A method for determining the content of astragaloside A in compound astragalus oral liquid using HPLC-ELSD

Abstract

This invention discloses a method for determining the astragaloside A content in compound astragalus oral liquid using HPLC-ELSD. The method includes the following steps: the prepared reference solution and test solution are separately analyzed using HPLC-ELSD, the chromatograms are recorded, and the astragaloside A content is calculated. In the HPLC-ELSD method, acetonitrile-water system is used as the mobile phase, the flow rate is 0.1~10 mL / min, the column temperature is 20~30℃, the injection volume is 1~20 μL, the theoretical plate value is not less than 10000 based on the astragaloside A peak, and the drift tube temperature of the ELSD detector is 96~102℃. The method provided by this invention has strong specificity, good robustness, and high precision, which is beneficial for improving the quality control of compound astragalus oral liquid production.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, specifically relating to a method for determining the content of astragaloside A in compound astragalus oral liquid using HPLC-ELSD. Background Technology

[0002] Compound Astragalus Oral Liquid is composed of eight medicinal herbs: Astragalus membranaceus (processed), Lycium barbarum, Cuscuta chinensis, processed Polygonum multiflorum, Ligustrum lucidum, Spatholobus suberectus, Angelica sinensis, and Poria cocos. It contains excipients such as honey and ethylparaben. It has the effects of tonifying the kidneys and spleen, replenishing qi and nourishing blood, and is suitable for middle-aged and elderly people suffering from a series of symptoms caused by kidney and spleen deficiency, such as fatigue, weakness in the lower back and knees, loss of appetite, insomnia, and dizziness. Astragaloside A, as the legally mandated content determination indicator for Astragalus membranaceus and its preparations, has the advantages of strong specificity, stable content, and quantifiable detection. In the production process of Compound Astragalus Oral Liquid, Ma Yinglong Pharmaceutical Group Co., Ltd. continuously optimizes the determination method according to the requirements of the "Guidelines for Validation of Analytical Methods for Drug Quality Standards" in the Chinese Pharmacopoeia. In the implemented new quality standard, the content determination method in the original drug standard was changed, and an HPLC-ELSD method was established to determine the content of astragaloside A in the preparation.

[0003] After careful examination of the prior art, the applicant found no relevant testing methods reported. Therefore, this invention is proposed. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a method for determining the content of astragaloside A in compound astragalus oral liquid using HPLC-ELSD, which has high specificity, good durability, and high precision.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for determining the astragaloside A content in compound astragalus oral liquid using HPLC-ELSD includes the following steps: determining the content of the prepared reference solution and test solution using HPLC-ELSD, recording the chromatograms, and calculating the astragaloside A content. In the HPLC-ELSD method, the acetonitrile-water system is used as the mobile phase, and the flow rate of the mobile phase is 0.1~10 mL / min; The column temperature is 20~30℃; The injection volume is 1~20 μL; The theoretical plate value, calculated based on the astragaloside A peak, should be no less than 10,000. The drift tube temperature of the ELSD detector is 96~102℃.

[0006] As a preferred embodiment of the technical solution of the present invention, the preparation method of the reference solution is as follows: take an appropriate amount of astragaloside A reference standard, accurately weigh it, add methanol to prepare a solution containing 0.5 mg per 1 mL, and the solution is obtained.

[0007] As a preferred embodiment of the present invention, the preparation method of the test solution is as follows: accurately measure the sample, extract it by shaking with water-saturated n-butanol multiple times, 30 mL each time, and combine the n-butanol solutions; wash it multiple times with ammonia solution, evaporate the n-butanol solution to dryness, dissolve the residue in methanol and transfer it to a volumetric flask, add methanol to the mark, shake well, and the test solution is obtained.

[0008] As a preferred embodiment of the technical solution of the present invention, in the acetonitrile-water system, the volume ratio of acetonitrile to water is (20~40):(60~80).

[0009] As a preferred embodiment of the technical solution of the present invention, in the acetonitrile-water system, the volume ratio of acetonitrile to water is 32:68.

[0010] As a preferred embodiment of the technical solution of the present invention, the flow rate of the mobile phase is 1 mL / min.

