A high performance liquid chromatography method for the analysis of olaratumab maleate

Abstract

The application relates to the technical field of analytical chemistry, in particular to a high-performance liquid chromatography analysis method for maleic acid olmutant, wherein the mobile phase in the high-performance liquid chromatography condition comprises mobile phase A and mobile phase B, the mobile phase A is a mixed solution, the mixed solution comprises trifluoroacetic acid, triethylamine and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.08-0.12%, the volume fraction of triethylamine is 0-0.3%, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A and the mobile phase B is 75:25-85:15. The trifluoroacetic acid and the triethylamine in the mobile phase are matched, the interaction between maleic acid olmutant 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 maleic acid olmutant on the chromatographic column and improve separation selectivity.

Description

Technical Field

[0001] This application relates to the field of analytical chemistry technology, specifically to a high-performance liquid chromatography (HPLC) method for the analysis of olalatinib maleate. Background Technology

[0002] Olapinib maleate belongs to the Janus kinase inhibitor class of drugs. It works by inhibiting various cytokines that induce itching and inflammation, as well as allergy-related cytokines, which are dependent on the activity of JAK1 and JAK3 enzymes. Studies have shown that olalapinib maleate can rapidly, effectively, and safely control itching caused by allergic and atopic dermatitis in dogs.

[0003] Generally, impurities are unavoidable in olatinib maleate raw materials. Detecting these impurities is crucial for quality control of olatinib maleate raw materials. High-performance liquid chromatography (HPLC) can be divided into two types: reversed-phase and normal-phase, determined by the relative polarity of the stationary and mobile phases. Reversed-phase HPLC uses a relatively weakly polar stationary phase and a more polar solvent as the mobile phase. It is commonly used to separate and detect non-polar and weakly polar compounds and has wide applications. Normal-phase HPLC uses a polar stationary phase and a relatively weakly polar solvent as the mobile phase. It is commonly used to separate and detect relatively strongly polar compounds.

[0004] The olatinib maleate raw material contains basic groups, which easily adsorb onto the residual silanol groups (-Si-OH) on the silica gel of the chromatographic column, resulting in peak tailing and affecting the determination of the olatinib maleate raw material. Summary of the Invention

[0005] To address the issue that the basic groups of olatinib maleate raw material affect its high-performance liquid chromatography (HPLC) determination, this application provides an HPLC analysis method for olatinib maleate. This method utilizes the combination of trifluoroacetic acid and triethylamine to hinder the interaction between olatinib maleate raw material and the chromatographic column, thereby improving the HPLC determination effect of olatinib maleate raw material.

[0006] This application provides a high-performance liquid chromatography (HPLC) method for the analysis of olalatinib maleate, employing the following technical solution:

[0007] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0008] Sample preparation: Take olatinib maleate raw material, dissolve and dilute it 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: The test sample solution was subjected to high-performance liquid chromatography (HPLC) for detection. The HPLC conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; the mobile phase consisted of mobile phase A and mobile phase B, wherein mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.08-0.12% for trifluoroacetic acid and 0-0.3% for triethylamine; mobile phase B was acetonitrile, and the volume ratio of mobile phase A to mobile phase B was 75:25-85:15; the column flow rate was 0.8-1.0 mL / min; the column temperature was 30-40℃; and the detection wavelength was 254-300 nm.

[0010] By employing the above technical solution, trifluoroacetic acid and triethylamine are added to the mobile phase. Trifluoroacetic acid significantly lowers the mobile phase pH, protonates the basic groups of olatinib maleate (such as pyrimidine cyclic amino groups), reduces its electrostatic attraction to silanol groups on the chromatographic column, and suppresses tailing. The trifluoromethyl group of trifluoroacetic acid also forms a hydrophobic ion pair with the protonated basic groups, prolonging the retention time and improving the resolution.

[0011] The lone pair electrons of the nitrogen atom in the triethylamine molecule preferentially bind to the silanol group, forming a shielding layer that blocks the secondary interactions between olatinib maleate and the stationary phase, further optimizing peak symmetry. Triethylamine can also form a buffer system with trifluoroacetic acid, allowing for precise pH control. In the mobile phase, triethylamine can counteract the strong acidity of trifluoroacetic acid, preventing excessively low pH from damaging the chromatographic column, while simultaneously stabilizing the retention time of the target analyte.

