Method for detecting related substances in key starting material A of enalastat

Abstract

The invention discloses a method for detecting related substances in a key starting material A of enalastat, and belongs to the technical field of analytical chemistry. The method comprises the following steps: preparing a system applicability solution, a test solution and a contrast solution; setting liquid chromatography detection conditions; and finally, respectively taking the system applicability solution, the test solution and the contrast solution, injecting into a liquid chromatograph, recording and analyzing a chromatogram, and calculating the content of each impurity. According to the method, the detection specificity is high, and known impurities and unknown impurities in the key starting material A of the enalastat can be effectively separated; the sensitivity is high, and the purity of the enalastat key starting material A and the content of various trace known impurities and unknown impurities in the enalastat key starting material A can be accurately quantified; the method fills the blank of detection of the purity of the key starting material A of the enalastat and the content of related substances in the key starting material A in the prior art, and provides a technical guarantee for quality control of an enalastat raw material medicine and a preparation thereof.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical chemistry, and particularly relates to a detection method for related substances in a key starting material A of enarodustat. BACKGROUND

[0002] Enarodustat, the INN name of which is Enarodustat, has a chemical name of N-[7-hydroxy-5-(2-phenylethyl)[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl]glycine, a molecular formula of C 17 H 16 N4O4, a molecular weight of 340.33, and a structural formula as shown in Formula I. As a HIF-PHI inhibitor of Me-Better, enarodustat has a significant therapeutic effect and smaller side effects compared to traditional EPO drugs, and can greatly improve the quality of life of patients while increasing the levels of HIF and erythropoietin; its preparation is mainly used in the clinical treatment of anemia in non-dialysis adult patients with chronic kidney disease (CKD), and the original preparation was first approved for listing in China in June 2023.

[0003]

[0004] The enarodustat bulk drug developed and produced by the applicant is obtained by using compound 5,7-dichloro-1,2,4-triazolo[1,5-a]pyridine-8-carboxylic acid methyl ester as a key starting material A, compound benzylmalonic acid diethyl ester as a key starting material B, and compound glycine methyl ester hydrochloride as a key starting material C, and sequentially performing chemical reactions such as a substitution reaction process, an ester hydrolysis and decarboxylation reaction process, an amide condensation reaction process, a hydrolysis and protonation reaction process, and a refining process. In the production and synthesis process of enarodustat, the purity and related substance level of the above key starting materials will directly affect the quality and stability of the bulk drug, including: ① the related substances existing in the starting materials may directly enter the intermediates or even the final bulk drug; ② the related substances existing in the starting materials may have side reactions with reaction reagents (such as catalysts and valuable reagents), producing new impurities with complex structures and being more difficult to remove; ③ insufficient purity of the starting materials may lead to incomplete main reactions, ultimately reducing the overall yield of the bulk drug and increasing the production cost; ④ if the purity fluctuation of the starting materials is large, different results may be produced under the same production process, so that the production process cannot be stably reproduced. Therefore, the detection and control of the purity and related substance level of the key starting materials are the first and most important gateway for the production of the bulk drug.

[0005] The compound 5,7-dichloro-1,2,4-triazolo[1,5-a]pyridine-8-carboxylic acid methyl ester is a key starting material A for producing the original drug of the company Enasidenib, and its English name is 5,7-dichloro-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid methyl ester, the molecular formula is C8H5Cl2N3O2, and the structural formula is shown as formula II. After searching, no related literature and reports on the detection method of the purity and related substances of the key starting material A (5,7-dichloro-1,2,4-triazolo[1,5-a]pyridine-8-carboxylic acid methyl ester) of Enasidenib have been found in the prior art. In order to strengthen the quality control of the key starting material A of Enasidenib, and further ensure the quality of Enasidenib, it is particularly important to provide a detection method of related substances in the key starting material A of Enasidenib.

[0006] SUMMARY

[0007] The technical problem to be solved by the present application is to provide a detection method of related substances in the key starting material A of Enasidenib, which is rapid, simple, effective, specific, has good separation degree, high sensitivity and good repeatability.

