Analysis and HPLC fingerprint map quality control detection method for chemical components in peony root wood core

Abstract

The invention discloses a chemical component analysis and HPLC fingerprint map quality control detection method for peony roots and wood cores, and belongs to the technical field of traditional Chinese medicine quality control. Comprising the following steps: firstly preparing a test sample and a mixed reference substance solution, then establishing an HPLC fingerprint map through HPLC detection, further identifying the components through an HPLC-MS / MS method, identifying that 39 components contain 33 common peaks, and finally carrying out quantitative analysis on 22 chemical components with good separation degrees by adopting an HPLC standard curve method. The HPLC fingerprint spectrum and HPLC-MS / MS component analysis method of the peony root wood cores are established, simultaneous quantitative detection of 22 chemical components is realized, the method has the advantages of high precision, high stability, high repeatability, good recovery rate and the like, a foundation is laid for quality control, development and utilization of the peony root wood cores, and the method has wide application prospects. The method solves the problem of lack of peony root wood core quality standard and component quantitative detection methods in the prior art.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality control technology, specifically to a method for chemical component analysis of peony root and its HPLC fingerprint quality control detection. Background Technology

[0002] The root heart of the peony is a plant of the Ranunculaceae family, peony. Paeonia suffruticosa The woody heartwood of the dried root of Andr. This product is derived from Moutan Cortex, listed in Part I of the Pharmacopoeia of the People's Republic of China (2020 edition), which is the root bark of the Ranunculaceae plant, Paeonia suffruticosa. Paeonia suffruticosa Andr., its roots are dug up in autumn, the fine roots and soil are removed, the rough bark is scraped off, and the woody core is extracted, dried, and becomes the woody core in the root of Peony (WCRP). The Chinese Pharmacopoeia records that peony bark has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis; current research shows that peony bark contains terpenes and their glycosides, phenols and their glycosides, organic acids and their esters, flavonoids, tannins, and other components, which have anti-inflammatory, anti-tumor, and lipid metabolism regulating effects.

[0003] However, WCRP is a processing byproduct of peony bark (scraped peony bark). Currently, there are no medicinal material standards or systematic studies. It is only speculated that it has the same chemical composition and similar pharmacological effects as peony bark. There is an urgent need to establish an HPLC fingerprint spectrum of WCRP and a quantitative detection method for its main chemical components. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a method for chemical composition analysis and HPLC fingerprint quality control detection of peony root core, thereby solving the existing problem of lacking quality standards and quantitative detection methods for peony root core.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: In a first aspect, the present invention provides an HPLC fingerprint quality control detection method for peony root heartwood, wherein the HPLC fingerprint is established through the following steps: (1) Take peony root heartwood powder and add it to methanol solution to prepare test solution; (2) Take 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallo-paeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neo-diosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol as reference standards, and add methanol to prepare a mixed reference solution; (3) The test solution obtained in step (1) and the mixed reference solution obtained in step (2) were subjected to HPLC detection and analysis to obtain the HPLC fingerprint of the peony root heartwood.

[0006] Furthermore, the preparation method of the test solution in step (1) specifically includes the following steps: First, mix the peony root heartwood powder with a methanol solution of 60%-80% by volume and weigh it. Then, sonicate it for 30-60 minutes, cool it, and weigh it again. Finally, make up the lost weight with a methanol solution of 60%-80% by volume, shake it well, filter it, and take the filtrate as the test solution. The concentration of peony root heartwood powder in the test solution was 8-12 mg / mL.

[0007] Preferably, the peony root heartwood powder is first mixed with a 70% methanol solution and weighed; then ultrasonically treated for 45 min, cooled and weighed again; finally, the weight loss is made up with a 70% methanol solution, shaken well, filtered, and the filtrate is taken as the test solution. The concentration of peony root heartwood powder in the test solution was 10 mg / mL.

[0008] Further, in step (2), the concentrations of 1-galloglucoside, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloglucoside, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloglucoside, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol in the mixed reference solution are all 0.8-1.2 mg / mL.

[0009] Preferably, in step (2), the concentrations of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol in the mixed reference solution are all 1.0 mg / mL.

