UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) chemical component of Zhuang medicine embelia

Abstract

The invention provides UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) chemical components of Zhuang medicine embelia parviflora and a fingerprint spectrum analysis method thereof, and belongs to the technical field of medicine analysis. The method comprises the following steps: step 1, setting detection conditions of a quadrupole electrostatic field orbitrap high-resolution mass spectrometry method; 2, setting fingerprint conditions; 3, chemical component identification result analysis; and 4, establishing the UPLC fingerprint chromatography of the embelia parviflora. According to the method, chemical components of embelia parviflora are analyzed by adopting a UPLC-Q-Exactive HRMS (High Resolution Mass Spectrometry-Quantitative Exactive High Resolution Mass Spectrometry) technology, and in consideration of serious interference of ion fragments, a spectrum library matching result can be seriously influenced, and the deduction of a mass spectrum splitting decomposition process can be interfered, so that only 10 components with the matching degree being greater than 85% are screened in an experiment, and the deduction of the mass spectrum splitting decomposition process is carried out.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to UPLC-MS chemical components of a Zhuang medicine Angelica sinensis vine and a fingerprint analysis method thereof. Background Art

[0002] Angelica vine is the dry aerial part of Embelia parviflora Wall. of the Myrsinaceae family. It is also known as Embelia parviflora Wall., Siejiqiang, Taku, and Yanhua Acid Wall. It is produced in Guangxi, Guangdong, Guizhou, Yunnan, Fujian and other regions. It is mostly distributed in dense forests in the mountains or forest edges and other places with fertile and moist soil. It can be picked throughout the year and used as medicine after drying. It can be used in combination to treat irregular menstruation, leucorrhea, chlorosis, infertility, etc. It has the effect of Angelica sinensis and is also used for back and leg pain, bone setting, and blood circulation. At present, the reports on the chemical components of Angelica vine mainly include: chemical component pre-experiment, property identification, total flavonoids extraction, total saponin extraction, response surface method optimization of polysaccharide extraction, volatile oil component research, fingerprint, catechin, gallic acid, protocatechuic acid, epicatechin and other component content determination, as well as blood-tonifying mechanism research and antioxidant effect. The chemical fingerprint of traditional Chinese medicine can fully reflect the overall situation of various chemical components in the complex mixed system of traditional Chinese medicine. The chemical components of Angelica Root have not been analyzed in detail in the prior art, and it is impossible to provide a reference for the development and utilization of Angelica Root. Therefore, it is necessary to design the UPLC-MS chemical components of the Zhuang medicine Angelica Root and its fingerprint analysis method. Summary of the invention

[0003] The purpose of the present invention is to provide a UPLC-MS chemical component of the Zhuang medicine Angelica dahurica and a fingerprint analysis method thereof, so as to solve the technical problem that the existing detailed analysis of the chemical components of Angelica dahurica cannot provide a reference for the development and utilization of Angelica dahurica.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] UPLC-MS chemical components of Zhuang medicine Angelica sinensis vine and fingerprint analysis method thereof, the method comprising the following steps:

[0006] Step 1: Setting the detection conditions of the quadrupole electrostatic field orbital trap high-resolution mass spectrometry method;

[0007] Step 2: Set fingerprint conditions;

[0008] Step 3: Analysis of chemical composition identification results;

[0009] Step 4: Establishment of UPLC fingerprint chromatogram of Angelica sinensis vine.

[0010] Furthermore, the detection conditions in step 1 include preparation of the test solution, preparation of the reference solution, and chromatography and mass spectrometry conditions;

[0011] Step 1.1: The process of preparing the test solution is as follows: 0.5 g of Angelica sinensis vine powder is passed through a No. 3 sieve, placed in a 50 mL conical flask, 20 mL of 50% ethanol is precisely added, ultrasonic extraction is performed for 20 min, centrifuged in a 13000 r / min centrifuge for 10 min, and filtered through a 0.22 μm microporous filter membrane to obtain the test solution;

[0012] Step 1.2: Preparation process of reference solution: accurately weigh a fixed amount of procyanidin B1, catechin, procyanidin B2 and epicatechin reference substances, add methanol to prepare a solution containing 0.46 mg of procyanidin B1, 1.46 mg of catechin, 0.408 mg of procyanidin B2 and 0.40 mg of epicatechin per 1 ml, as a mixed reference stock solution, take 1 ml of the above mixed reference stock solution and place it in a 10 ml volumetric flask, add methanol to the scale, and obtain a mixed reference solution containing 0.046 mg / ml of procyanidin B1, 0.042 mg / ml of catechin, 0.0408 mg / ml of procyanidin B2 and 0.040 mg / ml of epicatechin per 1 ml;

[0013] Step 1.3: The process of chromatography and mass spectrometry conditions is as follows: Chromatographic column: Thermo Gold C 18 Chromatographic column, flow rate of 0.3mL / min, injection volume of 1μL, column temperature of 35℃, gradient elution program: time is 0min, 0.1% formic acid-acetonitrile is 5%, 0.1% formic acid-water is 95%; time is 2min, 0.1% formic acid-acetonitrile is 5%, 0.1% formic acid-water is 95%; time is 42min, 0.1% formic acid-acetonitrile is 95%, 0.1% formic acid-water is 5%; time is 47min, 0.1% formic acid-acetonitrile is 95%, 0.1% formic acid-water is 5%; time is 47.1min, 0.1% formic acid-acetonitrile is 95%, 0.1% formic acid-water is 5%; time is 50min, 0.1% formic acid-acetonitrile is 5%, 0.1% formic acid-water is 95%;

[0014] UPLC-Q-Exactive LC-MS parameters: HESI source was used as the ion source, spray voltage: positive source 3.5 kV, negative source 3.2 kV, sheath gas volume flow rate: 30 μL / min, ion transfer tube temperature: 320 °C; auxiliary gas flow rate: 10 μL / min; auxiliary gas temperature: 300 °C;

[0015] Scanning method: FullMS / dd-MS 2mode, positive and negative ions are collected simultaneously, positive and negative ion scanning m / z range: 100-1500m / z, among which the first-level FullMS full scan selection resolution: 70000FWHM, the second-level scan selection resolution: 17500FWHM, specific ion scanning mode: off, vertex excitation: 2-8s, spray gas: nitrogen, collision gas: high-purity nitrogen.

