Method for detecting and analyzing plinabulin and related impurities thereof

Abstract

The invention provides a detection and analysis method of plinabulin and related impurities thereof. The detection and analysis method comprises the following steps: (1) preparing a to-be-detected sample solution; the sample to be detected is a chemical product containing plinabulin and / or related impurities thereof; and (2) detecting the to-be-detected sample solution by adopting high performance liquid chromatography, wherein the detection conditions are as follows: a, a chromatographic column: filling a C18 bonded silica gel chromatographic column resistant to a high-proportion water phase; the mobile phase is composed of a mobile phase A and a mobile phase B, the mobile phase A is a monopotassium phosphate buffer solution, and the mobile phase B is acetonitrile; c, the flow rate is 0.6 ml / min; and d, the detection wavelength is selected from 200 to 320 nm. According to the method, the separation of plinabulin from various related impurities can be realized.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis, specifically relating to a method for the detection and analysis of punabulin and its related impurities. Background Technology

[0002] Punabulin is a synthetic analogue of diketopiperazine phenylastidinium, found in marine and terrestrial Aspergillus species. It binds to or near the colchicine binding site on tubulin monomers, inducing tubulin depolymerization and monolayer permeability in vascular endothelial cells even at low concentrations. Therefore, punabulin exhibits favorable pharmacokinetic, pharmacodynamic, and safety characteristics in patients with advanced malignant tumors.

[0003] However, various impurities exist in punabulin products, some introduced by the raw materials and others newly generated during the synthesis reaction. It is well known that the impurity content in pharmaceutical products directly affects the quality and efficacy of the drug. Therefore, establishing a simple, rapid, economical, and practical method for detecting related substances in punabulin is particularly important for the quality control of punabulin. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a method for the detection and analysis of punabulin and its related impurities. This method can achieve the separation of punabulin from various related impurities.

[0005] To achieve the above objectives, the present invention provides a method for detecting and analyzing punabulin and its related impurities, comprising the following steps: (1) preparing a sample solution to be tested; wherein the sample to be tested is a chemical product containing punabulin and / or its related impurities; (2) detecting the sample solution to be tested using high performance liquid chromatography, wherein the detection conditions are:

[0006] a. Chromatographic column: C18 bonded silica gel column packed with high proportion of aqueous phase;

[0007] b. Mobile phase: Composed of mobile phase A and mobile phase B, wherein mobile phase A is potassium dihydrogen phosphate buffer and mobile phase B is acetonitrile;

[0008] c. Flow rate: 0.6 ml / min;

[0009] d. The detection wavelength is selected from 200-320nm;

[0010] The structure of Punablin is as follows:

[0011] According to a specific embodiment of the present invention, preferably, the potassium dihydrogen phosphate buffer solution is prepared by adjusting the pH to 3.5 with phosphoric acid.

[0012] According to a specific embodiment of the present invention, preferably, the molar concentration of the potassium dihydrogen phosphate buffer in the mobile phase A is in the range of 0.01-0.1 mol / L, more preferably, the molar concentration of the potassium dihydrogen phosphate buffer is 0.01 mol / L or 0.02 mol / L.

[0013] According to a specific embodiment of the present invention, preferably, the detection conditions further include a column temperature of 25°C-40°C, more preferably, the column temperature is 35°C.

[0014] According to a specific embodiment of the present invention, preferably, the chromatographic column is a Waters Xselect HSS T3 chromatographic column, more preferably, the chromatographic column is a Waters Xselect HSS T3 chromatographic column with a specification of 4.6mm × 150mm, 2.7μm or 3.5μm.

[0015] According to a specific embodiment of the present invention, preferably, the detection wavelength is selected from one or more combinations of 218nm, 248nm, and 230nm.

[0016] According to a specific embodiment of the present invention, preferably, the injection volume is selected from 2-100 μl, more preferably, the injection volume is 5 μl.