[0012] This application utilizes the combination of trifluoroacetic acid and triethylamine in the mobile phase to hinder the interaction between olatinib maleate and the chromatographic column, resulting in symmetrical peak shape and significantly improving peak tailing. Simultaneously, the two components form a buffer system. Furthermore, trifluoroacetic acid enhances the retention capacity of olatinib maleate on the chromatographic column, improving separation selectivity.

[0013] Preferably, the volume fraction of trifluoroacetic acid in the mixed solution is 0.1%, and the volume fraction of triethylamine is 0.1%.

[0014] Preferably, the volume ratio of mobile phase A to mobile phase B is 80:20.

[0015] Preferably, the column flow rate is 1.0 mL / min and the column temperature is 30 °C.

[0016] Preferably, the detection wavelength is 287nm.

[0017] By adopting the above technical solution, the test sample solution is more conducive to high-performance liquid chromatography operation under the above conditions.

[0018] Preferably, the mixed solution further includes disodium EDTA.

[0019] By employing the above-mentioned technical solution, residual metal ions in the chromatographic column may cause trifluoroacetic acid to hydrolyze and produce fluorides, and triethylamine to oxidize and produce oxides, interfering with the determination of olatinib maleate. Disodium EDTA can effectively chelate trace metal ions, preventing them from forming complexes with the basic groups of olatinib maleate and avoiding peak tailing.

[0020] Preferably, the concentration of disodium EDTA in the mixed solution is 170-670 mg / L.

[0021] By adopting the above technical solution, when the concentration of disodium EDTA is too low, the chelation effect on metal ions is poor, resulting in a decrease in column efficiency and an aggravation of peak tailing; when the concentration of disodium EDTA is too high, the baseline background is significantly increased, affecting the determination of olalatinib maleate. Therefore, after extensive research and experimental verification, the applicant finally determined that the concentration of disodium EDTA in the mixed solution of this application should be as described above.

[0022] Preferably, the mixed solution further includes trifluoroethanol.

[0023] By employing the above technical solution, trifluoroethanol can competitively occupy residual silanol groups (Si-OH) on the silica gel surface through hydrogen bonding, inhibiting the adsorption of basic groups of olatinib maleate and improving peak tailing. Trifluoroethanol and trifluoroacetic acid can synergistically protonate the amino group of olatinib maleate, reducing electrostatic adsorption.

[0024] Preferably, the volume fraction of trifluoroethanol in the mixed solution is 0.5-2%.

[0025] By adopting the above technical solution, when the volume fraction of trifluoroethanol is too low, the ability of trifluoroethanol to shield silanol groups is insufficient, and peak tailing is aggravated; when the volume fraction of trifluoroethanol is too high, the baseline background is significantly increased, affecting the determination of olatinib maleate. Therefore, after extensive research and experimental verification, the applicant finally determined that the concentration of trifluoroethanol in the mixed solution of this application should be as described above.

[0026] In summary, this application has the following beneficial effects:

[0027] 1. Because this application uses the combination of trifluoroacetic acid and triethylamine in the mobile phase, it hinders the interaction between olatinib maleate and the chromatographic column, 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 olatinib maleate on the chromatographic column and improve the separation selectivity.

[0028] 2. In this application, disodium EDTA is added to mobile phase A. Disodium EDTA can form a stable water-soluble complex with trace metal ions in the mobile phase, thereby blocking the catalytic oxidation reaction of metal ions and preventing them from forming a complex with the basic groups of olatinib maleate, thus avoiding peak tailing.

[0029] 3. In this application, trifluoroethanol is added to mobile phase A. Trifluoroethanol can competitively occupy the residual silanol groups (Si-OH) on the silica gel surface through hydrogen bonding, thereby inhibiting the adsorption of basic groups of olatinib maleate and improving peak tailing. Trifluoroethanol and trifluoroacetic acid can synergistically protonate the amino group of olatinib maleate and reduce electrostatic adsorption. Attached Figure Description

[0030] Figure 1 This is a chromatogram of the high performance liquid chromatography results from Example 1.

[0031] Figure 2 This is a high-performance liquid chromatography result chromatogram of Example 2.