[0016] Furthermore, the specific process of step 2 is:

[0017] Step 2.1: Sample preparation: The preparation of Angelica sphenanthera test and reference solutions is the same as that in Step 1.1 and Step 1.2;

[0018] Step 2.2: Chromatographic conditions: chromatographic column: Waters ACQUITY C18 column; mobile phase: acetonitrile-0.1% phosphoric acid, 0.5 ml / min gradient elution; detection wavelength: 230 nm; column temperature: 40°C; injection volume: 3 μL, gradient elution program: time 0 min, acetonitrile 5%, 0.1% phosphoric acid 95%; time 5 min, acetonitrile 5%, 0.1% phosphoric acid 95%; time 10 min, acetonitrile 10%, 0.1% phosphoric acid 90%; time 25 min, acetonitrile 11%, 0.1% phosphoric acid 89%;

[0019] Step 2.3: Precision investigation: Take the powder of Angelica sinensis vine medicinal material, pass it through a No. 2 sieve, weigh 0.50 g, accurately weigh it, prepare the Angelica sinensis vine medicinal material test solution according to the preparation method of step 1.1, and continuously inject it 6 times according to the chromatographic conditions of step 1.3. The relative retention time RSD values ​​of 15 common peaks in the liquid chromatogram of Angelica sinensis vine are analyzed and calculated to be in the range of 0.09% to 0.21%, and the relative peak area RSD values ​​are in the range of 2.29% to 3.00%, all of which are less than 3.00%, indicating that the instrument has good precision;

[0020] Step 2.4: Repeatability study: Take the powder of Angelica Root medicinal material, pass it through No. 2 sieve, weigh 0.50g, accurately weigh it, prepare 6 portions of Angelica Root medicinal material test solution according to the preparation method of step 1.1, inject it according to the chromatographic conditions of step 1.3, analyze and calculate the relative retention time RSD values ​​of 15 common peaks in the liquid chromatogram of Angelica Root, which are in the range of 0.05% to 0.47%, and the relative peak area RSD values ​​are in the range of 0.99% to 2.71%, all of which are less than 3.00%, indicating that the chromatographic method has good repeatability;

[0021] Step 2.5: Stability investigation: Take the Chinese angelica vine medicinal material, pass it through No. 2 sieve, the powder weight is 0.50g, accurately weighed, prepare the test solution according to the method of step 1.1, and inject and analyze at 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 20h, and 24h according to the chromatographic conditions of step 1.3. The results showed that the test solution was stable within 12 hours. After more than 12 hours, the RSD value of the relative retention peak area of ​​the chromatographic peak with a small area increased. The relative retention time RSD values ​​of the 15 common peaks in the Chinese angelica vine liquid chromatogram within 12h were in the range of 0.05% to 0.88%, and the relative peak area RSD values ​​were in the range of 1.01% to 2.94%, all of which were less than 3.00%, indicating that the test sample had good stability within 12h.

[0022] Furthermore, the specific process of step 3 is:

[0023] The total ion current of the Angelica vine sample was collected by UPLC-Q-Exactive HRMS. After library matching, fragment analysis and literature search, a total of 10 compounds were identified, among which proanthocyanidin B1, magnolia alkaloids, mannitol, sucrose, nicotinamide, adenosine, citric acid, and ethyl 3,4-dihydroxybenzoate were detected in Angelica vine for the first time. According to the current analysis of the chemical components of Angelica vine and the results of this instrumental analysis.

[0024] Furthermore, the specific process of step 4 is:

[0025] Step 4.1: UPLC fingerprints of different batches of Angelica sphenanthera vines. Take each batch of Angelica sphenanthera vine powder, pass it through a No. 3 sieve, weigh 0.50 g, accurately weigh it, prepare Angelica sphenanthera vine test solution according to the preparation method of step 1.1, and record the chromatograms of 11 batches of Angelica sphenanthera vines according to the chromatographic conditions of step 1.3 to establish the UPLC fingerprint of Angelica sphenanthera vines;

[0026] Step 4.2: Referring to the establishment of the fingerprint, the chromatographic peaks common to 11 origins were calibrated as common peaks using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software, and a total of 15 common peaks were calibrated. The chromatogram of Angelica sinensis vine from one of the origins was used as a reference spectrum to obtain the control fingerprint of Angelica sinensis vine and the superposition of UPLC chromatograms of Angelica sinensis vine from each origin;

[0027] Step 4.3: Identification of reference peaks. The peak times of peaks 1, 4, 5, 6 and 7 in the fingerprint are stable. One of the peaks is selected as the reference peak. The chromatogram of the reference substance is compared with the chromatogram of the sample. Peak 4 is identified as proanthocyanidin B1 and peak 5 is identified as catechin. In order to explore what the chromatographic peaks 6, 7 and other unknown peaks are, liquid chromatography-mass spectrometry is further used for analysis and identification.

[0028] Step 4.4: Chromatographic conditions, instrument: Waters Xevo G2-QTOF ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, column: ACQUITY UPLC BEH C 18 , column temperature 35 ° C, flow rate 0.3mL / min, gradient elution conditions: time 0min, 0.1% formic acid water 95%, acetonitrile 5%; time 4min, 0.1% formic acid water 95%, acetonitrile 5%; time 5min, 0.1% formic acid water 92%, acetonitrile 8%; time 8min, 0.1% formic acid water 88%, acetonitrile 12%; time 11min, 0.1% formic acid water 90%, acetonitrile 10%; time 13min, 0.1% formic acid water 92%, acetonitrile 8%; time 13.5min, 0.1% formic acid water 95%, acetonitrile 5%; time 14min, 0.1% formic acid water 95%, acetonitrile 5%;

[0029] Step 4.5: Mass spectrometry conditions, UPLC-MS ion source is ESI source, data acquisition is positive and negative ion mode, MS scan range: 50-1200Da, capillary voltage: 3.0KV, cone voltage: 40V, desolvation voltage: 80V, desolvation gas temperature: 300℃, desolvation gas flow rate: 600L / hr, cone gas flow rate: 50L / hr, nebulizer gas pressure: 7.0Ba, ionization mode: ES+;

[0030] Step 4.6: UPLC-QTOF MS result analysis, online analysis of the Angelica Root sample components, and obtaining a 278 nm chromatogram and a total ion current diagram;

[0031] Step 4.7: Reference peak identification. In the Angelica Root fingerprint, procyanidin B1, catechin, procyanidin B2, and epicatechin were identified as common components in the Angelica Root medicinal material. Among them, procyanidin B1 and procyanidin B2 were found in the Angelica Root medicinal material for the first time. In the UPLC chromatogram, the chromatographic peak symmetry of the four common components was between 0.95 and 1.05, the peak area was stable, and the separation was good. In view of the high response value of procyanidin B1, the largest peak area and the most characteristic, procyanidin B1 was selected as the reference peak of the fingerprint.

[0032] Step 4.8: Determination of common peaks and reference peaks, taking peak No. 4 in the fingerprint as the reference chromatographic peak, and the retention time and peak area of ​​the reference peak as the reference 1, respectively calculate the relative retention time and relative peak area of ​​the common peaks of 11 batches of Angelica dahurica;

[0033] Step 4.9: Similarity analysis: the fingerprint data of 11 different origins of Angelica dahurica vine medicinal materials were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the National Pharmacopoeia Committee. The similarity results of different origins were obtained by comparing with the control fingerprint of Angelica dahurica vine. The results showed that the similarity of the 11 batches of Angelica dahurica vine medicinal materials was greater than 0.9, indicating that the chemical composition of samples from different origins was consistent and the quality was stable.

[0034] Step 4.10: Cluster analysis. Cluster analysis is a statistical method that classifies samples or indicators in experiments and their individual characteristics. It can classify experimental research objects according to statistical variability.

