A preparation method of a test sample in a method for determining the content of astragalus and a method for determining the content of calycosin-7-glucoside in astragalus

Abstract

The application provides a preparation method of a test sample in a content determination method of Astragalus membranaceus, which comprises the following steps: a, taking Astragalus membranaceus medicinal material powder, and sieving; b, placing the powder into a stoppered conical flask, adding 20-100 ml of 1-6 % ammonia solution-methanol solution (4 ml of ammonia solution is taken, 50 % methanol is added to 100 ml, and shaking is performed), weighing, ultrasonic extraction (250 W, 40 kHz) is performed for 10-90 min, cooling, weighing again, supplementing the lost weight with 1-6 % ammonia solution-methanol solution, shaking, filtering, and taking the filtrate, so that the test sample is obtained. The application further provides a content determination method of calycosin-7-glucoside in Astragalus membranaceus. The optimized method is stable and feasible, simple in operation, high in extraction efficiency, short in analysis time, low in detection cost, and accurate in determination, and can provide a scientific basis for improving the content determination method of calycosin-7-glucoside in Astragalus membranaceus in Chinese Pharmacopoeia.

Description

Technical Field

[0001] This invention relates to a method for preparing a test sample in a method for determining the content of Astragalus membranaceus and a method for determining the content of verbenacisoflavone glucoside in Astragalus membranaceus. Background Technology

[0002] Astragalus is the dried root of *Astragalus membranaceus* (Fisch.) Bge. var. *mongholicus* (Bge.) Hsiao or *Astragalus membranaceus* (Fisch.) Bge., belonging to the legume family. It possesses numerous medicinal properties, including tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, generating fluids and nourishing blood, promoting blood circulation and relieving pain, promoting pus drainage and detoxification, and astringing sores and promoting tissue regeneration. The effective components of astragalus include saponins, flavonoids, and polysaccharides. Among them, calycosin-7-O-β-D-glucoside is one of the representative active substances of astragalus flavonoids. The 2025 edition of the Chinese Pharmacopoeia (hereinafter referred to as the Pharmacopoeia) stipulates that astragalus contains not less than 0.020% calycosin-7-O-β-D-glucoside on a dried basis. However, the pharmacopoeia method involves methanol reflux extraction for 4 hours, followed by evaporation of the filtrate and resolution of the residue. This method is cumbersome and time-consuming, and cannot meet the needs of rapid determination of large batches of samples.

[0003] In recent years, scholars have studied sample processing methods for the determination of flavonoid components such as verbenafil glucoside in Astragalus membranaceus. Sun Huanhuan et al. (Sun Huanhuan, Sun Haifeng, Chai Zhi, et al. Application of alkali treatment in the determination of trace control index components in Astragalus membranaceus [J]. Journal of Pharmaceutical Analysis, 2018, 38 (09): 1652-166) used 2% to 6% ammonia water in 70% ethanol solution followed by ultrasonic extraction twice. Although the extraction rates of verbenafil glucoside and gentianin were improved, the operation method was still relatively complicated and was not directly compared with the pharmacopoeia method. Shao Cai, Sun Hai, Guan Yiming, et al. Determination of Astragaloside A and Versiflavonoid Glucoside Content in Astragalus membranaceus by HPLC-MS [J / OL]. Special Products Research, 1-5 [2025-12-21]. https: / / doi.org / 10.16720 / j.cnki.tcyj.2024.190. Cui Yue, Gao Xing, Han Liqin, et al. Simultaneous Determination of Astragaloside A and Versiflavonoid Glucoside Content in Astragalus membranaceus by UFLC-MS / MS [J]. New Drugs and Clinical Pharmacology of Traditional Chinese Medicine, 2021, 32 (12): 1855-1858. Sun Yiyang, Rao Zhiwang, Guo Junling, et al. Determination of Content of 6 Active Components in Astragalus membranaceus and Correlation Study with Root Morphological Characteristics [J]. Modern Chinese Materia Medica, 2025, 27 (10): Literature from 1838-1846, etc., used 4% concentrated ammonia in methanol solution for extraction by heating and reflux, and simultaneously determined astragaloside A and verbascoside by HPLC-MS. However, this method relies on mass spectrometry detection and has limited applicability. Wang Dongbo, Zhao Manjia, Dai Yuntao, et al. Optimization of Astragalus analytical method and quality evaluation of Astragalus standard decoction [J]. Chinese Journal of Traditional Chinese Medicine, 2020, 45 (01): 119-123. Methanol was used as the extraction solvent. Gao Weicheng, Wang Xiaoping, Li Xinru, et al. Study on multi-index quality evaluation of Astragalus from different origins [J]. Journal of Shanxi University of Traditional Chinese Medicine, 2024, 25 (05): 508-513. 70% ethanol was used as the extraction solvent, and ultrasonic extraction was used to prepare the test solution of flavonoid components. Although the operation was simplified, the key parameters of the test sample preparation were not fully investigated, and the optimized method was not statistically compared with the pharmacopoeia method. Zhou Ke, Cai Wentao, Zhu Huimin, et al. Study on quality evaluation of Gansu Astragalus membranaceus slices based on different extraction methods and index component determination [J]. Journal of Nanjing University of Chinese Medicine, 2025, 41 (08): 1066-1074. The effects of water decoction and methanol reflux extraction on the determination of flavonoid components were compared. The results showed that water extraction was more conducive to the extraction of verbascoside and gentianoside, but the water extract contained more impurities, the subsequent purification steps were complicated, and there was also a lack of systematic optimization of extraction conditions.Although the above studies explored extraction methods of astragalus verbena isoflavone glucoside from different perspectives, they generally suffer from problems such as cumbersome operation, lack of systematic optimization of key parameters, or lack of direct comparison with pharmacopoeia standard methods. Summary of the Invention

