A method for establishing a UPLC-MS characteristic chromatographic fingerprint of peony flowers and a method for determining the contents of 16 characteristic components contained therein

Abstract

The present invention relates to a method for establishing a peony flower UPLC-MS characteristic chromatographic fingerprint, comprising: 1) preparing a mixed reference solution: 2) preparing a test solution: 3) performing chromatography and mass spectrometry on the mixed reference solution and the test solution, respectively, recording the DAD chromatogram and MS mass spectrum of the mixed reference solution and the test solution, loading the recorded chromatogram of the test solution into a traditional Chinese medicine chromatographic fingerprint similarity evaluation software, performing data shearing on the spectrum with a start time of 2 and an end time of 33, using S1 as a reference spectrum, and generating a UPLC control characteristic fingerprint after multi-point calibration and full spectrum peak matching. The present invention can comprehensively reflect the analytical method of the chemical component characteristics of different varieties of peony flowers, and evaluate and identify the intrinsic quality of each variety of peony flower samples, thereby providing a new chemical substance basis for further rational development and utilization of the plant resources.

Description

Technical Field

[0001] The invention belongs to the field of chromatogram technology, and particularly relates to a method for establishing a peony flower UPLC-MS characteristic chromatographic fingerprint. Background Art

[0002] Peony (Paeonia suffruticosa Andr.) is a perennial deciduous shrub of the genus Paeonia in the family Ranunculaceae. It is known as the "king of flowers" and is distributed in most parts of my country, especially in Luoyang, Heze, Tongling, Pengzhou, and other places where it is widely planted. In addition to being an ornamental plant, the root bark of peony is a famous Chinese medicine - Danpi, which has the effects of clearing heat and cooling blood, promoting blood circulation and dispersing blood stasis (Li Kai, Zhou Ning, Li Heyu. Research Progress on the Components and Functions of Peony Flowers and Peony Seeds [J]. Food Research and Development, 2012, 33(3): 228-230.). In addition, the Compendium of Materia Medica records that peony flowers are also a traditional Chinese medicinal material with a bitter taste and a neutral nature. They have the effects of clearing heat and detoxifying, and are mainly used to treat hidden fire in the blood and dryness and heat. Peony seeds are commonly used in folk medicine to treat low back and leg pain. In addition, peony seed oil was listed as a new resource food by the National Health Supervision Administration in 2011. It is rich in unsaturated fatty acids, plant sterols, trace elements and other physiologically active substances, with outstanding nutritional value, and has multiple health functions such as antioxidant, lipid-lowering, liver protection, and sun protection (Hou Tianlan, Wang Shunli, Mi Shengquan, et al. Research progress on the nutritional components and functional effects of peony seed oil [J]. China Oils and Fats, 2021, 4608: 51-55+71. Dong Limei, Zhao Xiujuan, Liu Yao, et al. Research progress on the chemical composition and development and utilization of different parts of oil-bearing peony [J]. Modern Food Science and Technology, 2021, 3710: 348-361. Zhang Kaiyue, He Chunling, Hou Xiaogai, et al. Research status of the economic value and landscape ecological value of oil-bearing peony [J]. Chinese Agricultural Science Bulletin, 2019, 3510: 66-71.). Modern pharmacological studies have shown that peony has the effects of lowering blood sugar, anti-inflammatory, analgesic, antibacterial, regulating the cardiovascular system, anti-tumor, scavenging free radicals, and antioxidant (Wang Xindi, Shi Xiaofeng, Wang Binli, et al. Research progress on the chemical components of peony [J]. Chinese Traditional Patent Medicine, 2018, 01(40): 177-182.).

[0003] In recent years, with the development of the peony industry, the functional activity value of peony flowers, leaves, and seeds, which are by-products of peony bark production, has attracted increasing attention and attention. Currently, research on the chemical composition of peony mainly focuses on the root, and the component types include flavonoids, phenols and phenolic glycosides, monoterpenes and their glycosides, triterpenes and their glycosides, stilbenes, organic acids, volatile oils, trace elements, etc. (Li Yuanyuan, Zheng Yan, Huang Junxiang, et al. Comprehensive Utilization Status and Industrial Development Analysis of Paeonia Suffruticosa [J]. Modern Chinese Medicine Research and Practice, 2012, 026(004): 83-85.). Peony flowers, as the main by-product of peony, are also rich in paeoniflorin, pedunculoids, flavonoids, polyphenols, polysaccharides, and other functional active ingredients with antioxidant, antibacterial, anti-inflammatory, and deodorizing properties. They are also produced in large quantities. If this resource is fully utilized and the added value of the peony industry is further increased, it will have great economic and social benefits.

[0004] With the development of various new and efficient separation and analysis technologies, many trace components and new or difficult-to-separate and identify components will continue to be discovered. At the same time, Chinese medicine fingerprints, as a modern identification and analysis technology, can not only comprehensively and holistically reflect the types and quantities of chemical substances in Chinese medicinal materials, but also serve as the main basis for identifying and controlling the quality of medicinal materials, providing scientific guidance for the quality evaluation of medicinal materials. At present, there are few studies on the material basis and quality control of the chemical components of peony flowers. For example, Hua Mei et al. (Hua Mei, Yuan Xiaolong, Wang Yi, Yang Wei, Chen Jian, Ma Huifen, Hu Yanli, Yang Yuming, Wang Juan. Study on the fingerprint of peony petals by high performance liquid chromatography [J]. Western Forestry Science, 2018, 47(01): 52-58.) used high performance liquid chromatography (HPLC) fingerprint technology to analyze and separate the chemical components in 9 fresh petal samples of different introduced peony varieties, and calibrated 6 common peaks, which can be used as the main basis for identifying the quality of peony petals from the introduced area. Yan Huijiao et al. (Yan Huijiao, Wang Zhiwei, Zhao Hengqiang, Geng Yanling, Zhao Wei, Wang Xiao. Study on HPLC characteristic spectrum of Heze peony and determination of 8 components [J]. Chinese Herbal Medicine, 2017, 48(09): 1866-1871.) established the HPLC fingerprint of Danfeng peony, identified 14 peaks, and attributed 8 components. However, they dried the fresh peony flowers at 50℃, which easily caused the loss and transformation of components. Zhang Guoqiang et al. (Zhang Guoqiang, Li Yun, Pan Jianzhong, Qiu Guoyu, Shi Xiaofeng, Li Zhijun, Ma Quhuan, Cheng Xianlong. HPLC Characteristic Chromatography of Paeonia lactiflora Pollen and Content Determination of Four Components [J]. Journal of Pharmaceutical Analysis, 2019, 39(11): 2020-2027.) established a characteristic chromatogram of Paeonia lactiflora pollen using HPLC, calibrated 16 common peaks, identified four components, and determined their contents. This method can be used as a quality control and content determination method for Paeonia lactiflora pollen. However, the above methods all use the retention time of the reference substance to identify the chromatographic peaks, which has low specificity and accuracy.

[0005] Currently, the Danfeng peony has been approved as a new food ingredient by the National Health and Family Planning Commission (Ministry of Health, 2013, No. 10). Whether other varieties also have the potential to be developed as new food ingredients is unclear. The method established in this study aims to compare the differences and characteristics of the chemical bases of 12 different peony varieties. This will provide a research foundation for further research on the functional activity value of different peony varieties and scientific guidance for the effective development of alternative varieties of Danfeng peony, which is of great significance. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of the prior art and provide a method for establishing a UPLC-MS characteristic chromatographic fingerprint of peony flowers. The present invention uses ultra-high performance liquid chromatography (UPLC) technology to establish characteristic fingerprints of multiple different varieties of peony flowers, and uses reference substance retention time, UV spectrum, and mass spectrum to identify and characterize chromatographic peaks, in order to obtain a more rapid, accurate, simple, and efficient analytical method that can comprehensively reflect the chemical component characteristics of different varieties of peony flowers. Different varieties of peony flower samples are identified through chromatographic identification, similarity evaluation, principal component analysis, and cluster analysis pattern recognition methods. A content determination method based on the selected characteristic index components is established to evaluate and identify the intrinsic quality of each variety of peony flower sample, thereby providing a new chemical substance basis for further rational development and utilization of this plant resource, providing a scientific basis for further in-depth research on the medicinal, edible, feed, and cosmetic values ​​of different varieties of peony flowers, and laying a theoretical foundation for the effective development of alternative varieties of "new food raw materials - Danfeng peony flowers."

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for establishing a UPLC-MS characteristic chromatographic fingerprint of peony flowers specifically comprises the following steps:

[0009] 1) Prepare mixed reference solution:

[0010] Accurately weigh appropriate amounts of 16 characteristic components, including gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, rhusin, peonyside C, benzoyloxidized paeoniflorin, and kaempferol, and dissolve them in solvent to prepare reference substance mother solutions; accurately measure appropriate amounts of the reference substance mother solutions and place them in volumetric flasks, dilute to the mark with the above-mentioned solvents, and mix thoroughly to obtain a mixed reference substance solution;

[0011] 2) Prepare the test solution:

[0012] Accurately weigh 0.4-0.6 g of peony pollen samples of different varieties into a stoppered conical flask, add 40-60 mL of a methanol-ethanol-water mixture, weigh the sample, perform ultrasonic extraction, weigh the sample again, make up the lost weight with the methanol-ethanol-water mixture, shake well, filter through an organic filter membrane, and place the sample in a sample bottle for testing.