[0035] The peak areas of the common peaks in the fingerprints of Angelica sinensis from different origins were quantified relative to the sample weight, and the SPSS22.0 statistical software was used for systematic clustering. The Ward method was used to cluster the samples, and the square Euclidean distance was used as the metric to obtain the cluster analysis results.

[0036] Step 4.11: Correlation matrix and principal component extraction analysis: using the principal component analysis method, the common peak areas of 11 batches of Angelica dahurica fingerprint spectra were imported into SPSS22.0 statistical software, and the data were standardized and subjected to principal component analysis;

[0037] Step 4.12: Eigenvector and principal component model. To obtain the expression of the common factors of the two selected principal components, use the conversion function in SPSS to calculate the variables. Open the Calculate Variables dialog box, enter A1 in the target variable, and enter B1 / SQRα1 in the numeric expression to obtain the eigenvector A1. In the formula, B1 represents the loading matrix and α1 represents the total eigenvalue. Similarly, the eigenvector A2 can be obtained.

[0038] Let F1 and F2 represent two common factors, and Z1 to Z15 represent the variables after standard normal transformation, and we can get the expression of the two component common factors:

[0039] F1=0.28*Z1+0.19*Z2+0.1*Z3+0.3*Z4+0.29*Z5+0.31*Z6+0.31*Z7+0.24* Z8+0.3*Z9+0.28*Z10+0.29*Z11+0.07*Z12+0.29*Z13+0.18*Z14+0.28*Z15

[0040] F2=-0.22*Z1-0.36*Z2+0.45*Z3-0.12*Z4-0.2*Z5+0.02*Z6-0.04*Z7+0.23* Z8+0.04*Z9-0.07*Z10+0.14*Z11+0.48*Z12+0.16*Z13+0.45*Z14-0.18*Z15

[0041] Taking the ratio of the two component factors to the sum of the total eigenvalues ​​of the extracted principal components as the weight coefficient, the principal component comprehensive model Z can be calculated as shown in the following formula:

[0042] Z=α1*Z1+α2*Z2

[0043] Where α is the component contribution rate, α1 = 0.10319, α2 = 0.3151;

[0044] Step 4.13: Comprehensive evaluation of the principal components of Angelica sinensis vine medicinal materials from different origins. Comprehensive evaluation of the Angelica sinensis vine medicinal materials is carried out based on the first two components. The comprehensive scores of Angelica sinensis vine medicinal materials from different origins are calculated according to the expression of the common factors of the two components in step 4.12 and the principal component comprehensive model Z.

[0045] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0046] (1) The present invention uses UPLC-Q-Exactive HRMS technology to analyze the chemical components of Angelica sinensis vine. Considering that the interference of ion fragments is serious, it may seriously affect the library matching results and interfere with the derivation of the mass spectrometry fragmentation process. Therefore, this experiment only screened 10 components with a matching degree greater than 85% and performed mass spectrometry fragmentation process derivation.

[0047] (2) By comparing with the reference substance, it was found that Angelica sinensis vine contains chemical components such as gallic acid, protocatechuic acid, catechin, and epicatechin. Gallic acid and catechin were screened out this time, but protocatechuic acid and epicatechin were not screened out. It is speculated that the separation degree did not meet the requirements, resulting in too many ion fragments, resulting in a low matching degree and failure to screen them out.

[0048] (3) When establishing the fingerprints of Angelica sinensis vines from different origins, the chromatographic conditions were investigated. Among them, the chromatographic column was screened according to the analysis effect, peak shape, separation degree, quantity, etc.; the mobile phase system was investigated, and acetonitrile-0.1% phosphoric acid was finally selected; the Angelica Root sample was 3D scanned in the wavelength range of 200-400nm. At 230nm, the number of chromatographic peaks was large, the response value was high, and the peak shape was sharp, so 230nm was selected as the measurement wavelength; the chromatographic peaks when the elution flow rates were 0.4ml / min, 0.5ml / min, 0.6ml / min, and 0.7ml / min were investigated respectively, and the comprehensive separation degree and chromatographic analysis time were considered, and finally the flow rate was determined to be 0.5ml / min; the separation of each chromatographic peak when the column temperature was 20℃, 25℃, 30℃, 35℃, and 40℃ was investigated respectively. In order to shorten the analysis time, ensure the separation effect, and improve the analysis efficiency, a column temperature of 40℃ was finally selected; the test solution was injected and analyzed, and the detection time was extended to 60min. As a result, no chromatographic peaks appeared after 25min, indicating that 25min was a suitable elution time.

[0049] (4) A detection method for the UPLC fingerprint of Angelica sinensis vine was established. The precision, repeatability and stability of the method were investigated by the relative retention time and relative peak area of ​​the common peaks. The RSD values ​​of all indicators were less than 3.0%, which met the requirements of item 9101 in Part IV of the 2020 edition of the Chinese Pharmacopoeia, indicating that the instrument had good precision, high repeatability and good stability within 12 hours. The fingerprint of Angelica sinensis vine was comprehensively evaluated using three methods: fingerprint similarity analysis, cluster analysis and principal component analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the total ion flow diagram of the Angelica sinensis vine test sample of the present invention;

[0051] Figure 2 This is the reference fingerprint spectrum of Angelica sinensis vine of the present invention;

[0052] Figure 3 It is the superimposed fingerprint spectrum of Angelica sinensis vine of the present invention;

[0053] Figure 4 This is the 278nm chromatogram of the Angelica sinensis vine sample of the present invention;

[0054] Figure 5 This is the base peak diagram under the positive ion mode of the present invention;

[0055] Figure 6 It is the base peak diagram of the sample and the standard of the present invention;

[0056] Figure 7 It is the UPLC chromatogram of the mixed reference substance of the present invention;

[0057] Figure 8It is a cluster analysis result diagram of the present invention;

[0058] Fig. 9 This is the scree plot of the PCA analysis of Angelica sinensis vine of the present invention. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0060] 1. Instruments and reagents

[0061] 1.1 Instrument

[0062] Thermo U3000 ultra-high performance liquid chromatograph-Q-Exactive (electrostatic field orbital trap) high-resolution mass spectrometer (Thermo Fisher Scientific, USA); Agilent 1290 high performance liquid chromatograph (Agilent, USA); Waters Xevo G2-QTOF ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer (Waters, USA); Waters ACQUITY C18 (3.0x100mm, 1.7μm) chromatographic column; Thermo Gold C18 chromatographic column (2.1mm×100mm, 1.9μm); SQP analytical balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.); Milli-Q Synergy ultrapure water instrument (Millipore, USA); KQ-500DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); TGL-16G high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory).

[0063] 1.2 Medicinal Materials

[0064] The angelica vine used in the experiment was collected from Jinxiu Yao Autonomous County, Guangxi and other places. It was identified by Associate Professor Guo Min of the Medicinal Botany Teaching and Research Section of the School of Pharmacy of Guangxi University of Chinese Medicine as the dried aerial parts of Embelia parviflora Wall. of the Myrsinaceae family. The medicinal material information is shown in Table 1.

