A detection method for determining the residual amount of a hydrolyzable impurity in apremilast

Abstract

The application discloses a detection method for determining the residual amount of easily hydrolyzed impurities in apremilast, which comprises the following steps: using a high performance liquid chromatograph, adopting polysaccharide derivatives as a filler (CHIRALPAK IH, 4.6mm*150mm, 5um), adopting a n-hexane-tetrahydrofuran (65:35) mobile phase, a flow rate of 1.0ml per minute, a column temperature of 30 DEG C, a detection wavelength of 230nm, and a sample injection volume of 50ul. The application adopts the polysaccharide derivative filler to determine the easily hydrolyzed impurities in the apremilast, has high separation efficiency, strong anti-interference ability, fast analysis speed and high detection sensitivity, and can effectively control the quality of the apremilast by determining the amount of the easily hydrolyzed impurities in the apremilast.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, and specifically to a method for determining the residual amount of easily hydrolyzable impurities in apromis. Background Technology

[0002] The easily hydrolyzable impurity in Apmisit is designated as TQ08310, which is a material required for the synthesis of Apmisit. The production process is as follows:

[0003]

[0004] The TQ08310 used in the production process of Apromis is prone to hydrolysis in aqueous systems, making accurate quantification impossible in conventional reversed-phase chromatography systems. However, even trace amounts of this impurity can affect the quality of the raw material. This patent utilizes polysaccharide derivatives as packing materials in an anhydrous system and employs a liquid chromatography-ultraviolet detector to accurately quantify their residues in Apromis. This has significant practical implications for the quality control of Apromis and its formulations. Summary of the Invention

[0005] The purpose of this invention is to establish a method for determining the residual amount of easily hydrolyzable impurities in apromis, which can effectively control them below the minimum limit in apromis and has important practical significance for the quality control of apromis and its preparations.

[0006] The technical solution of this invention is a liquid chromatography method for determining the residual amount of easily hydrolyzable impurities in apromid, characterized by comprising the following steps:

[0007] Take an appropriate amount of TQ08310 reference standard, accurately weigh it, dissolve it in 4 ml of tetrahydrofuran, and then quantitatively dilute it with solvent to prepare a solution containing approximately 1 µg of TQ08310 reference standard per 1 ml.

[0008] For the test solution, accurately weigh 12.5 mg of apromis and place it in a 25 ml volumetric flask. Dissolve the apromis in 4 ml of tetrahydrofuran and dilute quantitatively with solvent to prepare a solution containing approximately 0.5 mg per ml. Filter the solution to obtain the test solution.

[0009] For system suitability solution, accurately weigh appropriate amounts of apromisci reference standard and TQ08310 reference standard, dissolve them in 4 ml of tetrahydrofuran, and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of apromisci and 1 µg of TQ08310 per ml.

[0010] Chromatographic conditions: Solvent: n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.2-0.5); Mobile phase: n-hexane-tetrahydrofuran ratio of 63:37-68:32; Flow rate: 0.8-1.2 ml / min; Column temperature: 25-35℃; Detection wavelength: 225-235 nm; Column: packed with polysaccharide derivatives (CHIRALPAK IH, 4.6 mm × 150 mm, 5 μm).

[0011] Injection: Take 50 μl each of the reference solution, test solution, and system suitability solution, inject them into the liquid chromatograph, and record the chromatograms;

[0012] Based on the above conditions, calculate the linear regression equation between the concentration of the reference solution and the corresponding peak area. The correlation coefficient should not be less than 0.99. The peak shape of the reference solution should be symmetrical, and the theoretical plate number of the TQ08310 peak should be above 2000. If the test solution chromatogram contains a TQ08310 peak, its retention time should be consistent with that of the TQ08310 peak in the reference solution. In the blank solvent chromatogram, no TQ08310 peak should appear, indicating that the blank solution has no interference.

[0013] The concentration of TQ08310 in each gram of apromisc must not exceed 0.1%, and the concentration range of TQ08310 is 0.1~2.0µg / ml. Attached Figure Description

[0014] Figure 1 Specific chromatogram.

[0015] Figure 2 TQ08310 peak linear plot.

[0016] Figure 3 Comparison chart of column temperature change test results.

[0017] Figure 4 Comparison chart of flow velocity change test results.

[0018] Figure 5 Comparison chart of test results for wavelength change detection.

