六神丸指纹图谱及其6种指标成分含量测定的研究

Fingerprinting of Liushen pills and simultaneous determination of 6 index components

目的:建立六神丸高效液相色谱法HPLC指纹图谱,测定和蟾蜍它灵、远华蟾毒精、华蟾毒它灵、蟾毒灵、脂蟾毒配基、华蟾酥毒基的含量,为控制六神丸质量提供依据。方法:通过HPLC法建立六神丸指纹图谱,采用Waters Xbridge C18色谱柱(250 mm×4.6 mm,5μm);流动相为乙腈-0.5%磷酸水溶液,梯度洗脱;流速为1 mL·min^-1;检测波长为204 nm;柱温35℃。采用中药色谱指纹图谱相似度评价系统(2012版)对15批六神丸样品进行分析,同时测定6种指标成分含量。结果:建立了六神丸的指纹图谱,在204 nm检测波长下标定了22个共有峰,指纹图谱相似度均>0.90。所建立的6个指标成分同步测定的方法,各成分在各自质量浓度范围内与峰面积呈良好的线性关系(r2≥0.999 4),且平均回收率在95.23%~102.25%,RSD均<3.0%。结论:该研究所建立的六神丸指纹图谱和含量测定的方法简便、精密度、稳定性、重复性良好,能更全面反映六神丸的质量,可用于六神丸的质量控制。

Objective: To establish the high performance liquid chromatography(HPLC) fingerprint of Liushen pills and determine its six index components(gamabufotalin, telocinobufagin, cinobufotalin, bufalin, resibufogenin, and cinobufagin) by HPLC in order to provide a scientific basis for its quality control. Methods: HPLC method was used to establish the fingerprint of Liushen pills. The fingerprinting analysis was performed on a Waters Xbridge C18 column(250 mm×4.6 mm, 5 μm) using gradient elution with acetonitrile-0.5% phosphoric acid aqueous solution as mobile phase. The flow rate was 1 mL·min^-1;the detection wavelength was 204 nm;and the column temperature was set at 35 ℃. The fingerprint chromatogram was analyzed by the chromatographic fingerprint similarity evaluation system for tradition Chinese medicine(2012), and the contents of six index components were determined simultaneously. Results: The HPLC fingerprint of Liushen pills was successfully established and 22 common peaks were obtained at the wavelength of 204 nm. All 15 batches of Liushen pills showed a similarity of above 0.90 in HPLC fingerprint profiles. For the quantitive analysis method, the separation of six components showed good regression(r2≥0.999 4) with linear ranges, and the mean recoveries were in the range of 95.23%~102.25% with the RSD less than 3%. Conclusion: The established fingerprint method and quantitative analysis are highly specific and simple. It displays good precision, stability and repeatability, reflecting the quality of Liushen pills more comprehensively. The method can be used for quality control on Liushen pills.