[0065] Table 1 Information on the medicinal materials of Angelica sinensis vine

[0066]

[0067]

[0068] 1.3 Reagents

[0069] Proanthocyanidin B1 reference substance (Chengdu Medson Technology Co., Ltd., batch number: RP181114, purity: HPLC ≥ 98.0%), proanthocyanidin B2 reference substance (Chengdu Medson Technology Co., Ltd., batch number: RP181114, purity: HPLC ≥ 98.0%), catechin reference substance (China Food and Drug Inspection Institute, batch number: 110877-201604), epicatechin reference substance (Chengdu Medson Technology Co., Ltd., batch number: RP181114, purity: HPLC ≥ 98.0%); acetonitrile and formic acid were both chromatographically pure (Fisher, USA), and water was ultrapure water.

[0070] 2.1UPLC-Q-Exactive HRMS detection conditions

[0071] 2.1.1 Preparation of test solution

[0072] Take about 0.5 g of medicinal powder (passed through No. 3 sieve), accurately weigh, place in a 50 mL conical flask, accurately add 20 mL of 50% ethanol, weigh, ultrasonically extract for 20 minutes, take out and cool, then weigh, centrifuge in a 13000 r / min centrifuge for 10 minutes, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0073] 2.1.2 Preparation of reference solution

[0074] Accurately weigh appropriate amounts of proanthocyanidin B1, catechin, proanthocyanidin B2 and epicatechin reference substances, add methanol to prepare a solution containing 0.46 mg of proanthocyanidin B1, 1.46 mg of catechin, 0.408 mg of proanthocyanidin B2 and 0.40 mg of epicatechin per 1 ml as a mixed reference substance stock solution. Take 1 ml of the above mixed reference substance stock solution and place it in a 10 ml volumetric flask, add methanol to the scale to obtain a mixed reference substance solution containing 0.046 mg / mL of proanthocyanidin B1, 0.042 mg / mL of catechin, 0.0408 mg / mL of proanthocyanidin B2 and 0.040 mg / mL of epicatechin per 1 ml.

[0075] 2.1.3 Chromatographic and mass spectrometry conditions

[0076] Column: Thermo Gold C 18 Chromatographic column (2.1 mm × 100 mm, 1.9 μm); flow rate 0.3 mL / min; injection volume 1 μL, column temperature 35°C. Gradient elution program see Table 2.

[0077] Table 2 Gradient elution program

[0078]

[0079] UPLC-Q-Exactive LC-MS parameters: ion source adopts HESI source (heated ESI), spray voltage: 3.5 kV (positive source), 3.2 kV (negative source), sheath gas volume flow rate: 30 μL / min, ion transfer tube temperature: 320°C; auxiliary gas flow rate: 10 μL / min; auxiliary gas temperature: 300°C.

[0080] Scanning method: FullMS / dd-MS 2 mode, positive and negative ion collection at the same time, positive and negative ion scanning m / z range: 100-1500m / z, the first-level FullMS full scan selection resolution: 70000FWHM, the second-level scan selection resolution: 17500FWHM, specific ion scanning mode: "off", apex excitation: 2-8s, spray gas: nitrogen, collision gas: high-purity nitrogen.

[0081] 2.2 Fingerprint conditions

[0082] 2.2.1 Sample preparation method

[0083] The preparation of Angelica sphenanthera test and reference solutions is the same as the methods under "2.1.1" and "2.1.2".

[0084] 2.2.2 Chromatographic conditions

[0085] Column: Waters ACQUITY C 18 (3.0×100 mm, 1.7 μm) chromatographic column; mobile phase: acetonitrile-0.1% phosphoric acid, 0.5 ml / min gradient elution; detection wavelength: 230 nm; column temperature: 40°C; injection volume: 3 μL. Gradient elution see Table 3.

[0086] Table 3 Gradient elution program

[0087]

[0088] 2.2.3 Methodological considerations

[0089] 2.2.3.1 Precision investigation

[0090] Take about 0.50g of Angelica Root medicinal material powder (DGT-6, passed through No. 2 sieve), accurately weigh it, prepare Angelica Root medicinal material test solution according to the preparation method under "2.1.1", and continuously inject 6 times according to the chromatographic conditions under "2.1.3". The relative retention time RSD values ​​of 15 common peaks in the Angelica Root liquid chromatogram are analyzed and calculated. The RSD values ​​of the relative peak areas are in the range of 0.09% to 0.21%, and the RSD values ​​of the relative peak areas are in the range of 2.29% to 3.00%, all of which are less than 3.00%, indicating that the instrument has good precision.

[0091] 2.2.3.2 Repeatability study

[0092] Take about 0.50g of Angelica vine medicinal material powder (DGT-6, passed through No. 2 sieve), weigh it accurately, prepare 6 portions of Angelica vine medicinal material test solution according to the preparation method under "2.1.1", inject according to the chromatographic conditions under "2.1.3", and analyze and calculate the relative retention time RSD values ​​of 15 common peaks in the Angelica vine liquid chromatogram, which are in the range of 0.05% to 0.47%, and the relative peak area RSD values ​​are in the range of 0.99% to 2.71%, both of which are less than 3.00%, indicating that the chromatographic method has good repeatability.

[0093] 2.2.3.3 Stability study

[0094] Take about 0.50g of the powder of Angelica Root (No. DGT-6, passed through No. 2 sieve), accurately weigh, prepare the test solution according to the method of "2.1.1", and analyze the samples at 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 20h, and 24h according to the chromatographic conditions of "2.1.3". The results show that the test solution is relatively stable within 12 hours. After more than 12 hours, the RSD value of the relative retention peak area of ​​the chromatographic peak with a smaller area increases. The relative retention time RSD value of the 15 common peaks in the liquid phase chromatogram of Angelica Root within 12h is in the range of 0.05% to 0.88%, and the relative peak area RSD value is in the range of 1.01% to 2.94%, all less than 3.00%, indicating that the test sample has good stability within 12h.

[0095] 3 Results

[0096] 3.1 Chemical composition identification results

[0097] The total ion chromatogram of the Angelica sinensis vine sample collected by UPLC-Q-Exactive HRMS is shown in the figure below. Figure 1 As shown. After library matching, fragment analysis and literature search, a total of 10 compounds were identified. Among them, proanthocyanidin B1, magnolia alkaloids, mannitol, sucrose, nicotinamide, adenosine, citric acid, 3,4-dihydroxybenzoic acid ethyl ester, etc. were detected in Angelica Root for the first time. According to the current analysis of the chemical components of Angelica Root and the results of this instrumental analysis, it is very likely that Angelica Root contains the above components. The results are shown in Table 4.

[0098] Table 4 Identification of chemical components of Angelica sinensis vine

[0099]

[0100] 3.2 Establishment of UPLC fingerprint chromatogram of Angelica sinensis vine

[0101] 3.2.1 UPLC fingerprints of different batches of Angelica sinensis vine

[0102] Take about 0.50g of each batch of Angelica Root powder (passed through No. 3 sieve), weigh accurately, and prepare Angelica Root test solution according to the preparation method under "2.1.1". According to the chromatographic conditions under "2.2.2", record the chromatograms of 11 batches of Angelica Root, and establish the Angelica Root UPLC fingerprint.

