An ultra-high performance liquid chromatography-mass spectrometry detection method for Qingwen Hufei granules

Abstract

The present invention provides an ultra-high performance liquid chromatography-mass spectrometry detection method for Qingwen Hufei granules. The present invention adopts ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-QExactiveFocusMS / MS) technology. After comparison and confirmation with reference substances, the detection method of the present invention has identified 17 terpenoid compounds (including cleaved strychnine, cleaved strychnine and other components) and 28 phenylpropanoid compounds (1-O-caffeoylquinic acid, chlorogenic acid, 5-para-aromatic acid) in the Qingwen Hufei granules. The detection method of the present invention can quickly separate and identify the chemical components of traditional Chinese medicine in Qingwen Hufei granules by on-line separation and identification, providing a scientific basis for the research on its pharmacodynamic material basis, quality control and mechanism of action.

Description

Technical Field

[0001] The present invention relates to an ultra-high performance liquid chromatography-mass spectrometry detection method for Qingwen Hufei granules, belonging to the field of quality detection methods for traditional Chinese medicines. Background Art

[0002] This prescription, adapted from classic formulas such as "Yinqiao Powder," "Xuanmai Ganjue Decoction," and "Sijunzi Decoction," consists of 16 Chinese herbs: honeysuckle, forsythia, thunbergii bulb, isatis indigotica, bamboo leaf, platycodon, codonopsis, stir-fried atractylodes macrocephala, poria, scrophularia, ophiopogon, bitter apricot, capillaris, saposhnikovia divaricata, perilla leaf, and licorice. It has the effects of dispelling wind-heat, clearing the lungs and relieving cough, and strengthening the spleen and stomach. It is indicated for the initial stages of plague, characterized by fever, dry cough, and sore throat. The monarch herbs: honeysuckle, forsythia, and thunbergii bulb dispel wind-heat, clear heat and detoxify, and clear the lungs and relieve cough; isatis indigotica, bamboo leaf, and platycodon clear heat and detoxify, expelling pathogenic factors; codonopsis, stir-fried atractylodes macrocephala, and poria strengthen the acquired constitution, nourish the earth to generate energy, and aid the lungs in resisting pathogenic factors. These two groups of herbs are collectively referred to as assistant herbs. Scrophularia and Ophiopogon japonicus are sweet and cold, and the combination of salty and cold nourishes the source of sweat, implying the principle of "first calming the area that has not been affected by the pathogen." Bitter almonds descend lung qi and relieve coughs. Artemisia capillaris clears damp-heat in the triple energizer, and together with Saposhnikovia divaricata and Perilla frutescens, it works to disperse and eliminate dampness. These three groups of herbs collectively serve as adjuvants. Licorice clears heat and detoxifies, acting as the messenger of all the herbs.

[0003] However, there are currently no reports on the material basis of Qingwen Hufei Granules' efficacy, and the product's mechanism of action in preventing the novel coronavirus remains unclear. Therefore, it is necessary to conduct research on the product's material basis. Summary of the Invention

[0004] The present invention provides an ultra-high performance liquid chromatography-mass spectrometry detection method for Qingwen Hufei granules. The detection method of the present invention performs rapid online separation and identification of the chemical components of traditional Chinese medicine in Qingwen Hufei granules. A total of 17 terpenoid compounds, 28 phenylpropanoid compounds, 8 chromones, 29 total flavonoids, 18 organic acid compounds, 36 glycoside compounds, and 23 other compounds were identified in Qingwen Hufei granules, such as alkaloids, sugars, indoles, saponins, phenols, furans, cyclohexylethanol derivatives, etc., providing a reference basis for the study of its pharmacological material basis, quality control and mechanism of action.

[0005] The technical solution provided in the patent application of this invention is as follows:

[0006] An ultra-high performance liquid chromatography-mass spectrometry detection method for Qingwen Hufei granules, the detection method comprising the following steps:

[0007] ⑴. Chromatographic conditions: C18 column, column temperature 30-40°C, flow rate 0.1-0.5 mL min -1, mobile phase: methanol A-0.1% formic acid aqueous solution B, gradient elution: 0-46 min, 1% A-98% A;

[0008] (2) Mass spectrometry conditions are as follows: the ion source is a heated electrospray ion source (HESI), and positive and negative ion monitoring modes are adopted. The spray voltage in positive ion mode is 3-4 kV, and in negative ion mode, the spray voltage is 2-4 kV. The sheath gas volume flow rate is 30-50 flow units in both positive and negative ion modes, and the auxiliary gas flow rate is 8-12 flow units in both positive and negative ion modes. The capillary temperature is 310-330°C in both positive and negative ion modes, the nebulization temperature is 330-370°C, the maximum spray current is 80-120 A in both positive and negative ion modes, the S-lens RF level is 40-60, the scan mode is Full MS / dd-MS2, the Full MS resolution is 60,000-80,000, the ion source is HESI, the dd-MS2 resolution is 16,000-18,000, the scan range is m / z 60-1500, and the collision energy CE is set to 20-40 eV.

