Quality evaluation method of heart-fire-clearing agilawood eight-ingredient pill

Abstract

The invention discloses a quality evaluation method for heart-fire-clearing agilawood eight-ingredient pills. The quality evaluation method comprises the following steps: (1) establishing a fingerprint spectrum of each QCBP test sample solution and calibrating a common peak; (2) carrying out multi-dimensional quality difference evaluation and analysis on a plurality of QCBP test products; (3) if the quality difference exists, screening out key factors causing relatively large difference among the QCBP test samples from the calibrated common peaks of the QCBP test sample solutions; and (4) determining the content of each component causing the quality difference of the sample. According to the invention, based on HPLC-Q-Exactive-MS (High Performance Liquid Chromatography-Quantitative Exactive-Mass Spectrometry) technical component analysis, HPLC fingerprint spectrum establishment and HPLC-MS differential component quantitative evaluation research, a reference is provided for a quality evaluation system of commercially available QCBP.

Description

Technical Field

[0001] The invention relates to the technical field of quality evaluation of traditional Chinese medicine preparations, and in particular to a quality evaluation method for Qingxin Chenxiang Bawei Pills. Background Art

[0002] The original formula of Qingxin Chenxiang Bawei Pills (QCBP) is included in the Mongolian Medicine Volume, a standard issued by the Ministry of Education of my country. It is composed of eight herbs: agarwood, jujube, sandalwood, red sandalwood, safflower, nutmeg, rhizoma damiana, and Radix Adenophorae. It has the effects of clearing the heart and lungs, regulating qi, calming and tranquilizing the mind. It is mainly used to treat excessive heart and lung fire, chest tightness, and chest and rib pain. In QCBP, agarwood as the main drug has the effects of promoting qi and relieving pain, warming the middle and stopping vomiting; safflower promotes blood circulation and menstruation, dispersing blood stasis and relieving pain; jujube promotes qi, promotes blood circulation, nourishes the heart, and tranquilizes the mind; nutmeg warms the middle and promotes qi, astringes the intestines and stops diarrhea; sandalwood promotes qi and warms the middle, stimulates appetite and relieves pain; rhizoma damiana clears heat and resolves phlegm, calms the heart and calms the nerves, and awakens the mind; red sandalwood relieves pain, detoxifies and reduces swelling, stops bleeding and promotes tissue regeneration; and Radix Adenophorae nourishes yin and clears the lungs, benefits the stomach and promotes fluid production. Modern studies have shown that agarwood has multiple pharmacological effects such as sedation and hypnosis, analgesia, anti-inflammatory and antibacterial effects. The above eight herbs are used together to play the role of clearing the heart and lungs, regulating qi, and calming the mind.

[0003] Currently, only the content of individual characteristic components has been determined, and there is no literature report on the compositional qualitative analysis and comprehensive quality evaluation of QCBP. Summary of the invention

[0004] The invention aims to provide a quality evaluation method for Qingxin Chenxiang Bawei Pills.

[0005] The present invention uses HPLC-Q-Exactive-MS technology combined with literature data to identify and analyze the chemical components of commercially available QCBP, establishes fingerprints of 13 batches of commercially available QCBP, and conducts comprehensive quality evaluation of QCBP through cluster analysis (CA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and variance analysis, and quantitatively analyzes 4 differential components, providing a basis and reference for quality control of commercially available QCBP.

[0006] The purpose of the present invention is implemented by the following technical scheme: a method for evaluating the quality of Qingxin Chenxiang Bawei Pills, comprising the following steps: (1) establishing fingerprints of each QCBP test solution and calibrating common peaks; (2) performing multi-dimensional quality difference evaluation and analysis on a number of QCBP test samples; (3) if there is a quality difference, screening out the key factors that cause the large differences between the QCBP test samples from the calibrated common peaks of the QCBP test solutions; (4) determining the content of each component that causes the sample quality difference; wherein, (1) Establish fingerprints of each test solution and calibrate common peaks: Take several QCBP samples, prepare them into QCBP test solutions, perform chromatographic analysis on them, record the chromatograms, convert the collected chromatograms into CDF format files and import them into the Chinese medicine chromatographic fingerprint similarity evaluation system, select the chromatogram of any QCBP test solution and set it as the reference spectrum for fingerprint analysis, after multi-point correction and Marker peak matching, obtain the chromatogram overlay and reference fingerprint of each QCBP test solution, and calibrate several common peaks; after comparing with the spectrum of each single reference stock solution, multiple chromatographic peaks are identified; (2) Conduct multi-dimensional quality difference evaluation and analysis on several QCBP test products: conduct fingerprint similarity evaluation on the fingerprints established in step (1); conduct cluster analysis with several common peak areas as indicators; conduct principal component analysis with several common peak areas as variables; use orthogonal partial least squares discriminant analysis and variance analysis to analyze the differences in several common peak areas; and determine whether there are quality differences among several QCBP test products based on the above analysis results; (3) If there are quality differences, the key factors that cause the large differences between the QCBP test samples are screened out from the common peaks of the calibrated QCBP test sample solutions: evaluation method based on variable weight values [25-26] Each QCBP test product was analyzed. When the VIP value was > 1, it indicated that the component was the key factor causing the large differences among the QCBP test products; (4) Determination of the content of each component that causes the difference in sample quality: Weigh the QCBP test solution separately, and use the HPLC-MS quantitative analysis method to calculate the content of each component that causes the difference in sample quality in each sample, and determine the content difference of each component that causes the difference in sample quality in the QCBP test solution.

[0007] Specifically, in the step (1), 13 batches of commercially available QCBP samples from three manufacturers were taken; 20 common peaks were calibrated, of which 6 chromatographic peaks were identified, namely: peak 1 was gallic acid, peak 3 was protocatechuic acid, peak 5 was hydroxysafflor yellow A, peak 6 was agarwood tetraol, peak 7 was ferulic acid, and peak 10 was ellagic acid; the single reference substance stock solutions were ferulic acid stock solution, agarwood tetraol stock solution, gallic acid stock solution, hydroxysafflor yellow A stock solution, protocatechuic acid stock solution, quinic acid stock solution, kaempferol stock solution, costunolide stock solution, and rutin stock solution; In step (2), there were quality differences among 13 batches of commercially available QCBP samples from three manufacturers; In the step (3), there are 14 common peaks with VIP values ​​greater than 1, which are arranged from large to small: peak 2, peak 16, peak 15, peak 17, peak 11, ferulic acid, agarwood tetraol, peak 20, peak 18, gallic acid, peak 19, hydroxysafflor yellow A, peak 12, and peak 9; that is, these 14 chromatographic peaks are the key factors causing the quality differences of the 13 batches of samples; In the step (4), the contents of ferulic acid, agarwood tetraol, gallic acid and hydroxysafflor yellow A, which are key factors leading to sample quality differences, are measured to determine the content differences of the four components in the test solution.

