A method for high performance liquid chromatography analysis of tosedostat phosphate

Abstract

The application relates to the technical field of analytical chemistry, in particular to a high-performance liquid chromatography analysis method for tosedostat phosphate, wherein the mobile phase in the high-performance liquid chromatography condition comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution comprises trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.01-0.05%, the volume fraction of triethylamine is 0-0.3%, and the volume ratio of the mobile phase A and the mobile phase B is 50:50-55:45. The trifluoroacetic acid and the triethylamine in the mobile phase are matched, the interaction between tosedostat phosphate and a chromatographic column is hindered, the peak shape is symmetrical, and peak tailing is significantly improved; meanwhile, the two can also form a buffer system; in addition, the trifluoroacetic acid can enhance the retention capacity of tosedostat phosphate on the chromatographic column and improve separation selectivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analytical chemistry, in particular to a high performance liquid chromatography analysis method of tosedostat phosphate. BACKGROUND

[0002] Tosedostat phosphate is the first pet cancer tumor targeted drug in the world, which is often prepared into tosedostat tablets. It is a high selectivity multi-target tyrosine kinase receptor inhibitor, which can inhibit multiple tyrosine kinase receptors. It can produce anti-angiogenic and anti-tumor effects by blocking the blood and nutrient supply of tumor cell growth. Tosedostat phosphate has multiple targets and belongs to a broad-spectrum tumor targeted drug, which can be used for pet dogs and cats at the same time, and has good tolerance without toxic side effects caused by conventional chemotherapy.

[0003] Generally, impurity compounds will inevitably exist in tosedostat phosphate, and the detection of impurity compounds is of great significance for the quality control of tosedostat phosphate. High performance liquid chromatography is divided into reverse phase and normal phase, which is determined by the relative polarity of the stationary phase and the mobile phase. Among them, reverse phase high performance liquid chromatography: using a relatively weak stationary phase, and using a more polar solvent as the mobile phase, which is commonly used for separating and detecting non-polar and weakly polar compounds, and is widely used. Normal phase high performance liquid chromatography: using a polar stationary phase, and using a relatively weak solvent as the mobile phase, which is commonly used for separating and detecting relatively strong polar compounds.

[0004] Tosedostat phosphate contains basic amino groups, which is easy to be adsorbed by the silicon hydroxyl (-Si-OH) residues on the silica gel of the chromatographic column, resulting in peak tailing and affecting the determination of tosedostat phosphate. SUMMARY

[0005] In order to solve the problem that the basic amino group of tosedostat phosphate affects the high performance liquid chromatography determination, the present application provides a high performance liquid chromatography analysis method of tosedostat phosphate, which uses the combination of trifluoroacetic acid and triethylamine to hinder the interaction between tosedostat phosphate and the chromatographic column, and improves the high performance liquid chromatography determination effect of tosedostat phosphate.

[0006] The present application provides a high performance liquid chromatography analysis method of tosedostat phosphate, which adopts the following technical scheme:

[0007] A high performance liquid chromatography analysis method of tosedostat phosphate, comprising the following steps:

[0008] Preparation of sample: tosedostat tablets are taken, dissolved and diluted with a diluent to prepare a test sample solution; the diluent is consistent with the mobile phase used for high performance liquid chromatography detection;

[0009] Detection: Take the test sample solution to carry out high performance liquid chromatography detection, and the high performance liquid chromatography conditions are as follows: the filling material of the chromatographic column is octadecylsilane bonded silica gel; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine, an auxiliary agent and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.01-0.05%, the volume fraction of triethylamine in the mixed solution is 0-0.3%, the auxiliary agent includes at least one of EDTA tetrasodium and hexafluoroisopropanol; the volume ratio of the mobile phase A and the mobile phase B is 50:50-55:45; the column flow rate is 0.8-1.0 mL / min; the column temperature is 30-40 DEG C; and the detection wavelength is 220-254 nm.