[0004] This invention employs ultrasonic extraction combined with Box-Behnken response surface methodology to optimize the pretreatment method for determining verbenafil glucoside in Astragalus membranaceus. By measuring multiple batches of samples from different origins, the optimized results were compared with the pharmacopoeia method, establishing a simple and rapid sample pretreatment method for determining the content of verbenafil glucoside in Astragalus membranaceus.

[0005] This invention provides a method for preparing a test sample in the determination of Astragalus content, which includes the following steps:

[0006] a. Take the powdered Astragalus membranaceus and sift it;

[0007] b. Place the solution in a stoppered conical flask, add 20-100 ml of a 1-6% concentrated ammonia solution in 20-100% methanol (take 1-6 ml of concentrated ammonia solution, add 20-100% methanol to 100 ml, and shake well), weigh the solution, and extract using ultrasound (250 W, 40 kHz) for 10-90 min. Cool the solution, weigh it again, and replenish the lost weight with 20-100% methanol of the 1-6% concentrated ammonia solution. Shake well, filter the solution, and collect the filtrate.

[0008] In step a, the powder is passed through a No. 4 sieve.

[0009] In step b, the concentration of the concentrated ammonia solution is 4%; the concentration of the methanol solution is 50%; and the ultrasonic extraction time is 30 minutes.

[0010] This invention also provides a method for determining the content of verbascoside glucosinolates in Astragalus membranaceus, which includes the following steps:

[0011] a. Preparation of the reference solution:

[0012] Weigh 10 mg of verbascoside glucoside reference standard and place it in a 10 mL volumetric flask. Dissolve it in 50% methanol and dilute to the mark to prepare the reference standard stock solution. Then, accurately pipette 0.2 mL of the above reference standard stock solution into a 10 mL volumetric flask, dissolve it in 50% methanol and dilute to the mark, and shake well to obtain the final product.

[0013] b. Preparation of the test solution:

[0014] The test solution is prepared according to the preparation method of any one of claims 1-3;

[0015] c. Determine under the following chromatographic conditions:

[0016] Column: Agilent Eclipse plus C 18 (4.6 × 250 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.2% formic acid solution (B), gradient elution (0~20 min: 20%→40% mobile phase A, 80%→60% mobile phase B; 20~30 min: 40% mobile phase A, 60% mobile phase B); Detection wavelength: 260 nm; Flow rate: 1.0 mL / min; Column temperature: 35 ℃; Injection volume: 10 μL.

