Method for detecting impurities of warning structure in ziprasidone

Abstract

The invention relates to a method for detecting warning structure impurities in ziprasidone. The detection method comprises the step of detecting two warning structure impurities in ziprasidone by using a high performance liquid chromatography-mass spectrometry (LC-MS) and using a methanol-neutral to weakly alkaline buffer salt solution as a flowing phase. According to the detection method, the impurity A higher than 0.38 ppm and the impurity B-ZZ1 higher than 1.77 ppm in ziprasidone can be quantitatively detected, the practicability is high, and the detection process is simple and rapid.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analytical chemistry and relates to a method for detecting the quality of ziprasidone, and specifically relates to a method for detecting warning structural impurities in ziprasidone by separating and determining them using high performance liquid chromatography-mass spectrometry (LC-MS). Background Art

[0002] Ziprasidone is a new atypical antipsychotic. In vitro studies have shown that ziprasidone has an effect on dopamine D2, D3, 5-hydroxytryptamine 5HT 2A , 5HT 2C , 5HT 1A , 5HT 1D Ziprasidone has high affinity for D2, 5HT, and α1-adrenergic receptors, and has moderate affinity for histamine H1 receptors. It has no affinity for other tested receptors / binding sites including M cholinergic receptors. 2A , 5HT 1D Receptor antagonist to 5HT 1A Ziprasidone can inhibit the synaptic reuptake of 5-hydroxytryptamine and norepinephrine. Studies have shown that its anti-schizophrenia effect may be exerted through antagonism of D2 and 5HT2 receptors.

[0003] The synthetic route of Ziprasidone patent document CN1635892A is: Summary of the invention

[0004] According to the analysis of the synthesis route of ziprasidone, there may be warning impurities A and B-ZZ1 in ziprasidone. Impurity A is introduced by the residue of the intermediate product, and impurity B-ZZ1 is introduced by the by-product of the reaction of intermediate product B.

[0005]

[0006] Currently, there are no reports on the detection method of warning structural impurities in ziprasidone. In order to ensure the safety, effectiveness and quality control of the produced ziprasidone products, a detection method was established to conduct quantitative control of the warning structural impurities A and impurity B-ZZ1 in ziprasidone.

[0007] The present invention provides an effective method for detecting warning structure impurities A and impurity B-ZZ1 in ziprasidone. There are two problems in the control of warning structure impurities in ziprasidone: 1. Ziprasidone and its warning structure impurities have poor solubility. According to existing literature reports, ziprasidone is only slightly soluble in dimethyl sulfoxide and N,N-dimethylformamide. Impurities A and impurity B-ZZ1 have similar solubility to ziprasidone, and it is impossible to enrich the impurity level in the test sample by liquid-liquid extraction or solid phase extraction; 2. The limit of warning structure impurities is low, and the ultraviolet detection method cannot meet the specificity and sensitivity requirements.

[0008] In order to solve the above technical problems, the present invention provides a method for detecting warning structural impurities in ziprasidone, wherein the detection method adopts high performance liquid chromatography-mass spectrometry, and the detection method comprises the following steps: (1) Separating ziprasidone from impurity A and impurity B-ZZ1 using a neutral to weakly alkaline buffer solution and a methanol system; (2) Preparation of test solution: Weigh the ziprasidone sample, dissolve the sample with N,N-dimethylacetamide, dilute with water, precipitate the sample, filter, and take the filtrate as the test solution for later use; (3) Preparation of mixed reference solution: Weigh appropriate amounts of impurity A reference substance and impurity B-ZZ1 reference substance respectively, dissolve them in N,N-dimethylacetamide, and dilute with 50% to 70% by volume of N,N-dimethylacetamide aqueous solution to prepare a mixed reference solution for later use; (4) Take equal amounts of the test solution and mixed reference solution and inject them into a high performance liquid chromatography-mass spectrometer for quantitative determination.

