Establishment method of HPLC (High Performance Liquid Chromatography) specific chromatogram of cold and heat clearing granules

Abstract

The invention discloses an establishment method of an HPLC (high performance liquid chromatography) characteristic chromatogram of granules for treating common cold and clearing heat and a component content test thereof, and belongs to the technical field of pharmaceutical analys.According to the research, the HPLC characteristic chromatogram of the granules for treating common cold and clearing heat is established under the same chromatographic condition by adopting a high performance liquid chromatography, and the overall internal quality in a preparation is comprehensively reflected; the method for simultaneously determining the content of amygdalin, puerarin, cimicifugin and corydalis bungeana is established, methodology verifies that the method is simple, convenient and feasible, has good stability and repeatability, and can be used for detection in production process monitoring and quality evaluation of the cold and heat clearing granules to ensure that the quality of the cold and heat clearing granules meets the standard. The scheme not only scientifically restores the overall pharmacodynamic material basis of the compound preparation, but also provides a new generalizable normal form for quality control of traditional Chinese medicines with the advantages of low cost and high efficiency, and has milestone significance for promoting industry standardization and internationalization.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for establishing HPLC characteristic chromatograms of cold and fever-relieving granules and the determination of their component content. Background Technology

[0002] Cold and Fever Relief Granules are a common traditional Chinese medicine compound preparation, mainly used to treat symptoms such as wind-heat cold, sore throat, and fever. This preparation consists of 11 medicinal herbs, including Schizonepeta tenuifolia, Saposhnikovia divaricata, Bupleurum chinense, Pueraria lobata, Angelica dahurica, Perilla frutescens, Prunus armeniaca, Viola yedoensis, Platycodon grandiflorus, Mentha haplocalyx, and Phragmites communis. Due to the large number of herbs and the complexity of their components, quality control is correspondingly more difficult.

[0003] The current 2020 edition of the Chinese Pharmacopoeia, Volume I, primarily uses thin-layer chromatography (TLC) for identification of cold and fever-relieving granules, and only uses high-performance liquid chromatography (HPLC) to determine the content of puerarin in kudzu root. This quality control model covers a limited range of components, fails to fully reflect other key components related to efficacy, and cannot reflect the overall efficacy logic of the compound formula of "relieving exterior syndromes and dispersing cold + relieving cough and clearing heat." Furthermore, the current standard does not establish characteristic chromatograms or fingerprints, making it impossible to evaluate the overall consistency of component distribution through parameters such as the number of chromatographic peaks and relative retention times.

[0004] Currently, there are no reports in China regarding characteristic chromatograms and simultaneous quantitative methods for multiple indicator components in cold and fever-relieving granules. For example, the patent "A Method for Detecting the Content of Multiple Components in a Cold and Fever-Relieving Preparation" published by Guangzhou Baiyunshan only focuses on the content of 3-hydroxypuerarin, puerarin, 3-methoxypuerarin, and apigenin puerarin glycoside in kudzu root, without considering other medicinal material indicator components or the characteristic chromatograms of cold and fever-relieving granules, making it impossible to evaluate the overall consistency of component distribution. The patent "A Method for Determining the Content and Moisture in Cold and Fever-Relieving Granules" published by Shandong Jinzhang Longxiang Intelligent Technology Co., Ltd. uses near-infrared spectroscopy to determine the content and moisture of cold and fever-relieving granules, which differs from the mainstream HPLC content determination method. Furthermore, the measured content is a rather vague concept and lacks representativeness.

[0005] In summary, current literature only reports on the content determination of indicator components in 1-3 medicinal materials of cold and fever-relieving granules, or only on their characteristic spectra. A comprehensive quality control method that simultaneously covers multi-component quantification and overall characteristic spectrum evaluation has not yet been established. Therefore, to monitor the key indicator components of cold and fever-relieving granules, ensure consistent therapeutic effects, improve the compliance and international recognition of enterprises, and guarantee the safety and effectiveness of medication for patients, it is of great significance to establish a comprehensive and rapid method for detecting the quality of cold and fever-relieving granules through a multi-dimensional assessment. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for establishing HPLC characteristic chromatograms of cold and fever-relieving granules, so as to overcome the shortcomings of the current standard, which mainly relies on thin-layer identification, lacks overall characterization of multiple medicinal materials, and is difficult to fully reflect the quality of the preparation.

[0007] The second objective of this invention is to provide a method for simultaneously testing the content of four components in cold and fever-relieving granules, in order to solve the problem that the existing technology only measures the single indicator of puerarin, which cannot take into account other core active ingredients, resulting in one-sided quality control and difficulty in ensuring batch consistency.

[0008] The objective of this invention can be achieved through the following technical solutions: The ingredients of the Cold and Fever Relief Granules are Schizonepeta tenuifolia, Saposhnikovia divaricata, Bupleurum chinense, Pueraria lobata, Angelica dahurica, Perilla frutescens, Prunus armeniaca, Viola yedoensis, Platycodon grandiflorus, Mentha haplocalyx, and Phragmites communis.

