Quality control method and application of traditional Chinese medicine composition for treating arthralgia

Abstract

The invention provides a quality control method and application of a traditional Chinese medicine composition for treating arthralgia, and belongs to the technical field of traditional Chinese medicine detection. The quality control method comprises the following steps: (1) preparation of a test solution: taking a traditional Chinese medicine composition, grinding the traditional Chinese medicine composition, adding methanol for ultrasonic treatment, adding methanol, and filtering to obtain a subsequent filtrate; (2) preparing a reference substance solution, namely precisely weighing a paeonol reference substance, and mixing the paeonol reference substance with methanol; and (3) determination: respectively and precisely sucking the reference substance solution and the test solution, injecting into a liquid chromatograph, and determining to obtain the fingerprint spectrum. According to the method, the chromatographic peak area and the separation degree are used for quality evaluation of the traditional Chinese medicine composition, comprehensive monitoring of product quality is facilitated, oneness and one-sidedness of quality control are avoided, and the method is objective, comprehensive, good in accuracy, repeatable and high in accuracy. The traditional Chinese medicine composition has important significance in controlling the quality of the traditional Chinese medicine composition for treating arthralgia and ensuring the clinical curative effect.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a quality control method of a traditional Chinese medicine composition for treating arthralgia and an application thereof. Background Art

[0002] Joint pain is a common clinical disease or symptom, which is related to the natural damage and aging of articular cartilage, as well as long-term rheumatism, elevated uric acid, and long-term sitting. Different causes cause different diseases, such as rheumatoid arthritis or frozen shoulder induced by long-term cold and dampness, which is a group of diseases that mainly invade joints, bones, muscles, blood vessels, and soft tissues, or connective tissues; osteoarthritis caused by aging of articular cartilage is a wind-infected inflammation; and gouty arthritis caused by long-term hyperuricemia.

[0003] Joint pain diseases can be autoimmune diseases or chronic inflammatory diseases. The onset is relatively hidden and slow, and the course of the disease is long. The etiology of rheumatoid arthritis is complex and is related to allergic reactions after hemolytic streptococcal infection. Factors such as wind and cold, humidity, and overwork can trigger the onset of this disease. The common characteristics of this disease are swelling and pain in the affected muscles and joints, lameness, and the location of pain will shift. Osteoarthritis or periarthritis of the shoulder are related to wind and cold, humidity, and long-term labor, but they are non-infectious chronic inflammations. Regardless of whether it is related to infection, inflammation is a common pathological factor for various joint pains, and pain and inconvenience in movement are common clinical symptoms. Complications caused by long-term illness can lead to joint dysfunction and seriously affect the patient's quality of life. Chronic joint pain is difficult to cure, and it recurs over a long period of time and requires long-term medication. There is currently no specific drug in clinical practice. Non-steroidal anti-inflammatory drugs, analgesics, and glucocorticoids are mostly used for treatment in clinical practice. The medication is long-term, and strong adverse reactions are prone to occur. Chemical drugs will also develop drug resistance and poor patient compliance. Clinically, more effective drugs are needed to be selected or used alternately, especially local drug preparations with few adverse reactions and easy to use; especially modern Chinese medicine patches, which can achieve low allergic reactions, low systemic drug concentrations, and few adverse reactions, but high local drug concentrations, good efficacy, and long-term effects.

[0004] In order to solve the above technical problems, a Chinese patent with application number CN202310479559.6 discloses an invention patent for a Chinese medicine composition, preparation, preparation method and application for treating joint pain. The prior art provides the formula, preparation and preparation method of the above-mentioned Chinese medicine composition, but the quality control of the prepared product is required during the production process to ensure the stability and controllability of the product quality. At the same time, due to the particularity of quality control of Chinese medicine products, the quality control method is particularly required to be specific.

[0005] Traditional Chinese medicine has a wide range of sources and complex ingredients, which brings difficulties to its quality control and evaluation. The characteristic spectrum of traditional Chinese medicine refers to the spectrum that can reflect the overall characteristics of traditional Chinese medicine after proper treatment and using certain analytical methods. It is an important means of identifying the quality of traditional Chinese medicine. After more than ten years of research and application, this technology has become increasingly mature and has been more widely used in the quality control standards of traditional Chinese medicine.