[0009] ⑶. Preparation of sample solution: Take Qingwen Hufei granules, accurately weigh them, add 60-80% methanol, sonicate for 20-40 minutes, bring to room temperature, add 60-80% methanol to make up for the weight loss, filter, and take the filtrate;

[0010] ⑷. Preparation of reference solution: Weigh the reference solution, dissolve it in methanol and make up to volume. Prepare chlorogenic acid, liquiritin, scoparone, 5-O-methylvisamidoside, harpaoside, forsythiaside A, luteolin, 4,5-O-dicaffeoylquinic acid, forsythiaside, harpaoside, and cimicifugoside at concentrations of 0.1-0.15 mg mL -1 0.6~1.0mg·mL -1 , 0.08~0.13mg·mL -1 0.10~0.16mg·mL -1 0.13~0.17mg·mL -1 0.12~0.16mg·mL -1 0.14~0.18mg·mL -1 0.16~0.21mg·mL -1 0.13~0.17mg·mL -1 0.17~0.21mg·mL -1 , 0.19~0.23mg·mL -1 Reference substance stock solution;

[0011] ⑸. Establishment of a literature library: Comprehensive information from literature reports, ChemicalBook database, PubChem website, TCMSP database, ChemSpider database, etc., summarize the Chinese and English names, categories, references and structural identification information of the chemical components contained, including molecular formula, relative molecular mass, .mol ​​format structural formula, primary and multi-level mass spectrometry information, to establish a chemical component library of the entire medicinal material of Qingwen Hufei Granules;

[0012] (6) Compound identification: Based on the results of liquid chromatography-mass spectrometry (LC-MS / MS), Xcalibur 4.0 software was used to fit the molecular formula and obtain a database of primary information matching. This database was then repetitively screened against the self-built database of chemical components of Qingwen Hufei Granules. The fragment information of the screening database was inferred based on the secondary molecular information. Fragment ions that could not be matched were fragmented and analyzed using Thermo Fisher Scientific fragmentation software based on the .mol ​​format structural formula downloaded from the ChemSpider database. The fragmentation patterns of the compounds were further inferred based on the fragment ion information provided by reference substances, databases, references, Thermo Fisher Scientific fragmentation software, etc.

[0013] As a further preferred embodiment of the present invention, the C18 chromatographic column model in the chromatographic conditions of step (1) is ThermoAccucore aQ RP18, and the chromatographic column specifications are 2.1 mm×150 mm, 2.6 μm.

[0014] Preferably, in the chromatographic conditions of step (1), the column temperature is 35°C and the flow rate is set at 0.3 mL·min -1 Mobile phase: methanol A-0.1% formic acid aqueous solution B gradient elution, 0-3 min, 1% A; 3-20 min, 1%-35% A; 20-33 min, 35%-80% A; 33-35 min, 80% A; 35-44 min, 80%-98% A; 44-46 min, 98% A.

[0015] Preferably, the mass spectrometry conditions in step (2) are as follows: the spray voltage in positive ion mode is 3.5 kV, the spray voltage in negative ion mode is 3.0 kV, the sheath gas volume flow rate in both positive and negative ion modes is 40 flow units, the auxiliary gas flow rate in both positive and negative ion modes is 10 flow units, the capillary temperature in both positive and negative ion modes is 320 ° C, the nebulization temperature is 350 ° C, the maximum spray current in both positive and negative ion modes is 100 A, the S-lens RF level is 50, the scan mode is Full MS / dd-MS2, the FullMS resolution is 70,000, the ion source is HESI, the dd-MS2 resolution is 17500, the scan range m / z 80-1200; the collision energy CE is set to 30 eV.

[0016] Preferably, the methanol concentration in the preparation of the sample solution in step (3) is 70%.

[0017] Preferably, the ultrasonic time in the preparation of the sample solution in step (3) is 30 min.

[0018] Preferably, in the preparation of the reference solution in step (4), the mass concentrations of chlorogenic acid, liquiritin, scoparia lactone, 5-O-methylvisamidoside, harpagoside, forsythiaside A, luteolin, 4,5-O-dicaffeoylquinic acid, forsythiaside, harpagoside, and cimicifugoside are 0.135 mg mL -1 , 0.800mg·mL -1 , 0.118mg·mL -1 , 0.146mg·mL -1 , 0.157mg·mL -1 , 0.140mg·mL -1 , 0.164mg·mL -1 , 0.198mg·mL -1 , 0.157mg·mL -1 , 0.190mg·mL -1 , 0.215mg·mL -1 Reference stock solution.

[0019] In order to reflect the creativity of the technical solution of the present invention, some technical contents of the screening and optimization process of the chromatography and mass spectrometry are summarized as follows.