[0008] Specifically, the preparation method of the QCBP test solution is as follows: weigh the QCBP samples respectively, grind them into powder, sieve them, add a methanol solution with a mass percentage concentration of 70%-80% to dissolve them, seal them, and ultrasonicate them for 30min-50min, with an ultrasonic power of 60W-80W and an ultrasonic frequency of 50kHz-90kHz. After cooling to room temperature, add a methanol solution with a mass percentage concentration of 70%-80%, shake them well, and filter them to obtain each QCBP test solution.

[0009] Specifically, the mass volume ratio of the sieved commercially available QCBP sample powder to the methanol solution with a mass percentage concentration of 70%-80% is 0.1 g:1 mL~0.2 g:1 ml.

[0010] Specifically, in the step (1), the chromatographic analysis conditions are as follows: the mobile phase is methanol (A)-0.2% phosphoric acid aqueous solution (B), the flow rate is 1.0 mL / min, the column temperature is 35°C, the injection volume is 10 μL, and the detection wavelength is 254 nm; the gradient elution program is: 0-8 min, 15%→30% A; 8-17 min, 30%→35% A; 17-20 min, 35%→35% A; 20-25 min, 35%→40% A; 25-30 min, 40%~45% A; 30-50 min, 45%~60% A; 50-55 min, 60%→65% A; 55-65 min, 65%→65% A; 65-70 min, 65%~80% A; 70-80 min, 80%~15% A; 80-90 min, 15%~15% A.

[0011] Specifically, in the step (4), the HPLC-MS quantitative analysis method, the chromatographic column quantitative analysis conditions are: the mobile phase is methanol (A)-water (containing 0.1% formic acid) (B), the gradient elution program is: 0.01~0.5min, 15%~20%A; 0.5~1.0min, 20%~35%A; 1~3 min, 35%~60%A; 3.0~5.0min, 65%~75%A; 5.0~5.5min, 75%~95%A; 5.5~7.0min, 95%A; 7.0~7.01min, 95%~10%A; 7.01~9.5min, 10%A; volume flow rate 0.25mL / min; column temperature 35℃; injection volume 3μL.

[0012] Specifically, in the step (4), the HPLC-MS quantitative analysis method and the mass spectrometry quantitative analysis conditions are: using an electrospray ion source (ESI); positive and negative ion scanning; multiple reaction monitoring (MRM) mode; spray voltage 3.80 kV (+), 3.50 kV (-), nebulizing gas volume flow rate 3 L / min; heating gas volume flow rate 10 L / min; interface temperature 300°C; desolvation temperature 526°C; heating block temperature 400°C.

[0013] Advantages of the present invention: (1) The present invention is based on the component analysis of HPLC-Q-Exactive-MS technology combined with the establishment of HPLC fingerprint and the quantitative evaluation of HPLC-MS differential components, which provides a reference for the quality evaluation system of commercially available QCBP.

[0014] (2) The present invention investigated the characterization effects of samples with different ultrasonic extraction times, different mobile phases and different concentrations. The results showed that when the ultrasonic extraction time was 40 min, the mobile phase was methanol and 0.1% formic acid water as the mobile phase for gradient elution, and the sample was diluted 1000 times, the separation effect of each component, mass spectrum abundance and peak shape were better than those under other conditions.

[0015] (3) To ensure that the detection conditions are stable and feasible, and the HPLC fingerprint characterization information is comprehensive, the mobile phase, chromatographic column, and detection wavelength were investigated on the basis of HPLC-Q-Exactive-MS conditions. The experiment found that the detection wavelength was significantly higher when methanol and 0.2% phosphoric acid water were used as the mobile phase and the chromatographic column was Agilent Eclipse XDB-C 18 The fingerprint spectrum with a detection wavelength of 254 nm and a diameter of 4.6 mm × 250 mm (5 μm) can make each chromatographic peak achieve the most ideal peak shape and response value, and can more intuitively reflect the information of multi-component chromatographic peaks. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the total ion chromatogram of QCBP (S1) HPLC-Q-Exactive-MS.

[0018] Figure 2 is the MS of compound 5 2 Figure and possible cleavage pathways.

[0019] Figure 3 is the MS of compound 25 2 Figure and possible cleavage pathways.

[0020] Figure 4 is the MS of compound 39 2 Figure and possible cleavage pathways.

[0021] Figure 5 is the MS of compound 20 2 Figure and possible cleavage pathways.

[0022] Figure 6 MS of compound 27 2 Figure and possible cleavage pathways.

[0023] Figure 7 is the MS of compound 10 2 Figure and possible cleavage pathways.

[0024] Figure 8 is the MS of compound 1 2 Figure and possible cleavage pathways.

[0025] Fig. 9 The overlay of fingerprints of 13 batches of commercially available QCBP (A) and the reference fingerprint (B).

[0026] Fig.10 The chromatograms are compared between the single reference solution and the QCBP (S1) sample solution.

[0027] Fig.11 Cluster heat map analysis of 13 batches of commercially available QCBP.

[0028] Fig.12 The PCA score plots of 13 batches of commercially available QCBP samples.

[0029] Fig.13The OPLS-DA score plots for 13 batches of commercially available QCBP.

[0030] Fig.14 VIP maps of 20 common peaks of 13 batches of commercially available QCBP.

[0031] Fig.15 1 is the secondary mass spectrum of ferulic acid, 2 is the secondary mass spectrum of agarwood tetraol, 3 is the secondary mass spectrum of gallic acid, and 4 is the secondary mass spectrum of hydroxysafflor yellow A.

