A method for separating chemical components in a compound preparation of Wendan Decoction or a method for constructing a fingerprint

Abstract

The present application belongs to the quality detection technical field of traditional Chinese medicine preparation, and specifically provides a separation method of chemical components or a construction method of fingerprint of Wending Decoction compound preparation, which comprises the following steps: (1) preparation of Wending Decoction compound preparation test solution; (2) taking the Wending Decoction compound preparation test solution and detecting by high performance liquid chromatography, using octadecylsilane bonded silica gel as the filler, using acetonitrile-water solution containing formic acid as the mobile phase gradient elution, and obtaining the elution program through repeated tests, under the elution conditions of the present application, not only the number of common peaks is significantly increased, but also the separation degree of common characteristic peaks is obviously improved, and each common peak has good peak shape and no interference, so that the quality of Wending Decoction compound preparation can be more comprehensively, clearly and effectively detected.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a method for separating chemical components or constructing fingerprint spectra in a Wendan Decoction compound preparation. Background Technology

[0002] Wendan Decoction is recorded in the Tang Dynasty medical text *Essential Prescriptions Worth a Thousand Gold Pieces* (by Sun Simiao). The original text states: "For those suffering from insomnia due to weakness and restlessness after a serious illness, this is due to cold in the gallbladder; Wendan Decoction is appropriate." The prescription consists of two liang each of Pinellia ternata, Bambusa textilis, and Citrus aurantium, three liang of Citrus reticulata peel, four liang of fresh ginger, and one liang of Glycyrrhiza uralensis. Its effects include regulating qi and resolving phlegm, harmonizing the stomach and benefiting the gallbladder. It is indicated for gallbladder stagnation and phlegm disturbance syndrome, characterized by timidity, easily startled, dizziness, palpitations, restlessness, insomnia, and vivid dreams; or nausea, vomiting, hiccups, dizziness, and epilepsy. The tongue coating is white and greasy, and the pulse is wiry and slippery. As a classic phlegm-resolving formula, clinically, it focuses on the pathogenesis of "gallbladder stagnation and phlegm disturbance" and is often used to treat insomnia, coronary heart disease, hypertension, dizziness, depression, and other geriatric diseases. The formula uses Pinellia ternata, which is pungent and warm, to dry dampness, resolve phlegm, harmonize the stomach, and stop vomiting, serving as the principal herb. Bamboo shavings are used as the assistant herb, as they are sweet and slightly cold, clearing heat, resolving phlegm, relieving irritability, and stopping vomiting. The combination of Pinellia ternata and bamboo shavings, one warm and one cool, effectively resolves phlegm, harmonizes the stomach, stops vomiting, and relieves irritability. Tangerine peel is pungent, bitter, and warm, regulating qi, relieving stagnation, drying dampness, and resolving phlegm. Fructus Aurantii Immaturus is pungent, bitter, and slightly cold, lowering qi, guiding stagnation, eliminating phlegm, and removing masses. The combination of tangerine peel and Fructus Aurantii Immaturus, one warm and one cool, enhances the qi-regulating and phlegm-resolving effects. Poria cocos is added as an adjuvant to strengthen the spleen and drain dampness, thus preventing the generation of phlegm. Ginger is added during decoction to harmonize the spleen and stomach, and ginger also counteracts the toxicity of Pinellia ternata. Licorice is used as the guiding herb to harmonize all the herbs. Together, they achieve the effects of regulating qi, resolving phlegm, harmonizing the stomach, and promoting bile secretion.

[0003] Wendan Decoction consists of six Chinese medicinal herbs: Pinellia ternata, Bambusa textilis, Citrus aurantium, Citrus reticulata peel, Zingiber officinale, and Glycyrrhiza uralensis. Each herb contains key components that effectively exert the efficacy of Wendan Decoction. Therefore, controlling each component in this formula can provide a more comprehensive evaluation of its overall quality. However, the reference sample has lost the original morphological characteristics of the medicinal slices, and simple qualitative and quantitative analysis of indicator components is insufficient to reflect its quality.

[0004] Therefore, researching a new, effective, accurate, and comprehensive method for detecting the quality of Wendan Decoction compound preparations has become an urgent problem to be solved. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for separating chemical components or constructing fingerprint spectra in Wendan Decoction compound preparations. This method establishes fingerprint spectra of the Wendan Decoction compound preparation based on its characteristics, effectively separates each characteristic peak, increases the number of characteristic peaks, improves the separation effect, and can effectively, accurately, and comprehensively detect Wendan Decoction compound preparations.

[0006] Specifically, this invention discloses a method for separating chemical components or constructing fingerprint spectra in a Wendan Decoction compound preparation, comprising the following steps:

[0007] (1) Preparation of the test solution;

[0008] (2) The test solution was analyzed by high performance liquid chromatography. Octadecylsilane-bonded silica gel was used as the stationary phase, and acetonitrile-formic acid aqueous solution was used as the mobile phase for gradient elution. The gradient elution program included: 0→25min→40min→50min→75min→84min→90min→105min→110min. The volume percentage of acetonitrile in the mobile phase was: 0.5%→13%→15%→17%→20%→33%→40%→48%→90%.

[0009] According to any one of the construction methods of the present invention, step (1) includes: weighing the Wendan Decoction compound preparation, diluting it with a solvent, separating the solid and liquid, and taking the liquid, which is the test solution.

[0010] According to any one of the construction methods of the present invention, step (1) further satisfies any one or more of the following AC:

[0011] A. The volume ratio of the compound preparation of Wendan Decoction to the solvent is 3:5-20;

[0012] B. The solid-liquid separation is selected from centrifugation or filtration;

[0013] C. The solvent is selected from at least one of methanol, water and ethanol; preferably methanol.

