A detection method for rapidly identifying chemical components in Guipi mixture

Abstract

The application belongs to the field of traditional Chinese medicine preparation analysis, and specifically discloses a method for rapidly identifying main chemical components in Guipi mixture by using ultra-high performance liquid chromatography-quadrupole-electric field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive MS) technology, which realizes the purpose of rapidly qualitatively identifying 115 components such as phenylalanine, phthalide, flavonoids, ketones and terpenes in Guipi mixture. The method has the advantages of simplicity, rapidness, high efficiency and sensitivity, and can provide evidence for establishing the interaction between the components and the curative effect of Guipi mixture, and provide basis for the research on the safety and effectiveness of clinical medication.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation analysis, specifically relating to a method for rapidly identifying the main chemical components in Guipi Heji using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS). Background Technology

[0002] Traditional Chinese medicine (TCM) compound preparations are a major method and carrier of TCM clinical treatment, and are complex, multi-component chemical systems. Exploring the mysteries of these complex systems using modern science and technology is gradually providing a scientific basis for the quality control, mechanisms of action, and clinical application of major TCM products, and is a crucial link in ensuring the safety and effectiveness of TCM clinical medication.

[0003] Gui Pi Tang (Spleen-Nourishing Decoction) was first recorded in Yan Yonghe's *Ji Sheng Fang* (Formulas for Saving Lives) during the Song Dynasty. Gui Pi He Ji (Spleen-Nourishing Compound) is a compound Chinese medicine preparation based on Gui Pi Tang, made using modern pharmaceutical methods. The entire formula consists of twelve ingredients: Codonopsis pilosula, stir-fried Atractylodes macrocephala, prepared Astragalus membranaceus, prepared Glycyrrhiza uralensis, Poria cocos, prepared Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan aril, Angelica sinensis, Aucklandia lappa, jujube (pitted), and ginger. Ginseng, Astragalus membranaceus, Atractylodes macrocephala, Poria cocos, and Glycyrrhiza uralensis tonify the spleen and replenish qi; combined with Angelica sinensis, jujube, Ziziphus jujuba var. spinosa, and longan aril, which nourish blood and calm the mind, the entire formula works synergistically to replenish qi and blood, strengthen the spleen, and nourish the heart. Gui Pi He Ji is used to treat symptoms of deficiency of both heart and spleen, shortness of breath, palpitations, insomnia, dizziness, weakness, loss of appetite, metrorrhagia, and hematochezia. Clinical and pharmacological experiments have proven its efficacy. Studying the complex chemical composition system of Guipi Heji and establishing the interaction between its components and efficacy can help ensure the safety and effectiveness of clinical use. However, there are currently no reports on a systematic study of its chemical composition.

[0004] Modern research indicates that the efficacy of traditional Chinese medicine (TCM) is not due to a few "indicator components" or "major components," but rather the result of the synergistic effect of multiple components. The complex multi-component system of TCM makes component separation difficult, posing challenges to qualitative and quantitative identification. While traditional detection, analysis, and identification techniques such as thin-layer chromatography (TLC) and liquid chromatography (LC) can identify chemical or active ingredients relatively thoroughly and accurately, they are time-consuming, have significant limitations, are prone to errors, and cannot provide a comprehensive understanding of the chemical composition. Liquid chromatography-mass spectrometry (LC-MS) overcomes many of the limitations of traditional techniques, offering advantages such as speed, sensitivity, accuracy, and efficiency, playing a crucial role in qualitative identification across numerous fields.

[0005] This invention discloses a rapid detection method for chemical components in Guipi Heji (a traditional Chinese medicine formula). The aim is to provide a method for rapidly identifying the main chemical components in Guipi Heji using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS). This method enables the rapid identification of phenylpropanoids, phenyl peptides, flavonoids, and other chemical components in Guipi Heji. Rapid qualitative identification of 115 components, including ketones and terpenes, provides a basis for establishing the interaction between the components of Guipi Heji and its efficacy, and provides a basis for research on the safety and effectiveness of clinical use. Summary of the Invention

[0006] To overcome the limitations of existing technologies in determining multiple components of traditional Chinese medicine, this invention provides a rapid method for identifying chemical components in Guipi Heji (a traditional Chinese medicine compound). Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS) is used to analyze and identify the chemical components of Guipi Heji, obtaining quasi-molecular ion peaks and fragment ion peaks containing rich structural information. This allows for rapid and accurate identification of the structure of the components, providing a basis for research on the pharmacodynamic material basis of Guipi Heji.