[0019] Figure 6 Comparison chart of mobile phase change test results. Specific Implementation

[0020] Example 1: Solvent Screening

[0021] Instruments: High-performance liquid chromatograph, electronic balance

[0022] Solvents: 10% acetonitrile, 30% acetonitrile, 50% acetonitrile, 10% methanol, 30% methanol, 50% methanol, methanol

[0023] Preparation of reference standard stock solution: Take an appropriate amount of TQ08310 reference standard, accurately weigh it, and dissolve it in 10% acetonitrile, 30% acetonitrile, 50% acetonitrile, acetonitrile, 10% methanol, 30% methanol, 50% methanol, and methanol respectively. Then dilute quantitatively to prepare a solution containing approximately 100µg of TQ08310 reference standard per ml.

[0024] Column: The column is packed with octadecylsilane-bonded silica gel.

[0025] Mobile phase: Acetonitrile-water (30:70); the mobile phase is used as a diluent.

[0026] Column temperature: 30℃; Detection wavelength: 230nm

[0027] Flow rate: 1.0 ml / min; Injection volume: 50 μl; Injection time: 35 min

[0028] Using the above chromatographic conditions, TQ08310 reference standard stock solutions dissolved in different solvents were injected to investigate the peak elution of TQ08310.

[0029] Conclusion: Under these chromatographic conditions, TQ08310 was converted into its degradation product and eluted after being dissolved in different solvents in the stock solution of the reference standard under these conditions; this method is not applicable.

[0030] Example 2 Solvent Screening

[0031] Instrument: Electronic balance

[0032] Solvents: n-hexane, cyclohexane, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate, dichloromethane

[0033] Preparation of test solutions: Take 1 mg, 5 mg, 10 mg and 20 mg of apromiscalcium and place them in 10 ml volumetric flasks respectively. Dissolve them by sonication in hexane, cyclohexane, isopropanol, diethyl ether, tetrahydrofuran, ethyl acetate and dichloromethane respectively, and observe the dissolution.

[0034] Conclusion: Apromiscalcium is not completely soluble in n-hexane, cyclohexane, and isopropanol at a concentration of 0.1 mg / ml; it is not completely soluble in diethyl ether at a concentration of 0.5 mg / ml; its solubility in ethyl acetate and dichloromethane reaches 0.5 mg / ml; and its solubility in tetrahydrofuran reaches more than 2 mg / ml.

[0035] Example 3 Solvent Screening

[0036] Instrument: Electronic balance

[0037] Solvents: Tetrahydrofuran, Ethyl acetate, Dichloromethane

[0038] Preparation of reference solution: Take 1 mg and 5 mg of TQ08310 reference standard and place them in 10 ml volumetric flasks respectively. Dissolve them by sonication in tetrahydrofuran, ethyl acetate and dichloromethane respectively, and observe the dissolution.

[0039] Conclusion: TQ08310 has a solubility of over 0.5 mg / ml in tetrahydrofuran, ethyl acetate, and dichloromethane.

[0040] Example 4: Determination of Solvent

[0041] Instruments: Ultraviolet spectrophotometer, electronic balance

[0042] Solvents: Tetrahydrofuran, Ethyl acetate, Dichloromethane

[0043] Preparation of reference solution: Take 5 mg of TQ08310 reference standard and place it in a 10 ml volumetric flask. Dissolve and bring to volume with tetrahydrofuran, ethyl acetate, and dichloromethane respectively by ultrasonication. Dilute with the corresponding solvent to a solution of 0.5 ug / ml and perform ultraviolet scanning at a wavelength of 230 nm.

[0044] Conclusion: TQ08310 exhibits maximum absorption at 230 nm. Comparing the absorbance in the three solvents at the same concentration, the absorbance of TQ08310 in tetrahydrofuran is significantly better than that in ethyl acetate and dichloromethane. Therefore, tetrahydrofuran is ultimately used as the dissolving solvent to obtain the optimal sensitivity.

[0045] Example 5 Selection of mobile phase ratio

[0046] Instruments: High-performance liquid chromatograph, electronic balance

[0047] Solvents: Tetrahydrofuran, n-hexane

[0048] Mobile phase: n-hexane:tetrahydrofuran 80:20, n-hexane:tetrahydrofuran 50:50, n-hexane:tetrahydrofuran 65:35, also used as solvent;

[0049] Chromatographic conditions: Polysaccharide derivatives were used as the packing material; column temperature was 40℃; flow rate was 1.0 ml / min; wavelength was 230 nm; and injection volume was 50 μl.