[0013] 3) Perform chromatography and mass spectrometry on the mixed reference solution and the test solution, respectively, and record the DAD chromatograms, UV spectra, and MS spectra of the mixed reference solution and the test solution. Load the recorded chromatograms of the test solution into the Chinese medicine chromatographic fingerprint similarity evaluation software, cut the spectrum with the start time as 2 and the end time as 33, and use S1 as the reference spectrum. After multi-point calibration and full spectrum peak matching, generate the UPLC control characteristic fingerprint spectrum.

[0014] Specifically, in step 1), the solvent is methanol.

[0015] Furthermore, in the mixed reference solution of step 1), the mass concentrations of gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, rhubarb glycoside, peonyside C, benzoyl oxidized paeoniflorin, and kaempferol were 8.52 μg / ml, 9. .12μg / ml, 7.72μg / ml, 8.32μg / ml, 8.44μg / ml, 8.04μg / ml, 7.96μg / ml, 8.20μg / ml, 8.80μ g / ml, 8.08μg / ml, 8.36μg / ml, 8.16μg / ml, 8.28μg / ml, 8.72μg / ml, 7.68μg / ml, 7.80μg / ml.

[0016] Specifically, in step 2), the peony pollen sample is obtained by the following steps: fresh peony flowers of different varieties are separated, the stamens and sepals are placed on A4 paper, and freeze-dried in a freeze dryer (-55 to -80°C, 72 hours, vacuum degree <10Pa), and after taking out, the petals are individually crushed into powder and sieved to obtain peony pollen.

[0017] Further, in step 2), 0.5 g of peony pollen samples of different varieties are accurately weighed in a stoppered conical flask, 50 mL of a methanol-ethanol-water mixture is added, the weight is weighed, and ultrasonic extraction is performed at room temperature for 25-35 minutes. Ice cubes are placed during the ultrasonic process to keep the ultrasonic extraction at room temperature as much as possible. After completion, the weight is weighed again, and the lost weight is supplemented with a methanol-ethanol-water mixture. The mixture is shaken and filtered through a 0.22 μm organic filter membrane; the volume ratio of the methanol, ethanol, and water can be 3-5:2-4:3, and more preferably 4:3:3.

[0018] Specifically, the chromatographic conditions in step 3) are: chromatographic column: Agilent prosell 120SB C18, mobile phase gradient elution: acetonitrile (A)-0.1% formic acid water (B): 0-8 min, 2% A→12% A; 8-11 min, 12% A→16% A; 11-15 min, 16% A→20% A; 15-20 min, 20% A→25% A; 20-24 min, 25% A→45% A; 24-30 min, 45% A→65% A; 30-33 min, 65% A→90% A; 33 min-36 min, 90% A→2% A; 36-40 min, 2% A; flow rate: 0.4 mL / min, injection volume: 2 μl, column temperature: 30°C, wavelength: 270 (±2) nm.

[0019] Specifically, the mass spectrometry conditions in step 3) are: ion source: ESI source, scanning mode: multiple reaction monitoring mode and full scan mode (MRM and SCAN), positive and negative modes: ESI +- , drying gas temperature: 350℃, spray gas pressure: 275.8kPa, drying gas flow rate: 11L / min, capillary voltage: 4.0kV (ESI + )、3.5kV(ESI - ).

[0020] Furthermore, the full spectrum peak in step 3) matched 22 common chromatographic peaks, and 16 of the common peak chromatograms were identified based on the DAD chromatogram, UV spectrum and MS mass spectrum of the reference solution as follows: gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, wild sumac glycoside, peonyside C, benzoyl oxidized paeoniflorin, and kaempferol.

[0021] Ultra-high performance liquid chromatography (UPLC) technology, with its ultra-high pressure and ultra-high linear flow rate separation capabilities, can achieve the separation of multiple compounds in a very short time. Given the complex composition of natural products, the present invention utilizes the superior separation capabilities of UPLC combined with the structural information of compounds rich in mass spectrometry to identify and analyze the chemical and material composition of natural products. Furthermore, combined with the retention time and UV spectral information of the reference substance, the characteristic peak positions in the test solution to be tested are identified, making the qualitative results more accurate and reliable.

[0022] The method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower further includes step 4), wherein the similarity between the test solution and the reference spectrum of S1 is calculated using a Chinese medicine chromatographic fingerprint similarity evaluation software. When the similarity is not less than 0.9, the similarity is considered to be high with S1. Conversely, when the similarity is less than 0.9, the similarity is considered to be low with S1.

[0023] The above-mentioned method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower also includes step 5), using PCA analysis to perform principal component analysis on the fingerprints of different varieties of peony flower samples. The cumulative contribution of the two principal components reaches 46.36%, which contains almost half of the information, of which the contribution rate of the first principal component is 28.97%, and the characteristic value of the second principal component is 17.39%. The first and second principal components are projected to obtain a principal component projection diagram. The fingerprint data of different varieties of peony flower samples are clustered using HCA analysis. Based on the relative peak area, retention time and similarity of the common peaks in the test solution, SPSS 26.0 statistical analysis software is used, the inter-group linkage method is selected, the square Euclidean distance is used as the measurement interval, the number of samples is selected, and a systematic cluster analysis is performed. The samples are classified according to the cluster dendrogram and the inter-class distance.

[0024] The present invention also provides a method for determining the contents of 16 characteristic components in peony flowers, which comprises the following steps:

[0025] 1) Preparation of mixed reference solution: Same as in claim 1; then, using the initial mobile phase for stepwise dilution to prepare a series of mixed standard solutions, the initial ratio of the mobile phase being: acetonitrile: 0.1% formic acid water = 2:98 (volume ratio);

[0026] 2) Prepare the test solution:

[0027] 3) Subject the series of mixed standard solutions to chromatography and mass spectrometry, record the peak areas, and draw a standard curve with the concentration of each mixed standard solution as the abscissa and the peak area of ​​each characteristic component in the mixed standard solution as the ordinate; subject the test solution to chromatography and mass spectrometry and record the peak area of ​​each characteristic component, substitute it into the standard curve, and calculate the content of each characteristic component.

[0028] In the method for determining the content of 16 characteristic components in peony flowers, the steps 1) preparing a mixed reference solution and 2) preparing a test solution are carried out with reference to the steps 1) preparing a mixed reference solution and 2) preparing a test solution in the method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flowers described above, and are not repeated here.

[0029] The present invention also provides a method for identifying the quality of different varieties of peony flowers, and performs UPLC-MS / MS content determination on 16 representative index compounds of flavonoids, polyphenols, monoterpenes and their glycosides and aromatic acids contained therein. It uses cosmosin and benzoyloxypeoniflorin as identification quality markers of Zhaofen peony, uses benzoic acid as identification quality marker of Haihuang peony, uses wild sumac glycoside as identification quality marker of Roufurong peony, uses kaempferol as identification marker of Danfeng peony, uses oxypeoniflorin as identification quality marker of Xiangyu peony, uses paeoniflorin and peonyside C as identification quality markers of Heifu Lady peony, uses 1,2,3,6-MTG and benzoyloxypeoniflorin as identification quality markers of Shouanhong peony, uses paeoniflorin, 1,2,3,6-MTG, cosmosin and rhubarb glycoside were used as identification quality markers for Jingyu peony, gallic acid, paeoniflorin, 1,3,6-MTG, 1,2,3,6-MTG and 1,2,3,4,6-MTG were used as identification quality markers for Luoyanghong peony; gallic acid, 1,2,3,4,6-MTG and astragalin were used as identification quality markers for Xugang peony, methyl gallate, ethyl gallate and 1,2,3,4,6-MTG were used as identification quality markers for Huawang peony.

[0030] The present invention relates to a method for establishing a peony flower UPLC-MS characteristic chromatographic fingerprint. First, a mixed reference solution and a test solution are prepared. Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) is used to perform qualitative analysis on the mixed reference solution and the test solution, respectively. DAD chromatograms, UV spectra, and MS mass spectra of the mixed reference solution and the test solution are recorded. The recorded DAD chromatogram of the test solution is then loaded into traditional Chinese medicine chromatographic fingerprint similarity evaluation software. Data is sheared on the spectrum with a start time of 2 and an end time of 33. With S1 as a reference spectrum, a UPLC-DAD control characteristic fingerprint is generated after multi-point calibration and full spectrum peak matching. Characteristic peak identification and attribution verification are then performed. Finally, based on the relative peak area, retention time and similarity of the common peaks in the test solution as the data basis, SPSS statistical analysis software was applied, using the idea of ​​"dimensionality reduction" and with the help of orthogonal transformation, the original random variables with component correlation were transformed into new random variables with uncorrelated components, and multiple indicators were transformed into several complementary and related comprehensive indicators. PCA analysis was performed, and the first and second principal components were projected to obtain the principal component projection diagram to analyze and describe the correlation between the original multiple variables; then the inter-group connection method was selected, and the squared Euclidean distance was used as the measurement interval to perform system HCA analysis. Samples were classified according to the cluster dendrogram and inter-class distance. The UPLC fingerprint method of different varieties of peony samples established in the present invention can comprehensively reflect the new chemical component material basis characteristics of different varieties of peony, and can significantly evaluate and identify the intrinsic quality of each variety of peony from multiple angles, thereby providing a scientific basis for in-depth research on the medicinal, edible, feed and cosmetic values ​​of different varieties of peony, and also laying a theoretical foundation for the effective development of alternative varieties of "new food raw materials-Danfeng peony", which is of great significance.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This study established UPLC-MS characteristic chromatographic fingerprints for 12 different peony varieties. Through literature review, screening of an existing laboratory standard library, and mass spectrometry and spectral identification, a total of 26 components were initially screened. Combined with samples from 12 different peony varieties, 22 common peaks were identified. Further qualitative verification using mass spectrometry, spectral analysis, and retention values ​​led to the identification of 16 components. Instrument precision was evaluated, as well as sample stability and reproducibility, with favorable results. Similarity analysis was performed on the characteristic chromatographic fingerprints of the 12 peony petal samples using similarity evaluation software, and cluster analysis and principal component analysis were performed using SPSS software. The results showed that the chemical composition of the 12 peony varieties showed significant differences. Samples S2, S3, S4, and S9 were most similar to sample S1, with similarities exceeding 0.9. Samples S11, S12, and S10 were least similar to sample S1, with similarities less than 0.8. Sample S2 (Rou Furong, 0.985) and S3 (Xiangyu, 0.979) were most similar to sample S9 (Danfeng). Principal component analysis showed that the samples could be divided into two categories: S1-S9 were grouped as the first, and S10, S11, and S12 were grouped as the second. Among them, significant intra-class differences were observed in the first category. Analysis from the perspective of flower color revealed that S7 and S8 were similar and both had yellow flower colors, S3 and S9 had relatively small differences and both had white flower colors with pink, and S1 and S2 had the smallest differences and both had pink flower colors. In the second category, intra-class differences were relatively small, but S11 showed a discrete trend from S10 and S12, indicating that Luoyang Red peonies still differ significantly from Black Lady and Shou'an Red varieties, and the results were consistent with those from flower color analysis. Cluster analysis revealed that when the inter-class distance was 5, the samples could be divided into four categories: S1, S2, S3, S4, and S9, S5, S6, S7, and S8, S10 and S12, and S11, respectively. When the inter-class distance was 12.5, the samples could be divided into two categories: S10, S11, and S12, and the rest. This result was consistent with the classification results from principal component analysis.

