Fingerprint detection method for Puyuan Weigan capsules

Abstract

This invention discloses a fingerprint chromatogram detection method for Puyuan Hewei Capsules, belonging to the field of traditional Chinese medicine detection. The method includes: performing high-performance liquid chromatography (HPLC) on a system suitability solution, a reference solution, a medicinal material solution, and a test solution to obtain a chromatogram; based on the chromatogram, determining the chemical composition of each chromatographic peak in the fingerprint chromatogram, which is: peak 1 - caffeic acid, peak 2 - corydaline, peak 3 - chicoric acid, peak 6 - ammonium glycyrrhizate, peak 8 - α-cyperone, and peak 10 - 11-carbonyl-β-acetylbosuccinic acid. Peaks 2 and 4 are derived from Corydalis rhizome, peak 8 from Cyperus rhizome, peaks 7, 9, and 10 from frankincense, peaks 1, 3, and 5 from dandelion, and peak 6 from licorice. This invention, applied to the detection of Puyuan Hewei Capsules, solves the problems of complex operation and limited quality control indicators for Corydalis rhizome, Cyperus rhizome, and vinegar-processed frankincense in existing detection methods.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine testing, and in particular relates to a fingerprint spectrum detection method for Puyuan and Wei capsules. Background Technology

[0002] Puyuan Hewei Capsules are mainly composed of six Chinese herbs: Corydalis Rhizome, Cyperus Rhizome, Vinegar-processed Frankincense, Dandelion, Licorice Root, and Alum. Its main functions are to regulate qi, harmonize the stomach, and relieve pain. It is used for symptoms such as stomach distension and pain, belching and acid reflux, irritability, and hypochondriac distension, which are symptoms of qi stagnation in gastric and duodenal ulcers.

[0003] The current quality standards are included in Part I of the Chinese Pharmacopoeia. The standards include the identification of corydaline, α-cyperone, frankincense, dandelion and licorice, as well as the determination of the content of corydaline and ammonium glycyrrhizate.

[0004] However, existing testing methods are complex to operate and have limited quality control indicators for Corydalis, Cyperus, and Vinegar-processed Boswellia. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is that existing detection methods are complex to operate and have limited quality control indicators for Corydalis, Cyperus, and Vinegar-processed Boswellia. This invention proposes a fingerprint spectroscopy detection method for Corydalis and Stomach Capsules that provides accurate, stable, and reliable results.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a method for detecting the fingerprint spectrum of Puyuan and Wei capsules, comprising:

[0008] S1. Take corydaline, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylbosuccinic acid, and use pure methanol as the extraction solvent to prepare a mixed solution as a system suitability solution.

[0009] S2. Take caffeic acid, fumaric acid, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylbosuccinic acid, and use pure methanol as the extraction solvent to prepare their respective reference solutions.

[0010] S3. Take appropriate amounts of Corydalis Rhizome, Cyperus Rhizome, Frankincense, Dandelion, and Licorice. Add pure methanol to the granules prepared according to the prescription and preparation process of Puyuan Hewei Capsules, and filter to obtain a medicinal solution.

[0011] S4. Take different batches of Puyuan and Wei capsules, add pure methanol for treatment, and filter to obtain the test solution;

[0012] S5. Perform high-performance liquid chromatography (HPLC) on the system suitability solution, reference solution, medicinal material solution, and test solution to obtain chromatograms;

[0013] Based on the chromatogram obtained from S5, the chemical components of each chromatographic peak in the fingerprint spectrum were determined as follows: peak 1 is caffeic acid, peak 2 is fumaric acid, peak 3 is chicoric acid, peak 6 is ammonium glycyrrhizate, peak 8 is α-cyperone, and peak 10 is 11-carbonyl-β-acetylsalicylic acid.

[0014] Based on the chromatogram obtained from S5, the sources of each chromatographic peak in the fingerprint spectrum were determined. Peak 2 and peak 4 were derived from Corydalis Rhizome, peak 8 from Cyperus Rhizome, peaks 7, 9 and 10 from Frankincense, peaks 1, 3 and 5 from Dandelion, and peak 6 from Licorice Root.

[0015] In some embodiments, the chromatographic conditions for high-performance liquid chromatography (HPLC) detection in S5 are as follows:

[0016] A C18 column was used, with methanol as mobile phase A and 0.05% phosphoric acid solution as mobile phase B. The flow rate was 0.8-1.0 mL / min, the column temperature was 20-30℃, and the detection wavelength was 270 nm. Gradient elution conditions were applied.

