本项由国家自然科学基金（82130113）、国家自然科学基金（82204765）、四川省自然科学基金（2022NSFSC1470）、四川省博士后专项资助项目（TB2023020）和成都中医药大学杏林学者研究推广计划（BSH2021030）资助。这些资金在实验设备、实验材料、出版费用等方面提供支持。

1. 准备工作
<ol><li>样品制备<ol><li>使用 1/10,000 灵敏度天平在 3 mL 微量离心管中称取 0.25 g（干重）APB，加入 2.5 mL 甲醇，然后超声处理 30 分钟（功率 240 W，频率 40 kHz）。</li>
		<li>以1.2× g 离心5分钟，收集上清液，并通过孔径为0.22μm的膜过滤器过滤。</li>
	</ol></li>
	<li>实验材料的制备<ol><li>制备二维流动相，以乙腈为有机相B相，配置0.1%甲酸超纯水为水相A。</li>
		<li>进行两相过滤（0.22μm）并使用超声机（40kHz）超声15分钟。吹扫更换的流动相以去除气泡。本实验采用C18色谱柱作为1D色谱柱，亲水性色谱柱作为2D色谱柱，并将色谱柱安装到仪器上。</li>
	</ol></li>
	<li>调整适当的分流比<ol><li>将 2D-LC 仪器输出线通过三通连接到质量入口，将三通的另一端连接到分流线。将流速调节到合适的值，以确保进入质谱的流速为 0.3-0.5 mL/min。 注：由于 2D-LC 的第二维通常设置为 2 mL/min 以上，因此该流速对于 MS 来说太大，因此有必要进行分流。</li>
	</ol></li>
</ol>2. 2D-LC操作
<ol><li>双击 Instrument 1 Online 图标，化学工作站将自动与 1260 LC 通讯并进入工作站。</li>
	<li>对于2D-LC方法参数设置，请从View菜单中选择Method and Run Control屏幕以调用所需的接口，该接口通常会默认输入。点击进样器模块，右键点击进样方法，设置一维泵的进样体积、流速和流动相时间梯度。</li>
	<li>在“停止时间”下输入样本运行时间。单击主菜单“仪器”，然后单击下拉菜单中的“2D-LC 方法”。在 2D-LC 模式下选择 Comprehensive。在调制时间输入 2 分钟，在 2D 梯度停止时间输入 1.9 分钟。将流量设置设置为 2 mL。编辑 2D 渐变并设置为波长。编辑方法后，在“方法”菜单上选择“方法另存为”以命名新方法，然后单击“确定”。 注意： 外部输送泵需要手动设置流量。</li>
</ol>3. MS操作
<ol><li>打开真空泵的开关。打开氩气瓶主阀和分压阀，调节压力至0.3MPa左右。打开氮气阀。</li>
	<li>等待至少 8 小时以确保有足够的真空度用于实验条件。分析前检查氩气和氮气的气压是否足够高。</li>
	<li>要启动 MS 控制软件，请单击软件面板中的加热 SEI 源 并输入 MS 参数，包括加热器温度 （350 °C）、鞘流速 （35 arb）、辅助空气流速 （15 arb）、喷雾电压（正模式下为 3.8 KV，负模式下为 -2.5 KV）和毛细管温度 （275 °C）。单击 “应用 ”按钮以激活离子源。</li>
	<li>要设置 MS 方法，请输入值以配置采集时间、极性、质量范围、转移值数量、转移值持续时间等。设置 MS 运行时间（分钟）：93.00。设置 ScanEvent 详细信息：ITMS + c norm o （100.0-1200.0），CV=0.0v。ITMS + c norm Dep MS/MS，来自（1）的最强离子：活化类型：CID，所需最小信号：500，隔离宽度：2.00，归一化coll.能量：35.0，默认充电状态：3，活化Q：0.250，活化时间：93.000。要设置与数据相关的设置，请使用禁用的单独极性设置，顶部为中性损耗：3，顶部为产品：3。 单击“ 保存 ”以将设置配置为仪器方法。 注意：运行时间结束与 2D-LC 一致。</li>
	<li>单击“ 序列设置 ”按钮以打开序列表。在表单中输入样品类型、文件名、路径、样品ID、仪器方法、位置、进样量等信息。</li>
	<li>单击“ 保存 ”按钮记录序列列表，然后单击“ 开始分析 ”按钮设置并开始 MS 采集。单击 2D-LC 仪器上的 Run Control ，然后选择 Run Method。同时，MS 仪器还会单击 “运行 ”按钮。 注意：由于在此实验中，2D-LC 和 MS 不是由同一软件控制的，因此需要在两台仪器上分别设置它们。</li>
</ol>4. 分子网络操作
<ol><li>数据准备：导出 2D-HPLC-MS 原始质谱数据，并通过 ProteoWizard 的 Msconvert 将二次质谱数据转换为 mzXML 或 mzML 数据。（http://proteowizard.sourceforge.net/）。</li>
	<li>如需上传数据，请在官网下载WinSCP软件，并通过FTP协议将mzML数据上传到GNPS创建的账户。安装完成后，启动 WinSCP。</li>
	<li>连接到GNPS FTP服务器：在WinSCP界面的 会话 配置页面上，填写以下连接信息： 文件协议：选择FTP。对于主机名：输入 massive.ucsd.edu.，对于端口号：保留默认值 22。</li>
	<li>输入帐号和密码。填写完以上信息后，点击 登录 按钮，建立与GNPS FTP服务器的连接。</li>
	<li>通过打开网络浏览器并访问 GNPS 网站 （https://gnps.ucsd.edu/） 来创建分子网络。新用户需要注册一个帐户，然后登录。</li>
	<li>在GNPS网站主界面，点击顶部导航栏的 “任务” 选项卡，然后从下拉菜单中选择“ 创建新任务 ”。在任务创建页面上，单击“ 添加 文件”按钮，然后选择并上传数据文件。（例如，mzML 格式）。</li>
	<li>在“任务参数”选项卡中，设置生成 MN 的各种参数。这些参数包括峰值提取算法、峰值超程值、相似度计算方法等。根据需要进行适当的参数设置。单击页面底部的 “启动分析 ”按钮以运行任务。</li>
	<li>任务运行完成后，在任务列表中找到已创建的任务，单击 服务名称 ，查看分析结果。该网站提供 MN 图、替代品、共生网络和其他相关信息。</li>
</ol>