[0006] The characteristic spectrum has changed the previous identification mode of a single component or main spot. It can be used to identify the authenticity of Chinese medicinal materials, decoction pieces, extracts, and multiple medicinal flavors in preparation prescriptions, as well as to evaluate the authenticity of medicinal materials and the stability of preparation quality; the characteristic spectrum combined with multi-index component quantitative analysis can not only identify the authenticity of the product, but also determine the content of the main characteristic peaks. It is a feasible model for comprehensive control of the quality of Chinese medicine. However, the method of establishing the characteristic spectrum is not the same for different drugs, and different establishment methods may produce large differences in results. For the aforementioned Chinese medicine composition for treating joint pain, the prior art has not yet disclosed a quality control method with better performance. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a quality control method of a traditional Chinese medicine composition for treating arthralgia and its application.

[0008] In one aspect, the present invention provides a method for quality control of a traditional Chinese medicine composition for treating arthralgia.

[0009] Specifically, the Chinese medicine composition comprises: Cynanchum chinense, Angelica dahurica and Licorice; the quality control method of the Chinese medicine composition comprises the following steps:

[0010] (1) Preparation of the test solution: Take the Chinese medicine composition, grind it into powder, add methanol for ultrasonic treatment, then add methanol, filter and obtain the filtrate;

[0011] (2) Preparation of reference solution: accurately weigh the paeonol reference solution and mix it with methanol;

[0012] (3) Determination: Accurately pipette the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the fingerprint.

[0013] More specifically, in step (1), the methanol is 25-75% methanol; the dosage ratio of the Chinese medicine composition to methanol is: 0.5 g: 25-75 mL; and the duration of the ultrasonic treatment is 15-45 minutes.

[0014] In certain specific embodiments of the present invention, the methanol is 50% methanol, the dosage ratio of the traditional Chinese medicine composition to methanol is: 0.5 g: 50 mL; and the duration of the ultrasonic treatment is 30 min.

[0015] More specifically, in step (2), the dosage ratio of the paeonol reference substance to methanol is: 100 μg: 1 mL.

[0016] More specifically, in step (3), the liquid chromatograph uses octadecylsilane bonded silica gel as a filler and acetonitrile-water as a mobile phase.

[0017] Preferably, the volume ratio of the acetonitrile-water mobile phase is: 38-60:40-62.

[0018] In certain specific embodiments of the present invention, the volume ratio of the acetonitrile-water mobile phase is 60:40.

[0019] More specifically, the elution conditions of the liquid chromatograph in step (3) are: isocratic elution, column temperature 25-35° C., flow rate 0.9-1.1 mL / min.

[0020] In certain specific embodiments of the present invention, the elution conditions of the liquid chromatograph are: isocratic elution, column temperature 30° C., flow rate 1.0 mL / min.

[0021] More specifically, in step (3), the detection wavelength used in the determination step is 270-280 nm, and the theoretical plate number is not less than 5000 based on the paeonol peak.

[0022] In certain specific embodiments of the present invention, the detection wavelength used in the determination step is 274 nm.

[0023] Specifically, in the Cynanchum chinense used in this application, paeonol (C 9 H 10 O 3 ) content is not less than 6.28 mg / g.

[0024] Specifically, the preparation steps of the traditional Chinese medicine composition include: extracting 25-35 parts of Cynanchum chinense, 8-12 parts of Angelica dahurica and 2-5 parts of Licorice with alcohol; the alcohol extraction includes extracting 1-3 times with 30-95% ethanol at 50-75°C, each time for 1-2 hours, filtering, combining the filtrate and concentrating to obtain the composition.

[0025] On the other hand, the present invention provides an application of the above-mentioned quality control method in the quality control of a traditional Chinese medicine composition for treating joint pain, characterized in that the traditional Chinese medicine composition comprises: Cynanchum chinense, Angelica dahurica and Licorice.

[0026] Specifically, the similarity between the fingerprint of the test sample and the fingerprint of the reference sample is calculated according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, and a qualified product is one with a similarity greater than 90%.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The quality control method developed by the present invention can control the quality of the product prepared by the method to ensure the stability and controllability of the product quality. At the same time, due to the particularity of the quality control of traditional Chinese medicine products, the quality control method is particularly required to be specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the chromatogram of the test sample in Example 1 with the mobile phase of methanol-water (45:55);

[0030] Figure 2 The chromatogram of the test sample in Example 1 with the mobile phase of acetonitrile-water (45:55);

[0031] Figure 3 The chromatogram of the test sample in Example 1 with the mobile phase of acetonitrile-water (38:62);

