Use UNIFI natural product overall solution: identify the chemical components in unknown Chinese medicine tablets and speculate on the medicines they contain
Qiao Lirui 1 , Rob Lewis 2 , Alex Hooper 2 , James Morphet 2 , Tan Xiaojie 1 , Kate Yu 3
1. Waters Technology (Shanghai) Co., Ltd., Shanghai, China
2. Waters Corporation Mass Spectrometry Technology Center, Manchester, UK
3. Waters Corporation, Milford, MA, USA
Identification of chemical constituents in unknown Chinese medicine tablets using ultra-high performance liquid chromatography (ACQUITY UPLC® I-Class), quadrupole time-of-flight mass spectrometry (Xevo® G2-S QTof) and UNIFI® natural product solutions Contains herbs. This UNIFI's overall workflow for the identification of unknown components and the speculation of possible medicinal materials has greatly improved the accuracy and efficiency of the qualitative work of unknown Chinese medicine system components.
Background introduction:
Traditional Chinese medicine is an extremely complex material system, and its material basis is mainly the chemical composition of the medicine. Application Note "Using the UNIFI Total Solution to Analyze Chemical Components in Green Tea" (literature number zh), we have explained how to use the UNIFI total solution combined with the Chinese medicine database to quickly identify the chemical constituents in known medicinal samples. For such samples, it is only necessary to import relevant medicinal ingredients from the UNIFI Chinese medicine database, and compare the obtained data with them as search targets to obtain a list of matching components and confirm them. This workflow is clear in thinking and the steps are relatively simple.
However, in practice, people often need to analyze the chemical composition of some completely unknown samples, and speculate on the possible Chinese herbal medicines. This type of work is very difficult, and sometimes it is not even known where to start. Since the background information is known to be close to zero, even with a large amount of data, the analysis of the most common LC/MS instruments, how to narrow down the target range from huge information and quickly find useful information is still the biggest challenge faced by all researchers.
For the study of the composition of unknown Chinese medicine products, the traditional workflow is: the researchers manually extract each peak, and derive the possible molecular formula by the precise molecular weight. The online database is then retrieved according to the molecular formula, and the possible cleavage pathways need to be derived from the relevant secondary fragments to determine the structure. The research idea is from point to point. In this series of links, not only many steps require the direct intervention of researchers, but also the chemical background of the researchers is very high, and it is necessary to constantly find answers from the ocean of information.
The UNIFI natural product overall solution proposes a new set of ideas for solving problems in all aspects. It is based on the ultra-high performance liquid chromatography (ACQUITY UPLC I-Class) and quadrupole time-of-flight mass spectrometry (Xevo G2-S QTof). The sample is subjected to non-data-dependent MS E data collection, and the data obtained is combined with the UNIFI Chinese medicine database. For comparison, the matched component structure is confirmed by MassFragmentTM with its corresponding fragments, and finally the detailed information of the identified sample components is automatically displayed by a preset workflow template.
This application note uses the unknown drug tablet as an example to illustrate how to use the UNIFI natural product overall solution to identify the chemical components in unknown samples and speculate on possible medicinal materials.
Key words:
Ingredient analysis of unknown Chinese medicine products, speculation and confirmation of unknown Chinese medicine products, UNIFI natural product overall solution, UNIFI Chinese medicine database, UPLC/QTof MS, MS E , UNIFI natural product workflow, Danshen, Sanqi
Experimental conditions:
Sample processing:
Two tablets of the unknown Chinese medicine tablets were peeled off and coated into a powder. 500 mg was dissolved in 50 mL of MeOH:H 2 O (3:1), sonicated for 5 min, and filtered through a membrane of 0.45 μm for use.
