Characteristic chromatogram detection method and quality control method of cortex moutan medicinal material, cortex moutan charred decoction pieces or medicinal preparation

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, and relates to a characteristic map detection method and quality control method for cortex moutan medicinal materials, cortex moutan charred decoction pieces or medicinal preparations, comprising high performance liquid chromatography detection, and chromatographic conditions are as follows: an octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as a mobile phase A, and a 0.08-0.12% formic acid solution is used as a mobile phase B, and elution is performed according to a gradient program. The above method can comprehensively detect cortex moutan medicinal materials, cortex moutan charred decoction pieces or medicinal preparations, and can control the quality of cortex moutan medicinal materials, cortex moutan charred decoction pieces and end products. Meanwhile, the characteristic map method established exhibits rich material composition information, and characteristic peaks are obvious, multi-component information representation of various types of compounds is realized, and the cortex moutan medicinal materials, cortex moutan charred decoction pieces and medicinal preparations are comprehensively controlled in quality.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing technology, specifically to a method and quality control method for the detection of characteristic spectra of peony bark, charred peony bark slices, or pharmaceutical preparations. Background Technology

[0002] The chemical composition of peony bark is complex and diverse, and traditional Chinese medicine is a system in which complex components work together in a coordinated manner to exert therapeutic effects. Therefore, it is necessary to accurately control the quality of medicinal materials in a comprehensive and multi-faceted manner. After peony bark is charred, its chemical composition and pharmacological effects undergo significant changes. Currently, on the one hand, there is no comprehensive detection method that can simultaneously detect peony bark raw materials, charred peony bark slices, or pharmaceutical preparations, making it difficult to control the quality of peony bark raw materials, charred peony bark slices, and downstream products. On the other hand, existing characteristic chromatographic detection methods for peony bark raw materials and charred peony bark slices identify fewer characteristic components, some characteristic peaks are poorly separated, the response is low, and peak identification and localization are difficult, which is insufficient for quality control; in the characteristic chromatographic detection methods for peony bark pharmaceutical preparations, there are fewer characteristic peaks and fewer identified characteristic components, which is also insufficient for quality control. Based on this, the present invention provides a characteristic chromatographic detection method for peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparations, which provides a more comprehensive, objective and rapid quality evaluation method for peony bark medicinal material, peony bark charcoal slices and pharmaceutical preparations, and is of great significance for their comprehensive quality detection and overall quality control. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide a characteristic spectral detection method and quality control method for peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparations. The above method can simultaneously perform comprehensive detection on peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparations, and can control the quality of peony bark medicinal material, peony bark charcoal slices and downstream products. At the same time, the established characteristic spectral method shows rich information on the material composition and obvious characteristic peaks, realizing multi-component information characterization of various types of compounds, and playing a comprehensive quality control role in peony bark medicinal material, peony bark charcoal slices and pharmaceutical preparations.

[0004] Therefore, the present invention provides the following technical solution:

[0005] A method for detecting characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations, including detection using high-performance liquid chromatography (HPLC), with the following chromatographic conditions:

[0006] The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.08-0.12 v / v% formic acid solution as mobile phase B, eluted according to the following gradient:

[0007] From 0 to 10 min, the volume ratio of mobile phase A to mobile phase B was 7%:93%;

[0008] Over 10-15 minutes, the volume ratio of mobile phase A to mobile phase B changes from 7%:93% to 15%:85%.

[0009] Over 15-30 minutes, the volume ratio of mobile phase A to mobile phase B changes from 15%:85% to 30%:70%.

[0010] Over 30-45 minutes, the volume ratio of mobile phase A to mobile phase B is 30%:70% → 75%:25%.

[0011] After 45-46 minutes, the volume ratio of mobile phase A to mobile phase B changed from 75%:25% to 7%:93%.

[0012] Optionally, the chromatographic conditions include at least one of the following:

[0013] 1) The detection wavelength is 254nm;

[0014] 2) The flow rate is 0.9 ml / min - 1.1 ml / min;

[0015] 3) Column temperature is 28℃-32℃;

[0016] 4) The chromatographic column has the following specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm; optionally, the chromatographic column is selected from... XB-C18;

[0017] 5) The injection volume is 5-20 μl.

[0018] Optionally, the preparation of a reference solution may also be included:

[0019] Preparation of reference solution for reference medicinal material: Take peony bark as reference medicinal material, add solvent for extraction, filter for the first time, dilute with alcohol solvent, filter for the second time, and collect the filtrate;

[0020] Preparation of reference solution: Gallic acid and paeonol were added to 50 v / v% to 100 v / v% methanol solvent to prepare solutions containing 20-50 μg / ml of gallic acid and paeonol, respectively.

[0021] Optionally, in the preparation of the reference solution, the mass-volume ratio of peony bark reference material to solvent is 1.5g:25ml-100ml, preferably 1.5g:50ml, and the solvent is water;

[0022] Optionally, in the preparation of the reference solution of the control medicinal material, the extraction step is to extract by heating and reflux for 30-90 minutes, preferably 60 minutes;

[0023] Optionally, in the preparation of the reference solution of the reference medicinal material, the filtrate obtained from the first filtration is diluted at a volume ratio of 1:(1-4), preferably at a volume ratio of 1:2.

[0024] Optionally, the peony bark medicinal material, peony bark charcoal slices, or pharmaceutical preparation may include peony bark medicinal material, peony bark charcoal slices, peony bark charcoal standard decoction, or peony bark charcoal formula granules.

[0025] When the test sample is peony bark or charred peony bark slices, the test sample solution is prepared as follows: Take the test sample, add solvent to extract, filter, dilute with alcohol solvent, filter again, and take the filtrate; optionally, the mass-volume ratio of the test sample to the solvent is 1.5g:25ml-100ml, preferably 1.5g:50ml, and the solvent is water; and / or, the extraction step is reflux extraction for 30-90 minutes, preferably 60 minutes; and / or, in the preparation of the reference material solution, the filtrate obtained from the first filtration is diluted at a volume ratio of 1:(1-4), preferably 1:2.

[0026] When the test sample is a standard decoction of peony bark charcoal or peony bark charcoal formula granules, the preparation of the test sample solution is as follows: take the test sample, add solvent to extract, filter, and take the filtrate; optionally, the mass-volume ratio of the test sample to the solvent is 0.2g:10ml-100ml, preferably 1.5g:20ml, and the solvent is 25v / v%-75v / v%, preferably 50v / v% methanol solution; and / or, the extraction step is heating and reflux extraction for 30-60 minutes, preferably 30 minutes.

[0027] A method for determining the content of peony bark medicinal material, peony bark charcoal slices, or pharmaceutical preparations, comprising:

[0028] Take the test solution and the reference solution, and use the characteristic chromatographic detection method described above for peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparations.

[0029] Optionally, it includes the following steps: obtaining the characteristic spectrum of the test sample according to the characteristic spectrum detection method of the peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparation, and comparing the characteristic spectrum with the control characteristic spectrum;

[0030] The reference feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of standard samples of peony bark, charred peony bark slices or pharmaceutical preparations according to the feature spectrum detection method of peony bark, charred peony bark slices or pharmaceutical preparations.

[0031] Optionally, when testing peony bark, the reference characteristic chromatogram of peony bark includes 9 characteristic peaks. Peak 1 and peak 9 should correspond to the retention times of the corresponding reference peaks. Peak 1 is gallic acid, and peak 9 is paeonol. Peak 9 is used as reference peak S. The relative retention times of peaks 2 to 8 with peak S should be within ±10% of the specified values. The specified values ​​are: peak 2 0.45, peak 3 0.57, peak 4 0.66, peak 5 0.75, peak 6 0.81, peak 7 0.84, and peak 8 0.91.

[0032] When testing charred peony bark slices, the reference characteristic chromatogram of charred peony bark slices includes 9 characteristic peaks. Peak 1 and peak 9 should correspond to the retention times of the corresponding reference peaks. Peak 1 is gallic acid, peak 9 is paeonol, and peak 9 is reference peak S. The relative retention times of peaks 2 to 8 with peak S should be within ±10% of the specified values. The specified values ​​are: peak 2 0.20, peak 3 0.31, peak 4 0.45, peak 5 0.49, peak 6 0.50, peak 7 0.64, and peak 8 0.75.

[0033] When testing the standard decoction or granules of peony bark charcoal, the reference characteristic chromatograms of the standard decoction or granules of peony bark charcoal are as follows: peak 1 and peak 9 correspond to the retention times of the corresponding reference peaks. Peak 1 is gallic acid, and peak 9 is paeonol. Using peak 9 as the reference peak S, the relative retention times of peaks 2 to 8 with peak S are calculated to be within ±10% of the specified values. The specified values ​​are: peak 2 0.19, peak 3 0.28, peak 4 0.41, peak 5 0.45, peak 6 0.47, peak 7 0.63, and peak 8 0.74.

[0034] Optionally, in the reference characteristic spectrum of peony bark, peak 1: gallic acid; peak 2: p-hydroxybenzoic acid; peak 3: paeoniflorin; peak 4: β-1,2,3,4,6-pentagalloglucoside; peak 5: benzoic acid; peak 6: peony root extract C; peak 7: benzoyl paeoniflorin; peak 8: benzoyl paeoniflorin; peak 9: paeonol;

[0035] In the comparative characteristic chromatogram of charred peony bark slices, peak 1: gallic acid; peak 2: 5-hydroxymethylfurfural; peak 4: p-hydroxybenzoic acid; peak 8: benzoic acid; peak 9: paeonol;

[0036] In the comparative characteristic chromatogram of the standard decoction or formula granules of peony bark charcoal, peak 1: gallic acid; peak 2: 5-hydroxymethylfurfural; peak 4: p-hydroxybenzoic acid; peak 8: benzoic acid; peak 9: paeonol.

[0037] A method for distinguishing between raw peony bark and charred peony bark slices includes:

[0038] The steps are as follows: obtaining the characteristic spectrum of the test sample according to the characteristic spectrum detection method of peony bark medicinal material, peony bark charcoal slices or drug preparation, and comparing the characteristic spectrum with the control characteristic spectrum;

[0039] The reference feature spectrum is obtained by fitting the feature spectrum obtained by using at least a batch of standard samples of peony bark, peony bark charcoal slices or drug preparations according to the feature spectrum detection method of peony bark, peony bark charcoal slices or drug preparations.

[0040] The comparative feature maps include comparative feature maps of peony bark medicinal material and comparative feature maps of charred peony bark slices.