[0032] Figure 4 This is the chromatogram of the test sample in Example 1 with the mobile phase of acetonitrile-water (50:50);

[0033] Figure 5 The chromatogram of the test sample in Example 1 with the mobile phase of acetonitrile-water (60:40);

[0034] Figure 6 The chromatogram of the reference substance paeonol in the mobile phase of acetonitrile-water (60:40) in Example 2;

[0035] Figure 7 The chromatogram of the reference substance was used to verify the specificity of the method in Example 4;

[0036] Figure 8 The chromatogram of the test sample was examined for specificity of the methodological verification of Example 4;

[0037] Fig. 9 The specificity investigation for the methodological validation of Example 4 lacks the chromatogram of the negative sample of Cynanchum chinense;

[0038] Fig.10 This is the linear relationship diagram of Example 4. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0040] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary explanation of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0041] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0042] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Products from different manufacturers have no significant effect on the effects.

[0043] The term "peak area" refers to the area occupied by a single chromatographic peak on the chromatogram. It is the product of the chromatographic peak height (response signal) and width (time or distance), and is itself a dimensionless value.

[0044] The term "theoretical plate number" is an important parameter in chromatographic analysis, which comes from the plate theory. The plate theory compares the chromatographic column to a distillation tower, and regards the separation process of components in the chromatographic column as the fractionation process in the distillation tower, that is, the distribution equilibrium process of components within the plate interval. The theoretical plate number is a parameter that represents the separation ability or separation efficiency of the chromatographic column for the sample, usually represented by "N". The larger the N value, the higher the column efficiency, the stronger the separation ability, and the better the separation effect. The theoretical plate number is dimensionless, because it is a ratio, usually without a specific unit.

[0045] The term "Resolution" is a key concept in chromatography, which is used to measure the degree of separation of two adjacent chromatographic peaks in a chromatographic system. Resolution is a dimensionless value that describes the ability of two chromatographic peaks to be distinguished on a chromatogram. Specifically, resolution refers to the ratio of the distance between two adjacent chromatographic peaks to the sum of their peak widths on a chromatogram.

[0046] The calculation formula of separation degree is as follows:

[0047] Where: R is the separation, which is a dimensionless value; t 1 and t 2 are the retention times of the two chromatographic peaks; W 1 and W 2It is the width of two chromatographic peaks at the baseline, usually measured as the standard deviation (σ) or the width of the peaks at the baseline (usually expressed as 4σ or 0.05 times the peak area).

[0048] The prescription of the Chinese medicine composition for treating joint pain of the present invention is as follows: the raw materials of the composition are composed of 25-35 parts of Cynanchum chinense, 8-12 parts of Angelica dahurica and 2-5 parts of Licorice in parts by weight. According to the prescription composition and preparation process, Cynanchum chinense is used as the monarch drug in the prescription, and the main component paeonol is a heat-sensitive component, the content of which can reflect the degree of heating of the material in the production process, so the present invention selects paeonol as the content determination index.

[0049] As an example, a Chinese medicine composition prepared according to the method described in Example 1 of patent CN116763834B was used as a test sample to verify the effects of the following examples.

[0050] Example 1

[0051] Preparation of the test solution: Take 0.1 g of the Chinese medicine composition, accurately weigh it, put it in a 50 mL volumetric flask, add methanol to nearly the scale, ultrasonically treat (power 300 W, frequency 40 KHz) for 30 minutes, cool, add methanol to the scale, shake well, filter, and take the filtrate to obtain the solution.

[0052] Chromatographic conditions: Chromatographic column: Phenomenon Titank C18, particle size 5 μm, 250×4.6 mm, SN: 113AB20418; detection wavelength: 274 nm; column temperature 30°C; flow rate: 1.0 mL / min.

[0053] The mobile phases were composed of methanol: water (45:55), acetonitrile: water (45:55), acetonitrile: water (38:62), acetonitrile: water (50:50), and acetonitrile: water (60:40) in volume ratios, respectively, and the elution was performed isocratically.

[0054] Determination method: Accurately pipette 10 μL of the test solution, inject into the liquid chromatograph, measure, and record the spectrum.

[0055] The measurement results are as follows Figure 1-Figure 5 As shown in the figure, it can be seen from the results that compared with the methanol-water system and the acetonitrile-water system, the peak shape of paeonol measured by the acetonitrile-water system is better and the theoretical plate number is higher. Among the mobile phases with different ratios, the retention time of the paeonol peak measured by acetonitrile-water (60:40) is moderate, and the separation degree with adjacent impurities is better, so acetonitrile-water (60:40) is proposed as the mobile phase.

