Quality evaluation method of qingxin chenxiang bayawei pill

Abstract

The application discloses a method for evaluating the quality of Qingxin Chenxiang Baba Pills, which comprises the following steps: (1) establishing a fingerprint of each QCBP test solution and calibrating common peaks; (2) evaluating and analyzing the multi-dimensional quality differences of several QCBP test samples; (3) if there are quality differences, screening out key factors causing greater differences between the QCBP test samples from the common peaks of the calibrated QCBP test solution; and (4) determining the contents of each component causing the quality differences of the samples. The application is based on the component analysis of HPLC-Q-Exactive-MS technology, the establishment of HPLC fingerprint and the quantitative evaluation of HPLC-MS difference components, and provides a reference for the quality evaluation system of commercially available QCBP.

Description

Technical Field

[0001] This invention relates to the field of quality evaluation technology for traditional Chinese medicine preparations, specifically to a quality evaluation method for Qingxin Chenxiang Bawei Pills. Background Technology

[0002] Qingxin Chenxiang Bawei Pills (QCBP), originally recorded in the Mongolian Medicine Volume of the national standard issued by the Ministry of Health of my country, consists of eight herbs: agarwood, jujube, sandalwood, purple sandalwood, safflower, nutmeg, bamboo shavings, and north ginseng. It has the effects of clearing the heart and lungs, regulating qi, and calming the mind. It is mainly used to treat excessive heat in the heart and lungs, chest tightness, and chest and rib pain. In QCBP, agarwood, as the main ingredient, has the effects of regulating qi and relieving pain, warming the middle jiao and stopping vomiting; safflower invigorates blood circulation, promotes menstruation, disperses blood stasis and relieves pain; jujube regulates qi, invigorates blood, nourishes the heart, and calms the mind; nutmeg warms the middle jiao, regulates qi, astringes the intestines and stops diarrhea; sandalwood regulates qi, warms the middle jiao, invigorates the stomach and relieves pain; bamboo shavings clear heat and resolve phlegm, calm the mind and relieve convulsions, and open the orifices and awaken the mind; purple sandalwood relieves pain, detoxifies and reduces swelling, stops bleeding and promotes tissue regeneration; north ginseng nourishes yin, clears the lungs, benefits the stomach and promotes body fluid production. Modern research shows that agarwood has various pharmacological effects, including sedative-hypnotic, analgesic, anti-inflammatory, and antibacterial effects. When used together, these eight herbs work synergistically to clear the heart and lungs, regulate qi, and calm the mind.

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

[0004] The purpose of this invention is to provide a quality evaluation method for Qingxin Chenxiang Bawei Pills.

[0005] This invention uses HPLC-Q-Exactive-MS technology combined with literature data to identify and analyze the chemical components of commercially available QCBPs, establishing fingerprint chromatograms for 13 batches of commercially available QCBPs. A comprehensive quality evaluation of the QCBPs is conducted through cluster analysis (CA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and analysis of variance. Quantitative analysis of four differentially expressed components is also performed, providing a basis and reference for the quality control of commercially available QCBPs.

[0006] The purpose of this invention is implemented by the following technical solution: a quality evaluation method for Qingxin Chenxiang Bawei Pills, which includes the following steps: (1) establishing fingerprint spectra of each QCBP test sample solution and identifying common peaks; (2) conducting multi-dimensional quality difference evaluation and analysis on several QCBP test samples; (3) if quality differences exist, screening out the key factors that cause large differences between QCBP test samples from the identified common peaks of each QCBP test sample solution; (4) determining the content of each component that causes sample quality differences; wherein,

[0007] (1) Establish fingerprint chromatograms of each test sample solution and identify common peaks: Take several QCBP samples, prepare QCBP test sample solutions respectively, perform chromatographic analysis, record chromatograms, convert the collected chromatograms into CDF format files and import them into the Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system, select the chromatogram of any QCBP test sample solution as the reference chromatogram for fingerprint analysis, after multi-point correction and marker peak matching, obtain the chromatographic overlay chromatogram and reference fingerprint chromatogram of each QCBP test sample solution, and identify several common peaks; after comparison with the chromatograms of each single reference standard stock solution, multiple chromatographic peaks are identified;

[0008] (2) Multidimensional quality difference evaluation and analysis of several QCBP test samples: fingerprint spectrum similarity evaluation of the fingerprint spectrum established in step (1); cluster analysis with the peak area of ​​several common peaks as indicators; principal component analysis with the peak area of ​​several common peaks as variables; orthogonal partial least squares discriminant analysis and variance analysis of the differences in the area of ​​several common peaks; determine whether there are quality differences among several QCBP test samples based on the above analysis results;

[0009] (3) If quality differences exist, the key factors causing significant differences among the QCBP test samples are screened from the common peaks of each QCBP test sample solution: using the variable weight value evaluation method. [25-26] Analysis of each QCBP test sample revealed that when the VIP value was greater than 1, it indicated that the component was the key factor causing the large differences among the QCBP test samples.

[0010] (4) Determine the content of each component that causes the difference in sample quality: Weigh the QCBP test solution separately, use 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.

[0011] Specifically, in step (1), 13 batches of commercially available QCBP samples from 3 manufacturers were taken; 20 common peaks were calibrated, of which 6 chromatographic peaks were identified as follows: peak 1 is gallic acid, peak 3 is protocatechuic acid, peak 5 is hydroxysaffron yellow A, peak 6 is linalool, peak 7 is ferulic acid, and peak 10 is ellagic acid; the single reference standard stock solutions are ferulic acid stock solution, linalool stock solution, gallic acid stock solution, hydroxysaffron yellow A stock solution, protocatechuic acid stock solution, quinic acid stock solution, kaempferol stock solution, costone lactone stock solution, and rutin stock solution;

[0012] In step (2), there were quality differences among 13 batches of commercially available QCBP samples from 3 manufacturers.

[0013] In step (3), there are 14 peaks with VIP values ​​> 1, arranged from largest to smallest: peak 2, peak 16, peak 15, peak 17, peak 11, ferulic acid, linalool, peak 20, peak 18, gallic acid, peak 19, hydroxysaffron 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.

[0014] In step (4), the contents of ferulic acid, linalool, gallic acid and hydroxysaffron yellow A, which are the key factors that cause differences in sample quality, are determined to determine the differences in the contents of the four components in the test solution.