[0017] 0-10min, 25% A; 10-25min, 25% A→50% A; 25-40min, 50% A→70% A; 40-60min, 70% A→90% A; 60-70min, 90% A→95% A; 70-80min, 25% A.

[0018] In some embodiments, the method further includes: importing the chromatogram obtained in S5 into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine; performing data import, multi-point correction and data matching on the chromatogram of the test sample solution to generate a control fingerprint; and performing similarity analysis on the fingerprint chromatogram.

[0019] In some embodiments, the medicinal material solution and the test sample solution are subjected to heating and reflux or ultrasonic treatment before high-performance liquid chromatography detection.

[0020] In some embodiments, the chromatographic column is a Welch Ultimate XB-C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0021] In some embodiments, the flow rate is 1.0 mL / min.

[0022] In some embodiments, the column temperature is 25°C.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a fingerprint chromatogram detection method for Puyuan Hewei Capsules. Using this method, the similarity between the fingerprint chromatograms of 19 batches of Puyuan Hewei Capsules and a control chromatogram was evaluated. The results were all above 0.99, and the inter-batch similarity was above 0.96, indicating that the preparation process of Puyuan Hewei Capsules has good stability. A total of 10 common peaks were identified, and 6 of them were identified: peak 1 (caffeic acid), peak 2 (tetracycline), peak 3 (chicoric acid), peak 6 (ammonium glycyrrhizate), peak 8 (α-cyperone), and peak 10 (11-carbonyl-β-acetylbosuccinic acid). The sources of the 10 common peaks were determined: peaks 2 and 4 originated from Corydalis rhizome, peak 8 from Cyperus rhizome, peaks 7, 9, and 10 from frankincense, peaks 1, 3, and 5 from dandelion, and peak 6 from licorice. This provides an accurate and convenient detection method for the quality control of Puyuan Hewei Capsules. Attached Figure Description

[0025] Figure 1 Chromatograms of the test samples extracted with 50% methanol and methanol, respectively;

[0026] Figure 2 Chromatograms of the test samples treated by heating under reflux and ultrasonication, respectively;

[0027] Figure 3 Chromatograms of the test sample under different flow rate conditions;

[0028] Figure 4 Chromatograms of the test samples under different column temperature conditions;

[0029] Figure 5 Fingerprint chromatograms and control chromatograms of 19 batches of Puyuan Hewei Capsules;

[0030] Figure 6 Fingerprint chromatograms of the reference standard and Puyuan Hewei Capsules;

[0031] Figure 7 Fingerprint chromatograms of Corydalis Rhizome and Puyuan Hewei Capsule;

[0032] Figure 8 Fingerprint chromatograms of Cyperus rotundus and Pu Yuan He Wei capsules;

[0033] Figure 9 Fingerprint chromatograms of frankincense and Pu Yuan He Wei capsules;

[0034] Figure 10 Fingerprint chromatograms of dandelion and Puyuan Hewei capsules;

[0035] Figure 11 Fingerprint chromatograms of licorice and puyuan stomach-soothing capsules. Detailed Implementation

[0036] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0037] Based on the "Technical Guidelines for Characteristic Spectrum Research of Traditional Chinese Medicine Preparations", this invention conducted fingerprint spectrum research on Puyuan Hewei Capsules using high performance liquid chromatography. Multiple components and characteristic peaks in Corydalis Rhizome, Cyperus Rhizome, Vinegar-processed Boswellia Carterii, Dandelion, Alum, and Licorice were detected, and similarity evaluation was performed on 19 batches of preparations, providing a basis for quality control and standard improvement of this preparation.

[0038] This invention provides a method for detecting the fingerprint spectrum of Puyuan and Wei capsules, comprising:

[0039] S1. Take corydaline, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylbosuccinic acid, and use pure methanol as the extraction solvent to prepare a mixed solution as a system suitability solution.

[0040] S2. Take caffeic acid, fumaric acid, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylbosuccinic acid, and use pure methanol as the extraction solvent to prepare their respective reference solutions.