[0032] Figure 3 This is a high-performance liquid chromatography result chromatogram of Example 3.

[0033] Figure 4 This is a high-performance liquid chromatography result chromatogram of Example 4.

[0034] Figure 5 This is a high-performance liquid chromatography result chromatogram of Example 5.

[0035] Figure 6 This is a high-performance liquid chromatography result chromatogram of Example 6.

[0036] Figure 7 This is a high-performance liquid chromatography result chromatogram of Example 7.

[0037] Figure 8 This is a high-performance liquid chromatography result chromatogram of Example 8.

[0038] Figure 9 This is a high-performance liquid chromatography result chromatogram of Example 9.

[0039] Figure 10 This is a high-performance liquid chromatography result chromatogram of Example 10.

[0040] Figure 11 This is a high-performance liquid chromatography result chromatogram of Example 11.

[0041] Figure 12 This is the result of high performance liquid chromatography for Comparative Example 1.

[0042] Figure 13 This is the result of high performance liquid chromatography for Comparative Example 2.

[0043] Figure 14 This is a high-performance liquid chromatography result chromatogram of Example 12.

[0044] Figure 15 This is a high-performance liquid chromatography result chromatogram of Example 13.

[0045] Figure 16 This is a high-performance liquid chromatography result chromatogram of Example 14.

[0046] Figure 17 This is a high-performance liquid chromatography result chromatogram of Example 15.

[0047] Figure 18 This is a high-performance liquid chromatography result chromatogram of Example 16.

[0048] Figure 19 This is a high-performance liquid chromatography result chromatogram of Example 17. Detailed Implementation

[0049] The raw materials in this application include the following:

[0050] Trifluoroacetic acid: Use commercially available products with CAS number 76-05-1;

[0051] Triethylamine: Use commercially available product with CAS number 121-44-8;

[0052] Disodium EDTA: Uses commercially available product with CAS number 6381-92-6;

[0053] Trifluoroethanol: Use commercially available products with CAS number 75-89-8;

[0054] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0055] Example 1

[0056] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0057] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0058] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0059] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 1 .

[0060] Example 2

[0061] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0062] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0063] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0064] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine; mobile phase B was acetonitrile, with a volume ratio of 75:25 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 2 .

[0065] Example 3

[0066] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0067] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0068] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0069] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine; mobile phase B was acetonitrile, with a volume ratio of 85:15 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 3 .

[0070] Example 4

[0071] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0072] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0073] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0074] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 40 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 4 .

[0075] Example 5

[0076] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0077] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0078] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0079] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 254 nm. The detection results are shown in [Figure number missing]. Figure 5 .

[0080] Example 6

[0081] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0082] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0083] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0084] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 300 nm. The detection results are shown in [Figure number missing]. Figure 6 .

[0085] Example 7

[0086] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0087] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0088] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0089] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid and water, with a trifluoroacetic acid volume fraction of 0.08%; mobile phase B was acetonitrile, with a volume ratio of mobile phase A to mobile phase B of 80:20; isocratic elution was performed at a column flow rate of 1.0 mL / min; the column temperature was 40 °C; and the detection wavelength was 287 nm. The detection results are shown below. Figure 7 .

[0090] Example 8

[0091] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0092] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0093] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0094] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.12% for trifluoroacetic acid and 0.3% for triethylamine; mobile phase B was acetonitrile, and the volume ratio of mobile phase A to mobile phase B was 80:20; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 8 .

[0095] Example 9

[0096] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0097] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0098] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0099] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 0.8 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 9 .

[0100] The high performance liquid chromatography conditions and detection results of Examples 1-9 are shown in Table 1.

[0101] Table 1. High-performance liquid chromatography conditions and detection results for Examples 1-9

[0102]

[0103] Referring to Table 1, and comparing Examples 1-9, it can be seen that the above methods are all effective for the detection of olatinib maleate. This is because trifluoroacetic acid in the mobile phase significantly lowers the mobile phase pH, protonating the basic groups of olatinib maleate (such as the pyrimidine cyclic amino group), reducing its electrostatic attraction to the silanol groups on the chromatographic column, and suppressing tailing. Furthermore, the trifluoromethyl group of trifluoroacetic acid forms a hydrophobic ion pair with the protonated basic groups, prolonging the retention time and improving resolution.