[0010] Furthermore, the chromatographic column used for HPLC detection in step (3) is C18. 18 The column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The conditions for HPLC detection are as follows: Acetonitrile is the mobile phase A, and a 0.1% (v / v) aqueous solution of formic acid is the mobile phase B; The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The conditions for gradient elution are: 0-10 min, the volume fraction of mobile phase A is 7%-10%; 10-35 min, the volume fraction of mobile phase A is 10%-17%; 35-80 min, the volume fraction of mobile phase A is 17%-21%; 80-90 min, the volume fraction of mobile phase A is 21%-40%; 90-100 min, the volume fraction of mobile phase A is 40%-7%; 100-110 min, the volume fraction of mobile phase A is 7%.

[0011] A second aspect of the present invention provides a method for analyzing the chemical components in the root heartwood of peony, which uses HPLC-MS / MS to analyze the chemical components in the root heartwood of peony, and includes the following steps: Step 1: Take peony root heartwood powder and add it to methanol solution to prepare the test solution; Step 2: Take 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol as reference standards, and add methanol to prepare a mixed reference solution; Step 3: Perform HPLC-MS / MS analysis on the test solution obtained in Step 1 and the mixed reference solution obtained in Step 2 to complete the chemical composition analysis of peony root wood.

[0012] Furthermore, the preparation method of the test solution in step 1 specifically includes the following steps: First, mix the peony root heartwood powder with a methanol solution of 60%-80% by volume and weigh it. Then, sonicate it for 30-60 minutes, cool it, and weigh it again. Finally, make up the lost weight with a methanol solution of 60%-80% by volume, shake it well, filter it, and take the filtrate as the test solution. The concentration of peony root heartwood powder in the test solution was 8-12 mg / mL.

[0013] Preferably, the peony root heartwood powder is first mixed with a 70% methanol solution and weighed; then ultrasonically treated for 45 min, cooled and weighed again; finally, the weight loss is made up with a 70% methanol solution, shaken well, filtered, and the filtrate is taken as the test solution. The concentration of peony root heartwood powder in the test solution was 10 mg / mL.

[0014] Furthermore, in step 2, the concentrations of 1-galloglucoside, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol neoglycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloglucoside, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloglucoside, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol in the mixed reference solution are all 0.8-1.2 mg / mL.

[0015] Preferably, in step 2, the concentrations of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol in the mixed reference solution are all 1.0 mg / mL.

[0016] Furthermore, the chromatographic column used for HPLC detection in the HPLC-MS / MS method is C18. 18 The column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The conditions for HPLC detection are as follows: Acetonitrile is the mobile phase A, and a 0.1% (v / v) aqueous solution of formic acid is the mobile phase B; The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The conditions for gradient elution are: 0-10 min, the volume fraction of mobile phase A is 7%-10%; 10-35 min, the volume fraction of mobile phase A is 10%-17%; 35-80 min, the volume fraction of mobile phase A is 17%-21%; 80-90 min, the volume fraction of mobile phase A is 21%-40%; 90-100 min, the volume fraction of mobile phase A is 40%-7%; 100-110 min, the volume fraction of mobile phase A is 7%.

[0017] Furthermore, the mass spectrometry conditions in the HPLC-MS / MS method are as follows: Ion source: electrospray ionization source; ESI scanning mode: positive and negative ion switching scan; scanning range: 100.0-1500.0 m / z; collision gas: argon; sheath gas: nitrogen; auxiliary gas: nitrogen.

[0018] A third aspect of the present invention provides a method for quantitative analysis of 22 chemical components in the root heartwood of peony, comprising the following steps: S1: Take peony root heartwood powder and add it to methanol solution to prepare the test solution; S2: Take 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol as reference standards, and add methanol to prepare a mixed reference solution; S3: First, the mixed reference solution obtained in S2 is serially diluted and detected by HPLC. A standard curve is plotted with the mass concentration of each component as the abscissa and the peak area of ​​each component as the ordinate. Then, the test solution obtained in S1 is detected by HPLC under the same conditions. Finally, the content of each component in the root core of peony is calculated according to the standard curve.