[0015] Specifically, the preparation method of the QCBP test solution is as follows: weigh the QCBP sample, grind it into powder, sieve it, add a 70%-80% methanol solution to dissolve it, seal it and sonicate it for 30-50 minutes with an ultrasonic power of 60W-80W and an ultrasonic frequency of 50kHz-90kHz. After cooling to room temperature, add a 70%-80% methanol solution to make up the weight, shake well, and filter to obtain the QCBP test solution.

[0016] Specifically, the mass-to-volume ratio of the sieved commercially available QCBP sample powder to a 70%-80% methanol solution is 0.1g:1mL to 0.2g:1mL.

[0017] Specifically, in 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℃, the injection volume is 10 μL, and the detection wavelength is 254 nm; the gradient elution program is 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.

[0018] Specifically, in step (4), the HPLC-MS quantitative analysis method uses the following column conditions: mobile phase is methanol (A)-water (containing 0.1% formic acid) (B), gradient elution program: 0.01~0.5 min, 15%~20%A; 0.5~1.0 min, 20%~35%A; 1~3 min, 35%~60%A; 3.0~5.0 min, 65%~75%A; 5.0~5.5 min, 75%~95%A; 5.5~7.0 min, 95%A; 7.0~7.01 min, 95%~10%A; 7.01~9.5 min, 10%A; flow rate 0.25 mL / min; column temperature 35℃; injection volume 3 μL.

[0019] Specifically, in step (4), the HPLC-MS quantitative analysis method and the mass spectrometry quantitative analysis conditions are as follows: electrospray ionization (ESI) source; positive and negative ion scanning; multiple reaction monitoring (MRM) mode; spray voltage 3.80kV (+) and 3.50kV (-); nebulizer gas flow rate 3L / min; heating gas flow rate 10L / min; interface temperature 300℃; desolventizing temperature 526℃; heating block temperature 400℃.

[0020] Advantages of this invention:

[0021] (1) This invention provides a reference for the quality evaluation system of commercially available QCBP based on the combination of HPLC-Q-Exactive-MS technology for component analysis, HPLC fingerprinting, and HPLC-MS differential component quantitative evaluation.

[0022] (2) The present invention investigated the characterization effect of samples with different ultrasonic extraction times, different flow rates 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, mass spectrometry abundance and peak shape of each component were better than those under other conditions.

[0023] (3) To ensure stable and feasible detection conditions and comprehensive HPLC fingerprint characterization information, single-factor experiments were conducted on the mobile phase, chromatographic column, and detection wavelength based on the HPLC-Q-Exactive-MS conditions. The experiment found that using methanol and 0.2% phosphoric acid water as the mobile phase and an Agilent Eclipse XDB-C column were optimal. 18 The fingerprint spectrum (4.6mm×250mm, 5μm) with a detection wavelength of 254nm can make each chromatographic peak reach the most ideal peak shape and response value, and can more intuitively reflect the information of multi-component chromatographic peaks. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The total ion chromatogram of QCBP(S1) HPLC-Q-Exactive-MS is shown.

[0026] Figure 2 MS of compound 5 2 Figure and possible cleavage pathways.

[0027] Figure 3 MS of compound 25 2 Figure and possible cleavage pathways.

[0028] Figure 4 MS of compound 39 2 Figure and possible cleavage pathways.

[0029] Figure 5 MS of compound 20 2 Figure and possible cleavage pathways.

[0030] Figure 6 MS of compound 27 2 Figure and possible cleavage pathways.

[0031] Figure 7 MS of compound 10 2 Figure and possible cleavage pathways.

[0032] Figure 8 MS of compound 1 2 Figure and possible cleavage pathways.

[0033] Figure 9 The image shows an overlay of fingerprint spectra from 13 batches of commercially available QCBP (A) and a control fingerprint spectra (B).

[0034] Figure 10 The chromatograms are comparisons of a single reference solution and a QCBP (S1) sample solution.

[0035] Figure 11 Cluster heatmap analysis of 13 batches of commercially available QCBP.

[0036] Figure 12 PCA score chart for 13 batches of commercially available QCBP samples.

[0037] Figure 13The chart shows the scores for 13 batches of commercially available QCBP OPLS-DA.

[0038] Figure 14 This is a VIP chart for 20 QCBP products sold in 13 batches.

[0039] Figure 15 The figures show the secondary mass spectra of each component. In the figure, 1 is the secondary mass spectrum of ferulic acid, 2 is the secondary mass spectrum of linalool, 3 is the secondary mass spectrum of gallic acid, and 4 is the secondary mass spectrum of hydroxysafflower yellow A.

[0040] Figure 16 The figures show the MRM chromatograms of each component. In the figure, A represents the MRM chromatogram of each component of the standard, B represents the MRM chromatogram of each component of the test sample, and C represents the MRM chromatogram of each component of the blank solvent. 1 represents ferulic acid, 2 represents linalool, 3 represents gallic acid, and 4 represents hydroxysaffron yellow A. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] The original formula for Qingxin Chenxiang Bawei Pills (QCBP) is recorded in the Mongolian Medicine Volume of the national standard issued by the Chinese government. [1] This formula is composed of eight medicinal herbs: agarwood, jujube, sandalwood, rosewood, safflower, nutmeg, bamboo shavings, and north ginseng. It has the effects of clearing the heart and lungs, regulating qi, and calming the mind. It is mainly used to treat excessive heat in the heart and lungs, chest tightness, and chest and rib pain. In QCBP, agarwood, as the main ingredient, has the effects of regulating qi and relieving pain, warming the middle jiao and stopping vomiting; safflower invigorates blood circulation, promotes menstruation, disperses blood stasis and relieves pain; jujube regulates qi, invigorates blood, nourishes the heart, and calms the mind; nutmeg warms the middle jiao and regulates qi, astringes the intestines and stops diarrhea; sandalwood regulates qi, warms the middle jiao, invigorates the stomach and relieves pain; bamboo shavings clear heat and resolve phlegm, calm the mind and relieve convulsions, and open the orifices and awaken the mind; rosewood relieves pain, detoxifies and reduces swelling, stops bleeding and promotes tissue regeneration; north ginseng nourishes yin and clears the lungs, benefits the stomach and promotes body fluid production. Modern research shows that agarwood has sedative and hypnotic effects. [2-3] Analgesic effect [4] Anti-inflammatory and antibacterial [5] These herbs have various pharmacological effects. When used together, these eight herbs work synergistically to clear the heart and lungs, regulate qi, and calm the mind.