[0104] The lone pair electrons of the nitrogen atom in the triethylamine molecule preferentially bind to the silanol group, forming a shielding layer that blocks the secondary interactions between olatinib maleate and the stationary phase, further optimizing peak symmetry. Triethylamine can also form a buffer system with trifluoroacetic acid, allowing for precise pH control. In the mobile phase, triethylamine can counteract the strong acidity of trifluoroacetic acid, preventing excessively low pH from damaging the chromatographic column, while simultaneously stabilizing the retention time of the target analyte.

[0105] Comparison revealed that Examples 1, 5, and 6 had the lowest tailing factors. Therefore, Example 1 was selected as the preferred embodiment.

[0106] Example 10

[0107] Example 10 is based on the preparation method of Example 1, but with the following steps adjusted:

[0108] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0109] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0110] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0111] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, disodium EDTA, and water, with a volume fraction of 0.1% for both trifluoroacetic acid and triethylamine, and a concentration of 400 mg / L for disodium EDTA; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 10 .

[0112] Example 11 follows the preparation method of Example 10, but with the addition of tetrasodium EDTA instead of disodium EDTA. The test results are shown below. Figure 11 .

[0113] Comparative Examples 1-2 were prepared using the same method as in Example 10, but with the disodium EDTA replaced by other antioxidants. Specific adjustments are shown in Table 2. Detection results are shown below. Figure 12-13 .

[0114] Table 2. Types of antioxidants and test results in Examples 1, 10-11, and Comparative Examples 1-2.

[0115]

[0116] Referring to Table 2, and comparing Examples 1, 10-11, and 1-2, it is evident that not all antioxidants are suitable for this application. A comparison of the four antioxidants revealed that only disodium EDTA and tetrasodium EDTA are suitable for this application, with disodium EDTA showing superior performance compared to tetrasodium EDTA. This is because residual metal ions in the chromatographic column may cause trifluoroacetic acid hydrolysis to produce fluorides and triethylamine oxidation to produce oxides, interfering with the determination of olatinib maleate. Disodium EDTA effectively chelates trace metal ions, preventing them from forming complexes with the basic groups of olatinib maleate and avoiding peak tailing.

[0117] Tetrasodium EDTA has a similar chelating ability to disodium EDTA, but the proportion of mobile phase A in this application is too high. Tetrasodium EDTA is more alkaline and easily neutralizes trifluoroacetic acid, leading to an increase in pH, which in turn affects the detection of olatinib maleate.

[0118] Ascorbic acid can only reduce Fe 3+ To Fe 2+ Without chelation, Fe 2+ It can still adsorb basic groups, and the self-oxidation of ascorbic acid can increase baseline noise. Sodium bisulfite mainly inhibits oxidation by removing dissolved oxygen from the mobile phase and does not chelate metals. After decomposition, sodium bisulfite releases sulfur dioxide, which affects the determination.

[0119] Examples 12-13

[0120] Examples 12-13 are based on the preparation method of Example 10, but the concentration of disodium EDTA is adjusted, as shown in Table 3. The detection results are shown below. Figure 14 and Figure 15 .

[0121] Table 3. EDTA disodium concentration and detection results in Examples 1, 10 and 12-13

[0122]

[0123] Referring to Table 3, a comparison of Examples 1, 10, and 12-13 shows that as the concentration of disodium EDTA increases, the retention time and tailing factor initially decrease and then increase, while the percentage of measured content initially increases and then decreases. This is because as the concentration of disodium EDTA increases, its chelating effect is enhanced, ensuring the stability of trifluoroacetic acid and triethylamine, thus improving the assay results. However, when the concentration of disodium EDTA exceeds a certain range, it significantly increases the baseline background, affecting the assay of olalatinib maleate.

[0124] Examples 14-17

[0125] Example 14 is based on the preparation method of Example 1, but with the following steps adjusted:

[0126] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0127] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0128] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0129] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, trifluoroethanol, and water, with a volume fraction of 0.1% for trifluoroacetic acid, 0.1% for triethylamine, and 1% for trifluoroethanol; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 16 .

