基于UPLC-Q-Exactive Orbitrap MS结合化学计量学筛选蟾皮区别于蟾酥的差异成分及应用研究<sup>*</sup>

陈娟, 焦阳, 汪冰, 王琳, 薛菲, 解盈盈, 闫庆康, 李向, 林永强

doi:10.16155/j.0254-1793.2024.04.10

药物分析杂志 >

2024 , Vol. 44 >Issue 4: 628 - 636

DOI: https://doi.org/10.16155/j.0254-1793.2024.04.10

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基于UPLC-Q-Exactive Orbitrap MS结合化学计量学筛选蟾皮区别于蟾酥的差异成分及应用研究^*

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1.山东中医药大学,济南 250355;
2.山东省食品药品检验研究院国家药监局胶类产品质量评价重点实验室山东省中药标准创新与质量评价工程实验室中药配方颗粒共性技术山东省工程研究中心,济南 250101;
3.山东康源堂中药饮片股份有限公司,济宁 272000

第一作者 Tel:18369277698;E-mail:chenj7698@163.com

^**Tel:13864067104;E-mail:13864067104@163.com

收稿日期: 2023-09-27

网络出版日期: 2024-06-20

基金资助

^*山东省重点研发计划(重大科技创新工程)项目(2022CXGC010511、2021CXGC010511);泉城产业领军人才支持计划创新团队项目(MRJT2105);“新高校20”项目科研带头人工作室(202228096);国家重点研发计划-中医药现代化专项,National Key R&D Program of China (2023YFC3504102);国家药品监管科学体系建设重点项目(No. RS2024Z006)

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Differential components screening of Bofonis Corium and Bufonis Venenum based on UPLC-Q-Exactive Orbitrap MS couple with chemometrics and its application research^*

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1. Shandong University of Traditional Chinese Medicine, Jinan 250355, China;
2. Shandong Institute for Food and Drug Control, NMPA Key Laboratory for Quality Evaluation of Gelatin Products, Shandong Engineering Laboratory for Standard Innovation and Quality Evaluation of TCM, Shangdong Engineering Research Center for Generic Technologies of Traditional Chinese Medicine Formula Granules, Jinan 250101, China;
3. Shandong KangYuanTang Chinese Herbal Medicine Co., Ltd., Jining 272000, China

Received date: 2023-09-27

Online published: 2024-06-20

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摘要

目的: 应用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Exactive Orbitrap MS)结合化学计量学对蟾皮区别于蟾酥的差异成分进行研究和应用。方法: 采用Waters Atlantis T3(150 mm×2.1 mm,3 μm)色谱柱,以乙腈-0.1%甲酸溶液为流动相,进行梯度洗脱,流速0.3 mL·min^-1,柱温30 ℃,选用UPLC-Q-Exactive Orbitrap MS分别在正、负离子模式下采集数据,通过主成分分析(PCA)和正交偏最小二乘-判别分析(OPLS-DA)得到候选差异离子,通过Xcalibur 3.0数据处理系统提取差异离子的二级碎片信息,再使用AB SCIEX 6500^＋三重四极杆质谱的多反应监测模式(MRM)对候选差异离子进行专属性验证,应用专属性良好的离子对建立蟾酥中掺伪蟾皮的鉴别方法,并进行方法学考察。结果: 经查找验证,共找到5个可用于识别蟾皮的差异离子对(m/z: 377.5→243.2,172.1;330.4→170.9,127.0;313.4→201.2,171.0;452.4→280.2,298.2;614.7→332.4,281.3),应用差异离子信息建立了蟾酥中蟾皮的检查方法并进行了方法学考察。考察结果表明,所建方法具有较好的专属性、耐用性,各离子对在一定范围内均呈现良好的线性关系,相关系数均大于0.996 7,精密度试验RSD为1.3%~4.1%,重复性试验RSD为1.3%~3.2%,依据上述方法对5批蟾酥市场样品进行测定,结果有2批样品出现蟾皮成分但未超出拟定的5%掺伪上限。结论: 本研究报道了一种将UPLC-Q-Exactive Orbitrap MS和化学计量学相结合的快速寻找差异信息的方法,并应用差异信息建立了蟾酥中掺伪蟾皮的检查方法。

关键词： 蟾皮; 蟾酥; 化学成分; 差异成分; 高分辨质谱; 化学计量学; 主成分分析; 正交偏最小二乘-判别分析

本文引用格式

陈娟, 焦阳, 汪冰, 王琳, 薛菲, 解盈盈, 闫庆康, 李向, 林永强 . 基于UPLC-Q-Exactive Orbitrap MS结合化学计量学筛选蟾皮区别于蟾酥的差异成分及应用研究^*[J]. 药物分析杂志, 2024 , 44(4) : 628 -636 . DOI: 10.16155/j.0254-1793.2024.04.10

Abstract

Objective: To investigate and use the different constituents in Bofonis Corium distinguishing from Bufonis Venenum based on ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS) coupled with chemometrics. Methods: UPLC analysis was performed on a Waters Atlantis T3 (150 mm×2.1 mm, 3 μm) column with gradient elution of acetonitrile and 0.1% formic acid at a flow rate of 0.3 mL·min^-1. The column temperature was set at 30 ℃. UPLC-Q-Exactive Orbitrap MS was chosen for data collection in both positive and negative ion modes. The potential differential ions were obtained by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The Xcalibur 3.0 data processing system was utilized to extract the secondary fragmentation details of the differential ions. Subsequently, the specificity of differential ions was confirmed through the multiple reaction monitoring (MRM) mode of AB SCIEX 6500⁺ triple quadrupole mass spectrometry. Identification of adulterated Bofonis Corium in Bufonis Venenum was performed by applying the ion pairs with good specificity. Results: After the research and verification, a total of five distinction pairs (m/z: 377.5→243.2, 172.1; 330.4→170.9, 127.0; 313.4→201.2, 171.0; 452.4→280.2, 298.2 and 614.7→332.4, 281.3) were discovered, and a method for the examination of Bofonis Corium in Bufonis Venenum was established and methodologically investigated by applying the information of the different ions. The constructed method had reasonable specificity and durability. Each ion pair showed an excellent linear relationship within a specific range, with correlation coefficients above 0.996 7. The RSDs of the precision test were in the range of 1.3%-4.1%. The RSDs of the repeatability test were in the range of 1.3%-3.2%. Five batches of market samples of Bufonis Venenum were determined according to the above process, and two batches of samples showed the presence of Bofonis Corium components but did not exceed the proposed 5% adulteration limit. Conclusion: In this study, a rapid method for detecting discrepancy information based on UPLC-Q-Exactive Orbitrap MS combined with chemometrics is presented, and the discrepant information is applied to establish an identification method for adulterated Bofonis Corium in Bufonis Venenum.

Key words： Bofonis Corium; Bufonis Venenum; chemical constituents; differential component; high resolution mass spectrometry; chemometric; principal component analysis; orthogonal partial least squares discriminant analysis

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