A quality control method of a traditional Chinese medicine composition

Abstract

The present application relates to the technical field of traditional Chinese medicine analysis, and particularly relates to a quality control method of a traditional Chinese medicine composition. The quality control method comprises the following steps: (1) preparing reference substance solutions: preparing baicalin reference substance solutions and chlorogenic acid reference substance solutions respectively; (2) preparing test sample solutions: preparing a traditional Chinese medicine solution from the traditional Chinese medicine composition, precisely taking 2 mL of the traditional Chinese medicine solution into a 50 mL brown volumetric flask, adding methanol to the calibration mark, and mixing; (3) determining HPLC conditions: optimizing and determining the high performance liquid chromatography (HPLC) conditions of baicalin and chlorogenic acid respectively; and (4) determining methods: precisely taking 20 μL of each of the above reference substance solutions and test sample solutions, and injecting into a HPLC instrument for determination. The method can effectively and quickly control the quality of the traditional Chinese medicine composition.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for quality control of traditional Chinese medicine compositions. Background Technology

[0002] Currently, the prevention and control of swine fever mainly relies on vaccination, and there are no approved specific treatments. Drug treatment mainly involves the development of antiviral gene therapy drugs, biological products such as interferon and immunoglobulins, antibiotics and chemical drugs, as well as traditional Chinese veterinary medicine. Antiviral gene therapy drugs are currently in the laboratory research and development stage. Biological products such as interferon used clinically have problems such as high cost, unstable efficacy, and demanding storage conditions. Antibiotics and chemical drugs have caused drug residues and drug resistance problems due to their extensive use. Traditional Chinese veterinary medicine mainly involves drugs such as Qingwen Baidu San and Huanglian Jiedu San, which are used to relieve symptoms.

[0003] Traditional Chinese medicine (TCM) classifies swine fever as a "febrile disease," with its pathogenesis involving excessive heat and toxins, and simultaneous burning of qi and blood. Treatment should focus on clearing heat and detoxifying, cooling the blood and stopping bleeding, and strengthening the body's resistance to pathogens. However, highly effective TCM veterinary preparations specifically targeting swine fever are still scarce. This invention, through formula research, has yielded a scientifically formulated, effective, and quality-controllable TCM composition specifically for the effective prevention and treatment of swine fever. Furthermore, research on its quality control methods is being conducted, aiming to provide more precise quality control standards for effective TCM treatments of swine fever. Summary of the Invention

[0004] This invention provides a quality control method for a traditional Chinese medicine composition, mainly including a method for determining the content of baicalin and chlorogenic acid, which can effectively and quickly control the quality of the traditional Chinese medicine composition and solve the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: A method for quality control of a traditional Chinese medicine composition is provided, the composition being made from the following raw materials in parts by weight: 200-280 parts gypsum, 150-220 parts anemarrhena, 180-250 parts scutellaria, 100-160 parts honeysuckle, 180-250 parts isatis root, 100-160 parts indigo leaf, 80-120 parts forsythia, 80-120 parts scrophularia, 80-120 parts rehmannia, and 30-60 parts licorice. The quality control method for the traditional Chinese medicine composition includes a method for determining the contents of baicalin and chlorogenic acid using high performance liquid chromatography, the steps of which include: (1) Preparation of reference solutions: Prepare baicalin reference solution and chlorogenic acid reference solution separately for later use; (2) Preparation of test solution: Prepare the traditional Chinese medicine composition into a traditional Chinese medicine solution. Accurately weigh 2 mL of the traditional Chinese medicine solution, place it in a 50 mL brown volumetric flask, add methanol to the mark, mix well, and the solution is obtained. (3) Determination of HPLC conditions: The high performance liquid chromatography conditions for baicalin and chlorogenic acid were optimized and determined respectively; (4) Determination method: Accurately pipette 20 μL of the above reference solution and test solution into the high performance liquid chromatograph and determine.

[0006] Furthermore, the preparation steps of the traditional Chinese medicine solution in step (2) are as follows: (1) Weigh each raw Chinese medicine according to the aforementioned weight proportions, crush the raw gypsum, and cut the remaining raw Chinese medicine into Chinese medicine slices for later use; (2) Add 8-10 times the amount of water to the Chinese herbal medicine slices, soak for 0.5-1h, heat and decoct 3 times, 1-1.5h each time, combine the decoctions, filter to obtain the extract; (3) The filtrate is concentrated under reduced pressure at a temperature of 70-80℃ and a vacuum degree of -0.04--0.06MPa to a relative density of 1.12-1.18 to obtain a concentrated solution; (4) Let the concentrated liquid stand for 12-24 hours, take the supernatant and filter it through a 500-800 mesh filter cloth to obtain a clear filtrate; (5) Add 0.2%-0.4% preservative to the filtrate, add purified water to make up the volume, stir and mix well to obtain the final product.

