HPLC法同时测定款冬花中9个主要成分的含量

Simultaneous determination of nine major constituents in Farfarae Flos by HPLC

目的:建立HPLC-DAD法同时测定生药款冬花和蜜炙款冬花中绿原酸、反式咖啡酸、芦丁、金丝桃苷、反式阿魏酸、4,5-O-二咖啡酰基奎宁酸、2,2-二甲基-6-乙酰基苯并二氢吡喃酮、款冬酮和甲基丁酰-3,14-去氢-Z-款冬素酯9个主要成分的含量。方法:采用正交设计优化提取方法,以提取时间、方法、溶剂、溶剂用量为主要影响因素,以9个分析物的总量为评价指标。分析物的色谱分离用Dikma DiamonsilTMC18色谱柱(250 mm×4.6 mm,5μm)、Dikma Easy Guard C18保护柱(20 mm×4.6 mm,5μm);以乙腈(A)-0.03%三氟乙酸水溶液(B)为流动相,梯度洗脱,流速为1.0 mL.min-1;检测波长为240 nm;柱温为25℃。结果:确定最佳提取方法为1.00 g生药样品用20 mL 95%乙醇超声提取1 h。绿原酸、反式咖啡酸、芦丁、金丝桃苷、反式阿魏酸、4,5-O-二咖啡酰基奎宁酸、2,2-二甲基-6-乙酰基苯并二氢吡喃酮、款冬酮和甲基丁酰-3,14-去氢-Z-款冬素酯9个分析物的线性范围分别为62.5～2000μg.mL-1(r=0.9999),2.5～80μg.mL-1(r=0.9994),15.62～500μg.mL-1(r=0.9995),25～800μg.mL-1(r=0.9995),50～1600μg.mL-1(r=0.9992),37.5～1200μg.mL-1(r=0.9994),0.195～12.5μg.mL-1(r=0.9991),15.62～500μg.mL-1(r=0.9991),125～4000μg.mL-1(r=0.9995);平均加样回收率均在95.42%～105.0%范围内,RSD均在0.45%～4.5%范围内。结论:该方法准确,灵敏度高,重复性好,成本低,可用于款冬花中9个主要有效成分的含量测定。

Objective： To develop an HPLC - DAD method for simultaneous determination of nine main bioactive constituents including chlorogenic acid, trans - caffeic acid, rutin, hyperoside, trans - ferulic acid, 4,5 - O - dicaf- feoylquinic acid ,2,2 - dimethyl - 6 - acetylchromanonc, tussilagone, and 7β - （3＇ - ethylcrotonoyloxy ） - 1α - （ 2＇ - methylbutyryloxy） - 3,14 - dehydro - Z - notonipetranone, in Farfarae Flos （ the dried flower buds of Tussila- go farfara L. ;family Compositae）. Methods： Orthogonal array design was used to optimize the extracting process. The main influential factors of extraction efficiency were extracting time, method, as well as volume and ratio of sol- vent. The conditions of the extraction were evaluated by the total content of nine analytes. A Dikma DiamonsilYMc18 column（250 mm ×4.6 mm,5μm） with a Dikma Easy Guard Cls column （20 mm ×4.6 mm,5 μm） was used for chromatographic separation of the nine analytes. The mobile phase, consisting of solvent A （acetonitrile） and solvent B （0.03 % aqueous trifluoroacetic acid） , was programmed as a linear gradient. The flow rate was 1.0 mL min - 1 The absorbance was monitored at a wavelength of 240 nm. The column temperature was 25 ~C. Results： The analy- sis indicated that the optimum conditions for extraction of the nine analytes from the powdered crude drug Farfarae Flos were as follows ： dried and pulverized sample （ 1.00 g） was extracted from 20 mL of 95 % aqueous ethanol （20 - fold,v/w） under ultrasonic extractor for 1 h. The linearity was achieved in the range of 62.5 - 2000μg . mL-1 （r =0. 9999） for ehlorogenie acid,2. 580 μg . mL-1 （r =0. 9994） for trans - caffeic acid,15.62 -500μg . mL - 1 （ r = 0. 9995 ） for rutin,25 - 800μg . mL - 1 （ r = 0. 9995 ） for hyperoside,50 - 1600 μg . mL - J （ r = 0. 9992 ） for trans - fenllic acid, 37.5 - 1200μg . mL - 1 （ r = 0. 9994） for 4,5 - O - dicaffeoylquinie acid, 0.195 - 12.5 μg . mL - 1 （ r = 0. 9991 ） for 2,2 - dimethyl - 6 - acetylchromanone, 15.62 - 500 μg.mL - 1 （ r = 0. 9991 ） for tussil- agone,and 125 -4000μg . mL-l（r = 0.9995） for 7β - （ 3＇ - ethylerotonoyloxy） - 1 α - （ 2＇ - methylbutyryloxy） - 3,14 - dehydro - Z - notonipetranone. The average recoveries （ n = 3 ） were 95.42% - 105.0% with RSD of O. 45 % - 4.5 %. Conclusion： The method developed is accurate, sensitive, reproducible and cheap cost, which can be used for the determination of the above - mentioned nine bioactive constituents in Farfarae Flos.