[0043] There are currently no literature reports on the qualitative analysis of components and comprehensive quality evaluation of QCBP; only the content determination of individual characteristic components is available. [6-7]This study used HPLC-Q-Exactive-MS technology combined with literature data to identify and analyze the chemical components of commercially available QCBPs, established fingerprint chromatograms for 13 batches of commercially available QCBPs, and conducted a comprehensive quality evaluation of QCBPs through cluster analysis (CA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and analysis of variance. Quantitative analysis was performed on four differentially expressed components, providing a basis and reference for the quality control of commercially available QCBPs.

[0044] 1. Materials and Instruments

[0045] 1.1 Instruments

[0046] LC-2030C high-performance liquid chromatograph and its equipped LC-40BXR infusion pump, SIL-40C autosampler, CTO-40C column oven, CBM-20A system controller, and SPD-40V detector (Shimadzu Corporation, Japan); HPLC-Q-Exactive high-performance liquid chromatography-mass spectrometry system (Thermo Fisher Scientific); LC-MS 8045 high-performance liquid chromatography-triple quadrupole mass spectrometry system (Shimadzu Corporation, Japan); AP135 W 0.0001 g electronic balance (Shimadzu Corporation, Japan); BSA224S 0.0001 g balance (Sartorius Scientific Instruments Co., Ltd.); DS-7510DTH CNC ultrasonic cleaner (Shanghai Shengxi Ultrasonic Instruments Co., Ltd.).

[0047] 1.2 Test Drugs

[0048] We collected QCBPs from three manufacturers, totaling 13 batches: Manufacturer A (S1~S6, batch numbers 2211010, 2303024, 2112010, 2210003, 2304007, 2306053), Manufacturer B (S7~S9, batch numbers 220814, 220706, 221101), and Manufacturer C (S10~S13, batch numbers 230506, 210739, 230709, 210930), all with a specification of 1 g / 8 capsules.

[0049] Costus lactone (batch number: 111524-201911, purity ≥99.9%), quinic acid (batch number: 111717-201402, purity ≥98.5%), protocatechuic acid (batch number: 110809-202207, purity ≥98.5%), ferulic acid (batch number: 110773-202316, purity ≥99.3%), kaempferol (batch number: 110861-202214, purity ≥99.0%), linalool (batch number: 111980-201904, purity ≥98.6%), gallic acid (batch number: 110831-202407, purity ≥98.3%). Reference standards for rutin (batch number: 100080-202012, for identification purposes) and hydroxysaffron yellow A (batch number: 111637-202111, purity ≥98.8%) were all purchased from the National Institutes for Food and Drug Control; methanol, acetonitrile, and formic acid were all chromatographically pure; and water was Watson's distilled water.

[0050] 2. Methods and Results

[0051] 2.1 Preparation of standard solutions

[0052] Accurately weigh appropriate amounts of nine reference standards: ferulic acid, linalool, gallic acid, hydroxysaffron yellow A, protocatechuic acid, quinic acid, kaempferol, costone lactone, and rutin. Place them in different 10 mL volumetric flasks, dissolve and dilute to volume with methanol to prepare single reference standard stock solutions with a mass concentration of 551.00 μg·mL⁻¹. -1 366.00 μg·mL -1 159.00 μg·mL -1 447.00 μg·mL -1 481.00 μg·mL -1 521.00 μg·mL -1 339.00 μg·mL -1 699.00 μg·mL -1 392.00 μg·mL -1 Each of the above-mentioned mother liquors was precisely measured and placed in the same 10 mL volumetric flask, then diluted with methanol to obtain a mass concentration of 551.00 ng·mL⁻¹. -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 mixed standard solutions were stored at 4°C for later use.

[0053] 2.2 Preparation of test solution

[0054] Take 13 batches of commercially available QCBP, grind them into powder, pass them through a No. 4 sieve, weigh 1.000 g into 50 mL conical flasks, add 10 mL of 70% methanol solution, seal, sonicate for 50 min (power 80 W, frequency 50 kHz), let cool to room temperature, add 70% methanol solution to make up the weight, shake well, and filter through a 0.22 μm filter membrane to obtain the final product.

[0055] 2.3 Qualitative analysis by HPLC-Q-Exactive-MS

[0056] 2.3.1 High Performance Liquid Chromatography Conditions

[0057] The chromatographic column was an 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). Gradient elution was used: 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. The flow rate was 0.35 mL / min. -1 The column temperature was 35 ℃ and the injection volume was 10 μL.

[0058] 2.3.2 Mass Spectrometry Conditions

[0059] Using an electrospray ionization (ESI) source, in ESI + ESI - Detection was performed in ion detection mode using 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 transport tube temperature: 300 ℃ (+) and 400 ℃ (-), auxiliary gas temperature: 320 ℃, auxiliary gas volumetric flow rate: 30 L·min -1 .

[0060] 2.4 HPLC fingerprint analysis conditions

[0061] The chromatographic column was an Agilent Eclipse XDB-C. 18 (4.6 mm × 250 mm, 5 μm), mobile phase: methanol (A) - 0.2% phosphoric acid aqueous solution (B), flow rate: 1.0 mL / min, column temperature: 35 ℃, injection volume: 10 μL, detection wavelength: 254 nm; gradient elution program: 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. % A; 80~90 min, 15%~15% A.

[0062] 2.5 HPLC-MS Quantitative Analysis Conditions

[0063] 2.5.1 Chromatographic column

[0064] The column used was a 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; 1–3 min, 35%–60% A; 3.0–5.0 min, 65%–75% A; 5.0–5.5 min, 75%–95% A; 5.5–7.0 min, 95% A; 7.0–7.01 min, 95%–10% A; 7.01–9.5 min, 10% A; the flow rate was 0.25 mL / min; the column temperature was 35 ℃; and the injection volume was 3 μL.

[0065] 2.5.2 Mass Spectrometry

[0066] Electrospray ionization (ESI) was used; positive and negative ion scanning was employed; multiple reaction monitoring (MRM) mode was used; spray voltage was 3.80 kV (+) and 3.50 kV (-); nebulizing gas flow rate was 3 L / min; heating gas flow rate was 10 L / min; interface temperature was 300 °C; desolvation temperature was 526 °C; heating block temperature was 400 °C. Mass spectrometry parameters for each component are shown in Table 3, and secondary mass spectra for each component are shown in [Table 3]. Figure 15 The MRM chromatograms of each component are shown below. Figure 16 .

