Analysis method of chemical components in liujin qingwen granules

Abstract

The application discloses a method for analyzing chemical components in Liujin Qingwen granules, which is based on the ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry technology and belongs to the technical field of traditional Chinese medicine component analysis and detection. The determined chromatographic conditions can effectively separate the chemical components in the Liujin Qingwen granules, and the mass spectrometry response of each chemical component under the determined mass spectrometry conditions is high. The obtained mass spectrometry data are compared with reference substances, combined with a self-built database and mass spectrometry cracking rules to identify 117 chemical components, and a characteristic-based molecular network is constructed based on characteristic tables and MS / MS spectrum information by using a GNPS platform. The correlation of structural clusters in the molecular network is used to deduce potential compounds, and the chemical composition of the Liujin Qingwen granules is more comprehensively clarified, so as to provide chemical basis and theoretical support for effective component identification, quality evaluation and pharmacological mechanism research of the Liujin Qingwen granules.

Description

Technical Field

[0001] This invention relates to an analytical method for the chemical components in Liujin Qingwen granules, belonging to the field of traditional Chinese medicine component analysis and detection technology. Background Technology

[0002] Liujin Qingwen granules are composed of six traditional Chinese medicines: dandelion, honeysuckle vine, isatis root, indigo leaf, physalis, and dragon's tongue leaf. This formula has the effects of clearing heat and detoxifying, dispelling wind and relieving sore throat, and is a medication for treating influenza caused by wind-heat invading the lungs. Clinical application shows that Liujin Qingwen granules have outstanding "heat-clearing" effects, rapidly reducing fever and effectively improving sore throat, allowing "heat toxins" to be excreted through urination and defecation, demonstrating significant clinical advantages and characteristics. Due to the complexity of its composition, only six components have been identified using liquid chromatography; no systematic research reports on the chemical components of Liujin Qingwen granules have been found. This invention aims to establish a comprehensive analytical method for the chemical components of Liujin Qingwen granules to promote pharmacological research and expand clinical application of this drug. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the present invention provides an analytical method for the chemical components in Liujin Qingwen granules, which identifies 117 kinds of components including organic acids, flavonoids, steroids, phenylpropanoids, terpenoids, alkaloids and other components.

[0004] The present invention achieves the above objectives by adopting the following technical solutions:

[0005] The analytical method for the chemical components in Liujin Qingwen granules includes the following steps:

[0006] S1. Prepare sample solutions and reference solutions;

[0007] S2. Determine the chromatographic and mass spectrometry conditions:

[0008] The chromatographic conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; mobile phase A was 0.1% (v / v) formic acid aqueous solution, mobile phase B was acetonitrile, and the gradient elution program was as follows: 0–15 min: mobile phase A volume fraction decreased from 98% to 84% A; 15–35 min: 84% → 76% A; 35–50 min: 76% → 0% A; 50–55 min: 0% → 98% A; the flow rate was 0.3 mL / min. -1 The column temperature was 30℃, and the injection volume was 5 μL.

[0009] The mass spectrometry conditions were as follows: the ion source was an electrospray ionization source; the scanning modes were positive ion and negative ion modes; the capillary voltage was -3500 V in negative ion mode and +4000 V in positive ion mode; the nebulizer pressure was 35 psi, the drying gas temperature was 300 °C, and the flow rate was 8 L / min; the scanning range was mass-to-charge ratio m / z 50–3000, and the acquisition rate was 1.0 spectra / s.

[0010] S3. Measurement:

[0011] Sample solutions and reference solutions were collected, and mass spectrometry data of the sample solutions were acquired using an ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer.

[0012] S4. Match the mass spectrometry data with the self-built database to achieve the analysis and identification of the target chemical components.

[0013] Specifically, step S4 is as follows:

[0014] S4.1 By summarizing the information on the chemical components contained in the six raw medicinal materials of Liujin Qingwen Granules, a self-built database of chemical components of Liujin Qingwen Granules is constructed. The self-built database includes the name, molecular formula, molecular weight, parent ion m / z value and MS / MS fragment ion information of the chemical components.

[0015] S4.2 The mass spectrometry data obtained in step S3 were analyzed using Mass Hunter software to obtain information such as the retention time, precise molecular weight, and secondary mass spectrometry fragments of the compounds. The results were compared with a self-built database and the structures of the compounds were identified by referring to the reference standard data. Among them, 40 compounds were confirmed by comparison with 40 reference standards.

[0016] Furthermore, the method for analyzing the chemical components in the Liujin Qingwen granules also includes: S5. Constructing a molecular network:

[0017] The mass spectrometry data obtained in step S3 were processed using MS-DIAL software to complete the format conversion and export of the data required for FBMN analysis.