[0009] Optimally, in step (1), the neutral or weakly alkaline buffer salt solution can be an aqueous solution of ammonium formate, ammonium acetate or ammonium carbonate; optionally, the pH of the neutral or weakly alkaline buffer salt solution is 6-9; optionally, the pH can be adjusted to acidity with formic acid or acetic acid, or with aqueous ammonia to alkaline; the concentration of the neutral or weakly alkaline buffer salt solution is 2mM to 20mM, preferably 5mM; more preferably, it is a 5mM ammonium acetate solution.

[0010] Optimally, in step (1), the neutral or weakly alkaline buffered saline solution (5 mM ammonium acetate solution) is used as mobile phase A, and methanol is used as mobile phase B; preferably, gradient elution is performed; preferably, the procedure of gradient elution is:

[0011] Optimally, in step (2), the concentration of the test solution is 5 mg / ml to 20 mg / ml, optionally, 10 mg / ml.

[0012] Optimally, in step (2), the sample solution is prepared by using 50% to 70% by volume of N,N-dimethylacetamide to dissolve the sample, and using 30% to 50% by volume of water to dilute the sample solution to precipitate the sample, preferably, 50% by volume of N,N-dimethylacetamide and 50% by volume of water, respectively.

[0013] Optimally, in step (3), the concentration of each impurity in the mixed reference solution is 40 ng / ml to 160 ng / ml, and optionally, is 80 ng / ml.

[0014] Optimally, in step (4), 2 μl to 50 μl of the test solution and the mixed reference solution, optionally 20 μl each, are injected into the high performance liquid chromatography-mass spectrometry instrument.

[0015] Optimally, in step (4), the conditions of the quantitative detection method are: the chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler; the flow rate is 0.4 ml / min~0.6 ml / min; the column temperature is 35°C~45°C; the mass spectrometry conditions are: ESI negative ion source; selected ion detection mode; capillary voltage is 3000 V; fragmentation voltage is 70 V; m / z=208 ion is selected to detect impurity B-ZZ1, and m / z=242 ion is selected to detect impurity A.

[0016] Optimally, the detection limit of impurity A is 0.12ppm, and the quantification limit is 0.38ppm; the detection limit of impurity B-ZZ1 is 0.58ppm, and the quantification limit is 1.77ppm.

[0017] The invention has the advantages of simple operation, fast detection speed, short analysis time and high accuracy of detection result, and overcomes the difficulty that the existing analysis technology cannot detect the content of warning structure impurities in ziprasidone. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 What is shown is the HPLC-MS chart of the blank solution in Example 1; Figure 2 What is shown is the HPLC-MS chart of the reference substance solution in Example 1; Figure 3 The HPLC-MS diagram of the test solution in Example 1 is shown; Figure 4 The HPLC-MS graph of the spiked solution of the test sample in Example 1 is shown; Figure 5 Shown is the HPLC-MS graph of the sensitivity solution in Example 3; Figure 6 It shows the linear graph of impurity A in Example 3; Figure 7It shows the linear graph of impurity B-ZZ1 in Example 3; Figure 8 The HPLC-MS chart of the mixed solution in Comparative Example 1 is shown.

[0019] Fig. 9 The HPLC-MS chart of the mixed solution in Comparative Example 2 is shown. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0021] The present invention provides a method for detecting warning impurities A and impurity B-ZZ1 in ziprasidone, specifically a method for determining the warning structure in ziprasidone using high performance liquid chromatography-mass spectrometry. The sample is dissolved in N,N-dimethylacetamide, and then water is added to precipitate the sample. After filtering, low concentrations of ziprasidone warning impurities A and impurity B can be detected by LC-MS quantitative detection, which can further ensure the safety and quality controllability of the drug.

[0022] The method comprises: step (1) using a neutral to weakly alkaline buffer salt and a methanol system to separate ziprasidone and impurity A and impurity B-ZZ1; wherein the neutral to weakly alkaline buffer salt is prepared as follows: weighing 7.7 g of ammonium acetate, adding 100 ml of water to dissolve it, transferring 5 ml of the solution, adding 1000 ml of water to dilute it, and preparing a 5 mM ammonium acetate solution as mobile phase A; and taking pure methanol as mobile phase B.