[0032] Fig.16 Figure 1 is the MRM chromatogram of each component, Figure A is the MRM chromatogram of each component of the standard, Figure B is the MRM chromatogram of each component of the test sample, and Figure C is the MRM chromatogram of each component of the blank solvent. 1 represents ferulic acid, 2 represents agarwood tetraol, 3 represents gallic acid, and 4 represents hydroxysafflor yellow A. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0034] The original formula of Qingxin Chenxiang Bawei Pills (QCBP) is included in the Mongolian Medicine Volume, a standard issued by the Ministry of Health of my country. [1] It is composed of eight herbs: Agarwood, Ziziphus jujube, Sandalwood, Pterocarpus indica, Safflower, Nutmeg, Rhizoma Cibotii, and Radix Adenophorae. It has the effects of clearing the heart and lungs, regulating qi, calming and soothing the nerves. It is mainly used to treat excessive heart and lung fire, chest tightness, and chest and rib pain. In QCBP, agarwood as the main drug has the effects of promoting qi and relieving pain, warming the middle and stopping vomiting; Safflower promotes blood circulation and menstruation, disperses blood stasis and relieves pain; Ziziphus jujube promotes qi, promotes blood circulation, nourishes the heart, and soothes the nerves; Nutmeg warms the middle and promotes qi, astringes the intestines and stops diarrhea; Sandalwood promotes qi and warms the middle, stimulates appetite and relieves pain; Rhizoma Cibotii clears heat and resolves phlegm, calms the heart and calms the nerves, and opens the mind and refreshes the spirit; Pterocarpus indica relieves pain, detoxifies and reduces swelling, stops bleeding and promotes tissue regeneration; Radix Adenophorae nourishes yin and clears the lungs, benefits the stomach and promotes fluid production. Modern studies have shown that agarwood has a sedative and hypnotic effect [2-3] , analgesic effect [4] , anti-inflammatory and antibacterial [5] The above eight herbs are used together to play the role of clearing the heart and lungs, regulating qi, calming the mind and tranquilizing the mind.

[0035] There is no literature report on the qualitative composition and comprehensive quality evaluation of QCBP, only the content determination of individual characteristic components [6-7]In this study, the chemical components of commercially available QCBP were identified and analyzed by HPLC-Q-Exactive-MS technology combined with literature data, and the fingerprints of 13 batches of commercially available QCBP were established. The comprehensive quality evaluation of QCBP was carried out by cluster analysis (CA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and variance analysis, and the four differential components were quantitatively analyzed to provide a basis and reference for the quality control of commercially available QCBP.

[0036] 1 Materials and Instruments 1.1 Instrument LC-2030C high performance liquid chromatograph and its LC-40BXR infusion pump, SIL-40C autosampler, CTO-40C column oven, CBM-20A system controller, SPD-40V detector (Shimadzu Corporation, Japan); HPLC-Q-Exactive high performance liquid chromatography-mass spectrometry system (Thermo Fisher Scientific Inc.); LC-MS 8045 high performance liquid chromatography-triple quadrupole mass spectrometry (Shimadzu Corporation, Japan); AP135 W one-hundred-thousandth electronic balance (Shimadzu Corporation, Japan); BSA224S one-thousandth balance (Sartorius Scientific Instrument Co., Ltd.); DS-7510DTH CNC ultrasonic cleaner (Shanghai Shengxi Ultrasonic Instrument Co., Ltd.). 1.2 Drug testing QCBP from three manufacturers were collected, with a total of 13 batches, namely Manufacturer A (S1-S6, with batch numbers of 2211010, 2303024, 2112010, 2210003, 2304007, and 2306053), Manufacturer B (S7-S9, with batch numbers of 220814, 220706, and 221101), and Manufacturer C (S10-S13, with batch numbers of 230506, 210739, 230709, and 210930), with a specification of 1 g / 8 tablets.

[0037] Costunolide (Batch No.: 111524-201911, purity ≥99.9%), quinic acid (Batch No.: 111717-201402, purity ≥98.5%), protocatechuic acid (Batch No.: 110809-202207, purity ≥98.5%), ferulic acid (Batch No.: 110773-202316, purity ≥99.3%), kaempferol (Batch No.: 110861-202214, purity ≥99.0%), agarwood tetraol (Batch No.: 111980-201904, purity ≥98.6%), gallic acid (Batch No.: 110831-202407, purity ≥98.3%) %), rutin (batch number: 100080-202012, for identification), hydroxysafflor yellow A (batch number: 111637-202111, purity ≥98.8%) reference substances were purchased from China Food and Drug Inspection Institutes; methanol, acetonitrile and formic acid were all chromatographically pure; water was distilled water from Watsons.

[0038] 2 Methods and Results 2.1 Preparation of standard solution Accurately weigh appropriate amounts of nine reference substances, including ferulic acid, agarwood tetraol, gallic acid, hydroxysafflor yellow A, protocatechuic acid, quinic acid, kaempferol, costunolide, and rutin, and place them in different 10 mL volumetric flasks. Dissolve them in methanol and make up to volume to prepare a single reference substance stock solution, with a mass concentration of 551.00 μg mL -1 、366.00μg·mL -1 , 159.00μg·mL -1 , 447.00μg·mL -1 , 481.00μg·mL -1 , 521.00μg·mL -1 、339.00μg·mL -1 , 699.00μg·mL -1 、392.00 μg·mL -1 , accurately measure 100 μL of each of the above mother solutions and place them in the same 10 mL volumetric flask, add methanol to dilute, and obtain a mass concentration of 551.00 ng·mL -1 、366.00 ng·mL -1 、159.00 ng·mL -1 、447.00 ng·mL -1 、481.00 ng·mL -1 , 521.00 ng·mL -1 、339.00 ng·mL -1 、699.00 ng·mL -1 、392.00 ng·mL-1 All the mixed standard solutions were stored at 4°C for future use.

[0039] 2.2 Preparation of test solution Take 13 batches of commercially available QCBP, grind them into powder, pass them through a No. 4 sieve, weigh 1.000 g respectively into a 50 mL conical flask, add 10 mL of 70% methanol solution by mass percentage, seal, and sonicate for 50 min (power 80 W, frequency 50 kHz). After cooling to room temperature, add 70% methanol solution by mass percentage, shake well, and filter through a 0.22 μm filter membrane to obtain the product.