[0009] The method for establishing the HPLC characteristic chromatogram of cold and fever-relieving granules includes the following steps: (1) Accurately measure the cold and fever relief granules to prepare a cold and fever relief granules test solution; (2) Weigh amygdalin, puerarin, cimicifugain and corydaline respectively, and add methanol to prepare a mixed reference solution; weigh 5-O-methylvisamilol and add methanol to prepare a single reference solution. (3) Weigh out bitter almonds, kudzu root, saposhnikovia root, and bitter violet respectively, and obtain each negative sample solution by decoction; (4) The test solution, mixed reference solution, single reference solution and each negative sample solution of Ganmao Qingre Granules were analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Ganmao Qingre Granules.

[0010] Further, the high performance liquid chromatograph analysis parameters in step (4) are: column temperature 30℃, flow rate 1.0mL / min, and detection wavelength 210nm.

[0011] Furthermore, in the HPLC characteristic chromatogram of the cold and fever-relieving granules, the nine common peaks are: peak 1, peak 2 amygdalin, peak 3 puerarin, peaks 4, 5, 6, and 7 cimicifugain glycoside, peak 8 5-O-methylvisamidol glycoside, and peak 9 corydaline; with peak 3 as the reference peak, the relative retention times of each characteristic peak are as follows: peak 1 0.774, peak 2 0.827, peak 4 1.08, peak 5 1.11, peak 6 1.25, peak 7 1.31, peak 8 1.76, and peak 9 2.12.

[0012] The method for determining the content of ingredients in cold and fever-relieving granules includes the following steps: S1. Accurately measure the cold and fever relief granules to prepare a cold and fever relief granules test solution; S2. Weigh amygdalin, puerarin, cimicifugain, and corydaline respectively, and add methanol to prepare a mixed reference solution; weigh 5-O-methylvisamidol glycoside and add methanol to prepare a single reference solution. S3. Accurately pipette 5-20 μL each of the test solution of the cold and fever-relieving granules, the mixed reference solution, and the single reference solution, and inject them into the high performance liquid chromatograph for determination. Calculate the content of amygdalin, puerarin, cimicifugain, and corydaline in the test solution using the external standard method.

[0013] Furthermore, the analytical parameters of the high performance liquid chromatograph in S3 are as follows: column temperature 25-32℃, flow rate 0.8-1.0 mL / min, and detection wavelengths 210 nm (amygdalin), 250 nm (puerarin, cimicifugain), and 289 nm (corydaline).

[0014] Furthermore, the linear range for the determination of the components of the cold and fever-relieving granules was as follows: amygdalin: 45.82-229.09 μg / mL; puerarin: 78.02-390.1 μg / mL; cimicifugain: 6.11-30.56 μg / mL; corydaline: 5.95-29.76 μg / mL.

[0015] Furthermore, the preparation method of the cold and fever-relieving granule test solution is as follows: Accurately weigh 5.0g of cold and fever relief granules, add them to 25mL of solvent, sonicate for 15-60min, let stand at room temperature, filter, and take the filtrate to obtain the cold and fever relief granules test solution.

[0016] Furthermore, the solvent is one of a 70% volumetric methanol solution, methanol, a 70% volumetric ethanol solution, ethanol, and pure water.

[0017] Furthermore, the ultrasonic power is 900W and the ultrasonic frequency is 65kHz.

[0018] Furthermore, the solvent used in the preparation of the cold and fever relief granules test solution is preferably methanol, and the ultrasonic time is preferably 45 min.

[0019] Furthermore, the specific preparation method of the mixed reference solution is as follows: Amygdalin, puerarin, cimicifugoside, and corydaline were added to methanol to prepare a mixed reference solution with amygdalin concentration of 100 ug / mL, puerarin concentration of 200 ug / mL, cimicifugoside concentration of 15 ug / mL, and corydaline concentration of 15 ug / mL.

[0020] Furthermore, the specific preparation method of the single reference solution is as follows: 5-O-methylvesamidol glycoside was added to methanol to obtain a single reference solution with a concentration of 80 μg / mL.

[0021] Furthermore, the specific preparation methods for each negative sample solution are as follows: The prescribed amounts of bitter almond, kudzu root, saposhnikovia root, and bitter violet were pulverized. Then, three of them were selected and decocted twice with 8 times their weight of pure water, each time for 1 hour. The decoctions were filtered and combined. The decoctions were then diluted with methanol to obtain negative sample solutions lacking bitter almond, kudzu root, saposhnikovia root, and bitter violet. The negative sample solution lacking bitter almond was obtained by decocting kudzu root, saposhnikovia root, and bitter violet, which means that bitter almond was missing. Similarly, "missing" in other negative samples means that they were not present.