[0018] Data processed and exported using MS-DIAL software was submitted to the GNPS platform for FBMN analysis to generate a molecular network and complete the mass spectrometry analysis of Liujin Qingwen granules.

[0019] Furthermore, the mass spectrometry conditions in step S2 also include: real-time mass correction using standard solutions of ammonium trifluoroacetate, purine, and hexa(2,2,3,3-tetrafluoropropoxy)phosphazene. The m / z values ​​of ammonium trifluoroacetate in negative ion mode are 112.985587, and those of purine in positive ion mode are 121.050873. The m / z values ​​of hexa(2,2,3,3-tetrafluoropropoxy)phosphazene in positive ion mode are 922.009798, and in negative ion mode are 1033.988109.

[0020] The preparation method of the reference solution is as follows:

[0021] Take quinic acid, citric acid, succinic acid, phenylalanine, vanillic acid, protocatechuic acid, neochlorogenic acid, caffeic acid, 4-hydroxybenzoic acid, strychnine, strychnoside, chlorogenic acid, cryptochlorogenic acid, p-coumaric acid, isoharringtonine, quercetin-3-O-β-D-gentiobiglycoside, ferulic acid, quercetin, chicoric acid, 7-hydroxycoumarin, isoquercitrin, isocytisine, and isochlorogenic acid B, respectively. An appropriate amount of isoflavonic acid, isochlorogenic acid A, apigenin, isoflavonic acid C, apigenin, daidzein, luteolin, kaempferol, physalin G, isophysalin G, emodin, physalin O, physalin L, physalin A, physalin F, 4,7-didehydroneophysalin B, and apigenin reference standards were placed in a volumetric flask, methanol was added, and the reference standards were sonicated to completely dissolve them. The solution was then filtered through a 0.22 μm pore size filter membrane to prepare a mixed reference solution, which was stored for later use.

[0022] The sample solution was prepared as follows: Liujin Qingwen granules were ground into powder, an appropriate amount was weighed and added to a 75% methanol aqueous solution, ultrasonically extracted, cooled to room temperature, and the weight loss was made up with a 75% methanol aqueous solution. The sample solution was then filtered through a nylon microporous membrane and stored for later use.

[0023] More specifically, the sample solution was prepared as follows: Liujin Qingwen granules were ground into powder, 0.3g of sample was accurately weighed, 25 mL of 75% methanol aqueous solution was added, ultrasonic extraction was performed for 0.5 hours, and after cooling to room temperature, the weight loss was made up with 75% methanol aqueous solution. The sample was then filtered through a 0.22 μm nylon microporous membrane before testing.

[0024] Preferably, the chromatographic column used is a Waters ACQUITY UPLC HSS T3 column.

[0025] The Liujin Qingwen granules described in this invention are made from the following raw materials in parts by weight: 12-60 parts of honeysuckle vine, 12-60 parts of indigo leaf, 12-60 parts of isatis root, 12-60 parts of dandelion, 3-15 parts of physalis and 3-15 parts of dragon's tongue leaf.

[0026] The preparation method of the Liujin Qingwen granules includes the following steps:

[0027] (1) Take six medicinal materials: honeysuckle vine, indigo leaf, isatis root, dandelion, physalis and dragon tongue leaf, mix them well, soak them in water and decoct them, filter to obtain the first decoction and the dregs.

[0028] (2) Add water to the dregs and boil them again, then filter to obtain the second decoction;

[0029] (3) Combine the first decoction and the second decoction, and filter to obtain the third decoction;

[0030] (4) Heat and concentrate the third decoction, filter it, add maltodextrin and stevioside, dry and granulate to obtain the final product.

[0031] Furthermore, the preparation method of the Liujin Qingwen granules includes the following steps:

[0032] (1) Take six medicinal materials: honeysuckle vine, indigo leaf, isatis root, dandelion, physalis and dragon tongue leaf, mix them well, put them in a decoction pot, add water to soak them, the amount of water is 14 times the total amount of medicinal materials in this step, soak for 0.5 hours, then decoct for 0.5 hours, filter to obtain the first decoction.

[0033] (2) Add water to the decoction pot again, the amount of water is 12 times the total amount of medicinal materials in this step, the decoction time is 0.5h, and filter to obtain the second decoction;

[0034] (3) Combine the first decoction and the second decoction, and filter to obtain the third decoction;

[0035] (4) Heat and concentrate the third decoction to a relative density of 1.09-1.11, filter, add maltodextrin and stevioside, spray dry, and granulate the spray-dried powder after passing it through an 80-120 mesh sieve to obtain 100g of Chinese medicine composition No. 1 granules.

[0036] Characteristics: Brownish-yellow to brownish-red granules; slightly sweet, bitter taste.