[0056] Example 2

[0057] Take a sample of the preparation and examine it according to the chromatographic conditions explored in Example 1 to check the applicability of the chromatographic conditions.

[0058] Chromatographic conditions: Chromatographic column: Pheromones Titank C18, particle size 5 μm, 250×4.6 mm, SN: 113AB20422; acetonitrile-water (60:40) as mobile phase; detection wavelength: 274 nm; column temperature 30°C; flow rate: 1.0 mL / min.

[0059] Preparation of reference solution: Take paeonol reference substance (China National Institute for Food and Drug Control, batch number: 110708-201908, content is 99.8%), accurately weigh it, add methanol to make a solution containing 100 μg per 1 mL.

[0060] Determination method: Accurately aspirate 5 μL of reference solution, inject into liquid chromatograph, measure, and record the spectrum.

[0061] The measurement results are as follows Figure 6 As shown in the results, it can be seen that in the reference sample spectrum, the main peak and the adjacent chromatographic peaks are well separated and the peak shape is good, indicating that the chromatographic conditions are suitable for the detection of preparation samples.

[0062] Example 3

[0063] Chromatographic conditions: Chromatographic column: Pheromones Titank C18, particle size 5 μm, 250×4.6 mm, SN: 113AB20422; acetonitrile-water (60:40) as mobile phase; detection wavelength: 274 nm; column temperature 30°C; flow rate: 1.0 mL / min.

[0064] Preparation of reference solution: Take an appropriate amount of paeonol reference substance, weigh it accurately, and add methanol to make a solution containing 100 μg per 1 mL.

[0065] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively and inject into the chromatograph.

[0066] 1. Investigation of the extraction solvent of the test sample

[0067] Preparation of test solution: 4 portions of Chinese medicine composition (batch number: FSDT231009), 0.5 g each, accurately weighed, placed in a conical flask, accurately added 25% methanol, 50% methanol, 75% methanol, 50 mL of methanol respectively, weighed, ultrasonically treated (power 300 W, frequency 40 KHz) for 30 minutes, cooled, weighed, added corresponding extraction solvents to make up for the loss, shaken, filtered, and obtained. The test results are shown in Table 1:

[0068] Table 1 Extraction solvent investigation results

[0069] Extraction solvent 25% methanol 50% methanol 75% Methanol Methanol Content (mg / g) 13.40 13.61 13.59 13.81

[0070] From the experimental results, it can be seen that there is no significant difference in the content of paeonol in the test sample measured by methanol solutions of different concentrations, but when 50% methanol is used as the extraction solvent, the solution is better filtered, so 50% methanol is selected as the extraction solvent.

[0071] 2. Investigation of extraction time of test solution

[0072] Preparation of test solution: 3 portions of Chinese medicine composition (batch number: FSDT231009), 0.5 g each, accurately weighed, placed in a conical flask, accurately added 50 mL of 50% methanol, weighed, ultrasonically treated (power 300 W, frequency 40 KHz) for 15 minutes, 30 minutes, and 45 minutes, cooled, added 50% methanol to make up for the loss, shaken, filtered, and obtained. The test results are shown in Table 2:

[0073] Table 2 Extraction time investigation results

[0074] Extraction time (min) 15 30 45 Content (mg / g) 13.54 13.51 13.57

[0075] The experimental results show that there is no significant difference in the content of paeonol in the test samples measured at different ultrasonic extraction times. Taking into account the differences between batches in subsequent production, the ultrasonic extraction time is selected as 30 minutes.

[0076] 3. Investigation of the amount of solvent for extracting the test solution

[0077] Preparation of test solution: Take 3 portions of Chinese medicine composition (batch number: FSDT231009), 0.5 g each, accurately weigh, place in a conical flask, accurately add 50% methanol 25 mL, 50 mL, 75 mL, weigh, respectively, ultrasonically treat (power 300 W, frequency 40 KHz) for 30 minutes, cool, add 50% methanol to make up for the loss, shake well, filter, and obtain. The test results are shown in Table 3:

[0078] Table 3 Extraction solvent amount investigation results

[0079] Volume (mL) 25 50 75 Content (mg / g) 13.29 13.58 13.63

[0080] From the experimental results, it can be seen that there is no significant difference in the content of paeonol in the test sample measured with different extraction solvent amounts. When the extraction solvent amount is 25mL, the solution is difficult to filter. At the same time, in order to save solvent, the extraction solvent amount is selected to be 50mL.