[0017] The optimized method of this invention is stable, feasible, easy to operate, and has high extraction efficiency. While ensuring the accuracy of the determination, it also significantly shortens the analysis time and reduces the detection cost, and can provide a scientific basis for the improvement of the determination method of verbenarosflavonoid glucoside in Astragalus membranaceus in the Chinese Pharmacopoeia. Attached Figure Description

[0018] Figure 1 High-performance liquid chromatogram of verbascoside isoflavone glucoside (where 1. verbascoside isoflavone glucoside; A. reference solution; B. Astragalus sample solution; C. blank solvent).

[0019] Figure 2 Extraction method single-factor optimization results;

[0020] Figure 3 Response surface plot and contour plot showing the effect of the interaction of various factors on the content. Detailed Implementation

[0021] Example 1: Preparation method of the test sample in the Astragalus content determination method of the present invention.

[0022] Take about 1g of Astragalus powder (passed through a No. 4 sieve), weigh it accurately, place it in a stoppered conical flask, and accurately add 50ml of 50% methanol solution of 4% concentrated ammonia solution (take 4ml of concentrated ammonia test solution, add 50% methanol to 100ml, and shake well). Weigh it, extract it by sonication (250W, 40kHz) for 30 min, cool it, weigh it again, and make up the weight loss with 50% methanol solution of 4% concentrated ammonia solution. Shake well, filter it, and take the filtrate to obtain the product.

[0023] Example 2: Screening test of the preparation method of the test sample in the method for determining the content of Astragalus membranaceus in this invention.

[0024] 1. Instruments and reagents

[0025] 1.1 Instruments

[0026] LC-20D high-performance liquid chromatograph (Shimadzu Corporation, Japan); analytical balance of 0.01 g / L (Sartorius, BCE224i-1CCN); KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); RHP-400A high-speed multi-functional pulverizer (Zhejiang Ronghao Industry & Trade Co., Ltd.); HH-600 digital display constant temperature water tank (Changzhou Aohua Instrument Co., Ltd.).

[0027] 1.2 Drug Testing

[0028] Verbena isoflavone glucoside reference standard (Chengdu EFA Biotechnology Co., Ltd., batch number AZCL2014, purity >98%); acetonitrile, methanol (chromatographic grade, Merck); ammonia, methanol (analytical grade, Guangzhou Chemical Reagent Factory); water is ultrapure water.

[0029] Ten batches of Astragalus membranaceus medicinal materials were collected, numbered HQ01~HQ10. Among them, HQ01~HQ07 were produced in Datong, Shanxi Province, and HQ08~HQ10 were produced in Yulin, Shaanxi Province. They were identified as the dried roots of Astragalus membranaceus (Fisch.) Bge. var. mongholi-cus (Bge.) Hsiao, a legume.

[0030] 2. Methods and Results

[0031] 2.1 Determination method of verbascoside isoflavone glucoside

[0032] 2.1.1 Chromatographic conditions: Column: Agilent Eclipse plus C 18 (4.6 × 250 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.2% formic acid solution (B), gradient elution (0–20 min: 20% → 40% mobile phase A, 80% → 60% mobile phase B; 20–30 min: 40% mobile phase A, 60% mobile phase B); Detection wavelength: 260 nm; Flow rate: 1.0 mL / min; Column temperature: 35℃; Injection volume: 10 μL. See chromatogram below. Figure 1 .

[0033] 2.1.2 Preparation of the reference solution: Weigh approximately 10 mg of verbascoside glucoside reference standard and place it in a 10 mL volumetric flask. Dissolve it in 50% methanol and dilute to the mark to obtain the reference standard stock solution. Then, accurately pipette 0.2 mL of the above reference standard stock solution into a 10 mL volumetric flask, dissolve it in 50% methanol and dilute to the mark, and mix well to obtain the reference standard stock solution.

[0034] 2.1.3 Preparation of the test solution: Weigh approximately 1 g of the powder (passed through a No. 4 sieve) accurately, place it in a stoppered conical flask, and accurately add 50 ml of a 4% concentrated ammonia solution in 50% methanol (take 4 ml of concentrated ammonia test solution, add 50% methanol to 100 ml, and shake well). Weigh the sample, extract ultrasonically (250 W, 40 kHz) for 30 min, cool, weigh again, and replenish the lost weight with 4% concentrated ammonia in 50% methanol. Shake well, filter, and collect the filtrate to obtain the test solution.