[0014] According to any one of the construction methods of the present invention, the Wendan Decoction compound preparation is a compound preparation prepared using Pinellia ternata, bamboo shavings, immature bitter orange, tangerine peel, ginger, and licorice as raw materials according to conventional technical means. By weight, the Wendan Decoction compound preparation includes the following raw materials: 23-30 parts Pinellia ternata, 23-30 parts bamboo shavings, 23-30 parts immature bitter orange, 35-45 parts tangerine peel, 50-60 parts ginger, and 10-15 parts licorice. The fingerprint spectrum of these compound preparations can be constructed using the method of the present invention.

[0015] The Wendan Decoction compound preparations are preferably solid, semi-solid, and liquid preparations of Wendan Decoction, and more preferably at least one of Wendan Decoction decoction, Wendan Decoction granules, Wendan Decoction capsules, and Wendan Decoction tablets. The Wendan Decoction compound preparations can be prepared using conventional techniques, such as, but not limited to, the methods specified in the Chinese Pharmacopoeia.

[0016] Preferably, the Wendan Decoction compound preparation is a decoction obtained by decocting six Chinese herbs: Pinellia ternata, Bambusa textilis, Citrus aurantium, Citrus reticulata peel, Zingiber officinale, and Glycyrrhiza uralensis.

[0017] According to any one of the construction methods of the present invention, step (1) includes: taking 3 ml of Wendan Decoction compound preparation, diluting it to the mark in a 5 ml-20 ml volumetric flask, sealing it tightly, shaking it well, filtering it, and obtaining the product.

[0018] According to any one of the construction methods of the present invention, step (2) further satisfies any one or more of the following items 1)-3):

[0019] 1) The gradient elution program further includes: 110 min → 115 min → 120 min → 130 min, and the volume percentage of acetonitrile in the mobile phase is: 90% → 90% → 0.5% → 0.5%;

[0020] 2) The volume percentage of formic acid in the formic acid-containing aqueous solution is 0.05%-0.15%;

[0021] 3) The chromatographic conditions for high performance liquid chromatography also include: detection wavelength of 235-240 nm, preferably 237 nm; flow rate of 0.9-1.1 ml / min, preferably 0.9-1.0 ml / min; and column temperature of 25-33℃, preferably 30℃.

[0022] In some preferred embodiments, the construction method further includes the step of preparing a reference solution using at least one of glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, novohesperidin, 6-gingerol, and citrusin, and the step of obtaining a reference fingerprint chromatogram by detecting the reference solution using high performance liquid chromatography according to any of the above-described construction methods.

[0023] Reference solutions can be prepared using any one or more of the following reference standards: glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, novohesperidin, 6-gingerol, and citrus. When using two or more reference standards, the reference standards can be mixed to prepare a mixed solution, or a reference solution can be prepared separately.

[0024] According to any one of the construction methods of the present invention, the preparation method of the reference solution includes the following steps: taking glycyrrhizin, naringin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, nobiletin, 6-gingerol, and citrus reticulata reference standards, and adding solvent to prepare a reference solution containing 1-2500 μg of each reference standard per 1 ml; preferably, the solvent is selected from methanol or methanol-water solution; the volume fraction of methanol in the methanol-water solution is not less than 30%.

[0025] Each 1 ml of the reference solution contains glycyrrhizin 15.71–209.46 μg, naringin 32.02–640.37 μg, naringin 90.79–2269.71 μg, hesperidin 32.38–647.66 μg, neohesperidin 30–1761.12 μg, glycyrrhizic acid 17.85–357.09 μg, nobiletin 5.45–54.48 μg, 6-gingerol 4.15–49.81 μg, and citrus 4.24–42.43 μg.

[0026] Each reference solution can be prepared individually or mixed to prepare a mixed reference solution.

[0027] In some preferred embodiments, the method further includes constructing a control fingerprint chromatogram of the Wendantang compound preparation. Fingerprint chromatograms obtained from multiple batches of Wendantang compound preparation test samples are imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" calculation software to form a common pattern diagram, thereby generating a control fingerprint chromatogram of the Wendantang compound preparation. In some preferred embodiments, after generating the control fingerprint chromatogram of the Wendantang compound preparation using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software, the method further includes a step of marking common characteristic peaks.

[0028] At least 10 batches of Wendan Decoction compound preparations were used to obtain a control spectrum, for example, 10 batches, 15 batches, 20 batches, and 30 batches of Wendan Decoction compound preparations.

[0029] In some preferred embodiments, the fingerprint spectrum of the Wendan Decoction compound preparation has 30 common peaks: peak 14 is glycyrrhizin, peak 18 is naringin, peak 19 is naringin, peak 20 is hesperidin, peak 21 is neohesperidin, peak 26 is glycyrrhizic acid, peak 27 is norihesperidin, peak 28 is 6-gingerol, and peak 29 is citrus.

[0030] In certain preferred embodiments, the fingerprint chromatogram of the Wendan Decoction compound preparation has 30 common characteristic peaks, with the neohesperidin chromatographic peak as the reference peak, labeled as peak S. The relative retention times of each characteristic peak and peak S are within ±10% of a specified value; the specified values ​​for peaks 1-30 are, respectively: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, and 0.396. 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, 1.494.

[0031] This invention also provides the application of the method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation of this invention in the quality testing of Wendan Decoction compound preparation products.

[0032] The present invention also provides a method for quality testing of Wendan Decoction compound preparations, comprising constructing a fingerprint spectrum of the Wendan Decoction compound preparation product to be tested according to the fingerprint spectrum construction method of any one of the above-mentioned methods.

[0033] Specifically, the process includes comparing the fingerprint spectrum of the Wendantang compound preparation product to be tested with the fingerprint spectrum of the Wendantang compound preparation control; the fingerprint spectrum of the Wendantang compound preparation product to be tested is obtained by using the Wendantang compound preparation product to be tested according to any of the construction methods described in this invention, and the fingerprint spectrum of the Wendantang compound preparation control is the Wendantang compound preparation control fingerprint spectrum described in this invention.

