三叶青叶化学成分鉴定及其总黄酮含量测定研究

目的：建立超快速液相色谱-串联四极杆飞行时间质谱法（RRLC-Q-TOF-MS法）快速鉴定三叶青叶化学成分，根据鉴定的黄酮成分建立相适应的紫外-可见分光光度法（UV-Vis法），测定三叶青叶中总黄酮的含量。方法：RRLC-Q-TOF-MS法采用CORTECS C₁₈色谱柱（2.1 mm×100 mm，1.6 μm），流动相为乙腈（A）-0.1%甲酸水溶液（B），梯度洗脱，流速0.25 mL·min^-1，柱温45℃；飞行时间质谱采用负离子全扫描模式，数据采集范围m/z 50~1 000，通过一级质谱和二级质谱、相关文献及对照品推测鉴定化学成分。根据鉴定的黄酮进行3种总黄酮显色方法比较，确定三乙胺法显色，以UV-Vis法在400 nm波长处测定三叶青叶总黄酮含量。结果：采用RRLC-Q-TOF-MS共鉴定了三叶青叶中异荭草苷、荭草苷、异荭草素鼠李糖苷、荭草素鼠李糖苷、牡荆苷、异牡荆苷、牡荆素鼠李糖苷、异牡荆素鼠李糖苷共8个主要黄酮类成分；采用UV-Vis法测定三叶青叶总黄酮含量，以牡荆苷计，质量浓度在1.956~9.780 μg·mL^-1范围内与吸光度值呈良好线性关系（r=0.999 5），平均加样回收率99.8%（RSD=3.4%），三叶青叶总黄酮含量范围为13.38~28.67 mg·g^-1。结论：本文建立RRLC-Q-TOF-MS联用技术，为鉴定三叶青叶的黄酮成分提供了一种快速、高效的定性分析方法；建立以三乙胺显色的UV-Vis法定量分析三叶青叶总黄酮方法准确，为综合评价三叶青叶的质量提供参考。

Objective: To develop a qualitative analytical method by rapid resolution liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(RRLC-Q-TOF-MS)for identification of chemical components and an UV-Vis analytical method for determination of total flavonoids in Tetrastigma hemsleyanum Diels et Gilg leaves.Methods: The separation was performed on a CORTECSC₁₈(2. 1 mm×100 mm, 1. 6 μm) column with the mobile phase consisting of acetonitrile(A)and water containing 0. 1% formic acid(B)in gradient mode at a flow rate of 0. 25 mL·min^-1. The column temperature was set at 45℃. TOF-MS was used in negative scan mode. The data acquisition ranged from 50 to 1 000 and compounds were identified by MS spectrum, MS/MS spectrum, references and reference substances. Then triethylamine developer was chosen by comparing three UV specific visualisation methods of the identified flavonoids. With vitexin as the standard, triethylamine as the developer, UV-Vis method was applied to determination of total flavonoids at 400 nm.Results: 16 compounds including 8 organic acids(neochlorogenic,chlorogenic,cryptochlorogenic acid,caffeic acid,caffeoylquinic acid, feruloylquinic acid and two coumaroylquinic acids)and 8 flavonoids(isoorientin,orientin,isoorientin-2″ -O-rhamnoside, orientin-2″ -O-rhamnoside, vitexin, isovitexin, vitexin-2″ -O-rhamnoside and isovitexin-2″ -O-rhamnoside) in Tetrastigma hemsleyanum leaves were identified by RRLC-Q-TOF-MS. There was a good linear relationship between the concentration and absorbance of vitexin within the range of 1. 956-9. 780 μg·mL^-1(r=0. 999 5). The average recovery was 99. 8% with RSD of 3. 4%. The total flavonoids in the Tetrastigma hemsleyanum leaves ranged from 13. 38 to 28. 67 mg·g^-1.Conclusion: The results indicate that the new approach conbining RRLCQ-TOF-MS and UV-Vis is applicable in qualitative and quantitative quality control of medicinal leaves from Tetrastigma-hemsleyanum.