[0011] As a preferred embodiment of the technical solution of the present invention, the column temperature is 25°C; As a preferred embodiment of the technical solution of the present invention, the drift tube temperature of the ELSD detector is 100°C.

[0012] As a preferred embodiment of the technical solution of the present invention, the chromatographic column is a YMC-Triart C18.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The method for determining the astragaloside A content in compound astragalus oral liquid using HPLC-ELSD provided by this invention has high specificity, good robustness, and high precision.

[0014] The method for determining the astragaloside A content in compound astragalus oral liquid using HPLC-ELSD provided by this invention can meet the determination requirements of astragaloside A content by optimizing the mobile phase and various detection conditions, which is of great significance for controlling the production quality of compound astragalus oral liquid. Attached Figure Description

[0015] Figure 1 The spectrum of the test sample in the specificity test of Example 2; Figure 2 This is the chromatogram of the control standard in the specificity test of Example 2; Figure 3 The spectrum of the negative sample in the specificity test of Example 2; Figure 4 This is the chromatogram from the first test of the limit of quantitation experiment in Example 6; Figure 5This is the second test chromatogram for the limit of quantitation test in Example 6. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] In this invention, the sample and instrument information are as follows: Astragaloside A reference standard was purchased from the National Institutes for Food and Drug Control.

[0018] Compound Astragalus Oral Solution, produced by Mayinglong Pharmaceutical Group Co., Ltd.

[0019] Example 1 A method for determining the astragaloside A content in compound astragalus oral liquid using HPLC-ELSD includes the following steps: determining the prepared reference solution and test solution using HPLC-ELSD, recording the chromatograms, and calculating the astragaloside A content using the external standard two-point logarithmic equation. In the HPLC-ELSD method, octadecylsilane-bonded silica gel was used as the packing material, the column was YMC-TriartC18 (250×4.6mm, 5μm), the mobile phase was acetonitrile-water system (volume ratio 32:68), and the flow rate of the mobile phase was 1 mL / min. The column temperature is 25℃; Injection volume: 5 μL and 20 μL for reference solution, and 10 μL for test solution; The theoretical plate value, calculated based on the astragaloside A peak, is no less than 10,000. The drift tube temperature of the ELSD detector is 100℃; the mobile phase flow rate is 1 mL / min.

[0020] The preparation method of the reference solution is as follows: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.5 mg per 1 mL.

[0021] The preparation method of the test solution is as follows: Accurately measure 50 mL of this product (produced by Mayinglong Pharmaceutical Group Co., Ltd.), extract it 4 times with water-saturated n-butanol, 30 mL each time, combine the n-butanol solutions, wash twice with ammonia test solution, 30 mL each time, evaporate the n-butanol solution to dryness, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, add methanol to the mark, shake well, and the test solution is obtained.

[0022] Example 2 In this embodiment, system suitability and specificity tests were conducted. The specific methods are as follows: A reference solution and a test solution were prepared according to the method in Example 1, and a negative control solution was also prepared. Then, referring to the method in Example 1, 10 μL each of the test solution and negative control solution, and 5 μL of the reference solution were accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded.

[0023] Take another 20 μL of the reference solution and inject it 6 times consecutively. Calculate the RSD value of the peak area of ​​astragaloside A in the chromatogram of the reference solution.

[0024] The negative control solution is prepared as follows: Except for Astragalus membranaceus, the remaining ingredients are taken according to the aforementioned prescription ratio, and a negative sample without Astragalus membranaceus is prepared using the same method as for Compound Astragalus Oral Liquid. 50 mL of the negative sample is accurately measured and prepared using the same method as for the test solution.

[0025] In the above specificity test, the chromatogram of the test sample showed a component peak (retention time 20.782 min) at the same retention time as the main component peak (retention time 20.803 min) of the astragaloside A reference standard; the negative sample showed no chromatographic peak at this retention time, indicating no interference from the negative sample. See also Figures 1-3 .