[0007] Furthermore, the method for determining the baicalin content includes the following steps: S1. Preparation of baicalin reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.5 mg per 1 mL. S2. Preparation of test solution: Accurately measure 2 mL of the traditional Chinese medicine solution, place it in a 50 mL brown volumetric flask, add methanol to the mark, sonicate for 20 min, mix well, and the solution is ready. S3. Preparation of negative control solution: Prepare a negative control sample with Scutellaria baicalensis removed according to the prescription, and prepare a negative control solution according to the preparation method of the test solution; S4. HPLC conditions: Octadecylsilane-bonded silica gel was used as the packing material; methanol-0.4% phosphoric acid solution (50:50) was used as the mobile phase; the detection wavelength was 280 nm, the column temperature was [value missing], the flow rate was 30 °C, and the flow rate was 1 mL / min.

[0008] Furthermore, the theoretical plate number under the above chromatographic conditions, calculated based on the baicalin peak, shall not be less than 2500.

[0009] Furthermore, the method for determining chlorogenic acid content includes the following steps: S1. Preparation of chlorogenic acid reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, place it in a brown volumetric flask, and add 50% methanol to prepare a solution containing 40 μg per 1 mL. S2. Preparation of test solution: Accurately measure 2 mL of the traditional Chinese medicine solution, place it in a 50 mL brown volumetric flask, dilute to the mark with 50% methanol, and shake well to obtain the solution. S3. Preparation of negative control solution: Prepare a negative control sample with honeysuckle removed according to the prescription, and prepare a negative control solution according to the preparation method of the test solution; S4. HPLC conditions: Octadecylsilane-bonded silica gel was used as the packing material, acetonitrile-methanol-0.4% phosphoric acid water (10:6:84) was used as the mobile phase, the column temperature was 35℃, the detection wavelength was 327nm, and the flow rate was 1mL / min.

[0010] Furthermore, the theoretical plate number under the above chromatographic conditions, calculated based on the chlorogenic acid peak, shall not be less than 1000.

[0011] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 240 parts gypsum, 180 parts anemarrhena, 200 parts scutellaria, 130 parts honeysuckle, 200 parts isatis root, 130 parts indigo leaf, 100 parts forsythia, 100 parts scrophularia, 100 parts rehmannia, and 40 parts licorice.

[0012] Furthermore, the dosage form of the traditional Chinese medicine composition is oral liquid, granules, capsules, or powder.

[0013] Furthermore, the dosage form of the traditional Chinese medicine composition is an oral liquid, which is prepared by the aforementioned method for preparing traditional Chinese medicine solutions.

[0014] The beneficial effects of this invention are: This invention establishes a systematically optimized and validated HPLC quality control method for traditional Chinese medicine compositions used in the treatment of swine fever. Specifically, through the investigation of key parameters such as column screening, mobile phase gradient optimization, and detection wavelength determination, a technical solution with effective quality control is constructed to ensure stable and controllable product quality. Attached Figure Description

[0015] Figure 1 The HPLC chromatogram of baicalin of the present invention is shown below. Figure 2 This is the HPLC chromatogram of the traditional Chinese medicine composition of the present invention; Figure 3 This is the HPLC chromatogram of the negative control standard in the determination of baicalin content in this invention; Figure 4 Linear relationship graph of baicalin; Figure 5 This is the HPLC chromatogram of the chlorogenic acid reference standard of the present invention; Figure 6 This is the HPLC chromatogram of the test sample in the determination of chlorogenic acid content; Figure 7This is the HPLC chromatogram of the negative control standard in the determination of chlorogenic acid content; Figure 8 This is the standard curve for chlorogenic acid. Detailed Implementation

[0016] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific implementation methods.

[0017] Example 1

[0018] A method for quality control of a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 200-280 parts of gypsum, 150-220 parts of anemarrhena asphodeloides, 180-250 parts of scutellaria baicalensis, 100-160 parts of honeysuckle, 180-250 parts of isatis indigotica, 100-160 parts of indigofera tinctoria, 80-120 parts of forsythia suspensa, 80-120 parts of scrophularia ningpoensis, 80-120 parts of rehmannia glutinosa, and 30-60 parts of licorice.