[0041] S3. Take appropriate amounts of Corydalis rhizome, Cyperus rhizome, frankincense, dandelion, and licorice root, and granules prepared according to the prescription and preparation process of Puyuan Hewei Capsules. Add pure methanol for treatment and filter to obtain a medicinal solution. Specifically, take appropriate amounts of Corydalis rhizome, Cyperus rhizome, frankincense, dandelion, and licorice root, and granules prepared according to the prescription and preparation process of Puyuan Hewei Capsules. Mix well, grind finely, accurately weigh, add methanol for treatment, and filter to obtain a medicinal solution.

[0042] S4. Take different batches of Puyuan and Wei capsules, add pure methanol for treatment, and filter to obtain the test solution; specifically, take different batches of Puyuan and Wei capsules, mix them, grind them finely, accurately weigh them, add methanol for treatment, and filter to obtain the test solution.

[0043] S5. Perform high-performance liquid chromatography (HPLC) on the system suitability solution, reference solution, medicinal material solution, and test solution to obtain chromatograms;

[0044] Based on the chromatogram obtained from S5, the chemical components of each chromatographic peak in the fingerprint spectrum were determined as follows: peak 1 is caffeic acid, peak 2 is fumaric acid, peak 3 is chicoric acid, peak 6 is ammonium glycyrrhizate, peak 8 is α-cyperone, and peak 10 is 11-carbonyl-β-acetylsalicylic acid.

[0045] Based on the chromatogram obtained from S5, the sources of each chromatographic peak in the fingerprint spectrum were determined. Peak 2 and peak 4 were derived from Corydalis Rhizome, peak 8 from Cyperus Rhizome, peaks 7, 9 and 10 from Frankincense, peaks 1, 3 and 5 from Dandelion, and peak 6 from Licorice Root.

[0046] In the above technical solution, high-performance liquid chromatography (HPLC) was used to detect the system suitability solution, reference solution, medicinal material solution, and test solution. HPLC detection of the system suitability solution was used to confirm the stability of the instrument and system in each experiment. Verification showed that the RSD of the retention time and peak area of ​​each component in the system suitability solution was less than 2% in each experiment, proving the good suitability of the system. HPLC detection of the reference solution was used to identify characteristic peaks, the medicinal material solution was used to assign medicinal properties to characteristic peaks, and the test solution was used for similarity evaluation.

[0047] Using the chromatograms of the system suitability solution, reference solution, medicinal material solution, and test solution obtained from the above process, and the chromatograms of the test solution from multiple batches of finished products, the similarity was evaluated, and the results were all above 0.96. The characteristic peaks of the reference solution were identified, and 6 components were finally confirmed. The characteristic peaks of the medicinal material solution were assigned to medicinal flavors, and the 10 characteristic peaks originated from 5 medicinal materials in the prescription. The above results determined the fingerprint spectrum.

[0048] Based on the chromatogram obtained from S5, the chemical components of each chromatographic peak in the fingerprint spectrum were determined as follows: peak 1 is caffeic acid, peak 2 is fumaric acid, peak 3 is chicoric acid, peak 6 is ammonium glycyrrhizate, peak 8 is α-cyperone, and peak 10 is 11-carbonyl-β-acetylsalicylic acid.

[0049] Based on the chromatograms obtained from S5, the chromatographic peaks with the same retention time in the chromatograms of the test solution and the herbal solution belong to the herbal material. Common chemical components in the herbal material are identified, and the fingerprint spectra of the reference solution and the test solution are compared. Chromatographic peaks with the same retention time are the components corresponding to the reference standard. This allows for the determination of the source of each chromatographic peak in the fingerprint spectra. Peaks 2 and 4 originate from Corydalis Rhizoma, peak 8 from Cyperi Rhizoma, peaks 7, 9, and 10 from Boswellia Carterii, peaks 1, 3, and 5 from Taraxacum Officinale, and peak 6 from Glycyrrhizae Radix et Rhizoma. The difficulty in determining the source of each chromatographic peak in the fingerprint spectra lies in the attribution of the herbal properties of Taraxacum Officinale and the confirmation of the chromatographic peaks. Since there are thousands of species of Taraxacum Officinale with complex components, the herbal solution was ultimately prepared from the same batch of herbal material as the finished product. After fingerprint comparison, peaks 1, 3, and 5 were found to be common peaks and could be attributed to Taraxacum Officinale. Caffeic acid, chicoric acid, and luteolin are commonly used indicators for the analysis of dandelion components. Fingerprint analysis was performed using reference solutions of these three components, and the results were compared with the fingerprint spectrum of the test solution. The test solution chromatogram showed that luteolin was not detected. Peaks 1 and 3 had the same retention times as caffeic acid and chicoric acid, respectively, confirming that peak 1 was caffeic acid and peak 3 was chicoric acid. In some embodiments, the chromatographic conditions for high-performance liquid chromatography (HPLC) detection in S5 are as follows:

[0050] A C18 column was used, with methanol as mobile phase A and 0.05% phosphoric acid solution as mobile phase B. The flow rate was 0.8-1.0 mL / min, the column temperature was 20-30℃, and the detection wavelength was 270 nm. Gradient elution conditions were applied.

