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建立了同时测定甜叶菊中绿原酸、3,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸3种化合物的HPLC分析方法,并测定了3种化合物在根、茎、叶中的含量。色谱柱为Waters C18(4.6 mm×250mm,5μm),流动相为0.2%醋酸(A)和甲醇(B),流速:1 mL/min,检测波长:327 nm,柱温:40℃,洗脱条件为0~10 min 70%A;10~15 min 70%A-45%A;15~25 min 45%A,25~26 min 45%A-70%A;26~30 min 70%A。在此条件下,绿原酸、3,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸的线性范围分别为9.50~285.00、9.25~277.50、9.75~292。50μg/mL且在此范围内线性关系良好(R2>0.999),加标回收率介于96.42%~101.18%。从乙醇体积分数、料液比和超声提取时间3因素优化绿原酸提取条件,最佳条件为70%乙醇在料液比1:25的条件下超声提取60 min。对甜叶菊根、茎、叶中上述3种化合物含量进行分析,结果显示,甜叶菊根、茎、叶中绿原酸、3,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸的含量范围分别为0.59~7.56、2.43~23.92、1.12~21.21 mg/g。该方法准确度高,重复性好,可用于甜叶菊中3种绿原酸类化合物的同时测定。
A HPLC method was established for the simultaneous determination of three compounds, chlorogenic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid in Stevia rebaudiana. The three compounds were determined in roots and stems , The content of the leaves. The chromatographic column was Waters C18 (4.6 mm × 250 mm, 5 μm) with a mobile phase of 0.2% acetic acid (A) and methanol (B) at a flow rate of 1 mL / min and a detection wavelength of 327 nm at a column temperature of 40 ° C Conditions are 0 to 10 min 70% A; 10 to 15 min 70% A to 45% A; 15 to 25 min 45% A, 25 to 26 min 45% A to 70% A; 26 to 30 min 70% Under these conditions, the linear ranges of chlorogenic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were 9.50-285.00, 9.25-2727, 975-292.50μg / mL (R2> 0.999). The recoveries of spiked samples ranged from 96.42% to 101.18%. The extraction conditions of chlorogenic acid were optimized from the factors of ethanol volume fraction, solid-to-liquid ratio and ultrasonic extraction time. The optimal conditions were 70% ethanol and ultrasonic extraction for 60 min at a feed-liquid ratio of 1:25. The contents of these three compounds in the roots, stems and leaves of Stevia rebaudiana were analyzed. The results showed that chlorogenic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoyl The content of quinic acid ranged from 0.59 to 7.56, from 2.43 to 23.92 and from 1.12 to 21.21 mg / g, respectively. The method has high accuracy and repeatability and can be used for the simultaneous determination of three chlorogenic acids in stevia.