[0007] A rapid method for identifying chemical components in Gui Pi He Ji (a traditional Chinese medicine formula) includes the following steps:

[0008] Prepare test and reference solutions, and inject them into an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry (UHPLC-Q-Exactive MS) system for analysis. Rapid structural identification is performed by combining the chromatographic retention time, ultraviolet absorption spectral characteristics, molecular weight, and multi-stage fragmentation ion information of the reference solution.

[0009] Preferably, the preparation of the test solution includes the following steps:

[0010] Accurately pipette the Gui Pi He Ji sample, dissolve it in methanol, extract it by ultrasound, make up to volume, shake well, centrifuge, and filter the supernatant to obtain the test solution.

[0011] The reference standards were chlorogenic acid, ferulic acid, glycyrrhizin, verrucoside, and polygala tenuifolia. Ketone III, ellagic acid, spinosin, luteolin, codonopsis glycoside, 3,6'-disinoyl sucrose, gentiosin, ammonium glycyrrhizate, polygala saponin, atractylodes lactone I, ligustilide, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C.

[0012] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: octadecyl bonded silica column as stationary phase, 10 mmol / L ammonium acetate solution-acetonitrile as mobile phase gradient elution, detection wavelength of 320-330 nm, and flow rate of 0.2-0.4 ml / min.

[0013] The chromatographic column is a Waters CORTECS T3 C18 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.6 μm; or a Waters ACQUITYUPLC HSS T3 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm.

[0014] Preferably, the chromatographic column is a Waters CORTECS T3 C18 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.6 μm.

[0015] Preferably, the elution process is performed according to the following gradient table:

[0016]

[0017]

[0018] The detection wavelength is 324 nm and the flow rate is 0.2 ml / min.

[0019] The conditions for high-resolution mass spectrometry using a quadrupole-electrostatic track trap are as follows:

[0020] The ion source was a HESI source, with positive / negative ion switching mode, sheath gas flow rate of 30 arb, auxiliary gas flow rate of 8 arb, spray voltage of 3.8 kV, ion transmission tube temperature of 320℃, scanning mode of Full MS / dd-MS2, Full MS resolution of 70000, dd-MS2 resolution of 17500, scanning range of m / z 125-1875, MS / MS mode.

[0021] The technical solution of the present invention will be described in detail below:

[0022] A rapid method for identifying chemical components in Gui Pi He Ji (a traditional Chinese medicine formula) includes the following steps:

[0023] 1) Preparation of test solution: Accurately pipette Guipi Heji into a volumetric flask, dissolve in methanol, extract by sonication, make up to volume, shake well, centrifuge, and filter the supernatant to obtain the test solution.

[0024] 2) Preparation of reference solutions: Take chlorogenic acid, ferulic acid, glycyrrhizin, verbascoside, and polygala tenuifolia respectively. Ketone III, ellagic acid, spinosin, luteolin, codonopsis glycoside, 3,6'-disinoyl sucrose, gentiopicrin, ammonium glycyrrhizate, polygala saponin, atractylodes lactone I, ligustilide, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference standards were prepared into a 1 mg / ml stock solution with methanol. 1 ml of each was transferred to a 50 ml volumetric flask and diluted to volume with methanol to prepare a mixed reference solution.