[0019] The above-mentioned traditional Chinese medicine composition is prepared into an oral liquid formulation using the following method: (1) Weigh each raw Chinese medicine according to the aforementioned weight proportions, crush the raw gypsum, and cut the remaining raw Chinese medicine into Chinese medicine slices for later use; (2) Add 8-10 times the amount of water to the Chinese herbal medicine slices, soak for 0.5-1h, heat and decoct 3 times, 1-1.5h each time, combine the decoctions, filter to obtain the extract; (3) The filtrate is concentrated under reduced pressure at a temperature of 70-80℃ and a vacuum degree of -0.04--0.06MPa to a relative density of 1.12-1.18 to obtain a concentrated solution; (4) Let the concentrated liquid stand for 12-24 hours, take the supernatant and filter it through a 500-800 mesh filter cloth to obtain a clear filtrate; (5) Add 0.2%-0.4% preservative to the filtrate, add purified water to make up the volume, stir and mix well to obtain the final product.

[0020] The contents of baicalin and chlorogenic acid in the prepared oral liquid of traditional Chinese medicine were determined by the following steps: (1) Preparation of reference solutions: Prepare baicalin reference solution and chlorogenic acid reference solution separately for later use; (2) Preparation of test solution: Prepare the traditional Chinese medicine composition into a traditional Chinese medicine solution. Accurately weigh 2 mL of the traditional Chinese medicine solution, place it in a 50 mL brown volumetric flask, add methanol to the mark, mix well, and the solution is obtained. (3) Determination of HPLC conditions: The high performance liquid chromatography conditions for baicalin and chlorogenic acid were optimized and determined respectively; (4) Determination method: Accurately pipette 20 μL of the above reference solution and test solution into the high performance liquid chromatograph and determine.

[0021] Example 2: Determination of baicalin content in traditional Chinese medicine composition

[0022] I. Instruments and Chromatographic Conditions

[0023] Instruments: LC-20AD Shimadzu high performance liquid chromatograph, AB265-S dual-range 0.0001 g electronic balance, KQ5200DB ultrasonic instrument.

[0024] Chromatographic conditions: Based on the investigation and optimization of the main factors affecting the resolution and response value of the indicator component baicalin, the final chromatographic conditions were determined as follows: octadecylsilane-bonded silica gel as the packing material; methanol-0.4% phosphoric acid solution (50:50) as the mobile phase; and a detection wavelength of 280 nm. The theoretical plate number, calculated based on the baicalin peak, should be no less than 2500.

[0025] II. Preparation of reference solution, test solution and negative control solution

[0026] ① Preparation of baicalin reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.5 mg per 1 mL.

[0027] ② Preparation of the test solution: Accurately measure 2 mL of the oral liquid of traditional Chinese medicine in Example 1, place it in a 50 mL brown volumetric flask, add methanol to the mark, sonicate for 20 min, mix well, and the solution is ready.

[0028] ③ Preparation of negative control solution: Prepare a negative control sample with Scutellaria baicalensis removed according to the prescription preparation process, and prepare a negative control solution according to the test solution preparation method.

[0029] III. System adaptability test and blank test

[0030] Take 20 μL each of the baicalin reference solution, test solution, and negative control solution, and perform HPLC determination under the established chromatographic conditions. The chromatogram is shown in the figure. Figure 1 , Figure 2 and Figure 3 The chromatogram showed that the baicalin peak in the test sample had the same retention time as the reference sample, and baicalin was well separated from other components, while the negative control did not show this peak at the same retention time. The results indicate that with a theoretical plate number > 1000, the resolution R between the baicalin peak and its nearest neighbor peak > 1.5, and the negative control solution did not interfere with the determination of baicalin.

[0031] IV. Investigation of the linear relationship of baicalin

[0032] Different concentrations of baicalin solutions were prepared and measured sequentially under the established chromatographic conditions. The chromatograms and peak areas were recorded, and the results are shown in Table 1.

[0033] Table 1. Chromatographic peak area results of baicalin at different concentrations

[0034] A standard curve for baicalin was plotted with baicalin concentration on the x-axis and peak area on the y-axis. (See...) Figure 4 Performing linear regression on the data in Table 1 yields the linear regression equation: y = 7 × 10 7 x+375732, (r 2 =0.9998). The results showed that baicalin exhibited good linearity in the injection concentration range of 0.103-0.824 mg / mL.

[0035] V. Precision Test

[0036] The main focus was on the reproducibility of the instrument's measurement results. The standard solution was injected in a volume of 20 μL, and the injection was repeated six times. Chromatograms and peak areas were recorded, and the results are shown in Table 14-8. The experiment demonstrated that the instrument has good precision.