[0051] 0-10min, 25% A; 10-25min, 25% A→50% A; 25-40min, 50% A→70% A; 40-60min, 70% A→90% A; 60-70min, 90% A→95% A; 70-80min, 25% A.

[0052] In the above technical solution, a C18 chromatographic column was chosen because it is economical, readily available, and widely applicable, making it the preferred choice for liquid chromatography. The methodological feasibility has been verified, and this column will be used in subsequent studies. This invention uses methanol-0.05% phosphoric acid as the mobile phase because several different mobile phase systems, including acetonitrile-phosphoric acid, methanol-water, and methanol-phosphoric acid, were investigated. The results showed that methanol-0.05% phosphoric acid provided the best resolution. The above technical solution also specifies gradient elution conditions because these conditions result in a stable baseline, good resolution, and good peak shape.

[0053] In some embodiments, the chromatographic column is a Welch Ultimate XB-C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. This technical solution also specifies the brand and specifications of the chromatographic column because Waters columns have poor resolution under these chromatographic conditions; therefore, a 250 mm long column is selected to improve the resolution of characteristic peaks in the fingerprint chromatogram.

[0054] In some embodiments, the flow rate is 1.0 mL / min.

[0055] In some embodiments, the column temperature is 25°C.

[0056] In some embodiments, the method further includes: importing the chromatogram obtained in S5 into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine; performing data import, multi-point correction and data matching on the chromatogram of the test sample solution to generate a control fingerprint; and performing similarity analysis on the fingerprint chromatogram.

[0057] In some embodiments, the medicinal material solution and the test sample solution are subjected to heating and reflux or ultrasonic treatment before high-performance liquid chromatography detection.

[0058] This invention provides a fingerprint analysis method for Puyuan Hewei capsules. By investigating factors such as the mobile phase system, flow rate, column temperature, extraction solvent, and extraction method, a fingerprint analysis method was established and its methodology was validated. Repeatability, precision, and solution stability were evaluated using relative retention time and relative peak area. The results showed that the RSD values ​​for both relative retention time and relative peak area were less than 3.29%, indicating that the method is stable and reliable. Similarity evaluation was performed on the fingerprint spectra of 19 batches of Puyuan Hewei capsules and a control; the results were all above 0.99, and the inter-batch similarity was all below 0. The values ​​above 0.96 indicate that the preparation process of Puyuan Hewei Capsules has good stability. A total of 10 common peaks were identified, and 6 of them were identified: peak 1 is caffeic acid, peak 2 is corydaline, peak 3 is chicoric acid, peak 6 is ammonium glycyrrhizate, peak 8 is α-cyperone, and peak 10 is 11-carbonyl-β-acetylbosuccinic acid. The sources of the 10 common peaks were determined: peaks 2 and 4 are from corydalis, peak 8 is from cyperus, peaks 7, 9, and 10 are from frankincense, peaks 1, 3, and 5 are from dandelion, and peak 6 is from licorice.

[0059] To provide a clearer and more detailed description of the fingerprint spectrum detection method for Puyuan and Wei capsules provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0060] (1) Sources of instruments, reagents and samples

[0061] Instruments: LC-2030 high performance liquid chromatograph (Shimadzu Corporation, Japan); CPA225D balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); BSA224S-CW balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0062] Reagents: Methanol (Sinopharm Reagent Co., Ltd., batch number: 20240511); Phosphoric acid (Tianjin Guangfu Technology Development Co., Ltd., batch number: 20230302); Purified water (Hangzhou Wahaha Group Co., Ltd., batch number: 20241219).