[0026] In the system suitability test, the astragaloside A peak in the test solution was completely separated from adjacent component peaks, with a resolution R > 1.5, and the theoretical plate number calculated based on the astragaloside A peak was 18302. The RSD of the astragaloside A peak area in the reference solution obtained by six consecutive injections was 0.67%. The results are shown in Table 1.

[0027] Table 1 System Applicability Test Results Conclusion: This method has good specificity, and the chromatographic conditions used can meet the requirements for sample analysis.

[0028] Example 3 This embodiment examines the method for preparing the test solution to assess durability. The specific method is as follows: Based on the physicochemical properties of the analyte astragaloside A, the sample was purified using water-saturated n-butanol-water two-phase extraction. Therefore, the number of extraction cycles using water-saturated n-butanol was investigated. Test Solution I: Accurately measure 50 mL of this product, extract three times with 30 mL of water-saturated n-butanol each time, combine the n-butanol extracts, wash twice with 30 mL of ammonia solution each time, separate the n-butanol solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, dilute to the mark with methanol and shake well to obtain the test solution. Prepare two parallel solutions.

[0029] Test Solution II: Accurately measure 50 mL of this product and extract it four times with 30 mL of water-saturated n-butanol each time. Combine the n-butanol extracts and wash twice with 30 mL of ammonia solution each time. Separate the n-butanol solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, dilute to the mark with methanol, and mix well. Prepare two parallel solutions.

[0030] Test Solution III: Accurately measure 50 mL of this product, extract 5 times with 30 mL of water-saturated n-butanol each time, combine the n-butanol extracts, wash twice with 30 mL of ammonia solution each time, separate the n-butanol solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, dilute to the mark with methanol and shake well to obtain the test solution. Prepare two parallel solutions.

[0031] Reference solution: Accurately weigh 4.96 mg of astragaloside A, place it in a 10 mL volumetric flask, dissolve and dilute with methanol to the mark, and shake well to obtain the solution.

[0032] Accurately pipette 5 μL and 20 μL of the reference solution, and 10 μL of the test solution, respectively, and inject them into the liquid chromatograph, recording the chromatograms. Calculate the content of astragaloside A in the samples using the logarithmic equation with external standard two-point method. The results are shown in Table 2.

[0033] Table 2 Results of the investigation on the preparation method of the test sample The calculation formula is as follows: .

[0034] The results showed that after extraction with water-saturated n-butanol 3, 4, and 5 times, the average contents of astragaloside A in the product were 59.3 μg / mL, 62.8 μg / mL, and 60.8 μg / mL, respectively, with an RSD of 2.81% for the six samples. To ensure complete extraction, the samples were extracted with water-saturated n-butanol 4 times in this experiment.

[0035] Example 4 In this embodiment, the chromatographic conditions were investigated, and the specific methods are as follows.

[0036] (1) Investigation of different proportions of mobile phase components Based on the method of Example 1, the component ratio of the mobile phase, acetonitrile-water (32:68), was changed to (31:69) and (33:67), while other chromatographic conditions remained unchanged. The same test solution and reference solution were injected for analysis. The results are shown in Table 3.

[0037] Table 3 Results of investigation of different proportions of mobile phase components The results showed that when the acetonitrile-water ratio in the mobile phase was (31:69) and (32:68), the relative deviation of the astragaloside A content in the sample was 0.72%, indicating that it had no significant effect on the determination of astragaloside A content. However, when the acetonitrile-water ratio in the mobile phase was (31:69), the resolution between the main component peak and adjacent components was less than 1.5. This indicates that the proportion of acetonitrile in the mobile phase should not be increased. Therefore, attention should be paid to accurately controlling the component ratio of the mobile phase during the experiment.

[0038] (2) Investigation of different temperatures in the drift tube Based on the method of Example 1, the drift tube temperature was changed from 100℃ to 75℃ and 85℃, while other chromatographic conditions remained unchanged. The same test solution and reference solution were injected for analysis. The results are shown in Table 4.

[0039] Table 4. Results of the investigation at different temperatures in the drift tube. The results showed that when the drift tube temperature was 100℃, 75℃, and 85℃, the RSD value of the astragaloside A content in the sample was 1.00%, indicating that the drift tube temperature variation within the range of 75℃ to 100℃ had no significant effect on the astragaloside A content determination results.