[0103] 3.2.2 Establishment of reference fingerprint

[0104] In this experiment, the software "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) was used to calibrate the chromatographic peaks common to 11 different origins as common peaks. A total of 15 common peaks were calibrated in this study, and the chromatogram of Angelica sinensis vine from Jinxiu origin was used as a reference spectrum to obtain the reference fingerprint spectrum of Angelica sinensis vine ( Figure 2 ) and superposition of UPLC chromatograms of Angelica sinensis vines from different origins Figure 3 .

[0105] 3.2.3 Identification and selection of reference peaks

[0106] 3.2.3.1 Reference peak identification

[0107] Depend on Figure 2 It can be seen that the peaks 1, 4, 5, 6 and 7 in the fingerprint are stable in elution time and good in peak shape, so one of the peaks is considered to be selected as the reference peak. By comparing the reference chromatogram compared in this article with the sample chromatogram, it is determined that peak 4 is proanthocyanidin B1 and peak 5 is catechin. In order to explore what substances 6, 7 and other unknown chromatographic peaks are, the experiment further uses liquid chromatography-mass spectrometry for analysis and identification.

[0108] 3.2.3.2 Chromatographic conditions

[0109] Instrument: Waters Xevo G2-QTOF ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (American company); Chromatographic column: ACQUITY UPLC BEH C 18 (1.7μm2.1×100mm); column temperature 35℃; flow rate 0.3mL / min; elution conditions see Table 5.

[0110] Table 5 Gradient elution conditions

[0111]

[0112] 3.2.3.3 Mass spectrometry conditions

[0113] The UPLC-MS ion source was ESI source, and data acquisition was in positive and negative ion modes. MS scanning range: 50-1200Da, capillary voltage: 3.0KV, cone voltage: 40V, desolvation voltage: 80V, desolvation gas temperature: 300°C, desolvation gas flow rate: 600L / hr, cone gas flow rate: 50L / hr, nebulizer gas pressure: 7.0Ba, and ionization mode: ES+.

[0114] 3.2.3.4 UPLC-QTOF MS results analysis

[0115] The components of the Angelica sinensis vine sample were analyzed online quickly. The chromatogram at 278 nm showed Figure 4 , total ion current diagram is shown in Figure 5 .

[0116] 3.2.3.5 Reference peak identification

[0117] In the fingerprint of Angelica sinensis vine, proanthocyanidin B1, catechins, proanthocyanidin B2, and epicatechin were identified as the common components in Angelica sinensis vine, among which proanthocyanidin B1 and proanthocyanidin B2 were found in Angelica sinensis vine for the first time. In the UPLC chromatogram, the symmetry of the chromatographic peaks of the four common components was between 0.95 and 1.05, the peak areas were stable, and the separation was good. In view of the high response value of proanthocyanidin B1, the largest peak area and the most characteristic, proanthocyanidin B1 was selected as the reference peak of the fingerprint. The chromatogram of the mixed reference substance is shown in Figure 7 , Figure 7 Among them, 1. Proanthocyanidin B1 2. Catechin 3. Proanthocyanidin B2 4. Epicatechin. Figure 6 Among them, A. sample B. proanthocyanidin B2 C. proanthocyanidin B1 D. epicatechin E. catechin.

[0118] 3.2.3.6 Determination of common peaks and reference peaks

[0119] Taking peak No. 4 in the fingerprint as the reference chromatographic peak, and the retention time and peak area of ​​the reference peak as the benchmark 1, the relative retention times and relative peak areas of the common peaks of 11 batches of Angelica dahurica were calculated respectively. The results are shown in Tables 6 and 7.

[0120] Table 6 Angelica sinensis vine fingerprint - relative retention time of common peaks

[0121]

[0122]

[0123] Table 7 Angelica sinensis vine fingerprint - common peak relative peak area time

[0124]

[0125] 3.3 Similarity analysis: The fingerprint data of 11 different origins of Angelica dahurica medicinal materials were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) software of the National Pharmacopoeia Committee, and the similarity results of different origins were obtained by comparing with the control fingerprint of Angelica dahurica. The results showed that the similarity of the 11 batches of Angelica dahurica medicinal materials was greater than 0.9, indicating that the chemical components in samples from different origins were highly consistent and of stable quality.

[0126] 3.4 Cluster analysis Cluster analysis: It is a multivariate statistical method for classifying samples or indicators in experiments and their individual characteristics. It can classify experimental research objects according to statistical variability and is a multiple statistical analysis technique.

[0127] In this experiment, the peak area of ​​common peaks in the fingerprint spectra of Angelica sinensis from different origins was quantified relative to the sample weight, and SPSS22.0 statistical software was used for systematic clustering. The Ward method was used to cluster the samples, and the square Euclidean distance was used as the metric to obtain the cluster analysis results. The results showed that when d<10, the samples were clustered into 2 categories, of which Fusui and Rongxian were clustered into one category, and the rest of the samples from Jinxiu, Napo, Fangcheng, Babu, Fuchuan, Tiandeng, Zhongshan, Gongcheng, and Zhaoping were clustered into one category; when d<5, the samples were clustered into 4 categories, Gongcheng and Zhaoping were clustered into one category, Fusui was clustered into one category, Rongxian was clustered into one category, and the rest of the samples were clustered into one category, indicating that the regional differences of samples from several origins except Gongcheng, Zhaoping, Fusui, and Rongxian were small, and there were certain regional differences between Fusui and Rongxian and other origins. The clustering results are as follows Figure 8 As shown,

[0128] 3.5 Principal Component Analysis

[0129] 3.5.1 Correlation matrix and principal component extraction analysis

[0130] In this experiment, the principal component analysis (PCA) method was used to import the common peak areas of 11 batches of Angelica dahurica fingerprint spectra into SPSS22.0 statistical software, and the data were standardized and subjected to principal component analysis. The standardized data results are shown in Table 8, and the principal component analysis results are shown in Table 9.

[0131] Table 8 Normalized data results

[0132]

[0133] Table 9 Eigenvalues ​​and contribution rates of principal components

[0134]

[0135] PCA scree plot ( Fig. 9) shows that the slope of the first 1 to 3 factors is relatively steep, indicating that the eigenvalue of this part is large and contains more information. The slope of the scree plot after component 3 is also relatively gentle, and the eigenvalue of the flat part is small, and the information contained is also small. The initial eigenvalue contribution rate, with the eigenvalue greater than 1 as the extraction standard, found that the cumulative variance contribution rate of the first two factors was 89.798%, indicating that these two factors contain most of the information in the fingerprint spectrum. Combined with the PCA scree plot, two factors were finally selected as the main components, and the remaining 13 component factors contained very little information, so they were discarded. The principal component loading matrix reflects the contribution size and direction of each variable to the principal component. The larger the absolute value, the closer the correlation. Common factor 1 has a positive correlation with the other 13 components, and common factor 2 is positively correlated with components 3, 9, 11, 12, 13, and 14, and negatively correlated with the remaining components.