[0025] 3) Ultra-high performance liquid chromatography conditions: Waters CORTECS T3 C 18 Chromatographic column; mobile phase A: 2 mmol / L ammonium acetate solution, mobile phase B: acetonitrile; gradient elution according to the following table; detection wavelength: 324 nm; injection volume: 1 μL; flow rate: 0.2 ml / min; column temperature: 28-32℃;

[0026] 4) Quadrupole-electrostatic field orbital trap high-resolution mass spectrometry conditions:

[0027] The ion source used was a HESI source with positive / negative ion switching mode, sheath gas flow rate of 30 arb, auxiliary gas flow rate of 8 arb, spray voltage of 3.8 kV, ion transmission tube temperature of 320℃, scanning mode of Full MS / dd-MS2, Full MS resolution of 70000, dd-MS2 resolution of 17500, scanning range of m / z 125-1875, MS / MS mode;

[0028] 5) Determination method: The test solution is injected into ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry (UHPLC-Q-Exactive MS) for determination. The structure is rapidly identified by combining the chromatographic retention time, ultraviolet absorption spectral characteristics, molecular weight and multi-level fragmentation ion information of each component.

[0029] Preferably, step 1) involves ultrasonic extraction for 30 minutes.

[0030] Preferably, the chromatographic column in step 3) has a size of 2.1m × 100mm and a diameter of 1.6μm.

[0031] Preferably, the column temperature in step 3) is 30°C.

[0032] The gradient elution conditions for ultra-high performance liquid chromatography in this invention are as follows:

[0033]

[0034] The collected data were extracted using Thermo Xcalibur software. The retention time, UV absorption, molecular weight, molecular formula, and multi-level fragmentation ion information of different unknown components were retrieved and compared using the Chemical Book and Sci Finder websites. The data were also compared and analyzed using literature from PubChem, Mass Bank, and mz Cloud databases to qualitatively identify multiple components in Guipi Heji. The results are shown in Table 1.

[0035] This invention employs ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive MS) to perform chromatographic separation, mass spectrometry data acquisition, and structural identification of various components in Guipi Heji (a traditional Chinese medicine formula). Ultimately, phenylpropanoids, flavonoids, phthalides, and other compounds were identified. A total of 115 components, including ketones and terpenes, were identified, providing a basis and premise for further research on their pharmacodynamic material basis.

[0036] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0037] (1) On the one hand, this invention can avoid tedious and complicated sample pretreatment steps and retain the component information of the sample itself as much as possible; on the other hand, it can simultaneously obtain high-precision primary and secondary mass spectrometry scanning data of hundreds or thousands of components, which can save time and cost, and can also realize rapid, accurate and comprehensive analysis of several components, including trace components, which helps to study the complex chemical component system of Guipi Heji, establish the interaction relationship between components and efficacy, and ensure the safety and effectiveness of clinical medication.

[0038] (2) The ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap (UHPLC-Q-Exactive-Orbitrap-MS) technology of this invention can provide accurate, reliable and rich compound information. It has the advantages of high resolution, fast scanning speed, high separation efficiency and high sensitivity. It can simultaneously and accurately collect the mass-to-nucleus ratio of the parent ion and the mass number of fragment protons, and realize high-throughput and accurate qualitative and quantitative information of several compounds in Guipi Heji with a single injection.

[0039] (3) The ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap (UHPLC-Q-Exactive-Orbitrap-MS) technology of this invention is used to identify the components of Guipi Heji. By optimizing the unique ultra-high performance liquid chromatography conditions, the effective components of Guipi Heji are separated, avoiding mutual interference between the components, and laying the foundation for further rapid mass spectrometry identification analysis. Attached Figure Description

[0040] Figure 1: Chromatogram of the Guipi Heji test sample under chromatographic conditions (1);

[0041] Figure 2 : Chromatogram of the Guipi Heji test sample under chromatographic conditions (2);

[0042] Figure 3 : Chromatogram of the Guipi Heji test sample under chromatographic conditions (3);

[0043] Figure 4 Total ion chromatogram of mixed reference standards by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exductive MS);

[0044] Among them, 1. neochlorogenic acid; 2. chlorogenic acid; 3. cryptochlorogenic acid; 4. ferulic acid; 5. glycyrrhizin; 6. verbascoside; 7. Polygala tenuifolia. 8. Ketone III; 9. Ellagic acid; 10. Spinosin; 11. Luteolin; 12. Isochlorogenic acid B; 13. Isochlorogenic acid A; 14. Codonopsis pilosula glycoside; 15. 3,6'-Disinopyrosylsucrose; 16. Isochlorogenic acid C; 17. Mangosteen; 18. Polygala tenuifolia saponin; 19. Ammonium glycyrrhizate; 20. Atractylodes macrocephala lactone I; 21. Ligusticum striatum lactone;

[0045] Figure 5 Total ion chromatogram of Guipi Heji (a traditional Chinese medicine formula) obtained by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive MS).