[0033] The UPLC-MS characteristic fingerprint of peony flowers established in this study can effectively distinguish and identify peony flower samples. The differences in the material basis of the chemical components of peony flowers among different varieties can more comprehensively reflect the quality of peony flowers, and also provide a reliable identification method for different types or batches of peony flower samples. In the later stage, it is necessary to further accurately determine the content of the characteristic components of the samples to further compare the differences between them, providing data and method references for in-depth research and development of the functional value of different peony flowers, and also laying a theoretical foundation for the effective development of alternative varieties of "new food raw materials-Danfeng peony flowers". BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1The UV chromatogram (a: DAD graph) and total ion current (b: TIC graph) of the mixed reference solution in MRM mode; among them, 1: gallic acid; 2: methyl gallate; 3: oxidized paeoniflorin; 4: paeoniflorin; 5: ethyl gallate; 6: paeoniflorin; 7: 1,3,6-MTG; 8: benzoic acid; 9: 1,2,3,6-MTG; 10: 1,2,3,4,6-MTG; 11: astragaloside; 12: cosmosin; 13: rhubarb glycoside; 14: paeonol C; 15: benzoyloxypaeoniflorin; 16: kaempferol;

[0035] Figure 2 The UV spectrum of the mixed reference solution; the compounds represented by 1 to 16 in the figure are the same as above Figure 1 As shown;

[0036] Figure 3 The UV chromatogram (a: DAD graph) and total ion current (b: TIC graph) of the S1 sample in the multiple reaction monitoring (MRM) mode are shown in Figure 1. The compounds represented by 1 to 16 are the same as above. Figure 1 As shown;

[0037] Figure 4 To select the chromatographic peak diagram of different chromatographic columns;

[0038] Figure 5 The UV spectra of mixed reference solution at different wavelengths;

[0039] Figure 6 DAD spectra of the Huangguan sample at a wavelength of 270 nm (a: with reference (Ref = 360, 100), b: without reference (Ref = off));

[0040] Figure 7 Comparison of chromatographic peaks of different extraction solvents (a: 70% ethanol, b: 30% methanol, c: 50% methanol, d: 70% methanol, e: 90% methanol);

[0041] Figure 8 Comparison of chromatographic peaks of extraction solvents with different volume ratios (A: methanol: ethanol: water = 1:6:3, B: methanol: ethanol: water = 2:5:3, C: methanol: ethanol: water = 3:4:3, D: methanol: ethanol: water = 4:3:3, E: methanol: ethanol: water = 5:2:3, F: methanol: ethanol: water = 6:1:3);

[0042] Figure 9 This is the UPLC control characteristic fingerprint of the peony S1 sample;

[0043] Figure 10 UPLC-MS characteristic fingerprints of 12 peony samples (S1-S12);

[0044] Figure 11 The PCA projection diagram of 12 peony samples (S1-S12);

[0045] Figure 12 This is the HCA dendrogram of 12 peony flower samples (S1-S12);

[0046] Figure 13 This is the MRM spectrum of the characteristic components of the 16 target determinations. The compounds represented by 1 to 16 in the figure are the same as above. Figure 1 shown. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0048] 1 Materials and Methods

[0049] 1.1 Instruments and Equipment

[0050] Agilent G6460C UPLC-MS / MS triple quadrupole liquid-mass spectrometer system (Agilent Technologies, Inc., USA) equipped with a DAD detector;

[0051] Dura-CYL Dewar jar and nitrogen cylinder (Chart) (Henan Yuanzheng Technology Development Co., Ltd.);

[0052] ME20 1 / 10,000 electronic balance (Mettler, Switzerland);

[0053] AUW220D 1 / 100,000 electronic balance (Shimadzu Corporation, Japan);

[0054] FW-80 high-speed universal grinder (Tianjin Test Instrument Co., Ltd.);

[0055] KQ-500E ultrasonic extractor (Kunshan Ultrasonic Instrument Co., Ltd.);

[0056] CTFD-18S vacuum freeze dryer (Qingdao Yonghe Chuangxin Electronic Technology Co., Ltd.).

[0057] 1.2 Drugs and Reagents

[0058] Gallic acid (batch number 21012903, 99.48%), ethyl gallate (batch number 21060303, 99.66%), methyl gallate (batch number 17092604, ≥98%), paeoniflorin (batch number 21031706, 98.75%), quercetin (batch number 21032404, 98.64%), scutellarin (batch number 17061305, ≥98%), apigenin-7- O-β-D-glucopyranoside (cosmosin, batch number 19010203, ≥98%), oxidized paeoniflorin (batch number 21092304, 99.42%), paeoniflorin C (batch number 22031809, 99.02%), benzoyl oxidized paeoniflorin (batch number 220317, 98.02%), and paeoniflorin (batch number 20030203, 98.11%) were purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0059] 1,2,3,4,6-penta-O-galloyl-β-D-glucose (1,2,3,4,6-MTG, batch number DSTDW000102, 99.33%), 1,3,6-tri-O-galloyl-β-D-glucose (1,3,6-MTG, batch number DST210308-053, 98.41%), and astragaloside (batch number DSTDZ000102, 99.02%) were purchased from Chengdu Desite Biotechnology Co., Ltd.

[0060] 1,2,3,6-tetra-O-galloyl-β-D-glucose (1,2,3,6-MTG, batch number PCS3258-220301, 99.9%) was purchased from Chengdu PhytoPure Biotechnology Co., Ltd.

[0061] Benzoic acid (batch number 100419-201703, 99.9%) and kaempferol (batch number 110881-201611, 95.5%) were purchased from the China Food and Drug Administration.

[0062] Methanol, acetonitrile, and formic acid were of LC / MS grade; ultrapure water was Wahaha mineral water; other reagents were of analytical grade.

[0063] The 12 different varieties of peonies are: Zhaofen (S1, pink), Roufurong (S2, pink), Xiangyu (S3, white with pink), Jingyu (S4, pink and white), Xugang (S5, red), Huawang (S6, rose red), Haihuang (S7, yellow), Huangguan (S8, yellow), Danfeng (S9, jade white with pink), Heifuren (S10, deep purple-red), Luoyanghong (S11, purple-red), and Shouanhong (S12, deep purple-red).

[0064] 1.3 Methods

[0065] 1.3.1 Chromatographic conditions

[0066] Chromatographic column: Agilent prosell 120SB C18 (2.1×100mm, 2.7μm); mobile phase gradient elution: acetonitrile (A)-0.1% formic acid water (B): 0-8 min, 2% A→12% A; 8-11 min, 12% A→16% A; 11-15 min, 16% A→20% A; 15-20 min, 20% A→25% A; 20-24 min, 25% A→45% A; 24-30 min, 45% A→65% A; 30-33 min, 65% A→90% A; 33 min-36 min, 90% A→2% A; 36-40 min, 2% A, flow rate: 0.4 mL / min, injection volume: 2 μl, column temperature: 30°C, wavelength: 270 nm (no reference).

[0067] 1.3.2 Mass spectrometry conditions

[0068] Ion source: ESI source, scanning mode: multiple reaction monitoring mode and full scan mode (MRM and SCAN), positive and negative modes: ESI +- , drying gas temperature: 350℃, spray gas pressure: 275.8kPa, drying gas flow rate: 11L / min, capillary voltage: 4.0kV (ESI + )、3.5kV(ESI - ), the ion pairs used for qualitative and quantitative analysis, dwell time, collision energy and fragmentation voltage are shown in Table 1 below.

[0069] Table 1 Mass spectrometry analysis parameters of the analytes

[0070]

[0071] 1.3.3 Sample preparation

[0072] Fresh peony flowers from 12 varieties were collected, their stamens and calyxes separated, placed on A4 sheets, and freeze-dried in a freeze dryer (-55 to -80°C, 72 hours, vacuum <10 Pa). After removal, the petals were individually pulverized into powder using a grinder and passed through a No. 2 sieve to obtain peony pollen samples. These were then placed in sealed bags and set aside.