[0040] 2.3 HPLC-Q-Exactive-MS qualitative analysis 2.3.1 HPLC conditions The chromatographic column was Agilent ZORB-AX SB-Aq (4.6 mm×150 mm, 5 μm), and the mobile phase was methanol (A)-0.1% formic acid aqueous solution (B). The gradient elution was as follows: 0-5 min, 15%→20% A; 5-12 min, 20%→35% A; 12-16 min, 35%→60% A; 16-20 min, 60%→70% A; 20-25 min, 70%→85% A; 25-35 min, 85%→95% A; 35-40 min, 95%→95% A, and the flow rate was 0.35 mL min -1 , column temperature was 35 °C, and injection volume was 10 μL.

[0041] 2.3.2 Mass spectrometry conditions Using an electrospray ionization source (ESI), + 、ESI - Detection in ion detection mode, detection method is Full MS / dd-MS 2 , Full MS resolution is 70000, dd-MS 2 The resolution is 17500 and the scanning range is m / z 110~1200, collision energy set to 30 eV, spray voltage: 3.80 kV (+) and 3.20 kV (-), ion transfer tube temperature 300 ℃ (+) and 400 ℃ (-), auxiliary gas temperature 320 ℃, auxiliary gas volume flow rate 30 L·min -1 .

[0042] 2.4 HPLC fingerprint analysis conditions The chromatographic column is Agilent Eclipse XDB-C 18The column was eluted with 4% paraformaldehyde (A) and 0.2% phosphoric acid (B) as the mobile phase. The flow rate was 1.0 mL / min. The column temperature was 35 °C. The injection volume was 10 μL. The detection wavelength was 254 nm. The gradient elution program was as follows: 0-8 min, 15%→30% A; 8-17 min, 30%→35% A; 17-20 min, 35%→35% A; 20-25 min, 35%→40% A; 25-30 min, 40%~45% A; 30-50 min, 45%~60% A; 50-55 min, 60%→65% A; 55-65 min, 65%→65% A; 65-70 min, 65%~80% A; 70-80 min, 80%~15 % A; 80~90 min, 15%~15% A.

[0043] 2.5 HPLC-MS quantitative analysis conditions 2.5.1 Chromatographic columns The column was Shim-pack GIST-HP C18 (2.1 mm×100 mm, 3 μm); the mobile phase was methanol (A)-water (containing 0.1% formic acid) (B), with gradient elution: 0.01~0.5 min, 15%~20% A; 0.5~1.0 min, 20%~35% A; 1~3min, 35%~60% A; 3.0~5.0 min, 65%~75% A; 5.0~5.5 min, 75%~95% A; 5.5~7.0min, 95% A; 7.0~7.01 min, 95%~10% A; 7.01~9.5 min, 10% A; volume flow rate 0.25 mL / min; column temperature 35 ℃; injection volume 3 μL.

[0044] 2.5.2 Mass spectrometry Electrospray ion source (ESI) was used; positive and negative ion scanning; multiple reaction monitoring (MRM) mode; spray voltage was 3.80 kV (+), 3.50 kV (-), nebulizer gas volume flow rate was 3 L / min; heating gas volume flow rate was 10 L / min; interface temperature was 300 °C; desolvation temperature was 526 °C; heating block temperature was 400 °C. The mass spectrometry parameters of each component are shown in Table 3, and the secondary mass spectra of each component are shown in Fig.15 , MRM chromatograms of each component are shown in Fig.16 .

[0045] 2.6 Component Analysis 2.6.1 Data processing and analysis The chemical components, mass spectrometry fragment information and other literature data of Aquilaria sinensis, Fructus jujubae, Santalum album, Pterocarpus chinensis, Carthamus tinctorius, Myristica fragrans, Rhizoma Cibotii, and Glehnia littoralis in QCBP were searched through CNKI and PubMed data platforms, and a database including compound names, molecular formulas, multi-stage mass spectrometry fragments and other information was created. The total ion flow data of commercially available QCBP (S1 batch number 2211010) solution was obtained in positive and negative ion modes, and peak extraction and accurate relative molecular mass fitting were performed using Xcalibur 3.0 software. By comparing with the database, the error (δ) was selected within ±10 ppm (1 ppm=1×10 -6 ) were used to preliminarily identify the compounds in commercially available QCBP, of which 9 components were further confirmed by comparison with reference substances.

[0046] 2.6.2 QCBP chemical composition identification results Dilute the test solution 100 times according to item "2.2" and the mixed standard solution according to item "2.1", and perform injection analysis under the conditions of item "2.3" to obtain the total ion current diagram of the QCBP sample solution in positive and negative ion modes, as shown in the figure below: Figure 1 As shown, the sample data obtained were analyzed according to the method under "2.6.1". As a result, 70 chemical components were identified in the commercially available QCBP, including 7 fatty acids, 8 phenolic acids, 9 sesquiterpenes, 17 phenylpropanoids (4 simple phenylpropanoids, 5 coumarins, 8 lignans), 8 chromones, 14 flavonoids, and 7 other types (3 other components and 4 amino acids). The results are shown in Table 1. Table 1: HPLC-Q-Exactive-MS analysis results of the chemical composition of QCBP (S1)

[0047] Note: *） Compounds verified by reference 2.6.3 Fatty acids A total of 7 fatty acid compounds were identified in QCBP (S1). These compounds have a free -COOH quasi-molecular ion peak that is easy to lose an H ion and can be detected in negative ion mode. Take compound No. 5 (quinic acid) as an example: Compound No. 5 can be seen in negative ion mode [MH] - m / z 191.0561 quasi-molecular ion peak, with a deviation of 5.786×10 -6 The molecular formula of the product was fitted by Xcalibur 3.0 software as C7H 12 O6, the main fragment ions are m / z173.0089[MH-H2O] - , m / z 148.4393[MH-COOH] - , m / z 129.0108[MH-H2O-H2O-CO] - , m / z 111.0079[MH-H2O-H2O-CO-OH] - or [MH-COOH-2H2O] - , that is, the fragmentation rule of this compound in negative ion mode may be m / z 191.0561 → m / z 173.0089 → m / z 129.0108 → m / z 111.0079 or m / z 191.0561 → m / z 148.4393 → m / z 111.0079, through its cleavage rules and literature comparison [8,13] , confirming that compound No. 5 is quinic acid, its MS ２ Figure and possible cleavage pathways are shown in Figure 2 .

