Method for detecting toosendanin content in yin-nourishing and liver-soothing pharmaceutical composition

Abstract

The invention provides a method for detecting toosendanin content in a consistent decoction composition, which comprises the following steps: A) extracting a pharmaceutical composition to be detected by adopting a solvent to obtain a test solution; taking a toosendanin reference substance, and dissolving the toosendanin reference substance with a solvent to obtain a reference substance solution; (B) carrying out qualitative and quantitative analysis on the test solution and the reference solution by adopting a liquid chromatography-mass spectrometry method; the chromatographic conditions are as follows: the chromatographic column is C18; a mobile phase A is a 0.05-0.15% formic acid-water solution; a mobile phase B is acetonitrile; performing gradient elution; the mass spectrum condition is an electrospray ionization (ESI) negative ion mode. The method provided by the invention has the advantages of good detection accuracy, good precision and high sensitivity on the toosendanin content in the consistent decoction composition.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and in particular to a method for detecting the content of toosendanin in Yiguanjian composition. Background Art

[0002] Yiguanjian, from *Medical Formulae Purity* (by Qian Minjie in the Qing Dynasty), is one of the "List of Ancient Classic Famous Prescriptions (the First Batch)" announced by the state and also one of the "Key Information Tables of Classic Famous Prescriptions (7 Prescriptions)" announced by the state. The prescription consists of Glehnia littoralis, Ophiopogon japonicus, Angelica sinensis, Lycium barbarum, Rehmannia glutinosa, and Melia toosendan. Its functions and indications are: nourishing yin and soothing the liver. It is used for the syndrome of insufficiency of liver yin and stagnation of qi due to blood dryness. Chest and hypochondriac pain, acid regurgitation and bitter vomiting, dry throat and mouth, red tongue with little fluid, and thready and taut pulse. It can also treat hernia and mass accumulation. Yiguanjian and its modified prescriptions have significant curative effects on chronic liver diseases, stomach diseases, Sjogren's syndrome, diabetes, dry eye syndrome, tinnitus, etc. with the syndrome of deficiency of liver and kidney yin and stagnation of liver qi. This prescription is the first choice of basic prescription for nourishing yin and soothing the liver clinically. Its treatment fields involve digestive system diseases, infectious and communicable diseases, ophthalmic and otolaryngological diseases, gynecological diseases, etc. Clinically, it is mainly used to treat those with yin deficiency and liver depression, such as chronic hepatitis, chronic gastritis, gastric and duodenal ulcers, intercostal neuralgia, neurosis, etc.

[0003] The prescription contains Melia toosendan. There are literature reports on the liver, kidney and reproductive toxicity of Melia toosendan. Literature reports that toosendanin is the active ingredient in Melia toosendan, but there are also reports that it may be a toxic ingredient. The Chinese Pharmacopoeia stipulates the content range of Melia toosendan in the quality standard of Melia toosendan medicinal materials. There are literature reports on its qualitative identification, but there is no method for determining the content of toosendanin in Yiguanjian.

[0004] Therefore, it is very necessary to develop a method for determining the content of toosendanin in Yiguanjian composition with fast, simple, reproducible and stable results. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting the content of toosendanin in Yiguanjian composition. The method provided by the present invention has good detection accuracy, precision and high sensitivity for the content of toosendanin in Yiguanjian composition.

[0006] The present invention provides a method for detecting the content of toosendanin in Yiguanjian composition, including the following steps:

[0007] A) Extract the test drug composition with a solvent to obtain a test solution;

[0008] Take toosendanin reference substance and dissolve it with a solvent to obtain a reference solution;

[0009] B) Qualitatively and quantitatively analyze the test solution and the reference solution by liquid chromatography-mass spectrometry.

[0010] Chromatographic conditions: The chromatographic column is C18; mobile phase A: 0.05 - 0.15% formic acid - aqueous solution; mobile phase B: acetonitrile; gradient elution;

[0011] Mass spectrometry conditions: Electrospray ionization (ESI) negative ion mode.

[0012] The drug composition to be tested in the present invention includes but is not limited to the Yiguanjian composition.

[0013] The Yiguanjian composition provided by the present invention is the Yiguanjian composition well - known to those skilled in the art, and the present invention does not limit it. The Yiguanjian composition of the present invention preferably consists of the drug composition composed of 5.6 g of Glehnia littoralis, 5.6 g of Ophiopogon japonicus, 5.6 g of Angelica sinensis, 11.19 g of Lycium barbarum, 11.19 g of Rehmannia glutinosa, and 7.46 g of Toosendan fruit.