[0022] Furthermore, each negative sample solution was decocted according to the proportion of medicinal materials for Ganmao Qingre Granules in the 2020 edition of the Chinese Pharmacopoeia, and the corresponding decoction was diluted with methanol to the corresponding concentration of medicinal materials in the finished Ganmao Qingre Granules product.

[0023] Furthermore, the chromatographic conditions for the high-performance liquid chromatograph analysis are as follows: A 250 mm × 4.6 mm, 5 µm Agilent Eclipse XDB-C18 column was used as the chromatographic column, with octadecylsilane-bonded silica gel as the packing material, and gradient elution was performed using acetonitrile as mobile phase A and 0.1% (v / v) phosphoric acid aqueous solution as mobile phase B.

[0024] Further, the gradient elution settings are as follows: 0 min, A:B = 5%:95%; 0–15 min, A:B = 5%:95% → 10%:90%; 15 min, A:B = 10%:90%; 15–30 min, A:B = 10%:90% → 15%:85%; 30 min, A:B = 15%:85%; 30–50 min, A:B = 15%:85% → 25%:75%; 50 min, A:B = 25%:75%; 50–55 min, A:B = 25%:75%; At 55 min, A:B ratio was 25%:75% → 30%:70%; at 55 min, A:B ratio was 30%:70%; at 55–65 min, A:B ratio was 30%:70% → 60%:40%; at 65 min, A:B ratio was 60%:40%; at 65–65.1 min, A:B ratio was 60%:40% → 5%:95%; at 65.1 min, A:B ratio was 5%:95%; at 65.1–70 min, A:B ratio was 5%:95%; at 70 min, A:B ratio was 5%:95%.

[0025] Furthermore, the method for establishing the HPLC characteristic chromatogram of the cold and fever relief granules and the method for determining the content of the components of the cold and fever relief granules are used for quality detection and quality control of the cold and fever relief granules, and to determine whether the product quality is uniform and stable.

[0026] The beneficial effects of this invention are: 1. This invention provides a method for constructing a characteristic chromatogram of cold and fever-relieving granules. This method is simple to operate, has good reproducibility, and high precision. This invention also provides a method for determining the content of components in cold and fever-relieving granules, which can simultaneously test four component indicators. The combination of the two methods can be used for the quality detection and quality control of cold and fever-relieving granules, and can intuitively reflect the overall distribution of multiple components in the preparation (such as the number of chromatographic peaks and relative retention time). This solves the problem of single detection indicators and lack of overall evaluation in the current quality control system, and realizes the dual quality control of cold and fever-relieving granules by "overall + key components".

[0027] 2. This invention provides a simple, reproducible, and precise detection scheme by integrating qualitative analysis of characteristic spectra with quantitative testing of multiple components. It overcomes the limitations of single-component quality control in cold and fever-reducing granules, achieving for the first time the simultaneous detection of four key indicator components—amygdalin, puerarin, cimicifugain, and corydaline—along with their characteristic spectra. Furthermore, it exhibits good stability and repeatability, and can be used for monitoring the production process and evaluating the quality of cold and fever-reducing granules to ensure that the granules meet standards. This scheme not only scientifically restores the overall pharmacodynamic material basis of compound preparations but also provides a new, scalable paradigm for the quality control of traditional Chinese medicine with its low cost and high efficiency, marking a milestone in promoting industry standardization and internationalization. Attached Figure Description

[0028] The invention will now be further described with reference to the accompanying drawings.

[0029] Figure 1 This is the chromatogram of the mixed reference solution; Figure 2 The chromatogram of a negative sample solution lacking bitter almonds; Figure 3 The chromatogram of a negative sample solution of Viola yedoensis is shown. Figure 4 The chromatogram of the negative sample solution lacking Saposhnikovia divaricata is shown. Figure 5 This is the chromatogram of a negative sample solution lacking kudzu root; Figure 6 A comparison of the chromatograms of the test solution, mixed reference solution, and each negative sample solution of batch number 2504827 Cold and Fever Relief Granules; Figure 7 This is a chromatogram verifying the specificity of the cold and fever-relieving granules of this invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] The cold and fever relief granules used in this application are 16 batches of samples that have passed the current standard inspection. The specific batch numbers are shown in the table below:

[0032] Example 1: Preparation of Cold and Fever Relief Granules Test Solution

[0033] 1. Investigation of extraction solvents

[0034] Accurately weigh 5.0g of each of the five cold and fever-reducing granules (batch number 2504827), and add 25mL each of 70% methanol, methanol, 70% ethanol, ethanol, and pure water. Mix the solutions, sonicate at 900W and 60kHz for 60 minutes, remove, allow to stand at room temperature, and filter to obtain the test solutions of the cold and fever-reducing granules. Inject 5μL of each of the five test solutions for testing, and compare the peak area differences per unit mass of different solvents.