[0048] 2.6.4 Phenolic acids A total of 8 phenolic acid compounds were identified by QCBP (S1). These compounds have phenolic hydroxyl, hydroxyl, carboxyl and other substituents, which are easy to lose H2O, COOH and CH3 in negative ion mode, thus generating corresponding characteristic ion fragments. Take compound No. 25 (ferulic acid) as an example: Compound No. 25 can be seen in negative ion mode [MH] - m / z 193.0507 quasi-molecular ion peak, deviation is 6.137×10 -6 The molecular formula was fitted by Xcalibur 3.0 software as C 10 H 10 O4, the main fragment ions are m / z 178.0270[MH-CH3] - , m / z 149.0604[MH-COOH] - , m / z 134.0368[MH-COOH-CH3] - , that is, the fragmentation rule of this compound in negative ion mode may be m / z 193.0507 → m / z 178.0270 or m / z 193.0507 → m / z 149.0604 → m / z 134.0368, through its cracking rules and literature comparison [15,16] , confirming that compound No. 25 is ferulic acid, and its MS ２ Figure and possible cleavage pathways are shown in Figure 3 .

[0049] 2.6.5 Phenylpropanoids Phenylpropanoid compounds can be divided into simple phenylpropanoids, coumarins and lignans. In QCBP (S1), 4 simple phenylpropanoids, 5 coumarins and 8 lignans were identified. Take coumarin compounds as an example: this type of component is easy to lose CO, CO2 or H2O in positive ion mode. When there are substituents in the parent nucleus, CO and CO2 will be lost alternately. Take compound No. 39 (psoralen) as an example: Compound No. 39 can be seen in positive ion mode [M+H] + m / z 187.0385 quasi-molecular ion peak, deviation is -2.302×10 -6 The molecular formula was fitted by Xcalibur 3.0 software as C 11 H6O3, the main fragment ions are m / z 159.1160[M+H-CO] + , m / z 131.0853[M-CO-CO], m / z 143.0488[M+H-CO2] + , m / z 115.0542[M-CO2-CO], that is, the fragmentation rule of this compound in negative ion mode may be m / z 187.0385 → m / z 159.1160→ m / z 131.0853 or m / z 187.0385 → m / z 143.0488 → m / z 115.0542, through its cracking rules and literature comparison [11,17] , confirming that compound 39 is psoralen, its MS ２ Figure and possible cleavage pathways are shown in Figure 4 .

[0050] 2.6.6 Flavonoids A total of 14 flavonoid compounds were identified by QCBP (S1), which is also the most diverse compound in this qualitative analysis. The quality of the fragments produced by the flavonoid molecule after energy collision is closely related to the substituents on the A and B rings of the flavonoid. The abundance of fragments such as H2O and CO lost by the flavonoid molecular ion peak is also mainly affected by the substituents on the A and B rings. Take compound No. 20 (hydroxysafflor yellow A) as an example: Compound No. 20 can be seen in negative ion mode [MH] - m / z 611.1660 quasi-molecular ion peak, with a deviation of 6.958×10 -6 The molecular formula was fitted by Xcalibur 3.0 software as C 27 H 32 O4, the main fragment ions are m / z 491.1220[MH-C4H8O4] - , m / z 403.1064[MH-C6H 10 O5-CO-H2O] - , m / z 325.0738[MH-C2H4O2-H2O] - , m / z 283.0627[MH-C2H2O] - , m / z 205.0145[MH-C8H8O] - , that is, the fragmentation rule of this compound in negative ion mode may be m / z 611.1660 → m / z 491.1220 → m / z 325.0738 → m / z 283.0627 (or → m / z 205.0145) or m / z 611.1660 → m / z 403.1064 → m / z 325.0738 → m / z 283.0627 (or → m / z 205.0145), through its cleavage law and literature comparison [12,18] , confirming that compound No. 20 is hydroxysafflor yellow A, and its MS ２ Figure and possible cleavage pathways are shown in Figure 5 .

[0051] 2.6.7 Chromones A total of 8 chromone compounds were identified in QCBP (S1). After energy collision, chromone compounds often first remove one or more molecules of water, or decarbonylate, and then the chemical bonds between the chromone units are broken, and further fragmentation reactions occur on this basis. Take compound No. 27 (agarwood tetraol) as an example: Compound No. 27 can be seen in positive ion mode [M+H] + m / z 319.1150 quasi-molecular ion peak, deviation -8.225×10 -6 The molecular formula was fitted by Xcalibur 3.0 software as C 17 H 18 O6, the main fragment ions are m / z 301.1061[M+H-H2O] + , m / z 283.0945[M+H-H2O-H2O] + , m / z 255.1002[M+H-H2O-H2O-CO] + , m / z 227.1058[M+H-H2O-H2O-CO-CO] + , m / z 164.0462[M+H-H2O-H2O-CO-C7H8] + , that is, the fragmentation rule of this compound in positive ion mode may be m / z 319.1150 → m / z 301.1061 → m / z 283.0945 → m / z 255.1002 → m / z 227.1058 (or m / z 164.0462), through its cracking rules and literature comparison [9,19] , confirming that compound No. 27 is agarwood tetraol, and its MS ２ Figure and possible cleavage pathways are shown in Figure 6 .

[0052] 2.6.8 Sesquiterpenes A total of 9 sesquiterpenoids were identified in QCBP (S1). The skeleton of this type of compound is composed of 3 isoprene units and contains 15 carbon atoms. Compound No. 10 (dehydrocostus lactone) is used as an example: Compound No. 10 can be seen in positive ion mode [M+H] + m / z 231.1374 quasi-molecular ion peak, deviation is -2.537×10 -6 The molecular formula was fitted by Xcalibur 3.0 software as C 15 H18 O2, the main fragment ions are m / z 213.1264[M+H-H2O] + , m / z 185.1321[M+H-H2O-CO] + , m / z 157.1005[M+H-H2O-CO-C2H4] + , m / z 143.0851[M+H-H2O-CO-C2H4-CH2] + , that is, the fragmentation rule of this compound in positive ion mode may be m / z 231.1374 → m / z 213.1264 → m / z 185.1321 → m / z 157.1005 → m / z 143.0851, through its cracking rules and literature comparison [9,20] , confirming that compound No. 10 is dehydrocostus lactone, and its MS ２ Figure and possible cleavage pathways are shown in Figure 7 .