Liquid chromatography conditions:
Instrument: ACQUITY UPLC I-Class with FTN Sample Manager
Column: ACQUITY UPLC HSS T3, 2.1 x 100 mm, 1.8 μm, 40°C
Sample chamber temperature: 15 °C
Mobile phase A: water (0.1% formic acid)
Mobile phase B: acetonitrile
gradient:
time | Flow rate (ml/min) | Solvent A (%) | Solvent B (%) | curve |
0 | 0.6 | 90 | 10 | Start |
1 | 0.6 | 90 | 10 | 6 |
12 | 0.6 | 5 | 95 | 6 |
14 | 0.6 | 0 | 100 | 1 |
17 | 0.6 | 90 | 10 | 1 |
Mass spectrometry conditions:
Apparatus: Xevo G2-S QTof MS
Collection quality range: 100-1500 Da
Scanning time: 0.1 S
Acquisition mode: ESI+ and ESI-resolution mode, MS E
Lock mass: leucine enkephalin (LE) 1 ppm (0.3 S scan, interval: 15 S)
Capillary voltage: 3 KV (ESI+) / 2.5 KV (ESI-)
Taper hole voltage: 100V
Collision energy (eV): low CE: 6 / High CE: 20-50
Ionization source temperature: 120 °C
Desolvent temperature: 500 °C
Cone gas flow rate: 30 L/h
Desolvent gas flow rate: 1000 L/h
Data collection time: 17 min
result:
Ultra-high performance liquid chromatography (UPLC I-Class) and quadrupole time-of-flight mass spectrometry (Xevo G2-S QTof) were used to separate the chemical constituents of unknown drug tablets and to collect mass spectrometry data. The whole work flow of UNIFI natural products was combined with the whole Chinese medicine database for data processing. The software automatically identified 288 components, and 37 major components with high content were confirmed by MassFragment. Based on the correlation analysis between the chemical components and the medicinal materials, it is speculated that the unknown tablets may contain the medicinal materials Salvia miltiorrhiza and Sanqi. Through the network to search for known Chinese medicines containing this two-flavored medicine, the ingredients listed in the Chinese medicine database were again compared with the experimental data, confirming that the 59 main ingredients in the unknown Chinese medicine tablets are from Dan. Sanqi. Final conclusion: The tablet mainly contains only two kinds of herbs, Salvia miltiorrhiza and Radix Notoginseng, which may be Sanqi Danshen Tablets or Compound Danshen Tablets.
discuss:
The UNIFI natural product overall solution includes ACQUITY UPLC I-Class Ultra Performance Liquid Chromatography, Xevo G2-S QTof Quadrupole Time-of-Flight Mass Spectrometry and UNIFI software information platform with Chinese medicine database. The workflow for the analysis of known Chinese herbal ingredients has been elaborated in the application of the UNIFI overall solution to identify the chemical composition of green tea extract (part number zh). For completely unknown samples, it is necessary to add the medicinal materials inference based on the workflow, search for possible Chinese medicine prescriptions online, and re-introduction of relevant medicinal materials information from the UNIFI Chinese medicine database and re-confirm the steps. Figure 1 shows the workflow for complete identification of unknown components.
Figure 1 Workflow for identification of compound components in unknown drugs
Figure 2a is a UPLC/QTof MS base peak ion map (BPI) of the unknown Chinese medicine tablet displayed in a two-dimensional image. The UNIFI platform can also display the same result in a three-dimensional image (Fig. 2b). Compared with the two-dimensional map shown in Fig. 2a, the three-dimensional image information is richer and more realistic, and the composition distribution profile of the entire sample can be more intuitively understood. For example, from the figure we can quickly conclude that the molecular weight distribution of the chemical components of the sample is mainly concentrated in the range of 400-1000 Da. In addition to this, it is also possible to initially observe the coelution state of the compound in the entire sample.
This three-dimensional map is produced by UNIFI's unique Apex 3D image scanning. In addition to the intuitive effect, it is more helpful to improve the accuracy of the late quantitative and qualitative work, and has a strong advantage in the identification and elimination of noise.
Figure 2a UPLC/QTof MS base peak ion map (BPI) of unknown drug tablets
Figure 2b 3D spectrum of unknown drug tablets
The UNIFI natural product overall solution workflow has the following characteristics: 1. From the extraction of chromatographic peaks, the determination of molecular formula, the search of Chinese medicine database, the matching of secondary fragments to the preliminary identification of components, all are automatically completed by software; 2, researchers Only the fragmentation information that has been automatically given by MassFragment needs to be judged rationally. 3. If the suspected false positive or the components are not matched, the researchers will manually identify it.
Compared with the traditional research model, the UNIFI natural product overall solution will have a large amount of data screening work, that is, to find useful targets from the ocean of information. This has been turned into a software automation process by the researcher's manual operation of the neck, greatly reducing The workload of the researchers has greatly reduced the blindness of the work, which has greatly improved the efficiency.