[0014] The method for detecting the toosendanin content provided by the present invention first extracts the Yiguanjian composition with a solvent to obtain a test solution.

[0015] The preferred solvent in the present invention is methanol; the mass - volume ratio of the Yiguanjian composition to the solvent is (1 - 2) g: 50 mL.

[0016] The extraction method in the present invention is ultrasonic extraction; the time of ultrasonic treatment is 30 - 60 min. Preferably it is 30 min. Specifically, it can be 30 min, 40 min, 50 min or 60 min; or any point value between any two of the above.

[0017] The inventor of the present invention found that, compared with the reflux method, the extraction rate value of the ultrasonic method is higher than that of the reflux method, and the ultrasonic extraction method is convenient. Therefore, it is determined to select ultrasonic extraction as the preparation method. At the same time, within the above - mentioned ultrasonic time in the present invention, the extraction can be complete.

[0018] Take toosendanin reference substance, dissolve it with a solvent to obtain a reference solution. The preferred solvent is methanol; the injection concentration of the toosendanin reference substance in the present invention is 9.99096 μg / g - 499.548 μg / g, and the regression equation is y = 119260x + 22438 (r = 0.9995).

[0019] In the range of 9.99096 μg / g - 499.548 μg / g of toosendanin, there is a good linear relationship between the toosendanin concentration and the peak area.

[0020] The theoretical plate number of toosendanin is 13696, and the resolution is 7.54; this shows that the system suitability of this method is good and meets the current analysis requirements.

[0021] Qualitative and quantitative analysis of the test solution and the reference solution are carried out by liquid chromatography - mass spectrometry.

[0022] Chromatographic conditions: The chromatographic column is C18;

[0023] In one specific embodiment of the present invention, the chromatographic column is Aglient ZORBAX Eclipse Plus C18, 2.1×100 mm, 1.8 μm.

[0024] The inventor of the present invention found that the use of the above chromatographic column has good analysis effect.

[0025] The column temperature of the present invention is 35 - 45 °C; the flow rate is 0.2 - 0.4 mL per minute; the injection volume is 1 - 5 μL.

[0026] In one specific embodiment of the present invention,

[0027] the column temperature is 40 °C; the flow rate is 0.3 mL per minute; the injection volume is 2 μL.

[0028] The mobile phase A of the present invention is 0.05 - 0.15% formic acid - aqueous solution; mobile phase B: acetonitrile; gradient elution;

[0029] The specific gradient elution of the present invention is as follows:

[0030] 0 - 8 min: 70% A; 30% B;

[0031] 8 - 8.5 min: 70% A → 5% A; 30% B → 95% B;

[0032] 8.5 - 12.5 min: 5% A; 95% B;

[0033] 12.5 - 13 min: 5% A → 70% A; 95% B → 30% B;

[0034] 13 - 16 min: 70% A; 30% B.

[0035] Under the above gradient conditions of the present invention, toosendanin can be well separated, with high resolution and good stability.

[0036] The mass spectrometry conditions of the present invention: electrospray ionization (ESI) negative ion mode.

[0037] The mass spectrometry conditions are carried out in selected ion monitoring mode: select the ion with mass - to - charge ratio (m / z) 573 for detection;

[0038] The drying gas temperature is 250 °C, and the drying gas flow rate is 7 L / min; the nebulizer pressure is 30 psi,

[0039] The sheath gas temperature is 325 °C, the sheath gas flow rate is 11 L / min, capillary voltage: positive mode 3500, negative mode 3000; nozzle voltage: positive mode 0, negative mode 500;

[0040] Transmission voltage: 190, acceleration voltage: 3, dwell time: 200.

[0041] The qualitative and quantitative analysis described in the present invention is specifically as follows: The sum of the peak areas of toosendanin is used for qualitative and quantitative analysis.

[0042] The beneficial effects of the present invention are as follows:

[0043] (1) The establishment of the quantitative analysis method for toosendanin in the Yiguanjian composition of the present invention makes up for the blank of the quality control of the safety index in the composition containing Sichuan chinaberry fruit.

[0044] (2) The present invention uses liquid chromatography-mass spectrometry technology to determine the content of toosendanin in the Yiguanjian composition, effectively overcoming the problem that toosendanin has no absorption under ultraviolet conditions.