[0023] Step (2) preparing the test solution: weigh 100 mg of ziprasidone sample, accurately add 5 ml of N,N-dimethylacetamide, ultrasonically dissolve the sample, accurately add 5 ml of water, shake well, and precipitate the sample. Centrifuge the solution at 10,000 r / min for 5 minutes, filter the supernatant with a 0.22 μm organic filter, and use the filtrate as the test solution for later use; (3) Preparation of mixed reference solution: Weigh 10 mg of impurity A and B-ZZ1 reference respectively, place in the same 10 ml volumetric flask, dissolve and dilute to the mark with N,N-dimethylacetamide, shake well, the concentration is about 1 mg / ml; accurately pipette 0.2 ml of the solution, place in a 10 ml volumetric flask, dilute to the mark with N,N-dimethylacetamide, shake well, the concentration is about 20 μg / ml; accurately pipette 0.4 ml of the solution, place in a 10 ml volumetric flask, dilute to the mark with N,N-dimethylacetamide, shake well, the concentration is about 800 ng / ml; accurately measure 0.2 ml of the solution, accurately add 0.8 ml of N,N-dimethylacetamide and 1.0 ml of water respectively, mix well, the concentration is about 80 ng / ml, and the diluent contains about 50 volume % N,N-dimethylacetamide; (4) Take 20 μl of the test solution and the mixed reference solution respectively, and inject them into the high performance liquid chromatography-mass spectrometer for quantitative determination. The quantitative detection method conditions are as follows: the chromatographic column is a chromatographic column filled with octadecylsilane bonded silica gel; the flow rate is 0.4 ml / min~0.6 ml / min; the column temperature is 35℃~45℃; the mass spectrometry conditions are: ESI negative ion source; selected ion detection mode; capillary voltage is 3000V; fragmentation voltage is 70V; select m / z=208 ion to detect impurity B-ZZ1, and select m / z=242 ion to detect impurity A.

[0024] The preferred chromatographic column is Agilent ZORBAX Extend C18, with a specification of 3.0×100mm and a filler particle size of 3.5μm; the flow rate is 0.5ml / min; the column temperature is 40℃; the mass spectrometry conditions are: ESI negative ion source; selected ion detection mode; capillary voltage is 3000V; fragmentation voltage is 70V; m / z=208 ion is selected to detect impurity B-ZZ1, and m / z=242 ion is selected to detect impurity A.

[0025] The contents of impurity A and impurity B-ZZ1 were calculated by peak area using the external standard method.

[0026] According to the above method, the warning impurity A and impurity B-ZZ1 in ziprasidone were detected. The detection limit of impurity A was 0.12ppm and the quantification limit was 0.38ppm; the detection limit of impurity B-ZZ1 was 0.58ppm and the quantification limit was 1.77ppm; impurity A had good linearity in the concentration range of 24.2ng / ml~120.8ng / ml, and impurity B-ZZ1 had good linearity in the concentration range of 25.0ng / ml~124.9ng / ml. This detection method was verified by tests such as specificity, detection limit and quantification limit, system precision, accuracy, and linearity, proving that this method is accurate and reliable for detecting the warning impurity A and impurity B-ZZ1 in ziprasidone.

[0027] The present invention is further described below by taking a chromatographic condition as an example. Unless otherwise specified, the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies. The instrument used in the following examples is an Agilent 1260 HPLC + 6120 MS liquid-mass spectrometer, configured with a quaternary pump, a binary pump, an automatic sampler, a column oven, an ultraviolet detector, and a single quadrupole mass spectrometer.