[0136] Table 10 Principal component loading matrix

[0137]

[0138] 3.5.2 Eigenvector and principal component model

[0139] In order to obtain the expression of the common factors of the two selected principal components, the "Conversion" function in SPSS is used to calculate the variables. Open the "Calculate Variables" dialog box, enter A1 in the target variable, and enter "B1 / SQR(α1)" in the numerical expression to obtain the eigenvector A1, where B1 represents the load matrix; α1 represents the total eigenvalue. Similarly, the eigenvector A2 can be obtained, as shown in Table 11.

[0140] Table 11 Eigenvector (expression coefficient matrix)

[0141]

[0142] Let F1 and F2 represent two common factors, and Z1 to Z15 represent the variables after standard normal transformation, and we can get the expression of the common factors of the two principal components:

[0143] F1=0.28*Z1+0.19*Z2+0.1*Z3+0.3*Z4+0.29*Z5+0.31*Z6+0.31*Z7+0.24* Z8+0.3*Z9+0.28*Z10+0.29*Z11+0.07*Z12+0.29*Z13+0.18*Z14+0.28*Z15

[0144] F2=-0.22*Z1-0.36*Z2+0.45*Z3-0.12*Z4-0.2*Z5+0.02*Z6-0.04*Z7+0.23* Z8+0.04*Z9-0.07*Z10+0.14*Z11+0.48*Z12+0.16*Z13+0.45*Z14-0.18*Z15

[0145] Taking the ratio of the two principal component factors to the sum of the total eigenvalues ​​of the extracted principal components as the weight coefficient, the principal component comprehensive model Z can be calculated as shown in the following formula:

[0146] Z=α1*Z1+α2*Z2

[0147] (where α is the main component contribution rate, α1 = 0.10319, α2 = 0.3151)

[0148] 3.5.3 Comprehensive evaluation of the main components of Angelica sinensis vines from different origins

[0149] A comprehensive evaluation of Angelica sinensis vine medicinal materials was conducted based on the first two principal components. The comprehensive scores of Angelica sinensis vines from different origins were calculated according to the expressions of the common factors of the two principal components under "3.5.2" and the principal component comprehensive model Z. The results are shown in Table 12.

[0150] Table 12 Principal component scores and comprehensive scores

[0151]

[0152] The results show that the F1 score ranking of the same origin is not necessarily the same as the F2 score ranking, which means that the quality of a certain origin of medicinal materials should be evaluated by considering various indicators comprehensively.

[0153] 4 Discussion

[0154] (1) Previous studies have found that Angelica Root may contain sugars, glycosides, phenols, tannins, saponins, organic acids, flavonoids, triterpenes, steroids, volatile oils and fats. This time, UPLC-Q-Exactive HRMS technology was used to analyze the chemical composition of Angelica Root. Considering that the interference of ion fragments is serious, it may seriously affect the library matching results and interfere with the derivation of the mass spectrometry fragmentation process. Therefore, this experiment only screened 10 components with a matching degree greater than 85% and performed mass spectrometry fragmentation process derivation.

[0155] (2) By comparing with the reference substance, it was found that Angelica sinensis vine contains chemical components such as gallic acid, protocatechuic acid, catechin, and epicatechin. Gallic acid and catechin were screened out this time, but protocatechuic acid and epicatechin were not screened out. It is speculated that the separation degree did not meet the requirements, resulting in too many ion fragments, resulting in a low matching degree and failure to screen them out.

[0156] (3) When establishing the fingerprints of Angelica sinensis vines from different origins, the chromatographic conditions were investigated. Among them, the chromatographic column was screened according to the analysis effect, peak shape, separation degree, quantity, etc.; the mobile phase system was investigated, and acetonitrile-0.1% phosphoric acid was finally selected; the Angelica Root sample was 3D scanned in the wavelength range of 200-400nm. At 230nm, the number of chromatographic peaks was large, the response value was high, and the peak shape was sharp, so 230nm was selected as the measurement wavelength; the chromatographic peaks when the elution flow rates were 0.4ml / min, 0.5ml / min, 0.6ml / min, and 0.7ml / min were investigated respectively, and the comprehensive separation degree and chromatographic analysis time were considered, and finally the flow rate was determined to be 0.5ml / min; the separation of each chromatographic peak when the column temperature was 20℃, 25℃, 30℃, 35℃, and 40℃ was investigated respectively. In order to shorten the analysis time, ensure the separation effect, and improve the analysis efficiency, a column temperature of 40℃ was finally selected; the test solution was injected and analyzed, and the detection time was extended to 60min. As a result, no chromatographic peaks appeared after 25min, indicating that 25min was a suitable elution time.

[0157] (4) A detection method for the UPLC fingerprint of Angelica sinensis vine was established. The precision, repeatability and stability of the method were investigated by the relative retention time and relative peak area of ​​the common peaks. The RSD values ​​of all indicators were less than 3.0%, which met the requirements of item 9101 in Part IV of the 2020 edition of the Chinese Pharmacopoeia, indicating that the instrument had good precision, high repeatability and good stability within 12 hours. The fingerprint of Angelica sinensis vine was comprehensively evaluated using three methods: fingerprint similarity analysis, cluster analysis and principal component analysis.

[0158] The chemical components of Angelica sinensis vine were analyzed by UPLC-Q-Exactive HRMS technology, and the UPLC fingerprint of Angelica sinensis vine was established. 18 (2.1mm×100mm, 1.9μm), the mobile phase was 0.1% formic acid acetonitrile-0.1% formic acid aqueous solution, gradient elution, flow rate was 0.3ml / min, column temperature was 35℃, positive and negative ion acquisition mode, full scan and automatic triggering secondary mass spectrometry scanning mode were used. UPLC was used to study the fingerprint of 11 batches of Angelica dahurica medicinal materials from different origins, and the chromatographic column was Waters ACQUITY C 18(3.0×100mm, 1.7μm), the mobile phase was acetonitrile-0.1% phosphoric acid, gradient elution, flow rate of 0.5ml / min, column temperature of 40℃, detection wavelength of 230nm, injection volume of 3μL. The fingerprints of 11 batches of samples were analyzed by similarity analysis, cluster analysis and principal component analysis. Results: According to the obtained ion fragments, the library was matched, and a total of 10 compounds with high matching degree were detected, namely: mannitol, sucrose, citric acid, nicotinamide, gallic acid, protocatechuic acid, proanthocyanidin B1, catechin, magnolia alkaloids, and ethyl 3,4-dihydroxybenzoate. The common mode of UPLC fingerprint of Angelica sinensis vine was established, and 4 common peaks were identified by UPLC-QTOF MS, and the similarity was greater than 0.9. In the cluster analysis, when d<10, the samples were clustered into 2 categories, and when d<5, the samples were clustered into 4 categories. Conclusion: UPLC-Q-Exactive HRMS technology can be used to quickly and accurately identify the chemical components of Angelica sinensis. The established UPLC fingerprint of Angelica sinensis has good separation, short analysis time, high precision and good stability. It provides a scientific basis for the comprehensive quality control and quality evaluation of Angelica sinensis.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. UPLC-MS chemical components of the Zhuang medicine Angelica sinensis vine and its fingerprint analysis method, characterized by: The method comprises the following steps: Step 1: Setting the detection conditions of the quadrupole electrostatic field orbital trap high-resolution mass spectrometry method; Step 2: Set fingerprint conditions; Step 3: Analysis of chemical composition identification results; Step 4: Establishment of UPLC fingerprint chromatogram of Angelica sinensis vine.