[0050] Preparation of reference solutions: Take 5 mg of TQ08310 reference standard and place it in separate 10 ml volumetric flasks. Dissolve it in tetrahydrofuran separately, and then dilute to the mark with n-hexane:tetrahydrofuran 80:20, n-hexane:tetrahydrofuran 50:50, and n-hexane:tetrahydrofuran 65:35 respectively. Accurately measure 1 ml of the solution and place it in a 100 ml volumetric flask. Dilute to the mark with n-hexane:tetrahydrofuran 80:20, n-hexane:tetrahydrofuran 50:50, and n-hexane:tetrahydrofuran 65:35 respectively.

[0051] Preparation of test solution: Take 5 mg of apromiscalcium and place it in a 10 ml volumetric flask. Dissolve it in tetrahydrofuran and dilute to the mark with hexane:tetrahydrofuran 80:20, hexane:tetrahydrofuran 50:50, and hexane:tetrahydrofuran 65:35 respectively.

[0052] Using the above chromatographic conditions, the solvent, reference solution, and test solution were injected with n-hexane:tetrahydrofuran 80:20, n-hexane:tetrahydrofuran 65:35, and n-hexane:tetrahydrofuran 50:50, respectively.

[0053] Conclusion: None of the three mobile phases used as solvents interfered with the detection of TQ08310 and apromis. With a mobile phase of hexane:tetrahydrofuran 80:20, TQ08310 eluted at 5.6 min, while apromis failed to elute. With a mobile phase of hexane:tetrahydrofuran 65:35, TQ08310 eluted at 3.2 min, and apromis at 23 min, with a resolution of 27. With a mobile phase of hexane:tetrahydrofuran 50:50, TQ08310 eluted at 2.4 min, and apromis at 6.0 min, with a resolution of 5. Due to the wider peak of apromis, a mobile phase with higher resolution, namely hexane:tetrahydrofuran 65:35, was selected for subsequent experiments.

[0054] Example 6 Investigation of the amount of tetrahydrofuran added

[0055] Instruments: High-performance liquid chromatograph, electronic balance

[0056] Solvents: Tetrahydrofuran, n-hexane

[0057] Mobile phase: n-hexane:tetrahydrofuran 65:35, also used as solvent;

[0058] Chromatographic conditions: Polysaccharide derivatives were used as the packing material; column temperature was 40℃; flow rate was 1.0 ml / min; wavelength was 230 nm; and injection volume was 50 μl.

[0059] Preparation of reference solution: Take 5 mg of TQ08310 reference standard and place it in a 10 ml volumetric flask. Add 3 ml, 4 ml, and 5 ml of tetrahydrofuran respectively to dissolve the standard. Then, dilute to the mark with n-hexane:tetrahydrofuran 65:35. Accurately measure 1 ml of the solution and place it in a 100 ml volumetric flask. Add n-hexane:tetrahydrofuran 65:35 and dilute to the mark.

[0060] Preparation of test solution: Take 5 mg of apromiscalcium and place it in a 10 ml volumetric flask. Add 3 ml, 4 ml and 5 ml of tetrahydrofuran respectively to dissolve it. Then, dilute to the mark with n-hexane:tetrahydrofuran 65:35.

[0061] Using the above chromatographic conditions, with n-hexane:tetrahydrofuran 65:35 as the mobile phase, the reference solution and the test solution were injected separately.

[0062] Conclusion: When apromisit was dissolved in 3 ml of tetrahydrofuran and then diluted to volume with n-hexane:tetrahydrofuran at a ratio of 65:35, turbidity occurred. Solutions prepared by dissolving TQ08310 and apromisit in 4 ml and 5 ml of tetrahydrofuran, respectively, and then diluting with n-hexane:tetrahydrofuran at a ratio of 65:35, both showed good peak elution. The experiment demonstrates that dissolving in 4 ml or 5 ml of furan can meet the experimental requirements.

[0063] Example 7: Investigation of solution stability

[0064] Instruments: High-performance liquid chromatograph, electronic balance

[0065] Reagents: Tetrahydrofuran, n-hexane, acetic anhydride

[0066] Solvents: n-hexane-tetrahydrofuran (65:35), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.1), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.2), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.5)

[0067] Chromatographic conditions: A mobile phase of n-hexane:tetrahydrofuran 65:35 was used, with polysaccharide derivatives as the packing material. The column temperature was 40℃, the flow rate was 1.0 ml / min, the wavelength was 230 nm, and the injection volume was 50 μl.