使用分子联网的 2D-HPLC-MS 分析藏药植物 APB

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作者声明没有相互竞争的经济利益。

该实验的主要重点是在二维液相分离框架内优化部分方法。为了实现这一点，一种新型的柱下稀释调节剂被无缝集成到二维液相色谱（2D-LC）系统中。这项技术调整至关重要，因为它大大提高了分析藏药中存在的化学成分（称为APB）的熟练程度。结合 2D-HPLC-MS 技术，可以全面检查 APB 的复杂成分。通过利用二维分离技术的优点，我们在分辨率和灵敏度方面都取得了显著的提高。这转化为对更广泛的...

藏医药是中医不可分割的一部分，遵循藏医实践原则，用于疾病的预防和治疗1。然而，藏草药含有复杂的植物化学成分，其特点是含量波动很大。对基本生物活性元素的有限理解已成为藏医现代化的瓶颈2.液相色谱-质谱（LC-MS）的应用，将色谱法的强大分离能力与质谱法（MS）的高灵敏度相结合，在天然药物分析中得到了广泛的应用3。然而，由于分离机理单一的局限性，在一维液相色谱分离中，结构高度相似的组分往往会产生共洗脱。在随后的质谱分析中，由于高丰度组分引起离子抑制，低丰度共洗脱组分难以检测4。
2D-HPLC是二维高效液相色谱法的缩写，它是一种新颖的色谱方法，它使用一对色谱柱将不同的分离机制和谐地结合在一起。这包括正相色谱法与反相液相色谱法的融合或交替，以及亲水作用液相色谱法与反相液相色谱法的融合或交替反应5。通过合并这些互补的色谱特性，实现了增强的分离能力，有效地解决了复杂样品基质带来的挑战6。此外，通过将二维色谱与MS相结合，可以将2D-HPLC的强大分离能力与MS的高灵敏度检测能力充分融合，为复杂药物系统及其基本成分的研究提供支持7,8,9。
藏医通常具有多种成分和功能，其中活性成分通常以复杂的成分和最低浓度存在。通过将 2D-LC 作为强大的分离系统和 MS 作为极其灵敏的检测器集成在一起，可以更有效地解决复杂样品在分离和鉴定方面带来的挑战 10,11,12。这种融合极大地促进了对藏药化学成分的探索。
无论是传统的LC-MS还是2D-LC-MS，都可以获得大量的信息。然而，从如此海量的信息中提取复杂系统组件的结构信息一直是一个重大挑战。因此，研究人员开发了各种筛选和挖掘MS数据的方法。Global Natural Products Social Molecular Networking （GNPS） 是一个 MS/MS 数据组织和可视化平台，可以上传 2D-LC-MS 质谱数据13.每个光谱都被视为一个矢量，并使用余弦相似性与所有其他光谱进行比较。当两个光谱之间的相似性超过阈值时，它们就会在分子网络 （MN） 中连接起来。这可用于已知化合物的快速鉴定和各种未知天然产物的测定14。
藏药通常具有多种功能，但其成分复杂且浓度差异显著，难以有效阐明功能与物质基础的关系15。对藏医的深入研究需要对尽可能多的成分进行系统的描述。在本文的框架下，我们拟以APB在藏医学中的应用为研究对象，展示利用2D-HPLC-MS技术和MN技术系统研究藏药复杂化学成分的策略和过程。在二维色谱系统的构建中，我们将反相液相色谱法和亲水作用液相色谱法相结合，具有显著的分离机理，实现了对APB16复杂组分的更有效分离。此外，为了克服两维溶剂不相容性的问题，采用了柱前稀释调制模式。通过将2D-LC的强大分离能力与MS的高灵敏度检测能力相结合，可以更有效、更全面地获取APB中复杂组分的光谱信息。此外，通过MN技术对海量光谱信息的网络化和可视化，对APB的成分进行了系统分析。本研究所展示的策略和过程有望应用于其他藏药的研究，促进藏药物质基础的研究，对推进藏药资源化、提高藏药材质量控制标准具有重要意义17。整个实验过程如图 1所示。
在此介绍的实验中，在安捷伦二维液相色谱 （2D-LC） 系统中引入了一种新的柱前稀释调节剂18。通过添加独立的输送泵，改变了分析的流路，从而实现了 2D-LC 分析的高正交性。两个维度之间的耦合和切换由两个六通阀完成， 如图 2 所示。当一个样本环在第一维中填充时，另一个样本环将在第二个维度中进行分析。这意味着 1D 循环的填充时间和 2D 的运行时间相等。这需要二元泵在二维中生成的快速梯度。在分析整个流出物时，不会丢失任何峰。这对于未知样品的分析特别有用。它会导致大量的 2D 色谱图，需要将它们组合起来进行数据分析。

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			<td>Acetonitrile</td>
