Method for identifying chemical components in tablets for nourishing kidney, invigorating spleen and dispersing blood stasis

Abstract

The invention discloses a method for identifying chemical components in a kidney-nourishing spleen-invigorating blood stasis-dispersing tablet, belongs to the technical field of traditional Chinese medicine component detection, and realizes identification of various types of chemical components in the kidney-nourishing spleen-invigorating blood stasis-dispersing tablet through ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry. The method has the beneficial effects that the chemical compositions of the 11 traditional Chinese medicines except the mineral medicine concha haliotidis are represented through accurate molecular weight and characteristic fragment information of a high-resolution mass spectrum provided by the ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrum; the simple, rapid, accurate, sensitive and resolution-ratio identification of multi-source and multi-category chemical components in the kidney-nourishing, spleen-invigorating and blood stasis-dispersing tablet is realized, and a basis can be provided for the research on the pharmacodynamic material basis and the action mechanism of the medicine.

Description

Technical Field

[0001] The invention relates to the technical field of Chinese medicine component detection, and in particular to a method for identifying the chemical components of a Chinese medicine Zishen Jianpi Huayu tablet. Background Art

[0002] Zishen Jianpi Huayu Tablets are composed of twelve Chinese medicinal herbs, including wine-soaked cornus fruit, Panax notoginseng, rhubarb, Pueraria lobata, Astragalus, and Rehmannia glutinosa. They have the effects of nourishing yin and replenishing qi, removing blood stasis and purging heat, and promoting blood circulation and unblocking collaterals. They have significant efficacy in the clinical treatment of DR of qi and yin deficiency type, and can significantly improve the visual function of early patients, treat macular edema caused by DR, and balance systemic metabolism. Modern pharmacological studies have shown that Zishen Jianpi Huayu Tablets can inhibit the expression of inflammatory factors in patients' eyes, reduce the levels of VEGF and IL-6 in aqueous humor and serum, protect patients' retinal vascular endothelium, and delay or even improve retinal microvascular lesions. However, the material basis of the efficacy of this formula is still unclear, which to a certain extent limits the in-depth study of its mechanism of action and targets for the treatment of DR.

[0003] At present, there are few studies on the characterization of the chemical composition of Zishen Jianpi Huayu Tablets. Existing studies have not yet fully characterized the components of this compound and cannot provide strong data support for in-depth research on the material basis of its efficacy and mechanism of action. Summary of the invention

[0004] In order to overcome the problems that there are few studies on the characterization of the chemical composition of Zishen Jianpi Huayu Tablets in the prior art, and the existing studies on the characterization of the components of the compound are not comprehensive, the present invention provides a method for identifying the chemical components in Zishen Jianpi Huayu Tablets, comprising the following steps:

[0005] Step 1, dissolving the Zishen Jianpi Huayu Tablets in an extraction solvent to obtain a Zishen Jianpi Huayu Tablets solution, subjecting the Jianpi Huayu Tablets solution and the extraction solvent to ultrasonic mixing, centrifugation and microporous membrane filtration under the same conditions to obtain a test solution and a blank control solution;

[0006] Step 2, dissolving ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, notoginsenoside R1, puerarin, morroniside, loganin, 11-keto-boswellic acid, astragaloside IV, calycosin isoflavone glucoside, citric acid, ferulic acid, chlorogenic acid, aloe-emodin, rhein, rhein, rehmannia glutinosa D, (±)-liquiritigenin, and hyperoside as reference substances in an extraction solvent to obtain a reference substance solution;

[0007] Step 3, the test solution, blank control solution and reference solution are detected by ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry to obtain the retention time, parent ion mass-to-charge ratio and secondary fragment ion mass-to-charge ratio of each chemical component;

[0008] Among them, the chromatographic conditions of ultra-high performance liquid chromatography include:

[0009] Chromatographic column: C18 column;

[0010] Mobile phase: Phase A is 0.1% formic acid aqueous solution, phase B is acetonitrile; gradient elution is adopted with volume fraction 5-98% phase A, 2-95% phase B; flow rate: 0.4mL / min; column temperature: 40℃; injector temperature: 8℃; injection volume: 2μL;

[0011] Step 4, establish a compound database according to the names, molecular formulas, compound types, CAS number information and chemical structure files of the compounds contained in wine-boiled cornus fruit, raw rehmannia root, hawthorn, rhubarb, astragalus, Panax notoginseng, Millettia spatholobi, Pueraria lobata, vinegar frankincense, stir-fried silkworm and Rhizoma Polygoni Multiflori in Zishen Jianpi Huayu Tablets, establish a candidate compound list by matching and analyzing the compounds in the compound database with the original mass spectrometry data, filter the compounds in the candidate compound list, and identify the chemical components in Zishen Jianpi Huayu Tablets based on the retention time, parent ion mass-to-charge ratio and secondary fragment ion mass-to-charge ratio of each compound.

[0012] Preferably, the extraction solvent is a methanol-water solution with a volume ratio of 0-100%, and the concentrations of the extraction solvents used in the test solution, the blank control solution and the reference solution are equal.

[0013] Preferably, the ultrasonic mixing time is 30-60 min, the pore size of the microporous filter membrane is 0.2-0.45 μm, the concentration of the test solution is 90-110 mg / mL, and the storage temperature of the test solution and the blank control solution before use is 2-8°C.

[0014] Preferably, the concentration of the reference solution is 0.9-1.1 mg / mL, and the storage temperature of the reference solution before use is 2-8°C.