[0068] Preparation of reference solutions: Take 5 mg of TQ08310 reference standard and place it in a 10 ml volumetric flask. Dissolve it in 4 ml of tetrahydrofuran in each flask, and then dilute to the mark with n-hexane-tetrahydrofuran (65:35), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.1), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.2), and n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.5). Accurately measure 1 ml of the solution and place it in a 100 ml volumetric flask. Dilute to the mark with n-hexane-tetrahydrofuran (65:35), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.1), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.2), and n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.5).

[0069] Preparation of test solutions: Take 5 mg of apromiscalcium and place it in a 10 ml volumetric flask. Dissolve each flask in 4 ml of tetrahydrofuran. Then, add n-hexane-tetrahydrofuran (65:35), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.1), n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.2), and n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.5) respectively to the mark.

[0070] Using the above chromatographic conditions, the reference solution and the test solution were injected at different times.

[0071] Conclusions: For Apomitoxetine, the peak area showed no significant difference at different injection times in the four solvents. For TQ08310, the peak area showed a significant decreasing trend after different injection times in hexane-tetrahydrofuran (65:35) and hexane-tetrahydrofuran-acetic anhydride (65:35:0.1), but improved in hexane-tetrahydrofuran-acetic anhydride (65:35:0.1). For TQ08310, the peak area remained stable after different injection times in hexane-tetrahydrofuran (65:35:0.2) and hexane-tetrahydrofuran-acetic anhydride (65:35:0.5).

[0072] Example 8. Specificity of the method:

[0073] Chromatographic column: Using polysaccharide derivatives as packing material, Chiralpak IH (4.6×150mm, 5µm) or equivalent column.

[0074] Mobile phase: n-hexane-tetrahydrofuran (65:35); Column temperature: 30℃; Detection wavelength: 230nm

[0075] Flow rate: 1.0 ml / min; Injection volume: 50 μl; Injection time: 35 min

[0076] Blank solution: n-hexane-tetrahydrofuran-acetic anhydride (65:35:0.2) (solvent)

[0077] Accurately weigh appropriate amounts of apromisci and TQ08310 reference standards, dissolve them in 4 ml of tetrahydrofuran, and dilute with the solvent to prepare a mixed solution containing approximately 0.5 mg of apromisci and 1 µg of TQ08310 per ml. Record the chromatogram. The retention times of each component peak are shown in Table 1.

[0078] Table 1 Retention times of each substance

[0079]

[0080] Inject the test solution and record the chromatogram. The retention time and resolution data are shown in Table 2. Figure 1

[0081] Table 2 Retention time and resolution of each substance in the sample solution

[0082]

[0083] Conclusion: The test solution does not interfere with the detection of the analyte; the resolution between the Apomist TQ08310 peak and the Apomist peak should be greater than 1.5; the method has good specificity.

[0084] Example 9 Sample Injection Precision Detection

[0085] The chromatographic conditions were the same as in Example 8. An appropriate amount of TQ08310 reference standard was accurately weighed, dissolved in 4 ml of tetrahydrofuran, and quantitatively diluted with solvent to prepare a solution containing approximately 1 µg of TQ08310 reference standard per ml. This solution was used as the reference standard solution. The reference standard solution was injected five times consecutively, and the chromatograms were recorded. The peak area and retention time of TQ08310 were recorded for each injection, and the relative standard deviation was calculated. The experiment showed that the precision of this chromatographic system was good. The data are shown in Table 3.

[0086] Table 3 Precision Test

[0087]

[0088] Conclusion: After five consecutive injections of the TQ08310 reference solution, the peak area RSD of TQ08310 was 0.13%, indicating good precision; the peak retention time RSD of TQ08310 was 0.07%, which is less than 1.0%, indicating stable retention time. This method demonstrates good injection precision.

[0089] Example 10: Examination of Linear Relationships

[0090] The chromatographic conditions were the same as in Example 8. Appropriate amounts of the reference standard stock solution were accurately measured and placed in separate volumetric flasks. Solvents were added to prepare solutions containing 0.1 μg / ml, 0.4 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.2 μg / ml, 1.5 μg / ml, and 2.0 μg / ml of TQ08310, respectively. These solutions were injected separately, chromatograms were recorded, and peak areas were measured. Linear regression was performed on the peak area value (A) against the concentration (C), yielding a straight line. The results are shown in Table 4 and... Figure 2 .

