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

تحليل APB للنباتات الطبية التبتية باستخدام 2D-HPLC-MS مع الشبكات الجزيئية

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.

تسليط الضوء على المؤلف: دمج 2D-HPLC-MS والشبكات الجزيئية في تحليل الطب الطبيعي

تقنية 2D-HPLC-MS جنبا إلى جنب مع الشبكة الجزيئية لتحديد المكونات في بندول Aconitum الطب التبتي

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.

تحضير العينة ل 2D-HPLC-MS من بندول الأكونيتوم

ابدأ في تحضير العينة عن طريق وزن شجيرة البندول الأكونيتوم ، أو APB. باستخدام ميزان حساسية واحد في 10،000 ، قم بوزن 0.25 جرام من APB الجاف في أنبوب طرد مركزي صغير سعة ثلاثة ملليلترات. أضف 2.5 ملليلتر من الميثانول إليه ثم صوتنة لمدة 30 دقيقة بقوة 240 واط وتردد 40 كيلو هرتز.أجهزة الطرد المركزي عينة صوتنة في 1.2G لمدة خمس دقائق ، وجمع طاف. قم بترشيحه من خلال مرشح غشاء 0.22 ميكرون. بعد ذلك ، قم بإعداد المرحلة المتنقلة ثنائية الأبعاد باستخدام أسيتيل النتريل كمرحلة عضوية أو المرحلة B ، والمياه النقية للغاية التي تحتوي على 0.1٪ حمض الفورميك كمرحلة مائية أو المرحلة A.بعد إجراء الترشيح على مرحلتين من خلال مرشح 0.22 ميكرون ، تخضع المراحل المتنقلة لصوتنة بالموجات فوق الصوتية عند 40 كيلو هرتز لمدة 15 دقيقة.قم بتطهير المرحلة المتنقلة المستبدلة لإزالة الفقاعات. حدد عمودا C18 كعمود 1D وعمودا محبا للماء كعمود 2D ، وقم بتركيبهما على الجهاز. لضبط نسبة التحويلة المناسبة ، قم بتوصيل مخطط أداة 2D-LC من خلال T بمدخل الكتلة والطرف الآخر من T بخط محول.اضبط معدل التدفق على قيمة مناسبة لضمان معدل التدفق المناسب للطيف الكتلي.

Research

JoVE Journal

Medicine

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

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 ...

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

تنفيذ كروماتوغرافيا 2D-Liquid على عينات بندول الأكونيتوم لتعزيز الفصل المركب

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

قياس الطيف الكتلي بعد عملية 2D-LC على عينات بندول Aconitum

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

دمج تشغيل الشبكة الجزيئية في 2D-HPLC-MS لتحليل عينات بندول الأكونيتوم