[0028] The reagents used are as follows: Methanol: chromatographic grade; Merck; Lot No: I1231507234 N,N-Dimethylacetamide: analytical grade; Aladdin; G2204089 Ultrapure water: Wahaha purified water Ammonium acetate: chromatographically pure; ≥98%; ACS Impurity A: Content calculated as 100%, provided by Chongqing Liujiang Pharmaceutical Technology Co., Ltd. Impurity B-ZZ1: Content calculated as 100%, provided by Chongqing Liujiang Pharmaceutical Technology Co., Ltd. The LC-MS analysis conditions of the following examples are as follows: (1) Chromatographic column: Agilent ZORBAX Extend C18, 3.0×100 mm, 3.5 μm (2) Chromatographic conditions: 5 mM ammonium acetate as mobile phase A; methanol as mobile phase B; flow rate of 0.5 ml / min; column temperature of 40 °C; injection volume of 20 μl; gradient elution according to the following table:

[0029] (3) Mass spectrometry conditions: electrospray ionization (ESI) negative ion source; selected ion detection mode (SIM); drying gas temperature of 350°C; drying gas flow rate of 12 L / min; nebulizing gas pressure of 45 Psig; capillary voltage of 3000 V; fragmentation voltage of 70 V; m / z = 208 ion was selected to detect impurity B-ZZ1, and m / z = 242 ion was selected to detect impurity A; mass spectrometry acquisition time was 6-8.5 min.

[0030] Example 1: Specificity experiment Investigate whether other components in the blank solution and test solution interfere with the chromatographic peaks of impurity A and impurity B-ZZ1.

[0031] Blank solution: Take 5 ml of N,N-dimethylacetamide and 5 ml of water respectively and mix well.

[0032] Mixed reference substance mother solution: Weigh 10 mg each of impurity A reference substance and impurity B-ZZ1 reference substance, place them in the same 10 ml volumetric flask, dissolve and dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 1 mg / ml; accurately pipette 0.2 ml of the solution, place it in a 10 ml volumetric flask, dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 20 μg / ml; accurately pipette 0.4 ml of the solution, place it in a 10 ml volumetric flask, dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 800 ng / ml.

[0033] Mixed reference solution: Accurately measure 0.2 ml of mixed reference stock solution, add 0.8 ml of N,N-dimethylacetamide and 1.0 ml of water respectively, and mix well. Test solution: Weigh 100 mg of ziprasidone sample, accurately add 5 ml of N,N-dimethylacetamide and ultrasonically dissolve the sample, accurately add 5 ml of water, shake well, and the sample precipitates. Centrifuge the solution at 10,000 r / min for 5 minutes, filter the supernatant with a 0.22 μm organic filter, and use the filtrate as the test solution.

[0034] Test sample spiked solution: Weigh 100 mg of ziprasidone sample, accurately add 4 ml of N,N-dimethylacetamide and 1 ml of mixed reference solution respectively, dissolve the sample by ultrasonication, accurately add 5 ml of water, shake well, and precipitate the sample. Centrifuge the solution at 10,000 r / min for 5 minutes, filter the supernatant with a 0.22 μm organic filter head, and use the filtrate as the test sample spiked solution.

[0035] From the attached Figure 1 -Attached Figure 4 As can be seen from the results in Table 1, there are no interfering peaks in the blank solvent at the m / z=242 channel and the m / z=208 channel, at the peak positions of impurity A and impurity B-ZZ1; impurity A is detected in the test solution, and it satisfies the baseline separation with the adjacent peaks, while impurity B-ZZ1 is not detected, and there are no adjacent interfering peaks. All meet the acceptable standards.

[0036] Table 1 Specificity results

[0037] Example 2: System Precision Experiment Take the mixed reference substance solution of Example 1 and inject it continuously for 5 times, and record the chromatogram. Figure 5 , the peak area RSD and retention time RSD of impurity A and impurity B-ZZ1 were calculated respectively; the retention time RSD of impurity A was 0.06% and the peak area RSD was 0.34% after 5 consecutive injections, and the retention time RSD of impurity B-ZZ1 was 0.08% and the peak area RSD was 2.75% after 5 consecutive injections. The retention time RSD was less than 2% and the peak area RSD was less than 10%, both of which met the acceptable standards.