[0020] Optimization of chromatographic and mass spectrometric conditions in the detection conditions of the present invention

[0021] ① Chromatographic conditions

[0022] The mobile phase, injection volume, column temperature, and flow rate were investigated. This experiment selected the following LC / MS mobile phase compositions commonly used in the literature: methanol-0.1% formic acid aqueous solution, 0.1% formic acid acetonitrile-0.1% formic acid aqueous solution, and acetonitrile-0.1% formic acid aqueous solution for gradient elution. The optimal gradient conditions for each of the three mobile phase compositions were optimized. The optimal conditions for methanol A-0.1% formic acid aqueous solution B were: 0-3 min, 1% A; 3-20 min, 1%-35% A; 20-33 min, 35%-80% A; 33-35 min, 80% A; 35-44 min, 80%-98% A; and 44-46 min, 98% A. The optimal conditions for 0.1% formic acid acetonitrile A-0.1% formic acid aqueous solution B were: 0-10 min, 5% A; 10-25 min, 5%-45% A; 25-40 min, 45%-85% A; 40-55 min, 85%-90% A; 55-62 min, 90%-99% A; 62-65 min, 99% A. The optimal conditions for acetonitrile A-0.1% formic acid aqueous solution B were: 0-5 min, 3% A; 5-25 min, 3%-40% A; 40-55 min, 40%-75% A; 55-60 min, 75%-90% A; 60-65 min, 90%-97% A; 65-67 min, 97% A. The above three optimized mobile phases were compared. By comparing the base peak ion chromatograms and the number of primary quasi-molecular ions of the chemical components in Qingwen Hufei Granules in positive and negative ion modes, it was found that when methanol-0.1% formic acid aqueous solution was used as the mobile phase, it had a good response in both positive and negative ion modes, which could maximize the acquisition of rich mass spectrometric information on the components of Qingwen Hufei Granules.

[0023] During the chemical composition analysis, it was found that glycosides and organic acids had better responses in the negative mode, chromones had better responses in the positive mode, and phenylpropanoids, terpenes, and flavonoids had better responses in both positive and negative modes.

[0024] ② Mass spectrometry conditions

[0025] The beneficial effects of the detection method of the present invention are as follows: within the safe range of use of the mass spectrometry light source, the spray voltage, sheath gas volume, auxiliary gas flow rate, capillary temperature, atomization temperature, S-lens RF level, scan mode, Full MS resolution, dd-MS2 resolution, scan range, collision energy, etc. were investigated respectively. The scan mode and scan range are the important factors affecting the mass spectrometry results. Selecting the Full MS / dd-MS2 scan mode can simultaneously obtain primary information and secondary fragment information.

[0026] The selection was made based on the range of predicted molecular weight values ​​of the compounds in the database. The molecular weight of organic acid compounds is relatively small, generally around 100, and the molecular weight of fritillaryin compounds in fritillaria is relatively small, generally around 1000, so the scanning range m / z 80-1200 was selected.

[0027] By comparing the number of primary quasi-molecular ions scanned in positive ion mode, negative ion mode separately, and positive and negative ion mode simultaneously, and using positive and negative ion modes for sampling, scanning, and data collection respectively, the mass spectrum information of Qingwen Hufei Granules can be fully obtained.

[0028] ③. Sample preparation method

[0029] The sample concentration, extraction solvent, extraction method, extraction time, etc. of Qingwen Hufei Granules were investigated.

[0030] The sample concentration was investigated from low to high, with injection volumes ranging from 1ul to 3ul at 0.1mg / ml, 1.0mg / ml, 10mg / ml, 20mg / ml, and 40mg / ml. When the injection volume was 3ul for 20mg / ml or 1ul for 40mg / ml, more peaks appeared. In combination with the concept of energy conservation and environmental protection, the sample concentration was selected as 20mg / ml and the injection volume was 3ul.

[0031] The extraction solvents were pure water, 50% methanol, 70% methanol, and methanol. When 70% methanol was used as the extraction solvent, the extracted components were richer and more uniform.

[0032] The extraction methods of reflux extraction and ultrasonic extraction were investigated. The difference in the number of peaks was not large. In order to increase the repeatability of the method, simplify the process, save energy and protect the environment, the ultrasonic extraction method was selected.

[0033] The chromatographic peaks at extraction times of 20 min, 30 min, and 40 min were examined respectively. There was little difference in the peaks between 30 min and 40 min, and the peaks at 20 min were less, so the extraction time of 30 min was selected.