[0035] 2.2 Extraction Method: Single-Factor Optimization

[0036] 2.2.1 Selection of Extraction Method: Following the method described in section "2.1.3", a 50% methanol solution containing 4% concentrated ammonia was used as the extraction solvent, with an addition volume of 50 mL. Extraction was performed using ultrasonic extraction for 30 min, reflux extraction for 60 min, and extraction using a fully automated rapid solution extractor (extraction pressure 10 MPa, extraction temperature 120℃, preheating for 30 seconds, thermal equilibrium for 3 minutes, rinsing volume 20%, one extraction, and then adjusting the extract volume to 50 mL) for 15 min. The content was determined, and the results are shown in [Figure number missing]. Figure 2 (A). As shown in the figure, the content of verbascoside glucoside obtained by the three extraction methods is basically the same, but the ultrasonic extraction method is simple to operate and takes a moderate amount of time.

[0037] 2.2.2 Selection of Extraction Time: Following the method described in section "2.1.3", an extraction solvent of 50% methanol with 4% concentrated ammonia was used, with an addition volume of 50 mL. The mixture was ultrasonicated for 10, 30, 60, and 90 min respectively, and the content was determined. The results are shown below. Figure 2 (B). As shown in the figure, the content of verbascoside glucoside tends to stabilize after 30 min.

[0038] 2.2.3 Selection of methanol concentration: Following the method described in section "2.1.3", 50 mL of methanol solutions of different concentrations (20%, 50%, 80%, and 100%) with 4% concentrated ammonia were added respectively. The solutions were sonicated for 30 min, and the content was determined. The results are shown in [Figure 1]. Figure 2 (C). As shown in the figure, the content of verbascoside glucoside initially increases with increasing methanol concentration, but decreases after the methanol concentration exceeds 50%.

[0039] 2.2.4 Selection of Concentrated Ammonia Concentration: Following the method described in section "2.1.3", add 50 mL of 50% methanol solution containing 1%, 2%, 3%, 4%, 5%, and 6% concentrated ammonia, respectively. Sonicate for 30 min, then determine the content. Results are shown in [Figure 1]. Figure 2 (D). As shown in the figure, the content of verbascoside isoflavone glucoside increased significantly in the concentration range of 1% to 3%, the increasing trend of the content tended to stabilize after 4%, and the content decreased at 6%.

[0040] 2.2.5 Selection of Extraction Solvent Volume: Following the method described in section "2.1.3", a 50% methanol solution containing 4% concentrated ammonia was used as the extraction solvent. Volumes of 20, 40, 60, 80, and 100 mL were added, respectively. The ultrasonic treatment time was 30 min, and the content was determined. Results are shown below. Figure 2 (E). As shown in the figure, the component content increases within the range of 20–60 mL. This may be because, when the extraction solvent is within a certain liquid-to-solid ratio range, the saturation of verbascoside in the extract decreases with increasing liquid-to-solid ratio, resulting in a larger concentration gradient and accelerating its diffusion rate from the solid phase to the liquid phase, thereby increasing the extraction rate. When the volume reaches 60 mL or more, the extraction rate of verbascoside tends to stabilize.

[0041] 2.3 Response Surface Methodology for Optimized Extraction

[0042] 2.3.1 Response Surface Design

[0043] Based on the results of the single-factor experiments, using Design-Expert 13 software, with extraction time (A), concentrated ammonia concentration (B), and extraction solvent volume (C) as the factors to be investigated, the optimal experimental range for each factor was selected. With the content of verbena isoflavone glucoside (Y) as the response value, a Box-Behnken three-factor, three-level experimental design was carried out. The factor level table is shown in Table 1, and the experimental design and results are shown in Table 2. A total of 15 experiments were conducted, including 3 central replicate experiments to investigate experimental error, and the remaining experiments to investigate the interaction effects of each factor.

[0044] Experimental method: 1g of Astragalus membranaceus powder (passed through a No. 4 sieve) was accurately weighed and placed in a stoppered conical flask. According to the response surface methodology in Table 2, a specified volume and concentration of concentrated ammonia-methanol solution were added, and the weight was measured. The mixture was ultrasonically extracted (250W, 40kHz) for a specified time, cooled, and weighed again. The corresponding concentrated ammonia-methanol solution was added to make up the lost weight, shaken well, filtered, and the filtrate was collected to obtain 15 Astragalus membranaceus test solutions. The content of each solution was determined, and the results are shown in Table 2.