2. The UPLC-MS chemical components of the Zhuang medicine Angelica dahurica and its fingerprint analysis method according to claim 1, characterized in that: The test conditions in step 1 include preparation of the test solution, preparation of the reference solution, and chromatographic and mass spectrometric conditions; Step 1.1: The process of preparing the test solution is as follows: 0.5 g of Angelica sinensis vine powder is passed through a No. 3 sieve, placed in a 50 mL conical flask, 20 mL of 50% ethanol is precisely added, ultrasonic extraction is performed for 20 min, centrifuged in a 13000 r / min centrifuge for 10 min, and filtered through a 0.22 μm microporous filter membrane to obtain the test solution; Step 1.2: Preparation process of reference solution: accurately weigh a fixed amount of procyanidin B1, catechin, procyanidin B2 and epicatechin reference substances, add methanol to prepare a solution containing 0.46 mg of procyanidin B1, 1.46 mg of catechin, 0.408 mg of procyanidin B2 and 0.40 mg of epicatechin per 1 ml, as a mixed reference stock solution, take 1 ml of the above mixed reference stock solution and place it in a 10 ml volumetric flask, add methanol to the scale, and obtain a mixed reference solution containing 0.046 mg / ml of procyanidin B1, 0.042 mg / ml of catechin, 0.0408 mg / ml of procyanidin B2 and 0.040 mg / ml of epicatechin per 1 ml; Step 1.3: The process of chromatography and mass spectrometry conditions is as follows: Chromatographic column: Thermo Gold C 18 Chromatographic column, flow rate of 0.3mL / min, injection volume of 1μL, column temperature of 35℃, gradient elution program: time is 0min, 0.1% formic acid-acetonitrile is 5%, 0.1% formic acid-water is 95%; time is 2min, 0.1% formic acid-acetonitrile is 5%, 0.1% formic acid-water is 95%; time is 42min, 0.1% formic acid-acetonitrile is 95%, 0.1% formic acid-water is 5%; time is 47min, 0.1% formic acid-acetonitrile is 95%, 0.1% formic acid-water is 5%; time is 47.1min, 0.1% formic acid-acetonitrile is 95%, 0.1% formic acid-water is 5%; time is 50min, 0.1% formic acid-acetonitrile is 5%, 0.1% formic acid-water is 95%; UPLC-Q-Exactive LC-MS parameters: HESI source was used as the ion source, spray voltage: positive source 3.5 kV, negative source 3.2 kV, sheath gas volume flow rate: 30 μL / min, ion transfer tube temperature: 320 °C; auxiliary gas flow rate: 10 μL / min; auxiliary gas temperature: 300 °C; Scanning method: FullMS / dd-MS 2 mode, positive and negative ions are collected simultaneously, positive and negative ion scanning m / z range: 100-1500m / z, among which the first-level FullMS full scan selection resolution: 70000FWHM, the second-level scan selection resolution: 17500FWHM, specific ion scanning mode: off, vertex excitation: 2-8s, spray gas: nitrogen, collision gas: high-purity nitrogen.

3. The UPLC-MS chemical components of the Zhuang medicine Angelica dahurica and its fingerprint analysis method according to claim 2, characterized in that: The specific process of step 2 is: Step 2.1: Sample preparation: The preparation of Angelica sphenanthera test and reference solutions is the same as that in Step 1.1 and Step 1.2; Step 2.2: Chromatographic conditions: chromatographic column: Waters ACQUITY C18 column; mobile phase: acetonitrile-0.1% phosphoric acid, 0.5 ml / min gradient elution; detection wavelength: 230 nm; column temperature: 40°C; injection volume: 3 μL, gradient elution program: time 0 min, acetonitrile 5%, 0.1% phosphoric acid 95%; time 5 min, acetonitrile 5%, 0.1% phosphoric acid 95%; time 10 min, acetonitrile 10%, 0.1% phosphoric acid 90%; time 25 min, acetonitrile 11%, 0.1% phosphoric acid 89%; Step 2.3: Precision investigation: Take the powder of Angelica sinensis vine medicinal material, pass it through a No. 2 sieve, weigh 0.50 g, accurately weigh it, prepare the Angelica sinensis vine medicinal material test solution according to the preparation method of step 1.1, and continuously inject it 6 times according to the chromatographic conditions of step 1.

3. The relative retention time RSD values ​​of 15 common peaks in the liquid chromatogram of Angelica sinensis vine are analyzed and calculated to be in the range of 0.09% to 0.21%, and the relative peak area RSD values ​​are in the range of 2.29% to 3.00%, all of which are less than 3.00%, indicating that the instrument has good precision; Step 2.4: Repeatability study: Take the powder of Angelica Root medicinal material, pass it through No. 2 sieve, weigh 0.50g, accurately weigh it, prepare 6 portions of Angelica Root medicinal material test solution according to the preparation method of step 1.1, inject it according to the chromatographic conditions of step 1.3, analyze and calculate the relative retention time RSD values ​​of 15 common peaks in the liquid chromatogram of Angelica Root, which are in the range of 0.05% to 0.47%, and the relative peak area RSD values ​​are in the range of 0.99% to 2.71%, all of which are less than 3.00%, indicating that the chromatographic method has good repeatability; Step 2.5: Stability investigation: Take the Chinese angelica vine medicinal material, pass it through No. 2 sieve, the powder weight is 0.50g, accurately weighed, prepare the test solution according to the method of step 1.1, and inject and analyze at 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 20h, and 24h according to the chromatographic conditions of step 1.

3. The results showed that the test solution was stable within 12 hours. After more than 12 hours, the RSD value of the relative retention peak area of ​​the chromatographic peak with a small area increased. The relative retention time RSD values ​​of the 15 common peaks in the Chinese angelica vine liquid chromatogram within 12h were in the range of 0.05% to 0.88%, and the relative peak area RSD values ​​were in the range of 1.01% to 2.94%, all of which were less than 3.00%, indicating that the test sample had good stability within 12h.

4. The UPLC-MS chemical components of the Zhuang medicine Angelica dahurica and its fingerprint analysis method according to claim 1, characterized in that: The specific process of step 3 is: The total ion current of the Angelica vine sample was collected by UPLC-Q-Exactive HRMS. After library matching, fragment analysis and literature search, a total of 10 compounds were identified, among which proanthocyanidin B1, magnolia alkaloids, mannitol, sucrose, nicotinamide, adenosine, citric acid, and ethyl 3,4-dihydroxybenzoate were detected in Angelica vine for the first time. According to the current analysis of the chemical components of Angelica vine and the results of this instrumental analysis.