[0034] If the similarity between the fingerprint spectrum of the Wendantang compound preparation to be tested and the fingerprint spectrum of the control Wendantang compound preparation is not less than 0.85-0.95 (e.g., 0.90), the quality is qualified; if it is less than 0.85-0.95 (e.g., 0.90), it is unqualified. Specifically, the similarity is obtained by the software for evaluating the similarity of chromatographic fingerprint spectra of traditional Chinese medicine.

[0035] In this invention, 0.1% or 0.5% formic acid refers to an aqueous solution containing 0.1% or 0.5% formic acid by volume, respectively.

[0036] The technical solution of this invention has the following advantages:

[0037] 1. The method for separating chemical components or constructing fingerprint spectra in the Wendan Decoction compound preparation of the present invention uses octadecylsilane-bonded silica gel as the packing material and acetonitrile-formic acid aqueous solution as the mobile phase for gradient elution. The elution program is obtained through repeated experiments. Under the elution conditions of the present invention, not only is the number of common peaks significantly increased (up to 30), but the resolution of common characteristic peaks is also significantly improved. Moreover, each common peak has a good peak shape and is free from interference, thereby enabling more comprehensive, clear, and effective quality detection of the Wendan Decoction compound preparation.

[0038] 2. The method for separating chemical components or constructing fingerprint spectra in the Wendan Decoction compound preparation described in this invention involves collecting medicinal slices from different origins to prepare a decoction of Wendan Decoction. The decoction of Wendan Decoction established based on the pharmacopoeia is representative and referential. The fingerprint spectra method established based on the decoction of Wendan Decoction can characterize the herbal information of Pinellia ternata, bamboo shavings, immature bitter orange, tangerine peel, ginger, and licorice, and is applicable to the evaluation of other compound preparations of Wendan Decoction on the market.

[0039] 3. The method for separating chemical components or constructing fingerprint chromatograms in the Wendan Decoction compound preparation described in this invention involves preparing a reference solution and constructing a reference fingerprint chromatogram using at least one of glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, hesperidin, 6-gingerol, and tangeretin. This allows for the localization of 1-9 common peaks in the fingerprint chromatogram of the Wendan Decoction compound preparation, resulting in more accurate, stable, and reliable chromatograms. This indirectly demonstrates that the chromatographic conditions of this invention can effectively separate the nine active components in the Wendan Decoction compound preparation, namely glycyrrhizin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, hesperidin, 6-gingerol, and tangeretin, and the constructed fingerprint chromatogram has high reliability.

[0040] 4. The quality testing method for the Wendan Decoction compound preparation of the present invention constructs a fingerprint spectrum of the Wendan Decoction compound preparation product to be tested according to the fingerprint spectrum construction method of the present invention, and performs quality testing on the characteristic effective components of the Wendan Decoction compound preparation, so as to make the quality testing of the related preparations of the Wendan Decoction compound preparation more comprehensive and ensure the effectiveness and controllability of the quality of the related preparations of the Wendan Decoction compound preparation. Attached Figure Description

[0041] 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.

[0042] Figure 1 The fingerprint spectrum of the 15 batches of Wendan Decoction reference samples in Example 1;

[0043] Figure 2 This is the control fingerprint spectrum of the Wendan Decoction reference sample in Example 1;

[0044] Figure 3 This is a comparison chart of gradient 1 to gradient 2 under the optimization of chromatographic conditions in Example 2;

[0045] Figure 4 HPLC chromatograms of different flow rates in Example 2;

[0046] Figure 5 The HPLC chromatograms are for different column temperatures in Example 2;

[0047] Figure 6 The HPLC chromatograms of different mobile phases in Example 2 are shown below.

[0048] Figure 7The HPLC chromatogram obtained in the investigation experiment of the test sample solution preparation method in Example 3;

[0049] Figure 8 This is a 3D image of the test solution in Example 4;

[0050] Figure 9 This is a contour plot of the test solution in Example 4;

[0051] Figure 10 This is a comparison diagram of the fingerprint spectrum of Wendan Decoction in Example 5 and the decoction of each component herb.

[0052] Figure 11 The fingerprint spectrum of the Wendan Decoction reference sample in Example 5;

[0053] Figure 12 This is a comparison chart of the test solution and various reference standards in Example 5;

[0054] Figure 13 This is a graph showing the system suitability test results in Example 6;

[0055] Figure 14 The image shows the specific HPLC chromatogram of the Wendan Decoction reference sample in Example 6; from top to bottom, the images are: blank solvent, reference solution, tangerine peel negative, stir-fried immature bitter orange negative, tangerine peel-stir-fried immature bitter orange negative, and the test solution of the Wendan Decoction reference sample.

[0056] Figure 15 This is a graph showing the results of the stability test of the reference solution in Example 6;

[0057] Figure 16 The graph shows the results of the stability test of the test sample solution in Example 6;

[0058] Figure 17 This is a graph showing the repeatability test results in Example 6;

[0059] Figure 18 The following are the chromatographic results of different numbered columns in Example 6: 1 is Welch Ultimate-C18 (4.6×250mm, 5μm, ZD-LC-073); 2 is Welch Ultimate-C18 (4.6×250mm, 5μm, ZD-LC-062); 3 is Welch Ultimate-C18 (4.6×250mm, 5μm, ZD-LC-085).

[0060] Figure 19 The graph shows the results of different flow rate tests in Example 6; 1 represents a flow rate of 1.0 ml / min; 2 represents a flow rate of 0.9 ml / min; 3 represents a flow rate of 1.0 ml / min.

[0061] Figure 20The following are the results of acid tests at different concentrations in Example 6: 1 is acetonitrile-0.1% formic acid; 2 is acetonitrile-0.05% formic acid; 3 is acetonitrile-0.15% formic acid. Detailed Implementation

[0062] 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.