[0073] 1.3.4 Preparation of mixed reference solution

[0074] Accurately weigh 2.00 mg of 16 standard substances (gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, rhusin, peonyside C, benzoyl oxidized paeoniflorin, kaempferol) and place them in a 10 ml brown volumetric flask. Add 5 ml of chromatographic methanol to prepare the reference substance stock solution for later use.

[0075] Accurately measure 0.5 ml of the reference substance stock solution, place it in a 25 ml volumetric flask, and dilute to the mark to prepare mass concentrations of 8.52 μg / ml, 9.12 μg / ml, 7.72 μg / ml, 8.32 μg / ml, 8.44 μg / ml, 8.04 μg / ml, 7.96 μg / ml, 8.20 μg / ml, 8.80 μg / ml, 8.08 μg / ml, 8.36 μg / ml, 8.16 μg / ml, 8.28 μg / ml, 8.72 μg / ml, 7.68 μg / ml, and 7.80 μg / ml, respectively. Mix the reference substance solutions.

[0076] 1.3.5 Preparation of test solution

[0077] Accurately weigh 0.5 g of peony samples of 12 varieties in a stoppered conical flask, add 50 mL of methanol-ethanol-water mixture (methanol, ethanol, water volume ratio of 4:3:3), weigh the weight, and ultrasonically extract at room temperature (25 ° C) for 30 min. Ice cubes are added during the ultrasonic process to keep the ultrasonic extraction at room temperature as much as possible. After completion, weigh the weight again, make up the lost weight with methanol-ethanol-water mixture, shake well, and filter into a sample vial with a 0.22 μm organic filter membrane to obtain the test solution.

[0078] 2 Results and Discussion

[0079] 2.1 Methodological Review

[0080] 2.1.1 Specificity

[0081] The UV chromatogram (DAD diagram, see Figure 1 a), total ion current (TIC) diagram (see Figure 1 b) and UV spectrum (see Figure 2 The typical total ion chromatogram (TIC) and ultraviolet chromatogram (DAD) of the Zhaofen peony flower sample numbered S1 are shown in Figure 3As can be seen from the figure: the separation between the chromatographic peaks in the sample is good, and there is almost no interference at the target peak. In addition, the MS detector further qualitatively identifies the sample, and the specificity is even stronger, indicating that this method has good specificity.

[0082] 2.1.2 Precision test

[0083] Weigh the peony petal powder sample numbered S1 and prepare the test solution according to the conditions in "1.3.5". Pipette 2 μl of the test solution and inject it according to the chromatography and mass spectrometry conditions in "1.3.1" and "1.3.2". Repeat the injection 6 times and record the chromatogram.

[0084] The relative retention time RSDs of 11 components with relatively high responses (gallic acid, methyl gallate, oxidized paeoniflorin, ethyl gallate, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, wild sumac glycoside, and benzoyl oxidized paeoniflorin) were calculated and the results were 0.03%-0.25%, and the relative peak area RSDs were 0.14%-1.55%, indicating that the instrument has good precision. The specific results are shown in Table 2.

[0085] Table 2 Precision test (n=6)

[0086]

[0087] 2.1.3 Stability test

[0088] Weigh the peony petal powder sample numbered S1 and prepare the test solution according to the conditions in "1.3.5" in three parallel preparations. Pipette 2 μl of the test solution and inject the sample for determination at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic and mass spectrometric conditions in "1.3.1" and "1.3.2", respectively, and record the chromatogram.

[0089] The RSDs of the relative peak areas of 11 components with relatively high responses (gallic acid, methyl gallate, oxidized paeoniflorin, ethyl gallate, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, wild sumac glycoside, and benzoyloxidized paeoniflorin) were calculated to be 0.15%-3.03%, 1.31%-3.67%, 0.12%-3.61%, 0.44%-3.45%, 0.15%-3.68%, 0.40%-4.67%, 0.03%-4.10%, 0.12%-2.97%, 0.42%-3.74%, 0.24%-3.02%, and 0.42%-3.46%, respectively. The results indicate that the test solution has good stability when stored at room temperature in the dark for 24 hours. Detailed results are shown in Table 3. Therefore, the peony flower extract obtained in this experiment using a mixed solvent of methanol, ethanol, and water can be stably stored when stored at room temperature in the dark, providing better guidance for the sampling, transportation, storage, and identification of peony flower extract samples in the short term.

[0090] Table 3 Stability test (n=3)

[0091]

[0092]

[0093] 2.1.4 Repeatability test

[0094] Take 6 samples of Zhaofen peony petal powder numbered S1, prepare the test solution according to the conditions in "1.3.5", draw 2.0 μL of the test solution with a pipette, and then inject the sample according to the chromatography and mass spectrometry conditions in "1.3.1" and "1.3.2", and record the chromatogram.

[0095] The relative retention time RSDs of 11 components with relatively high responses (gallic acid, methyl gallate, oxidized paeoniflorin, ethyl gallate, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, wild sumac glycoside, and benzoyl oxidized paeoniflorin) were calculated and the results were 0.06%-0.20%, and the relative peak area RSDs were 1.04%-4.04%, indicating that the method has good repeatability. See Table 4 for details.

[0096] Table 4 Repeatability test (n=6)

[0097]

[0098] 2.2 Optimization of mass spectrometry conditions

[0099] Gallic acid, methyl gallate, and ethyl gallate met the requirements in both positive and negative modes during the optimization process; oxidized paeoniflorin, peonyside C, benzoyl oxidized paeoniflorin, and 1,2,3,6-MTG met the requirements in both negative mode (ESI - ) is much more efficient than the positive mode (ESI + ); During the optimization process, the positive and negative modes of paeoniflorin and 1,2,3,4,6-MTG were not good, but relative to ESI + The model is slightly better and it is easier to obtain [M+Na] + The precursor ion peak is not easy to obtain [M+H] + Ion peak, even if [M+H] + ion peak, but the abundance is low, it is not easy to obtain secondary fragment ions, which cannot meet the later qualitative and quantitative requirements. During the optimization process, 1,3,6-MTG - Relatively good in mode, easy to get [MH] + , while ESI + [M+Na] is easily obtained under this mode + The precursor ion peak, [M+H] + The ion abundance is low, and it is difficult to obtain secondary fragment ions; during the optimization process, ESI + The ion abundance is significantly stronger under the mode conditions.

[0100] The above 16 target components are flavonoids, polyphenols, monoterpenes and their glycosides and aromatic acid compounds, which have different structures and physicochemical properties. At the same time, considering that the mobile phase contains acidic medium, ESI can be further enhanced. + After a large number of experimental comprehensive analysis, the ionization efficiency of the positive-negative dual mode was selected for qualitative and quantitative research. Among them, oxidized paeoniflorin, peonyside C, benzoyl oxidized paeoniflorin, and 1,2,3,6-MTG were selected under ESI - Scan in ESI mode and select ESI for other components +Scanning in mode. After optimization, the corresponding product ions of each compound were as follows: gallic acid 81.1, 109.0, 152.9, 127.0; methyl gallate 153.0, 107.0, 126.0, 79.1; oxidized paeoniflorin 137.0, 92.9, 465.0, 292.5; paeoniflorin 117.1, 145.0, 117.1, 137.0; ethyl gallate 127.1, 152.9, 119.1, 70.7; paeoniflorin 341.0, 219.0, 342.1; 1,3,6-M TG174.9, 319.0, 386.7; benzoic acid79.1, 77.1, 51.1, 45.1; 1,2,3,6-MTG617.0, 169.0, 465.0, 313.0; 1,2,3,4,6-MTG471.1, 793.2; astragaloside287.0, 85.0, 69.1, 153.0; cosmosin271.0, 153.0, 119.0, 67.1; scutellarin271.0, 152.9, 119.0, 433.0; peonyside C 281.0, 137.0, 120.9, 93.0; benzoyloxypaeoniflorin 137.0, 93.0, 120.9, 377.3; kaempferol 69.1, 153.0, 121.0, 77.1. Among them, the one with the highest abundance was selected as the quantitative product ion, and the one with a higher abundance was selected as the qualitative product ion.That is to say, the ion pairs used for quantitative analysis of gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, rhubarb glycoside, peonyside C, benzoyl oxidized paeoniflorin, and kaempferol are: 17 1.0→109.0, 185.0→153.0, 495.2→137.0, 481.2→105.0, 199.1→127.1; 503.0→341.0; 659.1→174.9; 123.0→79.1; 787.1→617.0; 963.1→793.2; 449.0→287.0; 4 33.0→271.0; 579.2→271.0; 599.2→281.0; 599.2→137.0; 287.0→69.1; the ion pairs used for qualitative analysis are: 171.0→81.1, 185.0→107.0, 495.2→92.9, 481.2→133.0, 199.1→152.9 ;503.0→219.0;659.1→319.0;123.0→77.1;787.1→169.0;963.1→471.1;449.0→85.0;433.0→153.0;579.2→152.9;599.2→137.0;599.2→93.0;287.0→153.0.

[0101] 2.3 Column selection

[0102] In this experiment, four chromatographic columns, Agilent Eclipse plus C18 (4.6×100mm, 3.5μm), Agilent prosell120EC C18 (2.1×100mm, 2.7μm), Agilent Eclipse SB C18 (2.1×100mm, 1.8μm) and Agilentprosell 120SB C18 (2.1×100mm, 2.7μm), were selected to investigate the separation effect.