[0063] Reference standards: Caffeic acid (National Institutes for Food and Drug Control, batch number: 110885-201703); Corydaline (National Institutes for Food and Drug Control, batch number: 110726-202421); Chicoric acid (National Institutes for Food and Drug Control, batch number: 111752-202105); Ammonium glycyrrhizate (National Institutes for Food and Drug Control, batch number: 110731-202122); α-Cyperone (National Institutes for Food and Drug Control, batch number: 110748-202318); 11-Carbonyl-β-acetylbosuccinic acid (National Institutes for Food and Drug Control, batch number: 111760-202103).

[0064] There are a total of 19 batches of Puyuan Hewei Capsules, numbered S1-S19.

[0065] (2) Optimization of fingerprint spectrum analysis method

[0066] S1. Optimization of extraction solvent

[0067] This invention compares the high-performance liquid chromatograms of sample No. 1 under the same chromatographic instrument, column, and chromatographic conditions, using methanol and 50% methanol as extraction solvents.

[0068] Figure 1 The chromatograms of the test samples extracted with 50% methanol and methanol respectively show that when methanol is used, there are more peaks and the peak shapes are better, which is better than 50% methanol. Therefore, methanol was selected as the extraction solvent in the subsequent optimization process.

[0069] S2. Optimization of the sample solution treatment method

[0070] This invention compares the high-performance liquid chromatograms of sample No. 1 under different treatment methods, namely ultrasonication and heating reflux, using the same chromatogram, column, and chromatographic conditions.

[0071] Figure 2 The chromatograms of the test samples treated by heating and reflux and ultrasonication respectively show that the two treatment methods have no significant impact on the test results. Considering convenience, ultrasonication was chosen as the treatment method in the subsequent optimization process.

[0072] S3, Optimization of the gradient procedure

[0073] This invention employs a diode array detector to perform full-wavelength scanning of the test solution, examining the baseline, peak shape, and response values ​​at each wavelength. Results show a stable baseline and good peak shape at 270 nm. Several different mobile phase systems, including acetonitrile-phosphoric acid, methanol-water, and methanol-phosphoric acid, were investigated. The results showed that methanol-0.05% phosphoric acid achieved the best separation. Subsequent extensive experiments were conducted to screen for the optimal gradient elution program. The final gradient elution program, characterized by stable baseline, good separation, and good peak shape, is as follows: 0-10 min, 25% A; 10-25 min, 25% A → 50% A; 25-40 min, 50% A → 70% A; 40-60 min, 70% A → 90% A; 60-70 min, 90% A → 95% A; 70-80 min, 25% A.

[0074] S4, Flow rate optimization

[0075] This invention compares the same chromatograph and column, taking sample No. 1 as an example, with other chromatographic conditions being the same, and determines its high performance liquid chromatogram at different flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min.

[0076] Figure 3 The chromatograms of the test sample under different flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min are shown. It can be seen that the resolution of each common peak is good when the flow rate is in the range of 0.8-1.2 mL / min. In the subsequent optimization process, the flow rate of 1.0 mL / min was selected.

[0077] S5, Column Temperature Optimization

[0078] This invention compares the same chromatogram and column, taking sample No. 1 as an example, with other chromatographic conditions being the same, and measures its high performance liquid chromatogram at different column temperatures of 20℃, 25℃, and 30℃.

[0079] Figure 3 The chromatograms of the test sample under different column temperatures of 20℃, 25℃, and 30℃ are shown. It can be seen that the resolution of each common peak is good when the column temperature is in the range of 20-30℃. In the subsequent optimization process, the column temperature was selected as 25℃.

[0080] Example 1

[0081] S1. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; methanol was used as mobile phase A and 0.05% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 1; the detection wavelength was 270 nm; the flow rate was 1.0 mL / min; and the column temperature was 25 °C.

[0082] Table 1 Elution conditions

[0083]

[0084] S2. Take appropriate amounts of corydaline, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylbosuccinic acid, accurately weigh them, and add methanol to prepare a mixed solution containing 19.43 μg / mL of corydaline, 14.92 μg / mL of chicoric acid, 38.57 μg / mL of ammonium glycyrrhizate, 9.8 μg / mL of α-cyperone, and 34.97 μg / mL of 11-carbonyl-β-acetylbosuccinic acid per 1 mL. Shake well to obtain a system suitability solution.

[0085] S3. Take the contents of this product, mix well, grind finely, accurately weigh about 1.0g, place in a stoppered conical flask, accurately add 25mL of methanol, stopper tightly, weigh, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, take the filtrate to obtain the test solution.