[0063] Where specific experimental steps or conditions are not specified in the examples, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. The Wendan Decoction used in the following examples, embodiments, and comparative examples were all prepared according to the Chinese Pharmacopoeia process. Specifically: 27.6g of Pinellia ternata, 27.6g of Bambusa textilis, 27.6g of Citrus aurantium (fried with wheat bran), 41.4g of Citrus reticulata peel, 55.2g of Zingiber officinale, and 13.8g of Glycyrrhiza uralensis (fried) were taken, pulverized into coarse particles of 1-5mm, placed in a 3L earthenware pot, 1600ml of water was added, brought to a boil over high heat, and then simmered over low heat until reduced to 400ml. The decoction was passed through a 200-mesh sieve to obtain the decoction, which was used as the Wendan Decoction reference sample.

[0064] Example 1: Method for constructing fingerprint maps

[0065] This embodiment provides a method for separating chemical components or constructing fingerprint spectra in a Wendan Decoction compound preparation, including:

[0066] (1) Preparation of test solution: Take 3 ml of Wendan decoction, put it in a 10 ml volumetric flask, add methanol to dilute to the mark, stopper tightly, shake well, filter, and the solution is obtained.

[0067] (2) Preparation of reference solution: Take an appropriate amount of neohesperidin reference standard, weigh it accurately, and add methanol to prepare a solution containing 30 μg per ml.

[0068] (3) Detection by high performance liquid chromatography

[0069] High-performance liquid chromatography (HPLC) was used to detect the test solution and reference solution. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel (Welch Xtimate-C18; column length 25 cm, inner diameter 4.6 mm, particle size 5.0 μm) was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% formic acid was used as mobile phase B, eluted according to the gradient in the table below; the flow rate was 1 mL / min; the column temperature was 30 °C; and the detection wavelength was 237 nm. The injection volume was 10 μL.

[0070] Table 1 Elution gradient of mobile phase

[0071]

[0072] The common pattern of the fingerprint spectrum of Wendan Decoction was established using the method described above.

[0073] Fifteen batches of Wendan Decoction samples (Wendan Decoction decoction prepared according to the above method) were taken, and the drug fingerprint spectrum of Wendan Decoction was obtained according to the above method, as follows. Figure 1 As shown. AIA data from the chemical fingerprint chromatograms of 15 batches of Wendan Decoction were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software. Parameters were set as follows: data was cut off from solvent peaks within 0-3 minutes; S1 was used as the reference spectrum; the generation method was median; the time window was 0.1; multi-point correction was performed; peaks with an area greater than 0% of the total peak area were matched; a total of 30 common peaks were identified, forming a common pattern diagram, and a control spectrum was established. (See figure.) Figure 2 As shown in the figure, chromatographic peaks with good stability and suitable response values ​​from 15 batches of sample fingerprints were selected as common peaks, and a total of 30 common peaks were identified. Peak 21, the neohesperidin chromatographic peak, had suitable absorption and stable response, achieving baseline separation. Therefore, the neohesperidin chromatographic peak was selected as the reference peak and labeled as peak S.

[0074] pass Figure 1 and Figure 2 It can be seen that 30 common characteristic peaks are present in the comparison fingerprint spectrum. Taking the neohesperidin chromatographic peak as the reference peak, it is labeled as peak S. The relative retention times of each characteristic peak and peak S are as follows: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, 0.396, 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, 1.494.

[0075] The fingerprint chromatograms of 15 batches of Wendan Decoction were used, with the neohesperidin chromatographic peak as the reference peak, labeled as peak S. The relative retention times of each characteristic peak and peak S were within ±10% of the specified values. The specified values ​​for peaks 1-30 were as follows: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, 0.396, 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, and 1.494.

[0076] The fingerprint chromatogram of the test sample showed chromatographic peaks with the same retention times as the reference chromatogram. The chromatogram of the test sample was basically consistent with the fingerprint chromatogram of the reference sample, with 30 corresponding common peaks. According to the similarity evaluation system of chromatographic fingerprint chromatogram of traditional Chinese medicine, the similarity was calculated based on the common peaks. The results are shown in Table 2. The similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the reference sample was higher than 0.900. The difference between the fingerprint chromatograms of the reference samples from different batches was small when evaluated by similarity evaluation.

[0077] Table 2. Similarity results of fingerprint spectra of 15 batches of Wendan Decoction pharmaceutical preparations

[0078]

[0079] The construction method described in this embodiment effectively obtains fingerprint spectra with good separation of each characteristic peak. By selecting the S-peak neohesperidin as the internal reference peak in the fingerprint spectrum, the relative retention times of the common characteristic peaks 1-30 of the Wendan Decoction preparation can be determined. Therefore, it is possible to comprehensively and rapidly detect the Wendan Decoction preparation, which is beneficial for the comprehensive quality testing and overall quality control of the Wendan Decoction preparation, thereby helping to improve the safety and stability of the drug.

[0080] Example 2 Investigation of chromatographic conditions

[0081] 1. Preparation of the test solution: Accurately pipette 3 ml of the above-mentioned Wendan Decoction decoction into a 10 ml volumetric flask, dilute with methanol to the mark, stopper tightly, shake well, and filter to obtain the test solution.

[0082] 2. Optimization of chromatographic conditions

[0083] The test solution prepared in the first step of this embodiment was analyzed by HPLC according to the following method.

[0084] Method 1: Octadecylsilane-bonded silica gel was used as the packing material (Welch Xtimate-C18; column length 25 cm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid was used as mobile phase B, eluted according to the gradient in the table; the flow rate was 1 ml per minute; the column temperature was 30 °C; and the detection wavelength was 237 nm.

[0085] Table 3 Gradient elution program (Gradient 1)

[0086]

[0087]

[0088] Method 2: Octadecylsilane-bonded silica gel was used as the packing material (Welch Xtimate-C18; column length 25 cm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid was used as mobile phase B, eluted according to the gradient in the table; the flow rate was 1 ml per minute; the column temperature was 30 °C; and the detection wavelength was 237 nm.

