模拟体系中谷维素检测方法的建立

Development of a detection method for γ-oryzanol in simulation system

建立了高效液相色谱-紫外检测法,测定了模拟体系中谷维素4种主要组分的含量。以谷维素混合标准品为外标进行定量,利用异丙醇对样品进行溶解,色谱柱为Waters Symmetry C18 (4.6 mm×250 mm, 5μm),流动相为甲醇-乙腈(体积比1∶1),紫外检测波长为325 nm。结果表明:该方法在谷维素20~1 000μg/mL范围内线性良好,谷维素4种组分标准曲线回归方程的R^(2)分别为0.999 5、0.999 4、0.999 0、0.998 6;在样品重复性和稳定性试验中,谷维素4种主要组分保留时间和峰面积的RSD均小于5%,谷维素4种主要组分和总量的加标回收率分别为83.25%~119.74%、92.27%~110.87%;在米糠油应用试验中,在检测米糠油样品加标回收率时的RSD均小于5%。该方法重复性好、灵敏度高,结果准确可靠,可应用于测定模拟体系中谷维素4种主要组分。经初步研究,其在米糠油中的应用与模拟体系有相似的效果。

γ-Oryzanol is mainly composed of esters of trans-ferulic acid(trans-hydroxycinnamic acid) with phytosterols(sterols and triterpenic alcohols). Among these, cycloartenyl ferulate, 24-methylenecycloartenyl ferulate, campestanyl ferulate and β-sitosteryl ferulate are the main four components of γ-oryzanol, accounting for about 95%. UV spectrophotometry can only determine the total amount of γ-oryzanol, but high performance liquid chromatography can separate and determine the components of γ-oryzanol. In this work, a high performance liquid chromatography-ultraviolet detection method was developed for the determination of four main components of γ-oryzanol in a simulation system. The mixture of γ-oryzanol standards was used as an external standard for quantification and the sample was dissolved in isopropanol. Diphenyl ether and paraffin liquid(1∶ 1, V/V) were mixed as the simulated system. γ-Oryzanol was mixed in this system according to the mass ratio of 3%. The column was Waters Symmetry C18(4.6 mm×250 mm, 5 μm), and the mobile phase was methanol: acetonitrile(1∶ 1, V/V). Flow rate was 0.8 mL/min. Detection time was 50 min. The UV detection wavelength was set at 325 nm. The results showed that the method showed good linearity in the concentration range of 20-1 000 μg/mL. The standard curve equation of cycloartenyl ferulate was y=34 889x-63 757, the standard curve equation of 24-methylenecycloartenyl ferulate was y=34 813x-112 228, the standard curve equation of campestanyl ferulate was y=36 565x-67 528, the standard curve equation of β-sitosteryl ferulate was y=35 601x-32 177, and the correlation coefficients(R^(2)) of the standard curve regression equations of four main components were 0.999 5, 0.999 4, 0.999 0, and 0.998 6, respectively. The detection limit of cycloartenyl ferulate was 0.15 μg/mL. The detection limit of 24-methylenecycloartenyl ferulate was 0.15 μg/mL. The detection limit of campestanyl ferulate was 0.23 μg/mL. The detection limit of β-sitosteryl ferulate was 0.17 μg/mL. The RSD of the retention time and peak area of the four main components were less than 5% in the instrument precision and sample repeatability experiments. The RSD of the retention time and peak area of the four main components were less than 5% in the sample stability experiments. The recovery rates of four main components and total amount of γ-oryzanol were 83.25%-119.74% and 92.27%-110.87%, respectively. In the application of rice bran oil, the RSD of this recovery rate was less than 5%. This method could be applied to determine four main components of γ-oryzanol in a simulation system with good repeatability, high sensitivity and accurate results. The preliminary research showed that its application in rice bran oil has similar effects to the simulation system.