[0081] Example 4

[0082] 1. Exclusive inspection

[0083] Chromatographic conditions: Chromatographic column: Pheromones Titank C18, particle size 5 μm, 250×4.6 mm, SN: 113AB20422; acetonitrile-water (60:40) as mobile phase; detection wavelength: 274 nm; column temperature 30°C; flow rate: 1.0 mL / min.

[0084] Preparation of reference solution: Take an appropriate amount of paeonol reference substance, weigh it accurately, and add methanol to make a solution containing 100 μg per 1 mL.

[0085] Preparation of test solution: Take 0.5 g of the product, accurately weigh it, put it in a conical flask, accurately add 50 mL of 50% methanol solution, weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes, cool it, weigh it, add 50% methanol solution to make up for the lost weight, shake it well, filter it, and obtain it.

[0086] Preparation of negative sample solution lacking Cynanchum cynanchum: Take 0.5 g of negative sample lacking Cynanchum cynanchum and prepare it in the same way as the test solution to obtain the negative sample solution lacking Cynanchum cynanchum.

[0087] Determination method: Accurately pipette 5 μL of reference solution, test solution and negative sample solution lacking Cynanchum cyrtonema respectively, inject into chromatograph, and the determination result is as follows: Figure 7-Figure 9 As shown, Figure 7 Chromatogram of the reference substance, Figure 8 The chromatogram of the test product is Fig. 9 The chromatogram is for the negative sample of Cynanchum cyrtonema.

[0088] Result analysis: From the experimental results, it can be seen that in the chromatogram of the test sample, the same chromatographic peak was detected at the corresponding position of the paeonol reference substance (retention time 6.817min), and the separation degree between the main peak and the adjacent chromatographic peak was 11.83, which was greater than 1.5; the peak purity matching value was 1000, which was greater than 980; the spectral absorption of the main peak was consistent with that of the reference substance; in the chromatograms of the blank solution and the negative sample, no chromatographic peak was detected at the corresponding retention time of the paeonol peak. In summary, this method has good specificity.

[0089] 2. Instrument precision inspection

[0090] Preparation of reference solution: Take an appropriate amount of paeonol reference substance, weigh it accurately, and add methanol to make a solution containing 100 μg per 1 mL.

[0091] Determination method: Accurately draw 5 μL of the reference solution, inject it into the chromatograph, and inject 6 times continuously. The determination results are shown in Table 4:

[0092] Table 4 Instrument precision results

[0093]

[0094] The experimental results show that after 6 consecutive injections, the RSD of the paeonol peak area is less than 2.0%, and the retention time is less than 1.0%, indicating that the method has good precision.

[0095] 3. Linear relationship investigation

[0096] Preparation of reference stock solution: Take 12.5 mg of paeonol reference substance, weigh accurately, place in a 25 mL volumetric flask, add methanol to dissolve and dilute to scale, shake well, and obtain. (500 μg / mL)

[0097] Preparation of reference solution ①: Accurately pipette 2 mL of reference stock solution into a 50 mL volumetric flask, add methanol to dilute to the mark, shake well, and the solution is ready. (20 μg / mL)

[0098] Preparation of reference solution ②: Accurately pipette 2 mL of reference stock solution into a 20 mL volumetric flask, add methanol to dilute to the mark, shake well, and the solution is ready. (50 μg / mL)

[0099] Preparation of reference solution ③: Accurately pipette 2 mL of reference stock solution into a 10 mL volumetric flask, add methanol to dilute to the mark, shake well, and the solution is ready. (100 μg / mL)

[0100] Preparation of reference solution ④: Accurately pipette 2 mL of reference stock solution into a 5 mL volumetric flask, add methanol to dilute to the mark, shake well, and the solution is ready. (200 μg / mL)

[0101] Preparation of reference solution ⑤: Accurately pipette 3 mL of reference stock solution into a 5 mL volumetric flask, add methanol to dilute to the mark, shake well, and the solution is ready. (300 μg / mL)

[0102] Take the reference substance stock solution as reference substance solution ⑥. (500μg / mL)

[0103] Determination method: Accurately pipette 5 μL of reference solution ①-⑥ respectively, inject into liquid chromatograph, record each chromatogram, use concentration (μg / mL) as abscissa (X), peak area as ordinate (Y), draw a linear relationship graph, record regression equation, correlation coefficient (r). The determination results are shown in Table 5 and Fig.10 As shown:

[0104] Table 5 Paeonol linearity results

[0105] Reference solution Concentration μg / mL Average peak area Reference solution① 19.9919 500416 Reference solution② 49.9798 1260730 Reference solution ③ 99.9597 2524232 Reference solution④ 199.9194 5014530 Reference solution⑤ 299.8790 7554002 Reference solution ⑥ 499.7984 12552179

[0106] From the experimental results, it can be seen that the regression equation of the paeonol reference substance is y=25117.7267x+5032.8146, and the correlation coefficient r is 1.0000 greater than 0.9990, indicating that the paeonol reference substance has a good linear relationship in the range of 19.9919μg / mL-499.7984μg / mL.

[0107] Example 5

[0108] 1. Repeatability test

[0109] Preparation of reference solution: Take an appropriate amount of paeonol reference substance, weigh it accurately, and add methanol to make a solution containing 100 μg per 1 mL.

[0110] Preparation of test solution: Take 6 portions of the product (batch number: FST231009), 0.5 g each, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of 50% methanol, weigh the weight, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, cool, weigh the weight, add 50% methanol to make up for the loss, shake well, filter, and obtain.

[0111] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into the chromatograph, and determine as shown in Table 6.

[0112] Calculation formula:

[0113]

[0114] Where: c 对 is the concentration of the reference solution (μg / mL); A 对 A is the peak area of ​​the reference solution; 样 is the main peak area of ​​the test solution; V 稀 is the dilution factor of the test solution; is the average correction factor; M 样 is the sample size of the test product (g).

[0115] Table 6 Repeatability test results

[0116]

[0117] The content of paeonol in this product is lower than 100 mg / g. According to the method validation guidelines, the repeatability RSD should not exceed 2%. The average content of paeonol in 6 test solutions is 14.071 mg / g, and the RSD% is 0.26%, which is less than 2.0%, indicating that the method has good repeatability.

[0118] 2. Intermediate precision test

[0119] Different personnel used different brands of instruments (Thermo U3000 high performance liquid chromatograph) at different times to conduct intermediate precision tests on the content of paeonol according to the above repeatability test, and calculated the RSD% of 12 sets of repeatability data. The test results are shown in Tables 7 and 8:

[0120] Table 7 Intermediate precision test results

[0121]

[0122]

[0123] Table 8 Repeatability and intermediate precision results of 12 samples

[0124]

[0125] The paeonol content of this product is lower than 100 mg / g. According to the method validation guidelines, the repeatability RSD should not exceed 2%, and the intermediate precision RSD should not exceed 4%. The RSD% of the paeonol content of the 6 test samples was 0.23%, which was less than 2.0%; the repeatability and intermediate precision RSD% of the paeonol content of the 12 test samples was 0.38%, which was less than 4.0%, indicating that the intermediate precision of this method was good.

[0126] 3. Accuracy test

[0127] Preparation of reference solution: Take an appropriate amount of paeonol reference substance, weigh it accurately, and add methanol to make a solution containing 100 μg per 1 mL.

[0128] Preparation of the reference solution for recovery: Take 27 mg of paeonol reference substance, accurately weigh it, place it in a 200 mL volumetric flask, add 50% methanol to dissolve and dilute to the scale, shake well, and you have it. (0.135 mg / mL)

[0129] Preparation of test solution: Take 6 portions of this product (batch number: FST231009), 0.25g each, accurately weigh, place in a stoppered conical flask, accurately add 25mL of recovery reference solution, then accurately add 25mL of 50% methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40KHz) for 30 minutes, cool, weigh the weight, add 50% methanol to make up for the lost weight, shake well, filter, and obtain.

[0130] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into the chromatograph, and the determination results are shown in Table 9.

[0131] The calculation formula is the same as that in the repeatability test.

[0132] Table 9 Accuracy test results

[0133]

[0134] The experimental results show that the recovery rate and average recovery rate of the six test solutions are both in the range of 92%-105%, and the RSD% is less than 2.0%, which meets the requirements according to the method validation guidelines. The results show that the method has good accuracy.