[0041] The technical solution of this invention has the following advantages:

[0042] 1. The method for detecting the characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations provided by the present invention includes detection by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel, acetonitrile is used as mobile phase A, and 0.08-0.12 v / v% formic acid solution is used as mobile phase B. Elution is performed according to the following gradient: 0-10 min, the volume ratio of mobile phase A to mobile phase B is 7%:93%; 10-15 min, the volume ratio of mobile phase A to mobile phase B is 7%:93% → 15%:85%; 15-30 min, the volume ratio of mobile phase A to mobile phase B is 15%:85% → 30%:70%; 30-45 min, the volume ratio of mobile phase A to mobile phase B is 30%:70% → 75%:25%; 45-46 min, the volume ratio of mobile phase A to mobile phase B is 75%:25% → 7%:93%. The above method can simultaneously perform comprehensive testing on peony bark medicinal materials, peony bark charcoal slices, or pharmaceutical preparations, and can control the quality of peony bark medicinal materials, peony bark charcoal slices, and downstream products. At the same time, the established characteristic spectral method shows rich information on the material composition and obvious characteristic peaks, realizing multi-component information characterization of various types of compounds, and playing a comprehensive quality control role in peony bark medicinal materials, peony bark charcoal slices, and pharmaceutical preparations.

[0043] Furthermore, the above method can distinguish between peony bark medicinal material and peony bark charcoal slices. The components of peony bark before and after processing are significantly different. By selecting the characteristic peaks and specifying the relative retention time in this invention, peony bark medicinal material and peony bark charcoal slices can be clearly distinguished and their quality can be controlled.

[0044] Furthermore, the above method can be used to determine the content of known components in peony bark medicinal materials, peony bark charcoal slices, or pharmaceutical preparations. Specifically, it can determine gallic acid, p-hydroxybenzoic acid, paeoniflorin, β-1,2,3,4,6-pentagalloylglucose, benzoic acid, peony bark glycoside C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol in peony bark medicinal materials; and it can determine gallic acid, 5-hydroxymethylfurfural, p-hydroxybenzoic acid, benzoic acid, and paeonol in peony bark charcoal slices, peony bark charcoal standard decoction, or peony bark charcoal formulation granules.

[0045] Furthermore, using the method of this invention, a characteristic spectrum of peony bark was established, and nine common characteristic peaks were identified, all of which were identified as phenols, phenolic glycosides, and organic acids. After processing into charred peony bark slices, the composition changed significantly, and nine common characteristic peaks were identified. Five of these peaks were located and identified using liquid chromatography-mass spectrometry and reference standards, which were also mainly phenols and organic acids. Verification showed that each characteristic peak could be stably transferred from charred peony bark slices to standard decoctions and formula granules, enabling quality control of charred peony bark drug preparations. This provides a rapid and reliable detection method for the characteristic spectrum identification of peony bark, charred peony bark slices, and related preparations. Attached Figure Description

[0046] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0047] Figure 1 This is a comparative characteristic spectrum of the peony bark medicinal material in Example 1 of the present invention;

[0048] Figure 2 This is a characteristic spectrum of the reference medicinal material used in Example 1 of this invention;

[0049] Figure 3 This is the characteristic spectrum of the paeonol reference standard in Example 1 of the present invention;

[0050] Figure 4 This is the characteristic spectrum of the gallic acid reference standard in Example 1 of the present invention;

[0051] Figure 5 This is the characteristic chromatogram of the peony bark sample solution in "1. Specificity" of Embodiment 2 of the present invention;

[0052] Figure 6 This is the characteristic chromatogram of the peony bark reference material reference solution and the test sample solution in "1. Specificity" of Example 2 of the present invention;

[0053] Figure 7 This is the characteristic chromatogram of the paeonol reference solution and test solution in "1. Specificity" of Example 2 of the present invention;

[0054] Figure 8 This is the characteristic chromatogram of the gallic acid reference standard test solution in "1. Specificity" of Example 2 of the present invention;

[0055] Figure 9 This is the characteristic spectrum of the blank (50% methanol) in "1. Specificity" of Embodiment 2 of the present invention;

[0056] Figure 10 This is the characteristic spectrum of a flow rate of 0.9 ml / min in "6. Flow rate investigation" of Embodiment 2 of the present invention;

[0057] Figure 11 This is the characteristic spectrum of a flow rate of 1.0 ml / min in "6. Flow rate investigation" of Embodiment 2 of the present invention;

[0058] Figure 12 This is the characteristic spectrum of a flow rate of 1.1 ml / min in "6. Flow rate investigation" of Embodiment 2 of the present invention;

[0059] Figure 13 This is the characteristic spectrum of a column temperature of 28°C in "7. Column Temperature Examination" of Embodiment 2 of the present invention;

[0060] Figure 14 This is the characteristic spectrum of a column temperature of 30°C in "7. Column Temperature Examination" of Embodiment 2 of the present invention;

[0061] Figure 15 This is the characteristic spectrum of a column temperature of 32°C in "7. Column Temperature Examination" of Embodiment 2 of the present invention;

[0062] Figure 16 This is the characteristic spectrum of a concentration of 0.08% in "8. Investigation of mobile phase acid concentration" in Example 2 of the present invention;

[0063] Figure 17 This is the characteristic spectrum of a concentration of 0.10% in "8. Investigation of mobile phase acid concentration" in Example 2 of the present invention;

[0064] Figure 18 This is the characteristic spectrum of a concentration of 0.12% in "8. Investigation of mobile phase acid concentration" in Example 2 of the present invention;

[0065] Figure 19 This refers to the feature map in "11. Overall Examination" of Embodiment 2 of the present invention;

[0066] Figure 20 This is a comparative characteristic spectrum of the charred peony bark slices in Example 3 of the present invention;

[0067] Figure 21 This is a comparison of the comparative characteristic chromatograms of peony bark medicinal material and peony bark charcoal slices in Example 4 of the present invention; S1 is the comparative characteristic chromatogram of peony bark charcoal slices; S2 is the comparative characteristic chromatogram of peony bark medicinal material.

[0068] Figure 22 This is a comparative characteristic spectrum of the standard decoction of peony bark charcoal in Example 5 of the present invention;

[0069] Figure 23 These are characteristic chromatograms of three batches of peony bark charcoal formulation granules in Example 6 of the present invention; from bottom to top in the figure are: peony bark charcoal formulation granules 2310001Y, 2310002Y, and 2310003Y;

[0070] Figure 24 This is a comparative characteristic spectrum of the peony bark charcoal formulation particles in Example 6 of the present invention;

[0071] Figure 25 This is the characteristic spectrum of the blank (50% methanol) in "1. Specificity" of Example 7 of the present invention;

[0072] Figure 26 This is the characteristic chromatogram of the negative control solution (maltodextrin) in "1. Specificity" of Example 7 of the present invention;

[0073] Figure 27 This is the characteristic chromatogram of the negative control solution (betacyclodextrin) in "1. Specificity" of Example 7 of the present invention;

[0074] Figure 28 This is the characteristic chromatogram of the gallic acid reference solution in "1. Specificity" of Example 7 of the present invention;

[0075] Figure 29 This is the characteristic chromatogram of the paeonol reference solution in "1. Specificity" of Example 7 of the present invention;

[0076] Figure 30 This is the characteristic chromatogram of the peony bark reference medicinal material solution in "1. Specificity" of Example 7 of the present invention;

[0077] Figure 31 This is the characteristic spectrum of the peony bark charcoal formulation particles in "1. Specificity" of Embodiment 7 of the present invention;

[0078] Figure 32 This is the feature map of "5. Integrity" in Embodiment 7 of the present invention;

[0079] Figure 33 The characteristic chromatogram is the one with the flow rate set to 0.9 ml / min in "7. Flow rate investigation" of embodiment 7 of the present invention;

[0080] Figure 34 The characteristic chromatogram is the one with the flow rate set to 1.0 ml / min in "7. Flow rate investigation" of embodiment 7 of the present invention;

[0081] Figure 35 This is the characteristic spectrum of "7. Flow rate investigation" in Embodiment 7 of the present invention, where the flow rate is set to 1.1 ml / min. Detailed Implementation

[0082] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0083] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0084] instrument

[0085] Chromatograph 1: Waters 2695 chromatography system, including a quaternary gradient pump (Alliance2695 model), a 120-position high-performance autosampler, an original imported column oven, a Waters 2998 diode array UV detector, and an Empower chromatography management system;

[0086] Chromatograph 2: Agilent 1100 chromatography system, including G1312A binary pump, G1329A autosampler, G1314A VWD detector, G1316A column oven, and Agilent Chemstation chromatography workstation;

[0087] Chromatograph 3: LC-2030C 3D chromatography system, including a quaternary pump, diode array detector, 100-position liquid autosampler (with sample cooling), column oven, and Thermo Fisher Chromeleon 7 chromatography workstation;

[0088] Electronic analytical balances: MS205 DU (Mettler) 1 / 100,000 balance, FA2004 (Shanghai Sunny Hengping) 1 / 10,000 balance, LD610-2 (Shenyang Longteng Electronics) 1 / 1000 balance;

[0089] Column 1: Yuexu column. XB-C18, 4.6×250mm, 5μm; SN: 60220301261;

[0090] Column 2: Yuexu column. XB-C18, 4.6×250mm, 5μm; SN: 21706196;

[0091] Column 3: Yuexu column. XB-C18, 4.6×250mm, 5μm; SN: 211703538;

[0092] Column 4: Yuexu column. XB-C18, 4.6×250mm, 5μm; SN: 60220504602.

[0093] Drug testing

[0094] Reference material: Moutan bark (batch number: 121490-201102, China National Institutes for Food and Drug Control).

[0095] Reference standards: Gallic acid (batch number: PS012605, purity: 98.9%); Paeonol (batch number: PS012530, purity: 99.7%); p-hydroxybenzoic acid (batch number: PS160926-01, purity > 98%); Benzoic acid (batch number: PS020532, purity > 98%); 5-Hydroxymethylfurfural (batch number: PS020078, purity > 98%); Paeoniflorin (batch number: PS012832) The following reagents were used: β-1,2,3,4,6-pentagalloylglucose (batch number: PS011427, purity > 98%); paeonol C (batch number: PS011396, purity > 98%); benzoylpaeoniflorin (batch number: PS13011802, purity > 98%); and benzoylpaeoniflorin oxidase (batch number: PS000158, purity > 98%). All were sourced from Chengdu Pusi Biotechnology Co., Ltd. Reagents: Acetonitrile was chromatographic grade; methanol and formic acid were analytical grade; and water was ultrapure water.

[0096] Peony bark charcoal granules (batch numbers: 2310001Y, 2310002Y, 2310003Y). The preparation method is as follows: Take 5600g of peony bark charcoal slices, add water and decoct. Collect an appropriate amount of paeonol crystals or aromatic water (encapsulated with β-cyclodextrin for later use), filter, concentrate the filtrate into a clear extract, add the inclusion compound, dry, add an appropriate amount of excipients, mix well, granulate, and produce 1000g of the product.