[0037] The beneficial effects of the present invention include, but are not limited to:

[0038] The present invention provides an analytical method for the chemical components in Liujin Qingwen granules based on ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS), which identifies 117 kinds of components including organic acids, flavonoids, steroids, phenylpropanoids, terpenes, alkaloids and other components.

[0039] The chromatographic conditions determined in this invention can effectively separate the chemical components in Liujin Qingwen granules, and each chemical component exhibits high mass spectrometry response under the determined mass spectrometry conditions. This invention identifies 117 chemical components by comparing the obtained mass spectrometry data with reference standards, combining a self-built database, and analyzing mass spectrometry fragmentation patterns. Based on the feature table and MS / MS spectral information, a feature-based molecular network is constructed using the GNPS platform. Potential compounds are inferred through the correlation of structural clusters in the molecular network, thus more comprehensively elucidating the chemical composition of Liujin Qingwen granules and providing chemical basis and theoretical support for its effective component identification, quality evaluation, and pharmacological mechanism research. Attached Figure Description

[0040] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0041] Figure 1 Total ion chromatogram (TIC) of Liujin Qingwen Granules;

[0042] Figure 2 This is a structural diagram of the chemical components in Liujin Qingwen granules;

[0043] Figure 3 The cleavage modes of A caffeic tannic acid (32), B neochlorogenic acid (30), and C caffeic acid (31);

[0044] Figure 4 MS for caffeic acid (31) 2 Spectrum and fragmentation mode; Detailed Implementation

[0045] The present invention will be further described in detail below. However, it should be noted that the following specific embodiments are merely exemplary examples of the invention, and the scope of protection of the invention is not limited thereto. The scope of protection of the invention is defined only by the claims. It will be apparent to those skilled in the art that various other modifications and substitutions can be made to the embodiments of the invention within the scope of protection defined by the claims, and the same technical effects can still be achieved, thus achieving the ultimate technical objective of the invention.

[0046] Unless otherwise specified, all instruments and materials mentioned in this manual were purchased commercially.

[0047] 1. Chemical reagents and instruments

[0048] 1.1 Chemical reagents

[0049] LC-MS grade acetonitrile and formic acid were purchased from Fisher Chemical Company (Fairlawn, New Jersey, USA); ultrapure water was purchased from Watsons (Guangzhou, China); all other reagents were of analytical grade. Forty reference standards were purchased from Shanghai Zerun Biotechnology, Shanghai Hongyong Biotechnology, Shanghai Yuanye Biotechnology, Shanghai Yanni Biotechnology, Shanghai Naxian Biotechnology, and the National Institutes for Food and Drug Control, respectively. Liujin Qingwen granules were produced by Shandong Hongjitang Pharmaceutical Group.

[0050] 1.2 Instruments

[0051] An Agilent 1290 Infinity ultra-high performance liquid chromatography system (equipped with an autosampler, column oven, binary pump, and online vacuum degasser) was coupled with an Agilent 6530 high-resolution mass quadrupole time-of-flight mass spectrometer (Agilent Technologies, Inc.). Data acquisition was performed using Mass Hunter Qualitative Analysis B.08.00 software (Agilent Technologies, Inc.). An ACQUITY UPLC HSST3 column (2.1 × 100 mm, 1.8 μm, Waters Corporation) was used.

[0052] 2. Measurement method:

[0053] The measurement method provided by this invention includes the following steps:

[0054] S1. Preparation of reference solution and sample solution

[0055] Reference solutions: Take quinic acid, citric acid, succinic acid, phenylalanine, vanillic acid, protocatechuic acid, neochlorogenic acid, caffeic acid, 4-hydroxybenzoic acid, strychnine, strychnoside, chlorogenic acid, cryptochlorogenic acid, p-coumaric acid, isoharringtonine, quercetin-3-O-β-D-gentiobiglycoside, ferulic acid, quercetin, chicoric acid, 7-hydroxycoumarin, isoquercitrin, isocytisine, and isochlorogenic acid B, respectively. An appropriate amount of isoflavonic acid, isochlorogenic acid A, apigenin, isoflavonic acid C, apigenin, daidzein, luteolin, kaempferol, physalin G, isophysalin G, emodin, physalin O, physalin L, physalin A, physalin F, 4,7-didehydroneophysalin B, and apigenin reference standards were placed in a volumetric flask, LC-MS grade methanol was added, and the reference standards were sonicated until completely dissolved. The solution was then filtered through a 0.22 μm pore size filter membrane to prepare a mixed reference solution, which was stored at 4°C for later use.

[0056] Sample solution: Grind Liujin Qingwen granules into powder, accurately weigh 0.3 g of sample, add 25 mL of 75% methanol aqueous solution (v / v), ultrasonically extract for 0.5 hours, cool to room temperature, replenish the weight loss with 75% methanol aqueous solution, filter through a 0.22 μm nylon microporous membrane and then test.