[0089] Table 4 Gradient elution procedure (gradient 2)

[0090]

[0091] Method 3: Octadecylsilane-bonded silica gel was used as the packing material (Welch Xtimate-C18; column length 25 cm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid was used as mobile phase B, eluted according to the gradient in the table; the flow rate was 1 ml per minute; the column temperature was 30 °C; and the detection wavelength was 237 nm.

[0092] Table 5 Gradient elution procedure (gradient 3)

[0093]

[0094]

[0095] See results Figure 3 As shown, the chromatogram of the test sample in Method 3 has higher resolution and better peak shape than other chromatograms. Therefore, Method 3 is the optimal method for determining the fingerprint spectrum of reference samples.

[0096] 3. Flow velocity assessment

[0097] The test solution prepared in item 1 of this embodiment was analyzed by HPLC. Octadecylsilane-bonded silica gel was used as the packing material, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Elution was performed according to the gradient in Table 1, with flow rates of 0.9 ml, 1.0 ml, and 1.1 ml per minute, respectively. The column temperature was 30 °C, and the detection wavelength was 237 nm.

[0098] like Figure 4 As shown in the figure, the flow rate of 1.1 ml / min is used. The peak separation in the figure decreases, indicating that this flow rate is not suitable. 9.0-1.0 ml / min is suitable, and 1.0 ml / min is the optimal flow rate for the detection of chemical components in Wendan Decoction.

[0099] 4. Examination of column temperature

[0100] The test solution prepared in item 1 of this embodiment was analyzed by HPLC. Octadecylsilane-bonded silica gel was used as the packing material, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Elution was performed according to the gradient in Table 1. The flow rate was 1.0 ml per minute, the column temperature was 25℃, 30℃, and 33℃, and the detection wavelength was 237 nm.

[0101] like Figure 5 As shown in the figure, the peak separation decreases when the column temperature is 35℃, indicating that this column temperature is not suitable. 25-30℃ is more suitable, with 30℃ being the optimal temperature for detecting the chemical components of Wendan Decoction.

[0102] 5. Investigation of the mobile phase system

[0103] The test solution prepared in step 1 of this embodiment was analyzed by HPLC. Octadecylsilane-bonded silica gel was used as the packing material, acetonitrile as mobile phase A, and water, 0.1% phosphoric acid solution, 0.1% formic acid solution, and 0.1% acetic acid solution were used as mobile phase B, eluting according to the gradient in Table 1. The flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelength was 237 nm.

[0104] like Figure 6 As shown, when using acetonitrile-0.1% formic acid solution and acetonitrile-0.1% phosphoric acid solution as mobile phases, the amount of peak information is greater and the resolution of chromatographic peaks is better. However, in the acetonitrile-0.1% phosphoric acid solution system, the labeled chromatographic peaks are dense, the resolution is low, and it is easily affected by fluctuations in column temperature and flow rate, making it unstable. Therefore, acetonitrile-0.1% formic acid solution is the best mobile phase for the detection of chemical components in Wendan Decoction.

[0105] Example 3: Investigation of the preparation method of the test solution

[0106] Preparation of the test solution: Accurately pipette 3 ml of the decoction of Wendan Decoction into a 10 ml volumetric flask, and dilute to the mark with methanol, 50% methanol, 70% methanol, ethanol, 50% ethanol, and 70% ethanol, respectively. Seal tightly, shake well, and filter. Accurately pipette 10 μl of the filtrate and inject it into the liquid chromatograph for determination. The high-performance liquid chromatography method of Example 1 is used for detection.

[0107] Table 6 Results of the test sample preparation and determination

[0108]

[0109] Note: RT is the retention time, RRT is the relative retention time, A is the peak area, RPA is the relative peak area, and so on.

[0110] The data is shown in Table 6, and the comparison chart is as follows. Figure 7 As shown in the figure. By comparing the fingerprint spectra of different solvents, it was found that 30 peaks could be effectively separated under different solvents. Among them, the peak information content, peak response value and peak resolution of the methanol fingerprint spectrum were generally better than those extracted with 50% methanol, 70% methanol, ethanol, 50% ethanol and 70% ethanol. Taking all factors into consideration, methanol was tentatively selected as the extraction solvent for further research.

[0111] Example 4: Selection of Detection Wavelength

[0112] Accurately pipette 3 ml of the decoction of Wendan Decoction into a 10 ml volumetric flask, dilute to the mark with methanol, stopper tightly, shake well, and filter to obtain the test solution. Use a diode array detector to scan the test solution at wavelengths of 190–400 nm for analysis within this wavelength range. Figure 8 , Figure 9 It can be seen that the chromatographic peaks in the wavelength range of 190-240nm have high response values ​​and a large amount of information. However, the wavelength range of 203-230nm is near the cutoff wavelength and there are many impurity peaks, which cannot guarantee the simultaneous detection of more specific peaks of medicinal flavors. Therefore, considering the amount of peak information and the attribution of specific chromatographic peaks of medicinal flavors, 237nm was selected as the detection wavelength.

[0113] Example 5: Peak Assignment and Reference Selection

[0114] (1) Attribution of medicinal flavor peak

[0115] The decoction of single-herb medicinal slices was prepared according to the following method. Decoction of Pinellia ternata: Take 27.6g of Pinellia ternata, crush it into coarse particles of 1-5mm, put it in a 3L clay pot, add 1600ml of water, bring to a boil over high heat, simmer over low heat until 400ml remains, and pass the decoction through a 200-mesh sieve to obtain the decoction for later use.

[0116] Bamboo shavings decoction: Take 27.6g of bamboo shavings, crush them into coarse particles of 1-5mm, place them in a 3L clay pot, add 1600ml of water, bring to a boil over high heat, then simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction for later use.

[0117] Fructus Aurantii Immaturus (Zhishi) decoction: Take 27.6g of stir-fried Fructus Aurantii Immaturus (Zhishi) and crush it into coarse particles of 1-5mm. Place it in a 3L earthenware pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction for later use.