[0097] The preparation method of the standard decoction of peony bark charcoal is as follows: Take about 150g of peony bark charcoal, place it in a clay pot, add 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat, then simmer over low heat for 30 minutes, filter while hot through a 200-mesh filter cloth, add 8 times the amount of water to the dregs, bring to a boil over high heat, then simmer over low heat for 20 minutes, filter while hot through a 200-mesh filter cloth, combine the filtrates, freeze dry to obtain the standard decoction of peony bark charcoal (freeze-dried powder).

[0098] The batches of peony bark medicinal materials, peony bark charcoal slices, peony bark charcoal standard decoction, and peony bark charcoal formula granules in the following examples were provided by China Resources Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd.

[0099] Example 1

[0100] This embodiment provides a method for detecting the characteristic chromatograms of peony bark, including: detection by high-performance liquid chromatography (HPLC), with the following chromatographic conditions: the chromatographic column is packed with octadecylsilane-bonded silica gel (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm). XB-C18); using acetonitrile as mobile phase A and 0.1 v / v formic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on the paeonol peak, should not be less than 5000.

[0101] Table 1. Gradient elution program

[0102] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 7 93 10～15 7→15 93→85 15～30 15→30 85→70 30～45 30→75 70→25 45～46 75→7 25→93

[0103] Preparation of reference solutions: Take about 1.5g of peony bark reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60 minutes, cool, filter, transfer 5ml of the filtrate to a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate as the reference solution of the reference material; take gallic acid reference standard and add it to 50v / v% methanol solution to prepare a solution containing gallic acid concentration of 50ug / ml, take paeonol reference standard and add it to methanol solvent to prepare a solution containing paeonol concentration of 20ug / ml, as the reference solution.

[0104] Preparation of the test solution: Take about 1.5g of coarse powder of peony bark, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60 minutes, cool, filter, transfer 5ml of the filtrate to a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0105] Determination method: Accurately pipette 20 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0106] In this embodiment, 15 batches of Paeonia suffruticosa root bark were used to formulate a reference characteristic spectrum for Paeonia suffruticosa root bark. The detection method was carried out according to the above method, and characteristic spectra of 15 batches of Paeonia suffruticosa root bark were obtained. The results are shown in the table below.

[0107] Using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, the characteristic spectra of the above 15 batches of Moutan Cortex medicinal materials were used to generate reference characteristic spectra, as shown below. Figure 1 As shown, there are a total of 9 peaks, numbered 1-9 according to the chromatographic peak sequence. Literature review indicates that Paeonia suffruticosa mainly contains monoterpenes and their glycosides, phenols and their phenolic glycosides, and other components such as triterpenes, sterols and their glycosides, flavonoids, organic acids, and coumarins. Since the charred granule formulation of Paeonia suffruticosa mainly uses water extraction, and based on literature reports, its main components are mostly water-soluble, the chemical properties of each characteristic peak of Paeonia suffruticosa were analyzed. Reference standards were used to locate and identify the characteristic peaks. The analysis results are shown in the table below. Peak 1 was confirmed as gallic acid, peak 2 as p-hydroxybenzoic acid, peak 3 as paeoniflorin, peak 4 as β-1,2,3,4,6-pentagalloglucoside, peak 5 as benzoic acid, peak 6 as paeonol C, peak 7 as benzoyl paeoniflorin, peak 8 as benzoyl paeoniflorin, and peak 9 as paeonol. Based on the peak identification and selection results of the characteristic peaks of peony bark, paeonol was identified as having the largest characteristic peak response. Therefore, paeonol was used as the S peak in this characteristic chromatogram to calculate the relative retention times of each characteristic peak. It was stipulated that the reference characteristic chromatogram of peony bark should present 9 characteristic peaks, and this should be compared with the chromatogram of the reference herb (see...). Figure 2 The retention times of the nine characteristic peaks in the sample should correspond to the retention times of the corresponding reference peaks (see [reference]). Two of these peaks should correspond to the retention times of the corresponding reference peaks. Figure 3 and Figure 4 The retention times correspond to the peaks of the paeonol reference standard. Peak 9 is the S peak. Calculate the relative retention times of each characteristic peak and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.45 (peak 2), 0.57 (peak 3), 0.66 (peak 4), 0.75 (peak 5), 0.81 (peak 6), 0.84 (peak 7), and 0.91 (peak 8).

[0108] Table 2. Results of relative retention times of 15 batches of Paeonia suffruticosa root materials

[0109]

[0110]

[0111] Note: t is the retention time; t / ts is the relative retention time.

[0112] Table 3. Results of relative peak area of ​​15 batches of Paeonia suffruticosa root materials

[0113]

[0114] Note: A is the peak area; A / AS is the relative peak area.

[0115] Table 4. Analytical Results of Characteristic Peaks in Moutan Bark

[0116]

[0117] Furthermore, when using the method of this embodiment to conduct quality testing on peony bark, the test sample of peony bark is tested according to the aforementioned characteristic chromatogram detection method for peony bark. The chromatogram of the test sample is compared with the reference characteristic chromatogram. If the characteristic chromatogram of the test sample contains at least 9 characteristic peaks identical to those in the reference characteristic chromatogram, and the retention times correspond to the 9 characteristic peaks in the reference chromatogram of the reference medicinal material, with 2 peaks corresponding to the retention times of the corresponding reference peaks, and peak 9 corresponding to the paeonol reference peak is designated as peak S, the relative retention times of each characteristic peak and peak S are calculated. The relative retention times should be within ±10% of the specified values, which are: 0.45 (peak 2), 0.57 (peak 3), 0.66 (peak 4), 0.75 (peak 5), 0.81 (peak 6), 0.84 (peak 7), and 0.91 (peak 8). Then the quality of the test sample is qualified; otherwise, it is unqualified.

[0118] Example 2 Methodological Validation

[0119] 1. Exclusivity

[0120] Take blank (50 v / v % methanol), paeonol reference standard, gallic acid reference standard, peony bark reference material, and peony bark material (2106001) as the test sample, and test them according to the detection method in Example 1. Inject 20 μl of each sample for detection. The results are shown in the table below. Figures 5-9 As shown, no peak was emitted at the position corresponding to the reference peak under the detection conditions in the blank solvent. A chromatographic peak corresponding to the reference peak could be found in the test sample. In addition, all nine labeled peaks in the test sample solution could be found in the reference solution of the reference medicinal material, indicating that the method has good specificity.

[0121] Table 5. System adaptability parameters - Peony bark sample solution

[0122]

[0123] 2. Instrument precision

[0124] Approximately 1.5 g of the same batch of Paeonia suffruticosa root bark (2106001) was accurately weighed and prepared as a test sample according to the "Preparation of Test Sample Solution" method in Example 1. The sample was then tested according to the method in Example 1, with six consecutive injections. Chromatograms were recorded, and the RSD% of the relative retention time and relative peak area of ​​each labeled peak was calculated. The results are shown in the table below. The results indicate that the RSD% of the relative retention time and relative peak area of ​​the nine characteristic peaks are all less than 2.0%. Overall, the method demonstrates good precision and meets the requirements for characteristic chromatograms.

[0125] Table 6. Results of Instrument Precision-Relative Retention Time Test

[0126]

[0127] Table 7. Results of Instrument Precision-Relative Peak Area Test

[0128]

[0129] 3 Repeatability

[0130] Approximately 1.5g of the same batch of Paeonia suffruticosa root bark (2106001) was accurately weighed in six parallel portions. The test sample was prepared according to the "Preparation of Test Sample Solution" method in Example 1, and analyzed as described in Example 1. Chromatograms were recorded, and the relative retention time and relative peak area (RSD%) of each characteristic peak were calculated. The results are shown in the table below. The results indicate that the RSD of the relative retention time of all nine characteristic peaks was less than 2.0%, and the RSD% of the relative peak area was between 0% and 8.9%. Overall, the method demonstrates good repeatability and meets the requirements for characteristic chromatograms.

[0131] Table 8. Results of Repeatability-Relative Retention Time Test

[0132]

[0133]

[0134] Table 9. Results of Repeatability-Relative Peak Area Test

[0135]

[0136] 4. Intermediate precision

[0137] The same batch of Paeonia suffruticosa root bark (2106001) was used to observe the effects of random variations such as different dates, different analysts, and different instruments on repeatability. The test sample was prepared according to the "Preparation of Test Sample Solution" method in Example 1, and analyzed using the method in Example 1. Chromatograms were recorded, and peak 9 (paeonol) was designated as the S peak. The relative retention time and relative peak area RSD% of each labeled peak were calculated. The results are shown in the table below. The results indicate that the relative retention time RSD% of the nine characteristic peaks is less than 2.0%, and the relative peak area RSD% is between 0% and 39.4%. Overall, the intermediate precision of the method (under different dates, different analysts, and different instruments) is good and meets the requirements of characteristic chromatograms.

[0138] Table 10. Results of intermediate precision-relative retention time test

[0139]

[0140]

[0141] Table 11. Results of intermediate precision-relative peak area test

[0142]

[0143] 5. Solution stability

[0144] A sample solution of the same peony bark (2106001) was taken and tested according to Example 1. The sample was injected and detected at 0, 5, 12, 20 and 29 hours, and the chromatograms were recorded. Peak 9 (paeonol) was taken as the S peak. The relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in the table below. The results show that the relative retention time of the 9 characteristic peaks is less than 2.0%, and the relative peak area RSD% is between 0 and 7.3%, indicating that the sample solution is stable at room temperature for 29 hours.

[0145] Table 12. Results of Solution Stability-Relative Retention Time Test

[0146]

[0147] Table 13. Results of Solution Stability-Relative Peak Area Test

[0148]

[0149]

[0150] 6. Flow velocity investigation

[0151] The same batch of peony bark (2106001) was used in accordance with Example 1. Chromatographic conditions were set at flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively. Chromatograms were recorded, and the relative retention time and relative peak area (RSD%) of each characteristic peak were calculated. The results are shown in the table below. Figures 10-12 The results showed that the relative retention time (RSD%) of the nine characteristic peaks ranged from 0 to 8.1%, all within ±10% of the mean, and the relative peak area (RSD%) ranged from 0 to 24.5%. As can be seen from the figure, different flow rates have a certain influence on the relative retention time and relative peak area of ​​the chromatographic peaks. In order to ensure the separation and reproducibility of the characteristic chromatograms, 1.0 ml / min was selected as the optimal flow rate for the determination of the characteristic chromatograms of Paeonia suffruticosa.

[0152] Table 14. Results of the flow velocity-relative retention time test

[0153]

[0154] Table 15. Results of the Flow Velocity Study - Relative Peak Area Test

[0155]

[0156] 7. Column temperature investigation

[0157] The same test sample (2106001) was tested according to the method in Example 1. The column temperatures in the chromatographic conditions were set at 28℃, 30℃, and 32℃, respectively. Chromatograms were recorded, and the relative retention time and relative peak area (RSD%) of each labeled peak were calculated. The results are shown in the table below. Figures 13-15 The results indicate that the relative retention time (RSD%) of each characteristic peak is less than 2.0%, and the relative peak area (RSD%) is between 0 and 11.3%. Overall, the test exhibits good robustness under different column temperature conditions.