Figure 3 shows the results interface for the identification of unknown Chinese medicine tablet ingredients (Figure 3). 3a UNIFI natural product overall workflow template; 3b component identification list; 3c and 3b corresponding single component selected ion chromatogram; 3d and 3c corresponding mass spectrum
Figure 3 shows the results of the identification of the components of unknown Chinese medicine tablets after UNIFI software treatment. In the list of ingredients originally obtained (3a), the peak of the same retention time may contain multiple isomers. At this point, it is necessary to pass the compound's adduct ions, as well as its corresponding secondary fragment information (blue chart) 
Further investigation reveals the rationality of the identified compounds. For example, the chromatographic peak that is automatically identified by the software with a retention time of 7.54 minutes is ginsenoside Rb1 or Yesanchinoside E. Click on Figure 3d to see its corresponding enlarged view (Figure 4). Since MassFragment has automatically matched the secondary fragment structure of the compound, the researchers can visually and quickly observe the rationality of fragmentation. The cleavage of this compound from the cleavage of the glycosidic bond to the formation of the final protopanaxadiol aglycone fragment indicates that the structure of ginsenoside Rb1 is more reasonable. Therefore, ginsenoside Rb1 can be defined as Confirmed.
Figure 4 Matching results of MassFragment of the secondary fragment ions of the component ginsenoside Rb1
After confirmation of the above components, it can be seen that the unknown tablet contains ginsenoside, salvian phenol, tanshinone, notoginsenoside and other compound types, and the association between the components and the medicinal materials indicates that the unknown tablet may contain Raw materials Danshen and Sanqi. Through online online search, it was found that Chinese medicines that may contain these two kinds of herbs have Sanqi Danshen Tablets and Compound Danshen Tablets. Of course, other formulas other than these two herbs have been found, such as their compatibility with American ginseng or hawthorn.
The next step of the work has been transformed from the initial goalless screening to the analysis of the components of known medicinal materials. The process steps and application minutes are the same as “Applying the UNIFI overall solution to identify the chemical composition of green tea extract†(literature number zh). The target medicinal materials (danshen, notoginseng, American ginseng, hawthorn) were imported from the Chinese medicine database and compared with the experimental data. No chromatographic peaks matching the characteristic components of American ginseng and hawthorn were found (such as American ginseng saponin, Gynostemma saponin, Hawthorn saponin). It is further confirmed that the tablet does not contain American ginseng and hawthorn. At the same time, the main chromatographic peaks of the obtained samples were matched with the main components of Danshen and Sanqi (confirmed 59 main components), as shown in Table 1. Finally, it is concluded that the tablet contains Danshen and Panax notoginseng, and it is likely to be Sanqi Danshen Tablets or Compound Danshen Tablets.
Table 1 Summary report of component identification in unknown tablets obtained by importing natural product component information overview template
in conclusion:
This application note describes the overall workflow of the UNIFI Natural Product Total Solution for the identification of unknown components and the speculation of possible medicinal materials. This workflow is a process from initial untargeted screening to targeted analysis.
The UPLC/QTof MS sample analysis time is only 17 minutes. In the early stage, no target screening was used to identify 37 major chemical components, and the relevant medicinal materials were inferred to be Sanqi and Salvia. Through the network to search for known Chinese medicine prescriptions, the related medicinal materials (Salvia, Sanqi, American ginseng, Hawthorn) will be compared with the experimental data by targeted screening. As a result, there were 103 chemical constituents of Salvia miltiorrhiza and Sanqi listed in the Chinese medicine database, 59 of which were matched with the obtained data and confirmed, while the main components of the other two herbs could not be reasonably matched. Final conclusion: The unknown Chinese medicine tablets tested are most likely to be Sanqi Danshen Tablets or Compound Danshen Tablets.
The UNIFI natural product overall solution supplemented by the Chinese medicine database and combined with the automatic identification function of the component structure is a new solution for the analysis of complex components of traditional Chinese medicine. It provides a new idea for the complex formulation of complex traditional Chinese medicines, especially the components of completely unknown samples, and ultimately reduces the blindness of such research and greatly improves the efficiency.
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