[0045] Table 1 Factor Level Table

[0046]

[0047] Table 2. Response surface methodology design and measurement results

[0048]

[0049] 2.3.2 Model Fitting and Analysis of Variance

[0050] The multiple quadratic regression model was fitted between each factor and the response value using Design-Expert 11 software, resulting in the response surface model equation: Verbena isoflavone glucoside (μg / g) = 1340.50 + 68.87A + 3.89B + 5.66C - 9.42AB + 1.77AC + 4.30BC - 82.65A² - 61.47B² - 0.3250C². The results of the regression model variance analysis are shown in Table 3. The overall regression equation showed F = 93.93 and P < 0.0001, indicating statistical significance. The lack-of-fit term P was 0.0655, greater than 0.05, indicating no lack-of-fit factor, meaning the established regression equation could accurately fit the true level of verbena isoflavone glucoside in the experimental results. The F-value reflects the influence of each factor on the content, with A > C > B. The corrected coefficient of determination (Adj R) was analyzed by comparing the actual and predicted values. 2 The coefficient of variation (CV) was 0.9835, greater than 0.8, and the CV was 0.75%, indicating that only 1.65% of the variation in the predicted results of verbenafil isoflavone glucoside content was unexplained, showing a good fit; the Adeq Precision was 29.30, indicating low noise. Factors A and A2 in the verbenafil isoflavone glucoside model... 2 B 2 The p-value was less than 0.01, indicating that the effect on the content was extremely significant.

[0051] Table 3. Results of ANOVA for the Regression Model

[0052]

[0053] 2.3.3 Response Surface Analysis

[0054] Two-dimensional contour plots and three-dimensional response surface plots of pairwise factors for the generation of verrucoside glucoside content are shown in [the original text]. Figure 3 It can intuitively reflect the influence of factors and their interactions on the predicted content value. The results showed that the extraction time and concentrated ammonia concentration had a significant interaction on the content value of verbascoside; among the three factors, the extraction solvent volume had the least impact on the content value, which is consistent with the results of the analysis of variance.

[0055] 2.3.4 Determination and Verification of the Optimal Method

[0056] The Design-Expert 11 software was used to analyze the model, with the target value of verbascoside content set to the maximum. The software optimization yielded the optimal process conditions and predicted results: the optimal extraction method was 4.035% concentrated ammonia solution (v / v), 32.619 min of sonication, and 66.584 ml of solvent. The predicted verbascoside content was 1.354 mg / g. The actual operation determined the method to be 4% concentrated ammonia solution (v / v), 30 min of sonication, and 50 ml of solvent. Validation experiments yielded an average verbascoside content of 1.321 mg / g, with a relative error of 2.44% compared to the predicted value. This validates the reliability of the established regression model and the feasibility of the optimized method.

[0057] 2.4 Methodological Investigation of Content Determination

[0058] 2.4.1 Linearity Examination

[0059] Take the reference stock solution from section "2.1.2", prepare a series of reference solutions of different concentrations, and inject 10 μl of each solution. Plot a standard curve with peak area as the ordinate and reference concentration (μg / ml) as the abscissa. The results show that verbascoside glucoside exhibits a good linear relationship in the range of 5.33~53.3 μg / ml, with the regression equation Y=18499.9894X+869.2927 (r=0.9997).

[0060] 2.4.2 Precision Test

[0061] The same reference solution was injected six times. The RSD value of the peak area of ​​verbascoside glucoside was 0.53%, indicating that the instrument precision was good.

[0062] 2.4.3 Repeatability Test

[0063] Six test solutions were prepared in parallel using the same batch of Astragalus membranaceus (HQ01) according to the method described in section "2.1.3". These solutions were then injected and analyzed under the chromatographic conditions described in section "2.1.1", and the RSD values ​​of the contents were calculated. The results showed that the average content of verbascoside isoflavone glucoside was 1.321 mg / g, with an RSD value of 2.18%, indicating good repeatability of the method.

[0064] 2.4.4 Solution stability test

[0065] The same sample solution was taken and placed at room temperature. Injections were performed at 0, 2, 4, 6, 8, and 12 h, and the peak areas were recorded. The results showed that the RSD value of verrucoside isoflavone glucoside was 1.69%, indicating good stability of the sample solution within 12 h at room temperature.