[0019] Furthermore, in S1, the preparation method of the test solution specifically includes the following steps: First, mix the peony root heartwood powder with a methanol solution of 60%-80% by volume and weigh it. Then, sonicate it for 30-60 minutes, cool it, and weigh it again. Finally, make up the lost weight with a methanol solution of 60%-80% by volume, shake it well, filter it, and take the filtrate as the test solution. The concentration of peony root heartwood powder in the test solution was 8-12 mg / mL.

[0020] Preferably, the peony root heartwood powder is first mixed with a 70% methanol solution and weighed; then ultrasonically treated for 45 min, cooled and weighed again; finally, the weight loss is made up with a 70% methanol solution, shaken well, filtered, and the filtrate is taken as the test solution. The concentration of peony root heartwood powder in the test solution was 10 mg / mL.

[0021] Furthermore, in S2, the concentrations of 1-galloglucoside, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol neoglycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloglucoside, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloglucoside, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol in the mixed reference solution were all 0.8-1.2 mg / mL.

[0022] Preferably, in S2, the concentrations of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol in the mixed reference solution are all 1.0 mg / mL.

[0023] Furthermore, the chromatographic column used for HPLC detection in S3 is C12. 18 The column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The conditions for HPLC detection are as follows: Acetonitrile is the mobile phase A, and a 0.1% (v / v) aqueous solution of formic acid is the mobile phase B; The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The conditions for gradient elution are: 0-10 min, the volume fraction of mobile phase A is 7%-10%; 10-35 min, the volume fraction of mobile phase A is 10%-17%; 35-80 min, the volume fraction of mobile phase A is 17%-21%; 80-90 min, the volume fraction of mobile phase A is 21%-40%; 90-100 min, the volume fraction of mobile phase A is 40%-7%; 100-110 min, the volume fraction of mobile phase A is 7%.

[0024] The present invention has the following beneficial effects: This invention provides a method for establishing an HPLC fingerprint of peony root heartwood and a method for quantitative analysis of its chemical components. An HPLC fingerprint is established using HPLC detection, and the components are further identified using HPLC-MS / MS. 39 components were identified, containing 33 common peaks. Finally, 22 well-separated chemical components are quantitatively analyzed using an HPLC standard curve method. The established HPLC fingerprint revealed 36 common peaks, and the similarity of 13 batches of medicinal materials was higher than 0.90. Twenty-two chemical components were identified through identification, and these 22 chemical components were simultaneously quantitatively detected using high-performance liquid chromatography. The detection method has advantages such as high precision, high stability, strong repeatability, and good recovery rate, laying the foundation for the quality control and development of peony root heartwood. Attached Figure Description

[0025] Figure 1 HPLC reference chromatograms of 13 batches of peony root heartwood; Figure 2 HPLC superimposed chromatograms of 13 batches of peony root heartwood; Figure 3 The HPLC chromatogram of the mixed reference standards; Figure 4 The total ion current spectrum of peony root heartwood is shown, where A represents the negative ion mode and B represents the positive ion mode. Figure 5 The structural formulas of 22 chemical components in the root of peony are given. Detailed Implementation

[0026] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0027] Information on the control formulations used in the examples is shown in Table 1, and information on the source of the peony root heartwood used is shown in Table 2. Both were provided by Shaanxi Heyang Zhongzi Guoye Biotechnology Co., Ltd.

[0028] Table 1. Reference Standard Information

[0029] Table 2. Source information of peony root heartwood samples

[0030] Example 1: A method for establishing an HPLC fingerprint of peony root heartwood includes the following steps: (1) Preparation of the test solution After crushing WCRP, pass it through a No. 4 sieve. Accurately weigh 1 g of WCRP powder and place it in a stoppered conical flask. Accurately add 100 mL of 70% methanol and weigh it. Sonicate for 45 minutes, cool, and weigh it again. Make up the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0031] (2) Preparation of reference solution Accurately weigh 22 reference standards, including 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol, and dissolve them in methanol to prepare a mixed reference solution with a concentration of 1.00 mg / mL.