[0015] Preferably, the chromatographic column is Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm).

[0016] Preferably, the gradient elution is as follows: 0-1 min, 2-2% phase B; 1-13 min, 2-50% phase B; 13-21 min, 50-95% phase B; 21-24 min, 95-95% phase B; 24-24.1 min, 95-2% phase B; 24.1-27 min, 2-2% phase B.

[0017] Preferably, the mass spectrometry conditions of ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry include:

[0018] An electrospray ion source was used, and the positive and negative ion modes were scanned. Leucine enkephalin was used as a calibration solution for real-time calibration. The positive ion m / z was 556.2766, the negative ion m / z was 554.2620, and MSE full-time and full-information data were collected.

[0019] Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

[0020] Preferably, the chemical structure file is a mol file.

[0021] Preferably, step 4 includes:

[0022] A list of candidate compounds is established by matching and analyzing the compounds in the compound database with the original mass spectrometry data, and the compounds in the compound database are screened and the mass spectrometry fragmentation rules are annotated. Based on the compound database, the successfully matched compounds are recorded as identified compounds, and the unsuccessfully matched compounds are recorded as unknown compounds. The identified compounds and unknown compounds together constitute the candidate compound list.

[0023] Preferably, the parameters for extracting and matching the chromatographic peaks are:

[0024] Chromatographic peak extraction parameters: minimum retention time 0.5 min, maximum retention time 24.0 min, mass correction applied, intensity threshold 50 counts at high energy, intensity threshold 100 counts at low energy, high background noise filtering selected, maximum number of retained peaks 100,000;

[0025] Chromatographic peak matching parameters: target matching mass-to-charge ratio tolerance is 5.0ppm, structural fragment ion prediction function is applied, fragment ion matching tolerance is 2.0mDa, and in-source fragment ion matching is performed. The maximum number of candidate compounds used for screening and discovery is 50,000, and the number of candidate compounds retained after screening is 10,000.

[0026] Preferably, after filtering and screening the candidate compound list, the response of the well-matched compound obtained is not less than 10,000, the absolute value of the mass-to-charge ratio tolerance is not greater than 5.0 ppm, and the number of fragment ions is not less than 2.

[0027] Beneficial effects:

[0028] The beneficial effects produced by the technical solution of the present invention are as follows:

[0029] The present invention adopts ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC Q-TOF / MS) to collect chemical component information in Zishen Jianpi Huayu Tablets, and characterizes the chemical compositions of 11 traditional Chinese medicines except the mineral medicine Cassia tora by precise molecular weight and characteristic fragment information of high-resolution mass spectrum provided by ultra high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry based on the matching result of a self-built compound database, so as to realize the simple, rapid, accurate, sensitive and high-resolution identification of multi-source and multi-category chemical components in Zishen Jianpi Huayu Tablets, and provide a basis for the research on the material basis of the efficacy and the mechanism of action of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The TIC diagrams of Zishen Jianpi Huayu Tablets are shown in Figure 1. A is the TIC diagram in the negative ion mode, and B is the TIC diagram in the positive ion mode.

[0032] Figure 2 are the primary and secondary mass spectra of compound 74 (notoginseng saponin R1); wherein A is the primary mass spectrum of the compound, and B is the secondary mass spectrum of the compound;

[0033] Figure 3 are the primary and secondary mass spectra of compound 82 (ginsenoside Rg1); wherein A is the primary mass spectrum of the compound, and B is the secondary mass spectrum of the compound;

[0034] Figure 4 are the primary and secondary mass spectra of compound 134 (astragaloside IV); wherein A is the primary mass spectrum of the compound, and B and C are the secondary mass spectra of the compound;

[0035] Figure 5 are the primary and secondary mass spectra of compound 29 (puerarin); wherein A is the primary mass spectrum of the compound, and B and C are the secondary mass spectra of the compound;

[0036] Figure 6 are the primary and secondary mass spectra of compound 50 (calycosin isoflavone glucoside); wherein A is the primary mass spectrum of the compound, and B is the secondary mass spectrum of the compound;

[0037] Figure 7are the primary and secondary mass spectra of compound 175 (11-keto boswellic acid); wherein A is the primary mass spectrum of the compound, and BF is the secondary mass spectrum of the compound;

[0038] Figure 8 are the primary and secondary mass spectra of compound 138 (rhein); wherein A is the primary mass spectrum of the compound, and B and C are the secondary mass spectra of the compound;

[0039] Fig. 9 are the primary and secondary mass spectra of compound 155 (rhubarb); wherein A is the primary mass spectrum of the compound, and B and C are the secondary mass spectra of the compound;

[0040] Fig.10 are the primary and secondary mass spectra of compound 24 (morroniside); wherein A is the primary mass spectrum of the compound, and B is the secondary mass spectrum of the compound;

[0041] Fig.11 are the primary and secondary mass spectra of compound 34 (loganin); wherein A is the primary mass spectrum of the compound, and BD is the secondary mass spectrum of the compound;

[0042] Fig.12 are the primary and secondary mass spectra of compound 22 (chlorogenic acid); wherein A is the primary mass spectrum of the compound, and B and C are the secondary mass spectra of the compound;

[0043] Fig.13 are the primary and secondary mass spectra of compound 55 (verbascoside); wherein A is the primary mass spectrum of the compound, and B is the secondary mass spectrum of the compound;

[0044] Fig.14 are the primary and secondary mass spectra of compound 10 (gallic acid); wherein A is the primary mass spectrum of the compound, and B and C are the secondary mass spectra of the compound;

