Quality control method of traditional Chinese medicine composition

Abstract

The invention provides a quality control method of a traditional Chinese medicine composition, and belongs to the technical field of quality control of traditional Chinese medicine products. The method comprises the following steps: (1) mixing a traditional Chinese medicine composition with methanol, and carrying out ultrasonic treatment to obtain a test solution; (2) mixing ginsenoside Rb1 with methanol to obtain a reference solution; and (3) respectively measuring the fingerprints of the test solution obtained in the step (1) and the reference solution obtained in the step (2) by a liquid chromatography, and if the similarity of the fingerprints of the test solution and the reference solution is greater than or equal to 0.90, judging that the quality of the traditional Chinese medicine composition is qualified, and if the similarity of the fingerprints of the test solution and the reference solution is greater than or equal to 0.90, judging that the quality of the traditional Chinese medicine composition is qualified. The traditional Chinese medicine composition in the step (1) is composed of paecilomyces hepialid mycelium, ganoderma lucidum, ganoderma lucidum spore powder and American ginseng. The method provided by the invention has good repeatability, precision and stability, and can be used for comprehensively controlling the quality of the traditional Chinese medicine composition.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of traditional Chinese medicine products, and particularly to a quality control method for a traditional Chinese medicine composition. Background Art

[0002] Traditional Chinese medicine believes that: "When healthy qi exists inside the body, pathogenic factors cannot invade." The healthy qi of the human body includes qi, blood, yin, yang, etc., which can warm and nourish the human body and at the same time resist external pathogenic factors. Traditional Chinese medicine can regulate the immune function of the body by tonifying the healthy qi of the body. The traditional Chinese medicine composition provided by the Chinese patent "CN102228494B A traditional Chinese medicine composition for strengthening the healthy qi and consolidating the root, enhancing human immunity, anti-fatigue, anti-tumor, and delaying aging" obtained by the main inventor of the present application is composed of Paecilomyces hepiali mycelium (cordyceps mycelium, a substitute for Cordyceps sinensis), Ganoderma lucidum, broken-wall Ganoderma lucidum spores, and American ginseng. American ginseng in the formula has the effects of replenishing primordial qi, lung qi, spleen qi, heart qi, and kidney qi. At the same time, it can nourish yin and promote the production of body fluid, and is tonifying without being dry and hot; Ganoderma lucidum and Ganoderma lucidum spores have the effects of tonifying heart qi, nourishing heart blood, calming the mind, tonifying lung qi, regulating phlegm cough, strengthening the spleen and stomach, treating consumptive fatigue, and preventing aging. Cordyceps mycelium has the effects of tonifying lung qi, nourishing lung yin, benefiting kidney qi, and tonifying liver and kidney. When used together, these ingredients can tonify the five zang-organs, benefit primordial qi, tonify lung qi, spleen qi, heart qi, and liver and kidney; promote the production of body fluid, nourish heart blood, and calm the mind. The traditional Chinese medicine composition can strengthen the healthy qi and consolidate the root, enhance human immunity, anti-fatigue, anti-tumor, and delay aging by tonifying the qi of the zang-organs, tonifying qi and yin evenly, and resisting external pathogenic factors.

[0003] However, only the formula and preparation method of the traditional Chinese medicine composition are provided in the above patent document, and the content of how to effectively and comprehensively control the quality of this product is not involved. Based on the particularity of the quality control of traditional Chinese medicine products, providing a quality control method exclusive to this traditional Chinese medicine composition is an inevitable requirement to ensure the stability and controllability of product quality. Summary of the Invention

[0004] The purpose of the present invention is to provide a quality control method for a traditional Chinese medicine composition, which has good repeatability, precision, and stability, and can be used to comprehensively control the quality of the traditional Chinese medicine composition.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a quality control method for a traditional Chinese medicine composition, and the steps are as follows:

[0007] (1) Mix the traditional Chinese medicine composition with methanol and ultrasonicate to obtain a test solution.

[0008] (2) Mix ginsenoside Rb1 with methanol to obtain a reference solution.

[0009] (3) Determine the fingerprint spectra of the test solution obtained in step (1) and the reference solution obtained in step (2) by high performance liquid chromatography. If the similarity between the fingerprint spectra of the test solution and the reference solution is ≥ 0.90, it is determined that the quality of the traditional Chinese medicine composition is qualified.