[0046] Figure 6 The proposed cleavage pathway for 3,4-O-dicaffeoylquinic acid;

[0047] Figure 7 MS / MS spectrum of 3,4-O-dicaffeoylquinic acid;

[0048] Figure 8 : The proposed cleavage pathway of ligustilide;

[0049] Figure 9 MS / MS chromatogram of ligustilide;

[0050] Figure 10 The proposed cleavage pathway for gentianin;

[0051] Figure 11 MS / MS spectrum of gentianin;

[0052] Figure 12 Glycyrrhizic acid is presumed to have a cleavage pathway.

[0053] Figure 13 : MS / MS spectrum of glycyrrhizic acid;

[0054] Figure 14 Polygala tenuifolia Ketone III is presumed to have a cleavage pathway;

[0055] Figure 15 Polygala tenuifolia Ketone III MS / MS spectrum. Detailed Implementation

[0056] To provide a more comprehensive understanding of the implementation of this invention, typical implementation examples are provided below for further illustration. Furthermore, since the following embodiments are merely preferred embodiments of this invention, and their descriptions are quite specific and detailed, they should not be construed as limiting the scope of this invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the principles and concept of this invention, and these modifications and improvements should also fall within the protection scope of this invention.

[0057] Example 1: Screening of Ultra-High Performance Liquid Chromatography Conditions for Guipi Compound

[0058] 1. Preparation of test sample

[0059] Accurately pipette the Gui Pi He Ji (a traditional Chinese medicine formula) into a volumetric flask, dissolve it in methanol, extract it by sonication for 30 min, make up to volume, shake well, centrifuge, and take the supernatant and filter it through a 0.22 μm microporous membrane as the test solution.

[0060] 2. Liquid Chromatography Conditions

[0061] Inject the test solution into the high-performance liquid chromatograph under the following liquid chromatography conditions and record the chromatograms.

[0062] (1) Waters Cortecs T3 C 18 Chromatographic column (2.1m × 100mm, 1.6μm); mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile; gradient elution; detection wavelength: 324nm; injection volume: 1μl; flow rate: 0.2ml / min; column temperature: 30℃. Chromatography as shown... Figure 1 As shown.

[0063]

[0064] (2) Waters ACQUITYUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); mobile phase A: 10 mmol / L ammonium acetate solution, mobile phase B: acetonitrile; gradient elution; detection wavelength: 324 nm; injection volume: 1 μl; flow rate: 0.2 ml / min; column temperature: 30 ℃. The chromatogram is shown below. Figure 2 As shown.

[0065]

[0066] (3) Waters Cortecs T3 C 18 Chromatographic column (2.1m × 100mm, 1.6μm); mobile phase A: 10mmol / L ammonium acetate solution, mobile phase B: acetonitrile; gradient elution; detection wavelength: 324nm; injection volume: 1μl; flow rate: 0.2ml / min; column temperature: 30℃. Chromatography is shown below. Figure 3 As shown.

[0067]

[0068]

[0069] The chromatogram of the test sample under chromatographic conditions (1) is as follows: Figure 1 As shown, the chromatogram under chromatographic conditions (2) is as follows. Figure 2 As shown, the chromatogram under chromatographic conditions (3) is as follows. Figure 3 As shown. From Figure 1-3 It can be seen that under chromatographic conditions (1), Figure 1 Each chromatographic peak can be separated, but the number of peaks is small, meaning fewer substances are separated because all substances elute together within 5 minutes, making effective separation impossible. Chromatographic conditions (2) Figure 2 The substances cannot be effectively separated; under chromatographic conditions (3), more substances will emerge, more substances can be separated, and the separation degree is better.