[0158] Table 16. Results of Column Temperature Investigation - Relative Retention Time Test

[0159]

[0160] Table 17. Column Temperature Investigation - Relative Peak Area Test Results

[0161]

[0162] 8. Investigation of mobile phase acid concentration

[0163] The same test sample (2106001) was tested according to the method in Example 1. The concentrations of mobile phase B in the chromatographic conditions were 0.08%, 0.10%, and 0.12%, respectively. Chromatograms were recorded, and the relative retention times and relative peak areas (RSD%) of each characteristic peak were calculated. The results are shown in the table below. Figures 16-18 As shown, the results indicate that the relative retention time and relative peak area (RSD%) of the nine characteristic peaks are all less than 2.0%, suggesting that the variation of the mobile phase acid concentration within the range of 0.08% to 0.12% has little impact on the overall characteristic spectrum of Paeonia suffruticosa.

[0164] Table 18. Results of Acid Concentration Investigation - Relative Retention Time Test

[0165]

[0166] Table 19. Results of Acid Concentration Investigation - Relative Peak Area Test

[0167]

[0168] 9. Investigation using different instruments

[0169] The same test sample (2106001) was tested according to the method in Example 1. The tests were conducted on different brand instruments (Waters 2695, Agilent 1100, and Shimadzu LC-2030C 3D) under different chromatographic conditions. Chromatograms were recorded, and peak 9 (paeonol) was taken as the S peak. The relative retention time and relative peak area (RSD%) of the peaks were calculated. The results are shown in the table below. Figures 23-25 The results showed that the relative retention time (RSD%) of the nine characteristic peaks ranged from 0 to 2.8%, all within ±10% of the mean, and the relative peak area (RSD%) ranged from 0 to 39.1%. Overall, this method demonstrates good robustness on Waters, Agilent, and Shimadzu instruments.

[0170] Table 20. Results of Relative Retention Time Test for Different Brands of Instruments

[0171]

[0172] Table 21. Results of relative peak area tests for instruments of different brands

[0173]

[0174] 10. Chromatographic column investigation

[0175] Take the same test sample (2106001) and perform the test according to the method of Example 1, respectively, under the same chromatographic conditions (using the same type of...). The experiment was conducted on XB-C18 columns (4.6mm × 250mm, 5μm) from different batches (Column 1: SN: 60220301261; Column 2: SN: 21706196; Column 3: SN: 211703538). The results are shown in the table below. Figures 26-28As shown, the results indicate that the relative retention time (RSD%) of the nine characteristic peaks is in the range of 0–3.3%, all within ±10% of the mean, and the relative peak area (RSD%) is in the range of 0–26.4%, indicating that the column of this method has good robustness.

[0176] Table 22. Results of Relative Retention Time Tests for Different Columns

[0177]

[0178]

[0179] Table 23. Results of relative peak area tests for different chromatographic columns

[0180]

[0181] 11. Holistic Examination

[0182] The same test sample (2106001) was used and the method of Example 1 was followed, with the elution time extended by 1 time in the chromatographic conditions. A comprehensive study of the characteristic chromatograms was conducted. Results... Figure 19 As shown in the chromatogram, no other chromatographic peaks with obvious characteristics were eluted during the subsequent elution period, indicating that the characteristic peak information in the sample has been fully presented within the specified gradient elution time and will not affect subsequent injections.

[0183] Example 3

[0184] This embodiment provides a method for detecting the characteristic chromatograms of charred peony bark slices, including: detection by high-performance liquid chromatography (HPLC), with the following chromatographic conditions: the chromatographic column is packed with octadecylsilane-bonded silica gel (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm). XB-C18); using acetonitrile as mobile phase A and 0.1 v / v formic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on the paeonol peak, should not be less than 5000.

[0185] Table 24. Gradient elution program

[0186] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 7 93 10～15 7→15 93→85 15～30 15→30 85→70 30～45 30→75 70→25 45～46 75→7 25→93

[0187] Preparation of reference solutions: Gallic acid reference standard was added to 50 v / v% methanol solution to prepare a solution containing 50 μg / ml gallic acid. Paeonol reference standard was added to methanol solvent to prepare a solution containing 20 μg / ml paeonol. These were used as reference solutions.

[0188] Preparation of the test solution: Take about 1.5g of coarse powder of peony bark charcoal slices, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60 minutes, cool, filter, transfer 5ml of the filtrate to a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0189] Determination method: Accurately pipette 20 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0190] In this embodiment, 15 batches of charred peony bark slices were used to formulate a reference characteristic spectrum of charred peony bark slices. The detection method was carried out according to the above method, and characteristic spectra of 15 batches of charred peony bark slices were obtained. The results are shown in the table below.

[0191] Using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, the characteristic spectra of the above 15 batches of charred peony bark slices were used to generate reference characteristic spectra, as shown below. Figure 20 As shown, there are a total of 9 peaks, numbered 1-9 according to the chromatographic peak sequence. Relevant literature reports that peony bark mainly contains monoterpenes and their glycosides, phenols and their phenolic glycosides, and other components such as triterpenes, sterols and their glycosides, flavonoids, organic acids, and coumarins. Related studies have shown that during the high-temperature charring process of peony bark, the chemical components such as paeonol, gallic acid, paeoniflorin, quercetin, kaempferol, and isorhamnetin undergo varying degrees of quantitative changes due to the high temperature, and new components (such as 5-hydroxymethylfurfural) may also be generated. Since the peony bark charred granule formulation process mainly involves water extraction, and based on literature reports, its main components are mostly water-soluble. Reference standards were selected for localization and identification, and the analysis results are shown in the table below. Peak 1 was confirmed as gallic acid, peak 2 as 5-hydroxymethylfurfural, peak 4 as p-hydroxybenzoic acid, peak 8 as benzoic acid, and peak 9 as paeonol. Based on the peak identification and selection results of the characteristic peaks of charred peony bark, paeonol, which has a large peak response and is an easily obtainable reference standard, was selected as the reference standard S peak. The relative retention times of each characteristic peak were calculated. The reference characteristic spectrum of charred peony bark slices should show 9 characteristic peaks, of which 2 peaks should correspond to the retention times of the corresponding reference standard peaks. Peak 9, corresponding to the paeonol reference standard peak, is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.20 (peak 2), 0.31 (peak 3), 0.45 (peak 4), 0.49 (peak 5), 0.50 (peak 6), 0.64 (peak 7), and 0.75 (peak 8).

[0192] Table 25. Results of relative retention times of 15 batches of charred peony bark slices

[0193]

[0194]

[0195] Table 26, Relative peak area results of 15 batches of Cortex Moutan carbonized slices

[0196]

[0197]

[0198] Table 27, Analysis and detection results of characteristic peaks of Cortex Moutan carbonized slices

[0199]

[0200] Furthermore, when the quality of Cortex Moutan carbonized slices is detected by the method of this embodiment, the test sample of Cortex Moutan carbonized slices is carried out according to the above characteristic chromatogram detection method of Cortex Moutan carbonized slices. The obtained chromatogram of the test sample is compared with the control characteristic chromatogram. If at least 9 identical characteristic peaks in the control characteristic chromatogram appear in the characteristic chromatogram of the test sample, and 2 of the peaks should correspond to the retention times of the corresponding reference substance peaks of the reference substance respectively; the peak 9 corresponding to the paeonol reference substance peak is the S peak, and the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within the range of ±10% of the specified value. The specified values are: 0.20 (peak 2), 0.31 (peak 3), 0.45 (peak 4), 0.49 (peak 5), 0.50 (peak 6), 0.64 (peak 7), 0.75 (peak 8). Then the quality of the test sample is qualified, otherwise it is unqualified.

[0201] Example 4

[0202] This embodiment provides a method for distinguishing Cortex Moutan medicinal materials and Cortex Moutan carbonized slices, including:

[0203] Obtaining the characteristic chromatogram of the test sample according to the characteristic chromatogram detection method of Cortex Moutan medicinal materials in Example 1 and the characteristic chromatogram detection method of Cortex Moutan carbonized slices in Example 3, and comparing the obtained characteristic chromatogram with the control chromatogram of Cortex Moutan medicinal materials and the control characteristic chromatogram of Cortex Moutan carbonized slices.

[0204] Comparing from the control chromatogram of Cortex Moutan medicinal materials and the control characteristic chromatogram of Cortex Moutan carbonized slices, as Figure 21 it can be seen that Peak 1: gallic acid; Peak 2: p-hydroxybenzoic acid; Peak 3: paeoniflorin; Peak 4: β-1,2,3,4,6-pentagalloylglucose; Peak 5: benzoic acid; Peak 6: moutanpioside C; Peak 7: benzoylpaeoniflorin; Peak 8: benzoylpaeoniflorin; Peak 9: paeonol; Peak 10: 5-hydroxymethylfurfural; Peak 12: p-hydroxybenzoic acid; Peak 16: benzoic acid, which can significantly distinguish Cortex Moutan medicinal materials and Cortex Moutan carbonized slices and control their quality.

[0205] Example 5

[0206] This embodiment provides a method for detecting the characteristic chromatograms of a standard decoction of peony bark charcoal slices, including: detection by high-performance liquid chromatography (HPLC), with the following chromatographic conditions: the chromatographic column is packed with octadecylsilane-bonded silica gel (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm). XB-C18); using acetonitrile as mobile phase A and 0.1 v / v formic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on the paeonol peak, should not be less than 5000.

[0207] Table 28. Gradient elution program

[0208] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 7 93 10～15 7→15 93→85 15～30 15→30 85→70 30～45 30→75 70→25 45～46 75→7 25→93

[0209] Preparation of reference solutions: Take about 1.5g of charred peony bark reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60 minutes, cool, filter, transfer 5ml of the filtrate to a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate as the reference solution; take gallic acid reference standard and add it to 50v / v% methanol solution to prepare a solution containing 50ug / ml of gallic acid; take paeonol reference standard and add it to methanol solvent to prepare a solution containing 20ug / ml of paeonol, as the reference solution.

[0210] Preparation of the test solution: Take about 0.2g of lyophilized powder of peony bark charcoal standard decoction, place it in a stoppered conical flask, add 20ml of 50v / v% methanol solution, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0211] Determination method: Accurately pipette 20 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0212] In this embodiment, 15 batches of charred peony bark standard decoction were used to formulate a reference characteristic spectrum of the charred peony bark standard decoction. The detection method was carried out according to the above method, and the characteristic spectra of the 15 batches of charred peony bark standard decoction were obtained. The results are shown in the table below.