[0035] The results are shown in Table 1: Table 1. Peak areas of corresponding index components under different extraction solvents

[0036] As can be seen from the data recorded in Table 1, the main chromatographic peaks of the test sample extracted with methanol have larger peak areas per unit mass, indicating more complete extraction. Therefore, methanol is the preferred extraction solvent for the cold and fever relief granules test sample solution.

[0037] 2. Examination of extraction time

[0038] Accurately weigh 5.0g of each of the following eight cold and fever-reducing granules (batch number 2504827): add 25mL of methanol to each granule, mix, and sonicate at 900W power and 60kHz for 15min, 30min, 45min, and 60min respectively. Two samples are taken for each sonication time. The samples are then removed, allowed to reach room temperature, and filtered to obtain the cold and fever-reducing granule test solution. Inject 5μL of each of the five test solutions for testing, and compare the peak area differences per unit mass at different sonication times.

[0039] The results are shown in Table 2: Table 2. Peak areas of corresponding index components at different ultrasound times.

[0040] As can be seen from the data recorded in Table 2, the peak area per unit mass of the main chromatographic peak of the test sample extracted at 45 min and 60 min is larger, indicating more complete extraction, and the difference between the two is not significant. Based on the consideration of saving time and cost, the ultrasonic treatment time of the cold and fever relief granules test sample solution is preferably 45 min.

[0041] Example 2: Optimization of Chromatographic Conditions

[0042] Accurately weigh 5.0g of cold and fever relief granules (batch number 2504827), add 25mL of methanol, mix, sonicate at 900W power and 60kHz frequency for 45min, remove, place at room temperature, filter, and the cold and fever relief granules test solution is obtained. Amygdalin, puerarin, cimicifugoside, and corydaline were added to methanol to prepare a mixed reference solution with amygdalin concentration of 100 μg / mL, puerarin concentration of 200 μg / mL, cimicifugoside concentration of 15 μg / mL, and corydaline concentration of 15 μg / mL. 5-O-methylvisamidol was added to methanol to obtain a single reference solution with a concentration of 80 μg / mL. Accurately pipette 5 μL each of the cold and fever-reducing granules test solution, mixed reference solution, and single reference solution into a high-performance liquid chromatograph for determination. Six parallel samples were prepared using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.1% (v / v) phosphoric acid aqueous solution as mobile phase B, with gradient elution. The column temperature was 30℃, the flow rate was 1.0 mL / min, and the detection wavelengths were 210 nm (amygdalin), 250 nm (puerarin, cimicifugain), and 289 nm (corydaline). The test results are shown in Table 3. The gradient elution settings are as follows: 0 min, A:B = 5%:95%; 0–15 min, A:B = 5%:95% → 10%:90%; 15 min, A:B = 10%:90%; 15–30 min, A:B = 10%:90% → 15%:85%; 30 min, A:B = 15%:85%; 30–50 min, A:B = 15%:85% → 25%:75%; 50 min, A:B = 25%:75%; 50–55 min… A:B ratio is 25%:75% → 30%:70%; at 55 min, A:B ratio is 30%:70%; from 55 to 65 min, A:B ratio is 30%:70% → 60%:40%; at 65 min, A:B ratio is 60%:40%; from 65 to 65.1 min, A:B ratio is 60%:40% → 5%:95%; at 65.1 min, A:B ratio is 5%:95%; from 65.1 to 70 min, A:B ratio is 5%:95%; at 70 min, A:B ratio is 5%:95%.

[0043] Table 3 Peak areas of corresponding index components at different detection wavelengths

[0044] As can be seen from the data recorded in Table 3, puerarin, cimicifugain, and corydaline all showed absorption at 210 nm. Furthermore, cimicifugain and corydaline showed higher response at 210 nm than at 250 nm. At 210 nm, the separation of characteristic peaks was better, including all four index components. Therefore, this study ultimately decided to use 210 nm as the detection wavelength for the four index components and characteristic spectra.