[0103] The results showed that the chromatographic peak separation effect was the best when using the Prosell 120SB C18 column, the column pressure was low, the peak shape was sharp and symmetrical, especially the peaks at 10-12min and 15-17min had significant effects, as shown in Figure 2. Figure 4 At the same time, the use of ultra-high performance liquid chromatography columns to establish chromatographic fingerprints greatly shortened the analysis time and improved the analysis efficiency. Therefore, this experiment selected the Agilent Prosell 120SB C18 ultra-high performance liquid chromatography column as the best chromatographic column.

[0104] 2.4 Selection of mobile phase

[0105] When selecting the mobile phase, when methanol-water was selected as the mobile phase, the peak width of the chromatographic peak was found to be large, the retention time was long, and the elution pressure was large according to the generated chromatogram. The tolerance of the chromatographic column used to establish the fingerprint spectrum was poor. When acetonitrile-water was selected as the mobile phase, the chromatogram showed that it had good elution ability. Then a small amount of formic acid (0.1%, pH = 3-4) was added to the aqueous phase. According to the chromatogram, the peak shape of the chromatographic peak was found to be sharper than before, and the response value and ionization efficiency were increased. However, when too much formic acid buffer (0.2%, pH = 2-3) was added, the ion response value under negative mode scanning would be suppressed. In addition, the gradient elution method can fully elute the extracted components, providing a strong guarantee for the establishment of the fingerprint spectrum. At the same time, more impurities can be eluted to prevent interference from subsequent samples, further improving specificity and sensitivity.

[0106] Therefore, this experiment selected acetonitrile-0.1% formic acid aqueous solution as the mobile phase for gradient elution, which has a good peak shape, appropriate elution time, and fewer interference factors.

[0107] 2.5 Selection of column temperature and flow rate

[0108] 25°C, 30°C, 35°C, and 40°C were the four column temperatures selected for this experiment. Experimental findings indicate that increasing column temperature advances peak elution time, decreases the resolution of adjacent peaks, and increases response values. While maintaining satisfactory sensitivity, a comprehensive analysis determined that 30°C was the optimal column temperature.

[0109] When selecting flow rates, a comparison revealed that while analysis time would be further shortened with increasing flow rates, the separation of target components would decrease. Components of similar polarity, such as cosmosin and scutellarin, were particularly prone to overlap, preventing ideal separation. Furthermore, higher flow rates in the mass spectrometer reduced ionization efficiency. Furthermore, due to the higher flow rates, the drying airflow and pressure also needed to be increased, resulting in poor reproducibility and increased costs. Therefore, a flow rate of 0.4 mL / min was ultimately selected as the optimal analysis flow rate.

[0110] 2.6 Selection of detection wavelength

[0111] This experiment investigated the detection wavelengths of 254nm, 230nm, 270nm, 360nm and 90-400nm, and determined the optimal detection wavelength through full scanning. Figure 5): Under the condition of 270nm, the chromatographic peak shows more comprehensive information, and the target components have better separation and response sensitivity, so this experiment uses 270nm as the detection wavelength. However, after sample determination, it was found that the chromatograms of the sea yellow and yellow crown samples (see Figure 6 ) showed inverted peaks (negative peaks). These peaks disappeared after removing the reference (Ref = 360, 100). This may be due to the sample solution background value being lower than the mobile phase absorbance response value, or it may be due to the varying response of the solvent at different UV wavelengths, resulting in significant absorption differences. Therefore, 270 nm and no reference injection were ultimately selected.

[0112] 2.7 Selection of extraction solvent

[0113] Through consulting the literature, we know that peony contains a lot of water-soluble impurities. Considering the impact of three wastes on the environment, methanol or ethanol was selected as the extraction solvent. When selecting the extraction solvent, firstly, this experiment selected 4 different volume concentrations (30%, 50%, 70%, 90%) of methanol water solution for extraction. The results showed that (see Figure 7 ): With the increase of the volume proportion of methanol solvent, the response signal of each chromatographic peak gradually increased, but the signal of individual peaks (such as 1,2,3,4,6-MTG and other components) began to decrease when the proportion reached 90%. Therefore, 70% methanol aqueous solution was selected as the extraction solvent to further explore the optimal extraction ratio.

[0114] The team then explored the extraction solvent using a less toxic 70% ethanol solution. The results showed that the signal response values ​​for ethyl gallate and 1,2,3,4,6-MTG components were higher than those for 70% methanol, but methyl gallate was not extracted.

[0115] In order to extract more components from peony flowers and more comprehensively reflect the material basis of the chemical composition of peony flowers, this experiment finally selected three solvent mixtures with different volume ratios of methanol:ethanol:water (6:1:3, 5:2:3, 4:3:3, 3:4:3, 2:5:3, 1:6:3) to extract peony flower samples. The results showed that (see Figure 8 ): With the increase of the proportion of ethanol solvent, the responses of wild rhubarb glycoside, 1,2,3,4,6-MTG and astragaloside first increased and then weakened, the responses of cosmos glycoside first weakened and then increased, the responses of methyl gallate gradually weakened, and the responses of ethyl gallate gradually increased.

[0116] Taking all factors into consideration, the extraction solvent ratio of methanol:ethanol:water = 4:3:3 (volume ratio) was found to produce the most appropriate response signals for the extracted components. The resulting chromatographic fingerprint more comprehensively reflects the quality of the peony flowers. Furthermore, a slightly higher methanol ratio can reduce background interference in the mass spectrometry. Therefore, a 4:3:3 (volume ratio) methanol:ethanol:water ratio was ultimately selected as the optimal extraction solvent.

[0117] 2.8 Selection of extraction method

[0118] Condensation reflux extraction and ultrasonic extraction were the two extraction methods selected for this experiment. The results showed that the reflux method had slightly higher extraction efficiency, but the extraction time was at least 1 hour, which was inefficient. Different extraction times also affected the efficiency, and the equipment required was also high. Ultrasonic extraction, on the other hand, was quick and simple, with shorter extraction times, higher efficiency, and less stringent temperature requirements. Moreover, compared to reflux extraction, it was less prone to temperature-related transformations of unstable components. Therefore, considering economic efficiency, ultrasonic extraction was selected for this experiment.

[0119] 2.9 Identification of characteristic components

[0120] In this study, the chemical components of peony petal extract (S1) were systematically characterized and identified through literature review, screening of the existing laboratory standard library, and mass spectrometry identification. A total of 26 characteristic chemical components were preliminarily identified, as shown in Table 5.

[0121] Given the varying concentrations of ingredients in the samples and the difficulty in obtaining standard samples, we selected 16 components as reference substances. We further characterized and characterized the identified components using qualitative methods such as mass spectrometry, retention time, UV, and spectroscopy. The results showed that 16 components were clearly present in the 12 peony varieties and could be screened as quality control indicators for quality evaluation and functional activity verification.

[0122] Table 5 Identification of characteristic components

[0123]

[0124]

[0125] 2.10 Establishment of fingerprints of 12 peony varieties and similarity evaluation

[0126] 2.10.1 Establishment and analysis of characteristic fingerprints

[0127] Weigh the peony flower samples numbered S1-S12 and prepare them into test solutions according to the methods of "1.3.3 and 1.3.5". Use the "chromatographic conditions of 1.3.1 and mass spectrometry conditions of 1.3.2" to perform chromatography and mass spectrometry detection on the mixed reference solution and the test solution, respectively. Record the DAD chromatograms and MS mass spectra of the mixed reference solution and the test solution. Load the recorded DAD chromatograms of the test solution into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012 Edition). Cut the data of the spectrum with the start time as 2 and the end time as 33. Take S1 as the reference characteristic fingerprint. After multi-point correction and full spectrum peak matching, generate the UPLC-DAD reference characteristic fingerprint of the S1 sample ( Figure 9 ) and UPLC-DAD characteristic fingerprints of 12 peony samples ( Figure 10 The results showed that the characteristic spectra of the 12 peony samples were significantly different, but there were still 22 common peaks (chromatograms), and 16 chromatographic peaks were identified.

[0128] By comparing the characteristic spectra, 22 common peaks were matched in the full spectrum peaks. According to the DAD chromatogram and MS mass spectrum of the reference solution, 16 of the common peak chromatograms were further identified as: gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, rhubarb glycoside, peonyside C, benzoyloxypeaeoniflorin, and kaempferol. The chromatographic peaks of all 16 components were detected in all samples. However, peak 7, 1,3,6-MTG, had very low responses in samples S1, S3, S4, S5, S6, and S7, reaching trace levels (below the limit of quantification). Peak 14, paeonia suffruticosa C, also had low responses in samples S5 and S8. Peak 16, kaempferol, also had low responses in samples S7, S8, and S12. The responses of the 16 components varied significantly across the peony samples, serving as quality control indicators for the 12 peony varieties. Content determination allowed for further comparison of these differences, enabling quality identification and evaluation.

[0129] 2.10.2 Similarity Evaluation

[0130] Using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012 Edition), we calculated the similarity between the 12 peony flower samples (S1-S12) and the control fingerprint (S1). When the similarity was at least 0.9, the samples were considered highly similar to S1. Conversely, when the similarity was less than 0.9, the samples were considered less similar to S1. The similarity results for each sample are shown in Table 6.

[0131] Table 6 Similarity of fingerprints of 12 peony species

[0132]

[0133] The results in Table 6 indicate that samples S3 (0.978), S4 (0.953), S9 (0.946), and S2 (0.905) have the highest similarity to sample S1, reaching values ​​above 0.9, indicating strong regularity between the chromatographic peaks. The remaining samples have lower similarities to S1, with S11 (0.459), S12 (0.653), and S10 (0.744) showing the lowest similarity. Samples S2 (Rou Furong, 0.985) and S3 (Xiangyu, 0.979) have the highest similarity to sample S9 (Danfeng). This indicates that the chemical composition of peony petals varies significantly between different varieties. The content of the characteristic components in the samples will be accurately determined later to further compare their differences.