[0045] Fig.15 5 are the primary and secondary mass spectra of compound 5 (melibiose); wherein A is the primary mass spectrum of the compound, and B and C are the secondary mass spectra of the compound. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] This embodiment uses ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (UPLC-IMS-Q-TOF-MS) to establish a method for identifying the chemical components of Zishen Jianpi Huayu Tablets, and uses the precise molecular weight and characteristic fragment information of high-resolution mass spectrometry to characterize the chemical composition of 11 traditional Chinese medicines except the mineral medicine Shijueming (because the main components of Shijueming are inorganic substances such as inorganic salts and inorganic elements, and high-resolution mass spectrometry is mainly used for qualitative analysis of organic matter, so the chemical composition contained in Shijueming is not characterized in the present invention), providing a basis for the study of the drug's pharmacodynamic material basis and mechanism of action. The specific implementation is as follows:

[0048] A method for identifying chemical components in Zishen Jianpi Huayu tablets comprises the following steps:

[0049] Step 1, dissolving the Zishen Jianpi Huayu Tablets in an extraction solvent to obtain a Zishen Jianpi Huayu Tablets solution, subjecting the Jianpi Huayu Tablets solution and the extraction solvent to ultrasonic mixing, centrifugation and microporous membrane filtration under the same conditions to obtain a test solution and a blank control solution;

[0050] Step 2, dissolving ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, notoginsenoside R1, puerarin, morroniside, loganin, 11-keto-boswellic acid, astragaloside IV, calycosin isoflavone glucoside, citric acid, ferulic acid, chlorogenic acid, aloe-emodin, rhein, rhein, rehmannia glutinosa glycoside D, (±)-liquiritigenin, and hyperoside as reference substances in an extraction solvent to obtain a reference substance solution;

[0051] Step 3, the test solution, blank control solution and reference solution are detected by ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry to obtain the retention time, parent ion mass-to-charge ratio and secondary fragment ion mass-to-charge ratio of each chemical component;

[0052] Among them, the chromatographic conditions of ultra-high performance liquid chromatography include:

[0053] Chromatographic column: C18 column;

[0054] Mobile phase: Phase A is 0.1% formic acid aqueous solution, phase B is acetonitrile; gradient elution is adopted with volume fraction 5-98% phase A, 2-95% phase B; flow rate: 0.4mL / min; column temperature: 40℃; injector temperature: 8℃; injection volume: 2μL;

[0055] Step 4, establish a compound database according to the names, molecular formulas, compound types, CAS number information and chemical structure files of the compounds contained in wine-boiled cornus fruit, raw rehmannia root, hawthorn, rhubarb, astragalus, Panax notoginseng, Millettia spatholobi, Pueraria lobata, vinegar frankincense, stir-fried silkworm and Rhizoma Polygoni Multiflori in Zishen Jianpi Huayu Tablets, establish a candidate compound list by matching and analyzing the compounds in the compound database with the original mass spectrometry data, filter the compounds in the candidate compound list, and identify the chemical components in Zishen Jianpi Huayu Tablets based on the retention time, parent ion mass-to-charge ratio and secondary fragment ion mass-to-charge ratio of each compound.

[0056] Specifically, the extraction solvent is a methanol aqueous solution with a volume ratio of 0-100%, and the concentrations of the extraction solvents used in the test solution, the blank control solution and the reference solution are equal.

[0057] Specifically, the ultrasonic mixing time is 30-60 min, the pore size of the microporous filter membrane is 0.2-0.45 μm, the concentration of the test solution is 90-110 mg / mL, and the storage temperature of the test solution and the blank control solution before use is 2-8°C.

[0058] Specifically, the concentration of the reference solution is 0.9-1.1 mg / mL, and the storage temperature of the reference solution before use is 2-8°C.

[0059] Specifically, the chromatographic column is Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm).

[0060] Specifically, the gradient elution is: 0-1 min, 2-2% B phase; 1-13 min, 2-50% B phase; 13-21 min, 50-95% B phase; 21-24 min, 95-95% B phase; 24-24.1 min, 95-2% B phase; 24.1-27 min, 2-2% B phase.

[0061] Specifically, the mass spectrometry conditions of ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry include:

[0062] An electrospray ion source was used, and the positive and negative ion modes were scanned. Leucine enkephalin was used as a calibration solution for real-time calibration. The positive ion m / z was 556.2766, the negative ion m / z was 554.2620, and MSE full-time and full-information data were collected.

[0063] Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

[0064] Specifically, the chemical structure file is a mol file.

[0065] Specifically, step four includes:

[0066] A list of candidate compounds is established by matching and analyzing the compounds in the compound database with the original mass spectrometry data, and the compounds in the compound database are screened and the mass spectrometry fragmentation rules are annotated. Based on the compound database, the successfully matched compounds are recorded as identified compounds, and the unsuccessfully matched compounds are recorded as unknown compounds. The identified compounds and unknown compounds together constitute the candidate compound list.

[0067] Specifically, the parameters for extracting and matching the chromatographic peaks are:

[0068] Chromatographic peak extraction parameters: minimum retention time 0.5 min, maximum retention time 24.0 min, mass correction applied, intensity threshold 50 counts at high energy, intensity threshold 100 counts at low energy, high background noise filtering selected, maximum number of retained peaks 100,000;

[0069] Chromatographic peak matching parameters: target matching mass-to-charge ratio tolerance is 5.0ppm, structural fragment ion prediction function is applied, fragment ion matching tolerance is 2.0mDa, and in-source fragment ion matching is performed. The maximum number of candidate compounds used for screening and discovery is 50,000, and the number of candidate compounds retained after screening is 10,000.