[0008] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0009] A detection method of related substances in the key starting material A of Enasidenib, which adopts liquid chromatography, comprising the following steps:

[0010] 1) Prepare a system suitability solution, a test sample solution and a control solution for standby;

[0011] 2) Set the liquid chromatography detection conditions: use a chromatographic column with C18 as the filler; the gradient elution conditions are: 0-15 min, the volume content of mobile phase A is 90%-70%, and the volume content of mobile phase B is 10%-30%; 15-42 min, the volume content of mobile phase A is 70%, and the volume content of mobile phase B is 30%; 42-42.1 min, the volume content of mobile phase A is 70%-90%, and the volume content of mobile phase B is 30%-10%; 42.1-50 min, the volume content of mobile phase A is 90%, and the volume content of mobile phase B is 10%;

[0012] 3) Take the system suitability solution, the test sample solution and the control solution respectively, inject them into the liquid chromatograph, record and analyze the chromatogram, and calculate the content of each impurity.

[0013] Further, in step 1), the preparation method of the system suitability solution is as follows: taking the starting material A and the known impurity control sample in a proper amount, accurately weighing, dissolving in dimethyl sulfoxide and diluting to prepare a mixed solution containing about 2.5 μg of impurities and 0.5 mg of starting material A per 1 ml, and shaking to obtain.

[0014] Further, in step 1), the preparation method of the test solution is as follows: taking the starting material A in a proper amount, accurately weighing, dissolving in dimethyl sulfoxide and diluting to prepare a solution containing about 0.5 mg per 1 ml, and shaking to obtain.

[0015] Further, in step 1), the preparation method of the control solution is as follows: accurately taking the test solution in a proper amount, diluting with dimethyl sulfoxide to prepare a solution containing about 5 μg per 1 mL, and shaking to obtain.

[0016] Further, in step 2), the chromatographic column is selected from one of Agilent ZORBAX SB-C18, 100*4.6mm, 3.5 μm, Agilent ZORBAX SB-C18, 150*4.6mm, 5 μm, and Waters ACQUITY HSS C18SB, 150*4.6mm, 5 μm.

[0017] Further, in step 2), the mobile phase A is selected from one of formic acid, sodium formate-formic acid buffer solution, and sodium acetate-acetic acid buffer solution, and the pH is 2.5-5.5; and the mobile phase B is acetonitrile.

[0018] Further, in step 2), the flow rate is 0.6-1.4 ml / min.

[0019] Further, in step 2), the column temperature is 25-40℃.

[0020] Further, in step 2), the detection wavelength is 218-222 nm.

[0021] Further, in step 2), the injection volume is 10 μL.

[0022] Compared with the prior art, the present application has the following advantages:

[0023] This invention provides a method for detecting related substances in key starting material A of enanasstat, employing liquid chromatography. This method can rapidly detect the content of related substances in key starting material A of enanasstat, exhibiting high specificity and effectively separating known and unknown impurities in key starting material A of enanasstat. It also possesses high sensitivity, accurately quantifying the purity of key starting material A of enanasstat and the content of trace known and unknown impurities. This method fills the gap in the prior art for detecting the purity and related substance content of key starting material A of enanasstat, providing technical support for the quality control of enanasstat raw materials and their preparations. Attached Figure Description

[0024] Figure 1 This is a chromatogram of the solution used to detect the suitability of the system in Example 1 of this application;

[0025] Figure 2 This is the chromatogram of the test solution in Example 1 of this application;

[0026] Figure 3 This is the chromatogram of the control solution used in Example 1 of this application;

[0027] Figure 4 This is a linear relationship diagram of starting material A and impurity 1 in Example 2 of this application;

[0028] Figure 5 This is a linear relationship diagram of starting material A and impurity 2 in Example 2 of this application;

[0029] Figure 6 This is a linear relationship diagram of starting material A and impurity 3 in Example 2 of this application;

[0030] Figure 7 This is a linear relationship diagram of starting material A in Embodiment 2 of this application;

[0031] Figure 8 This is a linear relationship diagram of impurity 26 in Example 2 of this application. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0033] The structural formulas of the relevant substances in starting material A of ennasstatin are as follows:

[0034]

[0035]

[0036] Example 1

[0037] A detection method of related substances in a key starting material A of enasidenib, comprising the following steps:

[0038] 1) Instrument and sample selection: the liquid chromatograph is a Thermo U3000; the starting material A batch number is ENST2411001, and the manufacturer is Yangzhou Plins Pharmaceutical Technology Co., Ltd.