[0135] Example 6

[0136] 1. Stability study of test solution and reference solution

[0137] According to the test method of the repeatability test in Example 5, the test solution and the paeonol reference solution were sampled and measured at 0, 5, 10, 15, 20, 25, 30, and 35 hours, respectively, and the RSD (%) of the paeonol peak area was calculated. The test results are shown in Tables 10 and 11:

[0138] Table 10 Test solution stability results

[0139]

[0140]

[0141] The experimental results show that the peak area RSD% of the test solution within 35 hours is less than 2.0%, indicating that the test solution is stable within 35 hours.

[0142] Table 11 Paeonol reference solution stability results

[0143]

[0144] The experimental results show that the peak area RSD% of the paeonol reference solution within 35 hours is less than 2.0%, indicating that the paeonol reference solution is stable within 35 hours.

[0145] 2. Column temperature durability investigation

[0146] According to the test method of the repeatability test in Example 5, the influence of different column temperatures (25°C and 35°C) on the determination results was investigated, and compared with the average content results of 12 samples in the repeatability test and the intermediate precision test. The determination results are shown in Table 12:

[0147] Table 12 Column temperature durability results

[0148] temperature Average content (mg / g) Separation degree from adjacent impurity peaks RD% 30℃ 14.030 10.38 / 35℃ 13.924 9.79 0.38 25℃ 13.975 10.47 0.20

[0149] The experimental results show that when the column temperature is 25℃ and 35℃, the separation degree between the paeonol peak and the adjacent impurity peak in the chromatogram of the test sample is greater than 1.5, and the RD% of the paeonol content in the test sample between 25℃ and 30℃, and between 30℃ and 35℃ is less than 3.0%, indicating that the column temperature durability of this method is good.

[0150] 3. Flow rate durability inspection

[0151] According to the test method of the repeatability test in Example 5, the influence of different flow rates (0.9 mL / min and 1.1 mL / min) on the determination results was investigated, and compared with the average content results of 12 samples in the repeatability test and the intermediate precision test. The determination results are shown in Table 13:

[0152] Table 13 Flow rate durability results

[0153] Flow rate (mL / min) Average content (mg / g) Separation degree from adjacent impurity peaks RD% 1.0 14.030 10.33 / 0.9 13.924 10.42 0.38 1.1 13.841 10.16 0.68

[0154] The experimental results show that the separation degree between the paeonol peak and the adjacent impurity peaks in the chromatogram of the test sample at flow rates of 0.9 mL / min and 1.1 mL / min is greater than 1.5, and the RD% of the paeonol content at flow rates of 0.9 mL / min and 1.0 mL / min, and 1.0 mL / min and 1.1 mL / min is less than 3.0%, indicating that the flow rate durability of this method is good.

[0155] 4. Column durability inspection

[0156] According to the test method of the repeatability test in Example 5, the influence of different chromatographic columns on the determination results was investigated, and the average content results of 12 samples in the repeatability test and the intermediate precision test were compared. The determination results are shown in Table 14:

[0157] Table 14 Chromatographic column durability test results

[0158]

[0159] Note: Phenomenon Titank C18 column number: 113AB20422 is the chromatographic column used for verification experiments.

[0160] The experimental results show that the separation degree between the paeonol peak and the adjacent impurity peaks in the chromatogram of the test sample in chromatographic columns of different batches and different brands is greater than 1.5, and the RD% of the paeonol content measured by chromatographic columns of different batches and different brands is less than 3.0%, indicating that the chromatographic column of this method has good durability.

[0161] 5. Mobile phase ratio durability test

[0162] According to the test method of the repeatability test in Example 5, the influence of different mobile phase ratios (acetonitrile-water (58:42), acetonitrile-water (62:38)) on the determination results was investigated, and compared with the average content results of 12 samples in the repeatability test and the intermediate precision test. The determination results are shown in Table 15:

[0163] Table 15 Mobile phase ratio durability test results

[0164] Chromatographic columns Average content (mg / g) Separation degree from adjacent impurity peaks RD% Acetonitrile-water (60:40) 14.030 10.33 / Acetonitrile-water (58:42) 13.844 10.73 0.67 Acetonitrile-water (62:38) 13.870 9.62 0.57

[0165] From the experimental results, it can be seen that the separation degrees of the paeonol peak and the adjacent impurity peaks in the chromatogram of the test sample at flow rates of acetonitrile-water (58:42) and acetonitrile-water (62:38) are both greater than 1.5, and the RD% of the paeonol content in the flow rates of acetonitrile-water (58:42) and acetonitrile-water (60:40), and acetonitrile-water (60:40) and acetonitrile-water (62:38) are all less than 3.0%, indicating that the mobile phase ratio of this method has good durability.

