Detection method of butylphthalide related substances

Abstract

The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a detection method of butylphthalide related substances. A SUPELCOSILTML-DP chromatographic column is adopted to carry out liquid chromatographic analysis on a butylphthalide sample, accurate detection of sixteen related substances in butylphthalide and the content of the substances is achieved at the same time, separation of butylphthalide and impurities can be achieved, all impurity peaks do not interfere with a main peak, and all chromatographic peaks can be completely separated; the detection cost is low, the used mobile phase is simple, the preparation is convenient, and the time is saved. The detection method provided by the invention can meet the rapid detection of butylphthalide related substances, is high in accuracy and sensitivity and good in reproducibility and stability, can be used for quality control and comprehensive evaluation of butylphthalide, and provides reliable guarantee for improving and controlling the quality of butylphthalide.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly to a method for detecting related substances of butylphthalide. Background Art

[0002] Butylphthalide is a drug used for the treatment of mild to moderate acute ischemic stroke. During the production and storage of butylphthalide, some related substances may be generated, and these related substances may affect the safety and effectiveness of the drug. Currently, the synthetic route of butylphthalide is as follows:

[0003]

[0004] From the above synthetic route, the synthesis of butylphthalide includes three steps. Step 1: Raw material - 1 and raw material - 2 react under the conditions of potassium tert - butoxide and potassium hydroxide at a temperature of 0°C - 10°C to form intermediate - 1. Step 2: Intermediate - 1 reacts under the conditions of sodium borohydride at a temperature of 20°C - 30°C to form intermediate - 2. Step 3: Intermediate - 2 is refined into butylphthalide. During the production process, starting materials, intermediates, polymers, side - reaction products, degradation products during storage, etc. may all be introduced into the final product as impurities, affecting the product quality. Through research, some impurities and the reasons for their formation are as follows:

[0005] Butylphthalide will generate impurity A after hydrolysis and dehydrogenation; the residual impurities in intermediate - 1 undergo a reductive cyclization reaction under the reaction conditions of step 2 to generate impurity B, impurity C, impurity D, and impurity E; butylphthalide will generate isomeric impurities during storage, namely impurity F and impurity G; impurity H is an isomeric impurity of butylphthalide; the residual impurities in raw material - 1 hydrolyze under the reaction conditions of step 2 to generate impurity J; the residual impurities in intermediate - 1 participate in a reduction reaction under the reaction conditions of step 2 to generate impurity K, or impurity B hydrolyzes to generate impurity K; the residual impurities in intermediate - 1 undergo a reduction reaction under the reaction conditions of step 2 to generate impurity M, or impurity D hydrolyzes to generate impurity M; impurity A undergoes an oxidation - reduction reaction in step 2 to generate impurity N, or butylphthalide hydrolyzes to generate impurity N; the residual impurities in intermediate - 1 participate in a reduction reaction under the reaction conditions of step 2 to generate impurity O, or impurity E hydrolyzes to generate impurity O; butylphthalide undergoes oxidative degradation to generate impurity P; the structure of impurity A undergoes tautomerism, changing from a keto structure to an enol structure, and undergoes a ring - closing reaction under acidic conditions to generate impurity Q; the residual impurities in intermediate - 2 are impurity 20.

[0006] Among them, the specific molecular structures of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20 are as follows:

[0007]

[0008] Therefore, it is of great significance to study and develop a reasonable separation and detection method for determining the content of phthalide drugs and their related substances, which is of great importance for ensuring the quality of phthalide derivatives drugs, so as to ensure the effectiveness and safety of drugs and further realize the controllability of drug quality. Summary of the Invention

[0009] Aiming at the problem that the related substances of butylphthalide cannot be effectively detected in the prior art, the present invention provides a detection method for butylphthalide related substances. The detection method provided by the present invention can quickly and effectively separate and detect the impurities in butylphthalide, thus ensuring the safety of drugs and inhibiting the generation of side effects.