[0039] 2) Set the liquid chromatography conditions: the chromatographic column is Agilent ZORBAX SB-C18 (100*4.6 mm, 3.5 μm); the mobile phase A is 0.02% formic acid aqueous solution, which is prepared by taking 0.2 ml of formic acid into 1 L of ultrapure water, mixing, ultrasonic for 5-10 min, and the pH is 3.6; the mobile phase B is acetonitrile; the flow rate is 1.0 ml / min; the column temperature is 35 DEG C; the detection wavelength is 220 nm; the injection volume is 10 μL; the solvent is dimethyl sulfoxide; and the gradient elution program is shown in Table 1:

[0040] Table 1 Gradient elution program

[0041] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 15 70 30 42 70 30 42.1 90 10 50 90 10

[0042] 3) Preparation of sample solution:

[0043] System suitability solution: take 10 mg of the starting material A and 5 mg of the known impurity control, accurately weigh, dissolve and dilute with dimethyl sulfoxide to prepare a mixed solution containing about 2.5 μg of impurities and 0.5 mg of starting material A per 1 ml, shake well, and obtain; wherein the known impurities include starting material A impurity 1, impurity 26, starting material A impurity 2 and starting material A impurity 3;

[0044] Test sample solution: take an appropriate amount of starting material A, accurately weigh, dissolve with dimethyl sulfoxide and quantitatively dilute to prepare a solution containing about 0.5 mg per 1 ml, shake well, and obtain;

[0045] Control solution: accurately take an appropriate amount of test sample solution, dilute with dimethyl sulfoxide to prepare a solution containing about 5 μg per 1 mL, shake well, and obtain.

[0046] 4) Sample detection method

[0047] Inject the system suitability solution, the test solution and the control solution according to the chromatographic conditions described in the method description, and record the chromatogram. In the chromatogram of the system suitability solution, the separation degree between the impurity 26 peak and the main peak should be not less than 1.5; in the chromatogram of the test solution, if there are impurity peaks, the content of impurity 26 is calculated by the main component self-control method with the corrected peak area (multiplied by the correction factor 1.8), the contents of the known impurities starting material A impurity 1, starting material A impurity 2, starting material A impurity 3 and other unknown impurities are calculated by the main component self-control method without correction factor; the impurity peak with a peak area less than 0.05 times (0.05%) of the main peak area of the control solution is ignored.

[0048] From the system suitability chromatogram, the separation degrees between starting material A impurity 1, impurity 26, starting material A impurity 2, starting material A impurity 3 and each known impurity, and between the main peak and the adjacent impurity peak are good, indicating that the method has good specificity. Figure 1 From the system suitability chromatogram, the separation degrees between starting material A impurity 1, impurity 26, starting material A impurity 2, starting material A impurity 3 and each known impurity, and between the main peak and the adjacent impurity peak are good, indicating that the method has good specificity.

[0049] Figure 2 From the system suitability chromatogram, the separation degrees between starting material A impurity 1, impurity 26, starting material A impurity 2, starting material A impurity 3 and each known impurity, and between the main peak and the adjacent impurity peak are good, indicating that the method has good specificity.

[0050] From the system suitability chromatogram, the separation degrees between starting material A impurity 1, impurity 26, starting material A impurity 2, starting material A impurity 3 and each known impurity, and between the main peak and the adjacent impurity peak are good, indicating that the method has good specificity. Figure 3 From the system suitability chromatogram, the separation degrees between starting material A impurity 1, impurity 26, starting material A impurity 2, starting material A impurity 3 and each known impurity, and between the main peak and the adjacent impurity peak are good, indicating that the method has good specificity.

[0051] Example 2

[0052] The detection method of Example 1 was verified for method validation, which was verified from specificity, system precision, repeatability, quantitative limit and detection limit, linearity and range, accuracy, and durability.