[0034] The beneficial effects of the technical solution of the present invention are as follows:

[0035] ⑴. The present invention adopts ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-QExactive Focus MS / MS) technology. In the positive ion mode, based on the high-resolution mass spectrometry data of the compound, combined with a self-built database, reference to relevant literature and comparison with reference substances, the above data processing method is used to confirm the chemical components in Qingwen Hufei Granules. After comparison with reference substances, the detection method of the present invention has identified 17 terpenoid compounds (including broken oxidized strychnine, broken strychnine acid and other components) and 28 phenylpropanoid compounds (1-O-caffeoylquinic acid, chlorogenic acid, 5-p-coumarylquinic acid, 3-O-caffeoylquinic acid methyl ester, podocarpin, 1, 3-O-dicaffeoylquinic acid and other ingredients), 8 chromones (hyperoside), 29 total flavonoids (including hyperoside and rutin and other ingredients), 18 organic acid compounds (caffeic acid and other ingredients), 36 glycoside compounds, and 23 other compounds, such as alkaloids, sugars, indoles, saponins, phenols, furans, cyclohexylethanol derivatives, etc. The detection method of the present invention can effectively identify and comprehensively characterize the effective substance components in Qingwen Hufei Granules, and further classify the identified components by medicinal taste and category, providing a reference basis for subsequent quality control of the product.

[0036] (2) The detection method of the present invention resolved 565 components. After deduplication, it was determined that Qingwen Hufei Granules contained 445 components, including 193 components in the positive mode and 252 components in the negative mode. Those with fewer cleavage fragments and lack of secondary fragment molecular formula information were deleted, and a total of 158 components were screened out, including 39 from honeysuckle, 51 from forsythia, 1 from thunbergia, 10 from isatis indigotica, 9 from hylocereus grandiflora, 12 from codonopsis, 5 from stir-fried atractylodes, 6 from poria, 25 from figwort, 5 from bitter almond, 13 from scutellaria baicalensis, 18 from siler, 21 from perilla leaves, and 16 from liquorice. Among them, 12 components including caffeic acid, sucrose, 1-O-caffeoylquinic acid, chlorogenic acid, 5-p-coumarylquinic acid, 3-O-caffeoylquinic acid methyl ester, podocarpin, 4,5-O-dicaffeoylquinic acid, strychnine, strychnine-oxidized strychnine, hyperoside, and rutin responded in both positive and negative modes.

[0037] ⑶. The present invention selects the effective substance components that may be related to the anti-novel coronavirus infection and acute respiratory tract infection in winter and spring of Qingwen Hufei Granules: Atractylodes lactone I, strychnine, scopoletin, podocarpin, hyoscyamine, hyperoside, forsythiaside A and other components for cleavage pathway analysis, and determines the structure of the above chemical components. Among them, Atractylodes lactone I can inhibit the release of inflammatory factors and inflammatory mediators, and has anti-inflammatory effects; strychnine has a good antiviral effect; scopoletin has analgesic, anti-inflammatory and anti-tumor (lung cancer) effects; podocarpone can reduce the production of inflammatory factors such as IL-1, IL-2, IL-6, TNF-α under inflammatory stimulation, and play an anti-inflammatory role; pyrrol is a component of the chlorogenic acid ketone class, which has multiple effects such as antipyretic, analgesic, anti-inflammatory, anti-tumor, and anti-platelet aggregation. It is used to treat pediatric respiratory tract infections, allergic rhinitis, toothache, etc.; hyperoside can offset the induction of Mycoplasma pneumoniae infection and promote the proliferation of A549 cells infected with Mycoplasma pneumoniae, and can also significantly reduce the production of IL-8 and TNF-α, and can treat pneumonia through CCL5-CCR4 interaction; forsythiaside A can inhibit the inflammatory response of mice infected with H9N2 avian influenza virus, and can improve lung injury and immune function in newborn rats. The analysis of the cracking pathways of the above components can provide a theoretical basis for in-depth research on the material basis of the efficacy and quality control of Qingwen Hufei Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings described herein are used to provide further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0039] Figure 1 -Chromatogram of Qingwen Hufei Granules in positive ion mode;

[0040] Figure 2 -Chromatogram of Qingwen Hufei Granules in negative ion mode. DETAILED DESCRIPTION

[0041] In order to more fully understand the implementation of the present invention, an experimental example is listed below, and the present invention is further described below through typical embodiments.

[0042] Example 1 Ultra-high performance liquid chromatography-mass spectrometry detection method of the present invention

[0043] 1. Materials

[0044] An UltiMate 3000 ultra-high performance liquid chromatography system and a Q Exactive Focus mass spectrometer were both purchased from Thermo Fisher Scientific, Inc., USA, and a BT25S 1 / 100,000 electronic analytical balance was purchased from Sartorius Scientific Instruments (Beijing) Co., Ltd. Qingwen Hufei Granules were provided by the Preparation Center of Shaanxi Provincial Hospital of Traditional Chinese Medicine with batch number 20210901. The reference substances chlorogenic acid, glycyrrhizin, scoparia lactone, 5-O-methylvisamidoside, harpaoside, forsythoside A, luteolin, 4,5-O-dicaffeoylquinic acid, forsythiaside, harpaoside, and cimicifugoside were all purchased from Chengdu Kroma Biotechnology Co., Ltd. with batch numbers CHB201114, CHB201102, CHB201102, CHB201227, CHB201219, CHB201203, CHB201227, CHB210104, CHB201202, CHB201218, and CHB201107, respectively, with a purity of ≥98%. Water was ultrapure water, methanol was chromatographic grade, formic acid was mass spectrometry grade, and other reagents were of analytical grade.