[0070] Specifically, after filtering and screening the candidate compound list, the response of the well-matched compound obtained is no less than 10,000, the absolute value of the mass-to-charge ratio tolerance is no more than 5.0 ppm, and the number of fragment ions is no less than 2.

[0071] The following is a further review of the method for identifying the chemical components in the Zishen Jianpi Huayu Tablets in this embodiment through examples.

[0072] Example 1

[0073] The instruments and reagents used in this example are described below.

[0074] 1. Instrument

[0075] UPLC ultra-high performance liquid chromatograph (Waters Corporation, Milford, MA, USA); VionTM IMS-QTOF ion mobility quadrupole time-of-flight mass spectrometer (Waters Corporation, Milford, MA, USA); AS-20500AT ultrasonic cleaner (Tianjin Aotesense Instrument Co., Ltd., Tianjin, China); Quintix35-1CN one-hundred-thousandth balance (Sartorius Scientific Instrument (Beijing) Co., Ltd., Beijing, China); BCE224-1CCN one-ten-thousandth balance (Sartorius Scientific Instrument (Beijing) Co., Ltd., Beijing, China); Vortex Genius3 vortex mixer (Aika (Guangzhou) Instrument Equipment Co., Ltd., Guangzhou, China); ST8R high-speed refrigerated centrifuge (Thermo Fisher Scientific, Waltham, MA,, USA).

[0076] 2. Reagents

[0077] Acetonitrile and formic acid (Merck KGaA, Darmstadt, Germany) were all chromatography / mass spectrometry grade; deionized water was IQ 7000 system (Merck KGaA, Darmstadt, Germany) was used for purification. Zishen Jianpi Huayu Tablets were produced and provided by the First Affiliated Hospital of Guangzhou University of Chinese Medicine. The names, molecular formulas, manufacturers and other information of the 19 reference substances are shown in Table 1.

[0078] Table 1 Reference material information

[0079]

[0080] The following is the operation process of this embodiment:

[0081] Step 1: Preparation of test solution and blank control solution: weigh 1g of Zishen Jianpi Huayu Tablets, grind it into powder and dissolve it in 10mL of 75% methanol aqueous solution. After 45min of ultrasonic mixing of Zishen Jianpi Huayu Tablets solution, take 2mL for centrifugation, take 1mL of the supernatant after centrifugation and filter it through a 0.22μm microporous filter membrane to obtain a test solution with a concentration of 100mg / mL; after 45min of ultrasonic mixing of 2mL of 75% methanol aqueous solution, centrifuge, take 1mL of the supernatant after centrifugation and filter it through a 0.22μm microporous filter membrane to obtain a blank control solution. The test solution and the blank control solution were stored at 2-8℃ before use.

[0082] Step 2: Preparation of reference solution: weigh 2 mg of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, notoginsenoside R1, puerarin, morroniside, loganin, 11-keto-boswellic acid, astragaloside IV, calycosin isoflavone glucoside, citric acid, ferulic acid, chlorogenic acid, aloe-emodin, rhein, rhein, rehmannia glutinosa D, (±)-liquiritigenin, and hyperoside as reference substances, and dissolve them in 2 ml of 75% methanol aqueous solution to obtain reference solutions of each component with a concentration of 1 mg / mL. Each control group solution was stored at 2-8°C before use.

[0083] Step 3: Sample testing: ultra performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry is used to test the test solution, blank control solution and each reference solution to obtain high-resolution mass spectra of the test solution and the reference solution.

[0084] The chromatographic conditions for the detection were:

[0085] Chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm).

[0086] Mobile phase: A is 0.1% formic acid aqueous solution; B is acetonitrile; elution gradient: 0-1min, 2-2% B phase; 1-13min, 2-50% B phase; 13-21min, 50-95% B phase; 21-24min, 95-95% B phase; 24-24.1min, 95-2% B phase; 24.1-27min, 2-2% B phase; flow rate: 0.4mL / min; column temperature: 40℃; injector temperature: 8℃; injection volume: 2μL.

[0087] The mass spectrometry conditions during detection were:

[0088] An electrospray ion source was used, and the positive and negative ion modes were scanned. Leucine enkephalin was used as a calibration fluid for real-time calibration. The positive ion m / z was 556.2766, the negative ion m / z was 554.2620, and MSE full-time and full-information data were collected.

[0089] Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

[0090] Step 4: Chemical analysis and identification, including:

[0091] (1) Establish a compound database. By searching relevant literature and chemical database websites such as CNKI, Chemspider, Web of Science, PubChem and SciFinder, a compound database of the 11 herbs in Zishen Jianpi Huayu Tablets (except Semen Cassiae) was established, including the name, molecular formula, compound type, CAS number information and chemical structure file in mol file format. The established compound database was imported into the Library module of the software UNIFI and added to the target compound list.

[0092] (2) Establishing a list of candidate compounds: The software UNIFI was used to identify, match, and filter noise from the collected raw mass spectrometry data based on the compound database. The software was also able to automatically screen the compounds in Zishen Jianpi Huayu Tablets based on a specific database and annotate the mass spectrometry fragmentation patterns of the compounds.