[0053] 1. Specificity

[0054] System suitability solution: Take starting material A 10 mg and known impurity control 5 mg, accurately weigh, dissolve and dilute with dimethyl sulfoxide to prepare a mixed solution containing about 2.5 μg of impurities and 0.5 mg of starting material A per 1 ml, shake well; wherein the known impurities include starting material A impurity 1, impurity 26, starting material A impurity 2, starting material A impurity 3.

[0055] Test solution: Take starting material A, accurately weigh, dissolve and quantitatively dilute with dimethyl sulfoxide to prepare a solution containing about 0.5 mg per 1 ml, shake well.

[0056] Precisely take the test solution and the system suitability solution, inject into the liquid chromatograph, record the chromatogram, and the results are shown in Tables 2-3.

[0057] Table 2 System suitability solution results​

[0058]

[0059]

[0060] Table 3 Test solution results

[0061]

[0062] From the results of Tables 2-3, the separation degree between each known impurity is greater than 1.5, the separation degree is good, the separation degree between the main peak and the adjacent impurity peak meets the requirements, indicating that the method has good specificity.

[0063] 2. System precision

[0064] Take the system suitability solution under the specificity item, inject it into the liquid chromatograph, continuously determine it for 6 times, record the chromatogram, and the results are shown in Table 4.

[0065] Table 4 System precision test results

[0066]

[0067]

[0068] From the results of Table 4, the system suitability solution is continuously injected for 6 times, the RSD of the peak area and the retention time of each known impurity and the main component is less than 2.0%, the separation degree is greater than 1.5, which meets the requirements, indicating that the system precision of the method is good.

[0069] 3. Reproducibility

[0070] Test solution: Take an appropriate amount of starting material A, accurately weigh and determine, dissolve with dimethyl sulfoxide and quantitatively dilute to prepare a solution containing about 0.5 mg per 1 ml. Prepare 6 parallel samples.

[0071] Control solution: accurately take an appropriate amount of test solution, dilute with dimethyl sulfoxide to prepare a solution containing about 5 μg per 1 mL, shake well, and get it. Prepare 6 parallel samples.

[0072] Accurately take the test solution and the control solution, inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Table 5.

[0073] Table 5 Reproducibility test results

[0074]

[0075] From the results of Table 5, impurities 1, impurities 2, impurities 3 and impurities 26 of starting material A are not detected in 6 test solutions of starting material A, there is no significant difference in the maximum unknown single impurity and total impurity content, indicating that the method has good reproducibility.

[0076] 4. Quantification limit and detection limit

[0077] An appropriate amount of impurity 1 of starting material, impurity 2 of starting material A, impurity 3 of starting material A and impurity 26 and starting material A were precisely weighed, respectively, and were added to a solvent to prepare a test sample solution with a certain concentration, and were gradually diluted until the signal-to-noise ratio (S / N) was 10, which was the quantification limit, and the signal-to-noise ratio (S / N) was 3, which was the detection limit. Six quantification limit solutions were prepared in parallel, and the results are shown in Tables 6-7.

[0078] Table 6. Results of quantification limit test

[0079]

[0080]

[0081] Table 7. Results of detection limit test

[0082]

[0083] As shown in Tables 6-7, the detection limit concentrations of impurity 1 of starting material A, starting material A, impurity 26, impurity 2 of starting material A and impurity 3 of starting material A were 0.07531 μg / ml, 0.02483 μg / ml, 0.09955 μg / ml, 0.3575 μg / ml and 0.3559 μg / ml, respectively; the quantification limit concentrations of impurity 1 of starting material A, starting material A, impurity 26, impurity 2 of starting material A and impurity 3 of starting material A were 0.1506 μg / ml, 0.04966 μg / ml, 0.1991 μg / ml, 0.7149 μg / ml and 0.7117 μg / ml, respectively; the RSD values of peak areas of each component in the six quantification limit solutions were less than 10%, which met the requirements.