			<td>Fisher chemical</td>
			<td>F22M81203</td>
			<td>Mobile phase</td>
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			<td>Aconitum pendulum</td>
			<td>/</td>
			<td>/</td>
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			<td>Agilent 1290 Infinity (II) 2D-LC&#160;</td>
			<td>Agilent Technologies</td>
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			<td>Liquid chromatography</td>
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			<td>Chengdu Keen experimental equipment</td>
			<td>/</td>
			<td>1ml</td>
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			<td>EP tube</td>
			<td>Chengdu Keen experimental equipment</td>
			<td>/</td>
			<td>3ml</td>
		</tr>
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			<td>Liquid phase injection bottle</td>
			<td>Chengdu Keen experimental equipment</td>
			<td>/</td>
			<td>1.5ml</td>
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			<td>LTQ XL Mass Spectrometer</td>
			<td>Thermo Fisher</td>
			<td>LTQ21991</td>
			<td>Mass Spectrometer</td>
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			<td>Microporous membranes&#160;</td>
			<td>Chengdu Keen experimental equipment</td>
			<td>/</td>
			<td>0.22&#956;m</td>
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			<td>Ultimate XB-C18,5 &#956;m,2.1 x 200 mm</td>
			<td>Welch</td>
			<td>00201-31015</td>
			<td>Reversed-phase column</td>
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			<td>Ultrasonic Cleaner</td>
			<td>GT Sonic</td>
			<td>UGT20DEC048Y</td>
			<td>Ultrasonic Cleaner 240W 40KHz</td>
		</tr>
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			<td>XAmide,3 &#956;m,100A</td>
			<td>Dalian Mondi Technology</td>
			<td>D2019110601</td>
			<td>Hydrophilic column</td>
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2d-hplc-ms technology combined with molecular network for the identification of components in tibetan medicine aconitum pendulum

