基于UPLC-Q-TOF MS的广佛手指纹图谱建立及炮制前后成分差异分析

Construction of UPLC-Q-TOF MS Fingerprints for Fingered Citron and Its Ingredient Differences Analysis before and after Processing

采用超高效液相色谱-四极杆串联飞行时间质谱(UPLC-Q-TOF MS)建立生、制广佛手的指纹图谱,并通过化学模式识别方法分析两者的差异成分。采用Agilent Poroshell 120 EC-C18(3.0 mm×150 mm×2.7μm)色谱柱进行分离,以0.1%甲酸+5 mmol/L乙酸铵水溶液-乙腈为流动相进行梯度洗脱,离子源为电喷雾电离源(ESI),在正、负离子模式下采集数据。根据分子离子峰精确质量数、二级谱图等信息,结合文献和化合物数据库并经标准品验证,共鉴定生、制广佛手中37个化学成分,包括:10个黄酮及黄酮苷类、4个香豆素类、12个有机酸类、5个糖苷类、2个柠檬苦素类及4个其它类。将采集的UPLC-Q-TOF MS数据整理并导入“中药色谱指纹图谱相似度评价系统(2004A)”软件,分别建立生、制广佛手的指纹图谱,其中生广佛手确定了24个共有峰,识别和鉴定出19个峰成分;制广佛手确定了22个共有峰,识别和鉴定出14个峰成分。将数据预处理后,以SIMCA-P软件建立主成分分析(PCA)与正交偏最小二乘法判别分析(OPLS-DA)统计模型,分析生、制广佛手的差异成分。PCA结果显示生、制广佛手区分明显,表明生广佛手炮制后的化学成分发生变化;采用OPLS-DA法筛选显著性差异成分为:诺米林酸、5-异戊烯氧基-7-甲氧基香豆素、棕榈酸、油酸、水合氧化前胡素,其中5-异戊烯氧基-7-甲氧基香豆素的相对含量变化最显著,可作为区分生、制广佛手的化学标志物。该研究可为生、制广佛手的质量标准以及药效学研究提供数据支持。

An ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF MS)was applied to construct the fingerprints of raw Fingered Citron and processed Fingered Citron.Meanwhile,the chemical components differences between raw Fingered Citron and processed Fingered Citron were analyzed by chemical pattern recognition methods.The sample was firstly ultrasonically extracted with methanol,and then separated on an Agilent Poroshell 120 EC-C18(3.0 mm × 150 mm, 2.7 μm)column by gradient elution, with 0.1%formic acid +5 mmol/L ammonium acetate and acetonitrile as the mobile phases.The MS system with electrospray ionization(ESI)source was operated under positive and negative ion mode.Based on the accurate mass, fragment ions, retention behaviors, previous reports,net-work database and the fragmentation pathways,a total of 37 components in the raw Fingered Citron and the processed Fingered Citron were identified,including 10 flavonoids and flavonoid glycosides,4 coumarins,12 organic acids,5 glycosides,2 limonin and 4 other compounds.The fingerprints for the two Fingered Citrons were established,and analyzed by a similar evaluation system for chromatographic fingerprint of traditional Chinese medicine(version 2004 A,National Committee of Pharmacopoeia,China).The mass of each peak was accurately measured,and their chemical formula were generated via the MassHunter software.The MS/MS spectrum for each peak was compared with the mass database and literature,and their chemical structures were deduced based on the MS/MS fragment,and finally verified by the reference substance.In total,the UPLC-Q-TOF MS fingerprint for raw Fingered Citron contained 24 common peaks,of which 19 were identified.In addition,there were 22 common peaks in the UPLC-Q-TOF MS fingerprint for processed Fingered Citron,and 14 peaks were identified.The principal component analysis(PCA)and orthogonal partial-least squares discriminant analysis(OPLS-DA)were adopted to analyze these data of two herbal medicines via SIMCA-P software(pretreatment with Profinder software).The results of PCA and OPLS-DA showed that the two Fingered Citrons were significantly discriminated.Nomilin acid,5-isopentenyloxy-7-methoxycoumarin, palmitic acid, oleic acid and oxypeucedanin hydrate were identified, and screened as the chemical markers that attributed to classification.Among them,the content of 5-isopentenyloxy-7-methoxycoumarin changed prominently,and was chosen as the chemical marker for differentiation on raw Fingered Citron and processed Fingered Citron.This study provides a data support for the quality control of raw Fingered Citron and processed Fingered Citron.