黑种草子定性定量方法改进研究

目的：本文以异株五加甲苷为指标成分，对黑种草子定性定量方法进行改进研究。方法：采用改进后的TLC法和HPLC-ELSD法定性鉴别和定量测定异株五加甲苷。以硅胶HSG为薄层板，正丁醇-水-醋酸（2.5∶1∶0.5）为展开剂，10%硫酸乙醇试剂显色后在日光下检视，进行TLC定性分析。采用C₁₈色谱柱，以乙腈-乙酸铵系统为流动相进行梯度洗脱，ELSD为检测器，建立HPLC定量分析方法。同时按照2015年版《中华人民共和国药典》（以下简称中国药典）中质量控制方法对黑种草子药材进行处理，其结果与新建立的质量评价方法进行对比及相关性研究。结果：所建立的TLC方法斑点丰富、清晰、分离度好。异株五加甲苷在0.023~3.022 mg·mL^-1范围内线性关系良好（r=0.999 6），平均加样回收率（50%、100%、150%）分别为98.8%（RSD=1.8%）、101.4%（RSD=1.0%）及100.8%（RSD=1.9%），方法的精密度、重复性和稳定性试验RSD小于2.2%，HPLC-ELSD法定量异株五加甲苷方法学满足分析测定要求。16批样品分别采用新建与2015年版中国药典收载的方法进行含量测定，相关系数为0.952，判定结果一致。结论：新方法操作简便、省时、专属性高，并能提高检验效率、降低检测成本，为黑种草子质量标准的提升提供了依据。

Objective:To improve the qualitative and quantitative method of Nigellae Semen by selecting sieboldianoside A as the marker.Methods:Optimized TLC and HPLC-ELSD methods were used to identify and quantify sieboldianoside A respectively.The TLC method was developed by using 1-butanol-water-acetic acid (2.5:1:0.5)as developing solvent on HSG.The thin-layer plate was inspected under the condition of sunlight after coloration with 10% sulfuric acid in ethanol.HPLC analysis was performed on a reverse phase C₁₈ column and gradiently eluted with a mobile phase of acetonitrile-ammonium acetate.Evaporative light scattering detector (ELSD)was used as detector.At the same time,16 batches of samples were analyzed using methods adopted in ChP 2015,and the results were compared with the ones obtained by the optimized methods.Results:The established TLC method was characteristic of abundant and clear spots with good separation.Sieboldianoside A displayed ideal linearity within the range of 0.023-3.022 mg·mL^-1 (r=0.999 6).The average recoveries (50%,100% and 150%)of sieboldianoside A were 98.8% (RSD=1.8%),101.4% (RSD=1.0%)and 100.8% (RSD=1.9%),respectively.The RSD values of precision,repeatability and stability were all less than 2.2%. The methodology results of HPLC-ELSD quantitative determination method met the requirement of analysis determination.16 batches of samples were quantified by the old and new methods,and the results were the same as pearson's correlation coefficient of 0.952.Conclusion:The optimized method is easy,time-saving and specific,offering higher identification efficiency and lower examination costs,thus it provide experimental basis for the promotion of Nigellae Semen's standard.