[0057] S2. Determine the chromatographic and mass spectrometry conditions:

[0058] Chromatographic conditions: The mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B), with the following gradient elution program: 0–15 min, 98% → 84% A; 15–35 min, 84% → 76% A; 35–50 min, 76% → 0% A; 50–55 min, 0% → 98% A; flow rate 0.3 mL / min. -1 The column temperature was 30℃ and the injection volume was 5 μL.

[0059] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion modes; time-of-flight mass spectrometry parameters are as follows: capillary voltage -3500 V (negative ion mode) / +4000 V (positive ion mode), nebulizer pressure 35 psi, dry gas temperature 300℃, flow rate 8 L / min; mass spectrometry scan range m / z 50-3000, acquisition rate 1.0 spectra / s.

[0060] Real-time quality correction was performed using standard solutions of ammonium trifluoroacetate (negative ion mode m / z 112.985587), purine (positive ion mode m / z 121.050873), and hexa(2,2,3,3-tetrafluoropropoxy)phosphazene (positive ion mode m / z 922.009798, negative ion mode m / z 1033.988109).

[0061] S3. Determination: Take the sample solution and the reference solution, and use an ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer to acquire the mass spectrometry data of the sample solution;

[0062] S4. Match the mass spectrometry data with the self-built database to achieve the analysis and identification of the target chemical components, specifically including the following steps:

[0063] S4.1 Building a self-built database:

[0064] By searching the TCMSP database, PubMed database, Web of Science database, CNKI and Wanfang Data knowledge service platform, we obtained the chemical composition information of the six raw medicinal materials of Liujin Qingwen granules, and constructed a self-built database of the chemical composition of Liujin Qingwen granules based on the chemical composition information of the medicinal materials.

[0065] This self-built database contains the names, molecular formulas, molecular weights, parent ion m / z values, and MS / MS fragment ion information of chemical components.

[0066] S4.2 The mass spectrometry data obtained in step S3 were analyzed using Mass Hunter software to obtain information such as the retention time, precise molecular weight, and secondary mass spectrometry fragments of the compounds. The results were compared with a self-built database, and the structures of some compounds were identified by referring to the reference standard data. Among them, 40 compounds were confirmed by comparison with the reference standard.

[0067] Furthermore, the method for analyzing the chemical components in the Liujin Qingwen granules also includes: S5. Constructing a molecular network using a feature-based molecular network (FBMN) workflow on the GNPS platform:

[0068] The mass spectrometry data in Mass Hunter.D file format was converted to standard file format (.abf) using Abf Converter software, and then imported into MS-DIAL software.

[0069] The mass spectrometry data obtained in step S3 were processed using MS-DIAL software to complete the format conversion and export of the data required for FBMN analysis.

[0070] Data processed and exported using MS-DIAL software was submitted to the GNPS platform for FBMN analysis to generate a molecular network and complete the mass spectrometry analysis of Liujin Qingwen granules.

[0071] The key parameters for FBMN analysis were set as follows: precursor and fragment ion mass tolerance was 0.02 Da, cosine score threshold was set to ≥0.7, minimum number of matching fragment ions was set to ≥6, and TopK functionality was enabled, retaining only the 10 most similar connections for each node. Other parameters remained at GNPS default settings. The generated molecular network was visualized and further analyzed using Cytoscape 3.9.0 software.

[0072] 3. Results and Discussion

[0073] This invention uses mass spectrometry data of methanol extract of Liujin Qingwen granules. By comparing with reference standards and combining with a self-built chemical database and mass spectrometry fragmentation patterns, a total of 117 chemical components were identified.

[0074] The total ion current chromatogram (TIC) of Liujin Qingwen granules in negative ion mode is as follows: Figure 1 As shown, the separation of each chemical component (number of peaks, peak shape, retention time) is good. The structures of the chemical components are shown below. Figure 2 .