[0010] By adopting the technical scheme, trifluoroacetic acid and triethylamine are added to the mobile phase, the trifluoroacetic acid can inhibit the ionization of the basic amino group in the tosedostat phosphate, enhance the retention capacity of the tosedostat phosphate on the chromatographic column, avoid the peak shift or disappearance caused by insufficient retention, the trifluoromethyl of the trifluoroacetic acid can also form a hydrophobic ion pair with the basic group which has been protonated, prolong the retention time and improve the separation selectivity, and the trifluoroacetic acid can also protonate the silicon hydroxyl on the chromatographic column, block the non-specific adsorption between the residual silicon hydroxyl on the chromatographic column and the basic group of the tosedostat phosphate, and make the peak shape symmetrical.

[0011] The triethylamine can combine with the silicon hydroxyl on the chromatographic column preferentially, block the interaction between the tosedostat phosphate and the chromatographic column, and significantly improve the peak tailing, and the triethylamine can also form a buffer system with the trifluoroacetic acid and accurately control the pH, in the mobile phase, the triethylamine can offset the strong acidity of the trifluoroacetic acid, prevent the pH from being too low to damage the chromatographic column, and stabilize the retention time of the target substance.

[0012] The residual metal ions in the chromatographic column may cause the hydrolysis of the trifluoroacetic acid to generate fluoride and the oxidation of the triethylamine to generate oxide, which interferes with the determination of the tosedostat phosphate, and the EDTA tetrasodium can form a stable water-soluble complex with the trace metal ions in the mobile phase, block the catalytic oxidation reaction of the metal ions, and thus ensure the stable state of the trifluoroacetic acid and the triethylamine, in addition, the EDTA tetrasodium has a buffering capacity, can maintain the pH stability of the mobile phase, and hinder the pH fluctuation caused by the volatilization of the trifluoroacetic acid and the triethylamine.

[0013] The hexafluoroisopropanol can form a good synergistic effect with the trifluoroacetic acid and the triethylamine, the triethylamine provides a positive charge to electrostatically combine with the phosphate group of the tosedostat phosphate, the hexafluoroisopropanol stabilizes the ion pair through hydrogen bonding and reduces the hydrophobicity of the triethylamine, reduces the non-specific adsorption of the stationary phase, and thus significantly enhances the retention time of the tosedostat phosphate and improves the separation from impurities.

[0014] Trifluoroacetic acid as a strong acid inhibits the dissociation of silanol groups, reducing the adsorption of basic groups. Hexafluoroisopropanol further shields the active sites of residual silanol groups on the stationary phase through hydrogen bonds, and cooperates with trifluoroacetic acid to reduce the tailing of the pyrrolidine group of tosedostat phosphate.

[0015] The application hinders the interaction between tosedostat phosphate and the chromatographic column by using trifluoroacetic acid and triethylamine in the mobile phase, making the peak shape symmetrical and significantly improving peak tailing; at the same time, the two can also form a buffer system; in addition, trifluoroacetic acid can enhance the retention capacity of tosedostat phosphate on the chromatographic column and improve the separation selectivity; when EDTA tetrasodium or hexafluoroisopropanol is added to the mixed solution, it can form a good synergistic effect with trifluoroacetic acid and triethylamine, improving the determination effect of tosedostat phosphate.

[0016] Preferably, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, and the volume fraction of triethylamine is 0.1%.

[0017] Preferably, the volume ratio of the mobile phase A and the mobile phase B is 55:45.

[0018] Preferably, the column temperature is 40°C.

[0019] By using the above technical solution, the sample solution is more conducive to high performance liquid chromatography under the above conditions.

[0020] Preferably, the concentration of EDTA tetrasodium in the mixed solution is 38-114 mg / L.