[0084] 5. Linearity and range

[0085] Linear stock solution: an appropriate amount of impurity 1 of starting material A, starting material A, impurity 26, impurity 2 of starting material A and impurity 3 of starting material A reference substances were precisely weighed, respectively, and were added to a solvent to prepare a mixed solution containing about 30 μg of impurity 1 of starting material A, 30 μg of impurity 2 of starting material A, 30 μg of impurity 3 of starting material A, 50 μg of impurity 26 and 100 μg of starting material A per 1 mL, which was used as a linear stock solution. The linear series solutions were prepared according to Table 8.

[0086] Table 8. Preparation of linear solutions

[0087]

[0088] Take each linear series solution described above, inject liquid chromatograph, with concentration as abscissa, peak area as ordinate, do linear regression, results are shown in Tables 9-13 and Figures 4 to 8 .

[0089] Table 9 Starting material A impurity 1 linear test results

[0090]

[0091]

[0092] From Table 9 and Figure 4 The results show that the starting material A impurity 1 is linear in the range of 0.1506 μg / ml-3.012 μg / ml, the linear equation is y=0.6075x-0.0142, the correlation coefficient r is 0.9999, the intercept is less than 20% of the peak area of 100% label amount, and the linear relationship is good.

[0093] Table 10 Starting material A impurity 2 linear test results

[0094]

[0095] From Table 10 and Figure 5 The results show that the starting material A impurity 2 is linear in the range of 0.7149 μg / ml-2.860 μg / ml, the linear equation is y=0.6916x+0.0425, the correlation coefficient r is 0.9985, the intercept is less than 20% of the peak area of 100% label amount, and the linear relationship is good.

[0096] Table 11 Starting material A impurity 3 linear test results

[0097]

[0098] From Table 11 and Figure 6 The results show that the starting material A impurity 3 is linear in the range of 0.7117 μg / ml-2.847 μg / ml, the linear equation is y=0.4470x-0.0325, the correlation coefficient r is 0.9995, the intercept is less than 20% of the peak area of 100% label amount, and the linear relationship is good.

[0099] Table 12 Starting material A linear test results

[0100]

[0101]

[0102] From Table 12 and Figure 7The results show that the starting material A is linearly related in the range of 0.05236 μg / ml to 10.47 μg / ml, the linear equation is y=1.6056x+0.0189, the correlation coefficient r is 1.0000; the intercept is less than 20% of the peak area of 100% labeled amount; the linear relationship is good.

[0103] Table 13 Linear test results of impurity 26

[0104]

[0105] The results of Table 13 and Figure 8 The results show that the impurity 26 is linearly related in the range of 0.2508 μg / ml to 5.016 μg / ml, the linear equation is y=0.8714x-0.0034, the correlation coefficient r is 0.9999, the intercept is less than 20% of the peak area of 100% labeled amount, and the linear relationship is good.

[0106] 6. Accuracy

[0107] Mixed impurity control mother liquor: Take starting material A impurity 1, starting material A impurity 2, starting material A impurity 3 and impurity 26 control each appropriate amount, accurately weigh, add solvent to dissolve and dilute to prepare a mixed solution containing about 30 μg of starting material A impurity 1, 30 μg of starting material A impurity 2, 30 μg of starting material A impurity 3 and 50 μg of impurity 26 per 1 ml.

[0108] Test solution: Take starting material A, accurately weigh, add dimethyl sulfoxide to dissolve and quantitatively dilute to prepare a solution containing about 0.5 mg of starting material A per 1 ml.

[0109] Control solution: accurately measure the test solution, add solvent to dilute to prepare a solution containing about 5 μg per 1 ml.

[0110] Prepare the accuracy series solution according to the following Table 14.

[0111] Table 14 Preparation of accuracy solution

[0112]

[0113] 50% accuracy solution, 80% accuracy solution, 100% accuracy solution, 120% accuracy solution were prepared in triplicate, a total of 12 accuracy solutions were obtained, and 12 control solutions of accuracy solutions were prepared.

[0114] Accurately measure the mixed impurity solution, the control solution, the test solution and the accuracy solution, inject into the liquid chromatograph, record the chromatogram, and the recovery rate results are shown in Tables 15-18.