[0045] (3) The method provided by the present invention can effectively monitor the quality of different batches of Yiguanjian decoction composition, making its quality stable. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensively monitoring the quality of products. Description of the Drawings

[0046] Figure 1 Chromatogram of the reference substance in Example 1 of the present invention;

[0047] Figure 2 Chromatogram of the test sample in Example 1 of the present invention;

[0048] Figure 3 Linear relationship of toosendanin. Detailed Embodiments

[0049] The present invention provides a method for detecting the content of toosendanin in the Yiguanjian composition. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] In order to further illustrate the present invention, the following describes in detail a method for detecting the content of toosendanin in the Yiguanjian composition provided by the present invention in combination with embodiments.

[0051] Example 1

[0052] Preparation of the test solution: Weigh 2 g of the Yiguanjian composition accurately, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh, ultrasonicate for 60 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0053] Preparation of the reference solution: Weigh an appropriate amount of toosendanin reference substance accurately, dissolve it in methanol to prepare a solution containing 2 μg of toosendanin per 1 ml, which is the reference solution.

[0054] Assay method: Accurately pipette 2 μl each of the reference solution and the test solution, inject them into the liquid chromatography - mass spectrometry instrument for determination. Calculate based on the sum of the peak areas of the two toosendanin peaks.

[0055] Chromatographic conditions for high - performance liquid chromatography - mass spectrometry: Mobile phase A: 0.1% formic acid - aqueous solution; Mobile phase B: acetonitrile; Column temperature: 40 °C; Flow rate: 0.3 mL per minute; Detect the ion with a mass - to - charge ratio (m / z) of 573 under the negative - ion mode of electrospray ionization (ESI). The chromatographic column is Aglient ZORBAX Eclipse Plus C18, 2.1×100 mm, 1.8 μm; Gradient elution program

[0056]

[0057] Ion source: Electrospray ion source (AJS - ESI)

[0058] Scanning mode: Negative - ion scanning

[0059] Detection mode: Selected ion monitoring (SIM)

[0060] Source parameters: Set according to the regulations in the following table

[0061]

[0062] Mass spectrometry parameters

[0063]

[0064]

[0065] 1 Methodological investigation

[0066] 1.1 Specificity

[0067] Prepare a methanol blank solution, a toosendanin working standard solution with a concentration of 2 μg / mL, a test solution, and a negative control sample prepared according to the original prescription process withouttoosendan fruit. Inject them separately.

[0068] Determine the retention time and resolution of toosendanin. The resolution should be greater than 1.5. In the negative control chromatogram, no chromatographic peak with the same retention time as the toosendanin chromatographic peak was detected, and there was no interference from the negative control and the solvent.

[0069] Results: In the negative control chromatogram, no chromatographic peak with the same retention time as the toosendanin chromatographic peak was detected, and there was no interference from the negative control and the solvent. See Table 1 for details.

[0070] Table 1 Retention time and resolution of toosendanin

[0071]

[0072] 1.2 Linearity

[0073] Take 10.64 mg of toosendanin reference substance and dilute it successively with methanol to standard series solutions with concentrations of 0.1998 μg / mL, 0.4995 μg / mL, 0.9991 μg / mL, 1.9982 μg / mL, 4.9955 μg / mL, and 9.9910 μg / mL. Precisely pipette 2 μL of the above solutions and inject them into the liquid chromatography - mass spectrometry instrument respectively. Determine according to the chromatographic conditions in 2.2. Use the concentration (μg / mL) of the reference substance as the abscissa and the peak area as the ordinate to plot the standard curve, and calculate the regression equation and the correlation coefficient r (r ≥ 0.99).

[0074] Results: The regression equation was measured as y = 119260x + 22438 (r = 0.9995); the results showed that there was a good linear relationship between the toosendanin concentration and the peak area in the range of 9.99096 μg / g - 499.548 μg / g. See details Figure 3 。

[0075] 1.3 Instrument precision

[0076] Prepare a toosendanin working standard solution with a concentration of 2 μg / mL, inject the needle continuously 6 times, and record the toosendanin peak area and retention time.

[0077] The RSD of the toosendanin peak area for 6 needles ≤ 2.0%, and the RSD of the retention time ≤ 2.0%; the theoretical plate number of toosendanin should not be less than 8000; the resolution between toosendanin and its adjacent chromatographic peak should be greater than 1.5.

[0078] Results: The RSD value of the toosendanin peak area was 0.89% (n = 6), and the RSD value of the retention time was (peak 1 = 0.20%, peak 2 = 0.20%) (n = 6); the theoretical plate number of toosendanin was 13696, and the resolution was 7.54; it showed that the system suitability of this method was good and met the current analysis requirements. See Table 2 for details.