[0066] 2.4.5 Recovery Test

[0067] Six portions (approximately 0.5 g each) of the same batch of Astragalus membranaceus powder (HQ01) with known content were accurately weighed. A certain amount of verbascoside reference solution was accurately added, and the sample solution was prepared according to the method in section "2.1.3". The samples were injected for determination, and the recovery rate was calculated. The results are shown in Table 4. The average recovery rate of verbascoside was 98.13%, and the RSD% was 1.49%, indicating that the method has high accuracy.

[0068] Table 4 Results of the recovery rate experiment

[0069]

[0070] 2.5 Sample Measurement Results and Analysis

[0071] 2.5.1 Sample Measurement Results

[0072] Ten batches of Astragalus membranaceus samples from different origins were taken and processed according to the above optimized method and the pharmacopoeia method to obtain test solutions. The content of verbascoside isoflavone glucoside was determined, and the results are shown in Table 5.

[0073] Table 5. Determination of verbascoside content in Astragalus membranaceus by different methods

[0074]

[0075] 2.5.2 Statistical Analysis

[0076] The experimental design used different methods to determine the content of verbenafil isoflavone glucoside in the same sample of Astragalus membranaceus. The differences in content determined by the optimized method (ultrasonic extraction with 4% concentrated ammonia solution in 50% methanol solution for 30 min) and the pharmacopoeia method (refluxing extraction with 50% methanol for 4 h) were compared. SPSS 25.0 software was used to perform statistical analysis on the results corresponding to Table 5. The data conformed to a normal distribution. Paired t-tests were used to analyze the differences in content results between the methods. The results are shown in Table 6. P < 0.05, indicating that there is a significant difference between the results determined by the optimized method and the pharmacopoeia method. That is, the extraction rate of verbenafil isoflavone glucoside by the optimized method is higher than that by the pharmacopoeia method, and the extraction time is shorter, which has obvious advantages.

[0077] Table 6. Results of paired t-test analysis

[0078]

Claims

1. A method for preparing the test sample in a method for determining the content of Astragalus membranaceus, characterized in that: It includes the following steps: a. Take the powdered Astragalus membranaceus and sift it; b. Place the solution in a stoppered conical flask, add 20-100 ml of a 1-6% concentrated ammonia solution in 20-100% methanol solution, weigh it, and extract it using ultrasound (250W, 40kHz) for 10-90 min. Let it cool, weigh it again, and replenish the lost weight with a 1-6% concentrated ammonia solution in 20-100% methanol solution. Shake well, filter, and collect the filtrate to obtain the final product. The method for preparing the methanol solution is as follows: take 1-6 ml of concentrated ammonia test solution, add 20-100% methanol to 100 ml, and shake well.

2. The method for preparing the test sample in the method for determining the content of Astragalus membranaceus according to claim 1, characterized in that: The powder described in step a is passed through a No. 4 sieve.

3. The method for preparing the test sample in the method for determining the content of Astragalus membranaceus according to claim 1, characterized in that: The concentration of the concentrated ammonia solution in step b is 4%; the concentration of the methanol solution is 50%; and the ultrasonic extraction time is 30 minutes.

4. A method for determining the content of verbascoside glucoside in Astragalus membranaceus, characterized in that: It includes the following steps: a. Preparation of the reference solution: Weigh 10 mg of verbascoside glucoside reference standard and place it in a 10 mL volumetric flask. Dissolve it in 50% methanol and dilute to the mark to prepare the reference standard stock solution. Then, accurately pipette 0.2 mL of the above reference standard stock solution into a 10 mL volumetric flask, dissolve it in 50% methanol and dilute to the mark, and shake well to obtain the final product. b. Preparation of the test solution: The test solution is prepared according to the preparation method of any one of claims 1-3; c. Determine under the following chromatographic conditions: Column: Agilent Eclipse plus C 18 (4.6 × 250 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.2% formic acid solution (B), gradient elution (0~20 min: 20%→40% mobile phase A, 80%→60% mobile phase B; 20~30 min: 40% mobile phase A, 60% mobile phase B); Detection wavelength: 260 nm; Flow rate: 1.0 mL / min; Column temperature: 35 ℃; Injection volume: 10 μL.

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