[0070] Example 2: An analytical method for rapidly identifying the chemical components of Gui Pi He Ji (a traditional Chinese medicine formula).

[0071] 1. Instruments and reagents

[0072] 1.1 Instruments

[0073] Thermo Vanquish Flex ultra-high performance liquid chromatograph; Thermo Scientific Q Exactive quadrupole Orbitrap mass spectrometer; electronic analytical balance (Mettler-Toledo, Switzerland); XS204 electronic analytical balance (Mettler-Toledo, Switzerland); JY-15 ultrasonic cleaner (Hubei Dingtai Hengsheng Technology Equipment Co., Ltd.); Milli-Q ultrapure water system (Merck, France).

[0074] 1.2 Test Drugs

[0075] Chlorogenic acid, ferulic acid, glycyrrhizin, verbascoside, polygala Ketone III, ellagic acid, spinosin, luteolin, codonopsis glycoside, 3,6'-disinoyl sucrose, gentiopicrin, ammonium glycyrrhizate, polygala saponin, atractylodes lactone I, and ligustilide reference standards were all purchased from the China National Institutes for Food and Drug Control; neochlorogenic acid and cryptochlorogenic acid were purchased from Xinyang Zhongjian Metrology Biotechnology Co., Ltd.; isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were purchased from Chengdu Desite Biotechnology Co., Ltd. Guipi Heji was provided by Lunan Houpu Pharmaceutical Co., Ltd. (10ml / bottle); methanol and acetonitrile were chromatographic grade (Merck, USA); ammonium acetate (LC-MS) was purchased from Sigma-Aldrich Trading Co., Ltd.; the remaining reagents were analytical grade, and ultrapure water was prepared in-house.

[0076] 2 methods

[0077] 2.1 Preparation of the test solution:

[0078] Accurately pipette the Gui Pi He Ji (a traditional Chinese medicine formula) into a volumetric flask, dissolve it in methanol, extract it by sonication for 30 min, make up to volume, shake well, centrifuge, and take the supernatant and filter it through a 0.22 μm microporous membrane as the test solution.

[0079] 2.2 Preparation of the reference solution:

[0080] Chlorogenic acid, ferulic acid, glycyrrhizin, verbascoside, and polygala tenuifolia were taken separately. Ketone III, ellagic acid, spinosin, luteolin, codonopsis glycoside, 3,6'-disinoyl sucrose, gentiopicrin, ammonium glycyrrhizate, polygala saponin, atractylodes lactone I, ligustilide, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference standards were prepared into a 1 mg / ml stock solution with methanol. 1 ml of each was transferred to a 50 ml volumetric flask and diluted to volume with methanol to prepare a mixed reference solution.

[0081] 2.3 Ultra-high performance liquid chromatography conditions:

[0082] Waters Cortecs T3 C 18 Chromatographic column (2.1m×100mm, 1.6μm); mobile phase A: 10mmol / L ammonium acetate solution, mobile phase B: acetonitrile; gradient elution; detection wavelength: 324nm; injection volume: 1μl; flow rate: 0.2ml / min; column temperature: 30℃.

[0083]

[0084] 2.4 Quadrupole-Electrostatic Field Orbital Trap High-Resolution Mass Spectrometry Conditions:

[0085] The ion source used was a HESI source with positive / negative ion switching mode, sheath gas flow rate of 30 arb, auxiliary gas flow rate of 8 arb, spray voltage of 3.8 kV, ion transmission tube temperature of 320℃, scanning mode of Full MS / dd-MS2, Full MS resolution of 70000, dd-MS2 resolution of 17500, scanning range of m / z 125-1875, MS / MS mode.

[0086] 2.5 Determination Method:

[0087] The test solution was injected into ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry (UHPLC-Q-Exactive MS) for determination.