[0037] Table 2 Results of precision test for baicalin determination

[0038] VI. Repeatability Test

[0039] For samples from the same batch, six parallel test solutions were prepared according to the test solution preparation method. The solutions were then tested under the established chromatographic conditions. Chromatograms, peak areas, and baicalin content were recorded. The results are shown in Table 3. The experiment demonstrated good repeatability of the experimental method.

[0040] Table 3. Results of repeatability tests for baicalin determination

[0041] VII. Baicalin Recovery Test

[0042] Nine 1 mL aliquots of sample (baicalin content 15.5883 mg / mL) were accurately measured and placed in separate 50 mL volumetric flasks. Baicalin reference standard was added at 80%, 100%, and 120% of the baicalin content in the sample, respectively. The solutions were then diluted to 50 mL with a methanol-hydrochloric acid (100:1) mixture, shaken well, and allowed to stand. The supernatant was used as the test solution. The chromatographic conditions were established, and the chromatograms and peak areas were recorded. The baicalin content in the sample after adding the standard was determined, and the recovery rate was calculated using the following formula. The results are shown in Table 4. The results indicate that the experimental method is accurate.

[0043] Recovery rate =

[0044] Table 4 Results of the baicalin recovery test

[0045] 8. Intraday stability test

[0046] Samples from the same batch were prepared into test solutions according to the prepared solution method. The tests were conducted at 0, 1, 2, 4, 8, and 12 hours under the established chromatographic conditions. Chromatograms and peak areas were recorded, and the baicalin content was calculated. The results are shown in Table 5. The tests showed that the test solutions were stable within 12 hours.

[0047] Table 5 Results of intraday stability test for baicalin.

[0048] IX. Durability Test

[0049] Durability refers to the degree to which the measurement results remain unaffected by minor changes in measurement conditions. This experiment investigated the main factors affecting the determination of mobile phase composition, pH value, different brands of chromatographic columns, column temperature, flow rate, and detection wavelength in liquid chromatography.

[0050] ① Examination of detection wavelength

[0051] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSCL057260); Mobile phase: methanol-0.4% phosphoric acid solution (50:50); Detection wavelength: 280 nm; Flow rate: 1 mL / min; Column temperature: 30 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and measure under different detection wavelengths. Calculate the results, which are shown in Table 6.

[0052] Table 6. Results of baicalin determination under different detection wavelengths

[0053] Experimental results show that when the absorption wavelength is set to 280 nm with a fluctuation of 5 nm, the RSD of the content determination result is 0.086%, which has no significant effect. This indicates that when determining the content of baicalin, the detection wavelength between 275-285 nm can accurately determine its content.

[0054] ② Column temperature investigation

[0055] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: vscL057260); Mobile phase: methanol-0.4% phosphoric acid solution (50:50); Detection wavelength: 280 nm; Flow rate: 1 mL / min; Column temperature: 30 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and measure under different column temperatures. Calculate the results, which are shown in Table 7.

[0056] Table 7. Results of baicalin determination under different column temperatures

[0057] Experimental results show that when the column temperature is set at 30℃ with a fluctuation of 5℃, the RSD of the content determination result is 1.63%, which has no significant effect. This indicates that the content of baicalin can be accurately determined at a column temperature between 25-35℃.

[0058] ③ Flow velocity investigation

[0059] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: vscL057260); Mobile phase: methanol-0.4% phosphoric acid solution (50:50); Detection wavelength: 280 nm; Flow rate: 1 mL / min; Column temperature: 30 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and measure under different flow rate conditions. Calculate the results, which are shown in Table 8.

[0060] Table 8. Results of baicalin determination under different flow rates

[0061] Experimental results showed that a 20% fluctuation in the flow rate (1 mL / min) did not significantly affect the RSD of the content determination (0.82%). This indicates that a flow rate between 0.8 and 1.2 mL / min can accurately determine the content of baicalin.

[0062] ④ Investigation of the composition ratio of the mobile phase

[0063] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: vscL057260); Mobile phase: methanol-0.4% phosphoric acid solution (50:50); Detection wavelength: 280 nm; Flow rate: 1 mL / min; Column temperature: 30 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, and inject them separately. Measure and calculate the results under different mobile phase composition ratios. The results are shown in Table 9.

[0064] Table 9. Results of baicalin determination under different mobile phase composition ratios

[0065] Experimental results show that when the mobile phase ratio is set at 50:50, a fluctuation of 5% within ±1% results in an RSD of 3.29% for the content determination, which is not significantly affected. This indicates that when determining the content of baicalin, variations in the mobile phase ratio within ±5% can still result in relatively accurate determination of its content.

[0066] ⑤ pH test

[0067] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: vscL057260); Mobile phase: methanol-0.4% phosphoric acid solution (50:50); Detection wavelength: 280 nm; Flow rate: 1 mL / min; Column temperature: 30 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and measure under different pH conditions. Calculate the results, which are shown in Table 10.