[0213] Using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, the characteristic spectra of the above 15 batches of peony bark charcoal standard decoction samples were used to generate control characteristic spectra, as shown below. Figure 22As shown, there are 9 common peaks, and the chromatographic peak numbers 1 - 9 are arranged according to the order of the chromatographic peaks. The characteristic peak with a larger response is paeonol, so paeonol is used as the S peak of this characteristic chromatogram to calculate the relative retention time of each characteristic peak. It is stipulated that 9 characteristic peaks should be presented in the reference characteristic chromatogram of the carbonized cortex moutan standard decoction, and they should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, 2 peaks should respectively correspond to the retention times of the corresponding reference substance peaks of the reference substances; the peak 9 corresponding to the paeonol reference substance peak is the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: peak 2 is 0.19, peak 3 is 0.28, peak 4 is 0.41, peak 5 is 0.45, peak 6 is 0.47, peak 7 is 0.63, and peak 8 is 0.74.

[0214] Table 29. Relative retention times of the characteristic chromatograms of 15 batches of carbonized cortex moutan decoction pieces standard decoctions

[0215]

[0216]

[0217] Table 30. Relative peak areas of the characteristic chromatograms of 15 batches of carbonized cortex moutan decoction pieces standard decoctions

[0218]

[0219]

[0220] Furthermore, when the quality of the carbonized cortex moutan standard decoction is detected by the method of this embodiment, the test sample of the carbonized cortex moutan standard decoction is carried out according to the above-mentioned characteristic chromatogram detection method of the carbonized cortex moutan standard decoction. The obtained chromatogram of the test sample is compared with the reference characteristic chromatogram. If at least 9 identical characteristic peaks in the reference characteristic chromatogram appear in the characteristic chromatogram of the test sample, and 2 of them should respectively correspond to the retention times of the corresponding reference substance peaks of the reference substances; the peak 9 corresponding to the paeonol reference substance peak is the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.19 (peak 2), 0.28 (peak 3), 0.41 (peak 4), 0.45 (peak 5), 0.47 (peak 6), 0.63 (peak 7), and 0.74 (peak 8). Then the quality of the test sample is qualified, otherwise it is unqualified.

[0221] Example 6

[0222] This embodiment provides a method for detecting the characteristic chromatogram of carbonized cortex moutan formula granules, including: detecting by high performance liquid chromatography, and the chromatographic conditions are: the chromatographic column uses octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm, XB-C18); using acetonitrile as mobile phase A and 0.1 v / v formic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 254 nm. The theoretical plate number, calculated based on the paeonol peak, should not be less than 5000.

[0223] Table 31. Gradient elution procedure

[0224]

[0225]

[0226] Preparation of reference solutions: Take about 1.5g of charred peony bark reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60 minutes, cool, filter, transfer 5ml of the filtrate to a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate as the reference solution; take gallic acid reference standard and add it to 50v / v% methanol solution to prepare a solution containing 50ug / ml of gallic acid; take paeonol reference standard and add it to methanol solvent to prepare a solution containing 20ug / ml of paeonol, as the reference solution.

[0227] Preparation of the test solution: Take about 0.2g of peony bark charcoal formula powder, place it in a stoppered conical flask, add 20ml of 50v / v% methanol solution, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0228] Determination method: Accurately pipette 20 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0229] In this embodiment, three batches of Paeonia suffruticosa charcoal granules were used to formulate a reference characteristic spectrum for the Paeonia suffruticosa charcoal granules. The detection method was carried out according to the above method, and characteristic spectra of the three batches of Paeonia suffruticosa charcoal granules were obtained. The results are shown in the table below. Figure 22 .

[0230] Using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, the characteristic spectra of the above three batches of peony bark charcoal formula granules were used to generate control characteristic spectra, as shown below. Figure 24As shown, there are a total of 9 peaks, numbered 1-9 according to the order of the chromatographic peaks. Based on peak identification and reference standard localization, peak 1 was determined to be gallic acid, peak 2 to be 5-hydroxymethylfurfural, peak 4 to be p-hydroxybenzoic acid, peak 8 to be benzoic acid, and peak 9 to be paeonol. Paeonol, with a large peak response and readily available reference standard, was selected as the reference peak S, and the relative retention times of each characteristic peak were calculated. The reference characteristic chromatogram of the peony bark charcoal granules shows 9 characteristic peaks, among which peaks 1 and 9 should correspond to the retention times of the corresponding reference standard peaks. Peak 9, which responds to the paeonol reference peak, is the S peak. The relative retention times of peaks 2 to 8 with peak S are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.19 (peak 2), 0.28 (peak 3), 0.41 (peak 4), 0.45 (peak 5), 0.47 (peak 6), 0.63 (peak 7), and 0.74 (peak 8).

[0231] Table 32. Retention time and relative retention time of characteristic chromatograms of peony bark charcoal granules from three batches. a. Retention time

[0232] batch number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 2310001Y 6.039 8.329 12.998 18.787 20.417 20.859 26.75 30.705 41.033 2310002Y 6.058 8.353 13.029 18.820 20.446 20.892 26.777 30.741 41.051 2310003Y 6.030 8.317 12.977 18.776 20.408 20.851 26.745 30.702 41.042

[0233] b. Relative retention time

[0234] batch number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 2310001Y 0.147 0.203 0.317 0.458 0.498 0.508 0.652 0.748 1.000 2310002Y 0.148 0.203 0.317 0.458 0.498 0.509 0.652 0.749 1.000 2310003Y 0.147 0.203 0.316 0.457 0.497 0.508 0.652 0.748 1.000 mean 0.147 0.203 0.317 0.458 0.498 0.508 0.652 0.748 1.000 Minimum value 0.128 0.177 0.277 0.423 0.470 0.481 0.625 0.726 1.000 Maximum value 0.148 0.206 0.322 0.457 0.494 0.506 0.636 0.753 1.000 Mean -10% 0.132 0.183 0.285 0.412 0.448 0.457 0.587 0.673 0.900 Mean +10% 0.162 0.223 0.349 0.504 0.548 0.559 0.717 0.823 1.100 RSD% 0.4 0 0.2 0.2 0.2 0.2 0 0.1 0

[0235] Table 33. Peak area and relative peak area of ​​characteristic spectral graphs of 3 batches of Paeonia suffruticosa charcoal formulation granules. a. Peak area

[0236] batch number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 2310001Y 2487316 1236388 1396133 4109340 245568 2310002Y 2720384 1710920 1352000 4376428 258093 2310003Y 2633192 1304805 1362343 4270279 251942

[0237] a continuation, peak area

[0238] batch number Peak 6 Peak 7 Peak 8 Peak 9 2310001Y 520345 592390 547914 4240260 2310002Y 552663 1303842 571286 3332243 2310003Y 540217 706182 550292 5076851

[0239] b. Relative peak area

[0240]

[0241] Furthermore, when the method of this embodiment is used to conduct quality inspection on the Moutan Cortex Carbonis formula granules, the test sample of the Moutan Cortex Carbonis formula granules is carried out according to the above-mentioned characteristic chromatogram detection method of the Moutan Cortex Carbonis formula granules. The chromatogram of the test sample obtained is compared with the reference characteristic chromatogram. If at least 9 identical characteristic peaks in the reference characteristic chromatogram appear in the characteristic chromatogram of the test sample, and 2 of the peaks should respectively correspond to the retention times of the corresponding reference substance peaks of the reference substances; the peak 9 corresponding to the paeonol reference substance peak is the S peak, and the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within the range of ±10% of the specified values. The specified values are: 0.19 (peak 2), 0.28 (peak 3), 0.41 (peak 4), 0.45 (peak 5), 0.47 (peak 6), 0.63 (peak 7), 0.74 (peak 8). Then the quality of the test sample is qualified, otherwise it is unqualified.

[0242] Example 7 Methodological verification of Example 6

[0243] 1. Specificity

[0244] Take blank (50 v / v% methanol), negative control solution (maltodextrin), negative control solution (β-cyclodextrin), reference substance solution, reference solution of Moutan Cortex control medicinal material, and test sample of Moutan Cortex Carbonis formula granules, and conduct according to Example 6, record the chromatogram, and examine whether the negative control solution of Moutan Cortex Carbonis formula granules will cause interference. The results are as Figures 25-31 , the negative control solution did not show peaks at the positions corresponding to the reference peaks, the negative had no interference, and chromatographic peaks corresponding to the reference substance peaks could be found in the test sample, indicating that the method has good specificity.

[0245] 2. Instrument precision

[0246] Take about 0.2 g of the same batch of Moutan Cortex Carbonis formula granules (2310002Y), accurately weigh, prepare the test sample solution according to Example 6, and detect according to Example 6, and inject samples continuously for 6 times. The results are as follows in the table. The results show that the RSD values of the relative retention times of the 9 characteristic peaks are all less than 2.0%; the RSD values of the relative peak areas are between 0 and 16.5%. Judging comprehensively, the instrument precision of this method is good and meets the requirements of the characteristic chromatogram.

[0247] Table 34. Instrument precision - retention time and relative retention time a. Retention time

[0248] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Precision 1 6.701 9.219 14.427 19.909 21.449 21.946 27.738 32.170 42.024 Precision 2 6.706 9.238 14.468 19.922 21.455 21.954 27.731 32.159 42.004 Precision 3 6.660 9.161 14.363 19.867 21.407 21.903 27.683 32.098 41.979 Precision 4 6.660 9.167 14.364 19.860 21.403 21.899 27.668 32.080 41.964 Precision 5 6.656 9.150 14.328 19.828 21.369 21.864 27.659 32.063 41.966 Precision 6 6.640 9.146 14.340 19.843 21.383 21.880 27.667 32.062 41.937

[0249] b. Relative retention time [[ID=Z7]]

[0250] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Precision 1 0.159 0.219 0.343 0.474 0.510 0.522 0.660 0.766 1.000 Precision 2 0.160 0.220 0.344 0.474 0.511 0.523 0.660 0.766 1.000 Precision 3 0.159 0.218 0.342 0.473 0.510 0.522 0.659 0.765 1.000 Precision 4 0.159 0.218 0.342 0.473 0.510 0.522 0.659 0.764 1.000 Precision 5 0.159 0.218 0.341 0.472 0.509 0.521 0.659 0.764 1.000 Precision 6 0.158 0.218 0.342 0.473 0.510 0.522 0.660 0.765 1.000 mean 0.159 0.218 0.342 0.473 0.510 0.522 0.660 0.765 1.000 Minimum value 0.158 0.218 0.341 0.472 0.509 0.521 0.659 0.764 1.000 Maximum value 0.160 0.220 0.344 0.474 0.511 0.523 0.660 0.766 1.000 Mean -10% 0.143 0.196 0.308 0.426 0.459 0.470 0.594 0.689 0.900 Mean +10% 0.175 0.240 0.376 0.520 0.561 0.574 0.726 0.842 1.100 RSD% 0.4 0.4 0.4 0.2 0.2 0.2 0.1 0.2 0