[0045] 2 Methods

[0046] 2.1 Chromatographic conditions

[0047] A Thermo Accucore aQ RP18 column (2.1 mm × 150 mm, 2.6 μm) was used, with a column temperature of 35 °C and a flow rate of 0.3 mL min -1 , injection volume 3 μL, mobile phase methanol (A)-0.1% formic acid aqueous solution (B) gradient elution (0-3 min, 1% A; 3-20 min, 1%-35% A; 20-33 min, 35%-80% A; 33-35 min, 80% A; 35-44 min, 80%-98% A; 44-46 min, 98% A).

[0048] 2.2 Mass spectrometry conditions

[0049] The spray voltage in positive ion mode was 3.5 kV, and that in negative ion mode was 3.0 kV. The sheath gas volume flow rate was 40 flow units in both positive and negative ion modes, the auxiliary gas flow rate was 10 flow units in both positive and negative ion modes, the capillary temperature was 320°C in both positive and negative ion modes, the nebulization temperature was 350°C, the maximum spray current was 100 A in both positive and negative ion modes, the S-lens RF level was 50, the scan mode was Full MS / dd-MS2, the Full MS resolution was 70,000, the ion source was HESI, the dd-MS2 resolution was 17,500, the scan range was m / z 80-1200, and the collision energy CE was set to 30 eV.

[0050] 2.3 Preparation of sample solution

[0051] Take 0.2 g of Qingwen Hufei granules, accurately weigh it, add 10 mL of 70% methanol, ultrasonicate for 30 min (40 kHz, 400 W), let it cool to room temperature, make up for the weight loss with 70% methanol, filter through a 0.22 μm microporous membrane, and take the filtrate.

[0052] 2.4 Preparation of reference solution

[0053] Accurately weigh an appropriate amount of each reference substance, place it in a 10 mL volumetric flask, add methanol to dissolve and adjust the volume to prepare chlorogenic acid, liquiritin, scoparone, 5-O-methylvisamidoside, harpaoside, forsythiaside A, luteolin, 4,5-O-dicaffeoylquinic acid, forsythiaside, harpaoside, and cimicifugoside with a mass concentration of 0.135 mg·mL -1 , 0.800mg·mL -1 , 0.118mg·mL -1 , 0.146mg·mL -1 , 0.157mg·mL -1 , 0.140mg·mL -1 , 0.164mg·mL -1 , 0.198mg·mL -1 , 0.157mg·mL -1 , 0.190mg·mL -1 , 0.215mg·mL -1 The reference substance stock solution was stored in a refrigerator at 4°C.

[0054] 2.5 Compound identification

[0055] CNKI, PubMed, Web of Science, and other databases were consulted to search for research literature on the chemical components of the Qingwen Hufei Granules, including honeysuckle, forsythia, fritillaria thunbergii, isatis indigotica, loblolly bamboo, platycodon, codonopsis pilosula, stir-fried atractylodes, poria, figwort, ophiopogon japonicus, bitter apricot, capillaris, saposhnikovia divaricata, perilla leaf, and liquorice. Combining literature reports with information from the ChemicalBook database, the structural identification information of the chemical components, including molecular formula, relative molecular mass, structural formula, and primary and multi-level mass spectrometry data, was compiled to establish a chemical component library for Qingwen Hufei Granules.

[0056] Xcalibur 4.0 software was used to fit the molecular formula and match it with the self-built chemical component database of Qingwen Hufei Granules to preliminarily infer the molecular information. The formula was then compared with the ChemicalBook or ChemSpider database, and the chromatographic peaks were analyzed. The compound structure and its fragmentation pattern were further inferred based on the fragment ion information provided by reference substances, databases, and references.

[0057] 3. Test result data optimization

[0058] Those with fewer fragments and lacking molecular formula information for secondary fragments were deleted. A total of 158 components were screened (11 of which were confirmed by comparison with the reference), including 74 components in the positive mode (see Table 1) and 96 components in the negative mode (see Table 2). Among them, 39 components were found in honeysuckle, 51 in forsythia, 1 in thunbergia, 10 indigotica, 9 in loblolly bamboo, 12 in codonopsis, 5 in stir-fried atractylodes, 6 in poria, 25 in figwort, 5 in bitter apricot, 13 in capillaris, 18 in siler, 21 in perilla leaves, and 16 in glycyrrhiza. Twelve components, including caffeic acid, sucrose, 1-O-caffeoylquinic acid, chlorogenic acid, 5-p-coumarylquinic acid, methyl 3-O-caffeoylquinate, podocarpin, 4,5-O-dicaffeoylquinic acid, strychnine, strychnine-oxidized strychnine, hyperoside, and rutin, responded in both positive and negative modes.