[0068] Table 10 Results of baicalin determination under different pH conditions

[0069] Experimental results show that when the pH of the mobile phase fluctuates by 0.2, the RSD of the content determination result is 5.84%, which has no significant effect. This indicates that when determining the content of chlorogenic acid, the content can be accurately determined even when the pH of the mobile phase varies within ±0.2.

[0070] ⑥ Investigation of different chromatographic columns

[0071] Column 1: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSCL041370); Column 2: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSCL057260); Column 3: Waters C18 (4.6) 250 mm, 5 μm; SN: 0295360072382); Mobile phase: methanol-0.4% phosphoric acid solution (50:50); Detection wavelength: 280 nm; Flow rate: 1 mL / min; Column temperature: 30 ℃. Accurately pipette 20 μL each of the test solution and reference solution, inject them separately, and perform the determination using different chromatographic columns. Calculations are performed, and the results are shown in Table 11.

[0072] Table 11 Results of Baicalin Determination by Different Chromatographic Columns

[0073] Experimental results show that using different chromatographic columns to determine the content RSD is 2.96%, with no significant impact.

[0074] 10. Determination of baicalin content in samples

[0075] Twenty samples from ten batches of this traditional Chinese medicine solution were selected and tested according to the established chromatographic conditions. The chromatograms and peak areas were recorded. The baicalin content of each sample was calculated based on the peak area, and the results are shown in Table 12.

[0076] Table 12 Results of Baicalin Content Determination in Samples

[0077] XI. Determination of the limit for baicalin content

[0078] The measured results of the above 10 batches of 20 samples show that the average content of the 10 batches of samples is 13.113 mg / mL, and 80% of this content is 10.490 mg / mL. The lowest content of the batch is 9.774 mg / mL. Based on the measured data of the 10 batches of samples and taking into account the results of the drug stability test, the final limit for the content of baicalin in this product is specified as follows: This product contains baicalin (C... 21 H 18 O 11 The total amount should be no less than 9.0 mg per 1 mL.

[0079] (12) Conclusion

[0080] In the determination of baicalin content, optimized chromatographic conditions with high theoretical plate number and good resolution were established. Methodological tests, including precision studies, stability studies, repeatability studies, and recovery tests, verified the accuracy and reproducibility of the content determination method. Based on the content determination data from 20 samples in 10 batches and the results of the stability study, the final limit for baicalin content in this product was set as follows: This product contains baicalin (C... 21 H 18 O 11 The total amount should be no less than 9.0 mg per 1 mL.

[0081] Example 3: Determination of chlorogenic acid content in traditional Chinese medicine composition

[0082] I. Instruments and Chromatographic Conditions

[0083] Instruments: LC-20AD Shimadzu high performance liquid chromatograph, AB265-S dual-range 0.0001 g electronic balance, KQ5200DB ultrasonic instrument.

[0084] Chromatographic conditions: Based on the investigation and optimization of the main factors affecting the resolution and response value of the index component chlorogenic acid (including chromatographic column, column temperature, etc.), the final chromatographic conditions were determined as follows: octadecylsilane-bonded silica gel as the packing material, acetonitrile-methanol-0.4% phosphoric acid water (10:6:84) as the mobile phase, column temperature 35℃, detection wavelength 327nm, and the theoretical plate number calculated based on the chlorogenic acid peak should not be less than 1000.

[0085] II. Preparation of reference solution, test solution and negative control solution

[0086] ① Preparation of chlorogenic acid reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, place it in a brown volumetric flask, add 50% methanol to prepare a solution containing 40 μg per 1 mL (store below 10℃).

[0087] ② Preparation of the test solution: Accurately measure 2 mL of this product and place it in a 50 mL brown volumetric flask. Dilute to the mark with 50% methanol and shake well to obtain the test solution.

[0088] ③ Preparation of negative control solution: Prepare a negative control sample without honeysuckle according to the prescription preparation process, and prepare a negative control solution according to the test solution preparation method.

[0089] III. System adaptability test and blank test

[0090] Take 20 μL each of the chlorogenic acid reference solution, test solution, and negative control solution, and perform HPLC determination under the established chromatographic conditions. The chromatogram is shown in the figure. Figure 5 , Figure 6 and Figure 7 The chromatogram showed that the chlorogenic acid peak in the test sample had the same retention time as the reference sample, and chlorogenic acid was well separated from other components. The negative control did not show this peak at the same retention time. The results indicate that with a theoretical plate number > 1000, the resolution R between the chlorogenic acid peak and its nearest neighbor peak > 1.5, and the negative control solution did not interfere with the determination of chlorogenic acid.

