基于UPLC指纹图谱和一测多评法的虎杖药材质量评价

Quality Evaluation of Polygonum cuspidatum by UPLC Fingerprint and QAMS

目的:建立虎杖药材的超高效液相色谱(UPLC)指纹图谱,并测定药材中4种活性成分的含量,为虎杖药材的质量评价提供依据。方法:以色谱柱为Waters BEH C18(100 mm×2.1 mm,1.7μm)、流动相为乙腈-0.2%甲酸溶液(梯度洗脱)、流速为0.4 mL/min、柱温为40℃、检测波长为290 nm、进样量为1μL的色谱条件建立虎杖药材的UPLC指纹图谱,并采用相似度计算、聚类分析和正交偏最小二乘法判别分析方法对指纹图谱进行评价。以虎杖苷为内参物,测定白藜芦醇、大黄素-8-O-β-D-葡萄糖苷、大黄素的相对校正因子,建立一测多评法;再利用相对校正因子计算15批虎杖药材中上述4种成分的含量,并将一测多评法与外标法的结果进行比较。结果:建立了15批虎杖药材的UPLC指纹图谱,确定了12个共有峰,指认出了其中5个成分,分别为虎杖苷、白藜芦醇、大黄素-8-O-β-D-葡萄糖苷、大黄素和大黄素甲醚。15批虎杖药材指纹图谱的相似度为0.865~0.976;聚类分析将15批药材分为4类,并呈现一定的产地规律性;正交偏最小二乘法判别分析共确定了7个差异性标志物,差异显著性排序为峰7>大黄素-8-O-β-D-葡萄糖苷>白藜芦醇>峰8>虎杖苷>峰1>峰10。采用一测多评法测得的15批虎杖药材中白藜芦醇、大黄素-8-O-β-D-葡萄糖苷、大黄素的含量与外标法测得的含量的相对平均偏差均小于5.0%,说明两种方法无明显差异。结论:UPLC指纹图谱结合一测多评法可便捷、可靠地对虎杖药材进行质量评价;安徽和重庆所产虎杖药材的质量较优。

OBJECTIVE:To establish the UPLC fingerprint of Polygonum cuspidatum,and to determine the contents of four active ingredients and to provide reference for the quality evaluation of P.cuspidatum.METHODS:The determination was performed on Waters BEH C18 column(100 mm×2.1 mm,1.7μm)with mobile phase consisted of acetonitrile-0.2%formic acid(gradient elution)at flow rate of 0.4 mL/min.The column temperature was 40℃,and detection wavelength was 290 nm.The sample size was 1μL.The fingerprints were evaluated by similarity calculation,cluster analysis and orthogonal partial least square discriminant analysis(OPLS-DA).Using polydatin as internal standard,relative calibration factors of resveratrol,emodin-8-O-β-D-glucoside and emodin were determined to develop a method of QAMS.The contents of 4 above components in 15 batches of P.cuspidatum were calculated by relative calibration factors.The results of QAMS were compared with those of external standard.RESULTS:UPLC fingerprints of 15 batches of P.cuspidatum were established,and 12 common peaks were confirmed.Five components were identified,i.e.polydatin,resveratrol,emodin-8-O-β-D-glucoside,emodin,emodin methyl ether.The fingerprint similarity of 15 batches of P.cuspidatum was in the range of 0.865-0.976.According to cluster analysis,15 batches of P.cuspidatum were classified into 4 categories,showing certain regularity of origin.Seven markers were identified by OPLS-DA method.The order of difference significance was peak 7>emodin-8-O-β-D-glucoside>resveratrol>peak 8>polydatin>peak 1>peak 10.The relative deviation among the contents of resveratrol,emodin-8-O-β-D-glucoside and emodin in 15 batches of P.cuspidatum determined by QAMS and external standard method was less than 5.0%,indicating that there was no significant difference between the two methods.CONCLUSIONS:UPLC fingerprint combined with QAMS method is convenient and reliable for the quality evaluation of P.cuspidatum;the quality of P.cuspidatum produced in Chongqing and Anhui province is better.