[0075] The 117 chemical components are: mannitol (1), maltotriose (2), L-proline (3), hypoxanthine (4), quinic acid (5), lactose (6), malic acid (7), bindweed extract B3 (8), citric acid (9), plaintiff Yichun (10), xanthine (11), sweet orange flavonoids (12), uridine (13), succinic acid (14), zeaxanthin (15), adenine (16), guanosine (17), 2-methylcitric acid (18), adenosine (19), phenylalanine (20), vanillic acid (2) 1), Syringic acid (22), Protocatechuic acid (23), 5-hydroxy-2-indolone (24), Catechol (25), Isovaleric acid (26), Luteolin-7,4'-diglucoside (27), Guaiacin (28), 2-acetylpyrrole (29), Neochlorogenic acid (30), Caffeic acid (31), Caffeic tannic acid (32), 4-hydroxybenzoic acid (33), 4-hydroxyphenylacetic acid (34), Caffeic acid-4-O-β-D-glucopyranoside (35), Secologanic acid (36), strychnine (37), 2-methoxybenzoic acid (38), methyl caffeate (39), strychnine (40), 4-methylgentianic acid (41), chlorogenic acid (42), monomethyl adipic acid (43), forsythoside (44), 2,4-dihydroxybenzoic acid (45), fraxin (46), benzoic acid (47), swertiamarin (48), methyl 3-caffeoylquinic acid (49), cryptochlorogenic acid (50), methyl benzoate (51), physalin B (52), vanillin (53), indole (54), β-D-glucopyranoyl taraxacin (55), methyl 4-hydroxyphenylacetic acid (56), anthranilic acid (57), cynaric acid (5,8), p-coumaric acid (59), feruloylquinic acid (60), mongolicumin A (61), dimethyl adipate (62), 2-hydroxy-(2H)-1,4-benzoxazine-3(4H)-one (63), methyl 4-hydroxycinnamate (64) (+)-cantharides-3-α-O-β-D-glucopyranoside (65), isochoride (66), quercetin-3-O-β-D-gentiobiglycoside (67), ferulic acid (68), quercetin (69), 1-(3-indolyl)-2R,3-Dihydroxyacetone (70), Indole-3-carboxaldehyde (71), Loliolide (72), Chicoric acid (73), cis-Caftric acid (74), 7-Hydroxycoumarin (75), Protocatechuic aldehyde (76), Isovitexin (77), Isoquercitrin (78), Kaempferol-3-O-β-D-glucoside (79), Isocytisine (80), Isochlorogenic acid B (81), Quercetin-3-O-α-D-arabinofuranoside (82), Vitexin (83), Isoflavonic acid (84), Syringin-4-O-β-D-glucopyranoside (85), Isochlorogenic acid A (86), Apigenin (87), Syringin (88), Hesperidin (89), Apigenin-7-O-β-D-glucoside (90), Cephalanthrin A (91), isochlorogenic acid C (92), sennaol (93), 11β,13-dihydrotaraxacin (94), glucuronide (95), apigenin (96), daidzein (97), luteolin (98), kaempferol (99), sennain (100), 5α,6β-dihydroxy-25,27-dihydro-7-deoxyphylloid A (101), phylloidin G (102), geraniol (103), isophylloidin G (1) 04), emodin (105), physalin O (106), physalin L (107), physalin A (108), methyl isoeugenol (109), physalin F (110), physalin C (111), physalin B (112), orange-yellow piperamide (113), 4,7-didehydroneophysalin B (114), apigenin (115), 3',4',7-trimethylquercetin (116), oleanolic acid (117).

[0076] These include: 37 organic acids, 24 flavonoids, 11 steroids, 11 phenylpropanoids, 10 alkaloids, 9 terpenoids, 6 nucleosides, 3 sugars, 2 amino acids, 2 esters, and 2 quinones.

[0077] Taking organic acid compounds as an example, organic acid compounds are one of the main chemical components of Liujin Qingwen granules. This invention identified a total of 37 organic acid compounds (numbered 5, 7, 9, 14, 18, 21, 22, 23, 25, 26, 28, 30, 31, 32, 33, 34, 38, 41, 42, 43, 45, 47, 50, 53, 57, 58, 59, 60, 62, 68, 73, 74, 76, 81, 84, 86, and 92). These compounds mainly include three categories: succinic acid derivatives, benzoic acid derivatives, and caffeic acid derivatives.

[0078] Table 1 shows the predicted molecular formula, collision energy, retention time, precursor negative ion mass-to-charge ratio, and fragment ion information for each organic acid compound.