[0032] (3) HPLC detection Chromatographic conditions: The chromatographic column used was a ShimPack Scepter C1000. 18 Column (4.6 mm × 250 mm, 5 μm, Shimadzu (Shanghai) Laboratory Equipment Co., Ltd.); Gradient elution was performed using acetonitrile as mobile phase A and formic acid aqueous solution with a volume fraction of 0.1% as mobile phase B. The elution conditions were as follows: 0-10 min, mobile phase A volume fraction 7%-10%; 10-35 min, mobile phase A volume fraction 10%-17%; 35-80 min, mobile phase A volume fraction 17%-21%; 80-90 min, mobile phase A volume fraction 21%-40%; 90-100 min, mobile phase A volume fraction 40%-7%; 100-110 min, mobile phase A volume fraction 7%. The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the injection volume was 10 μL.

[0033] Optimization of HPLC chromatographic conditions: 1) Detection wavelength This invention uses HPLC detection of samples at wavelengths of 254, 270, and 330 nm. The results show that there are more chromatographic peaks, the baseline is the most stable, and the peak shape is better at a wavelength of 270 nm. Therefore, the detection wavelength of 270 nm was selected.

[0034] 2) Mobile phase This invention conducted sample injection analysis on mobile phase systems of methanol-water, methanol-0.2% (v / v) formic acid aqueous solution, methanol-0.4% (v / v) formic acid aqueous solution, acetonitrile-water, acetonitrile-0.1% (v / v) formic acid aqueous solution, and acetonitrile-0.2% (v / v) formic acid aqueous solution. The results showed that when acetonitrile-0.1% (v / v) formic acid aqueous solution was used as the mobile phase, the resulting chromatogram baseline was more stable. Therefore, the mobile phase of acetonitrile-0.1% (v / v) formic acid aqueous solution was selected.

[0035] 3) Column temperature The present invention conducted HPLC analysis of the samples at column temperatures of 25, 30 and 35°C. The results showed that the peak shape was optimal and the separation was good at a column temperature of 30°C, so the column temperature of 30°C was selected.

[0036] (4) Precision test Take the test solution obtained in step (1) and inject it 6 times consecutively for HPLC detection. Using paeonol as the reference peak, analyze and investigate the retention time and relative peak area of ​​the common peak.

[0037] The RSDs of the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the RSD of the relative retention time of each common peak was less than 2%, and the RSD of the relative peak area was less than 5%, indicating that the detection method of the present invention has high precision.

[0038] (5) Stability test Take the test solution obtained in step (1) and inject it at different time points (0, 2, 4, 8, 12 and 24 h) for HPLC detection. Use paeonol as the reference peak to investigate the relative retention time and relative peak area of ​​the common peak.

[0039] The RSDs of the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the RSD of the relative retention time of each common peak was <2%, and the RSD of the relative peak area was less than 5%, indicating that the detection method of the present invention has excellent stability.

[0040] (6) Repeatability test Take 6 samples of medicinal materials and prepare 6 groups of test solutions according to the method in step (1). Perform HPLC detection and analyze the relative retention time and relative peak area of ​​the common peaks respectively.

[0041] The relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the relative retention time RSD of each common peak was less than 2%, and the relative peak area RSD was less than 5%, indicating that the method of the present invention has good repeatability.

[0042] (7) Establishment of feature maps and similarity analysis Thirteen different WCRP samples (labeled S1-S13) were collected, and test solutions were prepared according to the method in step (1). HPLC detection was performed under the chromatographic conditions in step (3), and fingerprint spectra were recorded. Peak 31, using paeonol as a reference, was used to establish fingerprint spectra. The relative retention time and relative peak area RSD of the common chromatographic peaks were calculated. Similarity analysis was performed on the fingerprint spectra of the 13 batches of WCRP samples. The data from the chromatography workstation was imported into the traditional Chinese medicine fingerprint spectra similarity calculation software. Peak matching was performed on 33 selected common chromatographic peaks. The common pattern of the sample reference fingerprint spectra was calculated, and this common pattern was used as the standard (reference spectra are shown below). Figure 1 As shown, the HPLC overlay chromatograms of 13 batches of WCRP samples are as follows: Figure 2 As shown in the figure, an overall similarity evaluation is performed.

[0043] The results showed that the fingerprint patterns of S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, and S13 were similar to the control pattern. Figure 1 The similarity scores were 0.967, 0.976, 0.941, 0.965, 0.979, 0.904, 0.963, 0.968, 0.983, 0.980, 0.975, 0.969 and 0.983, respectively, all greater than 0.90, indicating good consistency in chemical composition.

