甜叶菊中莱苞迪苷D、莱苞迪苷A含量测定方法的优化及应用

为比较不同品系甜叶菊中甜味品质较好的莱苞迪苷D(RD)、莱苞迪苷A(RA)含量组成,在传统C18、HSS T3、Amide色谱柱中选择适宜固定相建立高效液相色谱(HPLC)方法进行测定与分析。结果表明,HSS T3柱对甜菊糖苷选择性较好,可同时分离RA、甜菜苷(ST)、莱苞迪苷F(RF)、莱苞迪苷C(RC)、甜茶苷(RBS)、莱苞迪苷B(RB)、甜菊双糖苷(SB),其HPLC分析参数为:流动相32%乙腈和68%磷酸水(0.01%),等度洗脱,柱温40℃,波长210 nm,进样量10 μL,流速1.0 mL·min^-1;Amide柱对RD分离能力最佳,其HPLC分析参数为:流动相76%乙腈和24%水,等度洗脱,柱温40℃,波长210 nm,进样量10 μL,流速0.8 mL·min^-1。 分析比较12个扦插培育的甜叶菊品系,以编号2甜叶菊中RD含量及其占比最高,提示以其为原材料可生产含RD较高的甜菊糖苷;编号3、5、7、11甜叶菊中具较有高含量的甜菊糖苷(主要为RA),提示这些品种富含RA且甜菊糖苷产量较高;编号1、8甜叶菊中RA+RD占比较高,提示以其为原材料的甜菊糖苷甜味品质较好。本研究所建立的HPLC法为甜叶菊中RD、RA分析研究提供了方法参考,含量分析结果可为实际应用中选择适宜甜叶菊品种提供依据。

Optimization and Application of Content Determination Method of Rebaudioside D and Rebaudioside A in Stevia rebaudiana Bertoni

In order to compare the content proportion of rebaudioside D (RD) and rebaudioside A (RA) in different varieties of Stevia Rebaudiana Bertoni, the optimized high performance liquid chromatography methods were established by selecting suitable stationary phase in C18, HSS T3 and Amide chromatographic column. The results showed that the selectivity of HSS T3 column to stevia glycosides was better, which could separate RA, ST, rebaudioside F (RF), rebaudioside C (RC), rubusoside (RBS), rebaudioside B (RB) and steviolbioside (SB) at the same time. The HPLC method employed was an isocratic mobile phase consisted of mixture of acetonitrile and sodium phosphate buffer (0.01%) with a ratio of 32.68(v∶v) at a flow rate of 1.0 mL·min^-1 and with 10 μL injections, and the detection wavelength was set at 210 nm and the column temperature at 40℃. Amide column showed the best separation ability for RD, the HPLC method employed was an isocratic mobile phase consisted of a 76:24 (v:v) mixture of acetonitrile and water at a flow rate of 0.8 mL·min^-1 and with 10 μL injections, the detection wavelength was set at 210 nm and the column temperature at 40℃. The HPLC results showed that the content proportion of RA and RD in 12 strains of Stevia Rebaudiana Bertoni was different. The content and proportion of RD in No. 2 were high, suggesting that this strain could be used as raw material to produce high content of RD. The content of stevia glycosides mainly RA in No 3, 5, 7 and 11 was higher, suggesting that these strains were rich in RA and high yield of steviol glycosides. The proportions of RA+RD in No. 1 and 8 were high, suggesting that steviol glycosides were of better sweetness quality in these raw materials. The HPLC methods established in this study could provide reference for the analysis of RD and RA in Stevia Rebaudiana Bertoni, and the results can provide reference and guidance for the selection of suitable Stevia rebaudiana Bertoni varieties in practical applications.