[0093] Peak extraction parameter settings: minimum retention time 0.5 min, maximum retention time 24.0 min, mass correction applied, intensity threshold at high energy 50 counts, intensity threshold at low energy 100 counts, high background noise filtering selected, maximum number of retained peaks 100,000.

[0094] Peak matching parameter settings: target matching mass-to-charge ratio tolerance is 5.0 ppm, structural fragment ion prediction function is applied, fragment ion matching tolerance is 2.0 mDa, and in-source fragment ion matching is performed.

[0095] The maximum number of candidate compounds for screening and discovery is 50,000, and the number of candidate compounds retained after screening is 10,000. The peak matching process is based on the established compound database. The successfully matched compounds are identified compounds, and the unsuccessfully matched compounds are unknown compounds, which together constitute the candidate compound list.

[0096] (3) Compound identification: The candidate compound list was filtered to obtain well-matched compounds with a response of not less than 10,000, a mass-to-charge ratio tolerance of not more than 5.0 ppm, and a number of fragment ions of not less than 2. The chemical components successfully identified in Zishen Jianpi Huayu Tablets were finally obtained by high-resolution mass spectrometry comparison with the test solution and the reference solution and literature review.

[0097] Through data acquisition in positive and negative ion modes, the total ion current of Zishen Jianpi Huayu Tablets is shown in Figure 1As shown. Based on the reference substances, database matching and literature comparison, a total of 186 chemical components were identified as shown in Table 2, including 58 saponins, 38 flavonoids, 24 terpenes, 15 anthraquinones, 11 iridoids, 11 organic acids, 10 phenylethanoid glycosides, 6 tannins, 4 sugars, 2 anthrones, 2 ionones, 1 phenylbutyrone, 1 stilbene, and 3 other types. Among the above components, 24 belonged to wine cornus fruit, 41 belonged to astragalus, 45 belonged to notoginseng, 17 belonged to vinegar frankincense, 21 belonged to rhubarb, 24 belonged to raw rehmannia, 3 belonged to rutin, 16 belonged to kudzu, 1 belonged to fried silkworm, 23 belonged to millettia reticulata, 20 belonged to hawthorn, and 31 components were derived from two or more medicinal flavors. Table 2 shows the compound identification results of Zishen Jianpi Huayu Tablets, where * indicates a compound confirmed by a reference substance, i.e., the chromatographic retention time and mass spectrometry fragmentation pattern of the compound in the sample are consistent with those of the reference substance.

[0098] Table 2 Compound identification results of Zishen Jianpi Huayu Tablets

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] (a) Saponin ingredients

[0111] The 58 saponin components identified in the prescription are mainly derived from Astragalus and Panax notoginseng, and the proportion of such components in Panax notoginseng is larger. According to the characteristics of their parent core chemical structure, the saponin components in Astragalus can be divided into cycloastragenol, soybean saponin B, etc., among which the representative compound is astragaloside. The saponins in Panax notoginseng can be roughly divided into ginsenosides, notoginseng saponins and aescin. Most of the saponin components have tetracyclic triterpenes as the parent core structure and are connected to different sugars. Due to the different connected sugar components, fragment ions after removing different sugar groups and aglycone fragment ions of saponins often appear during the mass spectrometry fragmentation process. The cleavage modes of sugar groups mainly include simple cleavage, rearrangement of terminal sugars, sugar group ring-opening cleavage, and sugar group cross-ring cleavage. In the cleavage process, ions with mass numbers less than sugar groups such as lost CO2, -C3H6O4, -C2H4O2, -C2H5O3, -CH5O2 and -CH2O3 may also be obtained. Saponins respond in both positive and negative ion modes, but the sensitivity is better in negative ion mode, that is, it also responds well to saponins with lower content. Compounds 74, 82 and 136 are used as examples to illustrate the typical mass spectrometry fragmentation characteristics of saponins. Compound 74 is detected in negative ion mode ( Figure 2 ) gave m / z 977.5336 [M+HCOOH-H] - and m / z 931.5281[MH] - The quasi-molecular ion peak of the product is m / z 799.4857[MH-Xyl] - 、637.4329[MH-Xyl-Glc] - and m / z 475.3799 [MH-Xyl-2Glc] - The fragmentation pattern was consistent with that reported in the literature. Compound 74 was confirmed to be notoginseng saponin R1 by reference substance verification. Compounds 64, 66, 89, 91, 97, 100, etc. in this series were also identified. Compound 82 was detected in negative ion mode ( Figure 3 ) gives the quasi-molecular ion peak m / z 845.4907 [M+HCOOH-H] - and m / z 799.4852[MH] - The characteristic fragment ions are mainly m / z 637.4323[MH-Glc]-, 619.4217[MH-Glc-H2O] - and m / z 475.3794 [MH-2Glc] - The fragmentation pattern was consistent with that reported in the literature. Compound 82 was confirmed to be ginsenoside Rg1 by reference substance verification. Compounds 81, 104, 111, 114, 122, etc. in the same series were also identified. Compound 134 was identified in negative ion mode ( Figure 4) gave m / z 829.4585 [M+HCOOH-H] - The quasi-molecular ion peaks of the molecule are mainly m / z 651.4101 [MH-C5H8O4] generated by sugar cleavage. - 、637.4313[MH-C3H6O3-2CO] - 、621.3921[MH-Glc] - and m / z 563.3582 [MH-Glc-C3H6O] generated by the loss of other substituents after deglycosylation - ,485.3263[MH-Glc-C3H6O-C2H6O3] - After literature comparison and reference verification, compound 134 was confirmed to be astragaloside IV, and the same type of compounds 127, 135, 146, 154, 158, etc. were all identified.