[0045] Example 3: Method for establishing the HPLC characteristic chromatogram of cold and fever-relieving granules, the steps are as follows: (1) Accurately weigh 5.0g of cold and fever relieving granules (batch numbers are 2504827, 202505002, 202505003, 202505004, 202505005, 202505006, 202505007, 202505008, 202505009, 202505010, 202505011, 202505012, 202505013, 202505014, 202505015, 202505016), add 25mL of methanol to each, mix, sonicate at 900W power and 60kHz frequency for 45min respectively, take out, place at room temperature, filter, and obtain the cold and fever relieving granules test solution; (2) Add amygdalin, puerarin, cimicifugain and corydaline to methanol to prepare a mixed reference solution with amygdalin concentration of 100ug / mL, puerarin concentration of 200ug / mL, cimicifugain concentration of 15ug / mL and corydaline concentration of 15ug / mL. Add 5-O-methylvesamiol to methanol to obtain a single reference solution with a concentration of 80μg / mL. (3) Pulverize bitter almond, kudzu root, saposhnikovia root, and bitter violet. Then, select three of them and add 8 times the mass of pure water to decoct twice, 1 hour each time. Filter and combine the decoctions. Take each decoction and make up to volume with methanol to obtain negative sample solutions for bitter almond, kudzu root, saposhnikovia root, and bitter violet. Each negative sample solution was decocted according to the proportion of medicinal materials in the Cold and Fever Relief Granules in the 2020 edition of the Chinese Pharmacopoeia, and the corresponding decoction was diluted with methanol to the corresponding concentration of medicinal materials in the finished Cold and Fever Relief Granules. (4) Set the chromatographic conditions: use octadecylsilane bonded silica gel as the packing material, acetonitrile as mobile phase A and 0.1% (v / v) phosphoric acid aqueous solution as mobile phase B, perform gradient elution, column temperature 30℃, flow rate 1.0 mL / min, and detection wavelength 210 nm. The gradient elution settings are as follows: 0 min, A:B = 5%:95%; 0–15 min, A:B = 5%:95% → 10%:90%; 15 min, A:B = 10%:90%; 15–30 min, A:B = 10%:90% → 15%:85%; 30 min, A:B = 15%:85%; 30–50 min, A:B = 15%:85% → 25%:75%; 50 min, A:B = 25%:75%; 50–55 min… A:B ratio is 25%:75% → 30%:70%; at 55 min, A:B ratio is 30%:70%; from 55 to 65 min, A:B ratio is 30%:70% → 60%:40%; at 65 min, A:B ratio is 60%:40%; from 65 to 65.1 min, A:B ratio is 60%:40% → 5%:95%; at 65.1 min, A:B ratio is 5%:95%; from 65.1 to 70 min, A:B ratio is 5%:95%; at 70 min, A:B ratio is 5%:95%.

[0046] (5) Take 5 μL each of the test solution of Ganmao Qingre Granules, the mixed reference solution, the single reference sample solution, and each negative control sample solution, and inject them into a high-performance liquid chromatograph for determination. Obtain the chromatograms of the test solution of Ganmao Qingre Granules, the mixed reference solution, and each negative control sample solution, as shown below. Figures 1-7 As shown, Figure 1 This is the chromatogram of the mixed reference solution. Figure 2 This is the chromatogram of a negative sample solution lacking bitter almonds. Figure 3 This is the chromatogram of a negative sample solution of Viola yedoensis. Figure 4 The chromatogram is for a negative sample solution lacking Saposhnikovia divaricata. Figure 5 This is the chromatogram of a negative sample solution lacking kudzu root. Figure 6 This is a comparison of the chromatograms of the test solution, mixed reference solution, and each negative control sample solution for batch number 2504827 Cold and Fever Relief Granules. Figure 6 S1 is the chromatogram of a negative sample solution lacking bitter almonds. Figure 6 S2 is the chromatogram of a negative sample solution lacking *Viola yedoensis*. Figure 6 S3 is the chromatogram of a negative sample solution lacking Saposhnikovia divaricata. Figure 6 S4 is the chromatogram of a negative sample solution lacking kudzu root. Figure 6 S5 is the chromatogram of the mixed reference solution. Figure 6 S6 is the chromatogram of the test solution of cold and fever relieving granules, batch number 2504827. Figure 6It can be seen that the chromatogram of the test solution of the cold and fever relief granules shows 9 characteristic peaks, with the corresponding peak of the puerarin reference as the S peak. The negative sample solution has no interference; the mixed reference solution is positive, and the retention time of the main component of the test solution is consistent with that of the reference solution. Using the National Pharmacopoeia Commission's Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicine, the chromatograms of the above 16 batches of cold and fever-relieving granules test solutions and mixed reference solutions were processed through data import, multi-point correction, and data matching to obtain specificity verification chromatograms for the cold and fever-relieving granules, as shown below. Figure 7 As shown, the chromatogram of the test solution of the cold and fever-relieving granules showed 9 characteristic peaks, with the corresponding peak of the puerarin reference as the S peak. The negative sample solution showed no interference; the mixed reference solution showed positive results, and the retention time of the main component of the test solution was consistent with that of the reference solution.

[0047] Example 4: Validation of the HPLC characteristic chromatographic method for cold and fever-reducing granules

[0048] 1. Precision assessment

[0049] Two testers tested two groups of cold and fever-relieving granules (batch number 2504827) according to the method in Example 3 and obtained characteristic spectra. The relative retention times of each peak on the characteristic spectra of the cold and fever-relieving granules were recorded. The test results are shown in Table 4. Table 4. Relative retention times of each peak on the characteristic spectrum of Ganmao Qingre Granules

[0050] As shown in Table 4, the cold and fever-reducing granules exhibit nine characteristic peaks. Using the corresponding peak of puerarin as a reference, the S peak is taken as the peak. The relative retention times of peaks 1 through 9 are within ±10% of the specified values. This indicates that the method for establishing the HPLC characteristic chromatogram of the cold and fever-reducing granules in this application has good intermediate precision.