[0017] According to a specific embodiment of the present invention, preferably, the relevant impurities include one or more combinations of impurities 1 to 17, wherein the structures of impurities 1 to 17 are as follows:

[0018]

[0019]

[0020] According to a specific embodiment of the present invention, preferably, the detection and analysis method employs gradient elution, and the initial volume fraction of mobile phase A during the detection process is 100%-80%, more preferably 95%, based on the volume fraction of mobile phase A.

[0021] According to a specific embodiment of the present invention, preferably, the mobile phase elution procedure in the detection process, based on the volume fraction of mobile phase A, includes:

[0022] 0min: 95%;

[0023] 25 min: 40%-50%;

[0024] 30min: 30%-40%.

[0025] According to a specific embodiment of the present invention, preferably, the mobile phase elution procedure during the detection process, based on the volume fraction of mobile phase A, further includes: 15 min: 55%-70%.

[0026] According to a specific embodiment of the present invention, preferably, the mobile phase elution procedure during the detection process, based on the volume fraction of mobile phase A, further includes: 35-40 min: 30%-20%.

[0027] According to a specific embodiment of the present invention, preferably, the mobile phase elution procedure during the detection process, based on the volume fraction of mobile phase A, is as follows:

[0028] 0min: 95%;

[0029] 25 min: 50%;

[0030] 30min: 30%;

[0031] 31 min: 95%;

[0032] 38 min: 95%;

[0033] or,

[0034] 0min: 95%;

[0035] 25 min: 40%;

[0036] 30min: 40%;

[0037] 31 min: 95%;

[0038] 38 min: 95%;

[0039] or,

[0040] 0min: 95%;

[0041] 15min: 55%;

[0042] 25 min: 40%;

[0043] 30min: 40%;

[0044] 31 min: 95%;

[0045] 38 min: 95%;

[0046] or,

[0047] 0min: 90%;

[0048] 25 min: 30%;

[0049] 30min: 30%;

[0050] 35 min: 20%;

[0051] 36 min: 90%;

[0052] 45min: 90%;

[0053] or,

[0054] 0min: 95%;

[0055] 25 min: 40%;

[0056] 28min: 40%;

[0057] 33 min: 30%;

[0058] 38min: 30%;

[0059] 39 min: 95%;

[0060] 45min: 95%.

[0061] According to a specific embodiment of the present invention, preferably, in step (1), punablin and / or its related impurities are prepared into the test sample solution using an acetonitrile-water solution; more preferably, the volume percentage of the acetonitrile-water solution is selected from 20% to 80%; and even more preferably, the volume percentage of the acetonitrile-water solution is selected from 20%, 40%, or 80%.

[0062] According to a specific embodiment of the present invention, preferably, in step (1), the product containing purnabulin and / or its related impurities is prepared into a test sample solution with a concentration of 5-200 μg / ml using an acetonitrile-water solution with a volume percentage of 20%-80%.

[0063] According to a specific embodiment of the present invention, preferably, in the detection and analysis method, the flow rate is 0.6 ml / min; the column temperature is 35°C; the detection wavelength is one or a combination of two or more of 218 nm, 230 nm and 248 nm; and the injection volume is 5 μl.

[0064] The present invention provides a method for the detection and analysis of punabulin and its related impurities. By rationally setting the chromatographic column packing and mobile phase, it can achieve the separation of multiple components in the sample to be tested. The method is highly specific and accurate, uses readily available and conventional reagents, and is simple and easy to operate, thus filling the gap in the analytical methods for punabulin and its impurities. Attached Figure Description

[0065] Figure 1 The chromatogram of Example 1;

[0066] Figure 2 The chromatogram of Example 2;

[0067] Figure 3 The chromatogram is for Example 3;

[0068] Figure 4 The chromatogram is for Example 4;

[0069] Figure 5 The chromatogram is for Example 4;

[0070] Figure 6 The chromatogram is for Comparative Example 1;

[0071] Figure 7 The chromatogram is for Comparative Example 2;

[0072] Figure 8 The chromatogram for Comparative Example 2 is shown below.

[0073] Figure 9 The chromatogram is for Comparative Example 2;

[0074] Figure 10 The chromatogram is for Comparative Example 3;

[0075] Figure 11 The chromatogram is for Example 5;

[0076] Figure 12 This is the chromatogram for Comparative Example 4;

[0077] Figure 13 The chromatogram is shown in Example 6.