[0091] Table 4 Linear Regression Data for TQ08310

[0092]

[0093] Conclusion: The linear equation for TQ08310 is y = 4.1200x - 0.0049. Within the concentration range of 0.1005~2.0097µg / ml, the peak area shows a good linear relationship with the concentration, with an R² of 0.9999 and an intercept deviation of -0.001, indicating a good linear relationship.

[0094] Example 11 Limit of Detection and Limit of Quantification

[0095] The chromatographic conditions were the same as in Example 8. The linear solution was accurately measured and serially diluted to appropriate factors before injection. The solution with a signal-to-noise ratio of approximately 10:1 was the limit of quantitation (LOQ) solution for TQ08310; the solution with a LQ / LOQ ratio of approximately 3:1 was the limit of detection (LOD) solution for TQ08310. The test results are shown in Tables 5 and 6.

[0096] Table 5 Results of Limit of Quantitation and Limit of Detection Tests

[0097]

[0098] Table 6 Results of Precision Test at the Limit of Quantification

[0099]

[0100] Conclusion: The limit of detection (LOD) of TQ08310 is 0.0502 μg / ml, equivalent to 0.010% of the sample concentration; the limit of quantitation (LOQ) is 0.1005 μg / ml, equivalent to 0.020% of the sample concentration; the RSD of the peak area of ​​the analyte solution for five consecutive injections is less than 10%, and the concentration of the analyte at the LOQ is not greater than the reporting limit. Under these chromatographic conditions, both the LOD and LOD meet the requirements.

[0101] Example 12 Repeatability Test:

[0102] The chromatographic conditions were the same as in Example 8. Six samples from the same batch were measured, and the relative standard deviations were obtained. The experimental results showed that the method had good reproducibility, as shown in Table 7.

[0103] Table 7 Repeatability Tests

[0104]

[0105] Conclusion: The absolute deviation of the content of each analyte in the six determinations was less than 20% of the quality standard limit, and the RSD of the content in the six determinations was 1.51%, which is less than 3.0%, indicating that the method has good repeatability.

[0106] Example 13 Recovery test:

[0107] The chromatographic conditions were the same as in Example 8. Samples from the same batch were added to volumetric flasks at amounts of 10%, 80%, 100%, and 120% of the TQ08310 limit, respectively. The solution was prepared according to the method described in the main text, and the chromatographic conditions were as described above. The recovery rate was calculated using the following formula. The experiment showed that the method has good accuracy, and the results are shown in Table 8.

[0108]

[0109] Table 8. Recovery Rate Test of TQ08310

[0110]

[0111] Conclusions: The recoveries of apromisc TQ08310 at the limits of quantitation were 96.53%–98.13%, with an average recovery of 97.38%; the recoveries at 80% concentrations were 100.24%–101.96%, with an average recovery of 100.88%; the recoveries at 100% concentrations were 100.11%–101.45%, with an average recovery of 100.88%; and the recoveries at 120% concentrations were 101.05%–102.36%, with an average recovery of 101.74%. The average recovery rate at all concentration levels was 100.22%, and the RSD% (n=24) was 1.8%. The recoveries at the limits of quantitation concentrations of apromisc TQ08310 were all between 70% and 130%, while the recoveries at other concentration levels were between 80% and 120%. The RSD for each concentration level was less than 10%, and the method accuracy was good.

[0112] Example 14 Durability Column Temperature Variation

[0113] The chromatographic conditions were the same as in Example 8, using the sample solution for testing. The column temperature was varied from 30°C to 25°C and 35°C, respectively. The test results are shown in Table 9. Figure 3 .

[0114] Table 9 Comparison of Column Temperature Change Test Results

[0115]

[0116] Conclusion: Under the above chromatographic conditions, the required separation effect can be achieved, and the detection deviation of TQ08310 meets the requirements. It can be seen that the column temperature variation within the allowable range of 25℃~35℃ has no effect on the separation and quantification of TQ08310.

[0117] Example 15 Durability Flow Rate Variation

[0118] The chromatographic conditions were the same as in Example 8, using the sample solution for testing. The flow rate was varied from 1.0 mL / min to 0.8 mL / min and 1.2 mL / min, respectively. The test results are shown in Table 10. Figure 4

[0119] Table 10 Comparison of Flow Velocity Change Test Results

[0120]

[0121] Conclusion: Under the above chromatographic conditions, the required separation effect can be achieved, and the detection deviation of TQ08310 meets the requirements. It can be seen that the variation of the flow rate within the allowable range of 0.8 mL / min to 1.2 mL / min has no effect on the separation and quantification of TQ08310.