[0051] 2. Repeatability test

[0052] The method described in Example 3 was used to test the cold and fever relief granules (batch number 2504827) and obtain their characteristic chromatograms. Six parallel experiments were conducted, and the relative retention times of each peak on the characteristic chromatograms of the cold and fever relief granules were recorded. The test results are shown in Table 5. Table 5. Repeatability test results of the method for establishing the HPLC characteristic chromatogram of cold and fever relief granules

[0053] As can be seen from the data recorded in Table 5, there are 9 characteristic peaks in the cold and fever relief granules. With the corresponding peak of puerarin as the reference, the S peak is taken as the peak. The relative retention times of peaks 1-9 are within ±10% of the specified value, and the RSD is 0.02%-0.04%, indicating that the method for establishing the HPLC characteristic spectrum of cold and fever relief granules in this application has good repeatability.

[0054] 3. Stability test

[0055] A test solution of cold and fever-relieving granules (batch number 2504827) was prepared according to the method in Example 3. The solution was injected at 0h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h for analysis. The relative retention times of each peak on the characteristic chromatogram of the cold and fever-relieving granules were recorded. The test results are shown in Table 6. Table 6. Stability test results of cold and fever relief granules solution

[0056] As can be seen from the data recorded in Table 6, the cold and fever relief granules exhibit 9 characteristic peaks. With the corresponding peak of puerarin as the reference, the S peak is taken as the peak. The relative retention times of peaks 1 to 9 are within ±10% of the specified value, indicating that the cold and fever relief granules test solution has good stability.

[0057] Example 5: Method for Determination of Component Content in Cold and Fever Relief Granules

[0058] S1. Accurately weigh 5.0g of cold and fever relief granules, add them to 25mL of methanol, sonicate for 45min, let stand at room temperature, filter, and take the filtrate to obtain the cold and fever relief granules test solution. S2. Add amygdalin, puerarin, cimicifugain and corydaline to methanol to prepare a mixed reference solution with amygdalin concentration of 100 ug / mL, puerarin concentration of 200 ug / mL, cimicifugain concentration of 15 ug / mL and corydaline concentration of 15 ug / mL. Add 5-O-methylvisamidol to methanol to obtain a single reference solution with a concentration of 80 μg / mL. S3. Accurately pipette 5 μL each of the test solution of cold and fever-relieving granules, the mixed reference solution, and the single reference solution, and inject them into the high-performance liquid chromatograph for determination. Calculate the content of amygdalin, puerarin, cimicifugain, and corydaline in the test solution using the external standard method.

[0059] The analytical parameters of the high performance liquid chromatograph in S3 are as follows: column temperature 30℃, flow rate 1.0 mL / min, and detection wavelengths 210 nm (amygdalin), 250 nm (puerarin, cimicifugain), and 289 nm (corydaline).

[0060] The chromatographic conditions for the high-performance liquid chromatography analysis are as follows: A 250 mm × 4.6 mm, 5 µm Agilent Eclipse XDB-C18 column was used as the chromatographic column, with octadecylsilane-bonded silica gel as the packing material, and gradient elution was performed using acetonitrile as mobile phase A and 0.1% (v / v) phosphoric acid aqueous solution as mobile phase B.

[0061] The gradient elution settings are as follows: 0 min, A:B = 5%:95%; 0–15 min, A:B = 5%:95% → 10%:90%; 15 min, A:B = 10%:90%; 15–30 min, A:B = 10%:90% → 15%:85%; 30 min, A:B = 15%:85%; 30–50 min, A:B = 15%:85% → 25%:75%; 50 min, A:B = 25%:75%; 50–55 min… A:B ratio is 25%:75% → 30%:70%; at 55 min, A:B ratio is 30%:70%; from 55 to 65 min, A:B ratio is 30%:70% → 60%:40%; at 65 min, A:B ratio is 60%:40%; from 65 to 65.1 min, A:B ratio is 60%:40% → 5%:95%; at 65.1 min, A:B ratio is 5%:95%; from 65.1 to 70 min, A:B ratio is 5%:95%; at 70 min, A:B ratio is 5%:95%.