[0251] Table 35. Instrument precision - peak area and relative peak area a. Peak area

[0252] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Precision 1 3077945 1944606 1545985 5201666 262757 442743 1667588 850033 3717770 Precision 2 3076959 1947194 1495352 5201121 263948 444635 1672881 850067 3708666 Precision 3 3063275 1937796 1535415 5187557 263894 439431 1668156 841198 3707479 Precision 4 3065312 1944259 1541188 5178362 261829 438291 1664782 842256 3703152 Precision 5 3055142 1939090 1532554 5176089 259701 437047 1664159 812070 3703525 Precision 6 3092040 1956912 1512754 5234516 266931 608911 1667993 820979 3587437

[0253] b. Relative peak area

[0254] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Precision 1 0.828 0.523 0.416 1.399 0.071 0.119 0.449 0.229 1.000 Precision 2 0.830 0.525 0.403 1.402 0.071 0.120 0.451 0.229 1.000 Precision 3 0.826 0.523 0.414 1.399 0.071 0.119 0.450 0.227 1.000 Precision 4 0.828 0.525 0.416 1.398 0.071 0.118 0.450 0.227 1.000 Precision 5 0.825 0.524 0.414 1.398 0.070 0.118 0.449 0.219 1.000 Precision 6 0.862 0.545 0.422 1.459 0.074 0.170 0.465 0.229 1.000 mean 0.833 0.528 0.414 1.409 0.071 0.127 0.452 0.227 1.000 RSD% 1.8 1.7 1.6 1.8 2.0 16.5 1.4 1.8 0

[0255] 3. Repeatability

[0256] Approximately 0.2 g of the same batch of peony bark charcoal granules (2310002Y) was weighed in parallel to form 6 portions, and the process was carried out according to Example 6. The results are shown in the table below, indicating that the relative retention time RSD values ​​of the 9 characteristic peaks are all less than 2.0%; the relative peak area RSD values ​​are within the range of 0 to 47.7%. Overall, the method has good repeatability and meets the requirements of the characteristic spectrum.

[0257] Table 36. Repeatability-Retention Time and Relative Retention Time a. Retention Time

[0258] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Repeatability 1 6.820 9.385 14.676 20.090 21.617 22.121 27.859 32.380 42.137 Repeatability 2 6.611 9.105 14.280 19.804 21.346 21.842 27.637 32.025 41.925 Repeatability 3 6.620 9.120 14.278 19.796 21.340 21.832 27.633 32.006 41.911 Repeatability 4 6.589 9.066 14.226 19.742 21.291 21.785 27.589 31.950 41.871 Repeatability 5 6.587 9.061 14.203 19.740 21.290 21.781 27.597 31.932 41.866 Repeatability 6 6.513 8.971 14.067 19.638 21.191 21.680 27.520 31.832 41.805

[0259] b. Relative retention time

[0260] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Repeatability 1 0.162 0.223 0.348 0.477 0.513 0.525 0.661 0.768 1.0000 Repeatability 2 0.158 0.217 0.341 0.472 0.509 0.521 0.659 0.764 1.0000 Repeatability 3 0.158 0.218 0.341 0.472 0.509 0.521 0.659 0.764 1.0000 Repeatability 4 0.157 0.217 0.340 0.471 0.508 0.520 0.659 0.763 1.0000 Repeatability 5 0.157 0.216 0.339 0.472 0.509 0.520 0.659 0.763 1.0000 Repeatability 6 0.156 0.215 0.336 0.470 0.507 0.519 0.658 0.761 1.0000 Mean % 0.158 0.218 0.341 0.472 0.509 0.521 0.659 0.764 1.0000 Minimum value 0.156 0.215 0.336 0.470 0.507 0.519 0.658 0.761 1.0000 Maximum value 0.162 0.223 0.348 0.477 0.513 0.525 0.661 0.768 1.0000 Mean -10% 0.142 0.196 0.307 0.425 0.458 0.469 0.593 0.688 0.9000 Mean +10% 0.174 0.240 0.375 0.519 0.560 0.573 0.725 0.840 1.1000 RSD% 1.4 1.3 1.2 0.6 0.5 0.5 0.2 0.4 0

[0261] Table 37. Repeatability - Peak Area and Relative Peak Area a. Peak Area

[0262]

[0263]

[0264] b. Relative peak area

[0265] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Repeatability 1 0.835 0.410 0.407 1.379 0.072 0.125 0.456 0.233 1.000 Repeatability 2 0.827 0.524 0.417 1.397 0.071 0.161 0.458 0.224 1.000 Repeatability 3 0.823 0.524 0.414 1.390 0.070 0.115 0.462 0.221 1.000 Repeatability 4 0.822 0.523 0.414 1.389 0.069 0.161 0.447 0.081 1.000 Repeatability 5 0.824 0.524 0.415 1.391 0.069 0.161 0.461 0.087 1.000 Repeatability 6 0.821 0.521 0.414 1.382 0.068 0.160 0.466 0.100 1.000 mean 0.825 0.504 0.414 1.388 0.070 0.147 0.458 0.158 1.000 RSD% 0.7 9.2 0.9 0.5 2.2 14.5 1.5 47.7 0

[0266] 4. Intermediate precision

[0267] The same batch of peony bark charcoal granules (2310002Y) was used, and repeatability tests were conducted by different personnel on different dates and with different equipment to investigate the effect of random variation factors (different dates, different analysts, and different instruments) on repeatability. The test was carried out according to Example 6, and the results are shown in the table below. It shows that the relative retention time RSD values ​​of the nine characteristic peaks are in the range of 0 to 7.6%, and all are within ±10% of the mean; the relative peak area RSD values ​​are in the range of 0 to 32.2%. Overall, the intermediate precision of the method (different dates, different analysts, and different instruments) is good and meets the requirements of the characteristic chromatogram.

[0268] Table 38. Intermediate Precision - Retention Time and Relative Retention Time a. Retention Time

[0269] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Intermediate precision 1 5.542 7.708 12.028 17.830 19.537 19.985 25.574 29.990 40.625 Intermediate precision 2 5.552 7.707 12.027 17.840 19.547 19.995 25.573 29.989 40.624 Intermediate precision 3 5.553 7.718 12.038 17.841 19.548 20.006 25.585 30.001 40.636 Intermediate precision 4 5.563 7.728 12.059 17.862 19.558 20.016 25.595 30.011 40.635 Intermediate precision 5 5.574 7.761 12.092 17.884 19.580 20.028 25.596 30.022 40.646 Intermediate precision 6 5.564 7.729 12.06 17.852 19.548 20.006 25.564 29.990 40.625

[0270] b. Relative retention time

[0271]

[0272]

[0273] Table 39. Intermediate Precision - Peak Area and Relative Peak Area a. Peak Area

[0274] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Intermediate precision 1 47.054 26.437 23.557 72.223 4.388 8.715 18.238 13.317 59.064 Intermediate precision 2 48.419 27.310 24.270 74.289 4.543 8.962 12.056 12.929 60.649 Intermediate precision 3 48.406 27.318 24.259 74.207 4.493 8.933 11.661 12.940 60.615 Intermediate precision 4 46.94 26.253 23.590 72.001 4.356 8.630 18.22 13.304 58.893 Intermediate precision 5 47.060 26.370 23.815 72.202 4.378 8.677 17.777 13.242 59.019 Intermediate precision 6 47.120 26.421 23.668 72.175 4.377 8.699 18.097 13.302 59.092

[0275] b. Relative peak area

[0276] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Intermediate precision 1 0.797 0.448 0.399 1.223 0.074 0.148 0.309 0.225 1.000 Intermediate precision 2 0.798 0.450 0.400 1.225 0.075 0.148 0.199 0.213 1.000 Intermediate precision 3 0.799 0.451 0.400 1.224 0.074 0.147 0.192 0.213 1.000 Intermediate precision 4 0.797 0.446 0.401 1.223 0.074 0.147 0.309 0.226 1.000 Intermediate precision 5 0.797 0.447 0.404 1.223 0.074 0.147 0.301 0.224 1.000 Intermediate precision 6 0.797 0.447 0.401 1.221 0.074 0.147 0.306 0.225 1.000 mean 0.811 0.476 0.407 1.306 0.072 0.147 0.364 0.189 1.000 RSD% 1.9 9.0 1.8 6.7 3.5 9.8 29.2 32.2 0

[0277] 5. Integrity

[0278] Take the same batch of peony bark charcoal granules (2310002Y), and implement it according to Example 6, extending the elution time by 1 time, to conduct a holistic study of the characteristic chromatograms. Figure 32 As shown, no other chromatographic peaks with obvious characteristics were eluted during the subsequent elution period, indicating that the characteristic peak information in the sample has been fully presented within the specified gradient elution time and will not affect subsequent injections.

[0279] 6. Solution stability

[0280] The same batch of peony bark charcoal granules (2310002Y) was used and tested according to Example 6. The samples were injected at 0h, 6.5h, 11h, 19.5h and 31h, and the chromatograms were recorded. The results are shown in the table below. The results show that the relative retention time RSD values ​​of the nine characteristic peaks are all less than 2.0%; the relative peak area RSD values ​​are between 0 and 36.7%, indicating that the test solution has good stability within 31h at room temperature.