[0010] To solve the above technical problems, the technical solution provided by the present invention is:

[0011] A detection method for butylphthalide related substances, wherein the butylphthalide related substances include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q and impurity 20, and the detection is carried out by high performance liquid chromatography, including the following steps:

[0012] (1) Preparation of test solution and mixed reference solution:

[0013] Preparation of test solution: Dissolve the butylphthalide sample in a solvent to obtain a test solution;

[0014] Preparation of mixed reference solution: Dissolve reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q and impurity 20 in a solvent to obtain a mixed reference solution;

[0015] (2) Detect the mixed reference solution and the test solution by high performance liquid chromatography. The chromatographic conditions of the high performance liquid chromatography include:

[0016] Chromatographic column: SUPELCOSIL TM LC-DP chromatographic column;

[0017] Use a mixed solution of acetic acid aqueous solution and acetonitrile as the mobile phase;

[0018] The elution program is isocratic elution.

[0019] The existing synthesis route of butylphthalide is as follows:

[0020]

[0021] Among them, step 1: Raw material - 1 and raw material - 2 react under the conditions of potassium tert - butoxide and potassium hydroxide at a temperature of 0°C - 10°C to form intermediate - 1; step 2: Intermediate - 1 reacts under the conditions of sodium borohydride at a temperature of 20°C - 30°C to form intermediate - 2; step 3: Intermediate - 2 is refined into butylphthalide.

[0022] The method for detecting impurities in butylphthalide provided by the present invention uses a chromatographic column of a specific model for liquid chromatography analysis of butylphthalide samples, achieving accurate detection of sixteen related substances including impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20 and their contents in butylphthalide. It can achieve the separation of butylphthalide and impurities, with no interference of each impurity peak on the main peak, and complete separation between each chromatographic peak; moreover, the detection cost is low, the mobile phase used is simple and convenient to prepare, saving time.

[0023] The detection method provided by the present invention can meet the rapid detection of related substances in butylphthalide, and has high accuracy and sensitivity, good reproducibility and stability. It can be used for the quality control and comprehensive evaluation of butylphthalide, providing a reliable guarantee for improving and controlling the quality of butylphthalide.

[0024] The specific molecular structures of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20 are as follows:

[0025]

[0026] Preferably, the solvent is acetonitrile.

[0027] Preferably, the mass concentration of the acetic acid aqueous solution is 0.1% - 0.2%.

[0028] More preferably, the mass concentration of the acetic acid aqueous solution is 0.2%.

[0029] More preferably, in the mobile phase, based on the total volume of the acetic acid aqueous solution and acetonitrile being 100%, the volume percentage of the acetic acid aqueous solution in the mobile phase is 68% - 72%, and the volume percentage of acetonitrile in the mobile phase is 28% - 32%.

[0030] More preferably, the volume percentage of the acetic acid aqueous solution in the mobile phase is 70%, and the volume percentage of acetonitrile in the mobile phase is 30%.

[0031] Preferably, the detection wavelength of the high - performance liquid chromatography method is 220nm - 240nm.

[0032] Further preferably, the detection wavelength of the high performance liquid chromatography is 230 nm.

[0033] Preferably, the column temperature of the high performance liquid chromatography is 25°C - 35°C.

[0034] Further preferably, the column temperature of the high performance liquid chromatography is 30°C.

[0035] Preferably, the flow rate of the high performance liquid chromatography is 0.8 mL / min - 1.2 mL / min.

[0036] Further preferably, the flow rate of the high performance liquid chromatography is 1.0 mL / min.

[0037] Preferably, the injection volume of the high performance liquid chromatography is 10 μL - 100 μL.

[0038] Further preferably, the injection volume of the high performance liquid chromatography is 10 μL. Description of the Drawings

[0039] Figure 1 It is the liquid chromatography diagram of the blank solvent in Example 2 of the present invention;

[0040] Figure 2 It is the liquid chromatography diagram of the test solution in Example 2 of the present invention;

[0041] Figure 3

[0042] Figure 4 It is the liquid chromatography diagram of the spiked test solution in Comparative Example 1 of the present invention;

[0043] Figure 5 It is the liquid chromatography diagram of the mixed solution in Comparative Example 2 of the present invention;

[0044] Figure 6 It is the liquid chromatography diagram of the mixed solution in Comparative Example 3 of the present invention;

[0045] Among them, peak 1 is impurity J, peak 2 is impurity K, peak 3 is impurity M, peak 4 is impurity B, peak 5 is impurity 20, peak 6 is impurity N, peak 7 is impurity C, peak 8 is impurity A, peak 9 is impurity O, peak 10 is impurity D, peak 11 is impurity P, peak 12 is impurity H, peak 13 is impurity G, peak 14 is impurity F, peak 15 is butylphthalide, peak 16 is impurity Q, peak 17 is impurity E, and peak 18 is acetonitrile. Detailed Embodiments

[0046] The technical solution of the present invention will be described clearly and completely below. It should be understood that the specific embodiments described herein can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention within the scope of the described embodiments.

