mass spectrometry following the 2d-lc operation on aconitum pendulum samples

Analyse der tibetischen Heilpflanze APB mittels 2D-HPLC-MS mit molekularer Vernetzung

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.

Autor im Rampenlicht: Integration von 2D-HPLC-MS und molekularer Vernetzung in der naturmedizinischen Analytik

2D-HPLC-MS-Technologie kombiniert mit molekularem Netzwerk zur Identifizierung von Bestandteilen in der tibetischen Medizin Aconitum-Pendel

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.

Massenspektrometrie nach der 2D-LC-Operation an Aconitum-Pendelproben

Schalten Sie zunächst den Schalter der Vakuumpumpe ein. Öffnen Sie das Hauptventil des Argonzylinders und das Druckteilerventil und stellen Sie den Druck auf etwa 0,3 Megapascal ein. Um die MS-Methode einzurichten, geben Sie die Laufzeit in Minuten bis 93,00 ein und geben Sie Werte ein, um die Erfassungszeit, die Polarität, den Massenbereich, die Anzahl der Übertragungswerte, die Dauer des Übertragungswerts und vieles mehr zu konfigurieren.Richten Sie die Scan-Ereignisdetails entsprechend den auf dem Bildschirm angezeigten Werten ein. Um datenabhängige Einstellungen festzulegen, verwenden Sie separate Polaritätseinstellungen, wenn diese deaktiviert sind. Legen Sie den neutralen Verlust innerhalb von top auf drei und die Produktmassenliste innerhalb von top auf drei fest.Speichern, um die Einstellungen als Instrumentenmethode zu konfigurieren. Klicken Sie auf die Schaltfläche Sequenzeinrichtung, um die Sequenztabelle zu öffnen. Geben Sie den Probentyp, den Dateinamen, den Pfad, die Proben-ID, die Gerätemethode, den Speicherort, die Injektionsmenge und andere Informationen in das Formular ein.Speichern Sie die Informationen, um die Sequenzliste aufzuzeichnen. Klicken Sie dann auf die Schaltfläche Analyse starten, um die MS-Erfassung einzurichten und zu starten.

mass-spectrometry-following-2d-lc-operation-on-aconitum-pendulum

Research

JoVE Journal

Medicine

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

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

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

Forschung

Medizin

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

Probenvorbereitung für die 2D-HPLC-MS des Aconitum-Pendels

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

Implementierung der 2D-Flüssigkeitschromatographie an Aconitum-Pendelproben zur verbesserten Trennung von Verbindungen

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

Integration des molekularen Netzwerkbetriebs in die 2D-HPLC-MS zur Analyse von Aconitum-Pendelproben