5. The UPLC-MS chemical components of the Zhuang medicine Angelica dahurica and its fingerprint analysis method according to claim 1, characterized in that: The specific process of step 4 is: Step 4.1: UPLC fingerprints of different batches of Angelica sphenanthera vines. Take each batch of Angelica sphenanthera vine powder, pass it through a No. 3 sieve, weigh 0.50 g, accurately weigh it, prepare Angelica sphenanthera vine test solution according to the preparation method of step 1.1, and record the chromatograms of 11 batches of Angelica sphenanthera vines according to the chromatographic conditions of step 1.3 to establish the UPLC fingerprint of Angelica sphenanthera vines; Step 4.2: Referring to the establishment of the fingerprint, the chromatographic peaks common to 11 origins were calibrated as common peaks using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software, and a total of 15 common peaks were calibrated. The chromatogram of Angelica sinensis vine from one of the origins was used as a reference spectrum to obtain the control fingerprint of Angelica sinensis vine and the superposition of UPLC chromatograms of Angelica sinensis vine from each origin; Step 4.3: Identification of reference peaks. The peak times of peaks 1, 4, 5, 6 and 7 in the fingerprint are stable. One of the peaks is selected as the reference peak. The chromatogram of the reference substance is compared with the chromatogram of the sample. Peak 4 is identified as proanthocyanidin B1 and peak 5 is identified as catechin. In order to explore what the chromatographic peaks 6, 7 and other unknown peaks are, liquid chromatography-mass spectrometry is further used for analysis and identification. Step 4.4: Chromatographic conditions, instrument: Waters Xevo G2-QTOF ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, column: ACQUITY UPLC BEH C 18 , column temperature 35 ° C, flow rate 0.3mL / min, gradient elution conditions: time 0min, 0.1% formic acid water 95%, acetonitrile 5%; time 4min, 0.1% formic acid water 95%, acetonitrile 5%; time 5min, 0.1% formic acid water 92%, acetonitrile 8%; time 8min, 0.1% formic acid water 88%, acetonitrile 12%; time 11min, 0.1% formic acid water 90%, acetonitrile 10%; time 13min, 0.1% formic acid water 92%, acetonitrile 8%; time 13.5min, 0.1% formic acid water 95%, acetonitrile 5%; time 14min, 0.1% formic acid water 95%, acetonitrile 5%; Step 4.5: Mass spectrometry conditions, UPLC-MS ion source is ESI source, data acquisition is positive and negative ion mode, MS scan range: 50-1200Da, capillary voltage: 3.0KV, cone voltage: 40V, desolvation voltage: 80V, desolvation gas temperature: 300℃, desolvation gas flow rate: 600L / hr, cone gas flow rate: 50L / hr, nebulizer gas pressure: 7.0Ba, ionization mode: ES+; Step 4.6: UPLC-QTOF MS result analysis, online analysis of the Angelica Root sample components, and obtaining a 278 nm chromatogram and a total ion current diagram; Step 4.7: Reference peak identification. In the Angelica Root fingerprint, procyanidin B1, catechin, procyanidin B2, and epicatechin were identified as common components in the Angelica Root medicinal material. Among them, procyanidin B1 and procyanidin B2 were found in the Angelica Root medicinal material for the first time. In the UPLC chromatogram, the chromatographic peak symmetry of the four common components was between 0.95 and 1.05, the peak area was stable, and the separation was good. In view of the high response value of procyanidin B1, the largest peak area and the most characteristic, procyanidin B1 was selected as the reference peak of the fingerprint. Step 4.8: Determination of common peaks and reference peaks, taking peak No. 4 in the fingerprint as the reference chromatographic peak, and the retention time and peak area of ​​the reference peak as the reference 1, respectively calculate the relative retention time and relative peak area of ​​the common peaks of 11 batches of Angelica dahurica; Step 4.9: Similarity analysis: the fingerprint data of 11 different origins of Angelica dahurica vine medicinal materials were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the National Pharmacopoeia Committee. The similarity results of different origins were obtained by comparing with the control fingerprint of Angelica dahurica vine. The results showed that the similarity of the 11 batches of Angelica dahurica vine medicinal materials was greater than 0.9, indicating that the chemical composition of samples from different origins was consistent and the quality was stable. Step 4.10: Cluster analysis. Cluster analysis is a statistical method that classifies samples or indicators in experiments and their individual characteristics. It can classify experimental research objects according to statistical variability. The peak areas of the common peaks in the fingerprints of Angelica sinensis from different origins were quantified relative to the sample weight, and the SPSS22.0 statistical software was used for systematic clustering. The Ward method was used to cluster the samples, and the square Euclidean distance was used as the metric to obtain the cluster analysis results. Step 4.11: Correlation matrix and principal component extraction analysis: using the principal component analysis method, the common peak areas of 11 batches of Angelica dahurica fingerprint spectra were imported into SPSS22.0 statistical software, and the data were standardized and subjected to principal component analysis; Step 4.12: Eigenvector and principal component model. To obtain the expression of the common factors of the two selected principal components, use the conversion function in SPSS to calculate the variables. Open the Calculate Variables dialog box, enter A1 in the target variable, and enter B1 / SQRα1 in the numeric expression to obtain the eigenvector A1. In the formula, B1 represents the loading matrix and α1 represents the total eigenvalue. Similarly, the eigenvector A2 can be obtained. Let F1 and F2 represent two common factors, and Z1 to Z15 represent the variables after standard normal transformation, and we can get the expression of the two component common factors: F1=0.28*Z1+0.19*Z2+0.1*Z3+0.3*Z4+0.29*Z5+0.31*Z6+0.31*Z7+0.2 4*Z8+0.3*Z9+0.28*Z10+0.29*Z11+0.07*Z12+0.29*Z13+0.18*Z14+0.28*Z15 F2=-0.22*Z1-0.36*Z2+0.45*Z3-0.12*Z4-0.2*Z5+0.02*Z6-0.04*Z7+0.2 3*Z8+0.04*Z9-0.07*Z10+0.14*Z11+0.48*Z12+0.16*Z13+0.45*Z14-0.18*Z15 Taking the ratio of the two component factors to the sum of the total eigenvalues ​​of the extracted principal components as the weight coefficient, the principal component comprehensive model Z can be calculated as shown in the following formula: Z=α1*Z1+α2*Z2 Where α is the component contribution rate, α1 = 0.10319, α2 = 0.3151; Step 4.13: Comprehensive evaluation of the principal components of Angelica sinensis vine medicinal materials from different origins. Comprehensive evaluation of the Angelica sinensis vine medicinal materials is carried out based on the first two components. The comprehensive scores of Angelica sinensis vine medicinal materials from different origins are calculated according to the expression of the common factors of the two components in step 4.12 and the principal component comprehensive model Z.

Images