[0078] Figure 14 The chromatogram is for Comparative Example 5;

[0079] Figure 15 This is the chromatogram of Comparative Example 6;

[0080] Figure 16 This is the chromatogram of Example 7. Detailed Implementation

[0081] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0082] The sources of impurities used in the following examples and comparative examples are as follows:

[0083] Impurities 1, 2, 4, 5, 6, 8, 9, 11, and 12 were purchased from Chengdu Zhengshanda Biomedical Technology Co., Ltd.; impurities 14, 16, and 17 are common reagents and can be obtained through regular purchase; impurity 3 was prepared according to the method of Example 1 in CN115531385A; impurity 10 was prepared according to the preparation method of heterocyclic compound C in CN106279039A; impurity 15 was prepared according to the method of Example 1 in CN107011331A; impurity 7 was prepared according to the preparation method of compound 3 in Journal of Antibiotics Volume:41 Issue:4 Pages:488-93; impurity 13 was prepared according to the steps for preparing compound AE in WO2023059605A1. Punabulin was prepared according to the preparation method of punabulin in the comparative example in CN116496257A.

[0084] Example 1

[0085] This embodiment provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0086] (1) Take appropriate amounts of impurities 2 to 6, dissolve and dilute them with diluent (20% acetonitrile-water solution) to prepare a solution containing 15 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of impurity 1 and reference stock solution, dissolve and dilute them with 20% acetonitrile-water solution to prepare a solution containing 1.5 μg of each of impurity 1, impurities 2-6 per 1 ml, as the system suitability solution;

[0087] (2) Octadecylsilane-bonded silica gel (Waters Xselect HSS T3, 4.6 mm × 150 mm, 3.5 μm) was used as the packing material, with 0.02 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid) as the mobile phase A and acetonitrile as the mobile phase B.

[0088] Gradient elution was performed according to the elution procedure shown in Table 1; the flow rate was 0.6 ml per minute; the detection wavelength was 230 nm; the column temperature was 35 °C; and the injection volume was 10 μl.

[0089] Table 1

[0090] Time (minutes) Mobile phase A (volume fraction, %) Mobile phase B (volume fraction, %) 0 95 5 25 50 50 30 30 70 31 95 5 38 95 5

[0091] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 1 , Figure 1 In the middle: Peak 1 corresponds to impurity 6, peak 2 corresponds to impurity 1, peak 3 corresponds to impurity 5, peak 4 corresponds to impurity 4, peak 5 corresponds to impurity 3, and peak 6 corresponds to impurity 2. (From...) Figure 1 As shown in the spectrum, all components can be separated at the baseline, and the separation degree meets the requirements.

[0092] Example 2

[0093] This embodiment provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0094] (1) Take appropriate amounts of impurities 1-3, 5-6, and 10, dissolve and dilute them with diluent (20% acetonitrile-water solution) to prepare a solution containing 150 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of impurity 8 and the reference stock solution, dissolve and dilute them with 20% acetonitrile-water solution to prepare a solution containing 1 mg of impurity 8 and 15 μg of each of the other impurities per 1 ml, as the system suitability solution;

[0095] (2) Octadecylsilane-bonded silica gel was used as the packing material (Waters Xselect HSS T3, 4.6 mm × 150 mm, 3.5 μm). 0.02 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was performed according to the elution program shown in Table 2. The flow rate was 0.6 ml per minute. The detection wavelength was 230 nm. The column temperature was 35 °C. The injection volume was 10 μl.

[0096] Table 2

[0097]

[0098]

[0099] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 2 , Figure 2 In the middle: Peak 1 corresponds to impurity 6, peak 2 corresponds to impurity 1, peak 3 corresponds to impurity 5, peak 4 corresponds to impurity 8, peak 5 corresponds to impurity 3, peak 6 corresponds to impurity 10, and peak 7 corresponds to impurity 2. (From...) Figure 2 As shown in the spectrum, all components can be separated at the baseline, and the separation degree meets the requirements.