[0044] (8) Identification of characteristic peaks The mixed reference solution obtained in step (2) and the blank control were subjected to HPLC detection and the chromatographic peaks were recorded. The HPLC chromatogram of the mixed reference solution is shown below. Figure 3 As shown, based on the results... Figure 1 The standard atlases in the database are compared and identified.

[0045] The results showed that among the characteristic peaks, peak 2 was 1-galloyl glucose, peak 3 was gallic acid, peak 6 was methyl gallate, peak 7 was hydroxypaeoniflorin, peak 9 was paeonol original glycoside, peak 10 was paeonol neoglycoside, peak 11 was paeoniflorin lactone glycoside, peak 14 was paeoniflorin, peak 15 was p-coumaric acid, peak 17 was ferulic acid, peak 18 was 1,2,3,6-tetra-O-galloyl glucose, peak 19 was ellagic acid, peak 20 was gallo-paeoniflorin, peak 21 was luteolin, peak 22 was 1,2,3,4,6-O-pentagalloyl glucose, peak 24 was diosmin, peak 25 was neo-diosmin, peak 26 was resveratrol, peak 27 was paeonol C, peak 28 was benzoyl paeoniflorin oxidized, peak 30 was benzoyl paeoniflorin, and peak 31 was paeonol.

[0046] Example 2: A method for analyzing the chemical components of peony root wood, using HPLC-MS / MS, specifically includes the following steps: The preparation methods for the test solution and the mixed reference solution are the same as in Example 1; The test solution and the mixed reference solution were analyzed by HPLC-MS / MS, and the HPLC detection conditions were the same as in Example 1. Mass spectrometry conditions are: Ion source: Electrospray ionization source ESI scanning mode: positive and negative ion switching scan; Detection method: Full mass / dd-MS2; Resolution: 70000 full mass 17500 dd-MS2; Scan range: 100.0-1500.0 m / z; Electrospray voltage: 3.2 kV (Positive, Negative) Capillary temperature: 300℃; Collision gas: High-purity argon, purity ≥ 99.999; Collision energy (N)CE: 30, 40, 60; Sheath gas: Nitrogen (purity ≥ 99.999%), 40 Arb; Auxiliary gas: Nitrogen (purity ≥99.999%), 15 Arb, 350℃; Data acquisition time: 110.0 min.

[0047] By confirming the test sample solution and the mixed sample solution with reference standards and analyzing high-resolution mass spectrometry data, 33 characteristic peaks and 6 non-characteristic peaks were identified. The results are as follows: Figure 4 As shown in Tables 3 and 4, the results indicate that the mass spectrometry identification results of the characteristic peaks are consistent with the identification results of the characteristic peaks in Example 1. Figure 4 (Structural formulas of 22 chemical components).

[0048] Table 3. LC-MS / MS identification results of WCRP (characteristic peaks 1-18)

[0049] Table 4. LC-MS / MS identification results of WCRP (characteristic peaks 19-33)

[0050] Example 3: An analytical method for 22 chemical components in peony root heartwood is disclosed. This embodiment of the method enables simultaneous quantitative detection of 22 chemical components in peony root heartwood, and specifically includes the following steps: In this embodiment, the preparation methods of the test solution and the mixed reference solution are the same as in Example 1, and the chromatographic conditions are the same as in Example 1.

[0051] (1) Specificity test The blank solvent, mixed reference solution, and test solution were injected and analyzed. The results showed that the blank control had no interference.

[0052] (2) Standard curve and linear range Weigh out 2, 10, 50, 250, and 1000 μL of the mixed reference solution, respectively, and place them in five 1 mL volumetric flasks. Add methanol to each flask to bring the volume to 1 mL, obtaining serially diluted mixed reference solutions. Then, perform HPLC analysis, record the peak areas, and investigate the linear relationship between the reference concentration (x) and peak area (y). The experimental results are shown in Table 5. In the table, R... 2 All values ​​are greater than 0.999, indicating a good linear relationship within the concentration range corresponding to the linear range.