[0079] Table 1

[0080] serial number Molecular formula CID / V RT / min Adust mass Observedmass MS / MS fragmentsadust mass Observedmass <![CDATA[5 b* ]]> <![CDATA[C7H 12 O6]]> 10 1.160 <![CDATA[191.0556[M-H] - ]]> 191.0558 <![CDATA[191.0556[M-H] - ]]> 191.0549 <![CDATA[111.0446[M-H-H2O-CO2-H2O] - ]]> 111.0440 <![CDATA[7 b,f ]]> <![CDATA[C4H6O5]]> 10 1.274 <![CDATA[133.0137[M-H] - ]]> 133.0143 <![CDATA[133.0137[M-H] - ]]> 133.0135 <![CDATA[89.0239[M-H-CO2] - ]]> 89.0241 <![CDATA[87.0082[M-H-CH2O] - ]]> 87.0086 <![CDATA[71.0133[M-H-CO2-H2O] - ]]> 71.0140 <![CDATA[9 d,f* ]]> <![CDATA[C6H8O7]]> 10 1.775 <![CDATA[191.0192[M-H] - ]]> 191.0198 <![CDATA[191.0192[M-H] - ]]> 191.0196 <![CDATA[129.0188[M-H-H2O-CO2] - ]]> 129.0189 <![CDATA[111.0082[M-H-H2O-CO2-H2O] - ]]> 111.0082 <![CDATA[14 d,f* ]]> <![CDATA[C4H6O4]]> 10 2.230 <![CDATA[117.0193[M-H] - ]]> 117.0195 <![CDATA[117.0193[M-H] - ]]> 117.0189 <![CDATA[99.0082[M-H-H2O] - ]]> 99.0086 <![CDATA[73.0295[M-H-CH2O2] - ]]> 73.0298 <![CDATA[18 b ]]> <![CDATA[C7H 10 O7]]> 10 3.347 <![CDATA[205.0349[M-H] - ]]> 205.0347 <![CDATA[205.0349[M-H] - ]]> 205.0349 <![CDATA[87.0082[M-H-C4H6O4] - ]]> 87.0077 <![CDATA[59.0133[M-H-C7H6O3] - ]]> 59.0131 <![CDATA[21 b,c,d* ]]> <![CDATA[C8H8O4]]> 10 5.055 <![CDATA[167.0345[M-H] - ]]> 167.0345 <![CDATA[167.0345[M-H] - ]]> 167.0353 <![CDATA[123.0446[M-H-CO2] - ]]> 123.0455 <![CDATA[22 b,d,f ]]> <![CDATA[C9H 10 O5]]> 10 5.237 <![CDATA[197.0450[M-H] - ]]> 197.0452 <![CDATA[197.0450[M-H] - ]]> 197.0460 <![CDATA[179.0344[M-H-H2O] - ]]> 179.0355 <![CDATA[135.0446[M-H-H2O-CO-O] - ]]> 135.0443 <![CDATA[123.0446[M-H-OCH2-CO2] - ]]> 123.0457 <![CDATA[23 a,b,c* ]]> <![CDATA[C7H6O4]]> 10 5.738 <![CDATA[153.0187[M-H] - ]]> 153.0186 <![CDATA[153.0187[M-H] - ]]> 153.0185 <![CDATA[109.0290[M-H-CO2] - ]]> 109.0293 <![CDATA[25 c ]]> <![CDATA[C6H6O2]]> 20 5.829 <![CDATA[109.0290[M-H] - ]]> 109.0293 <![CDATA[109.0290[M-H] - ]]> 109.0299 <![CDATA[91.0184[M-H-H2O] - ]]> 91.0188 <![CDATA[26 b,c,d ]]> <![CDATA[C8H8O4]]> 10 6.034 <![CDATA[167.0345[M-H] - ]]> 167.0348 <![CDATA[167.0345[M-H] - ]]> 167.0345 <![CDATA[123.0446[M-H-CO2] - ]]> 123.0443 <![CDATA[28 b ]]> <![CDATA[C7H8O2]]> 10 6.968 <![CDATA[123.0446[M-H] - ]]> 123.0449 <![CDATA[123.0446[M-H] - ]]> 123.0446 <![CDATA[93.0340[M-H-CH2O] - ]]> 93.0340 <![CDATA[77.0390[M-H-CH2O2] - ]]> 77.0395 <![CDATA[30 a,b,d* ]]> <![CDATA[C 16 H 18 O9]]> 20 7.424 <![CDATA[353.0873[M-H] - ]]> 353.0876 <![CDATA[353.0873[M-H] - ]]> 353.0867 <![CDATA[191.0556[M-H-C9H6O3] - ]]> 191.0555 <![CDATA[179.0345[M-H-C7H 10 O5] - ]]> 179.0342 <![CDATA[173.0450[M-H-C9H6O3-H2O] - ]]> 173.0459 <![CDATA[161.0239[M-H-C7H 10 O5-H2O] - ]]> 161.0248 <![CDATA[135.0447[M-H-C7H 10 O5-CO2] - ]]> 135.0443 <![CDATA[31 a,b,c* ]]> <![CDATA[C9H8O4]]> 10 7.560 179.0345 [MH]- 179.0342 <![CDATA[179.0345[M-H] - ]]> 179.0345 <![CDATA[135.0446[M-H-CO2] - ]]> 135.0450 <![CDATA[107.0497[M-H-CO2-CO] - ]]> 107.0497 <![CDATA[32 b,e ]]> <![CDATA[C 13 H 12 O9]]> 10 7.583 <![CDATA[311.0403[M-H] - ]]> 311.0408 <![CDATA[311.0403[M-H] - ]]> 311.0403 <![CDATA[179.0344[M-H-C4H4O5] - 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]]> 137.0238 <![CDATA[137.0239[M-H] - ]]> 137.0243 <![CDATA[109.0290[M-H-CO] - ]]> 109.0283 <![CDATA[81 a,b* ]]> <![CDATA[C 25 H 24 O 12 ]]> 10 21.432 <![CDATA[515.1190[M-H] - ]]> 515.1194 <![CDATA[515.1190[M-H] - ]]> 515.1216 <![CDATA[353.0873[M-H-C9H6O3] - ]]> 353.0885 <![CDATA[191.0556[M-H-2C9H6O3] - ]]> 191.0564 <![CDATA[179.0344[M-H-C9H6O3-C7H 10 O5] - ]]> 179.0346 <![CDATA[135.0446[M-H-C 16 H 16 O8-CO2] - ]]> 135.0448 <![CDATA[84 a,c,d* ]]> <![CDATA[C 10 H 10 O4]]> 10 21.728 <![CDATA[193.0501[M-H] - ]]> 193.0502 <![CDATA[193.0501[M-H] - ]]> 193.0499 <![CDATA[178.0266[M-H-CH3] - ]]> 178.0264 <![CDATA[134.0368[M-H-CH3-CO2] - ]]> 134.0365 <![CDATA[86 a,b* ]]> <![CDATA[C 25 H 24 O 12 ]]> 10 22.389 <![CDATA[515.1190[M-H] - ]]> 515.1192 <![CDATA[515.1190[M-H] - ]]> 515.1204 <![CDATA[353.0873[M-H-C9H6O3] - ]]> 353.0882 <![CDATA[191.0556[M-H-2C9H6O3] - ]]> 191.0555 <![CDATA[179.0344[M-H-C9H6O3-C7H 10 O5] - ]]> 179.0347 <![CDATA[135.0446[M-H-C 16 H 16 O8-CO2] - ]]> 135.0457 <![CDATA[92 a,b* ]]> <![CDATA[C 25 H 24 O 12 ]]> 10 25.145 <![CDATA[515.1190[M-H] - ]]> 515.1189 <![CDATA[515.1190[M-H] - ]]> 515.1158 <![CDATA[353.0873[M-H-C9H6O3] - ]]> 353.0849 <![CDATA[191.0556[M-H-2C9H6O3] - ]]> 191.0564 <![CDATA[179.0344[M-H-C9H6O3-C7H 10 O5] - ]]> 179.0328 <![CDATA[135.0446[M-H-C 16 H 16 O8-CO2] - ]]> 135.0437