[0100] Example 3

[0101] This embodiment provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0102] (1) Take appropriate amounts of impurities 1-3, 5-6, 8, 11-13, dissolve and dilute them with diluent (40% acetonitrile-water solution) to prepare a solution containing 15 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of impurity 10 and the reference stock solution, dissolve and dilute them with diluent to prepare a solution containing 1 mg of impurity 10 and 1.5 μg of the remaining impurities per 1 ml, as the system suitability solution;

[0103] (2) Octadecylsilane-bonded silica gel was used as the packing material (Waters Xselect HSS T3, 4.6 mm × 150 mm, 3.5 μm). 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B. The flow rate was 0.6 mL per minute; the detection wavelength was 230 nm; the column temperature was 35 °C; the injection volume was 5 μl; gradient elution was performed according to the elution program shown in Table 3.

[0104] Table 3

[0105] Time (minutes) Mobile phase A (volume fraction, %) Mobile phase B (volume fraction, %) 0 95 5 15 55 45 25 40 60 30 40 60 31 95 5 38 95 5

[0106] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 3 , Figure 3 In the middle: Peak 1 corresponds to impurity 6, peak 2 corresponds to impurity 1, peak 3 corresponds to impurity 5, peak 4 corresponds to impurity 8, peak 5 corresponds to impurity 11, peak 6 corresponds to impurity 13, peak 7 corresponds to impurity 3, peak 8 corresponds to impurity 10, peak 9 corresponds to impurity 12, and peak 10 corresponds to impurity 2. Figure 3 As shown in the spectrum, all components can be separated at the baseline, and the separation degree meets the requirements.

[0107] Example 4

[0108] This embodiment provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0109] (1) Take appropriate amounts of impurities 7-8, 10, 12-17, dissolve and dilute with diluent (80% acetonitrile-water solution) to prepare a solution containing 15 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of purnabulin and reference stock solution, dissolve and dilute with diluent to prepare a solution containing approximately 1 mg of purnabulin and 1.5 μg of each impurity per 1 ml, as the system suitability solution.

[0110] (2) Octadecylsilane-bonded silica gel was used as the packing material (Waters Xselect HSS T3, 4.6 mm × 150 mm, 3.5 μm). 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B. The flow rate was 0.6 mL per minute. The detection wavelengths were 206 nm and 248 nm. The column temperature was 40 °C. The injection volume was 5 μl. Gradient elution was performed according to the elution program shown in Table 4.

[0111] Table 4

[0112] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 25 40 60 28 40 60 33 30 70 38 30 70 39 95 5 45 95 5

[0113] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 4 (wavelength 248nm) and Figure 5 (wavelength 206nm) Figure 4 In the middle: Peak 1 corresponds to impurity 8, Peak 2 corresponds to impurity 13, Peak 4 corresponds to impurity 17, Peak 5 corresponds to impurity 14, Peak 6 corresponds to impurity 7, Peak 7 corresponds to impurity 10, Peak 8 corresponds to impurity 15, Peak 9 corresponds to impurity 12, and Peak 10 corresponds to Punabulin. Figure 5 Peak 3 corresponds to impurity 16. (From...) Figure 4 and Figure 5 As shown in the spectrum, all components can be separated at the baseline, and the separation degree meets the requirements.

[0114] Example 5

[0115] This embodiment provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0116] (1) Take appropriate amounts of impurities 7-8, 10, 12-15, dissolve and dilute with diluent (80% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of punabulin and the reference stock solution, dissolve and dilute with diluent to prepare a solution containing approximately 1 mg of punabulin and 15 μg of each impurity per 1 ml, as the system suitability solution;

[0117] (2) An octadecylsilane-bonded silica column (Waters Xbridge RP C18, 4.6 mm × 150 mm, 3.5 μm) was used with 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.6 mL per minute; the detection wavelength was 248 nm; the column temperature was 35 °C; the injection volume was 5 μl; gradient elution was performed according to the elution program shown in Table 5.