[0062] Example 6: Methodological Validation of the Ingredient Content of Cold and Fever Relief Granules

[0063] 1. Linearity Study: Accurately weigh 7.5 mg of cimicifugain and 7.5 mg of corydaline, and dilute to 50 ml with methanol. Then accurately weigh 6 mg of amygdalin and 10 mg of puerarin, dissolve them in 5 ml of the above mixed solution of cimicifugain and corydaline, and dilute to 25 ml with methanol. Shake well to obtain the reference stock solution. Then take 2 ml, 4 ml, 6 ml, and 8 ml of the stock solution respectively, and dilute to 10 ml to obtain test solutions of different gradients. Then inject and measure the samples. Plot a standard curve with peak area (Y) as the ordinate and injection volume (X) as the abscissa, and calculate the regression equation. The results show that each component exhibits a good linear relationship within the corresponding concentration range, as shown in Table 7 below. Table 7 Linear Relationship

[0064] As can be seen from the data recorded in Table 7, the linear range of amygdalin is 45.82-229.09 μg / mL; the linear range of puerarin is 78.02-390.1 μg / mL; the linear range of cimicifugoside is 6.11-30.56 μg / mL; and the linear range of corydaline is 5.95-29.76 μg / mL.

[0065] 2. Intermediate precision test: Two testers tested the content of corresponding components in the two groups of cold and fever relief granules (batch number 2504827) according to the method in Example 5. The test results are shown in Figure 8: Table 8. Intermediate precision test results of the method for determining the content of ingredients in cold and fever-reducing granules.

[0066] As can be seen from the data recorded in Table 8, the mean RSDs of the contents of amygdalin, puerarin, cimicifugoside, and corydaline are 2.5%, 1.5%, 3.7%, and 1.7%, respectively, indicating that the intermediate precision of this method is good.

[0067] 3. Repeatability test: Referring to Example 5, 6 parallel tests were conducted to test the content of the corresponding components in the cold and fever relief granules. The test results are shown in Table 9: Table 9. Repeatability test results of the method for determining the content of ingredients in cold and fever-reducing granules.

[0068] As can be seen from the data recorded in Table 9, the RSDs of amygdalin, puerarin, cimicifugoside, and corydaline are 0.6%, 0.8%, 1.0%, and 2.1%, respectively, indicating that the content testing method provided by the present invention has good repeatability.

[0069] 4. Solution stability study: The cold and fever-relieving granules (batch number 2504827) prepared in Example 5 were analyzed. The solutions were injected at 0h, 4h, 8h, 12h, 24h, 36h, and 48h, and the peak areas and retention times of the corresponding components were recorded. The test results are shown in Table 10. Table 10. Determination of component content in cold and fever-relieving granules and results of solution stability test.

[0070] As can be seen from the data recorded in Table 10, the RSDs of amygdalin, puerarin, cimicifugoside, and corydaline are 1.93%, 0.96%, 0.33%, and 1.86%, respectively, indicating that the solution stability of the content testing method provided by the present invention is good.

[0071] 5. Recovery rate test: Preparation of reference stock solution: Accurately weigh 5.6 mg of cimicifugain and 2.4 mg of corydaline, and add methanol to a final volume of 100 ml; then accurately weigh 7.6 mg of amygdalin and 27.2 mg of puerarin, and dissolve them in 25 ml of the above mixed solution of cimicifugain and corydaline, and add methanol to a final volume of 200 ml, shake well, and the solution is ready; Preparation of the test solution: Accurately weigh 5.0 g of cold and fever relieving granules (batch number 2504827), place them in a stoppered conical flask, accurately add 25 ml of reference stock solution, weigh the solution, sonicate (power 900 W, frequency 60 kHz) for 45 minutes, cool, weigh the solution again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the test solution; Then, six parallel test solutions were prepared, and the corresponding component contents were tested according to the method in Example 5. The test results are shown in Table 11. Table 11 Results of the recovery rate test of the sample spiking rate in the determination method of the content of ingredients in cold and fever-relieving granules.

[0072] As can be seen from the data recorded in Table 11, the RSDs of amygdalin, puerarin, cimicifugoside, and corydaline are 0.5%, 0.85%, 1.13%, and 0.72%, respectively, indicating that the recovery rate of the content testing method provided by this invention meets the requirements.

[0073] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for establishing the HPLC characteristic chromatogram of Ganmaoqingre granules, characterized in that, It comprises the following steps: (1) Precisely measure the cold-clearing granules, and prepare the cold-clearing granules test sample solution; (2) Respectively weigh the amygdalin, puerarin, and the other components, and add methanol to prepare the mixed control sample solution; weigh 5-O-methylvisamminol, and add methanol to prepare the single control sample solution; (3) Respectively weigh the amygdalin, puerarin, and the other components, and prepare the negative sample solutions by decocting; (4) Analyze the cold-clearing granules test sample solution, the mixed control sample solution, the single control sample solution, and the negative sample solutions by using the high-performance liquid chromatograph, and obtain the HPLC characteristic spectrum of the cold-clearing granules.