[0067] 2.6 Component Analysis

[0068] 2.6.1 Data Processing and Analysis

[0069] Literature on the chemical composition and mass spectrometry fragment information of agarwood, jujube, sandalwood, rosewood, safflower, nutmeg, bamboo shavings, and north ginseng in QCBP was searched through CNKI and PubMed data platforms. A database including compound names, molecular formulas, and multi-stage mass spectrometry fragments was created. Total ion current data of commercially available QCBP (S1 batch number 2211010) solution were obtained in positive and negative ion modes. Peak extraction and precise relative molecular mass fitting were performed using Xcalibur 3.0 software. By comparing with the database, the selection error (δ) was set to within ±10 ppm (1 ppm = 1 × 10⁻¹⁰ ppm). -6 The compounds within the range of ) were initially identified in commercially available QCBPs, and 9 of them were further confirmed by comparison with reference standards.

[0070] 2.6.2 QCBP Chemical Composition Identification Results

[0071] Following the procedures in section "2.2" (diluting the test solution 100-fold) and section "2.1" (preparing the mixed standard solution), perform injection analysis under the conditions described in section "2.3" to obtain the total ion chromatograms of the QCBP sample solution in both positive and negative ion modes. Figure 1 As shown, the sample data obtained were analyzed according to the method in section “2.6.1”. The results showed that 70 chemical components were identified in commercially available QCBP, including 7 fatty acids, 8 phenolic acids, 9 sesquiterpenes, 17 phenylpropanoids (4 simple phenylpropanoids, 5 coumarins, and 8 lignans), 8 chromones, 14 flavonoids, and 7 other components (3 other components and 4 amino acids). The results are shown in Table 1.

[0072] Table 1: HPLC-Q-Exactive-MS analysis results of chemical components in QCBP (S1)

[0073]

[0074] Note: *） Compounds verified by reference standards

[0075] 2.6.3 Fatty acids

[0076] Seven fatty acid compounds were identified in QCBP (S1). These compounds possess a free -COOH quasi-molecular ion peak that readily loses a H ion, allowing them to be detected in negative ion mode. Compound 5 (quinic acid) is used as an example: Compound 5 is visible as [MH] in negative ion mode. - m / z The quasi-molecular ion peak is at 191.0561, with a deviation of 5.786 × 10⁻⁶. -6 Xcalibur 3.0 software fitted its molecular formula as C7H 12 O6, the main fragment ions are m / z 173.0089 [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 pyrolysis pattern 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, based on its fragmentation pattern and literature comparison. [8,13] Compound No. 5 was confirmed to be quinic acid, and its MS... ２ See diagram and possible lysis pathways. Figure 2 .

[0077] 2.6.4 Phenolic acids

[0078] Eight phenolic acid compounds were identified in QCBP (S1). These compounds contain substituents such as phenolic hydroxyl, hydroxyl, and carboxyl groups. In negative ion mode, they readily lose H₂O, COOH, and CH₃, thus generating corresponding characteristic ionic fragments. Compound 25 (ferulic acid) is used as an example: Compound 25 shows [MH] in negative ion mode. - m / z The quasi-molecular ion peak is at 193.0507, with a deviation of 6.137 × 10⁻⁶. -6 Xcalibur 3.0 software fitted its molecular formula as C 10 H10 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 pyrolysis pattern 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 fragmentation pattern and in conjunction with literature comparison. [15,16] Compound 25 was confirmed to be ferulic acid, and its MS analysis showed... ２ See diagram and possible lysis pathways. Figure 3 .

[0079] 2.6.5 Phenylpropanoids

[0080] Phenylacetin compounds can be classified into simple phenylpropanoids, coumarins, and lignans. Four simple phenylpropanoids, five coumarins, and eight lignan compounds were identified in QCBP (S1). Taking coumarins as an example: these components readily lose CO, CO2, or H2O in positive ion mode; when their parent nucleus has substituents, CO and CO2 are lost alternately. Compound 39 (psoralen) is used as an example: compound 39 shows [M+H] in positive ion mode. + m / z The quasi-molecular ion peak is at 187.0385 Hz, with a deviation of -2.302 × 10⁻⁶. -6 Xcalibur 3.0 software fitted its molecular formula 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], meaning the cleavage pattern 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, based on its fragmentation pattern and literature comparison. [11,17] Compound number 39 was confirmed to be psoralen, and its MS... ２ See diagram and possible lysis pathways. Figure 4 .

[0081] 2.6.6 Flavonoids

[0082] Fourteen flavonoid compounds were identified in QCBP (S1), representing the largest number of compounds identified in this qualitative analysis. The mass of fragments produced after energy collisions of flavonoid molecules is highly dependent on the substituents on the A and B rings. The abundance of fragments such as H₂O and CO lost from the flavonoid ion peaks is also primarily influenced by the substituents on the A and B rings. Compound 20 (hydroxysaffron yellow A) is used as an example: Compound 20 is visible in negative ion mode [MH]. - m / z The quasi-molecular ion peak is at 611.1660, with a deviation of 6.958 × 10⁻⁶. -6 Xcalibur 3.0 software fitted its molecular formula 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 pyrolysis pattern 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 fragmentation pattern and in conjunction with literature comparison [12,18] Compound No. 20 was confirmed to be hydroxysaffron yellow A, and its MS... ２ See diagram and possible lysis pathways. Figure 5 .

[0083] 2.6.7 Chromones

[0084] Eight chromone compounds were identified in QCBP (S1). After energy collisions, chromone compounds often first lose one or more water molecules or decarbonyl groups, followed by the breaking of chemical bonds between chromone units, and further fragmentation reactions. Compound 27 (linalool) is used as an example: Compound 27 is visible in positive ion mode as [M+H]. + m / z The quasi-molecular ion peak is at 319.1150, with a deviation of -8.225 × 10⁻⁶. -6 Xcalibur 3.0 software fitted its molecular formula 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 pattern 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 fragmentation pattern and in conjunction with literature comparison [9,19] Compound No. 27 was confirmed to be linalool, and its MS... ２ See diagram and possible lysis pathways. Figure 6 .

[0085] 2.6.8 Sesquiterpenes

[0086] Nine sesquiterpenoid compounds were identified in QCBP (S1). These compounds have a skeleton consisting of three isoprene units containing 15 carbon atoms. Compound number 10 (dehydroauscin) is used as an example: Compound number 10 is visible in positive ion mode as [M+H]. + m / z The quasi-molecular ion peak is at 231.1374, with a deviation of -2.537 × 10⁻⁶. -6 Xcalibur 3.0 software fitted its molecular formula as C 15 H 18 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 pattern 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, based on its fragmentation pattern and literature comparison. [9,20] Compound 10 was confirmed to be dehydroauracetin lactone, and its MS... ２ See diagram and possible lysis pathways. Figure 7 .