[0118] Table 5

[0119] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 25 30 70 30 30 70 35 20 80 36 90 10 45 90 10

[0120] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 11 , Figure 11 In the middle: Peak 1 is impurity 8; Peak 2 is impurity 13; Peak 3 is impurity 14; Peak 4 is impurity 7; Peak 5 is impurity 10; Peak 6 is impurity 15; Peak 7 is impurity 12; Peak 8 is Punabulin. Figure 11 As shown in the spectrum, all components can be separated at the baseline, and the separation degree meets the requirements.

[0121] Example 6

[0122] This embodiment provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0123] (1) Take appropriate amounts of impurities 1-3, 5-6, 8-9, dissolve and dilute with diluent (20% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per ml, as the reference stock solution; take appropriate amounts of impurity 8 and the reference stock solution, dissolve and dilute with diluent to prepare a solution containing approximately 1 mg of impurity 8 and 15 μg of the remaining impurities per ml, as the system suitability solution;

[0124] (2) An octadecylsilane-bonded silica column (Waters Xselect HSS T3, 4.6 mm × 150 mm, 2.7 μm) was used with 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.6 mL per minute; the detection wavelength was 230 nm; the column temperature was 35 °C; the injection volume was 10 μl; gradient elution was performed according to the elution program shown in Table 6.

[0125] Table 6

[0126]

[0127]

[0128] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 13 , Figure 13 In the chromatogram shown, peak 1 represents impurity 6, peak 2 represents impurity 1, peak 3 represents impurity 5, peak 4 represents impurity 8, peak 5 represents impurity 3, peak 6 represents impurity 9, and peak 7 represents impurity 2. Figure 13 As shown in the spectrum, all components can be separated at the baseline, and the separation degree meets the requirements.

[0129] Example 7

[0130] This embodiment provides a method for detecting and analyzing punabulin and its related impurities, which includes the following steps:

[0131] (1) Take appropriate amounts of impurities 1-3, 8, 11-13, dissolve them with an appropriate amount of N,N-dimethylformamide, and dilute with a diluent (50% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per ml, as the reference stock solution; take appropriate amounts of impurity 10 and the reference stock solution, dissolve them with a diluent, and dilute to prepare a solution containing approximately 1 mg of impurity 10 and 15 μg of the remaining impurities per ml, as the system suitability solution;

[0132] (2) An octadecylsilane-bonded silica column (Waters Xselect HSS T3, 4.6 mm × 150 mm, 3.5 μm) was used with 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.6 ml per minute; the detection wavelength was 218 nm; the column temperature was 35 °C; the injection volume was 5 μl; and gradient elution was performed according to the elution program shown in Table 7.

[0133] Table 7

[0134] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 25 30 70 30 30 70 35 20 80 36 90 10 45 90 10

[0135] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 16 , Figure 16 In the chromatogram shown, peak 1 represents impurity 1, peak 2 represents impurity 8, peak 3 represents impurity 11, peak 4 represents impurity 13, peak 5 represents impurity 3, peak 6 represents impurity 10, peak 7 represents impurity 12, and peak 8 represents impurity 2. Figure 16 As shown in the spectrum, all components can be separated at the baseline, and the separation degree meets the requirements.

[0136] Comparative Example 1

[0137] This comparative example provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0138] (1) Take appropriate amounts of impurities 7-8 and 12-15, dissolve them in an appropriate amount of N,N-dimethylformamide, and dilute them with a diluent (80% acetonitrile-water solution) to prepare a solution containing 150 μg of each impurity per ml, which is used as the reference stock solution; take appropriate amounts of punabulin and the reference stock solution, dissolve them in a diluent, and dilute them to prepare a solution containing approximately 1 mg of punabulin and 1.5 μg of each impurity per ml, which is used as the system suitability solution;

[0139] (2) An octadecylsilane-bonded silica column (Waters Xbridge RP C18, 4.6 mm × 150 mm, 3.5 μm) was used with 0.1% phosphoric acid aqueous solution (pH 3.5) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.6 ml per minute; the detection wavelength was 210 nm; the column temperature was 35 °C; the injection volume was 5 μl; gradient elution was performed according to the elution program shown in Table 8;