[0081] Compounds 7, 9, and 18 are derivatives of compound 14 (succinic acid), with methyl, hydroxyl, and carboxyl substituents, respectively. In negative ion mode, 44... and 18 The loss of neutrality, forming and Characteristic fragment ions. Compounds 21, 22, 23, 26, 33, 38, 41, 45, and 57 are derivatives of compound 47 (benzoic acid), with methyl, hydroxyl, and methoxy substitutions at different positions on their benzene rings. Similar to succinic acid derivatives, these compounds readily undergo [further reactions]. and The loss of neutrality produces and Fragment ions.

[0082] Compounds 30, 32, 42, 50, 58, 73, 74, 81, 86, and 92 are derivatives or isomers of compound 31 (caffeic acid), and all produce product ions at m / z 179. Notably, compounds 42, 50, 58, 81, 86, and 92 produce typical fragment ions at m / z 191, indicating that they all contain quinic acid substituents. These compounds exhibit similar MS / MS fragmentation patterns: first, the quinic acid or caffeic acid group is lost, followed by further loss of... or ,generate and Fragment ions.

[0083] Figure 3 The cleavage modes of A. caffeic tannic acid (32), B. neochlorogenic acid (30), and C. caffeic acid (31) are described. Figure 4 Mass spectrum and fragmentation mode of caffeic acid (31).

[0084] Compounds 30 (tR = 7.424 min), 42 (tR = 10.385 min), and 50 (tR = 11.045 min) exhibited the same precursor ion peak in negative ion mode. And the molecular formulas are all This indicates that they are isomers. Characteristic fragment peaks at m / z 191, 179, 173, 161, and 135 were observed in their MS² spectra, corresponding to the following fragment ions: , , , and By comparing the peak order of the compounds reported in the literature and verifying with reference standards, the specific structures of the three isomers were finally determined.