[0053] Table 5. Linear equations, linear range, and detection limits

[0054] (3) Precision test A mixed reference solution was subjected to six consecutive HPLC analyses to determine its peak area. The RSD of the peak areas of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol neoglycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol was calculated. The results showed that the RSD of the peak areas of all 22 chemical substances was less than 2.0%, indicating high precision.

[0055] (4) Stability test The test solution (S1) was weighed and injected at 0, 2, 4, 8, 12 and 24 h for HPLC determination. The peak areas were measured, and the RSD of the peak areas of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol were calculated. The results showed that the RSD of the peak areas of the 22 chemical substances was less than 2.0%, indicating that the detection method of the present invention has high stability.

[0056] (5) Repeatability test Accurately weigh 1 g of the test sample (S1), prepare six test solutions and perform HPLC determination. Calculate the RSD of the contents of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol. The results show that the RSD of the contents of all 22 chemical substances is less than 3%, indicating that the detection method of this invention has good repeatability.

[0057] (6) Recovery rate Accurately weigh 0.5 g of the test sample (S1), weigh 6 portions, add different amounts of the mixed reference solution to each portion, and then prepare the test solutions separately for HPLC determination and calculate the recovery rate.

[0058] Experimental results show that the recovery rate of the sample addition is between 97% and 101%, indicating that the recovery rate of the method of the present invention is good.

[0059] (7) Detection of the content of 22 components in peony root heartwood samples Thirteen batches (S1-S13) of samples were prepared as test solutions, and HPLC was performed to determine and record the peak area. The content of 22 chemical components in the samples was calculated using the standard curve obtained in step (2). The experimental results are shown in Table 6. The abbreviations of the compounds in Table 6 represent the reference standards in Table 1.

[0060] Table 6. Results of content determination of 22 components in 13 batches of peony root wood.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A quality control method for HPLC fingerprinting of peony root heartwood, characterized in that, The HPLC fingerprint was established through the following steps: (1) Take peony root heartwood powder and add it to methanol solution to prepare test solution; (2) Take 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallo-paeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neo-diosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol as reference standards, and add methanol to prepare a mixed reference solution; (3) The test solution obtained in step (1) and the mixed reference solution obtained in step (2) were subjected to HPLC detection and analysis to obtain the HPLC fingerprint of the peony root heartwood.

2. The HPLC fingerprint quality control detection method for peony root heartwood according to claim 1, characterized in that, The preparation method of the test solution in step (1) specifically includes the following steps: First, mix the peony root heartwood powder with a methanol solution of 60%-80% by volume and weigh it. Then, sonicate it for 30-60 minutes, cool it, and weigh it again. Finally, make up the lost weight with a methanol solution of 60%-80% by volume, shake it well, filter it, and take the filtrate as the test solution. The concentration of peony root heartwood powder in the test solution was 8-12 mg / mL; In step (2), the concentrations of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol in the mixed reference solution are all 0.8-1.2 mg / mL.

3. The HPLC fingerprint quality control detection method for peony root heartwood according to claim 1, characterized in that, The chromatographic column used for HPLC detection in step (3) is C18. 18 The column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The conditions for HPLC detection are as follows: Acetonitrile is the mobile phase A, and a 0.1% (v / v) aqueous solution of formic acid is the mobile phase B; The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The conditions for gradient elution are: 0-10 min, the volume fraction of mobile phase A is 7%-10%; 10-35 min, the volume fraction of mobile phase A is 10%-17%; 35-80 min, the volume fraction of mobile phase A is 17%-21%; 80-90 min, the volume fraction of mobile phase A is 21%-40%; 90-100 min, the volume fraction of mobile phase A is 40%-7%; 100-110 min, the volume fraction of mobile phase A is 7%.

4. A method for analyzing the chemical components of peony root wood, characterized in that, The chemical components of peony root wood were analyzed using HPLC-MS / MS, including the following steps: Step 1: Take peony root heartwood powder and add it to methanol solution to prepare the test solution; Step 2: Take 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol as reference standards, and add methanol to prepare a mixed reference solution; Step 3: Perform HPLC-MS / MS analysis on the test solution obtained in Step 1 and the mixed reference solution obtained in Step 2 to complete the chemical composition analysis of peony root wood.