[0040] (3) Investigation of different column temperatures Based on the method of Example 1, the column temperature was changed from 30℃ to 22℃, 25℃, and 35℃, while other chromatographic conditions remained unchanged. The same test solution and reference solution were injected for analysis. The results are shown in Table 5.

[0041] Table 5 Results of the investigation at different column temperatures The results showed that at column temperatures of 25℃, 30℃, and 35℃, the resolution of the main component peak was greater than 1.5, and the RSD value of astragaloside A content in the sample was 3.94%, indicating that changes in column temperature within the range of 25℃ to 35℃ had little effect on the determination of astragaloside A content. However, at a column temperature of 22℃, the resolution of the astragaloside A peak in the chromatogram of the test solution was less than 1.5. This indicates that the column temperature should be controlled above 25℃; in this experiment, a column temperature of 30℃ was selected.

[0042] (4) Investigation of different flow velocities Based on the method of Example 1, the same test solution and reference solution (concentration of 0.496 mg / mL) were injected and analyzed at different flow rates (1.0 mL / min, 1.1 mL / min, 0.9 mL / min) and with other chromatographic conditions unchanged. The results are shown in Table 6.

[0043] Table 6 Results of the investigation at different flow velocities The results showed that the relative deviation of the astragaloside A content in the sample was 2.03% when the flow rate was 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, respectively. This indicates that small changes in flow rate have no significant effect on the determination of astragaloside A content.

[0044] (5) Investigation of different brands of chromatographic columns Based on the method of Example 1, two different brands of chromatographic columns were used, while other chromatographic conditions remained unchanged. The same test solution and reference solution were injected for analysis. The results are shown in Table 7.

[0045] Table 7 Results of the investigation of different brands of chromatographic columns The results showed that when using the two different brands of chromatographic columns, the relative deviation of the astragaloside A content in the sample was 4.85%, indicating that the method has good column robustness.

[0046] (6) Investigation of solution stability Based on the method of Example 1, 10 μL of the test solution and 20 μL of the reference solution, stored at room temperature, were accurately measured and injected for analysis at 0, 2, 4, 6, and 8 hours. The RSD values ​​of the logarithmic peak areas of astragaloside A measured five times within 8 hours were calculated, and the results are shown in Table 8.

[0047] Table 8 Results of solution stability study The results showed that the RSD values ​​of the peak areas of astragaloside A in the chromatograms of the test solution and the reference solution were 1.23% and 0.30%, respectively, n=5. This indicates that the test solution and the reference solution were basically stable at room temperature for 8 hours.

[0048] Example 5 Precision was examined in this embodiment.

[0049] (1) Repeatability Take the same batch of samples and prepare the test solution according to the method in Example 1. Prepare 6 test solutions in parallel.

[0050] Accurately measure 10 μL of the test solution, 5 μL of the reference solution, and 20 μL of the reference solution, and inject them for analysis. Calculate the RSD values ​​of astragaloside A content in the six test solutions. The results are shown in Table 9.

[0051] (2) Intermediate precision The analysis was performed by different analysts on different dates. The same batch of samples was used, and test solutions were prepared in parallel, following the method described in Example 1.

[0052] Based on the method of Example 1, 10 μL of the test solution, 5 μL of the reference solution, and 20 μL of the reference solution were accurately measured and injected for analysis. The RSD values ​​of astragaloside A content in the six test solutions were calculated, and then analyzed together with the six determination results under the repeatability section. The results are shown in Table 9.

[0053] Table 9 Precision Test Results The results showed that the average content of astragaloside A in the 6 test solutions in the repeatability test was 61.53 μg / mL, with an RSD of 4.27%; the content of astragaloside A in the 6 test solutions in the intermediate precision test was 61.84 μg / mL, with an RSD of 4.05%. The RSD of astragaloside A in the 12 test solutions in the precision test was 3.98%, indicating that the method has good precision.

[0054] Example 6 In this embodiment, linearity and range tests are conducted using the following method.