[0134] 2.10.3 PCA analysis

[0135] PCA analysis was performed on the fingerprints of 12 varieties of peony samples, and the first and second principal components were projected to obtain the principal component projection diagram, see Figure 11 The cumulative contribution of the two principal components reached 46.36%, nearly 50%, encompassing almost half of the information. The first principal component contributed 28.97%, and the second principal component had an eigenvalue of 17.39%, indicating varying degrees of association and similarity between species within the same family. Since all 12 peony varieties originate from Luoyang, Henan, and share similar natural environments, the cultivars differ significantly, leading to significant differences. They can be generally divided into two categories: S1-S9 are grouped as the first, and S10, S11, and S12 are grouped as the second. Within the first category, significant differences exist. Analysis of flower color reveals that S7 and S8 are similar and both exhibit yellow flower color. S3 and S9 differ slightly and both exhibit white flower color with pink. S1 and S2 differ minimally and both exhibit pink flower color. Within the second category, differences are relatively small, but S11 exhibits a divergent trend from S10 and S12, indicating that Luoyang Red peonies exhibit distinct differences from the Black Lady and Shou'an Red varieties. This finding is consistent with the flower color analysis.

[0136] 2.10.4 HCA analysis

[0137] Based on the relative peak area, retention time and similarity of the common peaks of 12 varieties of peony samples, SPSS 26.0 statistical analysis software was used to conduct a systematic cluster analysis with the inter-group linkage method and squared Euclidean distance as the measurement interval when the number of samples was 12.

[0138] According to the cluster dendrogram (see Figure 12). When the inter-cluster distance is 5, the samples can be divided into four categories: S1, S2, S3, S4, and S9 are in one category, S5, S6, S7, and S8 are in one category, S10 and S12 are in one category, and S11 is a separate category. When the inter-cluster distance is 12.5, the samples can be divided into two categories: S10, S11, and S12 are in one category, and the rest are in one category. This result is consistent with the classification divided by principal component analysis.

[0139] The above results show that the UPLC-MS characteristic fingerprint of peony flowers established in this study can effectively distinguish and identify peony samples, and can provide a reference for the comprehensive evaluation of the quality of peony flowers.

[0140] 2.11 Determination of 16 Characteristic Components in Different Peony Petals

[0141] 2.11.1 System adaptability test

[0142] Accurately weigh 2.00 mg of 16 standards (gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, rhusin, peonyside C, benzoyloxypeaeoniflorin, and kaempferol) and place them in 10-ml brown volumetric flasks. Add 5 mL of chromatographic methanol to prepare individual reference stock solutions, bring to volume, shake well, and set aside. Then, accurately pipette appropriate amounts of the individual reference stock solutions to prepare mixed standard solutions at concentrations of approximately 40.00 or 20.00 μg / ml. These solutions are then serially diluted with the initial mobile phase to prepare a series of mixed standard solutions.The initial ratio of the mobile phase was acetonitrile: 0.1% formic acid water = 2:98; the concentrations of gallic acid were: 21.19, 8.48, 4.24, 2.12, 1.06, 0.42, 0.21 μg / mL; the concentrations of methyl gallate were: 22.34, 8.94, 4.47, 2.23, 1.12, 0.45, 0.22 μg / mL; the concentrations of oxidized paeoniflorin were: 19.19, 7.68, 3.84, 1.92, 0.96, 0.38, 0.19 μg / mL; the concentrations of lactone glycosides were: 41.08, 16.43, 8.22, 4.11, 2. 05, 0.82, 0.41μg / mL; ethyl gallate: 21.03, 8.41, 4.21, 2.10, 1.05, 0.42, 0.21μg / mL; paeoniflorin: 39.44, 15.78, 7.89, 3.94, 1.97, 0.79, 0.39μg / mL; 1,3,6-MTG: 19.58, 7.83, 3.92, 1.96, 0.98, 0.39, 0.20μg / mL; benzoic acid: 40.96, 16.38, 8.19, 4.10, 2.05, 0.82, 0.41μg / mL; 1,2,3,6-MTG were: 21.56, 8.62, 4.31, 2.16, 1.08, 0.43, 0.22μg / mL; 1,2,3,4,6-MTG were: 20.06, 8.03, 4.01, 2.01, 1.00, 0.40, 0.20μg / mL; astragaloside were: 20.70, 8.28, 4.14, 2.07, 1.03, 0.41, 0.21μg / mL; cosmosin were: 19.99, 8.00, 4.00, 2.00, 1.00, 0.4 The serum levels of benzoyl peroxidase and kaempferol were 20.29, 8.11, 4.06, 2.03, 1.01, 0.41 and 0.20 μg / mL, respectively; the serum levels of benzoyl peroxidase and kaempferol were 21.59, 8.63, 4.32, 2.16, 1.08, 0.43 and 0.22 μg / mL, respectively; the serum levels of benzoyl peroxidase and kaempferol were 18.82, 7.53, 3.76, 1.88, 0.94, 0.38 and 0.19 μg / mL, respectively; the serum levels of kaempferol and kaempferol were 18.62, 7.45, 3.72, 1.86, 0.93, 0.37 and 0.19 μg / mL, respectively.

[0143] 2.0 μL of each of the above series of mixed standard solutions and the Zhaofen peony flower test solution numbered S1 were accurately aspirated and injected according to the chromatographic conditions and mass spectrometry conditions under "1.3.1" and "1.3.2". The 16 target components and their adjacent chromatographic peaks were well separated, and the theoretical plate numbers (N) were 8510, 223200, 303370, 622710, 100150, 567830, 251840, and 469070, respectively. , 985470, 1266220, 1268470, 1288490, 1502250, 1630930, 1416510, 3829190, except for gallic acid No. 1, the N values ​​of the other components are much greater than 7000. At the same time, the injection volume of the method is small, which reduces the chromatographic peak width and further increases the column efficiency. This shows that the UPLC method and ultra-high performance chromatographic column used in this experiment have high column efficiency, strong separation ability and good separation effect. In addition, this experiment also uses dynamic MRM mode for scanning, which makes qualitative and quantitative analysis more accurate, the system adaptability is stronger, and there is no interference with the target analyte. DAD and TIC chromatograms are shown respectively. Figure 1 and Figure 3 The MRM spectra of the 16 target components are shown in Figure 13 2.11.2 Linear relationship and detection limit

[0144] Accurately pipette the series of mixed standard solutions prepared under the above “2.11.1”. Inject 2.0 μL of each solution according to the chromatographic and mass spectrometric conditions under “1.3.1” and “1.3.2”, and measure the peak area. -1 ) as the abscissa and the peak area (Y) of each characteristic component in the mixed standard solution as the ordinate. Linear regression analysis was performed and a standard curve was plotted. The injection volume at which the peak area was approximately 3 times the noise level (S / N = 3) was defined as the lower limit of detection (LOD), and the injection volume at which the peak area was approximately 10 times the noise level (S / N = 10) was defined as the lower limit of quantification (LOQ). The results showed that the 16 target components exhibited good linearity within the selected concentration range, with low limits of detection, excellent stability, and enhanced accuracy. See Table 7 for details.

[0145] Table 7 Linear relationship, LOD and LOQ determined by external standard method

[0146]

[0147]

[0148] 2.11.3 Precision test

[0149] Accurately pipette the mixed reference solution (containing 2.12 μg·mL gallic acid) -1 , methyl gallate 2.23 μg·mL -1, oxidized paeoniflorin 1.92 μg·mL -1 , paeoniflorin 4.11 μg·mL -1 , ethyl gallate 2.10 μg·mL -1 , paeoniflorin 3.94 μg·mL -1 1,3,6-MTG 1.96 μg·mL -1 , benzoic acid 4.10 μg·mL -1 1,2,3,6-MTG 2.16 μg·mL -1 1,2,3,4,6-MTG 2.01 μg·mL -1 , astragaloside 2.07 μg·mL -1 , cosmosin 2.00 μg·mL -1 , wild sumac glycoside 2.03 μg·mL -1 , peonyside C 2.16μg·mL -1 , benzoyloxypaeoniflorin 1.88 μg·mL -1 , kaempferol 1.86 μg·mL -1 )2.0μL, and injected 6 times continuously according to the chromatographic and mass spectrometric conditions under "1.3.1" and "1.3.2". The RSD of the peak area of ​​16 target components was measured to be 0.12%-1.10%. The results showed that the instrument had good precision. See Table 8 for details.

[0150] Table 8 Precision test (n=6)

[0151]

[0152] Note: 1: Gallic acid; 2: Methyl gallate; 3: Oxidized paeoniflorin; 4: Peoniflorin; 5: Ethyl gallate; 6: Paeoniflorin; 7: 1,3,6-MTG; 8: Benzoic acid; 9: 1,2,3,6-MTG; 10: 1,2,3,4,6-MTG; 11: Astragaloside; 12: Cosmoside; 13: Rhus sutchuenensis; 14: Paeoniflorin C; 15: Benzoyloxypeeoniflorin; 16: Kaempferol

[0153] 2.11.4 Stability test

[0154] Accurately weigh 0.50 g of Zhaofen Peony Flower No. S1, prepare the test solution according to the method under "1.3.5", prepare three copies in parallel, accurately draw 2.0 μL, and inject and measure at 0, 1, 2, 4, 8, 12, and 24 h according to the chromatographic and mass spectrometric conditions under "1.3.1" and "1.3.2", respectively. The stability of the 16 target components was measured to be 97.99%-99.89%, 98.52%-100.60%, 99.03%-99.84%, 97.20%-100.03%, 98.81%-100.38%, respectively. %, 99.57%-99.90%, 96.68%-108.79%, 98.83%-100.19%, 98.91%-100.68%, 99.80%-100.18%, 99.14%-100.29%, 99.43%-100.06%, 98.27%-99.96%, 97.31%-99.58%, 98.61%-100.22%, 96.72%-100.43%, and the RSDs were all less than 7%, indicating that the test solution had good stability within 24 h. See Table 9 for details.