[0059] Table 1 UHPLC-Q Exactive Focus MS / MS identification of chemical components of Qingwen Hufei Granules (positive mode optimization results)

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] Note: ＊ : Confirmed by comparison with reference substances; JYH: honeysuckle, LQ: forsythia, ZBM: thunbergia, DQY: isatis indigotica, DZY: loblolly bamboo leaf, DS: codonopsis, CBZ: stir-fried atractylodes, FL: poria, XS: figwort, KXR: bitter apricot, YC: capillaris, FF: siler, ZSY: perilla leaf, GC: liquorice; a: organic acids, b: others; c: phenylpropanoids; d: terpenes; e: glycosides; f: chromones; g: flavonoids

[0072] Table 2 UHPLC-Q Exactive Focus MS / MS identification of chemical components of Qingwen Hufei Granules (negative mode optimization results)

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Note: ＊ : Confirmed by comparison with reference substances; JYH: honeysuckle, LQ: forsythia, ZBM: thunbergia, DQY: isatis indigotica, DZY: loblolly bamboo, DS: codonopsis, CBZ: stir-fried atractylodes, FL: poria, XS: figwort, KXR: bitter apricot, YC: capillaris, FF: siler, ZSY: perilla leaf, GC: liquorice; a: organic acids, b: others; c: phenylpropanoids; d: terpenes; e: glycosides; g: flavonoids

[0083] 3.1 Analysis of cleavage pathways

[0084] 3.1.1 Terpenes

[0085] There are 17 terpenoid compounds analyzed from Qingwen Hufei Granules, 9 in positive mode and 10 in negative mode. Two compounds, cleaved strychnine and cleaved strychnine acid, responded in both positive and negative modes. Taking Atractylodes lactone I as an example, the quasi-molecular ion peak of the eucalyptol sesquiterpenoids is m / z 233.1536 [M+H] + The corresponding compound molecular formula is C 15 H 20 O2 loses one molecule of H2O, and then loses one molecule of CO. In the secondary spectrum, the main fragment ion peak is 215.1430 [M+H-H2O] + 、187.1484[M+H-H2O-CO] + 、177.0912[M+H-C4H8] +、151.0755[M+H-C6H 10 ] + 、145.1012[M+H-H2O-CO-C3H6] + ,131.0857[M+H-H2O-CO-C3H6-CH2] + ,105.0699[M+H-H2O-CO-C3H6-CH2-C2H2] + ,91.0543[M+H-H2O-CO-C3H6-CH2-C2H2-CH2] + ,79.0543[M+H-H2O-CO-C3H6-CH2-C2H2-C2H2] + Combined with the relative molecular mass, this compound was confirmed to be Atractylodes lactone I.

[0086] Taking strychnine as an example, the quasi-molecular ion peak of iridoid terpenes is m / z 405.1391[M+H] + The corresponding compound molecular formula is C 17 H 24 O 11 , lost a molecule of glucose, and then lost another molecule of H2O. In the secondary spectrum, the main fragment ion peak is 243.0853 [M+H-Glc] + 、225.0752[M+H-Glc-H2O] + ,193.0490[M+H-Glc-H2O-CH3OH] + ,165.0548[M+H-Glc-H2O-C2H4O2] + Combined with the relative molecular mass, it was confirmed that this compound was oxidized strychnine.

[0087] 3.1.2 Phenylpropanoids

[0088] Twenty-eight phenylpropanoid compounds were isolated from Qingwen Hufei Granules, 18 of which were detected in positive mode and 16 in negative mode. Six compounds, including 1-O-caffeoylquinic acid, chlorogenic acid, 5-p-coumarylquinic acid, 3-O-caffeoylquinic acid methyl ester, podocarpine, and 1,3-O-dicaffeoylquinic acid, responded in both positive and negative modes. Scopolamine, for example, is a coumarin compound with a quasi-molecular ion peak of m / z 193.0495 [M+H] + The corresponding compound molecular formula is C 10 H8O4, in the secondary spectrum, the main fragment ion peak is 178.0263 [M+H-CH3] + ,165.0550[M+H-CO] + ,161.0236[M+H-CH3-OH] +,150.0314[M+H-CH3-CO] + ,149.0604[M+H-CO2] + ,137.0599[M+H-2CO] + ,133.0285[M+H-CH3-OH-CO] + ,122.0363[M+H-CH3-2CO] + , combined with the relative molecular mass, confirmed that this compound is scopoletin.

[0089] Taking podocarpin as an example, the quasi-molecular ion peak of lignans is m / z 359.1489 [M+H] + The corresponding compound molecular formula is C 20 H 22 O6, in the secondary spectrum, the main fragment ion peak is 341.1384 [M+H-H2O] + ,323.1281[M+H-H2O-H2O] + ,311.1269[M+H-H2O-CH2O] + , 137.0600[M+HC 12 H 14 O4] + ,122.0369[M+HC 12 H 14 O4-CH3] + ,109.0649[M+H-H2O-C 13 H 12 O4] + ,95.0492[M+HC 12 H 14 O4-C2H2O] + ,91.0542[M+H-H2O-CH2O-C 12 H 12 O4] + , combined with the relative molecular mass, it was confirmed that this compound was podocarpin.