本研究采用综合方法，利用2D-HPLC-MS技术结合分子网络，揭示了藏药植物APB错综复杂的化学成分。通过实施2D-HPLC，实现了复杂混合物的增强分离，从而能够分离出单个化合物以进行后续分析。分子网络方法进一步有助于阐明这些化合物之间的结构关系，有助于确定潜在的生物活性分子。这种综合策略有效地识别了植物中存在的各种化学成分。研究结果揭示了APB中多种化学成分，包括生物碱等。这项研究不仅促进了对这种传统藏药植物化学特征的理解，而且对其潜在的治疗特性提供了宝贵的见解。2D-HPLC-MS与分子网络的整合被证明是系统地探索和鉴定草药中复杂化学成分的有力工具，为天然产物发现领域的进一步研究和开发铺平了道路。

Tibetan medicine is an integral part of traditional Chinese medicine, adhering to the principles of Tibetan medical practices, and is used for disease prevention and treatment1. However, Tibetan herbal medicine contains complex plant chemical constituents characterized by significant fluctuations in content. Limited understanding of the fundamental bioactive elements has become a bottleneck in the modernization of Tibetan medicine2. The application of liquid chromatography-mass spectrometry (LC-MS), combining the strong separation power of chromatography with the high sensitivity of mass spectrometry (MS), has been widely used in natural medicine analysis3. However, due to the limitation of a single separation mechanism, components with highly similar structures tend to coelute in one-dimensional liquid chromatography separation. In subsequent mass spectrometric analysis, the low-abundance co-eluted components are difficult to detect due to ion suppression caused by high-abundance components4.
2D-HPLC, which stands for two-dimensional high-performance liquid chromatography, represents a novel chromatographic method that harmoniously combines different separation mechanisms using a pair of columns. This includes the fusion or alternation of normal-phase chromatography with reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with reversed-phase liquid chromatography5. By merging these complementary chromatographic properties, the enhanced separation capability is achieved, effectively addressing the challenges caused by complex sample matrices6. Furthermore, by coupling two-dimensional chromatography with MS, the powerful separation ability of 2D-HPLC and the high sensitivity detection capability of MS can be fully integrated, providing support for the study of complex drug systems and their fundamental constituents7,8,9.
Tibetan medicine commonly features a multifaceted array of components and functionalities, where active ingredients typically exist in intricate compositions and at minimal concentrations. By integrating 2D-LC as a robust separation system and MS as an exceedingly sensitive detector, the challenges posed by intricate samples in terms of separation and identification can be more effectively addressed10,11,12. This amalgamation substantially contributes to advancing the exploration of the chemical composition of Tibetan medicine.
Regardless of whether it is traditional LC-MS or 2D-LC-MS, a vast amount of information can be obtained. However, extracting structural information of complex system components from this massive amount of information has always been a significant challenge. Therefore, researchers have developed various methods for screening and mining MS data. Global Natural Products Social Molecular Networking (GNPS) is an MS/MS data organization and visualization platform where 2D-LC-MS mass spectrometry data can be uploaded13. Each spectrum is considered as a vector and compared to all other spectra using cosine similarity. When the similarity between two spectra exceeds a threshold, they are connected in a molecular network (MN). This can be used for the rapid identification of known compounds and the determination of various unknown natural products14.
Tibetan medicine usually has multiple functions, but its complex composition and significant concentration differences make it difficult to effectively elucidate the relationship between function and material basis15. An in-depth study of Tibetan medicine requires the systematic characterization of as many components as possible. In the framework of this study, we intend to use APB in Tibetan medicine as the research object to demonstrate the strategy and process of systematically studying the complex chemical components of Tibetan medicine using 2D-HPLC-MS technology and MN technology. In the construction of the 2D chromatographic system, we combined reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with significant separation mechanisms to achieve more effective separation of the complex components of APB16. In addition, to overcome solvent incompatibility between the two dimensions, an at-column dilution modulation mode was adopted. By combining the powerful separation capability of 2D-LC with the high sensitivity detection capability of MS, the spectral information of the complex components in APB is obtained more effectively and comprehensively. Furthermore, through the network and visualization of massive spectral information by MN technology, the components of APB are systematically analyzed. The strategy and process demonstrated in this study are expected to be applied to the study of other Tibetan medicines, promoting research on the material basis of Tibetan medicine, which is of great significance for advancing Tibetan medicine resources and improving quality control standards for Tibetan herbs17. The overall experimental process is shown in Figure 1.
In the experiment presented here, a new at-column dilution modulator was introduced into the Agilent two-dimensional liquid chromatography (2D-LC) system18. By adding an independent delivery pump, the flow path of the analysis was changed, resulting in a high orthogonality of the 2D-LC analysis. The coupling and switching between the two dimensions are accomplished by two six-port valves, as shown in Figure 2. When one sample loop is filled in the first dimension, another sample loop is analyzed in the second dimension. This means that the filling time of the 1D loop and the running time of the 2D are equal. This requires the fast gradient generated by the Binary pump in the second dimension. No peaks are lost when the entire effluent is analyzed. This is particularly helpful for the analysis of unknown samples. It results in a large number of 2D chromatograms that need to be combined for data analysis.