[0087] 2.6.9 Other Categories

[0088] Seven other compounds were identified in QCBP (S1), including four amino acids and three others. Compound 1 (5-hydroxymethylfurfural) is used as an example: Compound 1 is visible in positive ion mode [M+H]. + m / z The quasi-molecular ion peak is at 127.0388, with a deviation of -1.107 × 10⁻⁶. -6 Xcalibur 3.0 software fitted its molecular formula to C6H6O3, and the main fragment ions are: m / z 109.0283[M+H-OH] + , m / z 81.0338[M+H-OH-CO] + That is, the fragmentation pattern of this compound in positive ion mode may be... m / z127.0388→ m / z 109.0283→ m / z 81.0338, through its fragmentation pattern and in conjunction with literature comparison, [8,21] Compound No. 1 was confirmed to be 5-hydroxymethylfurfural, and its MS... ２ See diagram and possible lysis pathways. Figure 8 .

[0089] 2.7 Fingerprint spectrum study

[0090] 2.7.1 Precision Test

[0091] Take QCBP sample S1, prepare the test solution according to the method in section "2.2", inject it continuously for 6 injections under the chromatographic conditions in section "2.4", record the chromatogram, and take peak No. 3 (protocatechuic acid) as the reference peak. 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 instrument has good precision.

[0092] 2.7.2 Stability Test

[0093] Take QCBP sample S1 and prepare the test solution according to the method in section "2.2". Inject and determine the sample under the chromatographic conditions in section "2.4" at 0, 2, 4, 8, 12 and 24 h. Using peak No. 3 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.

[0094] 2.7.3 Repeatability Test

[0095] Six QCBP samples of S1 were taken, and test solutions were prepared according to the method in section "2.2". The samples were then injected and analyzed according to the chromatographic conditions in section "2.4". Peak 3 (gallic acid) was used as the reference peak. The results showed that the relative retention time RSD of the 20 common peaks in the sample was less than 0.10%, and the relative peak area RSD was less than 1.72%, indicating that the method has good repeatability.

[0096] 2.7.4 Establishment of fingerprint profile

[0097] Thirteen batches of commercially available QCBP from three manufacturers were collected. Test solutions for each batch were prepared according to the method described in section "2.2". The solutions were injected and analyzed under the chromatographic conditions described in section "2.4". Chromatograms were recorded, and the collected chromatograms were converted to CDF format files and imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version). Sample S1 was set as the reference chromatogram for fingerprint analysis (median method, time window set to 0.1 min). After multi-point correction and marker peak matching, the QCBP chromatographic overlay and reference fingerprint chromatograms were obtained. Figure 9 As shown, the results identified a total of 20 peaks. After comparison with the chromatograms of the individual reference stock solutions prepared in section 2.1, as follows... Figure 10 As shown, six chromatographic peaks were identified as gallic acid (peak 1), protocatechuic acid (peak 3), hydroxysaffron yellow A (peak 5), linalool (peak 6), ferulic acid (peak 7), and ellagic acid (peak 10).

[0098] 2.7.5 Similarity Analysis

[0099] Data from 13 batches of commercially available QCBP were imported into the Traditional Chinese Medicine Fingerprint Similarity Evaluation System (2012 version) for fingerprint similarity evaluation. The similarity results between the fingerprint spectra of the 13 batches of commercially available QCBP and the generated control spectra are shown in Table 2. The similarity of the 13 batches of commercially available QCBP ranged from 0.769 to 0.960, indicating that there were certain differences in the quality of the 13 batches of QCBP from the three manufacturers. Using peak 3 (protocatechuic acid) as the reference peak, the relative peak area RSD of the 20 common peaks of the 13 batches of QCBP was 28.0% to 96.0%, indicating that there were significant differences in the content of certain components among different batches of QCBP.

[0100] Table 2: Similarity Evaluation Results of 13 Batches of Commercially Available QCBPs

[0101]

[0102] 2.8 Chemical Pattern Recognition Analysis

[0103] 2.8.1 Cluster Analysis (CA)

[0104] Using the MicroBioinformatics online platform, the peak areas of 20 common peaks from 13 batches of commercially available QCBPs were used as indicators. The clustering method employed was the Complete clustering method, and the distance metric was set to Euclidean.

[22] The result is as follows Figure 11 As shown, the 13 batches of QCBP can be clustered into two categories: S1~S6 and S10~S13 are clustered into one category (manufacturers A and C), and S7~S9 are clustered into another category (manufacturer B), which is consistent with the similarity results.

[0105] 2.8.2 Principal Component Analysis (PCA)

[0106] Principal component analysis was performed using the peak areas of 20 common peaks from 13 batches of QCBP as variables, imported into SIMCA 14.1 software. The results are as follows: Figure 12 Manufacturers A and C are clearly separate from manufacturer B, and the result is consistent with the result of CA.

[0107] 2.8.3 Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0108] The differences in the areas of 20 common peaks in 13 batches of QCBP samples were analyzed using supervised OPLS-DA analysis with SIMCA 14.1 software. The cumulative explanatory power parameters of the constructed supervised model ( R 2 X , R 2 Y The predictive power parameters were 0.783 and 0.903, respectively. Q 2 The cum value 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, see the score chart. Figure 13 The samples from the three manufacturers clustered into one category, and the classification results were more obvious, further illustrating that there are quality differences in QCBPs produced by different manufacturers.

[0109] To identify components that cause significant inter-batch variability, the variable importance in projection (VIP) method was used to analyze 13 batches of samples. A VIP value > 1 indicates that the component is a key factor in inter-group classification. [25-26] .Depend on Figure 14 It can be seen that there are 14 peaks with a VIP value > 1, arranged from largest to smallest: 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), linalool (VIP=1.064), peak 20 (VIP=1.063), peak 18 (VIP=1.053), gallic acid (VIP=1.044), peak 19 (VIP=1.039), hydroxysaffron yellow A (VIP=1.033), peak 12 (VIP=1.030), and peak 9 (VIP=1.012). These 14 chromatographic peaks are the key factors causing the quality differences among the 13 batches of samples.

[0110] 2.9 Content Determination

[0111] 2.9.1 Examination of Linear Relationships

[0112] Accurately measure the following stock solutions prepared in section 2.1: 2 μL ferulic acid, 500 μL linalool, 500 μL gallic acid, and 700 μL hydroxysaffron yellow A. Place them in the same 10 mL volumetric flask and dilute with methanol to obtain mass concentrations of 11.02, 1830.00, 759.00, and 3129.00 ng·mL, respectively. -1 The mixed standard solution was prepared by precisely pipetting 100 μL, 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μL of the mixed standard solution sequentially into 1 mL volumetric flasks, diluting to the mark with methanol, and then injecting and measuring under the conditions described in section "2.3". Regression analysis was performed with the injection volume of the mixed standard solution as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 4, indicating that each component has a good linear relationship within its respective range.