[0112] (b) Flavonoids

[0113] A total of 38 flavonoid components were identified in the formula, mainly from Astragalus, Puerariae and Millettia reticulata. The parent core structure of this type of compound is chromone substituted with phenyl at different positions, which is prone to rearrangement, RDA cleavage, and loss of some neutral molecules such as CO and CO2 during mass spectrometry fragmentation. Taking compounds 29 and 50 as examples, the typical mass spectrometry fragmentation characteristics of flavonoid compounds are described. Compound 29 was detected in positive ion mode ( Figure 5 ) gives a quasi-molecular ion peak m / z 417.1189 [M+H] + , removing two molecules of water to form a fragment ion m / z 381.0972 [M+H-2H2O] + The sugar ring on the compound is cleaved to obtain a fragment ion m / z 297.0762 [M+H-C4H8O4] + and m / z 267.0657 [M+H-C5H8O4-H2O] + The fragmentation pattern was consistent with that reported in the literature. Compound 29 was confirmed to be puerarin by reference substance verification. Compounds 39, 40, 43, and 54 of the same series were also identified. Compound 50 was detected in negative ion mode ( Figure 6 ) gives the quasi-molecular ion peak m / z 491.1200 [M+HCOOH-H] - The characteristic fragment ions are mainly m / z 283.0614 [MH-Glc] - 、268.0378[MH-Glc-CH3] - and m / z 253.0508 [MH-Glc-CH2O] -The fragmentation pattern was consistent with that reported in the literature. Compound 50 was confirmed to be calycosin glucoside by reference substance verification. Compounds 45, 61, and 67 of the same series were also identified.

[0114] (c) Terpenoid components

[0115] A total of 24 terpenoid components were identified in the formula, mainly from vinegar frankincense and hawthorn. The terpenoid components in the formula are mainly pentacyclic triterpenoid compounds, most of which have β-configuration at the 3-position H and are mostly oxidized to -COOH or -CHO. The substituents on the parent nucleus are mainly hydroxyl, carboxyl, acetoxy, carbonyl, hydroxymethyl, methoxy, etc. The substituents on the parent nucleus are easily removed during the mass spectrometry fragmentation process, and some neutral molecules such as CO and CO2 are lost. Taking compound 175 as an example ( Figure 7 ), describing the typical mass spectrometry fragmentation characteristics of terpenoids. Compound 175 gave a quasi-molecular ion peak of m / z 471.3474 [M+H] in positive ion mode. + , loses one molecule of CO2 and forms a double bond to obtain a fragment ion m / z 425.3416 [M+H-CO2-H2] + On this basis, a molecule of H2O is lost to obtain the fragment ion m / z 407.3309 [M+H-CO2-H2-H2O] + The parent nucleus of the pentacyclic triterpene cleaved to form a more stable conjugated structure and obtained fragment ions m / z 347.2227, 287.2371 and 191.1799. The cleavage pattern was consistent with that reported in the literature. Compound 175 was confirmed to be 11-keto boswellic acid by reference substance verification. Compounds 181, 182, 183, 185, etc. in the same series were also identified.

[0116] (d) Anthraquinone components

[0117] A total of 15 anthraquinone components were identified in the formula, mainly from rhubarb and Millettia reticulata. The basic mother nucleus of this type of compound is anthraquinone, which often has hydroxyl, methyl, methoxy and carboxyl substituents. In the mass spectrometry fragmentation process, fragments are often produced by the breakage of substituents on the benzene ring, and free anthraquinone often obtains highly abundant ion peaks after desorbing two molecules of CO in sequence. Taking compounds 138 and 155 as examples, the typical mass spectrometry fragmentation characteristics of anthraquinone compounds are described. Compound 138 was detected in negative ion mode ( Figure 8 ) gives m / z 283.00250 [MH] - The quasi-molecular ion peak of the product is m / z 239.0351[MH-CO2]. - 、211.0402[MH-CO2-CO] - 、183.0449[MH-CO2-2CO] -The fragmentation pattern was consistent with that reported in the literature. Compound 138 was confirmed to be rhein by checking with reference substances. Compound 155 was detected in negative ion mode ( Fig. 9 ) gives a quasi-molecular ion peak m / z 269.0456 [MH] - The characteristic fragment ion is m / z 241.0502[MH-CO] - 、225.0557[MH-CO2] - 、197.0605[MH-CO2-CO] - 、181.0660[MH-CO2-CO-CH3] - Compound 155 was confirmed to be rhein by comparison with the literature fragmentation pattern and verification with reference substances. Based on similar fragmentation patterns, compounds 28, 33, 46, 48, 56, 65, 71, 73, 75, 95, 99, 119 and 128 in the same series were also identified.