[0038] Table 2 System precision results

[0039] Example 3: Linearity and detection limit Mixed reference solutions were prepared at 30%, 50%, 80%, 100%, and 150% of the limit as linear solutions. Different concentrations of linear solutions were sampled and tested, and the chromatograms were recorded. Linear regression analysis was performed with concentration (ng / ml) as the abscissa and peak area as the ordinate. The concentration and peak area results of impurity A and impurity B-ZZ1 are shown in Table 3, and the linearity graph is shown in Appendix Figure 6 and attached Figure 7The linear correlation coefficients R were all greater than 0.99, and the percentage of the Y-axis intercept to the 100% response value was less than 15%, which met the acceptable standards, indicating that the method had good linearity.

[0040] Table 3 Linearity results

[0041] The detection limit and quantification limit were calculated based on the standard deviation of the response value and the slope of the standard curve according to the formula LOD=3.3δ / S and LOQ=10δ / S. Where LOD: detection limit; LOQ: quantification limit; δ: deviation of the response value (standard deviation of the intercept of the standard curve); slope of the standard curve. The calculation results are shown in Table 4. The detection limit concentration of impurity A is 1.24ng / ml, which is equivalent to 0.12ppm of the sample concentration, and the quantification limit concentration is 3.76ng / ml, which is equivalent to 0.38ppm of the sample concentration, which is equivalent to 4.7% of the limit concentration; the detection limit concentration of impurity B-ZZ1 is 5.8ng / ml, which is equivalent to 0.58ppm of the sample concentration, and the quantification limit concentration is 17.7ng / ml, which is equivalent to 1.77ppm of the sample concentration, which is equivalent to 22.1% of the limit concentration. The quantification limits are all less than 30% of the limit concentration, which meets the acceptable standards.

[0042] Table 4 Calculation results of detection limit and quantification limit

[0043] Example 4: Accuracy Experiment Solution preparation: Mixed reference substance mother solution: Weigh 10 mg of impurity A and B-ZZ1 reference substance respectively, place in the same 10 ml volumetric flask, dissolve and dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 1 mg / ml; accurately pipette 0.2 ml of the solution, place in a 10 ml volumetric flask, dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 20 μg / ml; accurately pipette 0.4 ml of the solution, place in a 10 ml volumetric flask, dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 800 ng / ml.

[0044] Mixed reference solution: Accurately measure 0.2 ml of mixed reference stock solution, add 0.8 ml of N,N-dimethylacetamide and 1.0 ml of water respectively, and mix well. Test solution: Weigh 100 mg of ziprasidone sample, accurately add 5 ml of N,N-dimethylacetamide and ultrasonically dissolve the sample, accurately add 5 ml of water, shake well, and the sample precipitates. Centrifuge the solution at 10,000 r / min for 5 minutes, filter the supernatant with a 0.22 μm organic filter, take the filtrate as the test solution, and prepare 2 portions in parallel.

[0045] Test sample spiked solution: Weigh 100 mg of ziprasidone sample, accurately add 4 ml of N,N-dimethylacetamide and 1 ml of mixed reference solution respectively, dissolve the sample by ultrasonication, accurately add 5 ml of water, shake well, and precipitate the sample. Centrifuge the solution at 10,000 r / min for 5 minutes, filter the supernatant with a 0.22 μm organic filter head, take the filtrate as the test sample spiked solution, and prepare 5 portions in parallel.

[0046] The mixed reference solution, the test solution, and the test spiked solution were sampled and tested respectively, and the chromatograms were recorded. The recovery rates of impurity A and impurity B-ZZ1 were calculated by peak area according to the external standard method, and the results are shown in Tables 5 and 6. From the results in Tables 5 and 6, it can be seen that the recovery rate of impurity B-ZZ1 is between 91.1% and 97.4%, and the RSD is 2.48%; the recovery rate of impurity A is between 79.2% and 82.6%, and the RSD is 1.66%. The recovery rates are all between 70% and 125%, which meet the acceptable standards.