[0090] 3.1.3 Chromones

[0091] There are 8 chromone compounds analyzed from Qingwen Hufei Granules, all of which respond in positive mode. Taking chloranol as an example, the quasi-molecular ion peak is m / z 277.1071 [M+H] + The corresponding compound molecular formula is C 15 H 16 O5, in the secondary spectrum, the main fragment ion peak is 259.0966 [M+H-H2O] + ,244.0721[M+H-H2O-CH3]+ ,229.0498[M+H-H2O-2CH3] + ,217.0497[M+H-H2O-C3H6] + ,205.0498[M+H-C4H8O2] + ,177.0549[M+H-C4H8O2-CO] + ,189.0549[M+H-C4H8O3] + , combined with the relative molecular mass, it was confirmed that this compound was chelidonol.

[0092] 3.1.4 Flavonoids

[0093] There are 29 chromone compounds analyzed from Qingwen Hufei Granules, 15 of which are in positive mode and 16 in negative mode. Hyperoside and rutin respond in both positive and negative modes. Taking hyperoside as an example, the quasi-molecular ion peak is m / z 465.1028 [M+H] + The corresponding compound molecular formula is C 21 H 20 O 12 In the secondary spectrum, the main fragment ion peak is 303.0501[M+H-C6H 10 O5] + ,

[0094] 285.0389[M+H-C6H 10 O5-H2O] + ,165.0186[M+H-C6H 10 O5-C7H6O3] + ,153.0187[M+HC 14 H 16 O8] + ,137.0234[M+HC 14 H 16 O9] + , combined with the relative molecular mass, it was confirmed that this compound was hyperoside.

[0095] 3.1.5 Organic acids

[0096] There are 18 organic acid compounds analyzed from Qingwen Hufei Granules, 7 in positive mode and 12 in negative mode, among which caffeic acid responds in both positive and negative modes. Taking phenylalanine as an example, the quasi-molecular ion peak is m / z 166.0863 [M+H] + , the corresponding compound molecular formula is C9H 11 NO2, in the secondary spectrum, the main fragment ion peak is 148.0757 [M+H-H2O] +,120.0807[M+H-H2O-CO] +

[0097] 109.0649[M+H-C2H3ON] + ,103.0545[M+H-H2O-CO-NH3] + ,

[0098] 91.0543[M+H-C2H5O2N] + ,77.0385[M+H-HCOOH-NH3-C2H2] + ,

[0099] 51.0232[M+H-HCOOH-NH3-C2H2-C2H2] + Combined with the relative molecular mass, it was confirmed that this compound was phenylalanine.

[0100] 3.1.6 Glycosides

[0101] There are 36 glycoside compounds analyzed from Qingwen Hufei Granules, 4 in positive mode and 32 in negative mode. Taking forsythiaside A as an example, the quasi-molecular ion peak is m / z 623.1981[MH] - The corresponding compound molecular formula is C 29 H 36 O 15 In the secondary spectrum, the main fragment ion peak is 461.1641[MH-Caffeoyl] - ,443.1540[MH-Caffeoyl-H2O] - , 161.0236[MH-Rha-C8H8O2-Glu-H2O] - , combined with the relative molecular mass, confirmed that this compound is forsythiaside A.

[0102] 3.1.7 Other categories

[0103] In addition to the above compounds, 23 other types of compounds were analyzed from Qingwen Hufei Granules, such as alkaloids, sugars, indoles, saponins, phenols, furans, cyclohexylethanol derivatives, etc., 13 of which were in positive mode and 10 in negative mode. Among them, sucrose responded in both positive and negative modes.