[0010] The traditional Chinese medicine composition described in step (1) is composed of Paecilomyces hepiali mycelia, Ganoderma lucidum, Ganoderma lucidum spores, and American ginseng.

[0011] Preferably, the mass ratio of Paecilomyces hepiali mycelia, Ganoderma lucidum, Ganoderma lucidum spores, and American ginseng is 1 - 8:2 - 12:1 - 8:1 - 10.

[0012] Preferably, the mass percentage content of methanol in step (1) is 50 - 100%.

[0013] Preferably, the power of the ultrasonic wave in step (1) is 240 - 260W, the frequency of the ultrasonic wave is 30 - 50KHz, and the time of the ultrasonic wave is 20 - 40min.

[0014] Preferably, the mass percentage content of methanol in step (2) is 50 - 100%; the concentration of ginsenoside Rb1 in the reference solution is 0.4 - 0.6mg / mL.

[0015] Preferably, the injection volume of the test solution and the reference solution in step (3) is 5 - 15μL.

[0016] Preferably, the filler used in the determination by high performance liquid chromatography in step (3) is octadecylsilane chemically bonded silica, the column length of the chromatographic column used in the determination by high performance liquid chromatography is 250mm, the column inner diameter is 4.6mm, and the particle size is 5μm.

[0017] Preferably, in the determination by high performance liquid chromatography in step (3), acetonitrile is used as mobile phase A and phosphoric acid is used as mobile phase B, and the mass percentage content of phosphoric acid is 0.05 - 0.15%.

[0018] More preferably, the gradient elution conditions set in the determination by high performance liquid chromatography in step (3) are: 0 - 25min, 19 - 20% mobile phase A, 81 - 80% mobile phase B; 25 - 60min, 20 - 40% mobile phase A, 80 - 60% mobile phase B; 60 - 90min, 40 - 55% mobile phase A, 60 - 45% mobile phase B; 90 - 100min, 55 - 60% mobile phase A, 45 - 40% mobile phase B; 100.1 - 110min, 19% mobile phase A, 81% mobile phase B.

[0019] Preferably, the wavelength of the liquid chromatography measurement in step (3) is 200-205 nm, the column temperature is 38-42° C., and the flow rate is 0.8-1.2 mL / min.

[0020] The beneficial effects of the present invention compared with the prior art are:

[0021] 1. The present invention uses wavelength screening to make the peak area of ​​the final chromatographic peak high, the separation degree good, and the baseline stable; the preparation method of the test sample is determined by screening the extraction solvent, extraction method, extraction time and sample weight; by investigating its system applicability, repeatability, stability and durability, it is concluded that the detection method has good sensitivity, accuracy and precision, and the measurement results are accurate and reliable, which can effectively evaluate the product quality of the Chinese medicine composition provided in the Chinese patent "CN102228494B A Chinese medicine composition for strengthening the body, enhancing human immunity, resisting fatigue, resisting tumors, and delaying aging".

[0022] 2. The present invention selects 8 chromatographic peaks based on the chromatographic peak area and separation degree for evaluating the quality of the Chinese medicine composition. The selected 8 chromatographic peaks are conducive to comprehensive monitoring of the quality of the Chinese medicine composition product, avoiding the singleness and one-sidedness of quality control, and are of great significance for controlling the quality of the Chinese medicine composition and ensuring clinical efficacy.

[0023] 3. The present invention verifies the quality control method provided by the present invention through a number of experiments. The results show that the method provided by the present invention has good repeatability, precision and stability, and can be used to determine whether the quality of the Chinese medicine composition is qualified and comprehensively control the quality of the Chinese medicine composition, thereby ensuring the clinical effect of the Chinese medicine composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0025] Figure 1 It is the fingerprint spectrum measured at 203nm;

[0026] Figure 2 It is the fingerprint spectrum measured at 220nm;

[0027] Figure 3 It is the fingerprint spectrum measured at 254nm;

[0028] Figure 4 It is the fingerprint spectrum measured at 280nm;

[0029] Figure 5 It is the fingerprint obtained after extraction with methanol with a mass percentage content of 30%.

[0030] Figure 6 It is the fingerprint obtained after extraction with methanol with a mass percentage content of 100%.