[0021] By using the above technical solution, when the concentration of EDTA tetrasodium is too low, the chelating ability is insufficient, and free metal ions catalyze the oxidation of trifluoroacetic acid and triethylamine, causing column efficiency to decrease and peak tailing to increase; when the concentration of EDTA tetrasodium is too high, the baseline background is significantly improved, affecting the determination of tosedostat phosphate; therefore, after a large number of research and experimental verification, the applicant finally determines that the concentration of EDTA tetrasodium in the mixed solution of the application should be as above.

[0022] Preferably, the volume fraction of hexafluoroisopropanol in the mixed solution is 0.05-0.2%.

[0023] By using the above technical solution, when the volume fraction of hexafluoroisopropanol is too low, the synergistic effect of hexafluoroisopropanol with trifluoroacetic acid and triethylamine is insufficient; when the volume fraction of hexafluoroisopropanol is too high, the baseline background is significantly improved, affecting the quantitative determination of tosedostat phosphate; therefore, after a large number of research and experimental verification, the applicant finally determines that the volume fraction of hexafluoroisopropanol in the mixed solution of the application should be as above.

[0024] In summary, the application has the following beneficial effects:

[0025] 1、The application hinders the interaction of tosedostat phosphate with the chromatographic column by using the combination of trifluoroacetic acid and triethylamine in the mobile phase, making the peak shape symmetrical and significantly improving peak tailing; at the same time, the two can also form a buffer system; in addition, trifluoroacetic acid can enhance the retention ability of tosedostat phosphate on the chromatographic column and improve the separation selectivity;

[0026] 2、The application adds EDTA tetrasodium in the mobile phase B, EDTA tetrasodium can form a stable water-soluble complex with trace amounts of metal ions in the mobile phase, block the metal ion catalytic oxidation reaction, so as to ensure the stable state of trifluoroacetic acid and triethylamine;

[0027] 3、The application adds hexafluoroisopropanol in the mobile phase B, hexafluoroisopropanol can significantly enhance the retention time of tosedostat phosphate and improve the separation from impurities in cooperation with triethylamine; hexafluoroisopropanol further shields the active sites of residual silanol groups on the stationary phase through hydrogen bonds, and cooperates with trifluoroacetic acid to reduce the tailing of the pyrrolidine group of tosedostat phosphate. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the result graph of high performance liquid chromatography of example 1.

[0029] Figure 2 is the result graph of high performance liquid chromatography of example 2.

[0030] Figure 3 is the result graph of high performance liquid chromatography of example 3.

[0031] Figure 4 is the result graph of high performance liquid chromatography of example 4.

[0032] Figure 5 is the result graph of high performance liquid chromatography of example 5.

[0033] Figure 6 is the result graph of high performance liquid chromatography of example 6.

[0034] Figure 7 is the result graph of high performance liquid chromatography of example 7.

[0035] Figure 8 is the result graph of high performance liquid chromatography of example 8.

[0036] Figure 9 is the result graph of high performance liquid chromatography of example 9.

[0037] Figure 10 is the result graph of high performance liquid chromatography of example 10.

[0038] Figure 11 is the result graph of high performance liquid chromatography of example 11.

[0039] Figure 12 is a result graph of high performance liquid chromatography of Example 12.

[0040] Figure 13 is a result graph of high performance liquid chromatography of Example 13.

[0041] Figure 14 is a result graph of high performance liquid chromatography of Example 14.

[0042] Figure 15 is a result graph of high performance liquid chromatography of Example 15. DETAILED DESCRIPTION

[0043] The raw materials in the present application include the following parts:

[0044] Trifluoroacetic acid: commercially available product with CAS No. 76-05-1;

[0045] Triethylamine: commercially available product with CAS No. 121-44-8;

[0046] EDTA tetrasodium: commercially available product with CAS No. 64-02-8;

[0047] Hexafluoroisopropanol: commercially available product with CAS No. 920-66-1;

[0048] The present application is further described in detail below in combination with examples and comparative examples.

