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浙江大学学报（医学版）
浙江大学学报（医学版）  2022, Vol. 51 Issue (5): 534-543    DOI: 10.3724/zdxbyxb-2022-0347
专题报道     
基于HPLC-Q-Exactive MS的补阳还五汤化学成分快速识别及多成分含量测定
郑留燕,万浩芳,鲁一航,丁志山,李畅,万海同
浙江中医药大学 浙江省中医脑病重点实验室，浙江 杭州 310053
Rapid identification and quantitative determination of chemical compositions in Buyang Huanwu decoction based on HPLC-Q-Exactive mass spectrometry
ZHENG Liuyan,WAN Haofang,LU Yihang,DING Zhishan,LI Chang,WAN Haitong
Zhejiang Provincial TCM Key Laboratory of Encephalopathy, Zhejiang Chinese Medical University, Hangzhou 310053, China
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摘要：

目的：建立一种基于高效液相色谱串联四极杆轨道阱质谱（HPLC-Q-Exactive MS）的分析方法，对补阳还五汤中的化学成分进行快速鉴定，并以高效液相色谱紫外检测（HPLC-UV）对其中含量较高的成分进行定量检测。方法：通过文献检索建立补阳还五汤相关化合物数据库；使用HPLC-Q-Exactive MS系统在全扫描/数据依赖性二级质谱（dd-MS2）负离子模式下采集质谱信息；借助Compound Discoverer 3.0软件分析推断补阳还五汤中的化学成分；进一步建立HPLC-UV含量测定方法，对其芍药苷、没食子酸、羟基红花黄色素A、阿魏酸、毛蕊异黄酮苷、芒柄花苷、毛蕊异黄酮、芒柄花素等8种活性成分进行定量分析。结果：通过定性分析，共鉴定出补阳还五汤中178个化合物。HPLC-UV定量分析方法学考察结果显示，8个主要成分在各自浓度范围内线性关系良好（R2≥0.9990），精密度和稳定性相对标准偏差（RSD）均小于15%，加样回收率RSD为1.6%~2.4%。结论：本研究所建立的方法体系实现了补阳还五汤中成分的快速识别和主要成分的精确定量，其中芍药苷、羟基红花黄色素A和没食子酸含量较高，有望作为质控标志物。
关键词： 补阳还五汤;  化学成分;  高效液相色谱法;  高效液相色谱串联四极杆轨道阱质谱;  定量分析    
Abstract:

Objective: To establish an analytical method for rapid identification of chemical compositions and quantitative determination of major compositions in Buyang Huanwu decoction (BYHWD) based on high performance liquid chromatography-quadrupole orbitrap mass spectrometry (HPLC-Q-Exactive MS) and high performance liquid chromatography-ultraviolet detection (HPLC-UV). Methods: The mass spectrometry information was collected in Full MS/dd-MS2 negative ion mode with HPLC-Q-Exactive MS system; the chemical compositions of BYHWD were subsequently annotated with Compound Discoverer 3.0 software and a self-built in-house compound library. Eight major compositions (paeoniflorin, gallic acid, hydroxysafflor yellow A, ferulic acid, calycosin-7-glucoside, ononin, calycosin, formononetin) were picked out and their contents were quantitatively determined with HPLC-UV analysis. Results: A total of 178 compounds in BYHWD were tentatively identified. The results of HPLC-UV quantitative analysis showed that 8 compositions had a good linear relationship in their respective concentration range (R2≥0.9990), the relative standard deviations (RSD) of precision and stability were all less than 15%, and the recovery rate RSD was between 1.6% and 2.4%. Conclusions: The method established in this study can realize the rapid identification and accurate quantification of the major compositions in BYHWD. Paeoniflorin, hydroxysafflor yellow A and gallic acid may be used as quality control markers.
Key words: Buyang Huanwu decoction    Chemical composition    High performance liquid chromatography    HPLC-Q-Exactive MS    Quantitative analysis
收稿日期: 2022-06-25 出版日期: 2022-12-28
CLC:  R917  
基金资助: 国家重点研发计划（2019YFC1708600，2019YFC1708604）; 浙江省中医药科技计划（2021ZQ025）; 浙江省自然科学基金（LR19H280001）
通讯作者: 李畅,万海同   
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引用本文:

郑留燕,万浩芳,鲁一航,丁志山,李畅,万海同. 基于HPLC-Q-Exactive MS的补阳还五汤化学成分快速识别及多成分含量测定[J]. 浙江大学学报（医学版）, 2022, 51(5): 534-543.

ZHENG Liuyan,WAN Haofang,LU Yihang,DING Zhishan,LI Chang,WAN Haitong. Rapid identification and quantitative determination of chemical compositions in Buyang Huanwu decoction based on HPLC-Q-Exactive mass spectrometry. J Zhejiang Univ (Med Sci), 2022, 51(5): 534-543.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2022-0347    或    https://www.zjujournals.com/med/CN/Y2022/V51/I5/534

图1  补阳还五汤负离子模式下的基峰离子流图
图2  补阳还五汤中178种成分来源维恩图每个小节点代表一个成分, 每个蓝色大节点表示一味中药.

成　分

样品1

样品2

样品3

芍药苷

2.636

2.616

2.561

羟基红花黄色素A

1.191

1.196

1.214

没食子酸

0.838

0.835

0.796

阿魏酸

0.345

0.349

0.331

毛蕊异黄酮苷

0.276

0.280

0.283

芒柄花苷

0.266

0.270

0.287

毛蕊异黄酮

0.184

0.186

0.188

芒柄花素

0.042

0.042

0.043
表1  补阳还五汤中八个主要成分的含量
图3  芒柄花苷的二级质谱图和可能的裂解过程
图4  没食子酰芍药苷的二级质谱图及可能的裂解过程

成　分

回归方程

线性范围(mg/mL)

R2

检出限(μg/mL)

定量限(μg/mL)

芍药苷

y = 3339.1x + 169

0.2~1.4

0.9990

4.66

14.11

羟基红花黄色素A

y = 16255x–8.7

0.1~0.7

0.9995

1.28

3.88

没食子酸

y = 48823x + 135.14

0.1~0.7

0.9995

1.38

4.19

阿魏酸

y = 42804x + 41.5

0.05~0.35

0.9997

0.55

1.65

毛蕊异黄酮苷

y = 75349x–6.8286

0.02~0.14

0.9995

0.18

0.55

芒柄花苷

y = 38839x + 99.4

0.02~0.14

0.9990

0.45

1.38

毛蕊异黄酮

y = 103824x + 205.49

0.02~0.14

0.9991

0.75

2.28

芒柄花素

y = 143088x + 54.243

0.002~0.014

0.9991

0.19

0.57
表2  补阳还五汤八个主要成分定量测定方法的线性、检出限和定量限
图5  空白溶剂、混合对照品溶液、补阳还五汤供试品溶液的高效液相色谱图16：没食子酸；41：羟基红花黄色素A； 57：芍药苷；64：阿魏酸；69：毛蕊异黄酮苷；87：芒柄花苷；104：毛蕊异黄酮；133：芒柄花素.

成　分

精密度

稳定性

重复性 (n=6)

中间精密度(n=12)

芍药苷

3.2

3.6

0.5

羟基红花黄色素A

1.5

3.7

2.2

没食子酸

1.7

2.0

12.7

阿魏酸

1.7

4.2

2.0

毛蕊异黄酮苷

1.6

1.3

1.0

芒柄花苷

1.5

2.8

2.5

毛蕊异黄酮

0.9

0.7

1.0

芒柄花素

4.3

5.0

0.4
表3  补阳还五汤八个主要成分定量测定方法的精密度和稳定性
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