真武汤水提液HPLC特征图谱研究及指标成分的测定

HPLC Characteristics of Aqueous Extract of Zhenwu Decoction and Determination of Markers

目的:建立真武汤水提液HPLC特征图谱及同时测定方中苯甲酰新乌头原碱、苯甲酰乌头原碱、白术内酯Ⅲ、芍药苷、6-姜辣素5种主要指标成分的方法。方法:采用高效液相色谱法,Phenomenex Luna C18色谱柱(4.6 mm×250 mm,5μm),流动相0.2%磷酸水(A)-乙腈(B)梯度洗脱,检测波长230 nm,柱温30℃,流速1 mL·min-1,进样量20μL。结果:建立的特征图谱包含36个共有峰,各峰峰形、分离度好,明确并定量真武汤水提液中5种指标成分,芍药苷(1.410±0.024)g·L-1,苯甲酰新乌头原碱(0.066±0.001)g·L-1,苯甲酰乌头原碱(0.015±0.001)g·L-1,白术内酯Ⅲ(0.028±0.003)g·L-1,6-姜辣素(0.069±0.001)g·L-1。经方法学验证该方法具有良好线性关系芍药苷Y=25.907X+9.741(r=0.999 7)、苯甲酰新乌头原碱Y=15.265X-3.202(r=0.999 5)、苯甲酰乌头原碱Y=15.660X-4.699(r=0.999 1)、白术内酯ⅢY=63.730X-10.740(r=0.999 6)、6-姜辣素Y=21.645X-4.074(r=0.999 5),精密度(峰面积RSD分别为1.72%,1.76%,2.78%,1.20%,1.05%,保留时间RSD分别为0.81%,0.47%,0.43%,0.07%,0.05%),重复性(峰面积RSD分别为1.95%,2.56%,2.93%,2.65%,3.01%,保留时间RSD分别为0.03%,0.19%,0.28%,0.11%,0.12%),稳定性(峰面积RSD分别为1.05%,1.36%,2.89%,2.81%,2.64%,保留时间RSD分别为0.15%,0.12%,0.39%,0.04%,0.08%)和加样回收率(99.85%,101.67%,101.91%,99.82%,101.44%)。结论:建立的真武汤水提液特征图谱可同时对真武汤5种主要指标成分进行分析,可全面评价真武汤水提汤剂的质量。

Objective：This study was aimed to estabilish a high-performance liquid chromatography （HPLC） method for HPLC characteristics of aqueous extract of Zhenwu decoction （ZWT） and simultaneous determination of makers-characteristic chromatography,benzoylmesaconine,benzoylaconine,atractylenolide Ⅲ,paeoniflorin and 6-gingerol.Method：The separation was carried out on a Phenomenex Luna C18 （4.6 mm ×250 mm,5μm） column eluted with mobile phases of acetonitrile （A） and water containing 0.2％ phosphoric acid （B） in gradient mode at the flow rate of 1.0 mL ·min-1 The column temperature was maintained at 30 ℃ and the detection wavelength was set at 230 nm.Result：The obtained HPLC characteristics containing 36 common peaks,which were in well shape and high resolution.Five constituents of ZWT were analyzed and quantified paeoniflorin （1.410±0.024） g· L-1 benzoylmesaconine （0.066 ±0.001） g·L-1,benzoylaconine （0.015 ±0.001） g·L-1,atractylenolide Ⅲ （0.028 ±0.003） g ·L-1,6-gingerol （0.069 ± 0.001） g ·L-1,with the linear range as follows Y=25.907X＋9.741 （r=0.999 7）,Y=15.265X-3.202 （r=0.999 5）,Y=15.660X-4.699 （r=0.999 1）,Y=63.73X-10.740 （r =0.999 6）,Y=21.645X-4.074 （r =0.999 5）,respectively.The precision （1.72％,1.76％,2.78％,1.20％,1.05％ for RSD of peak area,0.81％,0.47％,0.43％,0.07％,0.05％ for RSD of retention time）,repeatability （1.95％,2.56％,2.93％,2.65％,3.01％ for RSD of peak area; 0.03％,0.19％,0.28％,0.11％,0.12％ for RSD of retention time）,stability （1.05％,1.36％,2.89％,2.81％,2.64％ for RSD of peak area; 0.15％,0.12％,0.39％,0.04％,0.08％ for RSD of retention time）,and average recovery （99.85％,101.67％,101.91％,99.82％,101.44％,respectively） of this method were all satisfied.Conclusion：The method established in this study could be used in simultaneous determination of the five makers of zhenwu decoction and could be used to control the quality of ZWT aqueous extract.