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

Análisis de la planta medicinal tibetana APB utilizando 2D-HPLC-MS con redes moleculares

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 destacado: Integración de 2D-HPLC-MS y redes moleculares en el análisis de medicina natural

Tecnología 2D-HPLC-MS combinada con una red molecular para la identificación de componentes en el péndulo de acónito de la medicina tibetana

Preparación de muestras para 2D-HPLC-MS de péndulo de acónito

Comience la preparación de la muestra pesando el casquillo de péndulo de acónito, o APB. Usando una balanza de sensibilidad de uno por 10.000, pese 0,25 gramos de APB seco en un tubo de microcentrífuga de tres mililitros. Agregue 2,5 mililitros de metanol y luego sonicase durante 30 minutos a una potencia de 240 vatios y una frecuencia de 40 kilohercios.Centrifugar la muestra sonicada a 1,2 G durante cinco minutos y recoger el sobrenadante. Fíltralo a través de un filtro de membrana de 0,22 micras. A continuación, prepare la fase móvil bidimensional utilizando nitrilo de acetilo como fase orgánica o fase B, y agua ultrapura que contenga ácido fórmico al 0,1% como fase acuosa o fase A.Después de realizar la filtración bifásica a través del filtro de 0,22 micras, someta las fases móviles a sonicación ultrasónica a 40 kilohercios durante 15 minutos.Purgue la fase móvil reemplazada para eliminar las burbujas. Seleccione una columna C18 como columna 1D y una columna hidrofílica como columna 2D, y móntelas en el instrumento. Para ajustar la relación de derivación adecuada, conecte el contorno del instrumento 2D-LC a través de una T a la entrada de masa y el otro extremo de la T a una línea derivada.Ajuste el caudal a un valor adecuado para garantizar el caudal adecuado para el espectro másico.

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

Research

JoVE Journal

Medicine

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

63 vistas. Comience la preparación de la muestra pesando el casquillo de péndulo de acónito, o APB. Usando una balanza de sensibilidad de uno por 10.000, pese 0,25 gramos de APB seco en un tubo de microcentrífuga de tres mililitros. Agregue 2,5 mililitros de metanol y luego sonicase durante 30 minutos a una potencia de 240 vatios y una frecuencia de 40 kilohercios.Centrifugar la muestra sonicada a 1,2 G durante cinco minutos y recoger el sobrenadante. Fíltralo a través de un filtro de membr...

Video: Preparación de muestras para 2D-HPLC-MS de péndulo de acónito  

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 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 ...