[0118] Decoction of dried tangerine peel: Take 41.4g of dried tangerine peel, crush it into coarse particles of 1-5mm, put it in a 3L clay pot, add 1600ml of water, bring to a boil over high heat, then simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction for later use.

[0119] Ginger decoction: Take 55.2g of ginger, crush it into coarse particles of 1-5mm, put it in a 3L clay pot, add 1600ml of water, bring to a boil over high heat, then simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction for later use.

[0120] Licorice decoction: Take 13.8g of licorice, crush it into coarse particles of 1-5mm, put it in a 3L clay pot, add 1600ml of water, bring to a boil over high heat, then simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction for later use.

[0121] The decoctions of each group of single-herb medicinal slices were prepared into test solutions according to the method in Example 1 and then tested.

[0122] The fingerprint spectrum of Wendan Decoction was compared with that of the decoction of each of its individual components. (See attached image.) Figure 10 .

[0123] The experimental results showed that chromatographic peaks 1, 2, 3, 4, 6, 7, 8, 10, 12, 17, 18, 19, 20, 21, 23, 24, 27, 29, and 30 belonged to stir-fried immature bitter orange; chromatographic peaks 2, 5, 6, 9, 8, 10, 18, 20, 23, 27, and 29 belonged to dried tangerine peel; chromatographic peaks 12 and 13 belonged to bamboo shavings; chromatographic peaks 28 and 30 belonged to fresh ginger; and chromatographic peaks 14, 15, 16, 20, 23, 24, 25, and 26 belonged to stir-fried licorice root.

[0124] (2) Characteristic peak identification

[0125] Accurately weigh an appropriate amount of neohesperidin reference standard and dissolve it in methanol to prepare a solution containing 30 μg of reference standard per 1 ml. Accurately weigh an appropriate amount of glycyrrhizin reference standard and dissolve it in methanol to prepare a solution containing 50 μg of reference standard per 1 ml. Accurately weigh an appropriate amount of naringin reference standard and dissolve it in methanol to prepare a solution containing 80 μg of reference standard per 1 ml. Accurately weigh an appropriate amount of naringin reference standard and dissolve it in methanol to prepare a solution containing 500 μg of reference standard per 1 ml. Accurately weigh an appropriate amount of hesperidin reference standard and dissolve it in methanol to prepare a solution containing 150 μg of reference standard per 1 ml. Accurately weigh an appropriate amount of glycyrrhizic acid reference standard and dissolve it in methanol to prepare a solution containing 50 μg of reference standard per 1 ml. Accurately weigh an appropriate amount of nobiletin reference standard and dissolve it in methanol to prepare a solution containing 10 μg of reference standard per 1 ml. Accurately weigh an appropriate amount of 6-gingerol reference standard and dissolve it in methanol to prepare a solution containing 10 μg of the reference standard per ml. Similarly, accurately weigh an appropriate amount of hesperidin reference standard and dissolve it in methanol to prepare a solution containing 10 μg of the reference standard per ml.

[0126] The above-mentioned reference solution and blank solvent (methanol) were tested under the chromatographic conditions of Example 1. The fingerprint chromatogram of the Wendan Decoction test sample was compared with the chromatogram of the reference solution. The results are shown in […]. Figure 12 As shown, peak 14 is glycyrrhizin, peak 18 is naringin, peak 19 is naringin, peak 20 is hesperidin, peak 21 is neohesperidin, peak 26 is glycyrrhizic acid, peak 27 is norihesperidin, peak 28 is 6-gingerol, and peak 29 is tangeretin.

[0127] (3) Selection of reference point

[0128] Among the known chromatographic peaks, peak 21, the neohesperidin peak, exhibits suitable absorption, stable response, baseline separation, and a retention time in the middle position. Figure 11 Therefore, the neohesperidin chromatographic peak was selected as the reference peak and labeled as peak S.

[0129] Example 6 Methodological Validation

[0130] (1) System applicability

[0131] The test solution prepared in Example 1 was injected six times consecutively according to the method in Example 1. The results showed that after six consecutive injections, the chromatographic peak retention time RSD was ≤2.0%, the peak area, theoretical plate number, and symmetry factor were moderate, the similarity was ≥0.900, and the system suitability was good (see Tables 7-12). Figure 13 .

[0132] Table 7 System Suitability Test Results - Retention Time

[0133]

[0134] Table 8 System Suitability Test Results - Peak Area

[0135]

[0136]

[0137] Table 9 System Suitability Test Results - Theoretical Plate Count

[0138]

[0139]

[0140] Table 10 System Suitability Test Results - Symmetry Factor

[0141]

[0142] Table 11 System Suitability Test Results - Separation Degree

[0143]

[0144]

[0145] Table 12 System Suitability Test Results - Similarity

[0146]

[0147] (2) Exclusivity

[0148] To investigate whether blank solvent (70% methanol solution), negative results for Pinellia ternata, Citrus reticulata peel, Citrus aurantium stir-fried with wheat bran, bamboo shavings, ginger, Glycyrrhiza uralensis stir-fried with wheat bran, and double negative results for Citrus reticulata peel and Citrus aurantium stir-fried with wheat bran interfered with the fingerprint spectrum of the Wendan Decoction reference sample, a negative decoction was prepared according to the following method.

[0149] Decoction of Pinellia ternata (for Yin deficiency): Take 27.6g of bamboo shavings, 27.6g of stir-fried immature bitter orange, 41.4g of dried tangerine peel, 55.2g of fresh ginger, and 13.8g of stir-fried licorice root. Grind them into coarse particles of 1-5mm, place them in a 3L earthenware pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction.

[0150] Chenpi Yin-type decoction: Take 27.6g of Pinellia ternata, 27.6g of bamboo shavings, 27.6g of stir-fried Citrus aurantium, 55.2g of ginger, and 13.8g of stir-fried licorice. Grind them into coarse particles of 1-5mm, place them in a 3L earthenware pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction.