[0049] Example 1

[0050] A high performance liquid chromatography analysis method of tosedostat phosphate includes the following steps:

[0051] Preparation of sample: Take tosedostat tablets 30 mg, accurately weigh and place in a 100 mL volumetric flask, add 80 mL of diluent, and ultrasonically treat for 30 min, take out and cool, dilute to the mark with diluent, shake well, filter through a 0.45 µm filter membrane, and use as the test sample solution; the diluent and the mobile phase are the same solution;

[0052] Detection: take 2 µL of the test sample solution for high performance liquid chromatography detection;

[0053] The high performance liquid chromatography conditions are as follows: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification: 4.6*150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume fraction of triethylamine in the mixed solution is 0.1%, and the volume ratio of the mobile phase A and the mobile phase B is 55:45; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40 DEG C; and the detection wavelength is 220 nm. The detection result is shown in Table 1. Figure 1 .

[0054] Example 2

[0055] A high performance liquid chromatography analysis method of tosedostat phosphate includes the following steps:

[0056] Preparation of sample: 30 mg of tosedostat tablets is accurately weighed, placed in a 100 mL volumetric flask, 80 mL of diluent is added, and ultrasonic treatment is performed for 30 min, then taken out and cooled, diluted to the mark with diluent, shaken uniformly, filtered through a 0.45 µm filter membrane, and used as a test sample solution; the diluent and the mobile phase are the same solution;

[0057] Detection: 2 µL of the test sample solution is subjected to high performance liquid chromatography detection;

[0058] The high performance liquid chromatography conditions are as follows: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification: 4.6*150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume fraction of triethylamine in the mixed solution is 0.1%, and the volume ratio of the mobile phase A and the mobile phase B is 50:50; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40 DEG C; and the detection wavelength is 220 nm. The detection result is shown in Table 2. Figure 2 .

[0059] Example 3

[0060] A high performance liquid chromatography analysis method of tosedostat phosphate includes the following steps:

[0061] Preparation of sample: 30 mg of tosedostat tablets is accurately weighed, placed in a 100 mL volumetric flask, 80 mL of diluent is added, and ultrasonic treatment is performed for 30 min, then taken out and cooled, diluted to the mark with diluent, shaken uniformly, filtered through a 0.45 µm filter membrane, and used as a test sample solution; the diluent and the mobile phase are the same solution;

[0062] Detection: Take 2 μL of the test sample solution for high performance liquid chromatography detection;

[0063] The high performance liquid chromatography conditions are: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification is 4.6*150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume fraction of triethylamine in the mixed solution is 0.1%, and the volume ratio of the mobile phase A and the mobile phase B is 50:50; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40°C; and the detection wavelength is 254 nm. The detection result is shown in Table 1. Figure 3 .

[0064] Example 4

[0065] A high performance liquid chromatography analysis method for tosedostat phosphate, comprising the following steps:

[0066] Preparation of sample: take tosedostat tablets 30 mg, accurately weigh, put into a 100 mL volumetric flask, add 80 mL of diluent, and ultrasonic treat for 30 min, take out and cool, dilute to the mark with diluent, shake well, filter through a 0.45 µm filter membrane, and use as a test sample solution; the diluent and the mobile phase are the same solution;

[0067] Detection: Take 2 μL of the test sample solution for high performance liquid chromatography detection;

[0068] The high performance liquid chromatography conditions are: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification is 4.6*150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume fraction of triethylamine in the mixed solution is 0.1%, and the volume ratio of the mobile phase A and the mobile phase B is 55:45; isocratic elution, the column flow rate is 0.8 mL / min; the column temperature is 40°C; and the detection wavelength is 220 nm. The detection result is shown in Table 2. Figure 4 .