[0047] The butylphthalide sample used in the following of the present invention is a self-made product, and its preparation process route is as follows:

[0048]

[0049] Among them, the reaction temperature of step 1 is 0°C - 10°C, and the reaction temperature of step 2 is 20°C - 30°C.

[0050] In the present invention, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20 are all self-made impurities, and the synthesis routes of each impurity are as follows:

[0051] Synthesis route of impurity A:

[0052]

[0053] Synthesis route of impurity B:

[0054]

[0055] Synthesis route of impurity C:

[0056]

[0057] Synthesis route of impurity D:

[0058]

[0059] Synthesis route of impurity E:

[0060]

[0061] Synthesis route of impurity F:

[0062] Synthesis route of impurity G:

[0063] Synthesis route of impurity H:

[0064]

[0065] Synthesis route of impurity J:

[0066]

[0067] Synthesis route of impurity K:

[0068] Impurity M:

[0069]

[0070] Impurity N:

[0071]

[0072] Impurity O:

[0073]

[0074] Impurity P:

[0075]

[0076] Impurity Q:

[0077]

[0078] Impurity 20:

[0079]

[0080] Example 1

[0081] High performance liquid chromatography conditions for the invention detection method:

[0082] Chromatographic column: SUPELCOSIL TM LC-DP chromatographic column (4.6 mm * 250 mm, 5 μm);

[0083] Mobile phase: An aqueous acetic acid solution with a mass concentration of 0.1% and acetonitrile in a volume ratio of 7:3 are used as the mobile phase;

[0084] Flow rate: 1.0 mL / min;

[0085] Injection volume: 10 μL;

[0086] Column temperature: 30 °C;

[0087] Detection wavelength: 230 nm;

[0088] The elution program is isocratic elution.

[0089] Blank solvent: Acetonitrile;

[0090] Test solution: Weigh an appropriate amount of butylphthalide sample, dissolve it with acetonitrile and dilute it to a test solution of 1 mg / mL butylphthalide;

[0091] Mixed reference substance solution: Weigh appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20, dissolve them with acetonitrile and dilute to a mixed reference substance solution with a concentration of 5 μg / mL. That is, in the mixed reference substance solution, the concentrations of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20 are all 100 μg / mL;

[0092] Spiked test sample solution: Weigh appropriate amounts of butylphthalide sample, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20, dissolve them with acetonitrile and dilute to a mixed solution containing approximately 1 mg of butylphthalide and 1 μg of each impurity in 1 mL.

[0093] Detection method: Accurately weigh 10 μL each of the test sample solution and the mixed reference substance solution, inject them into the liquid chromatograph for detection.

[0094] Example 2

[0095] Using the above high performance liquid chromatography method to detect the above blank solvent, test sample solution, and spiked test sample solution. Among them, the liquid chromatogram results of the blank solvent, test sample solution, and spiked test sample solution are as Figures 1-3 shown. The resolution of each impurity in the spiked test sample solution was tested, and the test results are shown in Table 1. From Figures 1-3 and Table 1, it can be seen that for the method for detecting impurities in butylphthalide provided in this example, the blank solvent does not interfere with the detection of each component, the resolution between each component meets the requirements, and the method has good specificity.

[0096] Table 1

[0097] Component Retention time (min) Resolution Impurity J 3.989 — Impurity K 4.245 0.894 Impurity M 6.511 7.496 Impurity B 7.479 3.236 Impurity 20 8.314 2.726 Impurity N 9.066 2.119 Impurity C 10.318 3.364 Impurity A 11.981 4.179 Impurity O 13.381 3.014 Impurity D 15.453 4.089 Impurity P 16.510 1.974 Impurity H 19.272 4.620 Impurity G 20.835 / 21.328 2.038 Impurity F 22.654 1.559 Butylphthalide 24.121 1.858 Impurity Q 32.960 8.255 Impurity E 38.789 4.340

[0098] Example 3

[0099] Specificity test:

[0100] Blank solvent: Take acetonitrile, inject it into the liquid chromatograph, and record the chromatogram;

[0101] Undecomposed solution: Take 25 mg of butylphthalide sample, accurately weigh it, place it in a 25 mL volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well, and detect it according to the related substances detection method provided in Example 1;

[0102] Thermal destruction sample: Take an appropriate amount of butylphthalide sample, accurately weigh it, place it in a water bath at 100 °C and heat for 2 h, dissolve it with acetonitrile and dilute to the mark, shake well, and detect according to the related substance detection method provided in Example 1;

[0103] Alkali destruction sample: Take an appropriate amount of butylphthalide sample, accurately weigh it, add 0.5 mL of 0.1 mol / L sodium hydroxide solution, let it stand for 5 min, neutralize it with 0.1 mol / L hydrochloric acid, dissolve it with acetonitrile and dilute to the mark, shake well, and detect according to the related substance detection method provided in Example 1;

[0104] Acid destruction sample: Take an appropriate amount of butylphthalide sample, accurately weigh it, add 2 mL of 1 mol / L hydrochloric acid solution, let it stand for 1 h, add an appropriate amount of sodium hydroxide solution to neutralize, dissolve it with acetonitrile and dilute to the mark, shake well, and detect according to the related substance detection method provided in Example 1;

[0105] Oxidation destruction sample: Take an appropriate amount of butylphthalide sample, accurately weigh it, add 2 mL of 30% hydrogen peroxide solution, let it stand at room temperature for 2 h, then dissolve it with acetonitrile and dilute to the mark, and detect according to the related substance detection method provided in Example 1;

[0106] Light destruction sample: Take an appropriate amount of butylphthalide sample that has been irradiated with light at dual wavelengths (254 nm and 365 nm) for 2 h, accurately weigh it, place it in a 10 mL volumetric flask, dissolve it with the mobile phase and dilute to the mark, shake well, and detect according to the related substance detection method provided in Example 1. The results are shown in Table 1-2:

[0107] Table 1 Degradation impurity spectrum analysis

[0108]

[0109] Table 2 Material balance data of the sample

[0110]

[0111]

[0112] As can be seen from Table 1-2, after thermal destruction of this product, the main degradation products are impurity 20 and impurity P; after alkali destruction, the main degradation product is impurity N; after acid destruction, the main degradation products are impurity N and impurity A; after oxidation destruction, the main degradation product is impurity P; there is no obvious increase in impurities after light destruction, and the degradation products can all be well separated from the main component peak. Through different specific tests, it is proved that the chromatographic system of the present invention can achieve baseline separation of the degradation products generated under various destruction conditions from the butylphthalide main peak, and can effectively detect these degradation substances, proving that the chromatographic method has high specificity. At the same time, the material balance of the butylphthalide sample was calculated, and the materials were basically balanced.

[0113] Example 4

[0114] Detection limit and quantification limit:

[0115] Appropriately weigh samples of butylphthalide, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20. Dissolve them in acetonitrile and serially dilute them step by step. Then, precisely measure appropriate volumes of each solution, inject 10 μL successively under the chromatographic conditions provided in Example 1, record the chromatograms, and calculate the quantification limits and detection limits of butylphthalide and each impurity based on signal-to-noise ratios of S / N≈3 and S / N≈10, respectively. The results are shown in Table 3:

[0116] Table 3

[0117]

[0118]

[0119] Test conclusion: The sensitivities of the main component and each impurity meet the detection requirements.

[0120] Example 5

[0121] Linearity investigation:

[0122] Appropriately weigh samples of butylphthalide, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20. Serially dilute them with acetonitrile to finally obtain solutions with different series of concentrations. Detect them under the chromatographic conditions of Example 1. The linear results are shown in Table 4 - 20.