[0118] (e) Iridoid components

[0119] A total of 11 iridoid components were identified in the prescription, mainly from Cornus officinalis and Rehmannia root. Iridoid components are a five-membered carbon ring and a six-membered oxygen heterocyclic monoterpene compound, generally existing in the form of glycosides, with multiple biological activities, and are often used as chemical classification markers. The common fragmentation patterns of iridoid compounds are loss of sugar groups, RDA cleavage, and neutral loss of H2O. In addition, there is also the breakage of ester bonds leading to loss of CH3OH and decarboxylation. This type of component responds better in negative ion mode. Taking compounds 24 and 34 as examples, the typical mass spectrometry fragmentation characteristics of iridoid compounds are described. Compound 24 was detected in negative ion mode ( Fig.10 ) gave m / z 451.1462 [M+HCOOH-H] - and m / z 405.1406[MH] - The quasi-molecular ion peak of the product is m / z 373.1334 [MH-CH3OH]. - 、243.0874[MH-Glc] - and m / z 155.0353 [MH-Glc-CH3OH-2CO] - The fragmentation pattern was consistent with that reported in the literature. Compound 24 was confirmed to be morroniside by reference. Compound 34 was Fig.11 ) gives a quasi-molecular ion peak m / z 435.1510 [M+HCOOH-H] - and m / z 389.1455[MH] - , fragment ion m / z 227.0924 [MH-Glc] -This indicates that one glucose molecule is lost, and on this basis one water molecule and CH3OH are removed to obtain the fragment ion m / z 177.0197 [MH-Glc-CH3OH-H2O] - After forming a double bond, the fragment ion m / z 175.0406 [MH-Glc-CH3OH-H2O-H2] is obtained. - The mass spectrum also showed the fragment ion m / z 127.0404 produced by RDA cleavage. After self-built database analysis, literature comparison and reference verification, compound 34 was identified as loganin. Based on similar cleavage rules, compounds 13, 16, 17, 21, 23, 31, 35, 42 and 63 in the same series were also identified.

[0120] (f) Organic acid components

[0121] A total of 11 organic acid components were identified in the formula, mainly derived from hawthorn, including phenolic acids, fatty acids and other organic acids. Organic acid components are often detected in negative ion mode in mass spectrometry, and common mass spectrometry fragmentation modes are dehydroxylation, carboxylation, carbonylation, etc. to form double bonds or conjugated structures. Taking compound 22 as an example ( Fig.12 ), which gave a quasi-molecular ion peak m / z 353.0880 [MH] in negative ion mode - , the ester bond breaks to form m / z 191.0563 [quinic acid-H] - and m / z 179.0350 [caffeic acid-H] - Quinic acid removes two molecules of water and one molecule of CO to form a double bond to obtain a fragment ion m / z 125.0249 [quinic acid-H-2H2O-CO-H2] - , caffeoyl dehydration forms fragment ion m / z161.0245 [caffeic acid-H-H2O] - , losing one molecule of CO2 to obtain the fragment ion m / z 135.0454 [caffeicacid-H-CO2] - , remove the propionic acid group on the benzene ring to obtain the fragment ion m / z 108.0219 [caffeic acid-H-C3H3O2] - After comparison with literature and verification of reference substances, compound 22 was identified as chlorogenic acid. Based on similar cleavage patterns and literature reports, compounds 1, 4, 6, 18, 19, 25, 26, 44, 170, and 178 were all identified.

[0122] (g) Phenylethanol glycosides

[0123] A total of 10 phenylethanol glycosides were identified in the formula, all of which were derived from Rehmannia glutinosa. Their basic skeletons were composed of phenylethanol, caffeic acid, glucose and rhamnose. In mass spectrometry detection, phenylethanol glycosides had a higher response in negative ion mode, and their fragmentation pathways were mainly neutral loss of caffeoyl (C9H6O3) or acetyl (C2H2O) after ester glycosidic bond cleavage, and neutral loss of glucose residues (C6H 10 O5) or rhamnose residues (C6H 10 O4). Taking compound 55 as an example ( Fig.13 ), and its quasi-molecular ion peak in negative ion mode is m / z 623.1978[MH] - , losing the caffeoyl group to obtain the fragment ion m / z 461.1659 [MH-C9H6O3] - The removed caffeoyl group forms a fragment ion m / z 161.0245 [MHC 21 H 34 O 11 ] - The compound 55 was confirmed as verbascoside by literature comparison. Based on the similar fragmentation rules, the compounds 36, 38, 47, 57, 58, 60, 69, 79, and 90 in the same series were identified by literature comparison.