[0031] Figure 7 It is the fingerprint obtained during reflux extraction.

[0032] Figure 8 It is the fingerprint obtained when ultrasonic extraction is carried out for 20 min.

[0033] Figure 9 It is the fingerprint obtained when ultrasonic extraction is carried out for 40 min.

[0034] Figure 10 It is the fingerprint measured when 0.3003 g of the traditional Chinese medicine composition is used.

[0035] Figure 11 It is the fingerprint measured when 0.6001 g of the traditional Chinese medicine composition is used.

[0036] Figure 12 It is the control fingerprint obtained according to the method in Example 1, wherein 2(S) is ginsenoside Rb1, and 1, 3, 4, 5, 6, 7, 8 are all identification peaks of the fingerprint. Detailed implementation manners

[0037] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0041] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are meant to include but not be limited to.

[0042] The present invention provides a method for quality control of a traditional Chinese medicine composition, and the steps are as follows:

[0043] (1) Mix the traditional Chinese medicine composition with methanol and ultrasonicate to obtain a test solution.

[0044] (2) Mix ginsenoside Rb1 with methanol to obtain a reference solution.

[0045] (3) Use liquid chromatography to separately determine the fingerprint spectra of the test solution obtained in step (1) and the reference solution obtained in step (2). If the similarity between the fingerprint spectra of the test solution and the reference solution is ≥ 0.90, it is determined that the quality of the traditional Chinese medicine composition is qualified.

[0046] In the present invention, the traditional Chinese medicine composition in step (1) is composed of Paecilomyces hepiali mycelia, Ganoderma lucidum, Ganoderma lucidum spore powder, and American ginseng; the traditional Chinese medicine composition is preferably the traditional Chinese medicine composition provided in the Chinese patent "CN102228494B A Traditional Chinese Medicine Composition for Strengthening the Vital Qi and Consolidating the Constitution, Enhancing Human Immunity, Anti-Fatigue, Anti-Tumor, and Anti-Aging"; the mass ratio of Paecilomyces hepiali mycelia, Ganoderma lucidum, Ganoderma lucidum spore powder, and American ginseng is preferably 1-8:2-12:1-8:1-10, more preferably 3-6:5-10:3-6:2-8, still more preferably 4-5:6-8:4-5:4-6, and even more preferably 4.5:7:4.5:5; the drug composed of Paecilomyces hepiali mycelia, Ganoderma lucidum, Ganoderma lucidum spore powder, and American ginseng preferably has the effects of strengthening the vital qi and consolidating the constitution, enhancing human immunity, anti-fatigue, anti-tumor, and anti-aging; the mass percentage content of methanol is preferably 50-100%, more preferably 60-80%, and still more preferably 70%; the power of the ultrasonic wave is preferably 240-260W, more preferably 245-255W, and still more preferably 250W; the frequency of the ultrasonic wave is preferably 30-50KHz, more preferably 35-45KHz, and still more preferably 40KHz; the time of the ultrasonic wave is preferably 20-40min, more preferably 25-35min, and still more preferably 30min; after the ultrasonic wave, filtration is preferably carried out, and when filtering, membrane filtration is preferably used, and the pore size of the membrane is preferably 0.4-0.5μm, more preferably 0.45μm.

[0047] In the present invention, the mass percentage content of methanol in step (2) is preferably 50-100%, more preferably 60-80%, and still more preferably 70%; the concentration of ginsenoside Rb1 in the reference substance solution is preferably 0.4-0.6mg / mL, more preferably 0.5mg / mL.

