HPLC法同时测定防风中6个主要成分的含量

目的:建立HPLC-DAD法同时测定中药防风中升麻苷、升麻素、5-O-甲基维斯阿米醇苷、印枳树皮苷、防风灵和亥茅酚苷6个主要有效成分的含量。 方法:采用正交设计优化提取方法,以提取时间、方法、次数、溶剂比为主要影响因素, 以6个分析物的总量为评价指标;分析物的色谱分离用Dikma Diamonsil^TM C₁₈ 色谱柱(250 mm×4.6 mm,5 μm)、Dikma Easy Guard C₁₈保护柱(20 mm×4.6 mm,5 μm),以甲醇-水为流动相,梯度洗脱,流速为1.0 mL·min^-1, 检测波长为254 nm,柱温为25℃。 结果:确定最佳提取方法为甲醇30倍量回流提取2次,每次2 h。6个分析物的线性范围分别为:升麻苷50~1000 mg·L^-1(r=0.9999), 升麻素10~200 mg·L^-1(r=0.9999),5-O-甲基维斯阿米醇苷20~500 mg·L^-1(r =0.9999),印枳树皮苷5~100 mg·L^-1(r=0.9995),防风灵5~100 mg·L^-1(r=0.9991),亥茅酚苷10~200 mg·L^-1(r =0.9995);平均加样回收率(n=3)均在95.6%~106.8%,RSD均在1.3%~4.8%。 结论:该方法准确,灵敏度高,重复性好,成本低,可用于防风中上述6个主要有效成分的含量测定。

Objective: To develop an HPLC-DAD method for simultaneous determination of six main bioactive constituents(prim-O-glucosylcimifugin,cimifugin,4'-O-β-D-glucosyl-5-O-methylvisamminol,marmesinin,sapodivarin,and sec-O-glucosylhamaudol)in the roots of Saposhnikovia divaricata(Turcz.)Schischk.(Saposhnikoviae Radix). Methods: The orthogonal array design was used to optimize the extracting process.The main influential factors of extraction efficiency were extracting time,method,times and solvent ratio.The conditions of the extraction were evaluated on the total content of the six analytes.A Dikma Diamonsil^TMC₁₈ column(250 mm×4.6 mm,5 μm)with a Dikma Easy Guard C₁₈ column(20 mm×4.6 mm,5 μm)was used for chromatographic separation of the six analytes.The mobile phase,consisting of methanol-water,was programmed for a gradient elution.The flow rate was 1.0 mL·min^-1,the detection wavelength was at 254 nm,and the column temperature was 25℃. Results: The analysis indicated that the optimum conditions of the six analytes were to reflux with 30 fold solvent methanol twice and for 2 hours at each time.The linearity was achieved in the range of 50-1000 mg·L^-1(r =0.9999)for prim-O-glucosylcimifugin,10-200 mg·L^-1(r =0.9999)for cimifugin,20-500 mg·L^–1(r =0.9999)for 4'-O-β-D-glucosyl-5-O-methylvisamminol,5-100 mg·L^–1(r =0.9995)for marmesinin,5-100 mg·L^-1(r =0.9991)for sapodivarin,and 10-200 mg·L^-1(r =0.9995)for sec-O-glucosylhamaudol.The average recoveries(n=3)were 95.6%-106.8% with RSD of 1.3%-4.8%. Conclusion: The method developed is accurate,highly sensitive,well reproducible and low cost,which can be used for the determination of the six above-mentioned bioactive constituents in Saposhnikoviae Radix.