[0113] Table 3: Mass Spectrometry Parameters of Each Component

[0114]

[0115] Table 4: Results of the linear relationship examination of the four components

[0116]

[0117] 2.9.2 Precision Test

[0118] Accurately measure the reference stock solution prepared in 2.1: ferulic acid, linalool, gallic acid, and hydroxysaffron yellow A, and prepare a mixed standard solution. The mass concentrations of ferulic acid, linalool, gallic acid, and hydroxysaffron yellow A in the mixed standard solution are 6.612, 183.000, 159.000, and 1251.600 ng·mL, respectively. -1 Under the conditions specified in section "2.3", the measurements were performed 6 times, and the RSDs of the peak areas of ferulic acid, linalool, gallic acid, and hydroxysaffron yellow pigment A were 4.24%, 4.26%, 0.63%, and 1.49%, respectively, indicating that the instrument has good precision.

[0119] 2.9.3 Repeatability Test

[0120] Six QCBP samples of S7 were taken, and test solutions were prepared according to the method in section "2.2". The samples were then injected and analyzed under the conditions in section "2.3". The RSDs of the peak areas of ferulic acid, linalool, gallic acid and hydroxysaffron yellow A were 4.46%, 1.63%, 1.61% and 4.06%, respectively, indicating that the method has good repeatability.

[0121] 2.9.4 Stability Test

[0122] Take QCBP sample S7 and prepare the test solution according to section "2.2". Under the conditions in section "2.3", the sample was injected for analysis at 0, 2, 4, 8, 12 and 24 h. The RSDs of the peak areas of ferulic acid, linalool, gallic acid and hydroxysaffron yellow A were 0.76%, 1.86%, 1.11% and 1.22%, respectively, indicating that the sample solution has good stability within 24 h.

[0123] 2.9.5 Recovery test

[0124] Accurately weigh 6 portions of QCBP (S7) with known component content, each approximately 0.500 g. Add reference standards to each portion at a ratio of approximately 100% of the component content in the sample. Prepare test solutions according to section “2.2”. Inject and determine the solutions under the conditions in section “2.3”. Calculate the average recovery rate and RSD of the four components. The results are shown in Table 5.

[0125] Table 5: Average recovery rate and RSD of the four components

[0126]

[0127] 2.9.6 Determination of Sample Content

[0128] Thirteen batches of samples were taken, and test solutions were prepared according to the method in section “2.2”. The samples were injected and determined under the conditions in section “2.3”, and the content was calculated. The results are shown in Table 6.

[0129] Table 6: Results of content determination of four components in 13 batches of QCBP samples (ng / g, n=3)

[0130]

[0131] 3. Discussion

[0132] High-performance liquid chromatography-quadrupole / electrostatic field orbitt trap high-resolution mass spectrometry (HPLC-Q-Exactive-MS) features high resolution (greater than 10,000 FWHM), high sensitivity, and high selectivity. [27-28]This invention investigated the characterization effects of different ultrasonic extraction times, different mobile phases, and different concentrations of samples. The results showed that a 40-minute ultrasonic extraction time, a gradient elution using methanol and 0.1% formic acid as the mobile phase, and a 1000-fold dilution of the sample all demonstrated superior separation, mass spectrometry abundance, and peak shape compared to other conditions. To ensure stable and feasible detection conditions and comprehensive HPLC fingerprint characterization information, single-factor analyses of the mobile phase, column, and detection wavelength were conducted based on HPLC-Q-Exactive-MS conditions. The experiments revealed that using methanol and 0.2% phosphoric acid as the mobile phase and an Agilent Eclipse XDB-C column yielded the best results. 18 The chromatogram (4.6 mm × 250 mm, 5 μm) with a detection wavelength of 254 nm can make each chromatographic peak reach the most ideal peak shape and response value, and can more intuitively reflect the information of multi-component chromatographic peaks.

[0133] The principal ingredient in QCBP is agarwood, among which agarotetraol is listed as an indicator component of agarwood in the 2020 edition of the Chinese Pharmacopoeia.

[29] Agaric tetraol is a chromone compound, and chromone compounds have antioxidant properties.

[30] Improve the body's immunity

[31] and inflammation regulation

[32] Based on HPLC-Q-Exactive-MS chemical composition analysis, eight chromone components were found in QCBP, providing a material basis for subsequent research. Through HPLC fingerprint similarity evaluation combined with CA, PCA, and OPLS-DA analysis, 13 batches of commercially available QCBP were clustered into two main categories (S1-S6 and S10-S13 clustered together (manufacturers A and C), and S7-S9 clustered together (manufacturer B)). The clustering within manufacturer A's S1-S6 was relatively dispersed, indicating significant intra-group differences. These intra-group differences may be related to factors such as the origin, processing, and harvesting of the medicinal materials. Using a variable weighting evaluation method, the 13 batches of samples were analyzed, revealing 14 common peaks with VIP values ​​>1. The contents of ferulic acid, linalool, gallic acid, and hydroxysaffron yellow A were determined by HPLC-MS, showing significant differences in the content of these four components.

[0134] In summary, this invention, based on HPLC-Q-Exactive-MS technology for component analysis combined with HPLC fingerprinting and HPLC-MS quantitative evaluation of differential components, provides a reference for the quality evaluation system of commercially available QCBPs.

[0135] 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 principles of the present invention should be included within the protection scope of the present invention.

[0136] References

[0137] [1] Drug Administration Bureau of the Ministry of Health of the People's Republic of China, Drug Standards of the Ministry of Health of the People's Republic of China: Mongolian Medicine Volume [S]. 1998: 163.

[0138] [2] Zhang Chenchen, Zhang Dongxu, Zhang Yufeng, et al. Study on quality markers of Mongolian medicine Nuangong Qiwei Pill based on "chemical composition-blood transmissible components-pharmacokinetics" [J]. Chinese Traditional and Herbal Drugs, 2023, 54(18): 5855-5866.

[0139] [3] Liu Jin, Lu Hanjin, Yang Shengtao, et al. Research progress on chemical composition of agarwood essential oil and its application in neuroprotection [J / OL]. Journal of Tropical Crops, 1-17 [2024-03-06].