[0053] 2.6.9 Other categories A total of 7 other compounds were identified in QCBP (S1), including 4 amino acids and 3 others. Compound 1 (5-hydroxymethylfurfural) was used as an example: Compound 1 showed [M+H] + m / z 127.0388 quasi-molecular ion peak, deviation is -1.107×10 -6 The molecular formula of the product was fitted by Xcalibur 3.0 software as C6H6O3. The main fragment ions are m / z 109.0283[M+H-OH] + , m / z 81.0338[M+H-OH-CO] + , that is, the fragmentation rule of this compound in positive ion mode may be m / z 127.0388 → m / z 109.0283 → m / z 81.0338, through its cracking rules and literature comparison [8,21] , confirming that compound 1 is 5-hydroxymethylfurfural, its MS ２ Figure and possible cleavage pathways are shown in Figure 8 .

[0054] 2.7 Fingerprint research 2.7.1 Precision test Take QCBP sample S1, prepare the test solution according to the method under "2.2", inject 6 times continuously according to the chromatographic conditions under "2.4", record the chromatogram, take peak 3 (protocatechuic acid) as the reference peak, and the calculation results show that the relative retention time RSD of the 20 common peaks is less than 0.47%, and the relative peak area RSD is less than 3.24%, indicating that the precision of the instrument is good.

[0055] 2.7.2 Stability test Take QCBP sample S1, prepare the test solution according to the method under "2.2", and inject the sample for determination at 0, 2, 4, 8, 12, and 24 h under the chromatographic conditions under "2.4". Peak 3 is used as the reference peak. The calculation results show that the relative retention time RSD of the 20 common peaks is less than 0.43%, and the relative peak area RSD is less than 1.09%, indicating that the sample solution has good stability within 24 h.

[0056] 2.7.3 Repeatability test Take 6 portions of S1 QCBP sample, prepare the test solution according to the method under "2.2", and analyze them according to the chromatographic conditions under "2.4". Peak 3 (gallic acid) is used as the reference peak. The results show that the relative retention time RSD of the 20 common peaks in the sample is less than 0.10%, and the relative peak area RSD is less than 1.72%, indicating that the method has good repeatability.

[0057] 2.7.4 Fingerprint establishment Thirteen batches of commercially available QCBP from three manufacturers were used to prepare test sample solutions of each batch according to the method in "2.2". Samples were injected and analyzed according to the chromatographic conditions in "2.4". Chromatograms were recorded. The collected chromatograms were converted into CDF format files and imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition). Sample S1 was set to perform fingerprint analysis with reference to the chromatogram (the method was the median method and the time window was set to 0.1 min). After multi-point calibration and Marker peak matching, the QCBP chromatogram overlay and reference fingerprint were obtained, as shown in Figure 2. Fig. 9 As shown in the figure, the calibration results showed that there were 20 peaks in total. After comparison with the spectra of the single reference stock solutions prepared in 2.1, Fig.10 As shown, six chromatographic peaks were identified, namely gallic acid (peak 1), protocatechuic acid (peak 3), hydroxysafflor yellow A (peak 5), agalate tetraol (peak 6), ferulic acid (peak 7), and ellagic acid (peak 10).

[0058] 2.7.5 Similarity Analysis The data of 13 batches of commercially available QCBP were imported into the Traditional Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition) for fingerprint similarity evaluation. The similarity results of the fingerprints of 13 batches of commercially available QCBP and the generated reference fingerprints are shown in Table 2. The similarity of the 13 batches of commercially available QCBP was between 0.769 and 0.960, indicating that there were certain differences in the quality of the 13 batches of QCBP from three manufacturers. Taking peak 3 (protocatechuic acid) as the reference peak, the relative peak area RSD of 20 common peaks of 13 batches of QCBP was 28.0%~96.0%, indicating that there were large differences in the content of certain components of different batches of QCBP.

[0059] Table 2: Similarity evaluation results of 13 batches of commercially available QCBP

[0060] 2.8 Chemical pattern recognition analysis 2.8.1 Cluster Analysis (CA) The Weishengxin online website platform was used to analyze the 20 common peak areas of 13 batches of commercially available QCBP. The clustering method was the completion method, and the distance metric was set to euclidean.

[22] , the results are as follows Fig.11 As shown in the figure, the 13 batches of QCBP can be clustered into two categories, S1~S6, S10~S13 are clustered into one category (Manufacturer A, C), and S7~S9 are clustered into one category (Manufacturer B), which is consistent with the similarity results.

[0061] 2.8.2 Principal Component Analysis (PCA) The 20 common peak areas of 13 batches of QCBP were used as variables and the principal component analysis was performed using SIMCA 14.1 software. The results are as follows: Fig.12 , manufacturers A and C are clearly separated from manufacturer B, and the results are consistent with those of CA.

[0062] 2.8.3 Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) SIMCA 14.1 software was used to select supervised OPLS-DA to analyze the differences in 20 common peak areas among 13 batches of QCBP products. The cumulative explanatory power parameters of the constructed supervised model were ( R 2 X , R 2 Y ) were 0.783 and 0.903 respectively, and the prediction ability parameters Q 2 (cum) is 0.805, and all three parameters are greater than 0.5, indicating that the OPLS-DA model has good explanatory power and stability. [23-24] Based on the above, the score diagram is shown in Fig.13 , the samples from the three manufacturers were clustered into one category, and the classification results were more obvious, which further indicated that there were quality differences among the QCBP produced by different manufacturers.

[0063] In order to screen out the components that cause large inter-batch differences, 13 batches of samples were analyzed using the variable importance in projection (VIP) evaluation method. When the VIP value is greater than 1, it indicates that the component is the key factor in the inter-group classification. [25-26] .Depend on Fig.14 It can be seen that there are 14 common peaks with VIP values ​​>1, which are arranged from large to small: peak 2 (VIP=1.367), peak 16 (VIP=1.100), peak 15 (VIP=1.096), peak 17 (VIP=1.095), peak 11 (VIP=1.090), ferulic acid (VIP=1.082), agarwood tetraol (VIP=1.064), peak 20 (VIP=1.063), peak 18 (VIP=1.053), gallic acid (VIP=1.044), peak 19 (VIP=1.039), hydroxysafflor yellow A (VIP=1.033), peak 12 (VIP=1.030), peak 9 (VIP=1.012). That is, these 14 chromatographic peaks are the key factors causing the quality differences of 13 batches of samples.