[0047] Table 5 Accuracy results of impurity A

[0048] Table 6 Accuracy results of impurity B-ZZ1

[0049] Example 5: Multiple batches of sample testing Solution preparation: Mixed reference substance mother solution: Weigh 10 mg of impurity A and B-ZZ1 reference substance respectively, place in the same 10 ml volumetric flask, dissolve and dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 1 mg / ml; accurately pipette 0.2 ml of the solution, place in a 10 ml volumetric flask, dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 20 μg / ml; accurately pipette 0.4 ml of the solution, place in a 10 ml volumetric flask, dilute to the scale with N,N-dimethylacetamide, shake well, the concentration is about 800 ng / ml.

[0050] Mixed reference solution: Accurately measure 0.2 ml of mixed reference stock solution, add 0.8 ml of N,N-dimethylacetamide and 1.0 ml of water respectively, and mix well. Test solution: Weigh 100 mg of ziprasidone sample, accurately add 5 ml of N,N-dimethylacetamide and ultrasonically dissolve the sample, accurately add 5 ml of water, shake well, and the sample precipitates. Centrifuge the solution at 10,000 r / min for 5 minutes, filter the supernatant with a 0.22 μm organic filter, take the filtrate as the test solution, and prepare 2 portions in parallel.

[0051] The mixed reference solution, test solution, and test spiked solution were sampled and tested respectively, and the chromatograms were recorded. The contents of impurity A and impurity B-ZZ1 were calculated by peak area using the external standard method. The results are shown in Table 7.

[0052] Table 7 Test results of multiple batches of samples

[0053] Comparative Example 1 This comparative example provides an LC-MS method for detecting the warning structure impurity A and the warning structure impurity B-ZZ1 in ziprasidone. The conditions of the comparative example LC-MS analysis method are as follows: (1) Chromatographic column: Shim Pack Velox Biphenyl 2.7 μm, 2.1 × 100 mm (2) Chromatographic conditions: 5 mM ammonium acetate as mobile phase A; methanol as mobile phase B; flow rate of 0.5 ml / min; column temperature of 40 °C; injection volume of 20 μl; gradient elution according to the following table:

[0054] (3) Mass spectrometry conditions: electrospray ionization (ESI) source; selected ion detection mode (SIM); drying gas temperature of 350°C; drying gas flow rate of 12 L / min; nebulizing gas pressure of 45 Psig; capillary voltage of 3000 V; fragmentation voltage of 70 V; m / z = 208 negative ion was selected to detect impurity B-ZZ1, m / z = 242 negative ion was selected to detect impurity A; m / z = 413 positive ion was selected to detect ziprasidone, and the mass spectrometry acquisition time was 5-20 min.

[0055] Solution preparation: Mixed solution: Weigh 10 mg each of impurity A reference substance, impurity B-ZZ1 reference substance and ziprasidone sample, place in the same 10 ml volumetric flask, dissolve and dilute to the scale with N,N-dimethylformamide, shake well, the concentration is about 1 mg / ml; accurately pipette 0.1 ml of the solution, place in a 10 ml volumetric flask, dilute to the scale with N,N-dimethylformamide, shake well, the concentration is about 10 μg / ml; accurately pipette 0.08 ml of the solution, place in a 10 ml volumetric flask, dilute to the scale with 50% volume of N,N-dimethylformamide aqueous solution, shake well, the concentration is about 80 ng / ml.

[0056] The mixed solution was sampled and tested. The results are shown in Table 8 and Appendix Figure 8 The retention time of ziprasidone, impurity A and impurity B-ZZ1 differed by more than 5 minutes, and the separation was good.

[0057] Table 8

[0058] Comparative Example 2 This comparative example provides an LC-MS method for detecting the analytical method of warning structure impurity A and warning structure impurity B-ZZ1 in ziprasidone, which is different from comparative example 1 only in that the mobile phase A is a 0.1% formic acid aqueous solution.

[0059] The mixed solution was sampled and tested. The results are shown in Table 9 and Appendix Fig. 9 , the retention time difference between ziprasidone and impurity A was less than 1 minute, and the separation between the two was significantly deteriorated.