[0155] Table 9 Stability test (n=3)

[0156]

[0157]

[0158] 2.11.5 Repeatability test

[0159] Accurately weigh 0.50 g of the Zhaofen peony flower sample numbered S1, prepare 6 parallel portions, prepare the test solution according to the method under "1.3.5", accurately aspirate 2.0 μL of each sample, and inject and determine according to the chromatographic and mass spectrometric conditions under "1.3.1" and "1.3.2". The average contents of the 16 target components were 2179.92, 669.45, 712.60, 175.42, 355.31, 1644.41, 14.67, 629.28, 362.11, 7493.55, 4138.31, 4380.96, 7466.67, 160.54, 847.24, and 75.65 mg·kg, respectively. -1 , RSD was 0.39%-3.14%, indicating that this method had good repeatability, as shown in Table 10.

[0160] Table 10 Repeatability test

[0161]

[0162]

[0163] Note: 1: Gallic acid; 2: Methyl gallate; 3: Oxidized paeoniflorin; 4: Peoniflorin; 5: Ethyl gallate; 6: Paeoniflorin; 7: 1,3,6-MTG; 8: Benzoic acid; 9: 1,2,3,6-MTG; 10: 1,2,3,4,6-MTG; 11: Astragaloside; 12: Cosmoside; 13: Rhus sutchuenensis; 14: Paeoniflorin C; 15: Benzoyloxypeeoniflorin; 16: Kaempferol

[0164] 2.11.6 Addition recovery test

[0165] Accurately weigh 0.25 g of Zhaofen peony flower numbered S1 and place it in a conical flask. Accurately add appropriate amounts of mixed control solutions of low, medium and high concentrations containing 16 target components to prepare quality control samples. Prepare 3 replicates for each addition value and process according to the method under the test sample preparation item. Accurately pipette 2.0 μL of each solution and inject and measure according to the chromatographic and mass spectrometry conditions under "1.3.1" and "1.3.2". Calculate the measured concentration and the measured value from the standard curve. Calculate the recovery and precision as (measured value - background value) / added value × 100%. The results are shown in Table 11. The data showed that the recovery rates of the method were 100.79% to 101.17%, 98.99% to 102.30%, 95.68% to 101.30%, 99.70% to 107.73%, 99.69% to 103.32%, 99.54% to 100.95%, 105.69% to 109.19%, 93.06% to 98.32%, and 96. The recoveries of the 16 target components in the samples met the requirements under these experimental conditions, indicating good precision.

[0166] Table 11 Addition recovery test (n=3)

[0167]

[0168]

[0169] 2.11.7 Content determination and result analysis

[0170] Accurately weigh 0.50 g of peony flower samples from 12 varieties and prepare them in triplicate. Prepare the test solution according to the method in "1.3.5" and accurately pipette 2.0 μL of each sample. Determine the peak area of ​​each characteristic component using the chromatographic and mass spectrometric conditions in "1.3.1" and "1.3.2." Substitute the peak area into the standard curve equation to calculate the content of the 16 characteristic components in the peony flower samples. The results are shown in Table 12.

[0171] Table 12 Content determination results (n=3)

[0172]

[0173] Note: 1: Gallic acid; 2: Methyl gallate; 3: Oxidized paeoniflorin; 4: Peoniflorin; 5: Ethyl gallate; 6: Paeoniflorin; 7: 1,3,6-MTG; 8: Benzoic acid; 9: 1,2,3,6-MTG; 10: 1,2,3,4,6-MTG; 11: Astragaloside; 12: Cosmoside; 13: Rhusin; 14: Paeoniflorin C; 15: Benzoyloxypeeoniflorin; 16: Kaempferol. TR: Trace qualitative detection.

[0174] The content of 16 components in 12 varieties of peony flowers (S1-S12) was determined. It was found that all samples contained 16 components with significant differences in content, ranging from 0.98 to 3.80 mg·g -1 , 0.30-3.34mg·g -1 , 0.19-1.75mg·g -1 , 0.012-0.32mg·g -1 , 0.20-0.62mg·g -1 , 0.42-5.61mg·g -1 , 0.014-0.044mg·g -1 , 0.32-2.02mg·g -1 , 0.36-1.23mg·g -1 , 5.23-14.12mg·g -1 , 0.75-7.69mg·g -1 , 1.32-4.77mg·g -1 , 4.31-9.48mg·g -1 , 0.0049-0.20mg·g -1 , 0.041-0.86mg·g -1 , 0.012-0.74mg·g -1Among the 12 varieties, the contents of paeoniflorin, 1,3,6-MTG, paeoniflorin C, benzoyloxypaeoniflorin, and kaempferol were relatively low, especially 1,3,6-MTG. S11 had the highest content (0.044 mg·g -1 ), S1, S3, S4, S5, S6, and S7 were qualitatively detected in trace amounts (below the limit of quantification), and the content levels of the remaining samples were similar. Gallic acid was found in the highest levels in samples S11 and S5, reaching 3.80 mg·g -1 and 3.75 mg·g -1 , methyl gallate in S6 (3.34 mg g -1 ) samples had the highest content, and oxidized paeoniflorin was found in S3 (1.75 mg·g -1 ) samples had the highest content, and the content of paeoniflorin was highest in S4 (0.32 mg·g -1 ) samples had the highest content, and ethyl gallate was found in S6 (0.62 mg·g -1 ) had the highest content, and paeoniflorin was found in S10 (5.51 mg·g -1 ) and S11 (5.61 mg·g -1 ) is higher in S7 (2.02 mg·g -1 ) was the highest in S4, S11, and S12, and the contents of 1,2,3,6-MTG were relatively high and comparable, reaching 1.22, 1.23, and 1.15 mg·g, respectively. -1 The content of 1,2,3,4,6-MTG in S5 was the highest, reaching 14.12 mg·g -1 , followed by S6 and S11 (12.61 mg·g -1 , 12.50 mg g -1 ), the content of astragalin in S5 was the highest, reaching 7.69 mg·g -1 , cosmosin in S4 (4.77 mg g -1 ) was the highest, followed by S1 (4.34 mg·g -1 ), wild sumac in S2 (9.52 mg g -1 ) had the highest content, followed by S4 (9.48 mg·g -1 ), the content of peonyside C was higher in S10, but was detected below the limit of quantification in S5 and S8, and benzoyloxypeonyside was higher in S1 (0.86 mg·g -1 ) and S12 (0.71 mg·g -1 ) had a higher content, and kaempferol was present in S9 (0.74 mg·g -1 ) had the highest content, and S7 had the lowest content, only 0.012 mg·g -1Whether this result is related to pollen collection and storage, and whether the stamens and pistils also contain such ingredients, requires further research and comparison.

[0175] Based on the above comparative analysis, cosmosin and benzoyloxypeoniflorin can be used as identification quality markers for Zhaofen peony, benzoic acid can be used as identification quality marker for Haihuang peony, wild sumac glycoside can be used as identification quality marker for Roufurong peony, kaempferol can be used as identification quality marker for Danfeng peony, oxidized paeoniflorin can be used as identification quality marker for Xiangyu peony, paeoniflorin and moutanside C can be used as identification quality markers for Heifu Lady peony, 1,2,3,6-MTG and benzoyloxypeoniflorin can be used as identification quality markers for Shouanhong peony, and the internal content of peony is 4. Ester glycosides, 1,2,3,6-MTG, cosmosin and rhubarb glycoside can be used as identification quality markers of Jingyu peony; gallic acid, paeoniflorin, 1,3,6-MTG, 1,2,3,6-MTG and 1,2,3,4,6-MTG can be used as identification quality markers of Luoyanghong peony; gallic acid, 1,2,3,4,6-MTG and astragalin can be used as identification quality markers of Xugang peony; methyl gallate, ethyl gallate and 1,2,3,4,6-MTG can be used as identification quality markers of Huawang peony.