[0123] Table 4 Linear test results of butylphthalide

[0124]

[0125] Table 5 Linear test results of impurity A

[0126]

[0127] Table 6 Linear test results of impurity B

[0128]

[0129] Table 7 Linear test results of impurity C

[0130]

[0131] Table 8 Linear test results of impurity D

[0132]

[0133] Table 9 Linear test results of impurity E

[0134]

[0135] Table 10 Results of the linearity test for impurity F

[0136]

[0137] Table 11 Results of the linearity test for impurity G

[0138]

[0139] Table 12 Results of the linearity test for impurity H

[0140]

[0141] Table 13 Results of the linearity test for impurity J

[0142]

[0143] Table 14 Results of the linearity test for impurity K

[0144]

[0145] Table 15 Results of the linearity test for impurity M

[0146]

[0147] Table 16 Results of the linearity test for impurity N

[0148]

[0149] Table 17 Results of the linearity test for impurity O

[0150]

[0151] Table 18 Results of the linearity test for impurity P

[0152]

[0153] Table 19 Results of the linearity test for impurity Q

[0154]

[0155] Table 20 Results of the linearity test for impurity 20

[0156]

[0157] Test conclusion: As can be seen from Table 4-20, the linear relationships of all components are good, indicating that the detection method provided by the present invention has a good linear relationship.

[0158] Example 6

[0159] Repeatability test investigation:

[0160] Repeatability was evaluated by preparing 6 samples of test solution containing each impurity at the limit concentration, testing them under the same conditions, and calculating the relative standard deviation of the impurity contents in the 6 samples of test solution.

[0161] Prepare the test solution according to the method provided in Example 1, prepare 6 samples in parallel according to the above preparation method, calculate the RSD% of the known impurities, single impurities and total impurities, and the specific results are shown in Table 21:

[0162] Table 21 Results of repeatability test

[0163]

[0164] Conclusion: The repeatability of butylphthalide and each impurity is good.

[0165] Example 7

[0166] Investigation on solution stability test:

[0167] Solution stability was confirmed for the test solution over a period of time.

[0168] Test solution: Weigh accurately 25 mg of butylphthalide, add appropriate amounts of the reference stock solutions of impurity B, impurity F, and impurity 20, and dissolve and dilute with acetonitrile to prepare a solution containing about 1.0 mg per 1 mL; inject and determine at 0 h, 2 h, 4 h, 9 h, and 16 h respectively, detect according to the chromatographic conditions and method provided in Example 1, and calculate its relative standard deviation based on the peak area. The results are shown in Table 22. After research, impurities E, G, H, J, K, M, N, and O were not detected in the butylphthalide sample, and these impurities are relatively stable and do not require further investigation.

[0169] Table 22 Results of solution stability test

[0170]

[0171]

[0172] Conclusion: The mixed sample of related substances is stable within 16 hours.

[0173] Example 8

[0174] Preparation of the mixed solution: Take butylphthalide, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q, and impurity 20 respectively, weigh accurately, dissolve with acetonitrile and dilute to prepare a solution containing about 1 μg of each impurity and 1 mg of butylphthalide per 1 mL. By changing the column temperature, flow rate, and wavelength, investigate the retention time of the main peak in the mixed sample and the separation of each impurity under different conditions. The specific results are shown in Tables 23 - 25.

[0175] Table 23 Test results at different flow rates

[0176]

[0177]

[0178] Table 24 Test results at different column temperatures

[0179]

[0180] Table 25 Test results at different wavelengths

[0181]

[0182]

[0183] Conclusion: After changing the column temperature, flow rate, and wavelength in this example, there is no obvious change in the resolution of butylphthalide and its various impurities, indicating that the chromatographic method provided by the present invention has good durability.

[0184] From Figures 1-4 and the methodological investigations provided in Examples 1 - 8, it can be seen that the detection method provided by the present invention can quickly and effectively separate and determine the related substances of butylphthalide, thereby achieving an effective detection of the content of the related substances of butylphthalide.

[0185] Comparative Example 1

[0186] Adopt the detection method of the quality standard WS1-(X - 124)-2005Z of butylphthalide. Use octadecylsilane chemically bonded silica gel as the filler; use methanol - water (65:35) as the mobile phase; the column temperature is 30 °C; the flow rate is 1.0 mL per minute; the detection wavelength is 280 nm; the injection volume is 20 μL; detect the spiked test solution provided in Example 1, record the chromatogram, and the results are shown in Figure 4 .

[0187] From Figure 4 it can be seen that when using the chromatographic conditions provided in Comparative Example 1 to detect the spiked test solution, the amount of impurities detected is small, and butylphthalide and various impurities cannot be effectively separated and detected.