[0048] In the present invention, the injection volume of the test solution and the reference solution in step (3) is preferably 5 - 15 μL, more preferably 8 - 12 μL, and even more preferably 10 μL; the filler used in the determination by liquid chromatography is preferably octadecylsilyl silica gel; the chromatographic column used in the determination by liquid chromatography is COSMOSIL C18-MS-II; the column length of the chromatographic column used in the determination by liquid chromatography is preferably 250 mm, the column inner diameter is preferably 4.6 mm, and the particle size is preferably 5 μm; in the determination by liquid chromatography, acetonitrile is preferably used as mobile phase A and phosphoric acid is used as mobile phase B, and the mass percentage content of the phosphoric acid is preferably 0.05 - 0.15%, more preferably 0.08 - 0.12%, and even more preferably 0.10%; the gradient elution conditions set in the determination by liquid chromatography are preferably: 0 - 25 min, 19 - 20% mobile phase A, 81 - 80% mobile phase B; 25 - 60 min, 20 - 40% mobile phase A, 80 - 60% mobile phase B; 60 - 90 min, 40 - 55% mobile phase A, 60 - 45% mobile phase B; 90 - 100 min, 55 - 60% mobile phase A, 45 - 40% mobile phase B; 100.1 - 110 min, 19% mobile phase A, 81% mobile phase B; more preferably 0 - 25 min, 19.5% mobile phase A, 80.5% mobile phase B; 25 - 60 min, 25 - 35% mobile phase A, 75 - 65% mobile phase B; 60 - 90 min, 45 - 50% mobile phase A, 55 - 50% mobile phase B; 90 - 100 min, 58% mobile phase A, 42% mobile phase B; 100.1 - 110 min, 19% mobile phase A, 81% mobile phase B; even more preferably 0 - 25 min, 19.5% mobile phase A, 80.5% mobile phase B; 25 - 60 min, 30% mobile phase A, 70% mobile phase B; 60 - 90 min, 48% mobile phase A, 52% mobile phase B; 90 - 100 min, 58% mobile phase A, 42% mobile phase B; 100.1 - 110 min, 19% mobile phase A, 81% mobile phase B; the wavelength for the determination by liquid chromatography is preferably 200 - 205 nm, more preferably 202 - 204 nm, and even more preferably 203 nm; the column temperature is preferably 38 - 42 °C, more preferably 40 °C; the flow rate is preferably 0.8 - 1.2 mL / min, more preferably 1.0 mL / min; after the determination by liquid chromatography, it is preferably to use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to calculate the similarity of the fingerprints of the two solutions.

[0049] Example 1

[0050] A method for quality control of a traditional Chinese medicine composition, the steps are as follows:

[0051] (1) Weigh 0.60 g of the traditional Chinese medicine composition provided in the Chinese patent "CN102228494B A traditional Chinese medicine composition for strengthening the healthy qi and consolidating the root, enhancing human immunity, resisting fatigue, anti-tumor, and delaying aging", place it in a 25 ml volumetric flask, add 50% methanol to make up the volume, ultrasonicate at 250 W and 40 KHz for 30 min, cool, add 50% methanol to make up the volume, shake well, and filter through a 0.45 μm filter membrane to obtain the test solution;

[0052] (2) Mix ginsenoside Rb1 with 100% methanol to obtain a reference solution with a concentration of 0.5 mg / mL of ginsenoside Rb1;

[0053] (3) Inject the test solution obtained in step (1) and the reference solution obtained in step (2) into the liquid chromatograph with an injection volume of 10 μL respectively. Use octadecylsilane chemically bonded silica gel as the filler, use COSMOSIL C18-MS-II as the chromatographic column (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm), use acetonitrile as mobile phase A, and 0.1% phosphoric acid as mobile phase B. The gradient elution conditions are set as follows: 0 - 25 min, 19 - 20% mobile phase A, 81 - 80% mobile phase B; 25 - 60 min, 20 - 40% mobile phase A, 80 - 60% mobile phase B; 60 - 90 min, 40 - 55% mobile phase A, 60 - 45% mobile phase B; 90 - 100 min, 55 - 60% mobile phase A, 45 - 40% mobile phase B; 100.1 - 110 min, 19% mobile phase A, 81% mobile phase B. Measure the fingerprint of the above solutions at a wavelength of 203 nm, column temperature of 40 °C, and flow rate of 1 mL / min. Use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to calculate the similarity of the fingerprints of the two solutions. If the similarity of the fingerprints of the test solution and the reference solution ≥ 0.90, it is determined that the quality of the traditional Chinese medicine composition is qualified.