[0140] Table 8

[0141] Time (min) Mobile phase A (volume fraction, %) Mobile phase B (volume fraction, %) 0 90 10 10 90 10 35 30 70 40 10 90 45 10 90 46 90 10 48 90 10

[0142] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 6 , Figure 6 In the chromatogram shown, peak 1 corresponds to solvent N,N-dimethylformamide + impurity 13 + impurity 8 (peak overlap); peak 2 is impurity 8 (crack in impurity 8); peak 3 is an unknown impurity; peaks 4 and 5 are impurity 14 (crack in impurity 14); peak 6 is impurity 7; peak 7 is impurity 12; peak 8 is impurity 15; and peak 9 is purnabulin.

[0143] according to Figure 6 It can be seen that the analytical method is not effective in separating impurity peaks, and there are peak splitting and overlap.

[0144] Comparative Example 2

[0145] This comparative example provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0146] (1) Take appropriate amounts of impurities 7-8, 10, 12-15, dissolve them with an appropriate amount of N,N-dimethylformamide, and then dissolve and dilute them with a diluent (80% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of punabulin and the reference stock solution, dissolve and dilute them with a diluent to prepare a solution containing approximately 1 mg of punabulin and 15 μg of each impurity per 1 ml, as the system suitability solution;

[0147] (2) An octadecylsilane-bonded silica column (Waters Xbridge RP C18, 4.6 mm × 150 mm, 3.5 μm) was used with 0.01 mol / L ammonium phosphate solution (pH 3.5) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.6 mL per minute; the detection wavelength was 248 nm; the column temperature was 35 °C; the injection volume was 5 μl; gradient elution was performed according to the elution program shown in Table 9.

[0148] Table 9

[0149] Time (min) Mobile phase A (volume fraction, %) Mobile phase B (volume fraction, %) 0 90 10 25 30 70 30 30 70 35 20 80 36 90 10 45 90 10

[0150] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 7 , Figure 7 In the chromatogram shown, peak 1 is impurity 8; peak 2 is impurity 13; peak 3 is impurity 14; peak 4 is impurity 7; peak 5 is impurity 10; peak 6 is impurity 15; peak 7 is impurity 12; and peak 8 is punabulin.

[0151] Using mobile phase A of 0.01 mol / L ammonium phosphate, and with other conditions remaining the same, injecting blank solvent (80% acetonitrile-water solution) yielded... Figure 8 The chromatogram mentioned above, Figure 8 The blank solvent showed a baseline bulge in the 12-14 min range, which is not conducive to adding other similar polarity impurities in subsequent methods and is also prone to integration misjudgment.

[0152] Using mobile phase A of 0.01 mol / L potassium dihydrogen phosphate, and with other conditions remaining the same, injecting blank solvent (80% acetonitrile-water solution) yielded... Figure 9 The chromatogram mentioned above, Figure 8 The results showed that in the blank control, there was no baseline bulge in the 12-14 min range, which did not affect the impurity peaks.

[0153] Therefore, when the mobile phase A is 0.01 mol / L potassium dihydrogen phosphate, the baseline interference is minimal and the separation effect is optimal.

[0154] Comparative Example 3

[0155] This comparative example provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0156] (1) Take appropriate amounts of impurities 7-8, 10, 12-15, dissolve and dilute with diluent (80% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of punabulin and the reference stock solution, dissolve and dilute with diluent to prepare a solution containing approximately 1 mg of punabulin and 15 μg of each impurity per 1 ml, as the system suitability solution;

[0157] (2) An octadecylsilane-bonded silica column (Waters Xbridge RP C18, 4.6 mm × 150 mm, 3.5 μm) was used with 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.6 mL per minute; the detection wavelength was 248 nm; the column temperature was 35 °C; the injection volume was 5 μl; gradient elution was performed according to the elution program shown in Table 10.

[0158] Table 10

[0159] Time (min) Mobile phase A (volume fraction, %) Mobile phase B (volume fraction, %) 0 90 10 25 30 70 30 30 70 35 20 80 36 90 10 45 90 10

[0160] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 10 , Figure 10 In the chromatogram shown, peak 1 is impurity 8; peak 2 is impurity 13; peak 3 is impurity 14; peak 4 is impurity 7; peak 5 is impurity 10; peak 6 is impurity 15; peak 7 is impurity 12; and peak 8 is punabulin.