[0069] Example 5

[0070] A high performance liquid chromatography analysis method for tosedostat phosphate, comprising the following steps:

[0071] Preparation of sample: Take 30 mg of tosedostat tablets, accurately weigh, place in a 100 mL volumetric flask, add 80 mL of diluent, and ultrasonic treat for 30 min, remove and cool, dilute to the mark with diluent, shake well, filter through a 0.45 µm filter, and use as the test sample solution; the diluent and the mobile phase are the same solution;

[0072] Detection: Take 2 µL of the test sample solution for high performance liquid chromatography detection;

[0073] The high performance liquid chromatography conditions are: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification 4.6 x 150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.01%, the volume fraction of triethylamine in the mixed solution is 0.3%, the volume ratio of mobile phase A and mobile phase B is 55:45; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40°C; the detection wavelength is 220 nm. The detection results are shown in Table 1. Figure 5 .

[0074] Example 6

[0075] A high performance liquid chromatography analysis method for tosedostat phosphate, comprising the following steps:

[0076] Preparation of sample: Take 30 mg of tosedostat tablets, accurately weigh, place in a 100 mL volumetric flask, add 80 mL of diluent, and ultrasonic treat for 30 min, remove and cool, dilute to the mark with diluent, shake well, filter through a 0.45 µm filter, and use as the test sample solution; the diluent and the mobile phase are the same solution;

[0077] Detection: Take 2 µL of the test sample solution for high performance liquid chromatography detection;

[0078] The high performance liquid chromatography conditions are: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification 4.6 x 150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.05%, the volume fraction of triethylamine in the mixed solution is 0.1%, the volume ratio of mobile phase A and mobile phase B is 55:45; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40°C; the detection wavelength is 220 nm. The detection results are shown in Table 2. Figure 6 .

[0079] Example 7

[0080] A high performance liquid chromatography analysis method of tosedostat phosphate, comprising the following steps:

[0081] Preparation of sample: take tosedostat tablets 30mg, accurately weigh, place in a 100mL volumetric flask, add 80mL diluent, and ultrasonic treat for 30min, take out and cool, dilute to the mark with diluent, shake well, filter through a 0.45µm filter membrane, and use as the test sample solution; the diluent and the mobile phase are the same solution;

[0082] Detection: take 2µL of the test sample solution for high performance liquid chromatography detection;

[0083] The high performance liquid chromatography conditions are: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification 4.6x150mm, 5µm); the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume ratio of mobile phase A and mobile phase B is 50:50; isocratic elution, column flow rate is 1.0mL / min; column temperature is 40℃; detection wavelength is 220nm. The detection results are shown in Figure 7 .

[0084] Example 8

[0085] A high performance liquid chromatography analysis method of tosedostat phosphate, comprising the following steps:

[0086] Preparation of sample: take tosedostat tablets 30mg, accurately weigh, place in a 100mL volumetric flask, add 80mL diluent, and ultrasonic treat for 30min, take out and cool, dilute to the mark with diluent, shake well, filter through a 0.45µm filter membrane, and use as the test sample solution; the diluent and the mobile phase are the same solution;

[0087] Detection: take 2µL of the test sample solution for high performance liquid chromatography detection;

[0088] The high performance liquid chromatography conditions are: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification 4.6x150mm, 5µm); the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume ratio of mobile phase A and mobile phase B is 50:50; isocratic elution, column flow rate is 1.0mL / min; column temperature is 30℃; detection wavelength is 220nm. The detection results are shown in Figure 8 .

[0089] The high performance liquid chromatography conditions and detection results of Examples 1-8 are shown in Table 1.