[0054] Example 2

[0055] A method for quality control of a traditional Chinese medicine composition, the steps are as follows:

[0056] (1) Weigh 0.60 g of the traditional Chinese medicine composition provided in the Chinese patent "CN102228494B A traditional Chinese medicine composition for strengthening the healthy qi and consolidating the root, enhancing human immunity, resisting fatigue, anti-tumor, and delaying aging", place it in a 25 ml volumetric flask, add 100% methanol to make up the volume, ultrasonicate at 240 W and 30 KHz for 40 min, cool, add 100% methanol to make up the volume, shake well, and filter through a 0.4 μm filter membrane to obtain the test solution;

[0057] (2) Mix ginsenoside Rb1 with 100% methanol to obtain a reference solution with a concentration of 0.4 mg / mL of ginsenoside Rb1;

[0058] (3) Inject the test solution obtained in step (1) and the reference solution obtained in step (2) into the liquid chromatograph with an injection volume of 15 μL respectively. Use octadecylsilane chemically bonded silica gel as the filler, with a column length of 250 mm, a column inner diameter of 4.6 mm, and a particle size of 5 μm. Use acetonitrile as mobile phase A and 0.05% phosphoric acid as mobile phase B. The gradient elution conditions are set as follows: 0 - 25 min, 19 - 20% mobile phase A, 81 - 80% mobile phase B; 25 - 60 min, 20 - 40% mobile phase A, 80 - 60% mobile phase B; 60 - 90 min, 40 - 55% mobile phase A, 60 - 45% mobile phase B; 90 - 100 min, 55 - 60% mobile phase A, 45 - 40% mobile phase B; 100.1 - 110 min, 19% mobile phase A, 81% mobile phase B. Measure the fingerprint of the above solutions at a wavelength of 200 nm, a column temperature of 38 °C, and a flow rate of 0.8 mL / min. Use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to calculate the similarity of the fingerprints of the two solutions. If the similarity of the fingerprints of the test solution and the reference solution is ≥ 0.90, it is determined that the quality of the traditional Chinese medicine composition is qualified.

[0059] Example 3

[0060] A quality control method for a traditional Chinese medicine composition, the steps are as follows:

[0061] (1) Weigh 0.60 g of the traditional Chinese medicine composition provided in the Chinese patent "CN102228494B A traditional Chinese medicine composition for strengthening the body resistance, enhancing human immunity, anti - fatigue, anti - tumor, and delaying aging", place it in a 25 - ml volumetric flask, add 60% methanol to make up the volume, sonicate at 260 W and 50 KHz for 20 min, cool, add 60% methanol to make up the volume, shake well, and filter through a 0.5 - μm filter membrane to obtain the test solution;

[0062] (2) Mix ginsenoside Rb1 with 60% methanol to obtain a reference solution with a concentration of 0.6 mg / mL of ginsenoside Rb1;

[0063] (3) Inject the test solution obtained in step (1) and the reference solution obtained in step (2) into the liquid chromatograph with an injection volume of 5 μL respectively. Use octadecylsilane chemically bonded silica gel as the filler, with a column length of 250 mm, a column inner diameter of 4.6 mm, and a particle size of 5 μm. Use acetonitrile as mobile phase A and 0.15% phosphoric acid as mobile phase B. The gradient elution conditions are set as follows: 0 - 25 min, 19 - 20% mobile phase A, 81 - 80% mobile phase B; 25 - 60 min, 20 - 40% mobile phase A, 80 - 60% mobile phase B; 60 - 90 min, 40 - 55% mobile phase A, 60 - 45% mobile phase B; 90 - 100 min, 55 - 60% mobile phase A, 45 - 40% mobile phase B; 100.1 - 110 min, 19% mobile phase A, 81% mobile phase B. Measure the fingerprint spectra of the above solutions at a wavelength of 205 nm, a column temperature of 42 °C, and a flow rate of 1.2 mL / min. Use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint spectra to calculate the similarity of the fingerprint spectra of the two solutions. If the similarity of the fingerprint spectra of the test solution and the reference solution is ≥ 0.90, it is determined that the quality of the traditional Chinese medicine composition is qualified.

[0064] Experimental Example 1 Selection of Detection Wavelength

[0065] Prepare the test solution according to the method in Example 1, perform full-wavelength scanning using a diode array detector (DAD detector), and compare the chromatographic information at wavelengths of 203 nm, 220 nm, 254 nm, and 280 nm. The results are as Figures 1 to 4 shown.

[0066] As Figures 1 to 4 can be seen, the chromatographic peaks at 203 nm are rich, and the resolution and peak shape of the chromatographic peaks are better than those at other wavelengths. Therefore, 203 nm is selected as the detection wavelength.