[0166] 6. Determination of content of multiple batches of samples

[0167] Take the pilot batch samples and test them according to the test method of the repeatability test in Example 5. The test results are shown in Table 16:

[0168] Table 16 Results of content determination of multiple batches of samples

[0169] batch number FSDT240221 FSDT240222 FSDT240223 Paeonol content (mg / g) 13.42 13.69 13.68

[0170] From the results, it can be seen that the average content of multiple batches of pilot samples of this product is 13.60 mg / g.

[0171] Example 7

[0172] According to the results of the above multiple batches of samples, the average content of Cynanchum chinense in this product is 13.60 mg / g in terms of paeonol. Considering that the current cumulative number of batches is small, the sources and origins of the Cynanchum chinense medicinal materials used in large-scale production are different, resulting in uneven content of Cynanchum chinense. Therefore, the content limit of this product is determined as follows: the content of Cynanchum chinense medicinal materials per gram × the limit of paeonol content in Cynanchum chinense × transfer rate × 80%. In the 2020 edition of the Chinese Pharmacopoeia, Cynanchum chinense is calculated as a dry product, and the total content limit of paeonol in Cynanchum chinense is 1.3%. The standard stipulates that the moisture content of Cynanchum chinense shall not exceed 15.0%. Therefore, the content limit of paeonol not calculated as a dry product is 1.3% × (1-15.0%) = 1.10%. The average transfer rate of paeonol is 105%, and each gram of Cynanchum chinense contains 0.68g / g. Therefore, the content limit of this product is calculated as follows: 1.10%×0.68g / g×105%×80%=6.28mg / g. Therefore, the content limit of paeonol (C 9 H 10 O 3 ) content shall not be less than 6.28 mg.

[0173] The above tests show that the present application has good stability, accuracy and precision, the measurement results are accurate and reliable, and can effectively evaluate the product quality of the traditional Chinese medicine composition for treating joint pain.

[0174] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A quality control method for a Chinese medicine composition for treating arthralgia, characterized in that: The Chinese medicine composition comprises: Cynanchum chinense, Angelica dahurica and Licorice; The quality control method comprises the following steps: (1) Preparation of the test solution: Take the Chinese medicine composition, grind it into powder, add methanol for ultrasonic treatment, then add methanol, filter and obtain the filtrate; (2) Preparation of reference solution: accurately weigh the paeonol reference solution and mix it with methanol; (3) Determination: Accurately pipette the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the fingerprint.

2. The quality control method according to claim 1, characterized in that: In step (1), the volume concentration of methanol is 25%-75%; the dosage ratio of the Chinese medicine composition to methanol is: 0.5g:25-75mL; and the duration of the ultrasonic treatment is 15-45 minutes.

3. The quality control method according to claim 1, characterized in that: The dosage ratio of the paeonol reference substance to methanol in step (2) is: 100 μg: 1 mL.

4. The quality control method according to claim 1, characterized in that: The liquid chromatograph described in step (3) uses octadecylsilane bonded silica gel as a filler; and the mobile phase is acetonitrile-water mobile phase.

5. The quality control method according to claim 4, characterized in that: The volume ratio of the acetonitrile-water mobile phase described in step (3) is: 38-60:40-62.

6. The quality control method according to claim 1, characterized in that: The elution conditions of the liquid chromatograph described in step (3) are: isocratic elution, column temperature 25-35° C., and flow rate 0.9-1.1 mL / min.

7. The quality control method according to claim 1, characterized in that: In step (3), the detection wavelength used in the determination step is 270-280 nm, and the theoretical plate number is not less than 5000 based on the paeonol peak.

8. The quality control method according to claim 1, characterized in that: The content of paeonol in the Cynanchum wilfordii is not less than 6.28 mg / g.

9. The quality control method according to claim 1, characterized in that: The preparation steps of the Chinese medicine composition include: extracting 25-35 parts of Cynanchum chinense, 8-12 parts of Angelica dahurica and 2-5 parts of Licorice root with alcohol; the alcohol extraction includes extracting 1-3 times with 30%-95% ethanol at 50-75°C, each time for 1-2h, filtering, combining the filtrate and concentrating to obtain the composition.

10. Application of the quality control method according to any one of claims 1 to 9 in the quality control of a traditional Chinese medicine composition for treating arthralgia, characterized in that: The traditional Chinese medicine composition comprises: Cynanchum chinense, Angelica dahurica and Licorice.

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