[0140] [4] Lai Yinfang, Ding Zongmiao, Hua Liping, et al. Progress and prospects of agarwood health products development [J]. Chinese Traditional and Herbal Drugs, 2023, 54(04): 1342-1352.

[0141] [5] Liu Xinyi, Wang Yali, Wang Hao, et al. GC-MS analysis of volatile oil components in leaves of four kinds of Aquilaria sinensis [J]. Journal of Tropical Crops, 2022, 43(01): 196-206.

[0142] [6] Na Shunbai Yila, Meng He. Determination of the content of agaric tetraol in Qingxin Chenxiang Bami San by HPLC [J]. Chinese Journal of Traditional and Folk Medicine, 2017, 26(12): 11-13.

[0143] [7] Chen Xiuqin, Lin Yan, Kang Shuanglong, et al. Determination of gallic acid content in Mongolian traditional Chinese medicine Bawei Qingxin Chenxiang San by reversed-phase high performance liquid chromatography [J]. Chinese Journal of Traditional Medicine, 2003, (04): 43-44.

[0144] [8] Yang Lumeng, Yang Lingjian, Jia Pu, et al. Analysis of chemical components of jujube pulp based on HPLC-Q-TOF-MS / MS [J]. Journal of the Second Military Medical University, 2016, 37(02): 159-166.

[0145] [9] Pan Xuanxuan, Song Fenyun, Li Hua. Chemical composition analysis of Chenxiang Huaqi Pill based on UPLC-Q-TOF / MS technology [J]. Chinese Traditional and Herbal Drugs, 2018, 49(13): 2984-2992.

[0146]

[10] Liang Fangyuan, Bu Ren, Yang Li, et al. Study on the role of jujube in Sanwei Tanxiang San based on spectrum-effect correlation analysis [J]. Modern Chinese Materia Medica, 2023, 25(06): 1218-1226.

[0147]

[11] Wang, Shuang et al. "A pre-classification strategy based on UPLC-Triple-TOF / MS for metabolic screening and identification of Radix glehniae inrats." Analytical and bioanalytical chemistryvol. 408, 26 (2016): 7423-36.

[0148]

[12] Liu Li, Duan Jin'ao, Tang Yuping. UHPLC-Q-TOF-MS analysis of chemical components of Angelica sinensis-Carthamus tinctorius and study on synergistic effect of the combination [J]. Chinese Journal of Traditional Chinese Medicine, 2017, 32(03): 996-1000.

[0149]

[13] Wang Yongli, Huang Guangjian, Liu Congjin, et al. Analysis of chemical components and rat tissue distribution of Huanglian Jiedu Decoction by UHPLC-Q-Exactive Orbitrap HRMS [J]. Chinese Traditional and Herbal Drugs, 2022, 53(22): 6985-7000.

[0150]

[14] Zeng Yujiao, Luo Yuting, Zhang Kun, et al. Study on chemical composition analysis of Tibetan medicine eight-flavor berberine capsule based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry [J]. Journal of Chengdu University of Traditional Chinese Medicine, 2023, 46(01): 32-40.

[0151]

[15] Zhao Mengyi, Bi Chenghao, Li Mengjiao, et al. Rapid analysis of components of Longsheng leech capsules based on UPLC-Q-TOF / MS technology [J / OL]. World Science and Technology - Modernization of Traditional Chinese Medicine: 1-20 [2024-06-28].

[0152]

[16] Wang Meng, Tian Wei, Zhen Yaqin, et al. Chemical composition analysis of Yi ethnic medicine ginger herb based on UPLC-Q-TOF-MS / MS technology [J]. Chinese Pharmaceutical Journal, 2022, 57(20): 1717-1725.

[0153]

[17] Li Jia, Zuo Lihua, Pei Jianying, et al. Identification of psoralen metabolites and analysis of metabolic pathways in rats based on UHPLC-Q-Orbitrap HRMS technology [J]. Journal of Shenyang Pharmaceutical University, 2021, 38(10): 1024-1032.

[0154]

[18] Cheng Yanfen, Du Kequn, Wu Yihan, et al. Study on the differences between compound decoction and single decoction of Taohong Siwu Decoction based on chemical characteristics and core efficacy analysis [J]. Chinese Traditional and Herbal Drugs, 2021, 52(19): 5879-5890.

[0155]

[19] Zhang Chenchen, Zhang Dongxu, Zhang Yufeng, et al. Study on quality markers of Mongolian medicine Nuangong Qiwei Pill based on "chemical composition-blood transmissible components-pharmacokinetics" [J]. Chinese Traditional and Herbal Drugs, 2023, 54(18): 5855-5866.

[0156]

[20] Yao Ziwei, Bian Lihua, Li Qiuyu, et al. Study on electrospray mass spectrometry fragmentation of three sesquiterpenoid components, namely naringinone, dehydrocostunolide and naringin H-3[J]. World Science and Technology - Modernization of Traditional Chinese Medicine, 2021, 23(07): 2353-2360.

[0157]

[21] Zhao Lulu, Sun Jiulan, Zhao Shiyang, et al. Qualitative and quantitative analysis of chemical components of rose petals based on UPLC-Q-Exactive Orbitrap-MS and UPLC-QQQ-MS [J / OL]. Modern Food Science and Technology, 1-24 [2024-07-17].

[0158]

[22] Li Jun, Zhang Qian, Wang Yuewu, et al. Quality evaluation of Mongolian medicine Naru-3-flavor pills based on HPLC-MS / MS and chemical pattern recognition analysis [J]. Chinese Journal of Hospital Pharmacy, 2024, 44(11): 1277-1284.

[0159]

[23] Zhang Shuai, Guo Xing, Li Yajing, et al. Quality evaluation of Taxus chinensis koji with different fermentation degrees based on fingerprinting and chemical pattern recognition [J]. Chinese Traditional and Herbal Drugs, 2024, 55(14): 4871-4881.

[0160]

[24] Zhang Hongjuan, Shi Yinji, Luo Yuqin, et al. Evaluation of the quality of Chelidonium majus from different producing areas based on HPLC fingerprinting and multi-index quantification combined with entropy weight TOPSIS method [J]. Chinese Traditional and Herbal Drugs, 2024, 55(12): 4194-4203.

[0161]

[25] Luo Yuqin, Wei Zheyang, Liu Geyu, et al. Evaluation of the quality of Chicorydalis yanhusuo at different growth stages based on HPLC fingerprinting combined with chemical pattern recognition and thin-layer chromatography bioautoradiography [J]. Chinese Traditional and Herbal Drugs, 2024, 55(08): 2746-2754.