[0067] Experimental Example 2 Selection of Extraction Solvent

[0068] According to the method in Example 1, measure the peak areas of 8 labeled peaks of the test solution (i.e., the traditional Chinese medicine composition) fixed with "50% methanol", "30% methanol", and "100% methanol" in step (1) respectively. The results are shown in Table 1 and Figure 5 、 Figure 6 shown.

[0069] Table 1 Peak Areas Measured by Different Extraction Solvents

[0070]

[0071] The results showed that the fingerprint spectra obtained by ultrasonic extraction with 30%, 50% and 100% methanol presented basically the same substance information, but the peak areas of each peak were more balanced and stable when extracted with 50% methanol. Therefore, 50% methanol was selected for extraction.

[0072] Test Example 3 Selection of Extraction Method

[0073] According to the method in Example 1, the peak areas of 8 labeled peaks of the test solution were measured respectively when extracted by "ultrasonic wave at 250W, 40KHz for 30min" and "heating under reflux at 80±2°C for 60min", and the relative standard deviation (RSD) of different labeled peaks was calculated. The results are shown in Table 2 and Figure 7 as follows.

[0074] Table 2 Peak Areas and Relative Standard Deviations Measured by Different Extraction Methods

[0075]

[0076] The results showed that the fingerprint spectra obtained by ultrasonic extraction and reflux extraction presented basically the same substance information, and the difference in peak areas of each peak was not significant (the relative standard deviation RSD was less than 3%). Considering that the ultrasonic extraction method is more convenient, the extraction method was selected as ultrasonic extraction.

[0077] Test Example 4 Selection of Extraction Time

[0078] According to the method in Example 1, the peak areas of 8 labeled peaks of the test solution were measured respectively with ultrasonic times of "30min", "20min" and "40min", and the relative standard deviation of each peak was calculated. The results are shown in Table 3 and Figures 8 to 9 as follows.

[0079] Table 3 Peak Areas and Relative Standard Deviations Measured by Different Extraction Times

[0080]

[0081] The results showed that the fingerprint spectra obtained by ultrasonic extraction for 20min, 30min and 40min presented basically the same substance information, and the difference in peak areas of each peak was not significant. Considering the stability of extraction, ultrasonic extraction for 30min was selected.

[0082] Test Example 5 Selection of the Quality of Traditional Chinese Medicine Composition

[0083] According to the method in Example 1, the peak areas of 8 labeled peaks of the test solution were measured respectively when using "0.3003g" and "0.6001g" of traditional Chinese medicine composition. The results are shown in Table 4 and Figures 10 to 11 as follows.

[0084] Table 4 Peak Areas Measured with Different Qualities of Traditional Chinese Medicine Composition

[0085]

[0086] The results showed that after converting to the same mass of the traditional Chinese medicine composition and comparing the peak areas, the peak areas of the identified peaks were basically the same, and the peak area of the traditional Chinese medicine composition with a mass of 0.60 g was more appropriate.

[0087] Experimental Example 6 Selection of Column Temperature

[0088] According to the method in Example 1, the peak areas and retention times of 8 identified peaks in the test solution were measured when the column temperature was "40 °C", "38 °C" and "42 °C" respectively. Taking Peak 2 as the reference peak, the relative peak areas and relative retention times of the remaining peaks were calculated, and the RSD was calculated. The results are shown in Tables 5 to 8.

[0089] Table 5 Peak Areas and Relative Standard Deviations Measured at Different Column Temperatures

[0090]

[0091] Table 6 Relative Peak Areas and Relative Standard Deviations at Different Column Temperatures

[0092]

[0093]

[0094] Table 7 Retention Times and Relative Standard Deviations Measured at Different Column Temperatures

[0095]

[0096] Table 8 Relative Retention Times and Relative Standard Deviations at Different Column Temperatures

[0097]

[0098] The results showed that the RSDs of the peak areas, relative peak areas, retention times and relative retention times of the 8 identified peaks at different column temperatures were all less than 3%, indicating that the method had good durability for different column temperatures.