[0064] 2.9 Content determination 2.9.1 Linear relationship investigation Accurately measure the reference stock solution prepared in 2.1: 2 μL ferulic acid, 500 μL agarwood tetraol, 500 μL gallic acid, 700 μL hydroxysafflor yellow A, place in the same 10 mL volumetric flask, add methanol to dilute, and obtain mass concentrations of 11.02, 1830.00, 759.00, and 3129.00 ng·mL -1 The mixed standard solution was accurately pipetted in 100μL, 200μL, 400μL, 600μL, 800μL, and 1000μL of the mixed standard solution in turn, placed in a 1mL volumetric flask, diluted to the scale with methanol, and injected under the conditions of "2.3". Regression analysis was performed with the injection volume of the mixed standard solution as the horizontal axis (X) and the peak area as the vertical axis (Y). The results are shown in Table 4, which shows that the linear relationship of each component is good within its respective range.

[0065] Table 3: Mass spectrometry parameters of each component

[0066] Table 4: Results of the linear relationship investigation of the four components

[0067] 2.9.2 Precision test Accurately measure the reference substance stock solution prepared in 2.1: ferulic acid, aloeswood tetraol, gallic acid, and hydroxysafflor yellow A to prepare a mixed standard solution. The mass concentrations of ferulic acid, aloeswood tetraol, gallic acid, and hydroxysafflor yellow A in the mixed standard solution are 6.612, 183.000, 159.000, and 1251.600 ng·mL, respectively. -1 , the peak areas of ferulic acid, agarwood tetraol, gallic acid and hydroxysafflor yellow A were measured 6 times under the conditions of item "2.3" and the RSDs were 4.24%, 4.26%, 0.63% and 1.49%, respectively, indicating that the instrument had good precision.

[0068] 2.9.3 Repeatability test Six portions of QCBP sample S7 were taken, and the test solution was prepared according to the method under "2.2". The samples were injected and analyzed under the conditions of "2.3". The RSDs of the peak areas of ferulic acid, agarwood tetraol, gallic acid and hydroxysafflor yellow A were 4.46%, 1.63%, 1.61% and 4.06%, respectively, indicating that the method has good repeatability.

[0069] 2.9.4 Stability test Take QCBP sample S7, prepare the test solution according to item "2.2", and analyze the samples at 0, 2, 4, 8, 12, and 24 h under the conditions of item "2.3". The RSDs of the peak areas of ferulic acid, agarwood tetraol, gallic acid, and hydroxysafflor yellow A were 0.76%, 1.86%, 1.11%, and 1.22%, respectively, indicating that the sample solution has good stability within 24 h.

[0070] 2.9.5 Sample recovery test Accurately weigh 6 portions of QCBP (S7) with known component content, each portion is about 0.500 g, and add the reference substance at a ratio of about 100% of the component content in the sample. Prepare the test solution according to "2.2" and inject and measure the samples under the conditions of "2.3". Calculate the average sample recovery and RSD of the four components. The results are shown in Table 5.

[0071] Table 5: Average recovery and RSD of the four components

[0072] 2.9.6 Sample content determination Take 13 batches of samples and prepare the test solution according to the method in "2.2". Inject and measure under the conditions in "2.3" and calculate the content. The results are shown in Table 6.

[0073] Table 6: Content determination results of 4 components in 13 batches of QCBP samples (ng / g, n=3)

[0074] 3 Discussion High performance liquid chromatography-quadrupole / electrostatic field orbital trap high resolution mass spectrometry (HPLC-Q-Exactive-MS) has the characteristics of high resolution (resolution greater than 10000 (FWHM)), high sensitivity, and high selectivity. [27-28] The present invention investigated the characterization effects of samples with different ultrasonic extraction times, different mobile phases and different concentrations. The results showed that when the ultrasonic extraction time was 40 min, the mobile phase was methanol and 0.1% formic acid water as the mobile phase for gradient elution, and the sample was diluted 1000 times, the separation effect of each component, mass spectrum abundance and peak shape were better than other conditions. In order to make the detection conditions stable and feasible, the HPLC fingerprint characterization information was comprehensive. Based on the HPLC-Q-Exactive-MS conditions, the mobile phase, chromatographic column and detection wavelength were investigated by single factors. The experiment found that when methanol and 0.2% phosphoric acid water were used as the mobile phase and the chromatographic column was Agilent Eclipse XDB-C 18 The chromatogram with a diameter of 4.6 mm × 250 mm, 5 μm and a detection wavelength of 254 nm can make each chromatographic peak achieve the most ideal peak shape and response value, and can more intuitively reflect the information of multi-component chromatographic peaks.

[0075] The main drug in QCBP is agarwood, of which agarwood tetraol is listed as an indicator component of agarwood in the 2020 edition of the Chinese Pharmacopoeia.

[29] Agarwood tetraol is a chromone component, which has antioxidant

[30] , improve the body's immunity

[31] and inflammation regulation

[32] , there are 8 chromone components in the chemical composition analysis results of QCBP based on HPLC-Q-Exactive-MS, which can provide a material basis for subsequent research. Through HPLC fingerprint similarity evaluation combined with CA, PCA and OPLS-DA analysis, 13 batches of QCBP on the market were clustered into two categories (S1~S6, S10~S13 were clustered into one category (A, C manufacturers), S7~S9 were clustered into one category (B manufacturer)), and the clustering in S1~S6 of manufacturer A was relatively scattered, indicating that the intra-group differences were large. The reasons for the intra-group differences may be related to the origin, processing, and harvesting of the medicinal materials used. The 13 batches of samples were analyzed by the variable weight value evaluation method, and 14 common peaks had VIP values ​​greater than 1. Among them, ferulic acid, agarwood tetraol, gallic acid, and hydroxysafflor yellow A were determined by HPLC-MS. The results showed that the contents of these four components were quite different.