[0162]

[26] Gao Tong, Zhang Xiaodan, Feng Tinghui, et al. Study on quality evaluation of Coix seed based on multi-modal recognition combined with UPLC fingerprinting and quantitative determination of multiple index components [J]. Chinese Traditional and Herbal Drugs, 2024, 55(08): 2755-2763.

[0163]

[27] Jiang Wei, Li Yin, Ma Xue, et al. Identification of chemical components of Xingpi Yang'er Granules [J]. Chinese Traditional and Herbal Drugs, 2024, 46(04): 1395-1401.

[0164]

[28] Yang Yantao, Li Zhuolun, Zhou Lin, et al. Identification of metabolites and metabolic pathway analysis of Shenkang injection in rats based on UHPLC-Q-Orbitrap HRMS technology [J]. Chinese Traditional and Herbal Drugs, 2024, 55(03): 730-745.

[0165]

[29] Chinese Pharmacopoeia [S]. Part I. 2020: 192.

[0166]

[30] LI CG, PAN L, HAN ZZ, et al. Antioxidative 2-(2-phenylethyl)chromones in Chinese eaglewood from Aquilaria sinensis. J Asian Nat Prod Res , 2019, 22(7): 639-646.

[0167]

[31] Qiu J, Yan Z, Tao K, et al. Sinomenine activates astrocyticdopamine D2 receptors and alleviates neuroinflammatory injury via the CRYAB / STAT3 pathway after ischemic stroke in mice. J Neuroinflammation , 2016, 13(1):263.

[0168]

[32] Yu Shuyan, Ma Yuheng, Wang Jianhua, et al. Research progress on the bioactivity of chromones and their derivatives [J]. Chinese Journal of Medicinal Chemistry, 2020, 30(05): 310-317.

Claims

1. A detection method of Qingxin Chenxiang Bayi Pills, characterized in that, It comprises the following steps: establishing each test solution fingerprint and calibrating common peaks: taking several QCBP samples, respectively preparing QCBP test solution, respectively performing chromatographic analysis, recording chromatogram, importing into traditional Chinese medicine chromatographic fingerprint similarity evaluation system after converting the collected chromatogram into CDF format file, selecting any QCBP test solution chromatogram as reference chromatogram for fingerprint analysis, after multi-point correction and Marker peak matching, obtaining chromatographic superposition of each QCBP test solution and control fingerprint, and calibrating 20 common peaks; after comparing with the chromatogram of each single control solution, 6 chromatographic peaks are identified, which are: No. 1 is gallic acid, No. 3 is protocatechuic acid, No. 5 is hydroxysafflor yellow A, No. 6 is linaloe, No. 7 is ferulic acid, and No. 10 is quercetin; the single control solution is ferulic acid stock solution, linaloe stock solution, gallic acid stock solution, hydroxysafflor yellow A stock solution, protocatechuic acid stock solution, quercetin stock solution, kaempferol stock solution, costunolide stock solution and rutin stock solution; The preparation method of the QCBP test solution is: respectively weighing QCBP samples, grinding into powder, sieving, dissolving in 70%-80% methanol solution, sealing, ultrasonicating for 30-50 min, ultrasonicating power is 60-80 W, ultrasonicating frequency is 50-90 kHz, cooling to room temperature, supplementing with 70%-80% methanol solution, shaking, filtering to obtain each QCBP test solution; The chromatographic analysis conditions are: mobile phase is methanol (A)-0.2% phosphoric acid aqueous solution (B), flow rate is 1.0 mL / min, column temperature is 35°C, injection amount is 10 μL, detection wavelength is 254 nm; gradient elution program: 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.

2. The method of claim 1, wherein the QXG8M is characterized by, The mass volume ratio of the sieved commercially available QCBP sample powder and 70%-80% methanol solution is 0.1 g:1 mL-0.2 g:1 mL.

3. The detection method for Qingxin Chenxiang Bawei Pill according to claim 1 or 2, characterized in that, It also includes the following steps: multi-dimensional quality difference evaluation and analysis of several QCBP test samples: fingerprint similarity evaluation of the established fingerprint; cluster analysis of several common peak areas as indicators; principal component analysis of several common peak areas as variables; orthogonal partial least squares discriminant analysis and variance analysis of the differences in several common peak areas; and determining whether there is a quality difference in several QCBP test samples through the above analysis results.

4. The method of claim 3, wherein the QXG8M is characterized by, It also includes the following steps: if there is a quality difference, screening the key factors that cause greater differences between QCBP test samples from the common peaks of the calibrated QCBP test sample solutions: analyzing each QCBP test sample by variable weight value evaluation method, when VIP value > 1, it indicates that the component is a key factor that causes greater differences between QCBP test samples. Specifically, if there is a quality difference in several QCBP test samples, and the common peaks with VIP value > 1 are 14, ranked from large to small, peaks 2, 16, 15, 17, 11, ferulic acid, linalyl tetrol, peak 20, peak 18, gallic acid, peak 19, hydroxysafflor yellow A, peak 12, peak 9; that is, these 14 chromatographic peaks are the key factors causing the quality difference of 13 batches of samples.

5. The method of claim 4, wherein the QXG8M is characterized by, It also includes the following steps: determining the content of each component causing sample quality difference: respectively weighing QCBP test sample solutions, using HPLC-MS quantitative analysis method, calculating the content of each component causing sample quality difference in each sample, and judging the content difference of each component causing sample quality difference in QCBP test sample solution. Specifically, the content of ferulic acid, linalyl tetrol, gallic acid and hydroxysafflor yellow A in the key factors causing sample quality difference is determined to judge the content difference of the four components in the test sample solution.

6. The method for detecting Qingxinchenxiabawei Pill according to claim 5, characterized in that, The HPLC-MS quantitative analysis method, the chromatographic column quantitative analysis conditions are: mobile phase is methanol (A) - water (containing 0.1% formic acid) (B), gradient elution program: 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. The HPLC-MS quantitative analysis method, the mass spectrometry quantitative analysis conditions are: using electrospray ion source (ESI); positive and negative ion scanning; multiple reaction monitoring (MRM) mode; spray voltage 3.80kV (+), 3.50kV (-), atomizing gas volume flow rate 3L / min; heating gas volume flow rate 10L / min; interface temperature 300℃; desolvation temperature 526℃; heating block temperature 400℃.

7. The method for evaluating the quality of Qingxinchenxiabawei Pill according to claim 6, characterized in that, ​ ​

Images