[0055] Preparation of reference stock solution: Accurately weigh 34.94 mg of astragaloside A, place it in a 25 mL volumetric flask, add methanol to the mark, and shake well.

[0056] Accurately measure 0.5 mL, 1 mL, 2 mL, 3 mL, and 5 mL of the reference stock solution and place them in five 10 mL volumetric flasks. Add methanol to the mark, shake well, and you will get five reference solutions of different concentrations for the linearity test.

[0057] Based on the method of Example 1, 20 μL of each of the linearity test reference solutions of various concentrations were precisely measured and injected for analysis. The regression equation was calculated with the logarithm of the astragaloside peak area as the ordinate and the logarithm of the astragaloside injection mass as the abscissa. The results are shown in Table 10.

[0058] Table 10 Results of Linearity and Range Tests Results: The regression equation for the logarithmic value of the injection mass and the logarithmic value of the peak area of ​​astragaloside A was: y = 1.6815x + 1.9106, with a correlation coefficient r = 0.9992 and n = 5. This indicates that within the injection mass range of 1.397 μg to 13.976 μg, the logarithmic value of the injection mass and the logarithmic value of the peak area of ​​astragaloside A exhibit a good linear relationship.

[0059] Example 7 In this embodiment, the limit of quantitation test is performed, and the specific method is as follows.

[0060] Based on the method of Example 1, the reference solution (concentration of 0.492 mg / mL) was gradually diluted until the signal-to-noise ratio (S / N) of the astragaloside peak in the chromatogram of the reference solution was about 10 (injection volume of 10 μL), thus obtaining the limit of quantification of astragaloside.

[0061] As a result, when the concentration of astragaloside A reference solution was 98.4 μg / mL, the signal-to-noise ratios of the two astragaloside A peak measurements were 11.0 and 13.1, respectively, indicating that the limit of quantification for astragaloside A by this method was 0.98 μg. See the results below. Figures 4-5 .

[0062] Example 8 This experiment uses a spiking recovery rate test to verify the accuracy of the method. The specific method is as follows.

[0063] Reference solution: Accurately weigh 4.92 mg of astragaloside A reference standard, place it in a 10 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and the solution is ready.

[0064] Reference stock solution: Accurately weigh 13.98 mg of astragaloside A reference standard, place it in a 250 mL volumetric flask, add 15 mL of n-butanol and sonicate to dissolve, add water to the mark, and shake well to obtain the solution.

[0065] Test solution: Accurately measure 25 mL of this product and place it in a separatory funnel. Accurately add 25 mL of the reference stock solution. Extract five times with 30 mL of water-saturated n-butanol each time. Combine the n-butanol extracts and wash twice with 30 mL of ammonia solution each time. Separate the n-butanol solution, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain a 100% spiked test solution. Prepare six parallel solutions. Accurately measure 10 μL of the test solution, 5 μL and 20 μL of the reference solution, respectively, and inject for analysis. Calculate the recovery rate. The results are shown in Table 11.

[0066] Calculation formula: Recovery rate = (Measured value - Content) / Amount added 100%.

[0067] Table 11 Results of the recovery rate test The results showed that the recoveries of the six 100% concentration spiked test solutions ranged from 86.77% to 99.5%, with an average recovery rate of 93.20%; the RSD was 5.47%, which was less than 6%, indicating that the method was accurate.

[0068] Example 9 In this embodiment, multiple batches of samples were measured, and the specific method is as follows.

[0069] Reference solution: Accurately weigh 4.77 mg of astragaloside A reference standard, place it in a 10 mL volumetric flask, dissolve and dilute with methanol to the mark, and shake well to obtain the solution.

[0070] Take ten batches of samples and prepare test solutions according to the method provided in Example 1.

[0071] Based on the method of Example 1, 10 μL of the test solution and 5 μL and 20 μL of the reference solution were accurately measured and injected for analysis. The sample content was calculated. The results are shown in Table 12.

[0072] Table 12 Results of Astragaloside A Content Determination in Ten Batches of Samples Example 10 In this embodiment, the limit for sample content is determined, and the specific method is as follows.