[0115] Table 15 Impurity 1 accuracy test results of starting material A

[0116]

[0117] Table 16 Impurity 26 accuracy test results

[0118]

[0119] Table 17 Impurity 2 accuracy test results of starting material A

[0120]

[0121] Table 18 Impurity 3 accuracy test results of starting material A

[0122]

[0123]

[0124] From the results of Tables 15-18, the average recovery of each impurity of the product at different concentrations is between 80% and 120%, and the RSD values meet the requirements, indicating that the method has good accuracy.

[0125] 7. Robustness

[0126] The effects of flow rate changes, column temperature changes, changes in the initial organic phase proportion of the mobile phase, acid concentration and type, wavelength changes, and changes in the chromatographic column on the detection results of related substances were investigated.

[0127] System suitability solution: 10 mg of starting material A and 5 mg of each known impurity reference substance were precisely weighed, dissolved and diluted with solvent to prepare a mixed solution containing about 2.5 μg of impurities and 0.5 mg of starting material A per 1 ml, and shaken to obtain the solution.

[0128] The system suitability solution was precisely measured, injected into the liquid chromatograph, and chromatograms were collected and recorded under different chromatographic conditions, as shown in Table 19. The preparation method of 0.1 mol sodium formate-formic acid buffer solution: 0.6 g of sodium formate and 3.0 ml of formic acid were weighed and dissolved in 1 L of ultrapure water, mixed, and ultrasonicated for 10-20 min, with a pH of 3.6; the preparation method of 0.1 mol sodium acetate-acetic acid buffer solution: 1 g of sodium acetate and 6.0 ml of acetic acid were weighed and dissolved in 1 L of ultrapure water, mixed, and ultrasonicated for 10-20 min, with a pH of 3.6.

[0129] Table 19 Robustness test results

[0130]

[0131]

[0132] From the results of Table 19, it can be seen that in the case where the chromatographic conditions are changed as described above, the resolution of each impurity in the system suitability solution meets the requirements, indicating that the method has good robustness.

[0133] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for detecting related substances in a key starting material A of enasidenib, characterized in that: Adopt liquid chromatography, including the following steps: ​ 1) preparation of system suitability solution, test solution and control solution, standby; 2) set liquid chromatography detection conditions: using C18 as the chromatographic column filler; Gradient elution conditions are: 0-15 min, the volume content of mobile phase A is 90%-70%, the volume content of mobile phase B is 10%-30%; 15-42 min, the volume content of mobile phase A is 70%, the volume content of mobile phase B is 30%; 42-42.1 min, the volume content of mobile phase A is 70%-90%, the volume content of mobile phase B is 30%-10%; 42.1-50 min, the volume content of mobile phase A is 90%, the volume content of mobile phase B is 10%; 3) respectively take system suitability solution, test solution and control solution, inject liquid chromatograph, record and analyze chromatogram, calculate the content of each impurity.

2. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 1), the preparation method of system suitability solution is: taking starting material A and known impurity control substance in appropriate amount, accurately weighing, dissolving and diluting to prepare a mixed solution containing about 2.5 μg of impurity and 0.5 mg of starting material A per 1 ml, shaking evenly.

3. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 1), the preparation method of test solution is: taking starting material A in appropriate amount, accurately weighing, dissolving and diluting to prepare a solution containing about 0.5 mg per 1 ml, shaking evenly.

4. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 1), the preparation method of control solution is: accurately taking test solution in appropriate amount, diluting with dimethyl sulfoxide to prepare a solution containing about 5 μg per 1 mL, shaking evenly.

5. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 2), the chromatographic column is selected from one of Agilent ZORBAX SB-C18, 100*4.6mm, 3.5μm, Agilent ZORBAX SB-C18, 150*4.6mm, 5μm, Waters ACQUITY HSS C18SB, 150*4.6mm, 5μm.

6. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 2), mobile phase A is selected from one of formic acid, sodium formate-formic acid buffer solution, sodium acetate-acetic acid buffer solution, with pH of 2.5-5.5; mobile phase B is acetonitrile.

7. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 2), the flow rate is 0.6-1.4 ml / min.

8. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 2), the column temperature is 25-40℃.

9. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 2), the detection wavelength is 218-222 nm.

10. The method for detecting related substances in the key starting material A of enasidenib according to claim 1, characterized in that: In step 2), the injection volume is 10 μL.

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