[0122] Example 16 Durability Detection Wavelength Change

[0123] The chromatographic conditions were the same as in Example 8, and the test was performed using a spiked sample solution. The detection wavelength was changed from 230 nm to 225 nm and 235 nm, respectively. The test results are shown in Table 11. Figure 5

[0124] Table 11 Comparison of Test Results for Wavelength Change Detection

[0125]

[0126] Conclusion: Under the above chromatographic conditions, the required separation effect can be achieved, and the deviation of TQ08310 detection amount meets the requirements. It can be seen that the variation of detection wavelength within the allowable range of 225nm to 235nm has no effect on the separation and quantification of TQ08310.

[0127] Example 17 Durability Flow Phase Proportion Change

[0128] The chromatographic conditions were the same as in Example 9, and the test was performed using a spiked sample solution. The mobile phase ratio was changed from n-hexane-tetrahydrofuran (65:35) to n-hexane-tetrahydrofuran (63:37) and n-hexane-tetrahydrofuran (68:32), respectively. The test results are shown in Table 12. Figure 6

[0129] Table 12 Comparison of Flow Velocity Change Test Results

[0130]

[0131] Conclusion: Under the above chromatographic conditions, the required separation effect can be achieved, and the detection deviation of TQ08310 meets the requirements. It can be seen that the variation in the mobile phase ratio within the allowable range has no effect on the separation and quantification of TQ08310.

Claims

1. A method for determining the residual amount of easily hydrolyzable impurities in apromis, characterized in that, Includes the following steps: Reference solution: Take an appropriate amount of TQ08310 reference standard, accurately weigh it, add 4-5 ml of tetrahydrofuran to dissolve it, and then quantitatively dilute it with solvent to prepare a solution containing about 1 μg of TQ08310 reference standard per 1 ml. Test solution: Weigh 12.5 mg of apromisex precisely, place it in a 25 ml volumetric flask, add 4-5 ml of tetrahydrofuran to dissolve it, then dilute quantitatively with solvent to prepare a solution containing approximately 0.5 mg per ml, filter, and the solution is obtained. System suitability solution: Take appropriate amounts of apromisci reference standard and TQ08310 reference standard, accurately weigh them, add 4-5 ml of tetrahydrofuran to dissolve them, and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of apromisci and 1 μg of TQ08310 per ml; Chromatographic conditions: Solvent: n-hexane-tetrahydrofuran-acetic anhydride, volume ratio 65:35:0.2–0.5; Mobile phase: n-hexane-tetrahydrofuran, volume ratio 63:37–68:32; Flow rate: 0.8–1.2 mL / min; Column temperature: 25–35 °C; Detection wavelength: 225–235 nm. Chromatographic column: CHIRALPAK IH column with polysaccharide derivatives as packing material, 4.6 mm × 150 mm, 5 μm; Injection: Take 50 μl each of the reference solution, test solution, and system suitability solution, inject them into the liquid chromatograph, and record the chromatograms; The concentration of TQ08310 in each gram of apromisc must not exceed 0.1%, and the concentration range of TQ08310 is 0.1~2.0 μg / ml.

2. The method for determining the residual amount of easily hydrolyzable impurities in apromidt according to claim 1, characterized in that, The solvents are n-hexane-tetrahydrofuran-acetic anhydride, with a volume ratio of 65:35:0.

2.

3. The method for determining the residual amount of easily hydrolyzable impurities in apromidt according to claim 1, characterized in that, The mobile phase is n-hexane-tetrahydrofuran, with a volume ratio of 65:

35.

4. The method for determining the residual amount of easily hydrolyzable impurities in apromidt according to claim 1, characterized in that, The flow rate was 1.0 ml / min.

5. The method for determining the residual amount of easily hydrolyzable impurities in apromidt according to claim 1, characterized in that, The column temperature is 30℃.

6. The method for determining the residual amount of easily hydrolyzable impurities in apromidt according to claim 1, characterized in that, The detection wavelength is 230nm.

7. The method for determining the residual amount of easily hydrolyzable impurities in apromidt according to claim 1, characterized in that, The reference solution, test solution, and system suitability solution were prepared by dissolving them in 4 ml of tetrahydrofuran and then diluting them to volume with solvent.

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