[0091] IV. Investigation of the linear relationship of chlorogenic acid

[0092] Chlorogenic acid solutions of different concentrations were prepared and measured sequentially according to the established chromatographic conditions. 20 μL was injected each time, and the chromatograms and peak areas were recorded. The results are shown in Table 13.

[0093] Table 13. Chromatographic peak area results for different concentrations of chlorogenic acid

[0094] A standard curve for chlorogenic acid was plotted with chlorogenic acid content on the x-axis and peak area on the y-axis. (See...) Figure 8Performing linear regression on the data in Table 13 yields the linear regression equation: y = 64100x + 39243, (r 2 =0.9995). The results show that the chlorogenic acid concentration exhibits a good linear relationship in the range of 10.32-104.8 μg / mL.

[0095] V. Precision Test

[0096] The main focus was on the reproducibility of the instrument's measurement results. 20 μL of the reference solution was injected, and the injection was repeated six times. The chromatograms and peak areas were recorded, and the results are shown in Table 14. The results indicate that the instrument has good precision.

[0097] Table 14 Results of Precision Test for Chlorogenic Acid Determination

[0098] VI. Repeatability Test

[0099] Samples from the same batch were prepared into six parallel test solutions according to the prepared solution method. Each solution was tested under the established chromatographic conditions, and the chromatograms, peak areas, and chlorogenic acid content were recorded. The results are shown in Table 15. The experiment showed that the test method had good repeatability.

[0100] Table 15 Results of repeatability test for chlorogenic acid determination

[0101] VII. Chlorogenic Acid Recovery Test

[0102] Nine 1 mL aliquots of the same sample with a known chlorogenic acid content (1.1531 mg / mL) were accurately measured and placed in separate 50 mL volumetric flasks. Chlorogenic acid reference standard was added at 50%, 100%, and 150% of the chlorogenic acid content in each sample, respectively. The solutions were diluted to the mark with 50% methanol and shaken well to obtain the test solutions. The chromatographic conditions were established, and the chromatograms and peak areas were recorded. The chlorogenic acid content was determined based on the peak area, and the recovery rate was calculated. The results are shown in Table 16. The results indicate that the experimental method has good accuracy.

[0103] Recovery rate =

[0104] Table 16 Results of Chlorogenic Acid Recovery Test

[0105] 8. Intraday stability test

[0106] Take the same sample and prepare the test solution according to the test solution preparation method. Perform the test under the established chromatographic conditions at 0, 1, 2, 4 and 12 h respectively. Record the chromatogram and peak area, and calculate the chlorogenic acid content. The results are shown in Table 17. The test shows that the test solution is stable within 12 h.

[0107] Table 17 Results of intraday stability test for chlorogenic acid

[0108] IX. Durability Test

[0109] ① Examination of the detection wavelength

[0110] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSCL057260); Mobile phase: acetonitrile-methanol-0.4% phosphoric acid water (10:6:84); Flow rate: 1 mL / min; Column temperature: 35℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and determine the chlorogenic acid content under different detection wavelengths. The results are shown in Table 18.

[0111] Table 18 Results of chlorogenic acid determination at different detection wavelengths

[0112] Experimental results show that when the absorption wavelength is set to 327 nm with a fluctuation of 5 nm, the RSD of the content determination is 0.37%, which has no significant impact. This indicates that when determining the content of chlorogenic acid, a detection wavelength between 322 and 332 nm can accurately determine its content.

[0113] ② Column temperature investigation

[0114] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSCL057260); Mobile phase: acetonitrile-methanol-0.4% phosphoric acid water (10:6:84); Detection wavelength: 327 nm; Flow rate: 1 mL / min; Column temperature: 35 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and determine the chlorogenic acid content under different column temperature conditions. The results are shown in Table 19.

[0115] Table 19 Results of chlorogenic acid determination at different column temperatures

[0116] Experimental results show that when the column temperature fluctuates by 5℃ around 35℃, the RSD of the content determination is 4.46%, which has no significant effect. This indicates that when determining the content of chlorogenic acid, a column temperature between 25-35℃ can accurately determine its content.

[0117] ③ Flow velocity investigation

[0118] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSCL057260); Mobile phase: acetonitrile-methanol-0.4% phosphoric acid water (10:6:84); Detection wavelength: 327 nm; Flow rate: 1 mL / min; Column temperature: 35 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and determine the chlorogenic acid content under different flow rate conditions. The results are shown in Table 20.