[0085] The above specific embodiments should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, any alternative improvements or modifications made to the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0086] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A method for analyzing the chemical components in Liujin Qingwen granules, characterized in that, Includes the following steps: S1. Prepare sample solutions and reference solutions; The sample solution was prepared as follows: Liujin Qingwen granules were ground into powder, an appropriate amount was weighed and added to 75% methanol aqueous solution, ultrasonically extracted, cooled to room temperature, and the weight loss was made up with 75% methanol aqueous solution. The solution was then filtered through a nylon microporous membrane before testing. The preparation method of the reference solution is as follows: weigh each reference standard, dissolve it in methanol, filter it through a nylon microporous membrane, and refrigerate it at 4℃ until use; among which, the reference standards are quinic acid, citric acid, succinic acid, phenylalanine, vanillic acid, protocatechuic acid, neochlorogenic acid, caffeic acid, 4-hydroxybenzoic acid, strychnine, strychnoside, chlorogenic acid, cryptochlorogenic acid, p-coumaric acid, isoharringtonine, and quercetin-3-O-β-D-gentianin. Glycosides, ferulic acid, quercetin, chicoric acid, 7-hydroxycoumarin, isoquercitrin, isocytisine, isochlorogenic acid B, isoflavone, isochlorogenic acid A, apigenin, isochlorogenic acid C, apigenin, daidzein, luteolin, kaempferol, physalin G, isophysalin G, emodin, physalin O, physalin L, physalin A, physalin F, 4,7-didehydroneophysalin B, apigenin; S2. Determine the chromatographic and mass spectrometry conditions: The chromatographic conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, and the gradient elution program was as follows: 0–15 min: 98% → 84% A; 15–35 min: 84% → 76% A; 35–50 min: 76% → 0% A; 50–55 min: 0% → 98% A; the flow rate was 0.3 mL / min. -1 The column temperature was 30℃, and the injection volume was 5 μL. The mass spectrometry conditions were as follows: the ion source was an electrospray ionization source; the scanning modes were positive ion and negative ion modes; the capillary voltage was -3500 V in negative ion mode and +4000 V in positive ion mode; the nebulizer pressure was 35 psi, the drying gas temperature was 300 °C, and the flow rate was 8 L / min; the scanning range was m / z 50–3000, and the acquisition rate was 1.0 spectra / s. S3. Measurement: Sample solutions and reference solutions were taken, and mass spectrometry data were acquired using an ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer. S4. Match the mass spectrometry data with the self-built database to achieve the analysis and identification of target chemical components; Step S4 is as follows: S4.1 By summarizing the information on the chemical components contained in the six raw medicinal materials of Liujin Qingwen Granules, a self-built database of chemical components of Liujin Qingwen Granules is constructed. The self-built database includes the name, molecular formula, molecular weight, parent ion m / z value and MS / MS fragment ion information of the chemical components. S4.2 The mass spectrometry data obtained in step S3 are analyzed using Mass Hunter software to obtain the retention time, precise molecular weight, and secondary mass spectrometry fragment information of the compound. The results are compared with the self-built database and the structure of the compound is identified by referring to the reference standard data. The Liujin Qingwen granules are made from the following raw materials in parts by weight: 12-60 parts of honeysuckle vine, 12-60 parts of indigo leaf, 12-60 parts of isatis root, 12-60 parts of dandelion, 3-15 parts of physalis and 3-15 parts of dragon's tongue leaf.

2. The method for analyzing the chemical components in Liujin Qingwen granules according to claim 1, characterized in that, Also includes: S5. Constructing a molecular network: The mass spectrometry data obtained in step S3 were processed using MS-DIAL software to complete the format conversion and export of the data required for FBMN analysis. The data processed and exported by MS-DIAL software was submitted to the GNPS platform for FBMN analysis to generate a molecular network and complete the mass spectrometry analysis of Liujin Qingwen granules.

3. The method for analyzing the chemical components in Liujin Qingwen granules according to claim 1, characterized in that, The mass spectrometry conditions in step S2 also include: real-time mass correction using standard solutions of ammonium trifluoroacetate, purine, and hexa(2,2,3,3-tetrafluoropropoxy)phosphazene. The m / z values ​​of ammonium trifluoroacetate in negative ion mode are 112.985587, and those of purine in positive ion mode are 121.050873. The m / z values ​​of hexa(2,2,3,3-tetrafluoropropoxy)phosphazene in positive ion mode are 922.009798, and those in negative ion mode are 1033.988109.

4. The method for analyzing the chemical components in Liujin Qingwen granules according to claim 1, characterized in that, The chromatographic column used was a Waters ACQUITY UPLC HSS T3 column.

5. The method for analyzing the chemical components in Liujin Qingwen granules according to claim 1, characterized in that, The preparation method of the Liujin Qingwen granules includes the following steps: (1) Take six medicinal materials: honeysuckle vine, indigo leaf, isatis root, dandelion, physalis and dragon tongue leaf, mix them well, put them in a decoction pot, add water to soak them, decoct them, and filter to obtain the first decoction liquid. (2) Add water to the decoction pot and boil again, then filter to obtain the second decoction; (3) Combine the first decoction and the second decoction, and filter to obtain the third decoction; (4) Heat and concentrate the third decoction, filter it, add maltodextrin and stevioside, dry and granulate to obtain the final product.

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