[0090] Table 1 High performance liquid chromatography conditions and detection results of Examples 1-8

[0091]

[0092] Referring to Table 1, it can be seen from Comparative Examples 1-8 that the above-mentioned methods can all be effectively used for the detection of tosedostat tablets. The reason is that: the trifluoroacetic acid in the mobile phase can inhibit the ionization of the basic amino group in tosedostat phosphate, enhance its retention capacity on the chromatographic column, and avoid peak shift or disappearance due to insufficient retention. The trifluoromethyl of trifluoroacetic acid will also form a hydrophobic ion pair with the protonated basic group, prolong the retention time and improve the separation selectivity. Trifluoroacetic acid also protonates the silicon hydroxyl group on the chromatographic column, blocking the non-specific adsorption between the residual silicon hydroxyl group on the chromatographic column and the basic group of tosedostat phosphate, making the peak shape symmetrical. Triethylamine can preferentially bind to the silicon hydroxyl group on the chromatographic column, blocking the interaction between tosedostat phosphate and the chromatographic column, and significantly improving the peak tailing. Triethylamine can also form a buffer system with trifluoroacetic acid to accurately control the pH. In the mobile phase, triethylamine can offset the strong acidity of trifluoroacetic acid to prevent the pH from being too low to damage the chromatographic column, while stabilizing the retention time of the target substance.

[0093] After comparison, it was found that the tailing factor of Example 1 was the lowest, and the determination content ratio was the highest. Therefore, Example 1 is preferred.

[0094] Example 9

[0095] Example 9 is based on the preparation method of Example 1, adjusted to the following steps:

[0096] A high performance liquid chromatography analysis method for tosedostat phosphate, comprising the following steps:

[0097] Preparation of sample: take tosedostat tablets 30 mg, accurately weigh, place in a 100 mL volumetric flask, add 80 mL of diluent, and ultrasonic treat for 30 min, take out and cool, dilute to the mark with diluent, shake well, filter through a 0.45 µm filter membrane, and use as the test sample solution; the diluent and the mobile phase are the same solution;

[0098] Detection: take 2 µL of the test sample solution for high performance liquid chromatography detection;

[0099] The high performance liquid chromatography conditions are as follows: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification is 4.6*150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine, EDTA tetrasodium and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume fraction of triethylamine in the mixed solution is 0.1%, the concentration of EDTA tetrasodium in the mixed solution is 80 mg / L, and the volume ratio of the mobile phase A and the mobile phase B is 55:45; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40°C; and the detection wavelength is 220 nm. The detection results are shown in Table 1. Figure 9 .

[0100] In the preparation method of Example 9, the EDTA tetrasodium is adjusted to other antioxidant types, and specific adjustments are shown in Table 2.

[0101] Table 2: Antioxidant types and detection results of Example 1, Example 9 and Comparative Examples 1-3

[0102]

[0103] Referring to Table 2, it can be seen from Comparative Example 1, Example 9 and Comparative Examples 1-3 that not any antioxidant is suitable for the present application. It is found by comparing the four antioxidants that only EDTA tetrasodium can be suitable for the present application, which can further reduce the retention time and the tailing factor and improve the determination content proportion. The reason is that: the residual metal ions in the chromatographic column may cause the hydrolysis of trifluoroacetic acid to produce fluoride and the oxidation of triethylamine to generate oxides, which interfere with the determination of tosedostat phosphate. And EDTA tetrasodium can form a stable water-soluble complex with trace metal ions in the mobile phase, block the metal ion catalytic oxidation reaction, and thus ensure the stable state of trifluoroacetic acid and triethylamine. In addition, EDTA tetrasodium has buffering capacity, which can maintain the pH stability of the mobile phase and prevent the pH fluctuation caused by the easy evaporation of trifluoroacetic acid and triethylamine.

[0104] The chelating ability of EDTA disodium is similar to that of EDTA tetrasodium, but the acetonitrile proportion of the mobile phase A of the present application is too high, which reduces the solubility of EDTA disodium in the mobile phase, and EDTA disodium may exist in local precipitation in the mobile phase, which interferes with the detection.

[0105] Vitamin C can only reduce Fe 3+ to Fe 2+ , and does not chelate, Fe 2+Still can adsorb basic groups, and ascorbic acid itself oxidation will increase the baseline noise. Sodium bisulfite is mainly to remove the dissolved oxygen in the mobile phase to inhibit oxidation, also not chelate metal. After the decomposition of sodium bisulfite, still release sulfur dioxide to affect the determination.