[0161] according to Figure 10 It can be seen that the analytical method is not effective in separating impurity peaks, and impurity peak 1 has cracks.

[0162] Comparative Example 4

[0163] This comparative example provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0164] (1) Take appropriate amounts of impurities 1-3, 5-6, and 9, dissolve and dilute them with diluent (20% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of impurity 8 and the reference stock solution, dissolve and dilute them with diluent to prepare a solution containing approximately 1 mg of impurity 8 and 15 μg of each of the other impurities per 1 ml, as the system suitability solution;

[0165] (2) An octadecylsilane-bonded silica column (Waters Xselect HSS T3, 4.6 mm × 150 mm, 2.7 μm) was used with 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.5 mL per minute; the detection wavelength was 230 nm; the column temperature was 35 °C; the injection volume was 10 μl; gradient elution was performed according to the elution program shown in Table 11.

[0166] Table 11

[0167]

[0168]

[0169] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 12 , Figure 12 In the chromatogram shown, peak 1 is impurity 6, peak 2 is impurity 6, peak 3 is impurity 1, peak 4 is impurity 5, peak 5 is impurity 8, peak 6 is impurity 3, peak 7 is impurity 9, and peak 8 is impurity 2.

[0170] Figure 12 In the chromatogram shown, impurity 6 shows cracks, indicating poor separation.

[0171] Comparative Example 5

[0172] This comparative example provides a method for the detection and analysis of punabulin and its related impurities, using a flow rate of 0.7 ml / min, which includes the following steps:

[0173] (1) Take appropriate amounts of impurities 1-3, 5-6, and 9, dissolve and dilute them with diluent (20% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per 1 ml, as the reference stock solution; take appropriate amounts of impurity 8 and the reference stock solution, dissolve and dilute them with diluent to prepare a solution containing approximately 1 mg of impurity 8 and 15 μg of each of the other impurities per 1 ml, as the system suitability solution;

[0174] (2) An octadecylsilane-bonded silica column (Waters Xselect HSS T3, 4.6 mm × 150 mm, 2.7 μm) was used with 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.7 mL per minute; the detection wavelength was 230 nm; the column temperature was 35 °C; the injection volume was 10 μl; gradient elution was performed according to the elution program shown in Table 12;

[0175] Table 12

[0176] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 25 40 60 30 40 60 31 95 5 38 95 5

[0177] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 14 , Figure 14 In the chromatogram shown, peak 1 is impurity 6, peak 2 is impurity 6, peak 3 is impurity 1, peak 4 is impurity 5, peak 5 is impurity 8, peak 6 is impurity 3, peak 7 is impurity 9, and peak 8 is impurity 2.

[0178] Figure 14 In the chromatogram shown, the peak of impurity 6 shows cracks, indicating poor separation.

[0179] Comparative Examples 4 and 5 show that when the flow rate is 0.5 ml / min and 0.7 ml / min, some impurity peaks show cracks, and good separation effect cannot be achieved.

[0180] Comparative Example 6

[0181] This comparative example provides a method for detecting and analyzing impurities related to punabulin, which includes the following steps:

[0182] (1) Take appropriate amounts of impurities 1-3, 8, 11-13, dissolve them in an appropriate amount of N,N-dimethylformamide, and dilute them with a diluent (50% acetonitrile-water solution) to prepare a solution containing approximately 150 μg of each impurity per ml, as the reference stock solution; take appropriate amounts of impurity 10 and the reference stock solution, dissolve them in a diluent, and dilute them to prepare a solution containing approximately 1 mg of impurity 10 and 1.5 μg of the remaining impurities per ml, as the system suitability solution;

[0183] (2) An octadecylsilane-bonded silica column (Waters Xbridge RP C18, 4.6 mm × 150 mm, 3.5 μm) was used with 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B; the flow rate was 0.6 mL per minute; the detection wavelength was 218 nm; the column temperature was 35 °C; the injection volume was 5 μl; gradient elution was performed according to the elution program shown in Table 13;