[0088] Mass spectrometry information of the chemical components in Guipi Heji in positive and negative ion modes was acquired using UHPLC-Q-Exactive-Orbitrap-MS. The data files were processed by Xcalibur software. Based on the m / z values ​​of the compounds, the software automatically calculated the molecular formulas and calculated the deviation between the measured relative molecular mass and the theoretical relative molecular mass. Molecular formulas with a deviation less than 0.005‰ were screened. Combined with nitrogen rules, the most likely elemental composition and molecular formula of the compounds were determined. Using the retention time, precise molecular weight, UV absorption, and primary and secondary fragmentation information provided by Xcalibur software, searches and comparisons were conducted using the Chemical Book and Sci Finder websites. Further comparisons and analyses were performed using literature from PubChem, Mass Bank, and mz Cloud databases to qualitatively identify multiple components in Guipi Heji.

[0089] 3 Results

[0090] Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive MS) was used to separate various components in Guipi Heji, acquire mass spectrometry data, and identify the structures of inherent components. Ultimately, phenylpropanoids, flavonoids, and phenyl peptides were identified. A total of 115 ketones, terpenes and other nitrogen-containing components were identified, providing a basis and premise for further research on their pharmacodynamic material basis; the results are shown in Table 1.

[0091] Total ion chromatogram of mixed reference standards by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive MS) is shown below. Figure 4 As shown, the total ion chromatogram of Guipi Heji obtained by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive MS) is as follows. Figure 5 As shown.

[0092] 3.1 Identification of Phenylpropanoid Compounds

[0093] Phenylacetins are a class of naturally occurring compounds consisting of a benzene ring linked to three straight-chain carbon atoms (C6-C3 groups). Their structure can contain one or more C6-C3 units, and they are widely distributed in plants. Compounds 3, 5, 7-14, 17, 20-21, 23-25, 29, 37, 40-43, 47, 50-52, 55-60, 62-65, 68, 77-79, and 82 in Table 1 belong to the phenylpropanoid class. Taking compound 40 as an example, this compound... R =34.82, producing m / z 515.1188 [MH] in negative ion detection mode. - Quasi-molecular ion peak and fragment peak of caffeoyl group (losing one molecule) m / z 353.0878 [MH-C9H6O3] - As a result, the fragment peaks lost one molecule of caffeoyl group and one quinoline yl group, respectively, yielding [MH-2C9H6O3] at m / z 191.0554. - and m / z 179.0342[MH-C9H6O3-C7H 10 O5] - Fragment peak. The fragment peak m / z is 191.0554 [MH-2C9H6O3]. - The process continues by losing one molecule of H₂O, producing m / z 173.0447 [MH-2C9H₆O₃-H₂O]. - The fragment peak m / z is 179.0342 [MH-C9H6O3-C7H]. 10 O5] - The loss of one more CO2 molecule produces m / z 135.0441 [MH-2C9H6O3-CO2]. - .

[0094] Based on comparison with reference standards and literature review, the compound is inferred to be 3,4-O-dicaffeoylquinic acid. R Compound 41 and t = 35.87 R Compound 43, with a concentration of 39.92, produces the same quasi-molecular ion peak and similar fragmentation patterns as compound 40, indicating that these three compounds are isomers. Based on comparisons, literature data, and retention times, it is inferred that compound 41 is 3,5-O-dicaffeoylquinic acid and compound 43 is 4,5-O-dicaffeoylquinic acid. The possible fragmentation pathway and secondary mass spectrum of compound 40 are shown below. Figure 6 , Figure 7 .

[0095] 3.2 Identification of phthalide compounds

[0096] Phenylphthalide is a class of heterocyclic compounds containing an isobenzofuranone skeleton. Compounds 39, 73, 95, and 115 in Table 1 belong to the phthalide class. In mass spectrometry analysis, phthalide dimers typically fragment into phthalide monomers first. Phenylphthalide monomers (characteristic ions are usually m / z 191) further lose H₂O, CO, CO₂, alkyl radicals, etc., during fragmentation, producing characteristic fragments. Taking compound 115 as an example, this compound... R =72.65, in positive ion detection mode, produces m / z 191.1065 [M+H] + The quasi-molecular ion peak, calculated from the precise mass number, indicates that the molecular formula of this compound is C. 12 H 14 O2. Secondary fragments have m / z 173.0906, m / z 163.1115, and m / z 145.1010, corresponding to [M+H-H2O] respectively. + [M+H-CO] + [M+H-CO-H2O] + In summary, based on further literature review and comparison with the chromatograms of reference samples, compound 115 is inferred to be ligustilide. The inferred fragmentation pathway and MS / MS chromatogram are shown below. Figure 8 , Figure 9 .

