经典名方固阴煎基准样品HPLC指纹图谱研究

HPLC fingerprinting study of classical formula Guyin Decoction

目的 建立经典名方固阴煎基准样品HPLC指纹图谱,结合多元统计分析对15批固阴煎基准样品进行质量评价,为其基准样品质量控制研究提供参考。方法 采用Waters SymmetryR C18(250 mm×4.6 mm,5μm)色谱柱,以乙腈-0.1%磷酸水溶液为流动相,梯度洗脱(0~10 min,0~5%乙腈;10~20 min,5%~10%乙腈;20~33 min,10%~11%乙腈;33~42 min,11%~18%乙腈;42~50 min,18%乙腈;50~80 min,18%~40%乙腈;80~100 min,40%~100%乙腈),体积流量1 mL/min,柱温40℃,检测波长230 nm,进样体积5μL,建立固阴煎基准样品HPLC指纹图谱,并进行方法学考察。采用“中药色谱指纹图谱相似度评价系统”(2012版)对15批固阴煎基准样品指纹图谱进行相似度评价,匹配共有峰,并对各共有峰进行归属研究。结合聚类分析、主成分分析(principal component analysis,PCA)与正交偏最小二乘-判别分析(orthogonal partial least squares-discriminant analysis,OPLS-DA)对共有峰结果进行统计学分析,评价不同批次固阴煎基准样品的质量差异,并找寻造成批间质量差异的主要成分及药味来源。结果 固阴煎基准样品HPLC指纹图谱方法学验证良好,15批固阴煎基准样品与对照指纹图谱的相似度均大于0.9。共标定了24个共有峰,经与混合对照品比对,鉴定11个色谱峰,分别为1号峰没食子酸、3号峰5-羟甲基糠醛、6号峰莫诺苷、7号峰绿原酸、9号峰马钱苷、11号峰甘草苷、12号峰金丝桃苷、13号峰毛蕊花糖苷、14号峰3,6′-二芥子酰基蔗糖、22号峰甘草酸、23号峰五味子醇甲。聚类分析和PCA可将15批基准样品分为2类,分析结果可互相印证。OPLS-DA(VIP>1)找到莫诺苷、5-羟甲基糠醛、绿原酸在内的11个差异性色谱峰,分别归属于单味药制远志、炒菟丝子、山茱萸、炙甘草和熟地黄。结论 建立的固阴煎基准样品HPLC指纹图谱专属性强、灵敏度高、分离度好、重复性、稳定性良好,除人参和炒山药以外均有表征,可为其后续制剂开发和质量控制研究提供参考。

Objective To establish HPLC fingerprints of the benchmark samples of the classical formula Guyin Decoction(GYJ) and to analyze and evaluate 15 batches of the benchmark samples by combining multivariate statistical analysis,so as to provide reference for the development of its benchmark samples.Methods A Waters SymmetryR C18(250 mm × 4.6 mm,5 μm) column was used with acetonitrile(A) and 0.1% phosphoric acid aqueous solution(B) as the mobile phase and gradient elution(0—10 min,0—5% A;10—20 min,5%—10% A;20—33 min,10%—11% A;33—42 min,11%—18% A;42—50 min,18% A;50—80 min,18%—40% A;80—100 min,40%—100% A) at a flow rate of 1 mL/min,column temperature of 40 ℃,detection wavelength of 230 nm,injection volume of 5 μL,to establish the HPLC fingerprints of the GYJ benchmark samples and conduct methodological investigation.The fingerprints of the 15 batches of GYJ samples were evaluated by using the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine”(2012 version) to match the shared peaks,and the attribution of the shared peaks was studied.The results were combined with cluster analysis,principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) to evaluate the quality differences among the batches of GYJ benchmark samples and to find the main components and source flavors that caused the quality differences between batches.Results The methodological validation of the HPLC fingerprints of the benchmark samples of GYJ was good,and the similarity between the fingerprints of the 15 batches of the benchmark samples and the control was greater than 0.9.A total of 24 shared peaks were found,and 11 peaks were identified by comparison with the mixed standards,namely,gallic acid(peak 1),5-hydroxymethylfurfural(peak 3),morroniside(peak 6),chlorogenic acid(peak 7),loganin(peak 9),liquiritin(peak 11),hyperoside(peak 12),verascoside(peak 13),3,6′-disinapoylsucrose(peak 14),glycyrrhizic acid(peak 22),schisandrin(peak 23).Cluster analysis and PCA analysis could classify the 15 batches of benchmark samples into two categories,and the results could be corroborated with each other.OPLS-DA(VIP > 1) excavated 11 differential peaks including morroniside,5-hydroxymethylfurfural,and chlorogenic acid,which were derived from Yuanzhi(Polygalae Radix,Glycyrrhizae Radix et Rhizoma decoction processed),fried Tusizi(Cuscutae Semen),Shanzhuyu(Corni Fructus),Zhigancao(Glycyrrhizae Radix et Rhizoma Praeparata cum Melle) and Shudihuang(Rehmanniae Radix Praeparata),respectively.Conclusion The established HPLC fingerprints of the benchmark samples of GYJ are highly specific,sensitive,well separated,reproducible and stable,and were characterized except for Ginseng Radix et Rhizoma and fried Dioscoreae Rhizoma,which can provide reference for its subsequent formulation development and quality control studies.