[0076] In summary, the present invention provides a reference for the quality evaluation system of commercially available QCBP based on the component analysis of HPLC-Q-Exactive-MS technology combined with the establishment of HPLC fingerprint and the quantitative evaluation study of HPLC-MS differential components.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims

1. A method for evaluating the quality of Qingxin Chenxiang Bawei Pills, characterized in that: The method comprises the following steps: (1) establishing fingerprints of each QCBP test solution and calibrating common peaks; (2) conducting multi-dimensional quality difference evaluation and analysis on several QCBP test solutions; (3) if there are quality differences, screening out the key factors that cause large differences between the QCBP test solutions from the common peaks of the calibrated QCBP test solutions; (4) determining the content of each component that causes the quality difference of the samples; wherein, (1) Establish fingerprints of each test solution and calibrate common peaks: Take several QCBP samples, prepare them into QCBP test solutions, perform chromatographic analysis on them, record the chromatograms, convert the collected chromatograms into CDF format files and import them into the Chinese medicine chromatographic fingerprint similarity evaluation system, select the chromatogram of any QCBP test solution and set it as the reference spectrum for fingerprint analysis, after multi-point correction and Marker peak matching, obtain the chromatogram overlay and reference fingerprint of each QCBP test solution, and calibrate several common peaks; after comparing with the spectrum of each single reference stock solution, multiple chromatographic peaks are identified; (2) Conduct multi-dimensional quality difference evaluation and analysis on several QCBP test products: conduct fingerprint similarity evaluation on the fingerprints established in step (1); conduct cluster analysis with several common peak areas as indicators; conduct principal component analysis with several common peak areas as variables; use orthogonal partial least squares discriminant analysis and variance analysis to analyze the differences in several common peak areas; and determine whether there are quality differences among several QCBP test products based on the above analysis results; (3) If there are quality differences, the key factors that cause the large differences among the QCBP test samples are screened out from the common peaks of the calibrated QCBP test sample solutions: the QCBP test samples are analyzed using the variable weight value evaluation method. When the VIP value is greater than 1, it indicates that the component is the key factor that causes the large differences among the QCBP test samples; (4) Determination of the content of each component that causes the difference in sample quality: Weigh the QCBP test solution separately, and use the HPLC-MS quantitative analysis method to calculate the content of each component that causes the difference in sample quality in each sample, and determine the content difference of each component that causes the difference in sample quality in the QCBP test solution.

2. The quality evaluation method of Qingxin Chenxiang Bawei Pills according to claim 1, characterized in that: In the step (1), 13 batches of commercially available QCBP samples from three manufacturers were taken; 20 common peaks were calibrated, of which 6 chromatographic peaks were identified, namely: peak 1 was gallic acid, peak 3 was protocatechuic acid, peak 5 was hydroxysafflor yellow A, peak 6 was agalon tetraol, peak 7 was ferulic acid, and peak 10 was ellagic acid; the single reference substance stock solutions were ferulic acid stock solution, agalon tetraol stock solution, gallic acid stock solution, hydroxysafflor yellow A stock solution, protocatechuic acid stock solution, quinic acid stock solution, kaempferol stock solution, costunolide stock solution, and rutin stock solution; In step (2), there were quality differences among 13 batches of commercially available QCBP samples from three manufacturers; In the step (3), there are 14 common peaks with VIP values ​​greater than 1, which are arranged from large to small: peak 2, peak 16, peak 15, peak 17, peak 11, ferulic acid, agarwood tetraol, peak 20, peak 18, gallic acid, peak 19, hydroxysafflor yellow A, peak 12, and peak 9; that is, these 14 chromatographic peaks are the key factors causing the quality differences of the 13 batches of samples; In the step (4), the contents of ferulic acid, agarwood tetraol, gallic acid and hydroxysafflor yellow A, which are key factors leading to sample quality differences, are measured to determine the content differences of the four components in the test solution.

3. The quality evaluation method of Qingxin Chenxiang Bawei Pills according to claim 1, characterized in that: The preparation method of the QCBP test solution is as follows: weigh QCBP samples respectively, grind them into powder, sieve them, add 70%-80% methanol solution with a mass percentage concentration to dissolve them, seal them, and perform ultrasound for 30min-50min, with an ultrasound power of 60W-80W and an ultrasound frequency of 50kHz-90kHz. After cooling to room temperature, add 70%-80% methanol solution with a mass percentage concentration to make up the weight, shake them well, and filter them to obtain the QCBP test solutions.

4. The quality evaluation method of Qingxin Chenxiang Bawei Pills according to claim 3 is characterized in that: The mass volume ratio of the sieved commercially available QCBP sample powder to the methanol solution with a mass percentage concentration of 70%-80% is 0.1g:1mL~0.2g:1ml.

5. The method for evaluating the quality of Qingxin Chenxiang Bawei Pills according to claim 1, characterized in that: In the step (1), the chromatographic analysis conditions are as follows: the mobile phase is methanol (A)-0.2% phosphoric acid aqueous solution (B), the flow rate is 1.0 mL / min, the column temperature is 35°C, the injection volume is 10 μL, and the detection wavelength is 254 nm; the gradient elution program is: 0-8 min, 15%→30% A; 8-17 min, 30%→35% A; 17-20 min, 35%→35% A; 20-25 min, 35%→40% A; 25-30 min, 40%~45% A; 30-50 min, 45%~60% A; 50-55 min, 60%→65% A; 55-65 min, 65%→65% A; 65-70 min, 65%~80% A; 70-80 min, 80%~15% A; 80-90 min, 15%~15% A.

6. The quality evaluation method of Qingxin Chenxiang Bawei Pills according to claim 1, characterized in that: In the step (4), the HPLC-MS quantitative analysis method, the chromatographic column quantitative analysis conditions are: the mobile phase is methanol (A)-water (containing 0.1% formic acid) (B), the gradient elution program is: 0.01~0.5min, 15%~20%A; 0.5~1.0min, 20%~35%A; 1~3 min, 35%~60%A; 3.0~5.0min, 65%~75%A; 5.0~5.5min, 75%~95%A; 5.5~7.0min, 95%A; 7.0~7.01min, 95%~10%A; 7.01~9.5min, 10%A; volume flow rate 0.25mL / min; column temperature 35℃; injection volume 3μL.

7. The method for evaluating the quality of Qingxin Chenxiang Bawei Pills according to claim 1, characterized in that: In the step (4), the HPLC-MS quantitative analysis method and the mass spectrometry quantitative analysis conditions are: using an electrospray ion source (ESI); positive and negative ion scanning; multiple reaction monitoring (MRM) mode; spray voltage 3.80 kV (+), 3.50 kV (-), and a nebulizer gas volume flow rate 3 L / min; Heating gas volume flow rate 10 L / min; interface temperature 300°C; desolvation temperature 526°C; heating block temperature 400°C.

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