基于HPLC-MS/MS和化学模式识别分析的蒙药那如-3味丸质量评价

Quality evaluations of Mongolian medicine Naru-3 Pills based upon HPLC-MS/MS and chemical pattern recognition analysis

目的:基于高效液相色谱-三重四极杆质谱(HPLC-MS/MS)建立同时测定那如-3味丸中9个成分的定量分析方法,结合化学模式识别比较不同批次间质量差异。方法:基于HPLC-MS/MS进行定量分析,色谱柱采用Shim-pack GIST-HP C18(2.1 mm×100 mm,3μm),流动相甲醇(A)-0.1%甲酸水(B),流速0.25 m L·min^(-1),柱温35℃,进样量3μL,正、负离子模式下多反应监测(MRM)方式定量分析16批次那如-3味丸中9个成分含量,并利用微生信科研绘图平台及SIMCA 14.1软件对含量数据进行聚类热图分析、主成分分析(principal component analysis,PCA)和正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA),进而筛选那如-3味丸质量差异标志物。结果:没食子酸、柯里拉京、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱、乌头碱、胡椒碱在线性范围内呈良好的线性关系(r≥0.9990),精密度、稳定性、重复性的RSD均小于3.0%,加样回收率范围在95.91%~102.92%。聚类热图和PCA分析将16批样品分为两大类,OPLS-DA法确定了6个差异标志物,分别为柯里拉京、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、乌头碱。结论:所建分析方法稳定、可行,HPLC-MS/MS结合化学计量学分析可全面评价不同厂家、不同批次间那如-3味丸差异,并筛选出差异组分,为该制剂工艺优化及质量评价提供参考。

OBJECTIVE To establish an HPLC MS/MS method for simultaneous determination of 9 chemical components in Naru-3 Pills and compare the quality variations between different batches of samples by chemometric analysis.METHODS Quantitative analysis was performed on Shim-pack GIST-HP C18(2.1 mm×100 mm,3μm)by HPLC MS/MS with a mobile phase of methanol(A)-0.1%formic acid aqueous solution(B).Flow rate was 0.25 mL·min^(-1),column temperature 35℃and volume of sample injection 3μL.The contents of 9 components in 16 batches of Naru-3 Pills were quantitatively analyzed by multi-reaction monitoring(MRM)under positive and negative ion modes.The contents data were processed by cluster heat map,principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)using bioinformatic platform and SIMCA14.1 software for screening the quality markers of Naru-3 Pills.RESULTS Gallic acid,corilagin,benzoyl⁃neoaconine,benzoylaconine,benzoylhypaconine,mesaconitine,hypaconitine,aconitine and piperine demonstrated excellent lin⁃ear relationships in a linear range(r≥0.9990).Relative standard deviations(RSDs)of precision,stability and reproducibility tests were all under 3.0%with a recovery range of 95.91%—102.92%.The 16 batches of samples were divided into two catego⁃ries by cluster heat map and PCA analysis.Corilagin,benzoylneoaconine,benzoylaconine,benzoylhypaconine,mesaconitine and aconitine were identified as differential markers by OPLS-DA.CONCLUSION The established analytical method is both stable and feasible.Combining HPLC-MS/MS and chemometrics,it may comprehensively evaluate the variations between different batches of Naru-3 Pills from different manufacturers.And key characteristic components are screened out for guiding the optimiza⁃tion of production processes and quality supervisions.