[0151] Fermented Citrus aurantium decoction (for treating cold-induced cold conditions): Take 27.6g of Pinellia ternata, 27.6g of Bambusa textilis, 41.4g of Citrus reticulata peel, 55.2g of fresh ginger, and 13.8g of stir-fried Glycyrrhiza uralensis. Grind them into coarse particles of 1-5mm, place them in a 3L earthenware pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction.

[0152] Bamboo shavings decoction: Take 27.6g of Pinellia ternata, 27.6g of Citrus aurantium stir-fried with wheat bran, 41.4g of Citrus reticulata peel, 55.2g of fresh ginger, and 13.8g of stir-fried Glycyrrhiza uralensis. Grind them into coarse particles of 1-5mm, place them in a 3L earthenware pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction.

[0153] Ginger Yin-type decoction: Take 27.6g of Pinellia ternata, 27.6g of bamboo shavings, 27.6g of stir-fried Citrus aurantium, 41.4g of dried tangerine peel, and 13.8g of stir-fried licorice root. Grind them into coarse particles of 1-5mm, place them in a 3L earthenware pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction.

[0154] Decoction of stir-fried licorice root (for yin deficiency): Take 27.6g of Pinellia ternata, 27.6g of bamboo shavings, 27.6g of stir-fried Citrus aurantium, 41.4g of dried tangerine peel, and 55.2g of fresh ginger. Grind them into coarse particles of 1-5mm, place them in a 3L earthenware pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction.

[0155] Decoction of Citrus reticulata peel and stir-fried Citrus aurantium: Take 27.6g of Pinellia ternata, 27.6g of bamboo shavings, 55.2g of ginger, and 13.8g of stir-fried licorice root. Grind them into coarse particles of 1-5mm, place them in a 3L clay pot, add 1600ml of water, bring to a boil over high heat, and simmer over low heat until reduced to 400ml. Pass the decoction through a 200-mesh sieve to obtain the decoction.

[0156] Accurately pipette 70% methanol solution, the test solution prepared in Example 1, the negative solutions of Pinellia ternata, Citrus reticulata peel, Citrus aurantium stir-fried with wheat bran, bamboo shavings, ginger, Glycyrrhiza uralensis, and Citrus reticulata peel-Citrus aurantium stir-fried double negative solution, respectively, and perform the determination according to the high performance liquid chromatography method of Example 1. The results are shown in the figure. Figure 14 This indicates that the blank solvent did not interfere with the common peak position. Among them, the negative solutions of Pinellia ternata, Citrus reticulata peel, Citrus aurantium stir-fried with wheat bran, Bambusa textilis, Zingiber officinale, Glycyrrhiza uralensis, and Citrus reticulata peel-Citrus aurantium stir-fried with wheat bran were prepared by the same method as the test solution in Example 1 using the decoction of each negative solution.

[0157] (3) Stability

[0158] Accurately pipette 3 ml of the Wendan Decoction reference sample. Following the preparation of the test solution in Example 1, take the novel hesperidin reference solution prepared in Example 1 and inject it at 0, 6, 12, 24, 42, 60, and 72 hours using the high-performance liquid chromatography method described in Example 1. Calculate the relative retention times of the remaining peaks using the S peak as a reference. Specific results are shown in Tables 13-15. Figures 15-16 The results showed that the retention time and peak area of ​​the reference solution were basically the same within 72 hours, and the relative retention time was basically the same (RSD≤2.0%). The relative retention times of each peak in the test solution were basically the same (RSD≤2.0%), with a similarity ≥0.900, indicating that the reference solution and the test solution had good stability within 72 hours.

[0159] Table 13 Results of stability test of reference solution - relative retention time

[0160]

[0161] Table 14 Results of Stability Test on Test Sample Solutions - Relative Retention Time

[0162]

[0163] Table 15 Stability Test Results - Similarity

[0164]

[0165] (4) Repeatability

[0166] Accurately pipette 3 ml of the Wendan Decoction reference sample, and prepare 6 samples according to the preparation and determination method of the test solution in Example 1. Using the S peak as a reference, calculate the relative retention times of the remaining peaks. Specific determination results are shown in Tables 16-17. Figure 17 The results showed that the relative retention times were basically consistent, with RSD ≤ 2.0% and similarity ≥ 0.900, indicating that the method had good repeatability.

[0167] Table 16 Repeatability Test Results - Relative Retention Time

[0168]

[0169]

[0170] Table 17 Repeatability Test Results - Similarity

[0171]

[0172] (5) Intermediate precision

[0173] Different determination times, different brands of high-performance liquid chromatographs (HPLC), and different experimental personnel (A and B) were selected. 3 ml of the Wendan Decoction reference sample was accurately pipetted, and the test solution preparation and determination were performed according to Example 1. Using the S peak as a reference, the relative retention times of the remaining peaks were calculated. Specific determination results are shown in Tables 18-19. The results show that the relative retention times were basically consistent, with RSD ≤ 2.0% and similarity ≥ 0.900, indicating good intermediate precision of this method.

[0174] Table 18 Intermediate Precision Test Results - Relative Retention Time

[0175]

[0176]

[0177] Table 19 Intermediate Precision Test Results - Similarity

[0178]

[0179] (6) Durability

[0180] (6) Stability

[0181] ① Different chromatographic columns

[0182] The same sample solution prepared by the method in Example 1 was used to compare the effects of different numbered chromatographic columns on the fingerprint chromatogram of the Wendan Decoction reference sample. Other tests were performed according to the chromatographic conditions described in section (3) of Example 1. The results are shown in the table below. Figure 18 The relative retention times were basically consistent, with RSD ≤ 3.0% and similarity ≥ 0.900, indicating that this method has good robustness to different chromatographic columns.