2. The method for establishing the HPLC characteristic chromatogram of the cold heat-clearing granules according to claim 1, characterized in that, In the HPLC characteristic spectrum of the cold-clearing granules, the 9 common peaks are: the 1st peak, the 2nd peak amygdalin, the 3rd peak puerarin, the 4th peak, the 5th peak, the 6th peak, the 7th peak cimifugin, the 8th peak 5-O-methylvisamminol, and the 9th peak cathayanin; and the relative retention times of the characteristic peaks are: the 1st peak 0.774, the 2nd peak 0.827, the 4th peak 1.08, the 5th peak 1.11, the 6th peak 1.25, the 7th peak 1.31, the 8th peak 1.76, and the 9th peak 2.12, with the 3rd peak as the reference peak. 3.The method for establishing the HPLC characteristic chromatogram of Ganmaoqingre granules according to claim 1, characterized by, The specific preparation method of the negative sample solutions is as follows: Grind the amygdalin, puerarin, and the other components, and then respectively select three of them to add 8 times of pure water to decoct twice, each time for 1 hour; filter and combine the decocting liquids, and then respectively dilute the decocting liquids with methanol to obtain the negative sample solution without amygdalin, the negative sample solution without puerarin, the negative sample solution without the other components, and the negative sample solution without the other components.

4. The method for determining the ingredient content of Ganmaoqingre granules, characterized in that, It comprises the following steps: S1, precisely measure the cold-clearing granules, and prepare the cold-clearing granules test sample solution; S2, respectively weigh the amygdalin, puerarin, and the other components, and add methanol to prepare the mixed control sample solution; weigh 5-O-methylvisamminol, and add methanol to prepare the single control sample solution; S3, respectively precisely take 5-20 μL of the cold-clearing granules test sample solution, the mixed control sample solution, and the single control sample solution, and inject them into the high-performance liquid chromatograph for determination; and the content of the amygdalin, puerarin, and the other components in the test sample solution is calculated by using the external standard method.

5. The method according to claim 4, wherein the components of the cold heat-clearing granules are determined by HPLC. The analysis parameters of the high-performance liquid chromatograph in S3 are: the column temperature is 25-32℃, the flow rate is 0.8-1.0 mL / min, and the detection wavelengths are 210 nm, 250 nm, and 289 nm.

6. The method of claim 4, wherein the components of the cold-clearing granules are determined by the following method. The linear range of the amygdalin is 45.82-229.09 μg / mL; the linear range of the puerarin is 78.02-390.1 μg / mL; the linear range of the cimifugin is 6.11-30.56 μg / mL; and the linear range of the cathayanin is 5.95-29.76 μg / mL.

7. The method according to claim 4, wherein the components of the cold heat-clearing granules are determined by HPLC. The preparation method of the cold-clearing granules test sample solution is as follows: Precisely weigh 5.0 g of the cold-clearing granules, add 25 mL of solvent, and ultrasonically treat for 15-60 min; then, place the mixture to room temperature, filter, and take the filtrate, and the cold-clearing granules test sample solution is obtained.

8. The method according to claim 7, wherein the components of the cold heat-clearing granules are determined by HPLC. The solvent is one of 70% methanol solution by volume, methanol, 70% ethanol solution by volume, ethanol and pure water.

9. The method of claim 4, wherein the components of the cold heat-clearing granules are determined by the following method. The specific preparation method of the mixed control solution is as follows: The amygdalin, puerarin, cimifugin and lithospermine are added into methanol to prepare the mixed control solution with the concentration of 100ug / mL of amygdalin, 200ug / mL of puerarin, 15ug / mL of cimifugin and 15ug / mL of lithospermine; The specific preparation method of the single control solution is as follows: The 5-O-methylvisamminol is added into methanol to obtain the single control solution with the concentration of 80ug / mL.

10. The method of claim 4, wherein the components of the cold heat-clearing granules are determined by the following method. The chromatographic conditions of the high performance liquid chromatograph analysis are as follows: Agilent Eclipse XDB-C18 with the size of 250mm*4.6mm and 5µm is used as the chromatographic column, octadecylsilane bonded silica gel is used as the filler, acetonitrile is used as the mobile phase A and the aqueous solution of phosphoric acid with the volume fraction of 0.1% is used as the mobile phase B for gradient elution; The gradient elution settings are as follows: 0min, A:B is 5%:95%; 0-15min, A:B is 5%:95%→10%:90%; 15min, A:B is 10%:90%; 15-30min, A:B is 10%:90%→15%:85%; 30min, A:B is 15%:85%; 30-50min, A:B is 15%:85%→25%:75%; 50min, A:B is 25%:75%; 50-55min, A:B is 25%:75%→30%:70%; 55min, A:B is 30%:70%; 55-65min, A:B is 30%:70%→60%:40%; 65min, A:B is 60%:40%; 65-65.1min, A:B is 60%:40%→5%:95%; 65.1min, A:B is 5%:95%; 65.1-70min, A:B is 5%:95%; 70min, A:B is 5%:95%.

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