高效液相色谱法测定甜叶菊糖中的甜菊苷和莱鲍迪苷A

Separation and identification of stevioside and rebaudiodside A in stevia by HPLC

建立甜叶菊糖中甜菊苷（简称为XT）和莱鲍迪苷A（简称为RA）的HPLC定量分析方法。色谱分离采用Kromasil NH2柱（250mm×4．6mm i．d．，5μm），以V（乙腈）：V（水）=75：25为流动相，采用质谱检测器鉴定甜叶菊糖中主要的成分。sT和RA质量浓度分别为9．0～287．6mg／L和3．9—126．1mg／L时线性关系良好，两者平均回收率分别为98．61％和97．40％，RSD分别为2．3％和1．3％（n=5）。本实验还利用经典的Eschweiler-clark甲基化反应将氨基柱的伯胺转变为叔胺，发现上述溶质保留减小，同时分离选择性减小，说明氨基与糖苷类化合物的氢键作用和偶极-偶极作用对分离有重要贡献。

A HPLC method for detennination of stevioside （ST） and rebaudiodside A （RA） in stevia was developed. The chromatographic analysis was performed on a Kromasil NH2 column by using acetonitrile-water （75：25, v/v） as mobile phase at a flow rate of 0.6 mL/min. Diode array detector （DAD） was used to monitor in the wavelength range from 210 to 400 nm. Meanwhile, the two components were identified by mass spectrometry. The calibration curves had good linearity in the range of 9.0～287.6 mg/L for ST and 3.9～126.1 mg/L for RA. The average recoveries of ST and RA were 98.6% （n =5, RSD=2.3%） and 97.4% （n =5, RSD= 1.3%）, respectively. The method is a convenient, rapid and effective method for separation and qualitatively analysis of stevia. In addition, the primary amino groups on the stationary phase were converted into tertiary amino groups via classical Eschweiler-clark methylafion reaction. The results showed that the retention time of the above compounds deceased with limited separation selectivity. It indicated that hydrogen-bonding and dipole-dipole interactions between the amino groups on the packing and steviosides played a significant role in the separation.