[0183] Table 20 Results of Chromatographic Columns with Different Numbers - Relative Retention Time

[0184]

[0185]

[0186] Table 21. Similarity of test results for chromatographic columns with different numbers

[0187]

[0188] ② Different flow velocities

[0189] The same sample solution prepared by the method in Example 1 was used to compare the effect of different flow rates on the robustness of the fingerprint chromatogram of the Wendan Decoction reference sample. All other chromatographic conditions were as described in section (3) of Example 1. The results are shown in the table below. Figure 19The relative retention times were basically consistent, with RSD ≤ 5.0% and similarity ≥ 0.900, indicating that the method has good robustness to different flow rates.

[0190] Table 22 Test Results at Different Flow Rates - Relative Retention Time

[0191]

[0192]

[0193] Table 23. Experimental Results at Different Flow Rates - Similarity

[0194]

[0195] ③ Different acid concentrations

[0196] The same sample solution prepared by the method in Example 1 was used to compare the effect of different flow rates on the robustness of the fingerprint chromatogram of the Wendan Decoction reference sample. All other chromatographic conditions were as described in section (3) of Example 1. The results are shown in the table below. Figure 20 Under different acid concentrations, the main common chromatographic peaks in the fingerprint chromatograms were sharp, symmetrical, well separated, and had basically consistent relative retention times, with RSD ≤ 3.0% and similarity ≥ 0.900, indicating that the method has good robustness to different acid concentrations.

[0197] Table 24 Results of the study at different acid concentrations - relative retention time

[0198]

[0199]

[0200] Table 25. Results of Experiments with Different Acid Concentrations - Similarity

[0201]

[0202] The fingerprint spectrum construction method of the present invention has good system applicability, stability, reliability, durability, specificity, high instrument precision, and good repeatability. It makes up for the shortcomings of the existing quality control methods of Wendan Decoction and has guiding significance for the quality detection and evaluation of this variety.

[0203] 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 constructing a fingerprint spectrum of a compound preparation of Wendan Decoction, characterized in that, Includes the following steps, (1) Preparation of the test solution, wherein the solvent of the test solution is methanol; (2) The test solution was analyzed by high performance liquid chromatography. Octadecylsilane-bonded silica gel was used as the stationary phase, and acetonitrile-formic acid aqueous solution was used as the mobile phase for gradient elution. The detection wavelength was 235-240 nm; the flow rate was 0.9-1.1 ml / min; the column temperature was 25-33℃; the gradient elution program included: 0→25 min→40 min→50 min→75 min→84 min→90 min→105 min→110 min, and the volume percentage of acetonitrile in the mobile phase was: 0.5%→13%→15%→17%→20%→33%→40%→48%→90%. The construction method also includes the step of preparing a reference solution using glycyrrhizin, naringin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, novohesperidin, 6-gingerol, and tangeretin; The fingerprint spectrum uses the neohesperidin chromatographic peak as a reference peak.

2. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 1, characterized in that, Step (1) includes: weighing the Wendan Decoction compound preparation, diluting it with methanol, separating the solid and liquid, and taking the liquid, which is the test solution.

3. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 2, characterized in that, Step (1) also satisfies any one or more of the following A and B: A. The volume ratio of the volume before dilution to the volume after dilution is 3:5-20; B. The solid-liquid separation is selected from centrifugation or filtration.

4. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 1 or 2, characterized in that, Step (2) also satisfies any one or more of the following terms 1)-3): 1) The gradient elution program further includes: 110 min → 115 min → 120 min → 130 min, with the volume percentage of acetonitrile in the mobile phase being: 90% → 90% → 0.5% → 0.5%; 2) The volume percentage of formic acid in the formic acid-containing aqueous solution is 0.05%-0.15%; 3) The chromatographic conditions for high performance liquid chromatography also include: detection wavelength of 237 nm; flow rate of 0.9-1.0 ml / min; and column temperature of 30 ℃.

5. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 1 or 2, characterized in that, The construction method also includes the step of obtaining the fingerprint spectrum of the reference solution by detecting the reference solution using high performance liquid chromatography.

6. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 5, characterized in that, The preparation method of the reference solution includes the following steps: take glycyrrhizin, naringin, naringin, hesperidin, neohesperidin, glycyrrhizic acid, novohesperidin, 6-gingerol, and citrus peel reference standards, add solvent to prepare a reference solution containing 1~2500µg of each reference standard per 1ml.

7. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 6, characterized in that, The solvent is selected from methanol or an aqueous methanol solution.

8. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 7, characterized in that, The volume fraction of methanol in the methanol-water solution is not less than 30%.

9. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 1 or 2, characterized in that, The fingerprint spectrum of the Wendan Decoction compound preparation has 30 common peaks: peak 14 is glycyrrhizin, peak 18 is naringin, peak 19 is naringin, peak 20 is hesperidin, peak 21 is neohesperidin, peak 26 is glycyrrhizic acid, peak 27 is norihesperidin, peak 28 is 6-gingerol, and peak 29 is citrus.

10. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to claim 1 or 2, characterized in that, The fingerprint spectrum of the Wendan Decoction compound preparation has 30 common characteristic peaks. The neohesperidin chromatographic peak is used as the reference peak and labeled as peak S. The relative retention time of each characteristic peak and peak S is within ±10% of the specified value. The specified values ​​of peaks 1 to 30 are as follows: 0.105, 0.249, 0.308, 0.325, 0.349, 0.371, 0.396, 0.406, 0.418, 0.469, 0.499, 0.526, 0.551, 0.663, 0.675, 0.682, 0.714, 0.813, 0.880, 0.926, 1.000, 1.220, 1.262, 1.287, 1.333, 1.365, 1.419, 1.427, 1.459, 1.

494.

11. The method for constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to any one of claims 1-10 is used in the quality testing of the Wendan Decoction compound preparation product.

12. A quality testing method for a compound preparation of Wendan Decoction, characterized in that, The fingerprint spectrum of the Wendan Decoction compound preparation to be tested is constructed by the method of constructing the fingerprint spectrum of the Wendan Decoction compound preparation according to any one of claims 1-10.

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