[0124] (h) Tannins, etc. and others

[0125] In addition to the above ingredients, a small amount of tannins, sugars, stilbenes, ionones and other compounds were identified in the prescription. Among them, the tannin compounds are derived from cornus officinalis and are galloyltannins. Their cleavage rules are neutral loss of galloyl (galloyl, 152), loss of the entire glucose group (162) and cross-ring excision cleavage of the sugar ring (C2H4O2, 60; C3H6O3, 90). Compound 10 ( Fig.14 ) The quasi-molecular ion peak in negative ion mode is m / z 169.0144 [MH] - , caused by the loss of CO2 and [MHC 21 H 34 O 11 ] - The fragment ion m / z 125.0247 [MH-CO2] was generated. - 、107.0140[MH-CO2-H2O] - , and compound 10 was confirmed to be gallic acid by literature comparison. By literature comparison, similar compounds 8, 11, 12, 14, and 15 were all identified. The sugar compounds in the prescription are mainly derived from Rehmannia glutinosa. The mass spectrometry fragmentation of sugars is mainly the loss of water, sugar moiety, and sugar moiety ring opening. Compound 5 ( Fig.15) The quasi-molecular ion peak in positive ion mode is m / z 381.0799 [M+K] + , and its main fragment ion is m / z 325.1129[M+H-H2O] + 、203.0524[M+H-C4H 12 O5] + 、145.0502[M+H-Glc-2H2O] + The compound 5 was confirmed to be Miertang by literature comparison. Compounds 2, 3, and 7 of the same series were also identified.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for identifying the chemical components in Zishen Jianpi Huayu Tablets, characterized in that: The following steps are involved: Step 1, dissolving the Zishen Jianpi Huayu Tablets in an extraction solvent to obtain a Zishen Jianpi Huayu Tablets solution, subjecting the Jianpi Huayu Tablets solution and the extraction solvent to ultrasonic mixing, centrifugation and microporous membrane filtration under the same conditions to obtain a test solution and a blank control solution; Step 2, dissolving ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, notoginsenoside R1, puerarin, morroniside, loganin, 11-keto-boswellic acid, astragaloside IV, calycosin isoflavone glucoside, citric acid, ferulic acid, chlorogenic acid, aloe-emodin, rhein, rhein, rehmannia glutinosa D, (±)-liquiritigenin, and hyperoside as reference substances in the extraction solvent to obtain reference substance solutions; Step three, detecting the test solution, the blank control solution and the reference solution by ultra performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry to obtain the retention time, parent ion mass-to-charge ratio, and secondary fragment ion mass-to-charge ratio of each chemical component; Among them, the chromatographic conditions of ultra-high performance liquid chromatography include: Chromatographic column: C18 column; Mobile phase: Phase A is 0.1% formic acid aqueous solution, phase B is acetonitrile; gradient elution is adopted with volume fraction 5-98% phase A, 2-95% phase B; flow rate: 0.4mL / min; column temperature: 40℃; injector temperature: 8℃; injection volume: 2μL; Step 4, establish a compound database according to the names, molecular formulas, compound types, CAS number information and chemical structure files of the compounds contained in wine cornus fruit, raw rehmannia root, hawthorn, rhubarb, astragalus, Panax notoginseng, Millettia spatholobi, Pueraria lobata, vinegar frankincense, stir-fried silkworm and Rhizoma Polygoni Multiflori in Zishen Jianpi Huayu Tablets, establish a candidate compound list by matching and analyzing the compounds in the compound database with the original mass spectrometry data, filter the compounds in the candidate compound list, and identify the chemical components in Zishen Jianpi Huayu Tablets based on the retention time, parent ion mass-to-charge ratio and secondary fragment ion mass-to-charge ratio of each compound.

2. The method for identifying chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The extraction solvent is a methanol aqueous solution with a volume ratio of 0-100%, and the concentration of the extraction solvent used in the test solution, the blank control solution and the reference solution is equal.

3. The method for identifying the chemical components in the Zishen Jianpi Huayu Tablet according to claim 2, characterized in that: The ultrasonic mixing time is 30-60 minutes, the pore size of the microporous filter membrane is 0.2-0.45 μm, the concentration of the test solution is 90-110 mg / mL, and the storage temperature of the test solution and the blank control solution before use is 2-8°C.

4. The method for identifying the chemical components in the Zishen Jianpi Huayu Tablet according to claim 2, characterized in that: The concentration of the reference solution is 0.9-1.1 mg / mL, and the storage temperature of the reference solution before use is 2-8°C.

5. The method for identifying chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The gradient elution is specifically as follows: 0-1 min, 2-2% B phase; 1-13 min, 2-50% B phase; 13-21 min, 50-95% B phase; 21-24 min, 95-95% B phase; 24-24.1 min, 95-2% B phase; 24.1-27 min, 2-2% B phase.

6. The method for identifying chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The mass spectrometry conditions for ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry include: An electrospray ion source was used, and the positive and negative ion modes were scanned. Leucine enkephalin was used as a calibration solution for real-time calibration. The positive ion m / z was 556.2766, the negative ion m / z was 554.2620, and MSE full-time and full-information data were collected. Mass spectrometry parameter settings: capillary voltage is 0.8 kV; cone voltage is 30 V; capillary temperature is 120 °C; auxiliary gas heating temperature is 450 °C; cone gas flow rate is 50 L / h; desolvation gas flow rate is 900 L / h; primary collision energy is 6 eV; secondary collision energy is 20-45 eV; scanning range is 50-1500 m / z.

7. The method for identifying chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: The step 4 includes: The candidate compound list is established by performing matching analysis on the original mass spectrometry data of the compounds in the compound database, and the compounds in the compound database are screened and the mass spectrometry fragmentation rules are annotated. Based on the compound database, the successfully matched compounds are recorded as identified compounds, and the unsuccessfully matched compounds are recorded as unknown compounds. The identified compounds and the unknown compounds together constitute the candidate compound list.

8. The method for identifying chemical components in Zishen Jianpi Huayu Tablets according to claim 7, characterized in that: The parameters for extracting and matching chromatographic peaks are: Chromatographic peak extraction parameters: minimum retention time 0.5 min, maximum retention time 24.0 min, mass correction applied, intensity threshold 50 counts at high energy, intensity threshold 100 counts at low energy, high background noise filtering selected, maximum number of retained peaks 100,000; Chromatographic peak matching parameters: target matching mass-to-charge ratio tolerance is 5.0ppm, structural fragment ion prediction function is applied, fragment ion matching tolerance is 2.0mDa, and in-source fragment ion matching is performed. The maximum number of candidate compounds used for screening and discovery is 50,000, and the number of candidate compounds retained after screening is 10,000.

9. The method for identifying chemical components in Zishen Jianpi Huayu Tablets according to claim 1, characterized in that: After filtering and screening the candidate compound list, the response of the well-matched compounds obtained is not less than 10,000, the absolute value of the mass-to-charge ratio tolerance is not greater than 5.0 ppm, and the number of fragment ions is not less than 2.

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