[0099] Experimental Example 7 Selection of Injection Volume

[0100] According to the method in Example 1, the peak areas and retention times of 8 identified peaks in the test solution were measured when the injection volume was "10 μL", "5 μL" and "15 μL" respectively. Taking Peak 2 as the reference peak, the relative peak areas, relative retention times and RSD of the remaining peaks were calculated. The results are shown in Tables 9 to 12.

[0101] Table 9 Peak Areas Measured at Different Injection Volumes

[0102]

[0103] Table 10 Relative peak areas and relative standard deviations at different sample injection volumes

[0104]

[0105] Table 11 Retention times measured at different sample injection volumes

[0106]

[0107] Table 12 Relative retention times and relative standard deviations at different sample injection volumes

[0108]

[0109] The results showed that the RSDs of the peak areas, relative peak areas, retention times, and relative retention times of the 8 identified peaks at different sample injection volumes were all less than 3%, indicating that the method had good durability for different sample injection volumes.

[0110] Test Example 8 Selection of chromatographic column

[0111] Detect the peak areas and retention times of the 8 identified peaks of the test solution when using chromatographic column 1: Cosmosil, C18-MS-II, 250×4.6 mm, 5 μm, Part / Serial: M05CLA25 / E176204; chromatographic column 2: Welch, XB-C18, 250×4.6 mm, 5 μm, lot number: 00201-31043; chromatographic column 3: Cosmosil CO-S-C18, 250×4.6 mm, 5 μm, lot number: 38202-41 respectively according to the method in Example 1. Taking peak 2 as the reference peak, calculate the relative peak areas and relative retention times of the remaining peaks, and calculate the RSD. The results are shown in Tables 13 to 16.

[0112] Table 13 Peak areas and relative standard deviations determined by different chromatographic columns

[0113]

[0114] Table 14 Relative peak areas and relative standard deviations of different chromatographic columns

[0115]

[0116] Table 15 Retention times and relative standard deviations determined by different chromatographic columns

[0117]

[0118] Table 16 Relative retention times and relative standard deviations of different chromatographic columns

[0119]

[0120] The results showed that the RSDs of the peak areas, relative peak areas, retention times and relative retention times of the 8 identified peaks for different chromatographic columns were all less than 3%, indicating that the method had good durability for different chromatographic columns.

[0121] Test Example 9 System Suitability Determination

[0122] Prepare the test solution according to the method in Example 1 and carry out the determination according to the method in Example 1, with parallel injection 6 times. Using peak 2 as the reference peak, calculate the peak areas, relative peak areas, retention times, relative retention times and RSDs of the remaining identified peaks. The results are shown in Tables 17 to 20.

[0123] Table 17 System Suitability Peak Areas and Relative Standard Deviations

[0124]

[0125] Table 18 System Suitability Relative Peak Areas and Relative Standard Deviations

[0126]

[0127] Table 19 System Suitability Retention Times and Relative Standard Deviations

[0128]

[0129]

[0130] Table 20 System Suitability Relative Retention Times and Relative Standard Deviations

[0131]

[0132] The results showed that the RSDs of the peak areas, relative peak areas, retention times and relative retention times of the 8 identified peaks were all less than 3%, indicating that the system suitability of the method was good.

[0133] Test Example 10 Repeatability Experiment

[0134] Prepare 6 test solutions according to the method in Example 1 and carry out the determination according to the method in Example 1 respectively. Using peak 2 as the reference peak, calculate the relative peak areas and relative retention times of the remaining identified peaks and calculate the RSDs. The results are shown in Tables 21 and 24.

[0135] Table 21 Peak Areas and Relative Standard Deviations Measured in the Repeatability Experiment

[0136]

[0137]

[0138] Table 22 Relative peak areas and relative standard deviations in the repeatability experiment

[0139]

[0140] Table 23 Retention times and relative standard deviations measured in the repeatability experiment

[0141]

[0142]

[0143] Table 24 Relative retention times and relative standard deviations in the repeatability experiment

[0144]

[0145] Results showed that the RSDs of the peak areas, relative peak areas, retention times, and relative retention times of the 8 labeled peaks were all less than 3%, indicating good repeatability of the method.

[0146] Test Example 11 Stability experiment

[0147] The same test solution was determined according to the method in Example 1 at 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h respectively. The retention times and peak areas of each labeled peak were recorded, and the relative retention times, relative peak areas, and RSD values were calculated. The results are shown in Tables 25 to 28.