[0188] Comparative Example 2

[0189] The difference from the chromatographic conditions provided in Example 1 is that the chromatographic column is replaced with a Venusil Xbp phenyl chromatographic column, with a model of 4.6×250 mm, 5 μm. Other chromatographic conditions and detection conditions are the same. The mixed solution is detected, and the chromatogram is recorded. The results are shown in Figure 5 and Table 26.

[0190] Preparation of the mixed solution: Take butylphthalide samples, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, weigh accurately, dissolve and dilute with acetonitrile to prepare a solution containing about 10 μg of each impurity and 100 μg of butylphthalide per 1 mL.

[0191] Table 26

[0192] Name Retention time (min) Resolution Impurity B 3.393 — Impurity C 4.855 4.405 Impurity D 8.426 8.724 Impurity G 12.829 7.916 Impurity F, Butylphthalide 15.424 3.830 Impurity E 30.996 16.844

[0193] From Figure 5 and Table 26, it can be seen that under the chromatographic conditions provided in Comparative Example 2, impurity F and the main peak of butylphthalide cannot be effectively separated, and the accurate determination of each impurity cannot be guaranteed.

[0194] Comparative Example 3

[0195] Replace the chromatographic column with an ES Industries.Inc.Caprisil Hexyl-Phenyl chromatographic column, with a model of 4.6×250 mm, 5 μm. Other chromatographic conditions and detection conditions are the same. The mixed solution is detected, and the chromatogram is recorded. The results are shown in Figure 6 and Table 27.

[0196] Preparation of the mixed solution: Take butylphthalide samples, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity Q, and impurity 20, weigh accurately, dissolve and dilute with acetonitrile to prepare a solution containing about 10 μg of each impurity and 100 μg of butylphthalide per 1 mL.

[0197] Table 27

[0198]

[0199]

[0200] Figure 6 and Table 27, it can be seen that under the conditions of Comparative Example 3, the resolution between impurity 20 and impurity B is 0.505, and the resolution between impurity A and impurity C is 0.531. The resolution is insufficient, and the accurate determination of each impurity cannot be guaranteed.

[0201] The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting related substances of butylphthalide, characterized in that, The related substances of butylphthalide include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q and impurity 20, and are detected by high performance liquid chromatography, which comprises the following steps: (1) Preparation of the test solution and the mixed reference solution: Preparation of the test solution: Dissolve the butylphthalide sample in a solvent to obtain the test solution; Preparation of the mixed reference solution: Dissolve the reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity M, impurity N, impurity O, impurity P, impurity Q and impurity 20 in a solvent to obtain the mixed reference solution; (2) Detect the mixed reference solution and the test solution by high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography include: Chromatographic column: SUPELCOSIL TM LC-DP chromatographic column; Use a mixed solution of aqueous acetic acid solution and acetonitrile as the mobile phase; The elution program is isocratic elution.

2. The detection method of the related substances of butylphthalide according to claim 1, characterized in that, The solvent is acetonitrile.

3. The detection method of the related substances of butylphthalide according to claim 1, characterized in that, The mass concentration of the aqueous acetic acid solution is 0.1%-0.2%.

4. The detection method of the related substances of butylphthalide according to claim 3, wherein The mass concentration of the aqueous acetic acid solution is 0.2%.

5. The detection method of the related substances of butylphthalide according to claim 1, wherein, In the mobile phase, based on the total volume of the aqueous acetic acid solution and acetonitrile being 100%, the volume percentage of the aqueous acetic acid solution in the mobile phase is 68%-72%, and the volume percentage of acetonitrile in the mobile phase is 28%-32%.

6. The detection method of the related substances of butylphthalide according to claim 5, wherein The volume percentage of the aqueous acetic acid solution in the mobile phase is 70%, and the volume percentage of acetonitrile in the mobile phase is 30%.

7. The detection method of the related substances of butylphthalide according to claim 1, characterized in that, The detection wavelength of the high performance liquid chromatography is 220nm-240nm.

8. The detection method of the related substances of butylphthalide according to claim 1, characterized in that, The column temperature of the high performance liquid chromatography is 25°C-35°C.

9. The detection method of the related substances of butylphthalide according to claim 1, wherein, The flow rate of the high performance liquid chromatography is 0.8mL / min-1.2mL / min.

10. The detection method of the related substances of butylphthalide according to claim 1, characterized in that, The injection volume of the high performance liquid chromatography is 10μL-100μL.

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