[0106] Examples 10-11

[0107] Examples 10-11 are based on the preparation method of Example 9, the concentration of EDTA tetrasodium is adjusted, see Table 3 for specific adjustment. The test results are shown in Table 4. Figure 10-11 .

[0108] Table 3 EDTA tetrasodium concentration and test results of Example 1 and Examples 9-11

[0109]

[0110] Referring to Table 3, it can be seen from Comparative Example 1 and Examples 9-11 that as the concentration of EDTA tetrasodium continuously increases, the retention time and the tailing factor show a trend of first decreasing and then increasing, and the determination content ratio shows a trend of first increasing and then decreasing. The reason is that as the concentration of EDTA tetrasodium continuously increases, the chelating effect of EDTA tetrasodium continuously increases, thereby ensuring the stable state of trifluoroacetic acid and triethylamine, and improving the determination effect. When the concentration of EDTA tetrasodium is too high, it significantly increases the baseline background, which affects the determination of tosedostat phosphate.

[0111] Examples 12-15

[0112] Example 12 is based on the preparation method of Example 1, which is adjusted as follows:

[0113] A tosedostat phosphate high performance liquid chromatography analysis method, comprising the following steps:

[0114] Preparation of sample: Take tosedostat tablets 30mg, accurately weigh, place in a 100mL volumetric flask, add 80mL diluent, and ultrasonic treat for 30min, take out and cool, dilute to the mark with diluent, shake well and filter through a 0.45µm filter membrane as the test sample solution; the diluent and the mobile phase are the same solution;

[0115] Detection: Take 2µL of the test sample solution for high performance liquid chromatography detection;

[0116] The high performance liquid chromatography conditions are as follows: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification: 4.6*150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine, hexafluoroisopropanol and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume fraction of triethylamine in the mixed solution is 0.1%, the volume fraction of hexafluoroisopropanol in the mixed solution is 0.1%, and the volume ratio of the mobile phase A to the mobile phase B is 55:45; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40°C; and the detection wavelength is 220 nm. The detection results are shown in Table 1. Figure 12 .

[0117] In the preparation method of Example 12, the volume fraction of hexafluoroisopropanol is adjusted, and the specific adjustment is shown in Table 4. The detection results are shown in Table 5. Figure 13-14 .

[0118] In the preparation method of Example 1, the following steps are adjusted:

[0119] A high performance liquid chromatography analysis method for tosedostat phosphate includes the following steps:

[0120] Preparation of a sample: 30 mg of tosedostat tablets is accurately weighed, placed in a 100 mL volumetric flask, 80 mL of diluent is added, and ultrasonic treatment is performed for 30 min. After cooling, the solution is diluted to the mark with diluent, shaken uniformly, filtered through a 0.45 µm filter membrane, and used as a test sample solution; the diluent and the mobile phase are the same solution;

[0121] Detection: 2 µL of the test sample solution is subjected to high performance liquid chromatography detection;

[0122] The high performance liquid chromatography conditions are as follows: the filling material of the chromatographic column is octadecylsilane bonded silica gel, specifically YMC-Triart C18 (specification: 4.6*150 mm, 5 µm); the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution includes trifluoroacetic acid, triethylamine, EDTA tetrasodium, hexafluoroisopropanol and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, the volume fraction of triethylamine in the mixed solution is 0.1%, the concentration of EDTA tetrasodium in the mixed solution is 80 mg / L, the volume fraction of hexafluoroisopropanol in the mixed solution is 0.1%, and the volume ratio of the mobile phase A to the mobile phase B is 55:45; isocratic elution, the column flow rate is 1.0 mL / min; the column temperature is 40°C; and the detection wavelength is 220 nm. The detection results are shown in Table 6. Figure 15 .