[0148] Table 25 Peak areas and relative standard deviations measured at different determination times

[0149]

[0150]

[0151] Table 26 Relative peak areas and relative standard deviations at different determination times

[0152]

[0153] Table 27 Retention times and relative standard deviations measured at different determination times

[0154]

[0155]

[0156] Table 28 Relative retention times and relative standard deviations at different determination times

[0157]

[0158] The results showed that the RSDs of the peak areas, relative peak areas, retention times, and relative retention times of the 8 identified peaks were all less than 3%, indicating that the test solution had good stability within 48 hours.

[0159] Determination of the fingerprint in Test Example 12

[0160] The fingerprint of the traditional Chinese medicine composition was determined according to the method in Example 1, and the results were as Figure 12 shown.

[0161] The results indicated that the chromatographic peaks of the traditional Chinese medicine composition were rich, and the resolution and peak shape of the chromatographic peaks were good.

[0162] It can be seen from the above examples that the present invention provides a comprehensive quality control method for a traditional Chinese medicine composition. The method has good repeatability, precision, stability, and durability, and can be used to control the quality of the traditional Chinese medicine composition.

[0163] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A quality control method for a traditional Chinese medicine composition, characterized in that, The steps are as follows: (1) Mix the traditional Chinese medicine composition with methanol and perform ultrasonic treatment to obtain a test solution; (2) Mix ginsenoside Rb1 with methanol to obtain a reference solution; (3) Use liquid chromatography to separately determine the fingerprint spectra of the test solution obtained in step (1) and the reference solution obtained in step (2). If the similarity between the fingerprint spectra of the test solution and the reference solution is ≥ 0.90, it is determined that the quality of the traditional Chinese medicine composition is qualified; The traditional Chinese medicine composition in step (1) is composed of Paecilomyces hepiali mycelia, Ganoderma lucidum, Ganoderma lucidum spores, and American ginseng.

2. The quality control method according to claim 1, wherein The mass ratio of the Paecilomyces hepiali mycelia, Ganoderma lucidum, Ganoderma lucidum spores, and American ginseng is 1 - 8:2 - 12:1 - 8:1 - 10.

3. The quality control method according to claim 1, wherein The mass percentage content of the methanol in step (1) is 50 - 100%.

4. The quality control method according to claim 1, characterized in that, The power of the ultrasonic treatment in step (1) is 240 - 260 W, the frequency of the ultrasonic treatment is 30 - 50 KHz, and the time of the ultrasonic treatment is 20 - 40 min.

5. The quality control method according to claim 1, characterized in that, The mass percentage content of the methanol in step (2) is 50 - 100%; the concentration of ginsenoside Rb1 in the reference solution is 0.4 - 0.6 mg / mL.

6. The quality control method according to claim 1, characterized in that The injection volume of the test solution and the reference solution in step (3) is 5 - 15 μL.

7. The quality control method according to claim 1, characterized in that The filler used in the liquid chromatography determination in step (3) is octadecylsilane chemically bonded silica gel. The column length of the chromatographic column used in the liquid chromatography determination is 250 mm, the column inner diameter is 4.6 mm, and the particle size is 5 μm.

8. The quality control method according to claim 1, wherein In the liquid chromatography determination in step (3), acetonitrile is used as mobile phase A and phosphoric acid is used as mobile phase B. The mass percentage content of the phosphoric acid is 0.05 - 0.15%.

9. The quality control method according to claim 8, wherein, The gradient elution conditions set in the liquid chromatography determination in step (3) are as follows: 0 - 25 min, 19 - 20% mobile phase A, 81 - 80% mobile phase B; 25 - 60 min, 20 - 40% mobile phase A, 80 - 60% mobile phase B; 60 - 90 min, 40 - 55% mobile phase A, 60 - 45% mobile phase B; 90 - 100 min, 55 - 60% mobile phase A, 45 - 40% mobile phase B; 100.1 - 110 min, 19% mobile phase A, 81% mobile phase B.

10. The quality control method according to claim 1, wherein The wavelength of the liquid chromatography determination in step (3) is 200 - 205 nm, the column temperature is 38 - 42 °C, and the flow rate is 0.8 - 1.2 mL / min.

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