[0176] 3 Conclusion

[0177] This study established a UPLC-MS characteristic chromatographic fingerprint for the petals of 12 peony varieties. The fingerprints demonstrated excellent resolution and reproducibility, effectively characterizing the chemical constituents of these different peony varieties. Twenty-six components were initially identified through literature review, screening of the laboratory's existing standard library, and qualitative mass spectrometry analysis. Full-spectrum fingerprint peak matching revealed 22 common peaks across the 12 peony varieties. These 16 components were further verified and assigned using reference material retention time, mass spectra, and spectra. Instrument precision was evaluated, along with sample stability and reproducibility, with favorable results. Similarity analysis was performed on the imported characteristic chromatographic fingerprints of the 12 peony varieties using similarity evaluation software, and PCA and HCA analyses were performed using SPSS software. The results showed significant differences in the chemical composition of the 12 peony varieties. Samples S2, S3, S4, and S9 were most similar to sample S1, with similarities exceeding 0.9. Samples S11, S12, and S10 were least similar to sample S1, with similarities less than 0.8. Sample S2 (Rou Furong, 0.985) and S3 (Xiangyu, 0.979) were most similar to sample S9 (Danfeng). PCA analysis showed that the samples could be divided into two categories: S1-S9 were grouped as the first, and S10, S11, and S12 were grouped as the second. Among them, there were significant differences within the first category. Combined with the analysis of flower color, S7 and S8 were similar and both had yellow flower colors. S3 and S9 had smaller differences and both had white flower colors with pink. S1 and S2 had the smallest difference and both had pink flower colors. The second category showed smaller differences, but S11 showed a discrete trend from S10 and S12, indicating that Luoyang Red peonies still have significant differences compared to Black Lady and Shou'an Red varieties, and the results were consistent with those of flower color. HCA analysis showed that when the inter-class distance was 5, the samples could be divided into three categories: S1, S2, S3, S4, and S9 were in one category, S5, S6, S7, and S8 were in another category, and S10, S11, and S12 were in another category. When the inter-class distance was 12.5, the samples could be divided into two categories: S10, S11, and S12 were in one category, and the rest were in another category. This result is consistent with the classification divided by principal component analysis.

[0178] Therefore, the UPLC-MS fingerprint of peony flowers established in this study can effectively distinguish and authenticate peony samples. The differences in the material basis characteristics of the new chemical components of peony petals between different varieties can more comprehensively reflect the quality of peony flowers and provide a reliable method for identifying different varieties or batches of peony flower samples. Furthermore, this study established a UPLC-MS / MS content determination method for 16 target components (comprising five categories: flavonoids, polyphenols, monoterpenes and their glycosides, phenolic acids, and aromatic acids). Using UPLC separation, retention time, MS ion pairs, and DAD data for simultaneous qualitative analysis, and MS ion quantification, this method provides more accurate and efficient qualitative and quantitative results. By comparing the content differences of these 16 components among 12 peony varieties, quality markers were further screened. This method provides data and methodological references for quality control and standardization of different peony varieties, and also provides a research foundation for in-depth research and development of the functional value of peony flowers, with scientific guidance significance.

Claims

1. A method for establishing a UPLC-MS characteristic chromatographic fingerprint of peony flowers, characterized in that: The steps include: 1) Prepare mixed reference solution: 16 characteristic components, gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-tri-O-galloyl-β-D-glucose, benzoic acid, 1,2,3,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, astragaloside, cosmosin, rhusin, peonyside C, benzoyloxidized paeoniflorin, and kaempferol, were accurately weighed respectively and dissolved in solvent to prepare reference substance mother solution; appropriate amount of reference substance mother solution was accurately measured and placed in a volumetric flask, the volume was made up to the mark with the above solvent, and mixed to obtain a mixed reference substance solution; 2) Prepare the test solution: Accurately weigh 0.4-0.6 g of peony flower samples of different varieties into a stoppered conical flask, add 40-60 mL of a methanol-ethanol-water mixture, weigh the sample, perform ultrasonic extraction, weigh the sample again, make up the lost weight with the methanol-ethanol-water mixture, shake well, filter through an organic filter membrane, and place in a sample injection bottle for testing. 3) Perform chromatography and mass spectrometry on the mixed reference solution and the test solution, respectively, and record the DAD chromatograms, UV spectra, and MS spectra of the mixed reference solution and the test solution. Load the recorded chromatograms of the test solution into the Chinese medicine chromatographic fingerprint similarity evaluation software, cut the spectrum with the start time as 2 and the end time as 33, and use S1 as the reference spectrum. After multi-point calibration and full spectrum peak matching, generate the UPLC control characteristic fingerprint spectrum; The chromatographic conditions in step 3) were as follows: chromatographic column: Agilent SB C18, mobile phase gradient elution: phase A: acetonitrile, phase B: 0.1% formic acid in water: 0-8 min, 2% A→12% A; 8-11 min, 12% A→16% A; 11-15 min, 16% A→20% A; 15-20 min, 20% A→25% A; 20-24 min, 25% A→45% A; 24-30 min, 45% A→65% A; 30-33 min, 65% A→90% A; 33-36 min, 90% A→2% A; 36-40 min, 2% A; flow rate: 0.4 mL / min, injection volume: 2 μL, column temperature: 30°C, wavelength: 270 nm; In step 3), the mass spectrometry conditions are as follows: ion source: ESI source, scan mode: multiple reaction monitoring mode and full scan mode, positive and negative modes: ESI +- , drying gas temperature: 350℃, spray gas pressure: 275.8 kPa, drying gas flow rate: 11 L / min, capillary voltage: ESI + 4.0kV, ESI - 3.5 kV.

2. The method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower according to claim 1, characterized in that: In step 1), the solvent is methanol; in the mixed reference solution, the mass concentrations of gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-MTG, benzoic acid, 1,2,3,6-MTG, 1,2,3,4,6-MTG, astragaloside, cosmosin, rhubarb glycoside, peonyside C, benzoyl oxidized paeoniflorin, and kaempferol are 8.52 μg / ml, 9.12 μg / ml, 7.72 μg / ml, 8.32 μg / ml, 8.44 μg / ml, 8.04 μg / ml, 7.96 μg / ml, 8.20 μg / ml, 8.80 μg / ml, 8.08 μg / ml, and 8.36 μg / ml, respectively. , 8.16μg / ml, 8.28μg / ml, 8.72μg / ml, 7.68μg / ml, 7.80μg / ml.

3. The method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower according to claim 1, characterized in that: In step 2), the peony pollen sample is obtained by the following steps: fresh peony flowers of different varieties are separated, the stamens and sepals are placed on A4 paper, and freeze-dried in a freeze dryer. After taking out, the petals are individually crushed into powder and sieved to obtain peony pollen.

4. The method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower as claimed in claim 3, characterized in that: In step 2), 0.5 g of peony pollen samples of different varieties are accurately weighed into a stoppered conical flask, 50 mL of a methanol-ethanol-water mixture is added, the weight is weighed, and ultrasonic extraction is performed at room temperature for 25-35 minutes. The weight is weighed again, and the lost weight is supplemented with a methanol-ethanol-water mixture. The mixture is shaken and filtered through a 0.22 μm organic filter membrane; the volume ratio of methanol: ethanol: water is 3-5:2-4:

3.

5. The method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower according to claim 1, characterized in that: Step 3) The full spectrum peak matched 22 common chromatographic peaks, and 16 of the common peak chromatograms were further identified based on the DAD chromatogram, UV spectrum and MS mass spectrum of the reference solution as follows: gallic acid, methyl gallate, oxidized paeoniflorin, paeoniflorin, ethyl gallate, paeoniflorin, 1,3,6-tri-O-galloyl-β-D-glucose, benzoic acid, 1,2,3,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, astragaloside, cosmosin, wild sumacin, peonyside C, benzoyloxypeneoniflorin, and kaempferol.

6. The method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower according to claim 1, characterized in that: The method further includes step 4), wherein the similarity between the test solution and the reference spectrum of S1 is calculated by using the similarity evaluation software of the Chinese medicine chromatographic fingerprint. When the similarity is not less than 0.9, it is considered to have a high similarity with S1.

7. The method for establishing the UPLC-MS characteristic chromatographic fingerprint of peony flower according to claim 1, characterized in that: It also includes step 5), using principal component analysis to perform principal component analysis on the fingerprint maps of different varieties of peony flower samples, and using cluster analysis to perform cluster analysis on the fingerprint map data of different varieties of peony flower samples; using the relative peak area, retention time and similarity of the common peaks in the test solution as the data basis, applying SPSS statistical analysis software, selecting the inter-group linkage method, and the square Euclidean distance as the measurement interval, selecting the number of samples, and performing systematic cluster analysis, and classifying the samples according to the cluster dendrogram and the inter-class distance.

8. A method for determining the contents of 16 characteristic components in peony flowers, comprising five types of components: flavonoids, polyphenols, monoterpenes and their glycosides, phenolic acids, and aromatic acid compounds, characterized in that: The steps include: 1) Prepare mixed reference solution: Same as claim 1; then dilute the initial mobile phase in series to prepare a series of mixed standard solutions; 2) Prepare the test solution: same as claim 1; 3) Subject the series of mixed standard solutions to chromatography and mass spectrometry, record the peak areas, and plot a standard curve with the concentration of each mixed standard solution as the abscissa and the peak area of ​​each characteristic component in the mixed standard solution as the ordinate; The test solution was subjected to chromatography and mass spectrometry detection and the peak area of ​​each characteristic component was recorded, substituted into the standard curve, and the content of each characteristic component was calculated; The chromatographic conditions in step 3) were as follows: chromatographic column: Agilent SB C18, mobile phase gradient elution: phase A: acetonitrile, phase B: 0.1% formic acid in water: 0-8 min, 2% A→12% A; 8-11 min, 12% A→16% A; 11-15 min, 16% A→20% A; 15-20 min, 20% A→25% A; 20-24 min, 25% A→45% A; 24-30 min, 45% A→65% A; 30-33 min, 65% A→90% A; 33-36 min, 90% A→2% A; 36-40 min, 2% A; flow rate: 0.4 mL / min, injection volume: 2 μL, column temperature: 30°C, wavelength: 270 nm; In step 3), the mass spectrometry conditions are as follows: ion source: ESI source, scan mode: multiple reaction monitoring mode and full scan mode, positive and negative modes: ESI +- , drying gas temperature: 350℃, spray gas pressure: 275.8 kPa, drying gas flow rate: 11 L / min, capillary voltage: ESI + 4.0kV, ESI - 3.5 kV.

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