sample preparation for 2d-hplc-ms of aconitum pendulum

Analyzing Tibetan Medicinal Plant APB Using 2D-HPLC-MS with Molecular Networking

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.

Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis

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

In this study, we employed a comprehensive approach utilizing 2D-HPLC-MS technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant APB. We want to establish a more efficient method for identifying the chemical composition of natural medicines. The main challenging of the current experiments is that the mass spectrometry section is not equipped with high-resolution mass spectrometry, resulting in the detection of different components.2D-LC-MS enables more accurate identification and the quantification of the compounds. Complex mixtures based on sample separation, combined with molecular network technology, it can be better process complex data information and it improves the efficiency and the accuracy of any size. Our laboratory will focus applying existing technologies to get the separation of natural, medicinal, chemical components.

Sample Preparation for 2D-HPLC-MS of Aconitum pendulum

Begin sample preparation by weighing the aconitum pendulum bush, or APB. Using a one by 10, 000 sensitivity balance, weigh 0.25 grams of dry APB in a three milliliter micro centrifuge tube. Add 2.5 milliliters of methanol to it and then sonicate for 30 minutes at a power of 240 watts and a frequency of 40 kilohertz.Centrifuge the sonicated sample at 1.2G for five minutes, and collect the supernatant. Filter it through a 0.22 micron membrane filter. Next, prepare the two-dimensional mobile phase by using acetyl nitrile as the organic phase or phase B, and ultra pure water containing 0.1%formic acid as the aqueous phase or phase A.After performing two-phase filtration through the 0.22 micron filter, subject the mobile phases to ultrasonic sonication at 40 kilohertz for 15 minutes.Purge the replaced mobile phase to remove the bubbles. Select a C18 column as the 1D column and a hydrophilic column as the 2D column, and mount them to the instrument. To adjust the appropriate shunt ratio, connect the 2D-LC instrument outline through a T to the mass inlet and the other end of the T to a shunted line.Adjust the flow rate to a suitable value to ensure the appropriate flow rate for the mass spectrum.

sample-preparation-for-2d-hplc-ms-of-aconitum-pendulum-

Research

JoVE Journal

Medicine

63 ビュー. トリカブト振り子ブッシュ(APB)の秤量からサンプル調製を開始します。1 x 10, 000 の感度バランスを使用して、3 ミリリットルのマイクロ遠心分離チューブに 0.25 グラムの乾燥 APB を計量します。それに2.5ミリリットルのメタノールを加え、次に240ワットの出力と40キロヘルツの周波数で30分間超音波処理します。超音波処理したサンプルを1.2Gで5分間遠心分離し、上清?...

ビデオ: トリカブトム振り子の2D-HPLC-MS用試料調製

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.

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

Implementing 2D-Liquid Chromatography on Aconitum pendulum Samples for Enhanced Compound Separation

To begin the 2D liquid chromatography, or 2DLC, double-click the instrument one online icon, and the chemical workstation will automatically communicate with the 1260LC and enter the workstation. For the 2DLC method parameter setting, click on the Injector module, right-click Method and set the injection volume, flow rate, and mobile phase time gradient of the 1D pump. Enter the sample runtime under Stop Time.Click on the main menu instrument and click 2DLC Method on the dropdown menu. Select Comprehensive in 2DLC mode. Enter two minutes at modulation time and 1.9 minutes at 2D gradient stop time.Set flow settings to two milliliters. Edit 2D gradient and set wavelengths. After editing the method, select Save Method As on the method menu to name the new method, and click OK.

Mass Spectrometry Following the 2D-LC Operation on Aconitum pendulum Samples

To begin, turn on the switch of the vacuum pump. Open the argon cylinder main valve and the pressure divider valve, and adjust the pressure to about 0.3 megapascals. To set up the MS method, enter the runtime in minutes to 93.00 and enter values to configure acquisition time, polarity, mass range, transfer value number, transfer value duration, and more.Set up scan event details according to the values displayed on the screen. To set data dependent settings, use separate polarity settings as disabled. Set neutral loss within top to three, and product mass list within top to three.Save to configure the settings as the instrument method. Click the sequence setup button to open the sequence table. Enter the sample type, file name, path, sample id, instrument method, location, injection amount, and other information in the form.Save the information to record the sequence listing. Then click the start analysis button to set up and start MS acquisition.

Integration of Molecular Network Operation to 2D-HPLC-MS for the Analysis of Aconitum pendulum Samples

To begin the data preparation, export 2D HPLC-MS raw mass spectral data. Next to connect to the GNPS FTP server in the WINSCP interface on the Session Configuration page, fill in the connection information. After filling in the information, click the Start button to establish a connection to the GNPS FTP server.Create molecular networks by opening a web browser and visiting the GNPS website. New users need to sign up for an account and then log in. On the main interface of the GNPS website, click Create Molecular Network under Data Analysis.On the Task Creation page, click the select Input Files button, then select and upload the data file. In the Workflow Option tabs, set the various parameters that generate the molecular network or MN.These parameters include the peak extraction algorithm, peak over travel value, similarity calculation method, and so on. After making the appropriate parameter settings as needed, click the Submit button at the bottom of the page to run the task.After the task runs, find the created tasks in the Jobs tab and click the workflow name to view the analysis results. The website provides MN diagrams, substitutes, symbiotic networks, and other related information. Four of the alkaloid components in the APB samples identified by the MN were aconitine, 14-benzoylaconine, 14-O-acetylneoline and hypaconitine.The four compounds had the same parent nucleus. Aconitine, 14-benzyl aconine, 14-benzoylaconine, and hypaconitine were similar, and only the substituents were different.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Chemistry

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Environment

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Biochemistry

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Cancer Research

Identifying Chemical Composition of &lt;em&gt;Gynura bicolor&lt;/em&gt; by UPLC-TOF Mass Spectrometry

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of &lt;em&gt;Gynura bicolor DC&lt;/em&gt; samples for High-Resolution Tandem Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...