逍遥丸HPLC特征指纹图谱研究及多指标成分定量分析

Fingerprint and multi-component determination of Xiaoyao pills by HPLC

目的:建立逍遥丸的HPLC指纹图谱,并测定其主要成分(绿原酸、芍药苷、甘草苷、阿魏酸、甘草酸和藁本内酯)的量,为逍遥丸的质量控制提供可靠方法。方法:采用C18色谱柱(4.6 mm×250 mm,5μm),以乙腈(A)-0.2%磷酸水溶液(B)为流动相,梯度洗脱(0~10 min,3%A;10~20 min,3%A→10%A;20~70 min,10%A→80%A),流速1.0 mL·min^(-1),检测波长326 nm(0~27 min,绿原酸)、230 nm(27~30.5 min,芍药苷)、280 nm(30.5~36 min,甘草苷、阿魏酸)、276 nm(36~52 min,甘草酸)、350 nm(52~70 min,藁本内酯),柱温35℃;建立逍遥丸HPLC指纹图谱,并对绿原酸、芍药苷、甘草苷、阿魏酸、甘草酸和藁本内酯主要成分进行含量测定。结果:在特征图谱研究中,共确定逍遥丸HPLC指纹图谱17个共有峰,通过与混合对照品比较,指认其中6个指标成分分别为绿原酸(4号峰)、芍药苷(6号峰)、甘草苷(8号峰)、阿魏酸(9号峰)、甘草酸(13峰)和藁本内酯(17号峰),利用相似度软件对12批样品指纹图谱进行分析,各批样品相似度均在0.99以上。在建立的色谱条件下试验6个成分分离度良好,方法精密度和重复性RSD均<2.0%,供试品溶液在24 h内稳定,各成分具有良好的线性关系和较宽线性范围;12批逍遥丸中绿原酸、芍药苷、甘草苷、阿魏酸、甘草酸和藁本内酯含量范围分别为12.254~12.325、10.375~10.449、5.571~5.645、5.066~5.140、2.006~2.080和2.667~2.741 mg·g^(-1)。结论:所建立的逍遥丸HPLC指纹图谱检测和定量测定分析方法可以有效地评价该制剂的质量。

Objective：To establish HPLC fingerprints of Xiaoyao pills and determine the main components（chlorogenic acid,paeoniflorin,liquiritin,ferulic acid,glycyrrhizic acid and ligustilide）,so as to provide reliable method for quality control.Methods：The analysis was carried out on an analytical column C18（4.6 mm×250 mm,5 μm）with gradient elution of acetonitrile（A）-0.2% phosphoric acid（B）（0-10 min,3%A;10-20 min, 3%A → 10%A;20-70 min,10%A → 80%A）at a flow rate of 1.0 mL·min^-1.The detection wavelength was set at 326 nm for chlorogenic acid（0-27 min）,at 230 nm for paeoniflorin（27-30.5 min）,280 nm for liquiritin and ferulic acid（30.5-36 min）,276 nm for glycyrrhizic acid（36-52 min）and 350 nm for ligustilide（52-70 min）.The column temperature was 35 ℃.The HPLC fingerprint of Xiaoyao pills was established,and the contents of chlorogenic acid,paeoniflorin,liquiritin,ferulic acid,glycyrrhizic acid and ligustilide were determined.Results：17 common peaks were found in fingerprints of Xiaoyao pills,6 of which were identified as chlorogenic acid（peak 4）,paeoniflorin（peak 6）,liquiritin（peak 8）,ferulic acid（peak 9）,glycyrrhizic acid（peak 13）and ligustilide（peak 17）by comparison with the reference substances.The similarities of 12 batches of samples were over 0.99.The six compounds were well separated under the chromatographic conditions.The RSD values for precision and repeatability were all less than 2.0% and the test solution was stable in 24 hours.All the compounds had good linearity and wide linear range.The content of chlorogenic acid,paeoniflorin,liquiritin,ferulic acid,glycyrrhizic acid and ligustilide in 12 batches of Xiaoyao pills were 12.254-12.325 mg·g^-1,10.375-10.449 mg·g^-1,5.571-5.645 mg·g^-1,5.066-5.140 mg·g^-1,2.006-2.080 mg·g^-1 and 2.667-2.741 mg·g^-1,respectively.Conclusion：The established fingerprints and assay methods offered effective evaluation approach for the control of Xiaoyao pills.