[0097] 3.3 Identification of Flavonoids

[0098] Flavonoids are a series of compounds consisting of two benzene rings linked by three carbon atoms, i.e., compounds with a C6-C3-C6 skeleton. Flavonoids are widely found in plants in nature and are plant secondary metabolites. In mass spectrometry analysis, RDA fragmentation and loss of neutral fragments are the basic fragmentation pathways. Compounds 16, 27, 30-31, 33, 36, 38, 44-46, 48-49, 53, 61, and 83 in Table 1 belong to the flavonoid class. R Taking compound 83 with m / z = 59.78 as an example, this compound produces [M+H] with m / z 269.0802 in positive ion mode. + The quasi-molecular ion peak, calculated from the precise mass number, indicates that the molecular formula of this compound is C. 16 H 12 In the secondary mass spectrometry, the molecular ion peak further fragments to produce several ion fragments, including m / z 254.0566, m / z 237.0542, m / z 213.0906, m / z 181.0646, m / z 118.0415, and m / z 107.0494. The fragment ion at m / z 237.0542 is obtained by the loss of CH3OH from the quasi-molecular ion peak, i.e., [M+H-CH3OH]. +The quasi-molecular ion peak simultaneously loses two neutral CO molecules, producing fragment ions at m / z 213.0906 [M+H-C2O2]. + Further loss of a CH3OH group produces m / z 181.0646 [M+H-C2O2-CH3OH]. + Fragmentation. The quasi-molecular ion peak lost C9H6O3 and a free radical CH3. · The m / z value of [M+H-C9H6O3] was 107.0494. + And m / z 254.0566[M+H-CH3] +· The fragment ion at m / z 254.0566 further underwent RDA fragmentation to produce a fragment ion at m / z 118.0415. Based on the above inference, and comparing with the chromatograms of reference materials and standards, compound 83 is inferred to be gentianin. The inferred fragmentation pathway and MS / MS chromatograms are shown below. Figure 10 , Figure 11 .

[0099] 3.4 Identification of Terpenoids

[0100] Terpenoids are widely found in nature. All compounds derived from the polymerization of isoprene have the molecular formula (C5H8). n All compounds with the general formula belong to the terpenoid class. This method identified several terpenoid components from Guipi Heji, including compounds 66-67, 69-72, 74-76, 80-81, 84-89, 91-94, 97-99, and 101-114. Taking compound 93 as an example, this compound t R =66.50, in negative ion detection mode, producing m / z 821.3951 [MH] - The quasi-molecular ion peak, whose molecular formula is deduced from the precise relative molecular mass, is C. 42 H 62 O 16 m / z 821.3951 [MH] - Remove C 30 H 46 O4 produces m / z 351.0575 [MHC] 30 H 46 O4] - Further removal of glucuronic acid residues produces m / z 193.0349 [MHC]. 30 H 46 O4-C6H6O5] - Then, removing one water molecule produces m / z 175.0240 [MHC]. 30 H 46 O4-C6H6O5-H2O] -After removing one water molecule, an m / z of 157.0130 [MHC] is produced. 30 H 46 O4-C6H6O5-2H2O] - Finally, decarboxylation produces m / z 113.0233 [MHC]. 30 H 46 [O4—C6H6O5-2H2O-CO2] - Based on the above cleavage process and comparison with the reference standard, the compound is inferred to be glycyrrhizic acid. The inferred cleavage pathway and MS / MS chromatogram are shown below. Figure 12 , Figure 13 .