[0086] S4. Determination method: Accurately pipette 10 μL of each solution and inject it into the liquid chromatograph for determination.

[0087] S5. Validation of analytical methods

[0088] Precision test

[0089] The solution obtained in step S3 was taken and measured 6 times consecutively according to the conditions and methods of steps S1 and S4. The 11-carbonyl-β-acetylbosuccinic acid peak No. 10 was used as the reference peak. The relative retention time of each common peak was calculated to be 0.01% < RSD < 0.08%, and the relative peak area was 0.08% < RSD < 0.51%, indicating that the method has good precision.

[0090] Repeatable experiments

[0091] Six test solutions were prepared in parallel according to step S3 using the same batch of Puyuan and Wei capsules. The solutions were determined according to the conditions and methods of steps S1 and S4. The 11-carbonyl-β-acetylbosuccinic acid peak No. 10 was used as the reference peak. The relative retention time of each common peak was calculated to be 0.01% < RSD < 0.06%, and the relative peak area was 0.50% < RSD < 3.29%, indicating that the method has good repeatability.

[0092] Stability test

[0093] The solution obtained in step S3 was tested at 0h, 3h, 6h, 9h, 12h, and 24h, respectively, according to the conditions and methods of steps S1 and S4. Using peak 10, 11-carbonyl-β-acetylbosuccinic acid, as the reference peak, the relative retention time of each common peak was calculated to be 0.01% < RSD < 0.10%, and the relative peak area was 0.36% < RSD < 2.40%, indicating that the test solution has good stability within 24h.

[0094] S5. Fingerprint mapping and similarity evaluation

[0095] Nineteen batches of Puyuan Hewei capsules were collected. Test solutions were prepared according to step S3, and the results were determined according to the conditions and methods of steps S1 and S4. Chromatograms were recorded, and data analysis was performed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. Using the chromatogram of sample S1 as the reference chromatogram, the median method was employed, with a time window set to 0.1 min. Mark peak matching was used to generate overlay chromatograms and a control chromatogram R. The results are shown in [Figure Number]. Figure 5 Ten common peaks were identified. The similarity between the chromatograms of each batch and the generated control chromatogram was above 0.99. The results are shown in the table below. The good similarity between batches indicates that the preparation process of Puyuan Hewei Capsules has good stability.

[0096]

[0097]

[0098] S5, Chromatographic Peak Identification

[0099] Accurately weigh appropriate amounts of caffeic acid, fumaric acid, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylspasmodic acid. Dissolve each in methanol to prepare solutions containing 8.18 μg / mL caffeic acid, 19.43 μg / mL fumaric acid, 14.92 μg / mL chicoric acid, 38.57 μg / mL ammonium glycyrrhizate, 9.8 μg / mL α-cyperone, and 34.97 μg / mL 1 mL of 11-carbonyl-β-acetylspasmodic acid. Shake well to obtain the reference solutions.

[0100] Take the above-mentioned reference solution and the solution obtained in step S3, and determine them according to the conditions and methods of steps S1 and S4. Record the chromatograms and compare them. Six components in the test solution were identified as peak 1 (caffeic acid), peak 2 (tetracycline), peak 3 (chicoric acid), peak 6 (ammonium glycyrrhizate), peak 8 (α-cyperone), and peak 10 (11-carbonyl-β-acetylbosuccinic acid). The results are shown in [Figure number missing]. Figure 6 .

[0101] S6, Chromatographic Peak Assignment

[0102] Take appropriate amounts of Corydalis Rhizome, Cyperus Rhizome, Frankincense, Dandelion, and Licorice Root, and prepare them into corresponding granules according to the prescription and preparation process of Puyuan Hewei Capsules. Mix them well, grind them into a fine powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, add 25mL of methanol accurately, stopper tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the medicinal material solution.

[0103] Take the above-mentioned medicinal material solution and the solution obtained in step S3, and determine them according to the conditions and methods of steps S1 and S4. Record the chromatograms and assign common peaks. Peaks 2 and 4 are derived from Corydalis Rhizome, peak 8 from Cyperus Rhizome, peaks 7, 9, and 10 from Frankincense, peaks 1, 3, and 5 from Dandelion, and peak 6 from Licorice Root. The results are shown in [Figure number missing]. Figure 7-11 .