[0123] Table 4 Volume fraction of hexafluoroisopropanol and addition of EDTA tetrasodium and detection results of Example 1 and Examples 12-15

[0124]

[0125] Referring to Table 4, it can be seen from Comparative Example 1 and Examples 12-15 that the addition of hexafluoroisopropanol in the mobile phase can also improve the determination results. The reason is that hexafluoroisopropanol can form a good synergistic effect with trifluoroacetic acid and triethylamine, in which triethylamine provides a positive charge to electrostatically bind with the phosphate group of the tosedostat phosphate, and hexafluoroisopropanol stabilizes the ion pair through hydrogen bonding and reduces the hydrophobicity of triethylamine, reducing non-specific adsorption of the stationary phase, thereby significantly enhancing the retention time of tosedostat phosphate and improving separation from impurities. Trifluoroacetic acid as a strong acid inhibits the dissociation of silanol groups and reduces the adsorption of basic groups. Hexafluoroisopropanol further shields the active sites of residual silanol groups on the stationary phase through hydrogen bonding, and cooperates with trifluoroacetic acid to reduce the tailing of the pyrrolidine group of tosedostat phosphate. When EDTA tetrasodium and hexafluoroisopropanol are added to the mobile phase at the same time, they can synergize with each other to improve the determination results of tosedostat phosphate.

[0126] As the volume fraction of hexafluoroisopropanol increases, the retention time and the tailing factor show a trend of first decreasing and then increasing, and the determination content ratio shows a trend of first increasing and then decreasing. The reason is that as the volume fraction of hexafluoroisopropanol increases, the synergistic effect of hexafluoroisopropanol with trifluoroacetic acid and triethylamine continuously improves, improving the determination effect. When the volume fraction of hexafluoroisopropanol is too high, it significantly raises the baseline background, affecting the determination of tosedostat phosphate.

[0127] The specific embodiments are merely illustrative of the present application, and are not intended to limit the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, and the modifications are within the scope of the present application as long as they are within the scope of the claims of the present application.

Claims

1. A high-performance liquid chromatography analysis method of tosedostat phosphate salt, characterized in that, The method comprises the following steps: Preparation of sample: take tocephinib tablets, dissolve and dilute with a diluent to prepare a test sample solution; the diluent is consistent with the mobile phase for high performance liquid chromatography detection; Detection: take the test sample solution for high performance liquid chromatography detection, and the high performance liquid chromatography conditions are as follows: the filling material of the chromatographic column is octadecylsilane bonded silica gel; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a mixed solution, the mixed solution comprises trifluoroacetic acid, triethylamine, an auxiliary agent and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.01-0.05%, the volume fraction of triethylamine in the mixed solution is 0-0.3%, and the auxiliary agent comprises at least one of EDTA tetrasodium and hexafluoroisopropanol; the volume ratio of the mobile phase A and the mobile phase B is 50:50-55:45; the column flow rate is 0.8-1.0 mL / min; the column temperature is 30-40 DEG C; and the detection wavelength is 220-254 nm.

2. The HPLC method for the analysis of tosedostat phosphonate salt according to claim 1, characterized in that: The volume fraction of trifluoroacetic acid in the mixed solution is 0.03%, and the volume fraction of triethylamine in the mixed solution is 0.1%.

3. The HPLC method for the analysis of tosedostat phosphonate salt according to claim 2, characterized in that: The volume ratio of the mobile phase A and the mobile phase B is 55:

45.

4. The HPLC method for the analysis of tosedostat phosphate salt according to claim 1, characterized in that: The column temperature is 40 DEG C.

5. The HPLC method for the analysis of tosedostat phosphate salt according to claim 1, characterized in that: The concentration of EDTA tetrasodium in the mixed solution is 38-114 mg / L.

6. The HPLC method for the analysis of tosedostat phosphate salt according to claim 1, characterized in that: The volume fraction of hexafluoroisopropanol in the mixed solution is 0.05-0.2%.

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