysis of a commercial crude β-sitosterol standard (purity ∼60% according to supplier). Different solvent systems were tested in shake-flask experiments, and the system n-hexane/methanol/aqueous silver nitrate solution (34/24/1, v/v/v) was finally used for HSCCC fractionation. About 50 mg phytosterols was injected and distributed into 57 fractions. Selected fractions were condensed and re-injected into the HSCCC system. This measure provided pure sitostanol (>99%) and β-sitosterol (∼99%), as well as a mixture of campesterol and stigmasterol without further phytosterols. An enriched HSCCC fraction facilitated the mass spectrometric analysis of further 11 minor phytosterols (after trimethylsilylation). It was also shown that the commercial product contained about 0.3% carotinoids which eluted without delay into an early HSCCC fraction and which were separated from the phytosterols.