[0281] Table 40. Solution Stability - Retention Time and Relative Retention Time a. Retention Time

[0282] Solution stability Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Solution stability 0h 6.820 9.385 14.676 20.090 21.617 22.121 27.859 32.380 42.137 Solution stability 6.5h 6.701 9.219 14.427 19.909 21.449 21.946 27.738 32.170 42.024 Solution stability 11h 6.640 9.146 14.340 19.843 21.383 21.880 27.667 32.062 41.937 Solution stability 19.5h 6.544 9.002 14.111 19.668 21.219 21.708 27.533 31.851 41.801 Solution stability 31h 6.475 8.908 13.971 19.573 21.127 21.613 27.468 31.750 41.742

[0283] b. Relative retention time

[0284] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Solution stability 0h 0.162 0.223 0.348 0.477 0.513 0.525 0.661 0.768 1.000 Solution stability 6.5h 0.159 0.219 0.343 0.474 0.510 0.522 0.660 0.766 1.000 Solution stability 11h 0.158 0.218 0.342 0.473 0.510 0.522 0.660 0.765 1.000 Solution stability 19.5h 0.157 0.215 0.338 0.471 0.508 0.519 0.659 0.762 1.000 Solution stability 31h 0.155 0.213 0.335 0.469 0.506 0.518 0.658 0.761 1.000 mean 0.158 0.218 0.341 0.473 0.509 0.521 0.660 0.764 1.000 Minimum value 0.155 0.213 0.335 0.469 0.506 0.518 0.658 0.761 1.000 Maximum value 0.162 0.223 0.348 0.477 0.513 0.525 0.661 0.768 1.000 Mean -10% 0.142 0.196 0.307 0.426 0.458 0.469 0.594 0.688 0.900 Mean +10% 0.174 0.240 0.375 0.520 0.560 0.573 0.726 0.840 1.100 RSD% 1.7 1.8 1.5 0.7 0.6 0.6 0.2 0.4 0

[0285] Table 41. Solution stability - relative peak area a, peak area

[0286] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0h 3111014 1527586 1515055 5135849 266952 465653 1699137 868525 3724064 6.5h 3077945 1944606 1545985 5201666 262757 442743 1667588 850033 3717770 11h 3092040 1956912 1512754 5234516 266931 608911 1667993 820979 3587437 19.5h 3061638 1944910 1548665 5180940 257313 597029 1639176 359766 3507259 31h 3013323 1927979 1528103 5077913 247146 585784 1595635 413239 3602415

[0287] b. Relative peak area

[0288] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0h 0.835 0.410 0.407 1.379 0.072 0.125 0.456 0.233 1.000 6.5h 0.828 0.523 0.416 1.399 0.071 0.119 0.449 0.229 1.000 11h 0.862 0.545 0.422 1.459 0.074 0.170 0.465 0.229 1.000 19.5h 0.873 0.555 0.442 1.477 0.073 0.170 0.467 0.103 1.000 31h 0.836 0.535 0.424 1.410 0.069 0.163 0.443 0.115 1.000 mean 0.847 0.514 0.422 1.425 0.072 0.149 0.456 0.182 1.000 RSD% 2.4 11.6 3.1 3.0 2.7 17.0 2.3 36.7 0

[0289] 7. Flow velocity investigation

[0290] The same batch of peony bark charcoal granules (2310002Y) was used, and the chromatographic process was carried out according to Example 6, with flow rates set to 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively. The results are shown in the table below. Figures 33-35 The results indicate that the relative retention time RSD values ​​of the nine characteristic peaks are in the range of 0 to 7.7%, and all are within ±10% of the mean; the relative peak area RSD values ​​are in the range of 0 to 34.3%. Based on the overall analysis, the method has good robustness in the flow rate range of 0.9 to 1.1 ml / min and meets the requirements of the characteristic spectrum.

[0291] Table 42. Flow rate-retention time and relative retention time a. Retention time

[0292]

[0293]

[0294] b. Relative retention time

[0295] Flow rate (ml / min) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0.9 0.149 0.207 0.323 0.458 0.494 0.507 0.636 0.753 1.000 1.0 0.136 0.190 0.296 0.439 0.481 0.492 0.630 0.738 1.000 1.1 0.128 0.178 0.278 0.424 0.471 0.481 0.625 0.726 1.000 mean 0.138 0.192 0.299 0.440 0.482 0.493 0.630 0.739 1.000 Minimum value 0.128 0.178 0.278 0.424 0.471 0.481 0.625 0.726 1.000 Maximum value 0.149 0.207 0.323 0.458 0.494 0.507 0.636 0.753 1.000 Mean -10% 0.124 0.173 0.269 0.396 0.434 0.444 0.567 0.665 0.900 Mean +10% 0.152 0.211 0.329 0.484 0.530 0.542 0.693 0.813 1.100 RSD% 7.7 7.6 7.6 3.9 2.4 2.7 0.9 1.9 0

[0296] Table 43. Flow velocity-peak area and relative peak area a. Peak area

[0297] Flow rate (ml / min) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0.9 54.114 30.468 27.453 79.824 4.267 6.935 13.073 10.804 66.272 1.0 47.054 26.437 23.557 72.223 4.388 8.715 18.238 13.317 59.064 1.1 43.638 24.591 22.201 68.36 3.999 8.333 8.852 8.286 54.303

[0298] b. Relative peak area

[0299] Flow rate (ml / min) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0.9 0.817 0.460 0.414 1.204 0.064 0.105 0.197 0.163 1.000 1.0 0.797 0.448 0.399 1.223 0.074 0.148 0.309 0.225 1.000 1.1 0.804 0.453 0.409 1.259 0.074 0.153 0.163 0.153 1.000 mean 0.806 0.454 0.407 1.229 0.071 0.135 0.223 0.180 1.000 RSD% 1.3 1.4 1.9 2.3 8.2 19.6 34.3 21.7 0

[0300] 8. Column temperature investigation

[0301] The same batch of peony bark charcoal granules (2310002Y) was used, and the chromatographic procedure was performed according to Example 6. The column temperatures were set to 28℃, 30℃, and 32℃, respectively. The results are shown in the table below. The relative retention time RSD values ​​of the nine characteristic peaks are in the range of 0–2.3%, and all are within ±10% of the mean. The relative peak area RSD values ​​are in the range of 0–23.9%. Overall, the method has good robustness in the column temperature range of 28–32℃ and meets the requirements of the characteristic chromatogram.

[0302] Table 44. Column Temperature-Retention Time and Relative Retention Time a. Retention Time

[0303] Column temperature (°C) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 28 5.724 7.889 12.38 18.3 19.91 20.316 25.916 30.364 40.881 30 5.542 7.708 12.028 17.83 19.537 19.985 25.574 29.99 40.625 32 5.446 7.644 11.857 17.51 19.313 19.814 25.393 29.809 40.55

[0304] b. Relative retention time

[0305]

[0306]

[0307] Table 45. Column Temperature-Peak Area and Relative Peak Area a. Peak Area

[0308] Column temperature (°C) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 28 48.078 26.809 23.499 76.423 4.153 8.922 12.243 13.077 59.42 30 47.054 26.437 23.557 72.223 4.388 8.715 18.238 13.317 59.064 32 50.697 27.073 23.483 75.291 4.398 8.781 12.661 12.174 59.678

[0309] b. Relative peak area

[0310] Column temperature (°C) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 28 0.809 0.451 0.395 1.286 0.070 0.150 0.206 0.220 1.000 30 0.797 0.448 0.399 1.223 0.074 0.148 0.309 0.225 1.000 32 0.850 0.454 0.393 1.262 0.074 0.147 0.212 0.204 1.000 mean 0.819 0.451 0.396 1.257 0.073 0.148 0.242 0.216 1.000 RSD% 3.4 0.7 0.8 2.6 3.2 1.1 23.9 5.1 0

[0311] 9. Investigation of mobile phase acid concentration

[0312] The same batch of peony bark charcoal formulation granules (2310002Y) was used, and the chromatographic process was carried out according to Example 6. The concentrations of mobile phase B in the chromatographic conditions were 0.08 v / v%, 0.10 v / v%, and 0.12 v / v%, respectively. The results are shown in the table below. The results show that the relative retention time RSD values ​​of the nine characteristic peaks are all less than 2.0%, and the relative peak area RSD values ​​are in the range of 0 to 50.6%, indicating that the variation of the mobile phase acid concentration in the range of 0.08 v / v% to 0.12 v / v% has little impact on the overall characteristic chromatogram of the peony bark charcoal formulation granules.

[0313] Table 46. Investigation of mobile phase acid concentration - retention time and relative retention time a. Retention time

[0314] Acid concentration (%) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0.08 5.508 7.652 11.78 17.764 19.481 19.918 25.508 29.902 40.633 0.10 5.542 7.708 12.028 17.83 19.537 19.985 25.574 29.99 40.625 0.12 5.561 7.737 12.238 17.881 19.577 20.036 25.582 30.009 40.633

[0315] b. Relative retention time

[0316]

[0317]

[0318] Table 47. Investigation of mobile phase acid concentration - peak area and relative peak area a. Peak area

[0319] Acid concentration (%) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0.08 48.508 27.113 24.015 73.93 4.551 9.184 8.54 12.335 59.868 0.10 47.054 26.437 23.557 72.223 4.388 8.715 18.238 13.317 59.064 0.12 48.657 26.455 24.811 74.028 4.331 8.879 8.047 12.348 60.038

[0320] b. Relative peak area

[0321] Acid concentration (%) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 0.08 0.810 0.453 0.401 1.235 0.076 0.153 0.143 0.206 1.000 0.10 0.797 0.448 0.399 1.223 0.074 0.148 0.309 0.225 1.000 0.12 0.810 0.441 0.413 1.233 0.072 0.148 0.134 0.206 1.000 mean 0.806 0.447 0.404 1.230 0.074 0.150 0.195 0.212 1.000 RSD% 1.0 1.4 1.9 0.6 2.8 2.0 50.6 5.2 0

[0322] 10. Instrumental Examination

[0323] The same batch of peony bark charcoal granules (2310002Y) was used and implemented according to Example 6. The chromatographic conditions were tested on different brand instruments (Waters 2695, Agilent 1100, and Shimadzu LC-2030C 3D). The results are shown in the table below. It shows that the relative retention time RSD values ​​of the nine characteristic peaks are in the range of 0 to 3.9%, and all are within ±10% of the mean; the relative peak area RSD values ​​are in the range of 0 to 33.1%, indicating that the method has good robustness on Waters, Agilent, and Shimadzu instruments.

[0324] Table 48. Instrument Examination - Retention Time and Relative Retention Time a. Retention Time

[0325] Different brands of instruments Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Waters 6.058 8.353 13.029 18.820 20.446 20.892 26.777 30.741 41.051 Shimadzu 5.552 7.707 12.027 17.840 19.547 19.995 25.573 29.989 40.624 Agilent 5.877 8.124 12.683 18.700 20.377 20.816 26.753 30.672 41.153

[0326] b. Relative retention time

[0327] Different brands of instruments Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 waters 0.148 0.203 0.317 0.458 0.498 0.509 0.652 0.749 1.000 Shimadzu 0.137 0.190 0.296 0.439 0.481 0.492 0.630 0.738 1.000 Agilent 0.143 0.197 0.308 0.454 0.495 0.506 0.650 0.745 1.000 mean 0.143 0.197 0.307 0.450 0.491 0.502 0.644 0.744 1.000 Minimum value 0.137 0.190 0.296 0.439 0.481 0.492 0.630 0.738 1.000 Maximum value 0.148 0.203 0.317 0.458 0.498 0.509 0.652 0.749 1.000 Mean -10% 0.129 0.177 0.276 0.405 0.442 0.452 0.580 0.670 0.900 Mean +10% 0.157 0.217 0.338 0.495 0.540 0.552 0.708 0.818 1.100 RSD% 3.9 3.4 3.5 2.3 1.9 1.9 1.9 0.8 0

[0328] Table 49. Instrumental Examination - Peak Area and Relative Peak Area a. Peak Area

[0329]

[0330]

[0331] b. Relative peak area

[0332] Different brands of instruments Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 waters 0.816 0.513 0.406 1.313 0.077 0.166 0.391 0.171 1.000 Shimadzu 0.798 0.450 0.400 1.225 0.075 0.148 0.199 0.213 1.000 Agilent 0.723 0.426 0.447 1.380 0.086 0.173 0.284 0.197 1.000 mean 0.779 0.463 0.418 1.306 0.079 0.162 0.291 0.194 1.000 RSD% 6.4 9.8 6.2 6.0 7.5 8.0 33.1 11.0 0

[0333] 11. Chromatographic column investigation

[0334] Take the same batch of peony bark charcoal granules (2310002Y) and implement it according to Example 6. Under the same chromatographic conditions, for the same type ( The experiment was conducted on XB-C18 columns (4.6mm × 250mm, 5μm) from different batches (Column 1: Serial Number: 60220301261; Column 2: Serial Number: 211703538; Column 3: Serial Number: 60220700617). The results are shown in the table below, indicating that the relative retention time RSD values ​​of the nine characteristic peaks are all less than 2.0%; the relative peak area RSD values ​​are in the range of 0–74.7%, indicating that the column robustness of this method is good.