[0104] Using the above method, 10 common peaks were identified in the fingerprint spectrum of Puyuan Hewei Capsules, and 6 of them were identified: caffeic acid, fumaric acid, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylboswellic acid. Among them, 11-carbonyl-β-acetylboswellic acid showed the best separation and stability. Using this as a reference peak, the relative retention time and relative peak area of ​​each common peak were good, indicating that the method is stable and reliable. A total of 19 batches of Puyuan Hewei Capsules were measured, and the fingerprint spectrum similarity was evaluated. The similarity between batches was above 0.96, indicating that... The preparation process of Puyuan Hewei Capsules has good stability. The chromatographic peak attribution results show that the most common peaks come from dandelion. According to the investigation, S1-S9 used the same batch of medicinal materials, and S10-S19 used the same batch of medicinal materials. The similarity evaluation results show that the similarity of 19 batches of Puyuan Hewei Capsules is 0.96-1.00, while the similarity of S1-S9 is 0.99-1.000, and the similarity of S10-S19 is 0.98-1.00. The similarity of preparations made from the same batch of medicinal materials is relatively high, which shows that the difference in the batch of medicinal materials can affect the similarity.

[0105] In summary, the fingerprint spectroscopy determination method established in this invention is stable and reliable, providing a basis for the quality control of formulations and the improvement of quality standards.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for detecting the fingerprint of Pugyuan Weigan Capsule, characterized in that, include: S1. Take corydaline, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylbosuccinic acid, and use pure methanol as the extraction solvent to prepare a mixed solution as a system suitability solution. S2. Take caffeic acid, fumaric acid, chicoric acid, ammonium glycyrrhizate, α-cyperone, and 11-carbonyl-β-acetylbosuccinic acid, and use pure methanol as the extraction solvent to prepare their respective reference solutions. S3. Take appropriate amounts of Corydalis Rhizome, Cyperus Rhizome, Frankincense, Dandelion, and Licorice. Add pure methanol to the granules prepared according to the prescription and preparation process of Puyuan Hewei Capsules, and filter to obtain a medicinal solution. S4. Take different batches of Puyuan and Wei capsules, add pure methanol for treatment, and filter to obtain the test solution; S5. Perform high-performance liquid chromatography (HPLC) on the system suitability solution, reference solution, medicinal material solution, and test solution to obtain chromatograms; Based on the chromatogram obtained from S5, the chemical components of each chromatographic peak in the fingerprint spectrum were determined as follows: peak 1 is caffeic acid, peak 2 is fumaric acid, peak 3 is chicoric acid, peak 6 is ammonium glycyrrhizate, peak 8 is α-cyperone, and peak 10 is 11-carbonyl-β-acetylsalicylic acid. Based on the chromatogram obtained from S5, the source of each chromatographic peak in the fingerprint spectrum was determined. Peak 2 and peak 4 were derived from Corydalis Rhizome, peak 8 from Cyperus Rhizome, peak 7, peak 9 and peak 10 from Frankincense, peak 1, peak 3 and peak 5 from Dandelion, and peak 6 from Licorice Root. The chromatographic conditions for high performance liquid chromatography (HPLC) detection in S5 are as follows: A C18 column was used, with methanol as mobile phase A and 0.05% phosphoric acid solution as mobile phase B. The flow rate was 0.8-1.0 mL / min, the column temperature was 20-30℃, and the detection wavelength was 270 nm. Gradient elution conditions were applied. 0-10min, 25% A; 10-25min, 25% A→50% A; 25-40min, 50% A→70% A; 40-60min, 70% A→90% A; 60-70min, 90% A→95% A; 70-80min, 25% A.

2. The method according to claim 1, wherein, Also includes: The chromatograms obtained from S5 were imported into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. The chromatograms of the test solution were processed by data import, multi-point correction and data matching to generate a reference fingerprint, and the fingerprint chromatograms were analyzed for similarity.

3. The method according to claim 1, wherein the method is characterized by, The medicinal material solution and the test sample solution were subjected to heating and reflux or ultrasonic treatment before high performance liquid chromatography detection.

4. The method according to claim 1, wherein, The chromatographic column was Welch Ultimate XB. C18 chromatographic column, with dimensions of 4.6mm × 250mm and 5μm.

5. The method according to claim 1, wherein the method is characterized by, The flow rate was 1.0 mL / min.

6. The method according to claim 1, wherein the method is characterized by, The column temperature is 25℃.

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