5. The method for chemical composition analysis of peony root wood according to claim 4, characterized in that, The preparation method of the test solution in step 1 specifically includes the following steps: First, mix the peony root heartwood powder with a methanol solution of 60%-80% by volume and weigh it. Then, sonicate it for 30-60 minutes, cool it, and weigh it again. Finally, make up the lost weight with a methanol solution of 60%-80% by volume, shake it well, filter it, and take the filtrate as the test solution. The concentration of peony root heartwood powder in the test solution was 8-12 mg / mL; In step 2, the concentrations of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol neoglycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol in the mixed reference solution are all 0.8-1.2 mg / mL.

6. The method for chemical composition analysis of peony root wood according to claim 4, characterized in that, In the HPLC-MS / MS method, the chromatographic column used for HPLC detection is C10. 18 The column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The conditions for HPLC detection are as follows: Acetonitrile is the mobile phase A, and a 0.1% (v / v) aqueous solution of formic acid is the mobile phase B; The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The conditions for gradient elution are: 0-10 min, the volume fraction of mobile phase A is 7%-10%; 10-35 min, the volume fraction of mobile phase A is 10%-17%; 35-80 min, the volume fraction of mobile phase A is 17%-21%; 80-90 min, the volume fraction of mobile phase A is 21%-40%; 90-100 min, the volume fraction of mobile phase A is 40%-7%; 100-110 min, the volume fraction of mobile phase A is 7%.

7. The method for chemical composition analysis of peony root wood according to claim 4, characterized in that, The mass spectrometry conditions for HPLC-MS / MS are as follows: Ion source: electrospray ionization source; ESI scanning mode: positive and negative ion switching scan; scanning range: 100.0-1500.0 m / z; collision gas: argon; sheath gas: nitrogen; auxiliary gas: nitrogen.

8. A quantitative analysis method for 22 chemical components in the root heartwood of peony, characterized in that, Includes the following steps: S1: Take peony root heartwood powder and add it to methanol solution to prepare the test solution; S2: Take 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin and paeonol as reference standards, and add methanol to prepare a mixed reference solution; S3: First, the mixed reference solution obtained in S2 is serially diluted and detected by HPLC. A standard curve is plotted with the mass concentration of each component as the abscissa and the peak area of ​​each component as the ordinate. Then, the test solution obtained in S1 is detected by HPLC under the same conditions. Finally, the content of each component in the root core of peony is calculated according to the standard curve.

9. The quantitative analysis method for 22 chemical components in the root heartwood of peony according to claim 8, characterized in that, In step S1, the preparation method of the test solution specifically includes the following steps: First, mix the peony root heartwood powder with a methanol solution of 60%-80% by volume and weigh it. Then, sonicate it for 30-60 minutes, cool it, and weigh it again. Finally, make up the lost weight with a methanol solution of 60%-80% by volume, shake it well, filter it, and take the filtrate as the test solution. The concentration of peony root heartwood powder in the test solution was 8-12 mg / mL; In S2, the concentrations of 1-galloyl glucose, gallic acid, methyl gallate, hydroxypaeoniflorin, paeonol original glycoside, paeonol new glycoside, paeoniflorin lactone glycoside, paeoniflorin, p-coumaric acid, ferulic acid, 1,2,3,6-tetra-O-galloyl glucose, ellagic acid, gallopaeoniflorin, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, diosmin, neodiosmin, resveratrol, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol in the mixed reference solution are all 0.8-1.2 mg / mL.

10. The method for quantitative analysis of 22 chemical components in the root heartwood of peony according to claim 8, characterized in that, The HPLC detection in S3 uses a C10 column. 18 The column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. The conditions for HPLC detection are as follows: Acetonitrile is the mobile phase A, and a 0.1% (v / v) aqueous solution of formic acid is the mobile phase B; The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The conditions for gradient elution are: 0-10 min, the volume fraction of mobile phase A is 7%-10%; 10-35 min, the volume fraction of mobile phase A is 10%-17%; 35-80 min, the volume fraction of mobile phase A is 17%-21%; 80-90 min, the volume fraction of mobile phase A is 21%-40%; 90-100 min, the volume fraction of mobile phase A is 40%-7%; 100-110 min, the volume fraction of mobile phase A is 7%.

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