Claims

1. An ultra-high performance liquid chromatography-mass spectrometry detection method for Qingwen Hufei granules, characterized in that: The detection method comprises the following steps: ⑴. Chromatographic conditions: C18 column, column temperature 30-40°C, flow rate 0.1-0.5 mL min -1 Mobile phase: methanol A-0.1% formic acid aqueous solution B, 0-3 min, 1% A; 3-20 min, 1%-35% A; 20-33 min, 35%-80% A; 33-35 min, 80% A; 35-44 min, 80%-98% A; 44-46 min, 98% A; (2) Mass spectrometry conditions are as follows: the ion source is a heated electrospray ion source (HESI), and positive and negative ion monitoring modes are adopted. The spray voltage in positive ion mode is 3-4 kV, and in negative ion mode, the spray voltage is 2-4 kV. The sheath gas volume flow rate is 30-50 flow units in both positive and negative ion modes, and the auxiliary gas flow rate is 8-12 flow units in both positive and negative ion modes. The capillary temperature is 310-330°C in both positive and negative ion modes, the nebulization temperature is 330-370°C, the maximum spray current is 80-120 A in both positive and negative ion modes, the S-lens RF level is 40-60, the scan mode is Full MS / dd-MS2, the Full MS resolution is 60,000-80,000, the ion source is HESI, the dd-MS2 resolution is 16,000-18,000, the scan range is m / z 60-1500, and the collision energy CE is set to 20-40 eV. ⑶. Preparation of sample solution: Take Qingwen Hufei granules, accurately weigh them, add 60% to 80% methanol, sonicate for 20 to 40 minutes, bring to room temperature, add 60% to 80% methanol to make up for the weight loss, filter, and take the filtrate; ⑷. Preparation of reference solution: Weigh the reference solution, dissolve it in methanol and make up to volume. Prepare chlorogenic acid, liquiritin, scoparone, 5-O-methylvisamidoside, harpaoside, forsythiaside A, luteolin, 4,5-O-dicaffeoylquinic acid, forsythiaside, harpaoside, and cimicifugoside at concentrations of 0.1-0.15 mg mL -1 0.6~1.0mg·mL -1 , 0.08~0.13mg·mL -1 0.10~0.16mg·mL -1 0.13~0.17mg·mL -1 0.12~0.16mg·mL -1 0.14~0.18mg·mL -1 0.16~0.21mg·mL -1 0.13~0.17mg·mL -1 0.17~0.21mg·mL -1 , 0.19~0.23mg·mL -1 Reference substance stock solution; (5) Establishment of a literature library: CNKI, PubMed, and Web of Science databases were consulted to retrieve research literature on the chemical components of the Qingwen Hufei Granule ingredients, including honeysuckle, forsythia, fritillaria thunbergii, isatis indigotica, loblolly bamboo, platycodon, codonopsis pilosula, stir-fried atractylodes, poria, figwort, ophiopogon japonicus, bitter apricot, capillaris, saposhnikovia divaricata, perilla leaf, and liquorice. Combining literature reports with information from the ChemicalBook database, the Chinese names, English names, categories, references, and structural identification information of the chemical components were summarized, including molecular formula, relative molecular mass, structural formula in mol format, and primary and multi-level mass spectrometry information, to establish a chemical component library for all medicinal materials in Qingwen Hufei Granule. (6) Compound identification: Based on the results of liquid chromatography-mass spectrometry, the molecular formula was fitted using Xcalibur 4.0 software to obtain a database of primary information matching, which was then repetitively screened with the self-built Qingwen Hufei Granules full-drug chemical component library. The fragment information of the screening library was inferred based on the secondary molecular information. The fragment ions that could not be matched were cleaved and fragment ion analyzed using the Thermo Fisher cleavage software based on the .mol ​​format structural formula downloaded from the ChemSpider database. The fragmentation rules of the compounds were further inferred based on the reference substances, databases, references, and the fragment ion information provided by the Thermo Fisher cleavage software.

2. The detection method according to claim 1, wherein In the chromatographic conditions of step (1), C 18 The chromatographic column model is ThermoAccucore aQ RP18, and the chromatographic column specifications are 2.1 mm × 150 mm, 2.6 μm.

3. The detection method according to claim 1, wherein The mass spectrometry conditions for step (2) are as follows: the spray voltage in positive ion mode is 3.5 kV, the spray voltage in negative ion mode is 3.0 kV, the sheath gas volume flow rate in both positive and negative ion modes is 40 flow units, the auxiliary gas flow rate in both positive and negative ion modes is 10 flow units, the capillary temperature in both positive and negative ion modes is 320°C, the nebulization temperature is 350°C, the maximum spray current in both positive and negative ion modes is 100 A, the S-lens RF level is 50, the scan mode is Full MS / dd-MS2, the Full MS resolution is 70,000, the ion source is HESI, the dd-MS2 resolution is 17,500, the scan range is m / z 80-1200; and the collision energy CE is set to 30 eV.

4. The detection method according to claim 1, wherein The methanol concentration in the preparation of the sample solution in step (3) is 70%.

5. The detection method according to claim 1, wherein The ultrasonic time in the preparation of the sample solution in step (3) is 30 min.

6. The detection method according to claim 1, wherein Preparation of reference solution in step (4): the mass concentrations of chlorogenic acid, liquiritin, scoparia lactone, 5-O-methylvisamidoside, harpaoside, forsythiaside A, luteolin, 4,5-O-dicaffeoylquinic acid, forsythiaside, harpaoside, and cimicifugoside were 0.135 mg mL -1 , 0.800mg·mL -1 , 0.118mg·mL -1 , 0.146mg·mL -1 , 0.157mg·mL -1 , 0.140mg·mL -1 , 0.164mg·mL -1 , 0.198mg·mL -1 , 0.157mg·mL -1 , 0.190mg·mL -1 , 0.215mg·mL -1 Reference stock solution.

Images