[0079] Table 2 Results of the instrument precision test

[0080]

[0081] 1.4 Repeatability

[0082] Prepare 6 portions of the test solution in parallel, inject the needles in parallel 2 times respectively, and record the peak area and retention time of toosendanin.

[0083] The RSD of the toosendanin content in 6 portions of the test solution is ≤2.0%, and the RSD of the retention time is ≤2.0%.

[0084] Results: The RSD value of the toosendanin content is 0.80% (n = 6), and the RSD values of the retention time are (peak 1 = 0.26%, peak 2 = 0.30%) (n = 6); the results show that this method has good repeatability, see Table 3 for details.

[0085] Table 3 Results of the repeatability test

[0086]

[0087]

[0088] 1.5 Intermediate precision

[0089] The same analyst prepares the toosendanin working standard solution with a concentration of 2 μg / mL and 3 portions of the test solution at two different times, injects the needles in parallel 2 times on the same instrument respectively, and records the peak area and retention time of toosendanin.

[0090] The same analyst prepares the toosendanin working standard solution with a concentration of 2 μg / mL and 6 portions of the test solution. Three portions are in a group, and inject the needles in parallel 2 times on two instruments respectively, and record the peak area and retention time of toosendanin.

[0091] Two analysts prepare the toosendanin working standard solution with a concentration of 2 μg / mL and 3 portions of the test solution respectively, inject the needles in parallel 2 times on the same instrument respectively, and record the peak area and retention time of toosendanin.

[0092] The RSD of the assay results for the three groups of intermediate precision is ≤2.0%.

[0093] Results: The RSD value of group a for the intermediate precision assay is 1.2% (n = 6), the RSD value of group b is 1.3% (n = 6), the RSD value of group c is 1.4% (n = 6), and the RSD value of the assay results for the three groups of intermediate precision is 1.5%. See Table 4 for details.

[0094] Table 4 Results of the intermediate precision test

[0095]

[0096] 1.6 Accuracy

[0097] Accurately weigh 6 portions of the reference samples of Yiguanjian, 1.0 g for each portion. Accurately add 0.15 mL of the working standard solution of toosendanin (1000 μg / mL) to each portion, mix well, and perform the operation according to the "2.1 Preparation Method of Test Solution" and calculate the recovery rate.

[0098] The recovery rate was 85% - 110%.

[0099] Results: The spiked recovery rates of toosendanin in the 6 samples at the concentration level of 150 μg / g were between 100% and 107%, meeting the requirements of quantitative analysis. See Table 5 for details.

[0100] Table 5 Results of Accuracy Test

[0101]

[0102]

[0103] 1.7 Durability

[0104] Prepare a blank methanol solution, a working standard solution of toosendanin at 2 μg / mL, and a test solution. Inject each solution in parallel 2 times, change the method according to the following conditions, and inject the samples in sequence after stabilization:

[0105] a Replace the same type of chromatographic column with different brands (or different batches);

[0106] Agilent ZORBAX Eclipse Plus C18, specification: 2.1×100 mm, 1.8 μm

[0107] Shimadzu Shim-pack GIST C18, specification: 2.1×100 mm, 2.7 μm

[0108] HORIZON Aurashell C18 / PFP, specification: 2.1×100 mm, 2.7 μm

[0109] b Change the column temperature to 35 °C and 45 °C

[0110] c Change the flow rate to 0.2 mL / min and 0.4 mL / min;

[0111] The theoretical plate number of toosendanin should not be less than 8000; the resolution between toosendanin and its adjacent chromatographic peaks should be greater than 1.5; the RSD of the content results of toosendanin under each condition should be ≤ 2.0%.

[0112] Results: Under the conditions of changing different parameters, the RSD of the content results of toosendanin was less than 2.0% and all could meet the determination requirements. See Table 6 for details.

[0113] Table 6 Results of Durability Test

[0114]

[0115]

[0116] 1.8 Solution Stability

[0117] Prepare a reference sample of Yiguanjian, and perform on - machine detection at 0h, 2h, 4h, 8h, 12h, and 24h respectively to investigate the stability of toosendanin in the sample solution.

[0118] The RSD of the toosendanin content result is ≤2.0%, and the RSD of the retention time is ≤2.0%.

[0119] Result: The RSD value of the stability content result of toosendanin in the sample solution for 24h is 2.0%, and the RSD values of the retention time are (peak 1 = 0.28%, peak 2 = 0.44%), indicating the stability of toosendanin in the sample solution for 24h. See Table 7 for details.