[0119] Table 20 Results of chlorogenic acid determination under different flow rates

[0120] Experimental results show that when the flow rate is set to 1 mL / min and fluctuates by 20%, the RSD of the content determination is 3.10%, which has no significant effect. This indicates that the content of chlorogenic acid in the sample can be accurately determined with a flow rate between 0.8 and 1.2 mL / min.

[0121] ④ Investigation of the composition ratio of the mobile phase

[0122] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: vscL057260); Mobile phase: detection wavelength 327 nm; Flow rate: 1 mL / min; Column temperature: 35 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, and inject them separately. Determine the chlorogenic acid content under different mobile phase composition ratios. The results are shown in Table 21.

[0123] Table 21 Results of chlorogenic acid determination under different mobile phase ratios

[0124] Experimental results show that when the mobile phase (acetonitrile-methanol-0.4% phosphoric acid solution) ratio is set to 10:6:84 with a fluctuation of 5%, the RSD of the content determination is 2.48%, which has no significant effect. This indicates that when determining the content of chlorogenic acid, the mobile phase ratio can be within ±5% to accurately determine its content.

[0125] ⑤ pH test

[0126] Column: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSCL057260); Mobile phase: acetonitrile-methanol-0.4% phosphoric acid water (10:6:84); Detection wavelength: 327 nm; Flow rate: 1 mL / min; Column temperature: 35 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and measure under different pH conditions. Calculations are performed, and the results are shown in Table 22.

[0127] Table 22 Results of chlorogenic acid determination under different pH conditions

[0128] Experimental results show that when the pH of the mobile phase fluctuates by 0.2, the RSD of the content determination is 5.52%, which has no significant effect. This indicates that when determining the content of chlorogenic acid, the mobile phase ratio can be within ±0.2 to accurately determine its content.

[0129] ⑥ Investigation of different chromatographic columns

[0130] Column 1: AgLient C18 (4.6) 250 mm, 5 μm; SN: VSC L0: 41370); Column 2: AgLient C18 (4.6) 250 mm, 5 μm; SN: vscL057260); Column 3: Waters C18 (4.6) 250 mm, 5 μm; SN: 0295360072382); Mobile phase: acetonitrile-methanol-0.4% phosphoric acid water (10:6:84); Detection wavelength: 327 nm; Flow rate: 1 mL / min; Column temperature: 35 ℃. Accurately pipette 20 μL each of the test solution and the reference solution, inject them separately, and determine the chlorogenic acid content using different chromatographic columns. The results are shown in Table 23.

[0131] Table 23 Results of chlorogenic acid determination using different chromatographic columns

[0132] The experimental results show that when different chromatographic columns are used, the RSD is 3.20%, which has no significant effect.

[0133] 10. Determination of chlorogenic acid content in samples

[0134] Twenty samples from ten batches of this product were selected and tested under the established chromatographic conditions. The chromatograms and peak areas were recorded. The chlorogenic acid content of each sample was calculated based on the peak area, and the results are as follows.

[0135] Table 24 Results of Chlorogenic Acid Content Determination in Samples

[0136] XI. Determination of Chlorogenic Acid Content Limits

[0137] The measured results of the above 10 batches of 20 samples show that the average content of the chlorogenic acid in the 10 batches of samples is 1.0715 mg / mL, and 80% of this content is 0.8572 mg / mL. The lowest content in one batch of samples is 0.7328 mg / mL. Based on the measured data of the 10 batches of samples and taking into account the results of the drug stability test, the final limit for the chlorogenic acid content in this product is set as follows: This product contains chlorogenic acid (C1) from honeysuckle. 16 H 18 According to O9), each 1mL shall not be less than 0.70mg.

[0138] XII. Conclusion

[0139] In the determination of chlorogenic acid content, optimized chromatographic conditions with high theoretical plate number and good resolution were established. Methodological tests, including precision studies, stability studies, repeatability studies, and recovery tests, verified the accuracy and reproducibility of the content determination method. Based on the content determination data from 10 batches and 20 samples, and the results of stability studies, the final limit for chlorogenic acid content in this product was set as follows: This product contains chlorogenic acid (C...) from honeysuckle. 16 H 18 According to O9), each 1mL shall not be less than 0.70mg.

[0140] The quality standards of the traditional Chinese medicine compositions in the above embodiments are shown in Table 25 below.

[0141] Table 25 Quality Standards for Oral Liquid Preparations of Traditional Chinese Medicine Compositions

[0142] The present invention has been described in detail above. The specific embodiments described above should not be construed as limiting the scope of protection of the present invention. Any alternative modifications or variations made to the embodiments of the present invention by those skilled in the art will fall within the scope of protection of the present invention. Any aspects of the present invention not described in detail are well-known techniques to those skilled in the art.