[0073] (1) Limit control of Astragalus membranaceus content The 2020 edition of the Chinese Pharmacopoeia, Part I, specifies the content of processed Astragalus membranaceus as follows: Calculated on a dried basis (moisture content not exceeding 10.0%), this product contains astragaloside A (C... 41 H 68 O 14 The content of astragaloside A in the medicinal materials must not be less than 0.060%. Since the implementation of the 2020 edition of the Pharmacopoeia standard for processed Astragalus membranaceus, the company has purchased nine batches of medicinal materials, with the highest content being 0.200% and the lowest being 0.119%. Considering the fluctuations in the content of medicinal materials in the market, the content of astragaloside A in the medicinal materials will be determined based on a 30% fluctuation between the lowest and highest historical values ​​of the content, i.e., the lower limit of astragaloside A content in the medicinal materials is 0.119%. (1-30%) = 0.083%, with an upper limit of 0.200%. (1+30%)=0.26%.

[0074] The 2025 edition of the Chinese Pharmacopoeia requires that the astragaloside A content in processed Astragalus membranaceus should not be less than 0.060%. The company plans to set the astragaloside A content range in processed Astragalus membranaceus to be 0.083% to 0.26%, which is higher than the pharmacopoeia standard requirement.

[0075] (2) Content limit control of compound astragalus oral liquid Each 1ml of Compound Astragalus Oral Solution is equivalent to 0.091g of processed Astragalus root. Based on the pharmacopoeia standard limit for processed Astragalus root, this product contains at least 54.6μg (0.091g) of astragaloside A. 0.060% 10 6= 54.6 μg). Based on the content of multiple batches of Compound Astragalus Oral Liquid collected and measured using the newly developed method, the average extraction transfer rate was 20.56%. The transfer rate range was calculated as ±30% of the average transfer rate. Therefore, the lower limit of the astragaloside A content range in Compound Astragalus Oral Liquid is 0.091. 0.083% 10 6 (20.56%) (0.7%) = 11.0, the upper limit of content is: 0.091 0.26% 10 6 (20.56%) 1.3) = 63.2.

[0076] The final formulation is: Each 1mL of this product contains astragaloside A (C... 41 H 68 O 14 The calculated value should be 11.0 μg to 63.2 μg.

[0077] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of individual raw materials in the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for determining the content of astragaloside A in compound astragalus oral liquid using HPLC-ELSD, characterized in that, The steps include: determining the content of astragaloside A in the prepared reference solution and test solution using HPLC-ELSD, recording the chromatograms, and calculating the content of astragaloside A. In the HPLC-ELSD method, the acetonitrile-water system is used as the mobile phase, and the flow rate of the mobile phase is 0.1~10 mL / min; The column temperature is 20~30℃; The injection volume is 1~20 μL; The theoretical plate value, calculated based on the astragaloside A peak, is no less than 10,000. The drift tube temperature of the ELSD detector is 96~102℃.

2. The method according to claim 1, characterized in that, The preparation method of the reference solution is as follows: Take the astragaloside A reference standard, accurately weigh it, add methanol to prepare a solution containing 0.5 mg per 1 mL.

3. The method according to claim 1, characterized in that, The preparation method of the test solution is as follows: accurately measure the sample, extract it several times by shaking with water-saturated n-butanol, and combine the n-butanol solutions; wash it several times with ammonia solution, evaporate the n-butanol solution to dryness, dissolve the residue in methanol and transfer it to a volumetric flask, add methanol to the mark, shake well, and the test solution is obtained.

4. The method according to claim 1, characterized in that, In the acetonitrile-water system, the volume ratio of acetonitrile to water is (20~40):(60~80).

5. The method according to claim 1, characterized in that, In the acetonitrile-water system, the volume ratio of acetonitrile to water is 32:

68.

6. The method according to claim 1, characterized in that, The mobile phase flow rate was 1 mL / min.

7. The method according to claim 1, characterized in that, The column temperature is 25℃.

8. The method according to claim 1, characterized in that, The drift tube temperature of the ELSD detector is 100℃.

9. The method according to claim 1, characterized in that, The chromatographic column is a YMC-Triart C18.

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