[0184] Table 13

[0185] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 25 30 70 30 30 70 35 20 80 36 90 10 45 90 10

[0186] (3) Take a system suitability solution, inject it under the conditions of step (2) for analysis, and then obtain the chromatogram. Figure 15 , Figure 15 In the chromatogram shown, peak 1 is N,N-dimethylformamide + impurity 1, peak 2 is impurity 8, peak 3 is impurity 11, peak 4 is impurity 13, peak 5 is impurity 3, peak 6 is impurity 10, peak 7 is impurity 12, and peak 8 is impurity 2.

[0187] Chromatography Figure 15 The results showed that impurity 1 completely overlapped with the solvent N,N-dimethylformamide, making it impossible to achieve a good separation effect.

Claims

1. A method for detecting and analyzing punabulin and its related impurities, comprising the following steps: (1) Prepare the sample solution to be tested; The sample to be tested is a chemical product containing purnabulin and / or its related impurities; (2) The sample solution to be tested was detected by high performance liquid chromatography under the following conditions: a. Chromatographic column: C18 bonded silica gel column packed with high proportion of aqueous phase; b. Mobile phase: Composed of mobile phase A and mobile phase B, wherein mobile phase A is potassium dihydrogen phosphate buffer and mobile phase B is acetonitrile; c. Flow rate: 0.6 ml / min; d. The detection wavelength is selected from 200-320nm.

2. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, The chromatographic column is a T3 type chromatographic column; Preferably, the chromatographic column is a Waters Xselect HSS T3 column with dimensions of 4.6 mm × 150 mm, 2.7 μm or 3.5 μm.

3. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, The relevant impurities include one or more combinations of impurities 1 to 17, and the structures of impurities 1 to 17 are as follows:

4. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, Based on the volume fraction of mobile phase A, the initial volume fraction of mobile phase A during the detection process is 100%-80%; Preferably, the initial volume fraction of mobile phase A is 95%.

5. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, The potassium dihydrogen phosphate buffer solution was prepared by adjusting the pH to 3.5 with phosphoric acid. Preferably, the molar concentration range of the potassium dihydrogen phosphate buffer solution is 0.01-0.1 mol / L; Preferably, the molar concentration of the potassium dihydrogen phosphate buffer solution is 0.01 mol / L or 0.02 mol / L.

6. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, Based on the volume fraction of mobile phase A, the mobile phase elution procedure in the detection process includes: 0min: 95%; 25 min: 40%-50%; 30min: 30%-40%.

7. The method for detecting and analyzing Punabulin and its related impurities according to claim 6, wherein, Based on the volume fraction of mobile phase A, the mobile phase elution program during the detection process includes: 15 min: 55%-70%; Preferably, the mobile phase elution program further includes: 35-40 min: 30%-20%.

8. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, Based on the volume fraction of mobile phase A, the mobile phase elution procedure during the detection process is as follows: 0min: 95%; 25 min: 50%; 30min: 30%; 31 min: 95%; 38 min: 95%; or, 0min: 95%; 25 min: 40%; 30min: 40%; 31 min: 95%; 38 min: 95%; or, 0min: 95%; 15min: 55%; 25 min: 40%; 30min: 40%; 31 min: 95%; 38 min: 95%; or, 0min: 90%; 25 min: 30%; 30min: 30%; 35 min: 20%; 36 min: 90%; 45min: 90%; or, 0min: 95%; 25 min: 40%; 28min: 40%; 33 min: 30%; 38min: 30%; 39 min: 95%; 45min: 95%.

9. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, In step (1), Punabulin and / or its related impurities are prepared into the test sample solution using acetonitrile-water solution; Preferably, the volume percentage of the acetonitrile-water solution is selected from 20% to 80%, more preferably 20%, 40% or 80%.

10. The method for detecting and analyzing Punabulin and its related impurities according to claim 1, wherein, The detection wavelength is selected from one or more combinations of 218nm, 248nm, and 230nm.

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