[0130] Examples 15-16 are based on the preparation method of Example 14, but the volume fraction of trifluoroethanol is adjusted, as shown in Table 4. Detection results are shown below. Figure 17 and Figure 18 .

[0131] Example 17 is based on the preparation method of Example 1, with the following steps adjusted:

[0132] A high-performance liquid chromatography (HPLC) method for the analysis of olatinib maleate includes the following steps:

[0133] Sample preparation: Weigh 30 mg of olatinib maleate raw material accurately, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well and filter through a 0.45 µm filter membrane to obtain the test sample solution; the diluent and mobile phase are the same solution;

[0134] Detection: Take 5 μL of the test sample solution for high performance liquid chromatography (HPLC) detection;

[0135] The high-performance liquid chromatography (HPLC) conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, specifically a Polypark C18-AQ (4.6 × 150 mm, 5 µm); the mobile phase consisted of mobile phase A and mobile phase B. Mobile phase A was a mixed solution containing trifluoroacetic acid, triethylamine, disodium EDTA, trifluoroethanol, and water, with a volume fraction of 0.1% for trifluoroacetic acid, 0.1% for triethylamine, 400 mg / L for disodium EDTA, and 1% for trifluoroethanol; mobile phase B was acetonitrile, with a volume ratio of 80:20 between mobile phase A and mobile phase B; isocratic elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 287 nm. The detection results are shown in [Figure number missing]. Figure 19 .

[0136] Table 4. Trifluoroethanol volume fraction and EDTA tetrasodium addition status and test results in Examples 1 and 14-17

[0137]

[0138] Referring to Table 4, a comparison of Examples 1 and 14-17 shows that adding trifluoroethanol to the mobile phase also improves the measurement results. This is because trifluoroethanol can competitively occupy residual silanol groups (Si-OH) on the silica gel surface through hydrogen bonding, inhibiting the adsorption of basic groups of olatinib maleate and improving peak tailing. Trifluoroethanol and trifluoroacetic acid can synergistically protonate the amino group of olatinib maleate, reducing electrostatic adsorption. When disodium EDTA and trifluoroethanol are added simultaneously to the mobile phase, they synergistically improve the measurement results of olatinib maleate.

[0139] As the volume fraction of trifluoroethanol increases, the retention time and tailing factor show a trend of first decreasing and then increasing, while the percentage of the measured content shows a trend of first increasing and then decreasing. This is because, with the increasing volume fraction of trifluoroethanol, the synergistic effect between trifluoroethanol and trifluoroacetic acid continuously improves the assay results. However, when the volume fraction of trifluoroethanol exceeds a certain range, excessively high concentrations significantly increase the baseline background, affecting the determination of olatinib maleate.

[0140] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A high-performance liquid chromatography method of analyzing olafaspirin maleate, characterized by, The method comprises the following steps: Preparation of sample: taking the raw material of olaratumab maleate, dissolving and diluting with a diluent to prepare a test sample solution; the diluent is consistent with the mobile phase for high performance liquid chromatography detection; Detection: taking 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 a mixed solution, the mixed solution comprises trifluoroacetic acid, triethylamine, disodium EDTA and water, the volume fraction of trifluoroacetic acid in the mixed solution is 0.1%, the volume fraction of triethylamine is 0.1%, the concentration of disodium EDTA is 170-670 mg / L, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 75:25-85:15; the column flow rate is 0.8-1.0 mL / min; the column temperature is 30-40 DEG C; and the detection wavelength is 254-300 nm.

2. The method of HPLC analysis of olaratumab maleate according to claim 1, characterized by: The volume ratio of the mobile phase A to the mobile phase B is 80:

20.

3. The method of HPLC analysis of olaratumab maleate according to claim 1, characterized in that: The column flow rate is 1.0 mL / min, and the column temperature is 30 DEG C.

4. The method of HPLC analysis of olaratumab maleate according to claim 1, characterized in that: The detection wavelength is 287 nm.

5. The method of HPLC analysis of olaratumab maleate according to claim 1, characterized in that: The mixed solution further comprises trifluoroethanol.

6. The method of HPLC analysis of olaratumab maleate according to claim 5, characterized by: The volume fraction of trifluoroethanol in the mixed solution is 0.5-2%.

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