Claims

1. A method for quality control of a traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 200-280 parts of gypsum, 150-220 parts of anemarrhena, 180-250 parts of scutellaria, 100-160 parts of honeysuckle, 180-250 parts of isatis root, 100-160 parts of indigo leaf, 80-120 parts of forsythia, 80-120 parts of scrophularia, 80-120 parts of rehmannia, and 30-60 parts of licorice. The quality control method for the traditional Chinese medicine composition includes a method for determining the contents of baicalin and chlorogenic acid using high performance liquid chromatography, the steps of which include: (1) Preparation of reference solutions: Prepare baicalin reference solution and chlorogenic acid reference solution separately for later use; (2) Preparation of test solution: Prepare the traditional Chinese medicine composition into a traditional Chinese medicine solution. Accurately weigh 2 mL of the traditional Chinese medicine solution, place it in a 50 mL brown volumetric flask, add methanol to the mark, mix well, and the solution is obtained. (3) Determination of HPLC conditions: The high performance liquid chromatography conditions for baicalin and chlorogenic acid were optimized and determined respectively; (4) Determination method: Accurately pipette 20 μL of the above reference solution and test solution into the high performance liquid chromatograph and determine.

2. The quality control method according to claim 1, characterized in that, The preparation steps of the traditional Chinese medicine solution in step (2) are as follows: (1) Weigh each raw Chinese medicine according to the aforementioned weight proportions, crush the raw gypsum, and cut the remaining raw Chinese medicine into Chinese medicine slices for later use; (2) Add 8-10 times the amount of water to the Chinese herbal medicine slices, soak for 0.5-1h, heat and decoct 3 times, 1-1.5h each time, combine the decoctions, filter to obtain the extract; (3) The filtrate is concentrated under reduced pressure at a temperature of 70-80℃ and a vacuum degree of -0.04--0.06MPa to a relative density of 1.12-1.18 to obtain a concentrated solution; (4) Let the concentrated liquid stand for 12-24 hours, take the supernatant and filter it through a 500-800 mesh filter cloth to obtain a clear filtrate; (5) Add 0.2%-0.4% preservative to the filtrate, add purified water to make up the volume, stir and mix well to obtain the final product.

3. The quality control method according to claim 1, characterized in that, The method for determining the baicalin content includes the following steps: S1. Preparation of baicalin reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.5 mg per 1 mL. S2. Preparation of test solution: Accurately measure 2 mL of the traditional Chinese medicine solution, place it in a 50 mL brown volumetric flask, add methanol to the mark, sonicate for 20 min, mix well, and the solution is ready. S3. Preparation of negative control solution: Prepare a negative control sample with Scutellaria baicalensis removed according to the prescription, and prepare a negative control solution according to the preparation method of the test solution; S4. HPLC conditions: Octadecylsilane-bonded silica gel was used as the packing material; methanol-0.4% phosphoric acid solution (50:50) was used as the mobile phase; the detection wavelength was 280 nm, the column temperature was [value missing], the flow rate was 30 °C, and the flow rate was 1 mL / min.

4. The quality control method according to claim 1, characterized in that, The method for determining chlorogenic acid content includes the following steps: S1. Preparation of chlorogenic acid reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, place it in a brown volumetric flask, and add 50% methanol to prepare a solution containing 40 μg per 1 mL. S2. Preparation of test solution: Accurately measure 2 mL of the traditional Chinese medicine solution, place it in a 50 mL brown volumetric flask, dilute to the mark with 50% methanol, and shake well to obtain the solution. S3. Preparation of negative control solution: Prepare a negative control sample with honeysuckle removed according to the prescription, and prepare a negative control solution according to the preparation method of the test solution; S4. HPLC conditions: Octadecylsilane-bonded silica gel was used as the packing material, acetonitrile-methanol-0.4% phosphoric acid water (10:6:84) was used as the mobile phase, the column temperature was 35℃, the detection wavelength was 327nm, and the flow rate was 1mL / min.

5. The quality control method according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 240 parts gypsum, 180 parts anemarrhena, 200 parts scutellaria, 130 parts honeysuckle, 200 parts isatis root, 130 parts indigo leaf, 100 parts forsythia, 100 parts scrophularia, 100 parts rehmannia, and 40 parts licorice.

6. The quality control method according to claim 1, characterized in that, The dosage form of the traditional Chinese medicine composition is oral liquid, granules, capsules or powder.

7. The quality control method according to claim 5, characterized in that, The dosage form of the traditional Chinese medicine composition is an oral liquid, which is prepared by the preparation method described in claim 2.

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