[0120] Table 7 Results of Stability Test

[0121]

[0122]

[0123] Investigation of the extraction method in Comparative Example 1 (concentration of reference solution: 2.2325 μg / ml)

[0124] (1) Take 1.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, heat under reflux for 1 hour, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the continuous filtrate, that is obtained.

[0125] (2) Take 2.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, heat under reflux for 1 hour, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the continuous filtrate, that is obtained.

[0126] (3) Take 1.0 g of the Yiguanjian reference sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the continuous filtrate, that is obtained.

[0127] (4) Take 2.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the continuous filtrate, that is obtained.

[0128] (5) Take 3.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 30 min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0129]

[0130]

[0131] Result: Comparing the reflux method with the ultrasonic method, the value of the ultrasonic method is higher than that of the reflux method, and the ultrasonic extraction method is convenient. Therefore, it is determined to select ultrasonic extraction as the preparation method. Comparing methods (3), (4), and (5), after increasing the solid-liquid ratio, the dissolution rate of toosendanin component becomes slower and the extraction is incomplete. Therefore, the sampling amount of the test sample is determined to be 1.0 - 2.0 g.

[0132] Comparative Example 2

[0133] Compare different ultrasonic times (concentration of reference solution: 1.9982 μg / ml)

[0134] (1) Take 1.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 20 min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0135] (2) Take 1.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 30 min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0136] (3) Take 1.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 45 min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0137] (4) Take 1.0 g of the Yiguanjian composition sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 60 min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0138]

[0139] Results: After comparing different ultrasonic times, the extraction rate was lower at 20 min of ultrasonication, and the extraction rates at 30 min, 45 min, and 60 min of ultrasonication were similar. For simplicity in the experimental process, the ultrasonic time of the sample was preferably 30 min.

[0140] 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 method for detecting the content of toosendanin in a Yiguanjian composition, characterized in that: The steps include: A) extracting the pharmaceutical composition to be tested with a solvent to obtain a test solution; Take toosendanin reference substance, dissolve it in a solvent, and obtain a reference substance solution; B) Conduct qualitative and quantitative analysis of the test solution and the reference solution by liquid chromatography-mass spectrometry; Chromatographic conditions: chromatographic column: C18; mobile phase A: 0.05~0.15% formic acid-water solution; mobile phase B: acetonitrile; gradient elution; Mass spectrometry conditions: electrospray ionization (ESI) negative ion mode.

2. The detection method according to claim 1, characterized in that: The gradient elution is specifically: 0~8min 70%A; 30%B; 8~8.5min 70%A→5% A; 30%B→95%B; 8.5~12.5min 5%A; 95%B; 12.5~13min 5%A→70% A; 95%B→30%B; 13~16min 70% A; 30% B.

3. The detection method according to claim 1, characterized in that: The chromatographic column is Aglient ZORBAX Eclipse Plus C18, 2.1×100 mm, 1.8 μm.

4. The detection method according to claim 1, characterized in that: The column temperature is 35-45°C; the flow rate is 0.2-0.4 mL per minute; and the injection volume is 1-5 μL.

5. The detection method according to claim 1, characterized in that: The mass spectrometry condition is carried out in a selected ion detection mode: ions with a mass-to-charge ratio (m / z) of 573 are selected for detection; The drying gas temperature is 250°C, the drying gas flow rate is 7L / min, and the atomizer pressure is 30psi. Sheath gas temperature 325°C, sheath gas flow 11 L / min, capillary voltage: 3500 in positive mode, 3000 in negative mode; nozzle voltage: 0 in positive mode, 500 in negative mode; Transfer voltage 190, acceleration voltage 3, dwell time 200.

6. The detection method according to claim 1, characterized in that: The injection concentration of the toosendanin reference substance is 9.99096 μg / g to 499.548 μg / g, and the regression equation is y=119260x+22438 (r=0.9995).

7. The detection method according to claim 1, characterized in that: The qualitative and quantitative analysis is specifically: qualitative and quantitative analysis is performed based on the sum of the two peak areas of toosendanin.

8. The detection method according to claim 4, characterized in that: The chromatographic conditions are as follows: column temperature is 40°C; flow rate is 0.3 mL per minute; injection volume is 2 μL.

9. The detection method according to claim 1, characterized in that: The solvent in step A) is methanol; the mass volume ratio of the Yi Guan Jian composition to the solvent is (1-2) g:50 mL.

10. The detection method according to claim 1, characterized in that: In step A), the drug composition to be tested is a Yi Guan Jian composition; The extraction method is ultrasonic extraction; the ultrasonic time is 30 to 60 minutes.

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