The Analysis Of Stigmasterol, β-sitosterol And Their Esterified Products

As an important group of natural activity substances, phytosterols are widely used for pharmaceutical trade, food industry, chemical industry and so on. In this research, two important sterols, stigmasterol andβ-sitosterol reacted with maleic anhydride. The sterols and the esterified products were characterized by element analysis (EA), infrared (IR) spectrometry and 1H nuclear magnetic resonance (NMR) spectrometry and analyzed by high performance liquid chromatography (HPLC) and gas chromatography (GC) respectively. The experimental result could offer the theoretic basis and technological parameter for the study of separation and derivatization process.Stigmasterol,β-sitosterol and their esterified products were characterized by several physico-chemical and spectroscopic methods such as EA, IR and 1H NMR spectrometry. The experimental results show that stigmasterol andβ-sitosterol react with maleic anhydride and the esterified products are stigmasteryl maleate andβ-sitosteryl maleate respectively.A gas chromatographic method for the analysis of sterols and steryl maleates was described. Separations were performed on a 6890 series gas chromatograph with a split injector on a capillary column DB^-1 (30m×0.25mm×0.25μm) with detection by flame ionization detection (FID). A temperature program with injection at 200℃, rising at 10℃·min^-1 to 285℃with 20min hold, was found optimal for a good separation of sterols and steryl maleates. The detector temperature was set at 290℃and the inlet temperature at 300℃. Nitrogen was used as the carrier gas at 3.0ml·min^-1. A volume of 0.1μl solution was injected with the split ratio 20:1.The two sterols, stigmasterol andβ-sitosterol, as well as stigmasteryl maleate andβ-sitosteryl maleate are well separated within 20min. Stigmasterol and stigmasteryl maleate,β-sitosterol andβ-sitosteryl maleate respond at the same time.The normalization method can be used for the quantitative analysis of stigmasterol andβ-sitosterol, stigmasteryl maleate andβ-sitosteryl maleate. The relative error of mass content is less than 7%.A validated and repeatable HPLC method with ultra-violet (UV) was developed for the analysis of two sterols, stigmasterol,β-sitosterol and two steryl maleates, stigmasteryl maleate andβ-sitosteryl maleate. The method was based on the separation of the four compounds on a C18 column (Hypersil ODS, 5μm, 150mm×4.6mm, id) using methanol as the mobile phase, and a flow rate of 0.7ml·min^-1 whin 15min. The temperature was maintained at 25℃and the injection volume was 20μl. UV detection was achieved in the range of 190-500nm, 210nm results were used for quantitative purposes.The order of elution of sterols and steryl maleates on C18 column is stigmasteryl maleate,β-sitosteryl maleate, stigmasterol andβ-sitosterol. On the basis of response principle, the polarity of steryl maleates is more powerful than sterols because of the structure of carboxyl group. Stigmasterol responses beforeβ-sitosterol because of the structure of double bonds.The qualitative and quantitative determination of sterols and steryl maleates is carried out using HPLC. The experimental results show that the external standard method can be used for the quantitative analysis of stigmasterol,β-sitosterol, stigmasteryl maleate,β-sitosteryl maleate and the sample in reaction process of sterols and maleic anhydride rapidly and accurately.Stigmasterol,β-sitosterol and their maleates are separated well on C18 column. Sterol and its maleate overlap on DB^-1 capillary column, only stigmasterol andβ-sitosterol, stigmasteryl maleate andβ-sitosteryl maleate can be separated. Accounting for the operation process and cost during analysis, GC method would be highly economical than HPLC method.