[0335] Table 50. Column Investigation - Retention Time and Relative Retention Time a. Retention Time

[0336] Different numbered chromatographic columns Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Column 1 5.919 8.286 12.895 18.712 20.383 20.824 26.562 30.954 41.109 Column 2 5.994 8.311 12.854 18.764 20.409 20.859 26.628 30.219 41.107 Column 3 6.058 8.353 13.029 18.82 20.446 20.892 26.777 30.741 41.051

[0337] b. Relative retention time

[0338] Different column numbering Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Column 1 0.144 0.202 0.314 0.455 0.496 0.507 0.646 0.753 1.000 Column 2 0.146 0.202 0.313 0.456 0.496 0.507 0.648 0.735 1.000 Column 3 0.148 0.203 0.317 0.458 0.498 0.509 0.652 0.749 1.000 mean 0.146 0.202 0.315 0.456 0.497 0.508 0.649 0.746 1.000 RSD% 1.4 0.3 0.7 0.4 0.3 0.3 0.5 1.3 0

[0339] Table 51. Chromatographic column investigation - peak area and relative peak area a. Peak area

[0340] Different chromatographic columns Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Column 1 2827591 1514843 1352515 4836699 139105 461677 296662 674988 3524806 Column 2 2770223 1503474 1401921 4866417 250877 544982 561707 273606 3404417 Column 3 2720384 1710920 1352000 4376428 258093 552663 1303842 571286 3332243

[0341] b. Relative peak area

[0342]

[0343]

[0344] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations, characterized in that, This includes detection using high-performance liquid chromatography (HPLC), with the following chromatographic conditions: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.08-0.12 v / v% formic acid solution as mobile phase B, eluted according to the following gradient: From 0 to 10 minutes, the volume ratio of mobile phase A to mobile phase B was 7%:93%. Over 10-15 minutes, the volume ratio of mobile phase A to mobile phase B changes from 7%:93% to 15%:85%. Over 15-30 minutes, the volume ratio of mobile phase A to mobile phase B changes from 15%:85% to 30%:70%. For 30-45 minutes, the volume ratio of mobile phase A to mobile phase B is 30%:70% → 75%:25%. After 45-46 minutes, the volume ratio of mobile phase A to mobile phase B changed from 75%:25% to 7%:93%. The detection wavelength is 254nm; The peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparations include peony bark medicinal material, peony bark charcoal slices, peony bark charcoal standard decoction or peony bark charcoal formula granules; When the test sample is peony bark or charred peony bark slices, the test sample solution is prepared as follows: take the test sample, add water to extract, filter, dilute with alcohol solvent, filter again, and take the filtrate. When the test sample is a standard decoction of peony bark charcoal or peony bark charcoal formula granules, the test sample solution is prepared as follows: Take the test sample, add a 25 v / v %-75 v / v % methanol solution to extract, filter, and take the filtrate. When the test sample is peony bark, the preparation of the reference solution is as follows: take the reference standards of gallic acid, p-hydroxybenzoic acid, paeoniflorin, β-1,2,3,4,6-pentagalloglucoside, benzoic acid, paeonol C, benzoyl paeoniflorin oxide, benzoyl paeoniflorin, and paeonol to prepare a reference solution; when the test sample is peony bark charcoal slices, peony bark charcoal standard decoction, or peony bark charcoal formula granules, the preparation of the reference solution is as follows: take the reference standards of gallic acid, 5-hydroxymethylfurfural, p-hydroxybenzoic acid, benzoic acid, and paeonol to prepare a reference solution.

2. The method for detecting the characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to claim 1, characterized in that, The chromatographic conditions include at least one of the following: 1) Flow rate: 0.9 ml / min - 1.1 ml / min; 2) Column temperature: 28℃-32℃; 3) The specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm; 4) The injection volume is 5-20 μl.

3. The method for detecting the characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to claim 1 or 2, characterized in that, This also includes the preparation of the reference solution: Preparation of reference solution for reference medicinal material: Take peony bark as reference medicinal material, add solvent for extraction, filter for the first time, dilute with alcohol solvent, filter for the second time, and collect the filtrate; And / or, preparation of reference solutions: Gallic acid and paeonol are added to 50 v / v%~100 v / v% methanol solvent to prepare solutions containing 20-50 μg / ml of gallic acid and paeonol respectively.

4. The method for detecting the characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to claim 3, characterized in that, In the preparation of the reference solution for the reference medicinal material, the mass-volume ratio of the reference medicinal material of peony bark to the solvent was 1.5g:25ml-100ml; And / or, in the preparation of the reference solution of the control medicinal material, the extraction step is to extract by heating and reflux for 30-90 minutes; And / or, in the preparation of the reference solution of the reference medicinal material, the filtrate obtained from the first filtration is diluted at a volume ratio of 1:(1-4).

5. The method for detecting the characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to claim 3, characterized in that, In the preparation of the reference solution for the reference medicinal material, the mass-volume ratio of the reference medicinal material peony bark to the solvent was 1.5g:50ml, and the solvent was water; And / or, in the preparation of the reference solution of the control medicinal material, the extraction step is to extract by heating and reflux for 60 minutes; And / or, in the preparation of the reference solution of the reference medicinal material, the filtrate obtained from the first filtration is diluted at a volume ratio of 1:

2.

6. The method for detecting the characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to any one of claims 1-2, characterized in that, When the test sample is peony bark or charred peony bark slices, the preparation of the test sample solution is as follows: the mass-volume ratio of the test sample to the solvent is 1.5g:25ml-100ml; and / or, the extraction step is reflux extraction for 30-90 minutes; and / or, in the preparation of the reference material solution, the filtrate obtained from the first filtration is diluted at a volume ratio of 1:(1-4). When the test sample is a standard decoction of peony bark charcoal or peony bark charcoal formula granules, the preparation of the test sample solution is as follows: the mass-volume ratio of the test sample to the solvent is 0.2g:10ml-100ml, and the solvent is a 50v / v% methanol solution; and / or, the extraction step is heating and reflux extraction for 30-60 minutes.

7. The method for detecting the characteristic chromatograms of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to claim 6, characterized in that, When the test sample is peony bark or charred peony bark slices, the test sample solution is prepared as follows: the mass-to-volume ratio of the test sample to the solvent is 1.5 g: 50 ml, and the solvent is water; and / or, the extraction step is reflux extraction for 60 minutes; and / or, in the preparation of the reference solution, the filtrate obtained from the first filtration is diluted at a volume ratio of 1:

2.

8. A method for determining the content of peony bark medicinal material, peony bark charcoal slices, or pharmaceutical preparations, characterized in that, include: Take the test solution and the reference solution, and respectively use the characteristic chromatographic detection method of peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparation as described in any one of claims 1-7.

9. A method for quality testing of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations, characterized in that, include: The steps of obtaining the characteristic spectrum of the test sample according to the characteristic spectrum detection method of peony bark medicinal material, peony bark charcoal slices or pharmaceutical preparation according to any one of claims 1-7, and comparing the characteristic spectrum with the control characteristic spectrum; The reference feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of standard samples of peony bark, charred peony bark slices or pharmaceutical preparations according to the feature spectrum detection method of peony bark, charred peony bark slices or pharmaceutical preparations as described in any one of claims 1-7.

10. The method for quality testing of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to claim 9, characterized in that, When testing peony bark, the reference characteristic chromatogram of peony bark includes 9 characteristic peaks. Peak 1 and peak 9 should correspond to the retention times of the corresponding reference standard peaks. Peak 1 is gallic acid, and peak 9 is paeonol. Peak 9 is used as the reference peak S. The relative retention times of peaks 2 to 8 with peak S should be within ±10% of the specified values. The specified values ​​are: peak 2 0.45, peak 3 0.57, peak 4 0.66, peak 5 0.75, peak 6 0.81, peak 7 0.84, and peak 8 0.

91. When testing charred peony bark slices, the reference characteristic chromatogram of charred peony bark slices includes 9 characteristic peaks. Peak 1 and peak 9 should correspond to the retention times of the corresponding reference peaks. Peak 1 is gallic acid, peak 9 is paeonol, and peak 9 is reference peak S. The relative retention times of peaks 2 to 8 with peak S should be within ±10% of the specified values. The specified values ​​are: peak 2 0.20, peak 3 0.31, peak 4 0.45, peak 5 0.49, peak 6 0.50, peak 7 0.64, and peak 8 0.

75. When testing the standard decoction or granules of peony bark charcoal, the reference characteristic chromatograms of the standard decoction or granules of peony bark charcoal are as follows: peak 1 and peak 9 correspond to the retention times of the corresponding reference peaks. Peak 1 is gallic acid, and peak 9 is paeonol. Using peak 9 as the reference peak S, the relative retention times of peaks 2 to 8 with peak S are calculated to be within ±10% of the specified values. The specified values ​​are: peak 2 0.19, peak 3 0.28, peak 4 0.41, peak 5 0.45, peak 6 0.47, peak 7 0.63, and peak 8 0.

74.

11. The method for quality testing of peony bark medicinal material, charred peony bark slices, or pharmaceutical preparations according to claim 10, characterized in that, In the comparative characteristic spectrum of peony bark, peak 1: gallic acid; peak 2: p-hydroxybenzoic acid; peak 3: paeoniflorin; peak 4: β-1,2,3,4,6-pentagalloglucoside; peak 5: benzoic acid; peak 6: peony root extract C; peak 7: benzoyl paeoniflorin; peak 8: benzoyl paeoniflorin; peak 9: paeonol. In the comparative characteristic chromatogram of charred peony bark slices, peak 1: gallic acid; peak 2: 5-hydroxymethylfurfural; peak 4: p-hydroxybenzoic acid; peak 8: benzoic acid; peak 9: paeonol; In the comparative characteristic chromatogram of the standard decoction or formula granules of peony bark charcoal, peak 1: gallic acid; peak 2: 5-hydroxymethylfurfural; peak 4: p-hydroxybenzoic acid; peak 8: benzoic acid; peak 9: paeonol.

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