作者聚焦：在自然医学分析中集成 2D-HPLC-MS 和分子网络

2D-HPLC-MS技术结合分子网络鉴定藏药乌头成分

利用APB作为模型生物，验证了2D-HPLC-MS技术结合MN方法的可行性。通过将 MS 原始数据导入到 MN 中，并将参数设置为默认值，可以生成 MN。MN 是一种可视化计算策略，可将完整的 LC-MS-MS 实验中检测到的所有分子离子以及这些分子离子之间的化学关系可视化13。
MN是基于化合物进入MS-MS后形成的二级质谱片段;结构相似的化合物在相同条件下产生相似的MS-MS离子碎?...

Watch this Scientific Journal Video about Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis at JoVE.com

本研究利用二维高效液相色谱-质谱联用（2D-HPLC-MS）技术结合分子网络，揭示了藏药植物 乌头-摆 布希（APB）的复杂化学成分。本文为系统探索和鉴定草药的复杂化学成分提供了详细的方案。

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate ...

在这项研究中，我们采用了一种综合方法，利用2D-HPLC-MS技术结合分子网络来揭示藏药植物APB的复杂化学成分。我们希望建立一种更有效的方法来识别天然药物的化学成分。目前实验的主要挑战是质谱部分没有配备高分辨率质谱法，导致检测出不同的组分。2D-LC-MS 可实现更准确的化合物鉴定和定量分析。基于样品分离的复杂混合物，结合分子网络技术，可以更好地处理复杂的数据信息，并提高任何大小的效率和准确性。我们的实验室将专注于应用现有技术来分离天然、药用和化学成分。

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2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

308 次观看.  Chengdu University of Traditional Chinese Medicine. 在这项研究中，我们采用了一种综合方法，利用2D-HPLC-MS技术结合分子网络来揭示藏药植物APB的复杂化学成分。我们希望建立一种更有效的方法来识别天然药物的化学成分。目前实验的主要挑战是质谱部分没有配备高分辨率质谱法，导致检测出不同的组分。2D-LC-MS 可实现更准确的化合物鉴定和定量分析。基于样品分离的复杂混合物，结合分子网络技术，可以更好地处理复...

视频: 作者聚焦：在自然医学分析中集成 2D-HPLC-MS 和分子网络

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.