[0101] 3.5 Identification of ketone compounds

[0102] Ketones exist in plants in the form of glycosides, and are mostly found in plants of the genus *Polygala*. Compounds 22, 26, 28, 32, 34, 54, 90, 96, and 100 in Table 1 belong to this genus. Ketone compounds. Taking compound 32 as an example, this compound t R =28.16, producing m / z 567.1360 [MH] in negative ion detection mode. - The quasi-molecular ion peak, whose molecular formula is deduced from the precise relative molecular mass, is C. 25 H 28 O 15 The quasi-molecular ion peak loses one molecule of apigenin, resulting in m / z 417.0835 [MH-C5H]. 10 O5] - Fragments. Further fragmentation produces more ionic fragments, primarily including m / z 345.0615, m / z 315.0509, m / z 297.0324, and m / z 272.0324, corresponding to [MH-C5H], respectively. 10 O5-C3H4O2] - ,[MH-C5H 10 O5-C3H4O2-CH2O] - ,[MH-C5H 10 O5-C3H4O2-CH2O-H2O] - ,[MH-C5H 10 O5-C6H9O4] - Based on the references and comparison with the chromatograms of the reference sample, compound 32 was inferred to be Polygala tenuifolia. Ketone III. Its inferred cleavage pathway and MS / MS chromatogram are shown below. Figure 14 , Figure 15 .

[0103] Table 1. Mass spectrometry information of chemical components of Guipi Heji under positive and negative ion modes.

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

Claims

1. A detection method for rapidly identifying chemical components in a Guipi mixture, characterized in that, The test sample solution and the control sample solution are prepared, the control sample and the test sample solutions are respectively injected into an ultra-high performance liquid chromatography-quadrupole-electrical field orbitrap high resolution mass spectrometry system for determination, and combined with the chromatographic retention time, ultraviolet absorption spectrum characteristics, molecular weight and multi-stage cleavage ion fragment information of the control sample, the structure identification is rapidly performed to obtain 115 kinds of compound components; the control sample is chlorogenic acid, ferulic acid, glycyrrhizin, calycosin-7-glucoside, polygala ketone III, ellagic acid, spinosin, luteoloside, party glycoside, 3,6'-di-glucosylsucrose, formononetin, ammonium glycyrrhizinate, polygalasaponin, atractylodes lactone I, ligusticum lactone, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C; the ultra-high performance liquid chromatography condition is that octadecyl bonded silica gel is used as a stationary phase, 10mmol / L ammonium acetate solution-acetonitrile is used as a mobile phase for gradient elution, and the detection wavelength is 320-330nm; The elution process is performed according to the following gradient table:

2. The detection method according to claim 1, characterized in that, The preparation of the test solution comprises the following steps: precisely pipetting the sample of Guipi mixture, dissolving with methanol, ultrasonic extraction, constant volume, shaking, centrifugation, filtering the supernatant, and taking it as the test solution.

3. The method of claim 1, wherein, The flow rate of the ultra-high performance liquid chromatography is 0.2-0.4 ml / min.

4. The detection method according to claim 3, characterized in that, The chromatographic column is a Waters CORTECS T3 C18 chromatographic column with a column specification of 2.1 mm*100 mm*1.6 μm, or a Waters ACQUITY UPLC HSS T3 chromatographic column with a column specification of 2.1 mm*100 mm*1.8 μm.

5. The detection method according to claim 4, characterized in that, The detection wavelength is 324 nm, and the flow rate is 0.2 ml / min.

6. The method of claim 1, wherein, The quadrupole-electric field orbitrap high-resolution mass spectrometry conditions are as follows: The ion source is HESI source, the positive / negative ion switching mode, the sheath gas flow rate is 30 arb, the auxiliary gas flow rate is 8 arb, the spray voltage is 3.8 kv, the ion transmission tube temperature is 320 DEG C, the scanning mode is Full MS / dd-MS2, the Full MS resolution is 70000, the dd-MS2 resolution is 17500, the scanning range is m / z 125-1875, and the MS / MS mode is Full MS / dd-MS2.

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