[0060] Table 9

[0061] Based on the above comparative examples, it can be seen that the pH value of mobile phase A has a significant effect on the separation of ziprasidone and warning structure impurities and the response of impurity B-ZZ1. When the mobile phase A selects an acidic buffered salt solution, compared with the embodiment, the separation of ziprasidone and impurity A is poor, and the response of impurity B-ZZ1 is lower.

Claims

1. A method for detecting warning structural impurities in ziprasidone, characterized in that: The detection method adopts high performance liquid chromatography-mass spectrometry, and the detection method comprises the following steps: (1) Separating ziprasidone from impurity A and impurity B-ZZ1 using a neutral to weakly alkaline buffer solution and a methanol system; (2) Preparation of test solution: Weigh the ziprasidone sample, dissolve the sample with N,N-dimethylacetamide, dilute with water, precipitate the sample, filter, and take the filtrate as the test solution for later use; (3) Preparation of mixed reference solution: Weigh appropriate amounts of impurity A reference substance and impurity B-ZZ1 reference substance respectively, dissolve them in N,N-dimethylacetamide, and dilute with 50% to 70% by volume of N,N-dimethylacetamide aqueous solution to prepare a mixed reference solution for later use; (4) Take equal amounts of the test solution and mixed reference solution and inject them into a high performance liquid chromatography-mass spectrometer for quantitative determination.

2. The detection method according to claim 1, characterized in that: In the step (1), the neutral or weak alkaline buffer salt solution may be ammonium formate, ammonium acetate or ammonium carbonate aqueous solution; optionally, the pH of the neutral or weak alkaline buffer salt solution is 6-9; optionally, the pH is adjusted to acidity with formic acid or acetic acid, or with ammonia water; the concentration of the neutral or weak alkaline buffer salt solution is 2mM to 20mM, preferably 5mM; more preferably, it is 5mM ammonium acetate solution; Optionally, the neutral or weakly alkaline buffer salt solution is used as mobile phase A, and methanol is used as mobile phase B to perform gradient elution.

3. The detection method according to claim 1, characterized in that: In the step (2), the concentration of the test solution is 5 mg / ml to 20 mg / ml, optionally 10 mg / ml.

4. The detection method according to claim 1, characterized in that: In the step (2), the sample solution is prepared by using 50% to 70% by volume of N,N-dimethylacetamide to dissolve the sample, and using 30% to 50% by volume of water to dilute the sample solution to precipitate the sample, preferably, 50% by volume of N,N-dimethylacetamide and 50% by volume of water.

5. The detection method according to claim 1, characterized in that: In the step (3), the concentration of each impurity in the mixed reference solution is 40 ng / ml to 160 ng / ml, and optionally, is 80 ng / ml.

6. The detection method according to claim 1, characterized in that: In the step (4), 2 μl to 50 μl of the test solution and the mixed reference solution, optionally 20 μl each, are injected into the high performance liquid chromatography-mass spectrometry instrument.

7. The detection method according to any one of claims 1 to 6, characterized in that: In the step (4), the quantitative detection method conditions are: selecting m / z=208 ions to detect impurity B-ZZ1, and selecting m / z=242 ions to detect impurity A.

8. The detection method according to any one of claims 1 to 6, characterized in that: In the step (4), the quantitative detection method conditions are as follows: the chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler; the flow rate is 0.4 ml / min~0.6 ml / min; the column temperature is 35°C~45°C; the mass spectrometry conditions are: ESI negative ion source; selected ion detection mode; capillary voltage is 3000V; and fragmentation voltage is 70V.

9. The detection method according to any one of claims 1 to 8, characterized in that: The detection limit of impurity A is 0.12ppm and the quantification limit is 0.38ppm.

10. The detection method according to any one of claims 1 to 9, characterized in that: The detection limit of impurity B-ZZ1 was 0.58 ppm, and the quantification limit was 1.77 ppm.

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